Microcrystalline chitosan

Preparation of Microcrystalline chitosan(MCCh)/tricalcium phosphate complex withHydroxyapatite in sponge and fibre form for

hard tissue regeneration.

By

Luciano Pighinelli

Research Institute of Textile Chemistry/Physics, Dornbirn, Austria

Faculty of Chemistry and Pharmacy, Leopold-Franzens University of Innsbruck andInstitute of Biopolyners and Chemical Fibres, Lodz, Poland.

2012

ABSTRACT

Bone repair or regeneration is a common and complicated clinical problem in

orthopaedic surgery. The importance of natural polymers and calcium phosphates

composites has grown significantly over the last two decades due to their renewable and

biodegradable source, increasing the knowledge and functionality of composites in

technological and biomedical applications.

This work present chitosan and a new method to obtain derivatives such as

MCCh/ß-TCP complex with nano and micro size of calcium phosphates in different

forms (sponge, fibres), including chemical characterization, mechanical properties,

particle size, morphology, solution stability, biodegradation, bioactivity and also a new

method to obtain nanoceramic formation in chitosan salt solution.

All sponge preparations with MCCh/ß-TCP complex have a well-shaped 3-

dimensional interconnected and homogeneous pore structure with high porosity, to

ensure a biological environment conducive to cell attachment and proliferation

providing a passage of nutrient flow. In the fibre form, the presence of HAp/ -TCP

nanoparticles in the solution of chitosan has a beneficial effect on the production of

modified chitosan fibres with a lower cost of process, improving the mechanical

properties, such as tensile strenght, in wet conditions. The complex in sponge form is

susceptible to hydrolytic and enzymatic degradation with good mechanical properties

after 60 days of degradation, showing also a bacteriostatic activity and a bactericidal

activity against Escherichia coli and Staphylococcus aureus.

These materials can be used in the future for medical applications as a base for

scaffold production, as implants in regenerative medicine.

TABLE OF CONTENTS

Abstract……………………………………………………….……………...................2

Chapter 1 Introduction and literature review

1.1. Introduction……………………………………………………………08

1.2. Hard tissue regeneration………...……………..……………………..09

1.2.1. Bones histology and bone structure ………………….……………………...10

1.2.2. Tissue repair…………………………………………………………….…….13

1.2.3. Bone remodelling…………………………………..………………………....14

1.2.4. Regulations of bone cell function and bone turnover………………………15

1.2.5. Mechanical properties………………………………………………………..17

1.2.6. Biomaterials for hard tissue regeneration………………………………......18

1.3. Chitosan

1.3.1. Origin and general properties…………………………….….18

1.3.2. Microcrystalline chitosan…………………………………….24

1.3.3. Medical applications………………………………………….24

1.4. Calcium Phosphates

1.4.1. General properties of hydroxyapatite……………………….29

1.4.2. General properties of tricalcium phosphate………………...32

1.4.3. Medical applications………………………………………......36

1.4.4. References………………………………………………………37

1.5. Aim and scope of the thesis…………………………………………...48

Chapter 2 Properties of chitosan material

2.1. Introduction……………………………………………………….…..41

2.2. Materials……………………………………………………………....42

2.3. Methods

2.3.1 Preparation of chitosan hydrochloride salt…………………42

2.3.2. Analytical methods....................................................................43

2.3.3. Assessment of physical-mechanical properties of chitosan

hydrochloride salt in film form………………………………………………49

2.4. Results and discussions

2.4.1. Results of analytical methods of chitosan hydrochloride salt

solution…………………………………………………………………………49

2.4.2. Results for Mechanical properties from chitosan hydrochloride

salt in film form……………………………………………………………….50

2.5. Conclusions…………………………………………...……………….51

2.6. References…………………………………………………………......51

Chapter 3 Preparation of Microcrystalline chitosan (MCCh)/tricalcium phosphate

complex in sponge form.

3.1. Introduction………………………………………………………...….53

3.2. Materials…………………………………………………………...…..56

3.3. Methods

3.3.1 Schema of preparation MCCh/ tricalcium phosphate

complex………………………………………………………....56

3.3.2. Analytical methods…………………………………………….59

3.3.3. Preparation of MCCh/ ß-TCP composite in film form……...60

3.3.4. Preparation of MCCh/ ß-TCP complex in sponge form….....60

3.3.5. Infrared Spectroscopy of the complex………………………..60

3.3.6. Determination particles size, morphology of commercial ß-

TCP powder…………………………………………………………………………...61


3.4.1. Elaboration of the quantitative and qualitative MCCh/ ß-TCP

complex………………………………………………………………………………...61

3.4.2. FTIR study..................................................................................61

3.4.3. Particles size, morphology of commercial ß-TCP powder….67

3.4.4. SEM of composite MCCh/ ß-TCP complex in film form…...69

3.4.5. SEM of MCCh/ ß-TCP complex in sponge form…………….72

3.5. Conclusions…………………………………………………………….73

3.6. References……………………………………………………………...74


complex with Hydroxyapatite in sponge form.

4.1. Introduction............................................................................................78

4.2. Materials.................................................................................................84

4.3. Methods

4.3.1 Preparation of composite in sponge form................................84

4.3.2. Powder complex preparation....................................................85

4.3.3. SEM study...................................................................................86

4.3.4. Infrared Spectroscopy................................................................86

4.3.5. Determination of Ca and P in commercial HAp and ß-TCP

powders and inthe composites………………………………………………………..86

4.3.6. Determination of particles size of commercial HAp poder…87

4.3.7. WAXS- Diffraction of ß-TCP and HAp poder………………87

4.3.8. Mechanical properties of composites........................................87


4.4.1. Elaboration of the quantitative and qualitative of composites

in sponge form…………………………………………………………………………88

4.4.2. Composites with different ratios of ethanol to prepare sponge

form…………………………………………………………………………………….90

4.4.3. FTIR study of the commercial HAp………………………….91

4.4.4. FTIR study of the composites………………………………....93

4.4.5. WAXS investigation of ß-TCP and HAp powder…………... 95

4.4.6. Analysis of the particles size and morphology of the HAp

poder…………………………………………………………………………………...96

4.4.7. SEM study of the composites in sponge form……………..…98

4.4.8. Physical-mechanical tests of composite in sponge form…...105

4.4.9. Determination of Ca and P content in composites…………107

4.5. Conclusions...........................................................................................108

4.6. References.............................................................................................109


complex with Hydroxyapatite in fibre form.

5.1. Introduction..........................................................................................115

5.2. Materials...............................................................................................116

5.3. Methods

5.3.1 Methods of manufacture composite chitosan fibres………..116

5.3.2. Preparation of Chitosan Spinning Solution Containing HAp,

ß-TCP and HAp/ß-TCP Nanoparticles……………………………………………..117

5.3.3. Wet Spinning of Chitosan Fibres Containing HAp, ß-TCP

and HAp/ ß –TCP Nanoparticles…………………………………………………...118

5.3.4. Analytical Methods..................................................................118


5.4.1. Preparation of Chitosan Solutions Containing -TCP, HAp

and HAp/ -TCP………………………………………………………………...……119

5.4.2. Rheology of Chitosan Solutions Modified with HAp/ -TCP

Nanoparticles………………………………………………………………………...124

5.4.3. Investigation into the Spinning of Chitosan Fibres Modified

with HAp, -TCP and HAp/ -TCP…………………………………………………125

5.4.4. Mechanical Properties of Chitosan Fibres Modified with

HAp, -TCP and HAp/ -TCP……………………………………………...……….126

5.4.5. FTIR Examination of Chitosan Fibres Modified with HAp, -

TCP and HAp/ -TCP Nanoparticles………………………………………...……..127

5.4.6. Morphology and Chemistry of Chitosan Fibres Modified with

HAp, -TCP and HAp/ -TCP Nanoparticles………………………………………131

5.5. Conclusions...........................................................................................134

5.6. References.............................................................................................136

Chapter 6 Degradation and bioactivity of the composite

6.1. Introduction..........................................................................................141

6.2. Experimental Section

6.2.1. Materials...................................................................................143

6.3. Methods

6.3.1 Preparation of the MCCh/ß-TCP complex…………………143

6.3.2. Preparation of the composites in sponge form……………..143

6.3.3. SEM study of the composites in sponge form………………144

6.3.4. Mechanical properties..............................................................144

6.3.5. Assessment of the degradability of the composites……..….144

6.3.6. Bioactivity.................................................................................145

6.4. Results and discussions........................................................................145

6.4.1. Biodegradation mass loss........................................................146

6.4.2. SEM study of the composites in sponge form………………147

6.4.3. Mechanical properties of the sponges………………………151

6.4.4. Bioactivity.................................................................................152

6.5. Conclusions...........................................................................................153

6.6. References.................................................................................154

Conclusions...................................................................................................................158

1.1. INTRODUCTION

Every year, millions of people are suffering from bone disease cause by trauma,

tumor, bone fractures or defects and unfortunately some of them are dying due to

insufficient optimal bone substitute and/or treatment.

Much attention has been given to the use of different materials that could be

used as a base material for scaffold devices and as modification tools for currently used

biomedical devices that improve hard and soft tissue regeneration and/or reinforcement

efficacy, also to expand the feasibility of combined controlled drug release and tissue

engineering, tissue formation in regenerative therapy in the field of periodontics,

orthopaedics, cancer and plastic surgery, and veterinary [1,2,3,4]. According to a new

market research report, ‘Global Biomaterial Market’ (2009-2014), published by Markets

and Markets (http://www.marketsandmarkets.com), the total global biomaterial market

is expected to be worth US$58.1 billion by 2014, growing by 15.0% from 2009 to 2014.

The U.S. market is the largest geographical segment for biomaterials and is expected to

be worth $22.8 billion by 2014, with 13.6% from 2009 to 2014. Europe is the second

largest segment and is expected to reach $17.7 billion by 2014 with 14.6%, while the

Asian market size is estimated to increase by 18.2% from 2009 to 2014. The biomaterial

market today has already exceeded $28 billion [7]. Biomaterials are defined as natural

or man-made materials that are used directly as a supplement and/or a replacement for

functions of the living tissues in human body. Two important criteria that a biomaterial

is required to have are biocompatibility and biofunctionality [5,6,8].

The properties of bone in health and disease attract much attention. With an ever

greater proportion of population needing medical devices for hard tissue regeneration

and/or replacement, health systems of all countries are pressured. Musculoskeletal

disorder affected by aging, diseases, micro and/or macrofractures and demineralization

contributes and improves the suitability and development of new materials and methods

for hard tissue engineering.

1.2. Hard Tissue Regeneration

The properties of bone in health and disease justifiably attract much attention.

With an ever greater proportion of the population surviving into the third and fourth age

groups, the pressure on the health and welfare systems of our countries is set to rise

even further. Aging is a musculoskeletal disorder, which is expected to happen as an

underlying trend, but when combined with other skeletal complications, it compounds

problems even further. [8]

The wide range of biomaterials available is a reflection of the current diversity

related to the use of several materials, whether naturals and/or artificial, and as for the

various synthesis techniques or processing methodologies. This condition allows the

obtaining of materials with various macro and microstructures, which can cause

different mechanical, physical and chemical properties for the same material. Despite

the huge scientific production involving bone repair in tissue engineering, there is no

consensus defining the best technique yet, or the best synthesis or processing material,

considering the difficulty in reproducing the bone tissue properties as a whole.

Therefore, the knowledge of how the bone tissue reacts before a new material

and how this material behaves during the preparation is extremely important in new

technologies assessment. This chapter emphasizes concepts that serves as support for

this study, as the detail of the issues concerning to bone issues; natural polymers such as

chitin and later the chitosan, calcium phosphates and other materials that can be

relevantly used to obtain biomaterials to human tissue repair.

1.2.1. Bones histology and bone structure

The bone is a connective tissue characterized by a mineralized extracellular matrix,

hematopoietic and adipose tissue, blood vessels and nerves. [1]

Bone formation, or osteogenesis, occurs through osteoprogenitor cells that are

derivated of mesenchymal stem cells. These ones can differentiate into several types of

cells involved in bone physiology:

Osteocytes: is the mature bone cell wrapped up by bone matrix and responsible by

keep it, synthesizing it or absorbing it when needed. Each osteocyte occupies its place

histologically known as gap and project cellular extensions by canals that connect to

gaps beside, so that it can produce a network of interconnected cells. The lifespan of an

osteocyte is estimated in 25 years. [1.2]

Osteoblast: is a bone-forming differentiated cells, which produces the bone matrix

organic part composed of type I collagen, glycoproteins and proteoglycan (phosphate

concentrated). It also acts some way in the mineralization of bone matrix [1,2]

Osteoclast: it participates in the bone resorption and remodeling process. It is huge

and multinucleated cells, widely branched, derived from monocytes that cross the blood

capillaries. The activity of this cell is controlled by calcitonin and parathormone and it

is not embryologically related to osteoblasts, but to the mononuclear hematopoietic

progenitor cells, which are monocytes same lineage. [1,2,5].

Bone matrix: bone matrix consists of a fundamental substance, highly mineralized,

in which numerous collagen fibers are embedded, usually arranged in parallel

arrangement. In mature bone, the matrix is moderately hydrated, being 10% of its mass

the water; in its dry weight, 60% is composed of inorganic materials, mineral salts

(hydroxyapatite and amorphous calcium phosphate), 30% of collagen and protein and

carbohydrates, especially in conjunction with glycoproteins. The proportions of several

components vary with the age, location and metabolic condition. The aspect that

differentiates bone from other tissues is the mineralization of its matrix, producing an

extremely rigid tissue capable to provide support and protection to the organism. The

mineral that is deposited in this matrix is calcium phosphate in the form of

hydroxyapatite crystals in addition to phosphorus, and other mineral elements, which

may undergo mobilization of the bloodstream by bone matrix [1,2,5,7]. This

mechanism serves both to maintain bone function and to regulate calcium and

phosphorus concentrations in the organism, thus maintaining the homeostatic regulation

[6].

In the early stages of bone formation, prior to mineralization, the matrix is named

osteoid. In adult bones, there is a small amount of osteoid, reflecting the bone local

remodeling in which the mineralization is performed and rapidly the deposition of the

organic matrix occurs. In some diseases, such as rickets, the mineralization is impaired,

and the amount of osteoids is much higher to healthy parameters [3.4].

Histologically, there are two variants of the bone tissue: compact bone tissue (dense

and cortical) and trabecular bone tissue (spongy and marrow) containing the same types

of cells and intercellular substance, being different from each other by the arrangement

of these elements and amount of no marrow space. Because they are widely

vascularized and innervated, the bones have great capacity for regeneration. The bone

tissue receives vascularization of about 20% of cardiac output [1,2,3,6].

Figure 1 - structure of spongy bone [1].

The compact bone has no marrow space, but it has canals which protect nerves andblood vessels known as Haversian canals which are perpendicularly interconnected bycanals known as Volkmann, thus forming what is known as Harvers or Harversiansystem. Around each Harvers canal it is possible to observe several concentriclamellae of intercellular substance and bone cells, as shown in Figure 2 [1].

Figure 2-Structure of compact bone.

Concentric lamellae which circulate a Harvers canal determine the basic unit of

bone named osteon. The spongy bone is composed of large marrow spaces, giving the

appearance of porous bone, formed by spicules and trabeculae [1,2].

Compact Bone spongybone

Anatomically there are two types of bones: the ones that are longs as tibia, femur

and humerus, or the ones that are short and flat: skull, scapula, mandible and

hipbones [1,2,3].

The maturity of mineralized bone tissue is reached around 30 years old, starting

after this period a progressive loss of mineralized tissue and developing the disease

known as osteoporosis [4.9].

The membrane covering the bone is called periosteum and contains

osteoprogenitor cells and may undergo transformation to osteoblasts and to assist bone

repair. The bone itself has a structural architecture to support compressive forces. The

intraosseous vessels are protected by the rigid structure of the bone, avoiding trauma

such as bone infarction by extrinsic compression [6.10].

1.2.2. Tissue repair

Bone tissue is among the most specialized tissues in the body due the presence

of unique characteristics, which combines structural resistance to the ability for

regeneration. The bone shows an ability to repair from fractures without the presence of

any scar. The mechanism of this repair pattern is considered a summary of osteogenesis

occurred in the embryo during the growth period [1,2,3,6].

The bone repair is a regenerative process highly complex and essentially a

repetition of developmental events. This process has a lot of similarities with the repair

of soft tissues. Both of them show similar phases in cell replacement process:

inflammation, proliferation and remodeling phases. The cells involved in this process

include polymorph nuclear leukocytes, macrophages, fibroblasts, endothelial cells and

bone matrix. There is also the participation of different types of proteins and an active

genetic expression restoring the natural integrity of the bones [4,8,9]. When the bone

suffers any injury, blood vessels produce a local hemorrhage which leads to clot

formation, promoting an intense local inflammatory reaction. During repairing, the cells

and the battered bone matrix and the clot are removed by phagocytosis. Concomitantly,

the periosteum is responsible for proliferation of connective tissue fibroblasts and

osteoprogenitor cells will form a new cell tissue. After one week of the trauma the

tissue formed around the lesion is transformed into immature bone tissue by the

connective tissue cells change by means of osteoblasts which start the production of

bone matrix [3,5,6,]. The action of osteoclasts in bone repair has several simultaneous

aspects and stages. One of them relates to the occurrence of variation in the pressure

zone, where the levels of oxygen and carbon dioxide are different than usual, due to

compression of blood vessels or tearing of them due the trauma. This makes the

osteoclasts come from other adjacent areas or are inactive stimulated to undergo

processing in monocytes, osteoclasts [1,3,5,6].

The cells found in the early stages of repair processes contain a range of

chemical mediators that are stored within cells and released during this process. The

release of these agents causes changes in cell membrane and in the covering in ions

transport into the cell. The release of chemical agents stimulates the presence of

fibroblasts and endothelial cells at the injured site. Endothelial cells are essential for the

formation of new capillaries, which transport nutrients and remove metabolites in the

repairing tissue [1,3,6].

1.2.3. Bone remodelling

Throughout adult life, the skeleton undergoes a continuous repair and renewal

process. Bone remodelling is a surface-dependent phenomenon: the turnover rate in

trabecular bone may be up to ten times greater than in cortical bone, reflecting the

largest surface area presented by the former tissue. Mineralised bone matrix is resorbed

by osteoclasts and replaced in plywood-like layers, or lamellae, by groups of

osteoblasts. This sequence of events is tightly coordinated both temporally and spatially.

Under normal circumstances in young adults, remodelling activity keeps overall

bone mass relatively constant. However Aging, the menopause and many other

pathophysiological states can alter the balance of the turnover process, such that

reabsorption begins to outstrip formation, leading to net bone loss and ultimately

osteoporosis. This could be due not only to enhance osteoclastic resorption but also to

reduce osteoblastic function. Trabecular bone sites, for example, in the vertebral bodies

or in the ends of the long bones, are particularly susceptible to remodelling imbalances,

due to the relatively high turnover rate [4,7,9].

Bone growth, turnover and repair involve high levels of cellular activity, and

require an effective blood supply. This is in contrast with adult cartilage, an essentially

primitive, avascular tissue with low cellularity and turnover rates. The importance of the

vascular supply of bone, with its attendant network of fine nerve fibres is perhaps

insufficiently recognised. Disruption of the blood supply to bone, for example because

of inflammation, infection, tumours or fractures will result in hypoxia and acidosis, and

may have profound negative consequences [4,6,8].

1.2.4. Regulations of bone cell function and bone turnover

Some of the classical systemic actions of hormones on bone may be mediated at

tissue level via local production of growth factors and cytokines, and these effects may

be mediated in turn by agents such as prostaglandins. The systemic and local actions on

bone cells of simple inorganic moieties such as hydrogen ions and molecular oxygen

(Arnett et al, 2003), phosphate (Yates et al, 1991) or nitric oxide (Ehrlich & Lanyon,

2001) also appear to be of considerable importance [1,2,4,6,9].

The strain resulting from mechanical loading is a key regulator of remodelling in

some parts of the skeleton. The long bones and the vertebral bodies appear to require

modest but regular loading cycles in order to maintain their mass. The mass and

strength of bones in normal individuals is ultimately determined by the need to resist the

loads and deformations resulting from the most extreme normal activities (for example,

jumping off a wall 1-2 metres high on to a hard surface) [9].

Growth factors are a group of biological mediators which regulate important

cellular events in tissue repair and cell proliferation, including differentiation,

chemotaxis and matrix formation. The TGF-pi and TGF-P2 proteins distinguish among

growth and transforming factors (TGF) involved in connective tissue repair, in general,

and bone regeneration, being its most important function the chemotaxis and

mitogenesis of osteoblasts precursors and its ability to stimulate collagen matrix

deposition in wound and bone repair [3,6.9].

The tension on the tissue is an important factor to proliferation and evolution of

the tissue to be repaired. Considering the bone tissue reaction to the movement, it can be

stated that the relative motion causes a tension, thus generating a deformation up to the

tolerance levels supported by the tissue repair [1,2,3,9].

When the motion increases and tension overcome the limits of the tissue, a

instability condition can be generated. If the tension is low, there is insufficient

mechanical induction tissue to tissue differentiation; this can often result in grafted

material resorption, for example. Several studies have shown decreased bone mass \

calcium concentration in the bone in gravityless environments, confirming these

discoveries [4.6].

In recent decades, experimental studies of cell culture have attempted to define

which molecule or system is responsible for translating the language of physical

stimulus into biological language. It is important to emphasize that although these

studies explain isolated phenomena, it cannot be extrapolated to what happens to the

tissue the organism as a whole [9].

1.2.5. Mechanical properties

Bone is a damageable, viscoelastic composite and most of all a living material

capable of self-repair and thus exhibits a complex repertoire of mechanical properties.

From a mechanical perspective, the rigidity and strength of a structure is determined not

only by the amount of material but even more importantly by the arrangement of the

material in its space. The cortical structure and microstructure contribute to the whole

bone mechanical competence and weakness or fragility and the cortical thickness and

area are strong predictors of bone strength and resistance to damages or fractures [3,6].

Collagen fibrils are responsible for tensile strength and toughness, while the

crystalline structure provides compressive strength, and the microdamage accumulated

during the years may weaken cortical bone tissue and contributes to increased

susceptibility to fracture [7]. Mechanical properties of cortical bone depend on the size

and distribution of the mineral crystals. Crystal size increases by the addition of ions

and by the circulating proteins, as well as bone diseases, drugs, diet and age. Generally

the crystals are smaller in young bones and larger in mature bones. As we find these

bones in the same organism, this represents the optimal situation for good resistance to

load [3,7].

Age affects the biomechanical properties of bone in animals and humans.

However, the so-called Aging effect is of primary importance to humans who suffer the

effects of senescence as they survive into old age [8,10].

1.2.6. Biomaterials for hard tissue regeneration

The success of a bone scaffold as measured in vivo is determined by its ability to

stimulate and aid in both the onset and completion of bone defect repair. Because the

only control parameters that can be affected prior to implantation are the incorporation

of growth factors, cell seeding and architecture modification, optimization of the

scaffold must be completed prior to use and must encompass at the very least

mechanical stability for its load bearing application. This optimization requires a

complete knowledge of the system the scaffold will be interacting with [8-10].

1.3. Chitosan

1.3.1. Origin and general properties

Chitin it is a natural (polysaccharide), abundant and renewable biomaterial,

being the second most abundant in nature after cellulose. It is the primary structural

component of the outer skeletons of crustaceans, and of many other species such as

molluscs, insects and fungi. The role played by chitin is similar to those played by

cellulose in plants and collagen in higher animals. It is a reinforcing material, which

occurs in three polymorphic forms, , and -chitin. Where hardness is needed, -chitin

is found; where flexibility is required, and -chitin occur. Chitin is inert in aqueous

environment [24].

The chitin derivative is chitosan, which appears to be a good candidate for

wound-dressing and for hard and soft tissue regeneration. Chitosan is prepared from

chitin to obtain a more reactive polymer. In preparing chitosan, ground shells are treated

with alkali and acid to remove proteins and minerals, respectively, after which the

extracted chitin is deacetylated to chitosan by alkaline hydrolysis at high temperature.

Preparation of chitosan from crustacean-shell waste, for example, from shrimp

(Pandalus borealis), is economically feasible and ecologically desirable because large

amounts of shell waste are available as a product and/or waste of the food industry.

Production of chitosan from these is inexpensive and easy [23, 24]. Structures of

cellulose, chitin and chitosan are showed in Figure 3.

Figure 3. Structures of cellulose, chitin and chitosan [23].

Like cellulose, it is a glucose-based unbranched polysaccharide. It differs from

cellulose at the C-2 carbon by having an acetamido group instead of a hydroxyl group.

Chitosan is a partially deacetylated polymer of acetyl glucosamine obtained after

alkaline deacetylation of chitin. Polyaminosaccharides, especially chitosan (poly(ß-

(1,4)-2-amino-2-deoxy-D-glucopyranose)) and its derivatives, are characterized by

excellent biostimulating properties that facilitate reconstruction and vascularisation of

damage tissues, also compensate the shortcomings of cells components, which are

conductive for small scar forming. This cationic property is the basis of many of the

potential applications of chitosan that can be considered as a linear polyelectrolyte with

a high charge density which can interact with negatively charged surfaces, like proteins

and anionic polysaccharides [23, 24, 25, 26, 27, 28, 29].

Currently, chitosan is been used also in water treatment, cosmetic, drug and

medicine manufacturing, food additives, semi-permeable membranes and the

development of biomaterials. One of the most important limits to determine chitosan is

the degree of acetylation and the molecular weight, which vary in molecular weight

(from about 10,000 to 2 million Dalton) this characteristic is directly related to the

hydrogen bonding existing in this biopolymer, affecting its structure, solubility,

reactivity and the viscosity [15, 23, 24, 30, 31].

Chitosan as well as its derivatives like MCCh are often used for the preparation

of biodregradeble biomaterials. Chitosan of acetylation degree over 80% and average

molecular weight around 350kDa demonstrated the highest level of activity. In the

Figure 4, a comparation between structures of the chisotan and cellulose monomers is

shown.

Figure 4. Structure of glucosamine (chitosan monomer) and glucose (cellulose

monomer) [20].

Chitosan is insoluble at neutral and alkaline pH, but forms water-soluble salts

with inorganic and organic acids including glutamic, hydrochloric, lactic and acetic

acids. Upon dissolution in acidic media, the amino groups of the polymer become

protonated rendering the molecule positively charged. Recently, a defined degree of

deacetylation and depolymerization has been attached to chitosan derivatives as

important, because of their significantly different physicochemical properties [23, 24,

27]. The degree of acetylation represents the proportion of N-acetyl-D-glucosamine

units with respect to the total number of reactive units. The properties of chitosan (pKa

and solubility) can be modified by changing the degree of deacetylation and formulation

properties such as the pH and ionic strength. At neutral pH, most chitosan molecules

will lose their charge and precipitate from solution. Chitosan exhibits a variety of

physicochemical and biological properties, therefore it has found numerous applications

in various fields such as waste and water treatment, agriculture, fabric and textiles,

cosmetics, nutritional enhancement, and food processing. In addition to its lack of

toxicity and allergenicity, its biocompatibility, biodegradability and bioactivity make it a

very attractive substance for diverse applications as a biomaterial in pharmaceutical and

medical fields [23, 31, 32, 43].

Chemical derivatization of chitosan provides good materials for promoting new

biological activities and for modifing its mechanical properties. The primary amino

groups on the molecule are reactive and provide a mechanism for side group attachment

using a variety of mild reaction conditions. The general effect of addition of a side chain

is to disrupt the crystal structure of the material and hence to increase the amorphous

fraction. This modification generates a material with lower stiffness and often altered

solubility, but the precise nature of changes in chemical and biological properties

depends on the nature of the side group. In addition, the characteristic features of

chitosan such as being cationic, hemostatic and insoluble at high pH, can be completely

reversed by a sulfation process, which can render the molecule anionic and water-

soluble, and also introduce anticoagulant properties [15, 23, 24,36].

The variety of groups which can be attached to chitosan is almost unlimited, and

side groups can be chosen to provide specific functionality, change biological properties

or modify physical properties. Due to its high molecular weight and a linear unbranched

structure, chitosan is an excellent viscosity-enhancing agent in acidic environments. It

behaves as a pseudoplastic material exhibiting a decrease in viscosity with increasing

rates of shear. The viscosity of chitosan solution increases with an increase in chitosan

concentration, decrease in temperature and with increasing degree of deacetylation,

which is a structural parameter also influencing physiochemical properties such as the

molecular weight, the elongation at break and the tensile strength. Viscosity also

influences biological properties such as wound-healing properties and osteogenesis

enhancement as well as biodegradation by lysozyme [23,24, 30, 31, 35].

Chitosan, which is polycationic in acidic environments, possesses an ability to

form gels because it is hydrophilic and can retain water in its structure. The acetylation

of chitosan in hydroalcoholic media allows the selective modification of the free amino

groups and is responsible for a process of gelation. It has been shown that the charge

density of the chain segments is an essential parameter for the formation of gels and all

factors that lower this parameter favor deswelling and reversibility. The high hydration,

the physicochemical and physical properties, as well as the polyelectrolyte behavior of

this kind of gel allow applications such as bioactive dressing for wound healing. Gels

can also be used as a slow release drug-delivery system [23, 24, 30, 33].

The solubility of chitosan can be sharply reduced by cross-linking the

macromolecules with covalent bonds using for example glutaraldehyde. Swelling of the

films, for exemple, decreases when increase the amount of cross-linking agent added.

When chitosan is intended for contact with serous liquids, sterility becomes

necessary. Heat is often employed to facilitate polymer processing and to sterilize the

pharmaceutical and medical products. However, exposure to high temperatures can

change the physical properties of chitosan, affecting its aqueous solubility, rheology,

and appearance. Chitosan films were found to be less hydrophilic when autoclaved at

121 0C for 1h 30min. This provoque a reduction in solubility that was related to the

formation of the anhydrous crystal polymorph observed in chitosan samples heated in

the presence of water. Unlike gamma irradiation, which caused main chain scissions and

a dose dependent decrease in viscosity [10, 19, 23, 24, 31, 34, 36].

While increasing the ionic strength, the counter-ions would screen the

protonated amine group and make the molecule contracted. Strong intramolecular

hydrogen bonding was formed in solution because of the large number of OH and acetyl

groups in the chitosan molecular chains. Additionally, the hydrophobic properties in

chitosan, acetyl groups and glucosidic rings can play a significant role on aggregation in

the formation of hydrophobic interaction

The conformation changes of chitosan in solution are attributed to the intra/inter

molecular forces interaction, and thus suggesting that the conformation change has a

relation with surface tension, charge surface distribution. This cationic property is the

basis of many of the potential applications of chitosan that can be considered as a linear

polyelectrolyte with a high charge density which can interact with negatively charged

surfaces, like proteins. The superior tissue compatibility, biofunctionality and non-

antigenic property make chitosan and derivatives an ideal material for medical

application in tissue regeneration [6, 18, 24, 36, 37].

Polysaccharides such as chitosan, in particular, have some excellent properties.

They have one of the largest and widest ranges of medical applications:

nontoxicity (monomer residues are not hazardous to health), water solubility or high

swelling ability by simple chemical modification, stability to pH variations, and so on.

There are some disadvantages, such as low mechanical properties, temperature

and chemical instability, which, in some cases, can appear as an advantage [35, 36].

1.3.2. Microcrystalline chitosan

Struszczyk H. M. 2003, showed “the preparation of microcrystalline chitosan

(MCCh), according to Polish Patent P-281975, 1989”. MCCh is a modified chitosan

form elaborated based on the aminoglucose macromolecule aggregation method; it’s

characterized by special properties of initial chitosan such as biocompatibility,

bioactivity, non-toxic, hydrophility with same extraordinary behavior like direct film-

forming and creation of molecular and super-molecular structure during its

manufacture. This form of chitosan is very suitable in medical application, especially

for wound dressings and drug delivery. However, application of microcrystalline

chitosan form shows resistance to dissolution at neutral pH, as well as prolongation of

the biodegradation due to the relatively high crystallinity of the formed biocomposites

[8, 16].

1.3.3. Medical applications

Medical Applications and bioactivity of chitosan (being the result of several

properties: biodegradability, biocompatibility, antibacterial, antifungal and antiviral

activity, high adhesivity, film-fibre forming, non-toxicity, high miscibility, high

chemical reactivity, high ability for creation of hydrogen and ionic bonds,

biostimulation of natural resistance, by controlling and improving bioactivity) make this

biomaterial an excellent substrate. Those properties make it susceptible for preparation

and modification of a modern generation of scaffolds for tissue regeneration. The

application of chitosan depends upon the useful form of the copolymer for different

places to use [24, 36, 38, 39, 40, 42, 43].

The major limitations in the use of the chitin and chitosan for designing medical

devices are [40].

· the collection of the raw material;

· difficulty to obtain reproducible products with different raw materials;

· constantly high cost of production;

· the absence of validated process and products of biopolymer manufacture;

· no standardization of product quality and product assessment methods for chitin

and chitosan.

In orthopaedic uses, the enzymatic degradability associated to its structural

similarity to extracellular matrix glycosaminoglycans makes chitosan an attractive

biopolymer for bone tissue repair. Numerous bone filling materials have been

developed in which chitosan is used in combination with calcium phosphates,

essentially as a binding agent, or associated to biological molecules. Additionally, its

versatility to be processed into injectable, porous and membrane forms without using

toxic solvents makes chitosan an interesting material to be used as a non-protein

temporary scaffold, for bone regeneration. Presently, an increasing number of

anchorage-dependent cells, including bone cells, are being cultured on 2-D and 3-D

chitosan-based matrices, for regenerative therapies.

Biocomposities as polymeric materials used in implants with the criteria of the

proper choice of polymers. These criteria cover the structure of a polymer and other

materials, porosity and surface properties and biodegradability process; they make the

chitosan and calcium phosphates a good choice to work as a scaffold [5, 6, 19, 38, 44].

Due to its high molecular weight and a linear unbranched structure, chitosan

shows to be an excellent viscosity-enhancing agent in acidic environments. It behaves

as a pseudoplastic material exhibiting a decrease in viscosity with increasing rates of

shear. The viscosity of chitosan solution increases with an increase in chitosan

concentration, decrease in temperature and with increasing degree of deacetylation,

which is a structural parameter also influencing physiochemical property such as the

molecular weight, the elongation at break and the tensile strength. Viscosity also

influences biological properties such as wound-healing properties and osteogenesis

enhancement as well as biodegradation by lysozyme [3]. Table 1 shows the biomedical

applications and bioactive properties of chitosan and derivatives.

Table 1. Biomedical applications and bioactive properties of chitosan [3].

ARTIFICIAL SKIN

Surgical sutures

Artificial blood vessels

Controlled drug release

Contact lens

Eye humour fluid

Bandages, sponges

Burn dressings

Blood cholesterol control

Anti-inflammatory

Tumor inhibition

Anti-viral

Dental plaque inhibition

Bone healing treatment

Wound healing accelerator

Hemostatic

Antibacterial

Antifungal

Weight loss effect

Tissue regeneration is related with cellular interactions of chitosan with

mammalian tissues, which have been positive from the tissue repair and regeneration

standpoint. One of chitosan’s most promising features is its excellent ability to be

processed into porous structures for use in cell transplantation and tissue regeneration.

Porous chitosan structures can be formed by freezing and lyophilizing chitosan

solutions in suitable molds. The mechanical properties of chitosan scaffolds depend

largely on the pore sizes and pore orientations [3]. Such scaffolds can enhance bone

repair by supporting the proliferation of osteoblastic cells as well as their differentiation.

Table 2, collects the main characteristic properties of the natural biopolymers of

interest in this area: it should be underlined that chitosan has the capacity to form

complexes with both inorganic and biochemical substances. In turn, the inorganic

complexes favor correct biomineralization, and chitosan-glycosaminoglycan complexes

concentrate and retain growth factors.

Table 2. Favorable and unfavorable properties of natural biopolymers prepared for

applications in regenerative medicine (pharmaceutical and medical grades) [1].

Chitosan Unique cationic behavior. Hydrophilic surface promoting cell adhesion,

proliferation and differentiation. High filmogenicity. Good biocompatibility and

good host response. High biochemical significance in hemostasis, angiogenesis,

macrophage activation, fibroblast proliferation control. Biodegradability by

lysozyme and other enzymes. Bactericidal/bacteriostatic activity. Mechanical

weakness. Capacity to maintain a predefined shape after cross-linking.

Silk fibroin Slow degradability, versatility in processing, remarkable mechanical strength.

Genetically tailorable composition and aminoacid sequence. Residual sericin may

cause biocompatibility problems.

Collagen Low antigenicity and good cell-binding properties. Collagen type I (the most

abundant extracellular matrix protein) supports cell adhesion and proliferation;

integrin-mediated adhesion to collagen type I enhances osteogenic differentiation

of human bone marrow mesenchymal stem cells. Low biomechanical stiffness

and rapid biodegradation. mesenchymal

Hyaluronan Absence of immunogenic properties. Easy chain size manipulation. Interactions

with cell-surface receptors. Production through large-scale microbial

fermentation. Its anionic surface does not promote cell attachment and tissue

formation. Very soluble in water. Quick degradation by lysozyme and other

enzymes

Alginate Cross-linking under very mild conditions. Suitable for gel injection. Mechanical

weakness. Difficulties in handling and sterilization. Variety of Structures

Starch Inexpensive. In vivo degradation has not been fully assessed yet

Bacterial cellulose High purity, nanofibrous structure, high tensile strength and good

biocompatibility. Small pore size. Unclear in vivo behavior

Dextran Susceptible to chemical modification, suitable for designing of scaffolds with

specific sites for cell recognition. Shortcomings typical of hydrogels. Needs

modification to enhance cell adhesion

Polymer-hydroxyapatite blends have been reported to be easily handled during surgery,

a moldable or injectable material being more easily applied than pure hydroxyapatite powder or

granules. Major disadvantages of those biodegradable systems are their considerably inferior

mechanical strength, when compared to natural bone. This limits application to high load

bearing parts of the human skeleton [7]. Several new forms of chitosan-based dressings are

elaborated in form of hydrogels, films, microspheres, sponges and so on. One of the

most promising chitosan derivatives is MCCh that is a modified chitosan form

elaborated and based on the aminoglucose macromolecule aggregation method. This

form of chitosan is very suitable for medical application, especially for wound dressings

and drug-delivery [32, 37, 41, 42, 44].

1.4. Calcium Phosphates

1.4.1. Hydroxyapatite general properties

Calcium phosphates, particularly hydroxyapatite and beta-tricalcium phosphate,

as biomaterials had a significant application in the past and, in those days, as materials

for bone replacement. Its applications include, for example, prostheses for replacement,

materials coating or support in parts of the "Scaffold" [1, 2, 10, 11].

The bone consists of 69 wt% calcium phosphate (mainly hydroxyapatite), 21%

collagen, 9% water and 1% other constituents. It has a composite nature which is built

up of mainly ceramic (hydroxyapatite) and polymer (collagen), with a complex

hierarchical microstructure very difficult to mimic, which gives most of the superior

mechanical properties to bone [3].

Recent developments in artificial bone field include ceramics, which are bioinert

(such as alumina and zirconia), resorbable (such as tricalciumphosphate), and bioactive

(hydroxyapatite). Different phases of calcium phosphate ceramics are used depending

upon whether a resorbable or bioactive material is desired. Many applications in hard

tissue have been repoted in the past and continue to be nowadays, such as, for example,

replacements for hips, knees, teeth, tendon and ligaments and repair for peridontal

disease, maxillofacial reconstruction, augmentation and stabilization of the jaw bone,

spinal fusion and bone repair after tumor surgery, the tissue bonding between ceramic

and soft tissues besides the hard tissue can be notice using bioactive ceramics [11, 12].

The most common biomaterial used in the past years in hard tissue regeneration

was Hydroxyapatite (HAp), because it is the major inorganic compound in mammalian

hard tissue and is highly recognized and used for its biocompatibility, not expensive and

abundant. It has been incorporated into a wide variety of biomedical devices including

dental implants, biodegradable scaffolds, and other types of orthopaedic implants in

different parts of the skeleton [1,13, 14].

Hydroxyapaptite (Ca10(Po4)3OH), has been widely used in the present due to its

chemical similarity to bone and good biocompatibility in the physiological

environmental, as well as compatibility with synthetic and natural polymers such as

polysaccharides and/or proteins like collagen, creating a functional biomaterial for

medical and veterinary application. HAp has been shown to stimulate osteoconduction

and is a material that can be integrated into bone without provoking an immune reaction

[15, 16, 17, 18, 19]. The biological response to HAp implants is influenced by factors

such as the properties of the HAp powder including the grain size or any decomposition

of the HAp powder [15, 16, 17, 18, 19]. The structural configuration of HAp including

the size and morphology of the particles within the fabricated scaffold has been shown

to be critical in allowing osteoconduction and bone growth into the scaffolds whilst also

allowing the transfer of nutrients through the scaffold [15, 16, 17, 18].

The challenge of hard tissue engineering is to develop a suitable bone scaffold

with sufficient porosity and mechanical strength to allow cell adhesion, migration,

growth and proliferation resulting in good integration with surrounding tissues. A

number of materials have been used for bone tissue engineering, including synthetic and

natural polymers, bioglass and a variety of calcium phosphate ceramics [15, 17, 18].

Surface with a positive charge promotes cell adhesion due to its negative charge,

it is able to chemically bond with positively charged polysaccharides and/or HAp with

negative charge like proteins and/or another calcium phosphate as a -TCP, forming a

stronger scaffold material [20, 21].

The biodegradation of calcium phosphates, including HAp, may represent a

combination of the following items [1, 11, 21, 22 ] :

1 - physical: abrasion, fracture, disintegration, shape, porosity, surface area,

crystallinity and grain size.

2 - chemical: dissolution, local increase of Ca and P on the surface, composition

of the material.

3 - method: reduction of pH caused by cellular activity, resulting in increased

rate of degradation due to dissolution.

4 - biological: includes pH involving cell involvement, infections or diseases,

degree of bone contact, bone type, specimens, sex, age, hormonal and genetic [1].

Studies suggest that the mechanism of degradation of dense ceramics of calcium

phosphate which exhibit high crystallinity is mainly dissolution by extracellular fluids

[1].

It has been claimed that the behavior of the apatite family upon immersion in a

simulated body fluid was structure and composition dependent. The powders dissolution

rate is dependent largely on the crystallinity level, phase composition, microstructure,

surface area and density [7, 10].

1.4.2. General properties of tricalcium phosphate

Calcium phosphates are chemical compounds of special interest in many

interdisciplinary fields of science, including geology, chemistry, biology and medicine.

According to the literature, the initial attempts to establish their chemical

composition were performed by J. Berzelius in the middle of the 19th century and

constitute a major family of inorganic materials currently in use in dental and

orthopaedic reconstructive medicine, specifically; hydroxyapatite (HAp) and -

tricalcium phosphate (ß-TCP) were developed as bioceramics in the early 1980s and,

nowadays, are the most common calcium phosphates used in medical applications.

Despite their relative importance, both ceramics show a number of drawbacks

that reduce their clinical performance. The biodegradation of HAp in physiological

environments is too low to achieve the optimal formation of bone tissue.

On the other hand, ß-TCP shows fast release of Ca2+ and PO43- ions when

exposed to physiological fluids and could be considered as bioactive [3, 19, 20].

The biological performance of biphasic mixtures is controlled by the dissolution

profile of the mixture. Selecting the appropriate blend of both calcium phosphates, the

mixture gradually dissolves in the physiological environment, releasing Ca2+ and PO43-

ions and inducing the bioactive behavior. The material that remains during dissolution

acts as a template for the newly formed bone. Tricalcium phosphate is called a

resorbable ceramic, and it is believed that it binds to bone and then is resorbed and

replaced by bone. It has been reported that the bioresorbability is due to dissolution and

phagocytosis. It has been considered that -TCP makes contact with bone directly,

suggesting mainly mechanical bonding. Synthetic -tricalcium phosphate (ß-TCP),

Ca3(PO4)2, is a material with a high potential for bioapplications. In particular,

composites made of ß-TCP and HA, the so-called biphasic calcium phosphates (BCPs),

which combine the excellent bioactivity of HA with the good bioresorbability of ß-TCP,

are interesting candidates for medical applications such as bone replacement [1,5] .

By definition, all calcium orthophosphates consist of three major chemical

elements: calcium (oxidation state +2), phosphorus (oxidation state +5) and oxygen

(reduction state. – 2), as a part of orthophosphate anions. These three chemical elements

are present in abundance on the surface of our planet: oxygen is the most widespread

chemical element of the Earth's surface (~ 47 mass %), calcium occupies the fifth place

(~ 3.3 – 3.4 mass %) and phosphorus (~ 0.08 .– 0.12 mass %) is among the first twenty

chemical elements most widespread on our planet [20]. In addition, the chemical

composition of many calcium orthophosphates includes hydrogen, either as part of an

acidic orthophosphate anion (for example, HPO42- or H2PO4-), hydroxide (for example,

Ca10(PO4)6(OH)2) and/or incorporated water (for example, CaHPO4·2H2O). Diverse

combinations of CaO and P2O5 (both in the presence of water and without it) provide a

large variety of calcium phosphates, which are distinguished by the type of the

phosphate anion: ortho- (PO43-), meta- (PO3

-), pyro- (P2O74-) and poly- ((PO3)n

n-). In

the case of multi-charged anions (orthophosphates and pyrophosphates), calcium

phosphates are also differentiated by the number of hydrogen ions attached to the anion.

Examples include mono- (Ca(H2PO4)2), di- (CaHPO4), tri- (Ca3(PO4)2) and tetra-

(Ca2P2O7) calcium phosphates [19, 20, 21].

-TCP ( -tricalcium phosphate, -Ca3(PO4)2; the chemically correct name is

calcium phosphate tribasic beta) cannot be precipitated from aqueous solutions. It is a

high temperature phase, which can only be prepared at temperatures above 800 ºC by

thermal decomposition of CDHA or by solid-state interaction of acidic calcium

orthophosphates, e.g., DCPA, with a base, e.g., CaO. Apart from the chemical

preparation routes, ion-substituted -TCP can be prepared by calcining of bones: such

type of -TCP is occasionally called. “bone ash”. In -TCP, there are three types of

crystallographically nonequivalent PO43- groups located at general points of the crystal,

each type with different intratetrahedral bond lengths and angles. At temperatures above

~ 1125 ºC, -TCP transforms into a high-temperature phase -TCP.

Being the stable phase at room temperature, -TCP is less soluble in water than

-TCP. Furthermore, the ideal structure contains calcium ion vacancies that are too

small to accommodate calcium ions, but allow for the inclusion of magnesium ions,

which thereby stabilize the structures. Pure -TCP never occurs in biological

calcifications. In biomedicine, -TCP is used in calcium orthophosphate bone cements.

In combination with HA, -TCP forms a biphasic calcium phosphate that are

widely used as a bone substitution bioceramics. Pure -TCP is added to some brands of

toothpaste as a gentle polishing agent. Multivitamin complexes with calcium

orthophosphate are widely available in the market and -TCP is used as the calcium

phosphate there. In addition, it serves as a texturizer, bakery improver and anti-

clumping agent for dry powdered food (flour, milk powder, dried cream, cocoa

powder). In addition, -TCP is added as a dietary or mineral supplement to food and

feed, where it is marked as E341 according to the European Classification of Food

Additives. Occasionally, it might be used as inert filler in pelleted drugs. Other

applications comprise porcelains, pottery, enamel, using as a component for mordants

and ackey, as well as a polymer stabilizer [19, 20, 21].

ACP (amorphous calcium phosphate, CaxHy(PO4)z·nH2O, n = 3 .– 4.5; 15 –

20% H2O) is often encountered as a transient phase during the formation of calcium

orthophosphates in aqueous systems. Usually, ACP is the first phase precipitated from a

supersaturated solution prepared by rapid mixing of solutions containing ions of

calcium and orthophosphate. ACP is thought to be formed at the beginning of the

precipitation due to a lower surface energy than that of OCP and apatites. The

amorphization level of ACP increases with the concentration increasing of Ca+2 and

PO4- containing solutions, as well as at a higher solution pH and a lower crystallization

temperature. A continuous gentle agitation of as precipitated ACP in the mother

solution, especially at elevated temperatures, results in a slow recrystallization and

formation of better crystalline compounds, such as CDHA. The lifetime of ACP in

aqueous solution was reported to be a function of the presence of additive molecules

and ions, pH, ionic strength and temperature.

In medicine, pure ACP is used in calcium orthophosphate cements and as a

filling material in dentistry. Bioactive composites of ACP with polymers have

properties suitable for use in dentistry and surgery. Due to a reasonable solubility and

physiological pH of aqueous solutions, ACP appeared to be consumable by some

microorganisms and, due to this reason; it might be added as a mineral supplement to

culture media. Non-biomedical applications of ACP comprise its using as a component

for mordants and ackey. In food industry, ACP is used for syrup clarification.

Occasionally, it is used as inert filler in pelleted drugs. In addition, ACP is used

in glass and pottery production and as a raw material for production of some organic

phosphates. For further details on ACP, interested readers are referred to specialized

reviews [19, 20, 21].

1.4.3. Medical applications

These substrates, when modified by the addition of cells or biomolecules, are

able to stimulate regeneration in a shorter time; they can function as hybrid materials,

further enhancing in vivo tissue formation femur, knee, teeth, tendons, ligaments,

materials for repairs due to problems of periodontics, neurosurgery and for filling bone

cavities after tumour surgery [2, 19, 20].

These substrates or supports can give two mechanisms to improve the

regeneration [1] :

1 - give support permissive for cell migration and adhesion and growth outside the

host;

2 - as a vehicle for controlled release of drugs that promote growth and survival

during regeneration.

Bone has a varied arrangement of material structures at many length scales

which work in concert to perform diverse mechanical, biological and chemical

functions; such as structural support, protection and storage of healing cells, and

mineral ion homeostasis. Scale is important to determine the ideal scaffold bone

architecture as the structure of the natural bone hierarchical and complex structure, in

Table 3, showed the the most used calcium phosphates for medical applications [33, 21,

23]. The calcium phosphates are fully supported by the physiological environment in

bone replacement, enabling rates of resorption and replacement very favorable. HAp is

more similar to natural bone than other calcium phosphate like -TCP, (Ca3(Po4)2);

however, the resorption of HAp is extremely low in comparation to ß-TCP [7, 21].

Table 3. existing calcium orthophosphates and their major properties [19].

[a] These compounds cannot be precipitated from aqueous solutions. [b] Cannot be measured precisely. However, the following values were found: 25.7±0.1 (pH = 7.40), 29.9±0.1 (pH = 6.00), 32.7±0.1 (pH = 5.28). The comparative extend of dissolution in acidic bufferis:ACP>> -TCP>> -TCP>>CDHA>>HA>>FA. [c] Stable at temperatures above 100°C.

[d] Always metastable. [e] Precipitated HÁ (PHA). [f] Existence of OA remains questionable.

1.4.4. References

1. Riccardo A.A. Muzzarelli (2011). Chitosan composites with inorganic,

morphogenetic proteins and stem cells for bone regeneration, Carbohydrate

Polymers 83, 1433-1445

2. Misiek D J, Kent JM, Carr RF. (1984). Soft tissue responses to hydroxyapatite

particles of different shapes. J Oral Maxillof Surg;42:150-60.

3. Oktay Yildirim. (2004). Preparation and Characterization of Chitosan /Calcium

Phosphate Based Composite Biomaterials, Master Of Science Dissertation, zmir

Institute of Technology zmir, Turkey.

4. Vert M, Li MS, Spenlehauer G, Guerin P. (1992). Bioresorbability and

biocompatibility of aliphatic polyesters. J Mater Sci;3:432-46.

5. Sevda Snel, Susan J. McClure. (2004). Potential applications of chitosan in

veterinary medicine, Advanced Drug Delivery Reviews 56, 1467- 1480.

6. Dorozhkin Sergey V. (2009). Review Nanodimensional and Nanocrystalline

Apatites and Other Calcium Orthophosphates in Biomedical Engineering,

Biology and Medicine, Materials, 2, 1975-2045.

7. Wilmington, Delaware, September 1 /PRNewswire/ - New market research

report, 'Global biomaterials Market (2009-2014)', published by Markets and

Markets(http://www.marketsandmarkets.com),(http://www.prnewswire.co.uk/cgi

/news/release?id=264557).

8. Muzzarelli C., Riccardo A.A. Muzzarelli. (2002). Natural and artificial chitosan-

inorganic composites, Journal of Inorganic Biochemistry 92 89-94.

9. Junqueira LC, Carneiro J, Long JA. (1986). Bone, Basic Histology. 5th Ed.

Norwalk, Conn: Appçeton-Cenury-Crofts;140-65.

10. Adler C. Bones and bone tissue: normal anatomy and histology. Bone Diseases.

2000. New York, NY: Springer-Verlag; 1-30.

11. McCarthy EF, Frassica FJ. Anatomy and physiology of the bone. Pathology of

Bone and Joint Disorders. Philadelphia, Pa: WB Saunders; 1998: 25-50

12. Mundy GR. (1987). Bone resorption and turnover in health disease. Bone;8

(suppl 1):S 9-16. [medline]

13. McHugh KP, Shen Z, Crotti TN. (2007). Role of cell-matrix interactions in

osteoclastc differentiation. Adv Exp Med Biol.;602:107-11. [medline]

14. Marks SC Jr, Propoff SN. (1988). Bone cell biology: the regulation of the

development, structure and function in the skeleton. Am J Anat.183(1):1-44.

[medline]

15. Leblond CP. (1989). Synthesis and secretion of collagen by cells of connective

tissue, bone and dentin. Anat Rec; 224(2): 123-28. [medline]

16. Bouxsein ML, Myburgh KH, van der Meulen MC, Lindenberger E, Marcus R.

(1994). Age-related differences in crosssectional geometry of the forearm bones

in healthy women. Calcif Tissue Int; 54: 113-8.

17. Burr DB, Martin RB, Schaffler MB, Radin EL. (1985). Bone remodelling in

response to in vivo fatigue microdamage. J Biomech; 18: 189-200.

18. Zioupus P. (2001). Aging Human Bone: actors Affecting Its Biomechanical

Properties and the Role of Collagen. JOURNAL OF BIOMATERIALS

APPLICATIONS Volume 15 - January; 187-229.

19. Dorozhkin Sergey V. (2011). Medical Application of Calcium Orthophosphate

Bioceramics, BIO, 1, 1-51.

20. Dorozhkin Sergey V. (2009). Review Calcium Orthophosphates in Nature,



Apatites and Other Calcium Orthophosphates in Biomedical Engineering,


1.5. Aim and scope of research

The biomaterial development in hard tissue engineering is a multidisciplinary area,

which has been improving in the past years with many published literature and patents

related about new materials, methods of fabrications and applications. The aim of this

research is focusing in develop a new material and method to obtain an alternative

biomaterial containing micro and nano ceramic with natural polymer as a chitosan in

different shapes that can be used in regenerative medicine.

Chapter 2.

Properties of chitosan material

2.1. Introduction

Bone repair or regeneration is a common and complicated clinical problem in

orthopaedic surgery, and much attention has been given to the use of different materials

and methods that could be employed as a base material for scaffold devices and as

modification tools for currently used biomedical devices improving hard and soft tissue

regeneration and/or reinforcement efficacy, also to expand the feasibility of combined

controlled drug release and tissue engineering, tissue formation in regenerative therapy

in the field of periodontics, orthopaedics, cancer and plastic surgery, veterinary [1, 2, 3,

4].

Chitosan, presented in Chapter 1, has been used in biomedical applications and

exhibits a variety of physicochemical and biological properties, in addition to its lack of

toxicity and allergenicity, biocompatibility, biodegradability and bioactivity make it a

very attractive substance for diverse applications as a biomaterial in pharmaceutical and

medical fields, especially in tissue regenerative therapy. Chitosan is the N-deacetylated

derivative of chitin, the second most abundant polysaccharide in nature after cellulose.

Depending on the chitin source and the methods of hydrolysis, chitosan varies greatly in

its molecular weight (MW) and degree of deacetylation (DD). The typical DD of

chitosan is over 70%, making it soluble in some aqueous acidic solutions [5,6,7, 8].

While increasing the ionic strength, the counter-ions would screen the

protonated amine group and make the molecule contracted. Strong intra/intermolecular

hydrogen bonding was formed in solution because of the large number of OH- and

acetyl groups in the chitosan molecular chains. Additionally, the hydrophobic properties

in chitosan, acetyl groups and glucosidic rings can play a significant role on aggregation

in the formation of hydrophobic interaction. The conformation changes of chitosan in

solution are attributed to the intra/intermolecular force interaction, and thus suggested

that the conformation change has a relation with surface tension, charge surface

distribution [7, 8, 9, 10]. This cationic property is the basis of many of the potential

applications of chitosan that can be considered as a linear polyelectrolyte with a high

charge density which can interact with negative charged surfaces, like proteins. The

superior tissue compatibility, biofunctionality and non-antigenic property make chitosan

and its derivatives an ideal material for medical application in tissue regeneration [8, 9,

10]

The aim of this study was to prepare chitosan hydrochloride salt from two

different chitosan producers from Norway and Brazil, comparing results to continuous

studies using analytical methods for characterization and mechanical properties in the

film form.

2.2. Materials

- chitosan ChitoClear FG-90 (Primex, Norway (Pandalus borealis))

- chitosan Polymar (Brazil (Pandalus borealis))

- hydrochloric acid 37,8 %, pure p.a., manufactured by POCh

2.3. Methods

2.3.1 Preparation of chitosan hydrochloride salt

First, a suitable amount of inorganic acid (hydrochloric acid) was added to

chitosan in -form (2.4 g) suspended in distilled water (150 cm3) to give 0.2% final

concentration of chitosan hydrochloride salt. Next, 50 cm3 of distilled water were added

and stirred for 1 hour. To the chitosan solution, 1% aqueous NaOH to pH 5.5 volume of

240 cm3was added dropwise, slowly, under stirring.

Figure 1 shows that the batches of preliminary chitosan were transformed into

the salt form of chitosan according to method elaborated in the Institute of Biopolymers

and Chemical Fibres, Poland.

Figure 1. Scheme of chitosan salt preparation

2.3.2. Analytical methods

a) The average molecular weight of chitosan was determined according to the

viscometric method using 0.0365 ÷ 0.0400 g ± 0.0001 g on dry weight of chitosan;

sample prepared from microcrystalline chitosan or chitosan gel sample is dissolved

in 15 cm3 of the solvent in measuring flask with volume of 25 cm3 using a

laboratory shaker. The flask is filled up to 25 cm3 with the solvent and the obtained

solution is filtered using G-4 Schott filtering funnel. 10 cm3 of the chitosan solution

is transferred into dilution viscometer and conditioned for 15 min. at 25°C ± 0.1°C in

a thermostat. The flow time of the solution through the 1st capillary is measured.

Repeat the measurement 3 times. Further measurements are similarly done for four

consecutive dilutions of the chitosan solution. Each time, add 5 cm3 of the solvent to

the viscometer, mix thoroughly. Keep the vessel with the solvent in the thermostat to

avoid thermostatical conditioning of the diluted solutions. Elaborated on the base

of: R.A.A. Muzzarelli, “Chitin”, Pergamon Press, 1978.

For each concentration of the solution, calculate the reduced viscosity ηred from

0

0

tc

ttnred ×

−=η

where: tn - flow time of solution with the actual concentration, (sec)

t0 - flow time of pure solvent (sec)

c - amount of chitosan in 100 cm3 of diluted solution

The limiting viscosity number is expressed by the equation:

redc ηη 0lim][ →=

The limiting viscosity number is determined graphically from the dependence of

reduced viscosity and concentration of chitosan solution by extrapolating the curve up

to zero concentration. The viscometric average molecular weight of chitosan is

determined on the base of the Mark-Houwink equation:

[η] = K × ⎯Mva

where:

[η] - the limiting viscosity number

K, a - empirical constants, K = 8.93 × 10-4; a = 0,71

Measurement accuracy: ± 1.5%

b) The water retention value of chitosan (WRV) is determined by submerging 0.5g

± 0.0001g of chitosan in 50 cm3 of distilled water [6]. Next, it is centrifuged for

10 min at 4000 rpm. The weight of the sample is determined after centrifuging

(m1) and drying to constant weight after 20 hours at 105°C ± 1°C (m0). The

water retention value (WRV, %) is found from the equation: Measurement

accuracy: ± 1.0%. Elaborated on the base of: R. Ferrus, et al. Cell. Chem.

Technol. 11, 633, 1977.

WRV =0

01

m

mm − ×100

where:

m1- weight of samples after centrifuged [g],

m0 - weight of samples after drying [g]

c) Deacetylation degree of microcrystalline chitosan or chitosan gel is determined

by the potentiometric titration method using glass and calomel electrodes. 0.10-

0.15 g ± 0.0001 g (m) sample of chitosan, on a dry weight, prepared from

microcrystalline chitosan or chitosan gel is dissolved in 10 cm3 of 1 wt %

aqueous acetic acid in a measuring flask with volume of 100 cm3 (V). The flask

is filled up to 100 cm3 with anhydrous acetic acid. 20 cm3 of obtained solution

(v) is transferred into a measuring vessel; 20 cm3 of anhydrous acetic acid and

10 cm3 of 1,4-dioxane are added. The measuring vessel containing the

investigated solution is placed on the magnetic stirrer and the glass indicator

electrode is immersed. The modified calomel electrode is immersed in a second

measuring vessel with saturated solution of potassium chloride in anhydrous

acetic acid. Both measuring vessels are connected by a salt bridge.

Solution of perchloric acid in 1,4 dioxane with determined concentration of

approx. 0.1 mol/dm3 (n) is added from a burette to the investigated solution, and

the electromotive power (EMP) is determined. The value of perchloric acid

volume (V1) corresponding to the neutralization point of amine groups is found

from the dependence between EMP and the volume of perchloric acid solution

used. Content of amine groups in chitosan (-NH2) is calculated from the

following equation: Elaborated on the base of: K. H. Bodek; Acta Polonica

Pharmaceutica - Drug Research 52, 4, 337, 1995.

(-NH2) =vm

VVn

××× 1

where:

n - Determined normality of HClO4 (perchloric acid) solution (for example n =

0.1118 mol/dm3)

m - Chitosan sample weight (g) (± 0.0001 g)

V1 - Volume of perchloric acid solution at neutralization point (cm3)

V - Volume of dissolved chitosan solution (100 cm3)

v - Determined sample volume (20 cm3)

Deacetylation degree (DD) of chitosan as a percentage ratio between the actual

and theoretical content of amine group is calculated from the following

equation:

DD =( )

( )LTHEORETICANH

NH

2

2

−−

=( )

211.62NH−

x 100 [%]

Measurement accuracy: ± 2%

d) Chitosan ash content - The quartz crucible is heated in the furnace at 800 °C to

constant weight. After cooling to room temperature in the exsiccator, the

crucible is weighed on the analytical balance. Approx. 2 g ± 0.0001 g of dry

chitosan sample prepared from microcrystalline chitosan or chitosan gel is

placed in the crucible. The crucible with the sample is preheated on an electric

heater to about 200 °C. After preheating, the chitosan sample is burned in the

furnace at 800 °C for about 3 h, to the constant weight. After cooling to room

temperature, the burned sample is weighed. Chitosan ash content (X, %) is

calculated from the following equation: Elaborated on the base of: ISO 3451-1

(1997).

1002

1 ×=m

mX

where:

m1 - weight of ash (g), (± 0.0001 g)

m2 - weight of dry sample (g), (± 0.0001 g)

Measurement accuracy: ± 10%

e) The moisture content in chitosan and MCCh is determined by a weight method

drying samples to constant weight at temperature of 105 ± 1oC. The moisture

content (M) is calculated from the following equation:

M =1

01

m

mm −×100

where:

m1- weight of samples before drying [g],


f) Reaction medium (pH). Equipment, pH-meter CP-315 M (producer: Elmetron,

Poland), accuracy ± 0.01. The reaction medium is controlled using a suitable

pH-meter. Measurement accuracy: 0.01 pH ± 1 scale interval with

temperature control.

g) The determination of dynamic viscosity of polymer solutions by Brookfield was

performed by rotating the spindle immersed in a liquid measuring - Spindle

(cone). The spindle is coupled to a calibrated spindle. The strength of resistance,

resulting from the viscosity of the fluid stops the rotating spindle and causes

deformation of the spring, whose degree is measured electronically. For the

determination of dynamic viscosity of the initial chitosan solution to prepare 1%

solution of chitosan having moisture in 1% acetic acid or, in the case of

chitosan, after deproteinization 1% solution in dry weight in 1% acetic acid.

Before measuring viscosity, the viscometer has to be reset. After resetting the

viscometer spindle shall be secured by screwing it into the threaded ends of the

handle axis. Before measuring viscosity, adjust the distance between the cone

(stem) and the plate or the bottom of a measurement vessel. The test solution of

0.5 or 2 cm3 (depending on the stem) should be placed in the measurement

vessel for 15 min at 25 °C ± 1.0 °C. After 15 min, measure the viscosity of the

solution and adjust the speed so that the torque value was above 90% (the

smallest measurement error). The result marked the dynamic viscosity read from

the screen viscometer is given in [mPa • s] or [cp]. Tangential stresses are given

in [N/m2] or [dyna/cm2], shear rate is given in [1 / s]. Accuracy of the method:

± 2%.

2.3.3. Assessment of physical-mechanical properties of chitosan hydrochloride salt

in film form.

The mechanical properties of the film form was determined in the Accredited

Laboratory of Metrology at IBWCh (certificate No. AB 388) by using INSTRON (type

5544) in accordance with the following standards:

PN-ISO 2602:1994 (Statistical interpretation of test results)

PN-EN 527-3:1998 (tests conditions)

For product in film form:

ISO 4593:1999 (thickness)

PN-EN 527-1:1998 (breaking strength)

PN- EN ISO 1798:2009 (tensile, and elongation at break)


2.4.1. Results of analytical methods of chitosan hydrochloride salt solution.

The analytical results comparing chitosan hydrochloride salt solution (2%) from

Primex and Polymar are showed in Table 1.

Table 1. Characterization of Chitosan: Primex FG-90 (Norway), and Polymar (Brazil).

Tests Norway, Primex,

Chito-Clear FG90

Brazil

Polymar

Viscosimetric average

molecular weight [kD]

342.1 113.3

Moisture content [%] 12.77 11.32

Water retention value [%] 43.45 49.76

Dynamic Viscosity [cP] 63.11 13.51

Deacetylation degree [%] 83.20 76.9

Ash content [%] 0.40 3.1

Salt solution concentration [%] 2.0 2.0

pH 0.95 0.93

Based on the results presented in Table 1, it has been found that ash content in

chitosan Polymar (Brazil) is too high, also the low molecular weight, which is directly

related to mechanical properties performance, the lower dynamic viscosity related also

with mechanical properties and processing and, finally, the lower deacetylation degree

related to bioactivity and degradability, giving the free amino groups available, these

results showed that the Primex FG-90 (Norway) was a good choice for further studies.

2.4.2. Results for Mechanical properties from chitosan hydrochloride salt in film

form.

The resuts in Table 2, present a higher mechanical properties of chitosan

hydrochloride salt from Primex, such as max load and tensile strength, are directly

related to the higher average molecular weight (Mv) showed in the literature. The

viscosity of chitosan solution increases with an increase in chitosan concentration;

decrease in temperature and with increasing degree of deacetylation, which is a

structural parameter (such as the molecular weight) and mechanical properties (such as

elongation at break and tensile strength) [1,7,9].

Table 2. Mechanical properties of films obtained from the chitosan salts

Producer Max Load

[N]

Elongation at

break [%]

Tensile strength

[MPa]

Film thickness

[mm]

Primex 33.8 13.0 49.03 0.042

Polymar 9.8 12.6 16.62 0.038

2.5. Conclusions

Based on the presented results, it has been found that the ash content in chitosan

Polymar (Brazil) is too high; therefore, the molecular weight and viscosity showed to

lower than the chitosan from Primex (Norway).

The mechanical properties of chitosan hydrochloride salt in film form depend on

the kind of initial chitosan used, molecular weight and kind of chemical degradability.

A better performance of Primex FG-90 (Norway) showed a good choice for future work

and continuous studies related to preparation of different chitosan forms (sponges and

fibres) as a base for scaffolds for hard tissue regeneration.

2.6. References

1) Oktay Yildirim (2004). Preparation and Characterization of Chitosan /Calcium


Institute of Technology zmir, Turkey. August.

2) Misiek D J, Kent JM, Carr RF. (1984). Soft tissue responses to hydroxyapatite

particles of different shapes. J Oral Maxillof Surg;42:150-60.

3) Vert M, Li MS, Spenlehauer G, Guerin P. (1992). Bioresorbability and


4) Riccardo A.A. Muzzarelli. (2009). Chitins and chitosans for the repair of

wounded skin, nerve, cartilage and bone, Carbohydrate Polymers 76, 167-182.

5) Sevda Snel, Susan J. McClure. (2004). Potential applications of chitosan in

veterinary medicine, Advanced Drug Delivery Reviews 56, 1467- 1480.

6) Francis Suh J.-K., Howard W.T. Matthew. (2000). Application of chitosan-

based polysaccharide biomaterials in cartilage tissue engineering: a review,

Biomaterials 21, 2589-2598.

7) Marguerite Rinaudo. (2006). Chitin and chitosan: Properties and applications,

Prog. Polym. Sci. 31 603-632.

8) Alberto Di Martino, Michael Sittinger, Makarand V. Risbud. (2005). Review

Chitosan:A versatile biopolymer for orthopaedic tissue-engineering,


9) Harish Prashanth K.V., R.N.Tharanathan. (2007). Chitin/chitosan: modifications

and their unlimited application potential-an overview, Trends in Food Science &

Technology 18, 117-131.

10)Struszczyk H. M. (2003). The effect of the preparation method on the

physicochemical properties of microcrystalline chitosan (MCCh). ed., Progress

on Chemistry and Application of Chitin and its Derivatives, PTChit., vol. IX,

p.179-186.

Chapter 3.

Preparation of Microcrystalline chitosan (MCCh)/tricalcium phosphate complex in

sponge form.

3.1. Introduction

Different types of materials have been developed from chitosan and calcium

phosphates that are biocompatible, osteoconductive, resorbable and osteoinductive. The

wide range of materials available for hard tissue regeneration reflects methods of

production and application to reconstruct part of the skeleton [1, 2, 3].

All methods may achieve restoration and/or replacement. They all possess

inherent limitations, such as use site, donor-site morbidity, an obligatory graft

resorption phase, contour irregularities, insufficient autogenous resources, disease

transmission, major histocompatibility, structural failure, stress shielding, infection and

many others problems [4,5].

Most of scaffolds can be classified based on their component materials: natural

scaffolds, synthetic scaffolds and mineral-based scaffolds [6, 7, 26, 27].

Natural scaffolds are a combination of naturally occurring materials such as

collagen, alginate, agarose, chitosan, fibrin glue, and so on. Natural scaffolds have

proven successful at stimulating new tissue growth, for example, bones. However, while

natural biodegradable materials integrate well with endogenous tissues, they lack the

structural integrity required for scaffolds used in load bearing locations. For example,

chitosan scaffolds are both biodegradable and biocompatible, but are mechanically

weak, limiting their use as scaffolds for bone regeneration.

Synthetic Scaffolds are most commonly used bioabsorbable polymers are alpha-

hydroxy acids such as polyglycolide (PGA), polylactides (PLA), polycaprolactone

(PCL), polydioxanone (PDS), and their copolymer variants. Alpha-hydroxy acid-based

materials are designed to hydrolytically degrade in situ and then be converted to CO2,

and water by tissue macrophages.

Mineral-Based Scaffolds, polycrystalline ceramic scaffolds are designed to

synthetically replicate the calcium phosphate found in the extracellular matrix of normal

bone, thus providing an osteoconductive substrate for bone formation. The chemical

anisotropic materials most often utilized to construct polycrystalline ceramics are

hydroxyapatite (HAp) and tricalcium phosphate (TCP). Bone formation invariably starts

on the surface of a ceramic and then proceeds through the interconnected pore system

toward the centre of the construct.

Tricalcium phosphate is called a resorbable ceramic, and it is believed that it

binds to bone and is then resorbed and replaced by bone. It has been reported that the

bioresorbability is due to dissolution and phagocytosis. It has been considered that ß-

TCP makes contact with bone directly, suggesting mainly mechanical bonding.

Synthetic ß-tricalcium phosphate (ß-TCP), Ca3(PO4)2 is a compound with a high

potential for bioapplications. In particular, composites made of ß-TCP and HAp, the so-

called biphasic calcium phosphates (BCPs), which combine the excellent bioactivity of

HAp with the good bioresorbability of ß-TCP, are interesting candidates for medical

applications such as, for example, materials for bone replacement [8, 9, 10, 11] .

Many factors can affect the performance of the scaffolds, such as mechanical

properties, including the chemical composition of the polymer, rate of organic/inorganic

part, molecular weight distribution, the crystalline/amorphous ratio, the size and shape

of the implant and the particles involved in the scaffold, polymer processing methods,

implantation site, and the age/metabolic condition of the patient. On the other hand,

mineral-based biomaterials, such as calcium phosphate ceramics, are attractive as bone

grafts substitutes due to their unlimited supply and ease of sterilization and storage [12,

13, 14]. The porosity of a scaffold influences fibrovascular infiltration and, in turn,

determines the extent of bone in growth, cells nutrient flow and attachment. Studies

showed that pore sizes smaller than 15 to 50 µm result in fibrous tissue invasion, pore

sizes from 50 to 150 µm encourage osteoid formation, and pore sizes greater than 150

µm encourage the formation of mineralized bone. The literature shows that the optimal

pore size is considered to be between 150 and 500 µm, that it’s also related with

mechanical properties, stability and inflammatory response of the scaffolds [1, 14, 15,

16].

It appeared that the resorption of calcium phosphate biomaterials is beneficial to

bone formation realising Calcium and phosphate ions that have been considered as the

origin of bioactivity working. The gradual degradation at the same rate in which the

new bone is formed, providing the appropriate initial mechanical properties, and thus

encouraging a new bone to form in the region, is the task of the scaffold studies in hard

tissue regeneration [17]. The release of ionically bound phosphate functionalities from

MCCh/complex, under alkaline conditions, possibly contributed to the formation of

calcium phosphate precursor sites, due to the chelation of calcium ions from the primer

solution. A multi-layered porous mineral structure of the composites with partially

carbonated and poorly crystalline apatite was formed on the surface and inner surface of

MCCh/ß-TCP complex [18, 19].

In orthopaedics, the enzymatic degradability associated to scaffold structure

similar to extracellular matrix with glucosamine groups makes MCCh an attractive

biopolymer for bone tissue repair. Numerous bone filling materials have been

developed in which chitosan and its derivatives are used in combination with calcium

phosphates, essentially as a binding agent, or associated to biological signalling

molecules. In addition, its versatility to be processed into different shapes makes MCCh

with calcium phosphates an interesting material to be used as a non-protein temporary

scaffold, for bone regeneration [18, 19, 20]. The aim of the research is to obtain a new

method and material combining a natural-/ mineral-based scaffold in sponge form that

could be a base for a scaffolds production in hard tissue regeneration. Characterized by

chemical analytical methods, structure of the raw material and the complex in sponge

form according with the Polish patent application P 393758, 2011 [21].

3.2. Materials

1) Chitosan ChitoClear FG-90 (Primex, Norway) characterized by deacetylation degree

(DD) = 83.2%, water retention value (WRV) = 43.45%, molecular weight =342.1 kDa,

Moisture content =12.77%.

2) Tricalcium phosphate (ß-TCP), (Ca3 (Po4)2) - Sigma Aldrich Lab., Germany.

3) Plasticizer -Glycerol (C3H8O3) 99%, pure p.a., Sigma-Aldrich, Germany.

4) Hydrochloridric acid, 37.8% p.a., manufactured by Fluka.

5) Composite with MCCh/ ß-TCP water retention value (WRV) = 480.77 %, Moisture

content = 12.06%.

3.3. Methods

3.3.1 Preparation of the MCCh/ tricalcium phosphate complex.

Tricalcium chitosan/orthophosphate microcrystalline complex and tricalcium

chitosan/orthophosphate microcrystalline complex production method according to the

invention and Polish patent application number P 393758, 2011 [21].

Tricalcium chitosan/orthophosphate microcrystalline complex is a gel-like

suspension of viscometrically determined average molecular weight of 100,000-

250,000, rate of secondary swelling of 500 - 600% and pH= 7.2-7.4, containing 0.5-4.5

wt% tricalcium chitosan/orthophosphate complex, including 0.05-1.93 wt% of

tricalcium orthophosphate together with the microcrystalline chitosan:tricalcium

orthophosphate ratio from 90:10 to 57:43 wt%. In the production, showed in the Figure

1, it is represented the scheme of preparation of the MCCh/ß-TCP complex according

to the invention; the following method has been used: the equivalent part quantity of

weight of tricalcium orthophosphate solution of 0.5-1.5 wt% concentration in

hydrochloric acid of 0.7-1.8 wt% concentration is entered into chitosan aqueous

suspension containing 2.0 wt% of polymer of viscometrically determined average

molecular weight of 200,000-400,000 and deacetylation degree of 70-90%. The mixture

is being homogenized for 10-15 minutes with rotary speed of 400-600 rev/min. The

obtained solution is filtrated, and the filtrate is entered into the mixer, to which then

(constantly mixing) 870-910 of weight of sodium hydroxide aqueous solution of 0.4

wt% concentration are added to obtain pH = 7.7-7.8. The tricalcium

chitosan/orthophosphate microcrystalline complex obtained in suspension form is left

for 24 hours at 5-7 °C temperature. Then, it is purified with demineralised water up to

pH = 7.2-7.4 showed in Figure 1.

Figure 1. Scheme of preparation MCCh/ ß-TCP complex [21].

Manufacture process of MCCh/ ß-TCP complex is based on several physical-

chemical and chemical phenomena, such as neutralization, coagulation, agglomeration

of glucosamine macromolecules with calcium phosphate affecting the properties of final

product. The choice of ß-TCP is due to its lower solubility and better overall

performance in the physiological environment in comparison to the -form [8, 3].

3.3.2. Analytical methods

a) The water retention value of microcrystalline chitosan (WRV) is determined by

submerging 0.5g ± 0.0001g of MCCh in 50 cm3 of distilled water. Next, it is

centrifuged for 10 min at 4000 rpm. The weight of the sample is determined

after centrifuging (m1) and after drying to constant weight after 20 hours at

105°C ± 1°C (m0). The water retention value (WRV, %) is found from the

equation: Measurement accuracy: ± 1.0%. Elaborated on the base of: R.

Ferrus, et al. Cell. Chem. Technol. 11, 633, 1977.

WRV =0

01

m

mm − ×100

where:

m1 - weight of samples after centrifugation [g],


b) The moisture content in MCCh is determined by a weight method drying

samples to constant weight at temperature of 105 ± 10C. The moisture content

(M) is calculated from following equation:

M =1

01

m

mm −×100

where:

m1- weight of samples before drying [g],


3.3.3. Preparation of MCCh/ ß-TCP composite in film form

The films before and after purification process were prepared from a mixture

composed of an aqueous suspension of MCCh/ß-TCP complex. The samples without

glycerol were poured on Teflon plates, and dried at room temperature to obtain samples

in film form. The film form was prepared because was an easy and inexpensive way to

study the dissolution of the ß-TCP particles before and after purification process and

also the possible high contraction of the volume and size in a dry complex film to study

the attraction forces between the characteristic charge surface of MCCh(+) and ß-TCP(-),

and to study the best concentration of glycerol content used as a plasticizer.

3.3.4. Preparation of MCCh/ ß-TCP complex in sponge form.

The sponges were prepared from a mixture composed of an aqueous suspension

of MCCh/ß-TCP, according with Table 1. The preparation was first carefully

homogenized and, next, freeze-dried in a lab freeze dryer ALFA 1-4 made by Christ Co

in the temperature range from (-25) to 10 0C and vacuum from 0.1 to 0.53 mbar during

20 to 24 hours depending on the charge size. Drying accomplished that way resulted in

the preparation of sponges with a smooth surface without defects.

3.3.5. Infrared Spectroscopy of the complex

Fourier transform infrared spectroscopy (FTIR) is a non-destructive technique

that was used to identify the functional groups through their chemical bonds, which

generate a spectrum of infrared bands characteristic of each connection type. The

infrared analysis was performed on a range from 500 to 4000 cm-1, resolution 4.0 cm-1

with a Spectrum Genesis Series. Samples were prepared for analysis with KBr, in the

form of tablets, to verify the presence of functional characteristic groups in the ß-TCP:

P-O, O-H, P-O-H, H-O-H and for MCCh material the functional groups: NH2, Amide I,

amide II, according with Table 1.

3.3.6. Determination of particles size and morphology of commercial ß-TCP

powder.

The particle size of ß-TCP powder was determined by a technique using laser

particle size analyser Sympatec Hellos H1330, type BF (sympatec GmbH, Clausthal,

Germany). This analysis was a cooperation made between Institute of Biopolymers and

Chemical Fibres (IBWCh) in Lodz, Poland, and Thuringian Institute of Textile and

Plastics Research (TITK), Rudolstadt, Germany. The morphology of the used powder

and distribution in the polymer matrix in sponge and film forms was observed using a

scanning electron microscope (SEM) - FEI Quanta 200, USA.


3.4.1 Elaboration of the quantitative and qualitative MCCh/ ß-TCP complex

The composition of samples is showed in Table 1.

Table 1. The quantitative and qualitative composition of MCCh/ ß-TCP complex

Symbol of sample Weight proportion of components

MCCh/ ß-TCP(suspension)

Glycerol

Sample 1 1 0

Sample 2 1 0.5

3.4.2. FTIR study

The main peaks of energy vibration in transmission mode are showed in Figure

2, identified in the ß-TCP characteristic functional groups of orthophosphate, hydroxyl,

and phosphate, and the carbonate group characteristic of synthetic calcium phosphate

material showed in traces. The presence of carbonate suggests that, in some commercial

ß-TCP and other calcium phosphates such as a HAp, CaO and Ca(0H)2 are used to get

an ideal stoichiometric relation between Ca / P in the material. It is known that synthetic

ß-TCP is more stable and more soluble in human body than HAp, among the calcium

phosphates. These features can increase the regeneration of bone tissue in the presence

of an implant scaffold. Thus, it is desirable that the material replacements for bones

present some solubility to allow this regeneration, which can be achieved with

carbonated calcium phosphates [2, 6, 17].

In Table 2, it is showed the FTIR study in transmission mode of commercial ß-

TCP, the absence of bands at 460 and 740 (cm-1) and an isolated band, 600 cm-1;

characteristic of -TCP, indicating that the starting material is only composed of ß-TCP,

also shows a small amount of CO3-2 at 1454 cm-1. The ß-TCP is easily identified through

the presence of a broad band in 900-1200 cm-1, characteristic of the symmetric mode (P-

O-P) assigned to antisymmetric stretching of P-O. Since the peak at 1118 cm-1 is

characteristic of a non-degenerate deformation of hydrogen groups - OPO 3 - H, O-PO3,

of common ions HPO, the presence of this group may be a consequence of the

interaction of water molecules in the structure, changing some neighbors in the

crystalline part, showed in the spectrum of energy vibration [ 2, 6, 17].

Table 2. Localization of peaks of a commercial ß-TCP in transmission FTIR.

Figure 2. FTIR spectrum of the commercial ß-TCP

The commercial ß-TCP in powder form, showed in a symmetric stretching at

3338 cm-1 and stretching in 1650 cm-1, is presented by O-H, H-O-H group, referring a

water adsorbsion by the powder characteristic of calcium phosphate family. The band

observed at 968 cm-1 of low intensity, corresponding to non-degenerate symmetric

stretching of P-O bonds of phosphate groups. The bands 1041, 1081 and 1098 (cm-1)

represent the asymmetric stretch modes, respectively, the P-O bonds of phosphate

Mode of vibrations Wave number of peaks, cm-1 Mode of groups

Antisymmetric stretching

Symmetric stretching

1041, 1081, 1098

968, 1118

P-O of PO4

Symmetric stretching 3338 O-H

Antisymmetric stretching 1454, 1428 CO of C=O

Stretching 1650 H-O-H

groups. The peaks of MCCh/ ß-TCP complex, before purification process showed in

Table 3 and Figure 3, confirm the presence of functional groups NH2 (1539 cm-1),

Amide I (1648 cm-1), Amide II (1557 cm-1) in the material and the main peaks of energy

vibration (1026, 1086 cm-1) identified the C-O skeletal vibrations stretch of saccharide

structure showed, respectively, in Figure 3 before purification process and Figure 4 after

purification process.

Table 3. Localization of peaks of the MCCh/ ß-TCP complex before purification

process.


Symmetric stretching 1648 Amide I C=O

bending 1539 -NH2

stretching 1506 -NH2

1557 Amide II

1318 Amide III

Anti-symmetric strechingbridge

Skeletal vibration stretchingof saccharide structure

1154

1026, 1086

C-O-C

C-O

stretching3298, 3500 N-H

stretching3434 O-H

stretching1000, 1100 P-O, PO

Figure 3. FTIR spectrum of the MCCh/ ß-TCP complex before purification process

In the spectrum of energy vibration in transmission mode showing the MCCh/ß-

TCP complex before purification process peaks that are observed in the Figure 3, the

peak with 1648 (cm-1) corresponds to a stretch modes of amide I, associated with energy

level and the kind of structure that are linked with the type of bound NH2 (1539, 1506

cm-1) and Amide II. In 3434 cm-1, it is also observed the stretch of OH- group peak

referring to the way they stretch and water absorption. The band observed at 1154 cm-1,

of high intensity, corresponding an anti-symmetric stretching of C-O-C bond groups.

Bands 1026 and 1086 (cm-1) represent the symmetric stretch modes,

respectively, with the C-O bonds being characteristic of the saccharide structure groups.

The Figure 4 shows the FTIR energy vibration in transmission mode, the spectra of

MCCh/ß-TCP complex after purification process and exhibits the same characteristic

bands, such as a strong and broad peak between 1154 cm-1 and 1026 cm-1 and 1086 cm-1

that refer to the skeletal vibrations stretch of saccharide structure and a peak at 1636 cm-

1 attributed to the free primary amino group (NH2) in MCCh. The main peaks of energy

vibration identified in the ß-TCP also was observed in the complex with characteristic

functional groups of orthophosphate at 1100 cm-1, hydroxyl, phosphate (HPO) at 1000

cm-1 , (OH)- at 3430 cm-1 , the last one referring a water absorption. A broad band

appears from 1665 cm-1 corresponding to the H-O-H stretching absorption band in the

samples, showing a fair agreement with the literature [23, 24, 25].

Table 4. Localization of peaks of the MCCh/ ß-TCP complex after purification process.


stretching 1636 Amide I C=O

bending 1538 -NH2

stretching 1506 -NH2

1557 Amide II

1316 Amide III

antisymmetric stretching

Symmetric stretching

1000

1100

P-O

PO4-3

stretching 3430 O-H

Antisymmetric stretching 862 P-O-H


Figure 4. FTIR spectrum of the MCCh/ ß-TCP complex after purification process

The chemical interactions between the inorganic and organic constituents in the

composite probably take place via the ionic bonding between ion phosphate and the

amino group of chitosan. The FTIR study of complex before and after purification

process showed a very similar peak of the same product showed in Figure 5, suggesting

that the free amino groups from microcrystalline chitosan bonded with the phosphate

groups of tricalcium phosphate creating a complex and validating the method of

preparation of MCCh/ ß-TCP complex, also notice a little decrease in the initial calcium

phosphate amount, which comes from several washings (purification process) with

purified and demineralised water.

Figure 5. FTIR spectrum of the MCCh/ ß-TCP complex before and after purificationprocess

3.4.3. Particles size and morphology of commercial ß-TCP powder.

The aim of this study was to determine the particles size of commercial ß-TCP

in the form of powder. Before analysis, the material used was dried with vacuum at

105oC for 24h to remove moisture and was separated to measure the particles size

showed in Table 5 and to study the grain morphology of commercial ß-TCP, showed in

the figure 6.

Figure 6. Respective particles size and distribution of of the commercial ß-TCP and

SEM microphotography of grain morphology.

The measurements of particles size showed in Table 5, showing a specified

surface area covered by commercial ß-TCP at 2,53 e+04 cm2/g and average particle size

of 4.48 m. It was found less than 10% of the tricalcium phosphate particles in nano-

size, which also indicates a faster and easier release of calcium and phosphate ions in

physiological environmental following the literature [25].

Table 5. Particles size and distribution of commercial ß-TCP.

POWDERS ß-TCP

Particles size (90%), ( m) 12.78



Superficial area (cm2/g) 2.53 e+04

The analysis of particles size using laser particle size analyser Sympatec Hellos

H1330, type BF, showed that a standard deviation cannot be given so easy. The particle

size depends firstly on particle shape (aspect relation). Most calculation models refer

to ball-shaped bodies. The more the particles deviate from this model, the more the

deviation rises. Secondly, the broadness (width) of particle size distribution has an

influence on the standard deviation using this method. In a case where this distribution

covers only a narrow part, for instance 2 to 5 µm, then the deviation is lower than 10%.

However, the measurement itself is very precise. The measurement of the powders used

in this research had procedure of three-times for each sample and receives deviations of

results lower than 2 %, the samples of both powders had a range from 0.5 to 50 µm. The

analysis showed a ß-TCP grain size fits in the range 3.0 - 9.0 m with fair agreement

with the literature. Notice the morphology of the ß-TCP powder is more spherical

showing a great ability to agglomeration and cluster formation, which could be

explained by a non-homogenous charge distribution on the surfaces and high ability for

water adsorption characteristic of this material and showed in the FTIR study.

Typically, the very small particles tend to agglomerate due to the increased

intensity of the attractive forces over the repulsive forces, due to charge distribution on

the surfaces. There are many factors related with the performance of the biocomposite

for hard tissue regeneration that reflect in the preparation of suspension, mechanical

properties, calcium and phosphate ions released in the physiological environment,

particles size, shape, particles distribution and also ratio of inorganic part in the polymer

matrix [21, 22, 25].

3.4.4. SEM study of composite MCCh/ ß-TCP complex in film form.

The objective of the investigation was to study the dissolution of the ß-TCP

particles before and after purification process and also the possible high contraction of

the volume and size in a dry complex film, as well as to study the best concentration of

glycerol content used as a plasticizer and finally to estimate the suitability of the

MCCh/ß-TCP complex in film forms and the ß-TCP distribution in the polymer matrix

by SEM analysis. The Figure 7, 8, 9 and 10 showed presence of MCCh/ß-TCP before

and after purification.

x500 magnification x1000 magnification

Figure 7. SEM microphotographies, cross-section view of films, MCCh/ß-TCP complex

before purification process


Figure 8. SEM microphotographies, film surface, MCCh/ß-TCP complex before purification

process


Figure 9. SEM microphotographies, cross-section view of films, MCCh/ß-TCP complex after

purification process

Figure 10. SEM microphotographies, film surface, MCCh/ß-TCP complex after purification

process

The preparation of films showed in Figures 7, 8, 9, 10; there is presence of ß-TCP in the

composites before and after purification process with incomplete dissolution. The particles of

ß-TCP aggregates well in the polymer matrix with a homogeneous distribution and clusters

size reduction after purification process showed in Figure 9 and 10. In the suspension, no

precipitation was found, suggesting a good stability and an ionic bonding between the amino

groups and ion phosphate groups, also a good inter/intra hydrogen bonding in the complex.

Notice that films without glycerol used as a plasticizer reduces the size around 3X, suggesting

a very high electrostatic interaction between MCCh and ß-TCP in a complex formation. The

addition of glycerol (0.5 wt-%) in the complex suspension reduces the contraction, thus

improving the film elasticity; glycerol acts increasing the polymer chains mobility, thus

making the film less fragile with more elongation.

3.4.5. The SEM study of MCCh/ ß-TCP complex in sponge form.

The Figures 11 and 12 show the structure of MCCh/ ß-TCP complex after purification

process in sponge form according with Table 1 preparation.


Figure 11. SEM microphotographies, surface of MCCh/ß-TCP complex without glycerol.


Figure 12. SEM microphotographies, surface of MCCh/ß-TCP complex with glycerol.

The sponges showed the presence of ß-TCP particles in the polymer matrix after

purification process. The composite sponge without plasticizer did not show a smooth

surface with more agglomeration and bigger size of cluster formation in the entire area

of the polymer matrix. The sponges with glycerol as a plasticizer increase the

interconnected porosity with homogenous size and shape (round shape) of the pores

with good and homogenous distribution of the calcium phosphate in the polymer matrix.

The preparation of composites in sponge form with glycerol (0.5 wt-%) formed

smooth surface without defects and well-shaped 3-dimensional structure with highly

number of interconnected porous.

3.5. Conclusion

The FTIR study of the composites, according with Table 1, showed that both

components, MCCh and ß-TCP, existed in the composite before and after purification

process, which can be seen as well in the SEM study of the films, which suggests a

validation of the method to obtain MCCh/ ß-TCP complex.

The SEM analysis showed a ß-TCP more round shape and the grain size fits mainly

in the range 1.0 - 9.0 m, average particle size of 4.48 m ± 0.54 µm.

The SEM images showed that the composites have a homogeneous distribution of

the ß-TCP particles with reducing cluster size formation after purification process. The

calcium phosphate particles were uniformly dispersed in the amino-glucose

macromolecule aggregation (coagulation process) and no precipitation of the calcium

phosphate was noticed in the samples showing the good stability of the suspension. The

study showed the feasibility of freeze drying method in the preparation of the MCCh/ ß-

TCP complex sponges, resulting in a 3-dimensional porous network structure that can

be used in future as a base for scaffolds production.

3.6. References

1) Oktay Yildirim (2004). Preparation and Characterization of Chitosan /Calcium



2) Rumi Fujita, Atsuro Yokoyama, Yoshinobu Nodasaka, Takao Kohgo, Takao

Kawasaki. (2003). Ultrastructure of ceramic-bone interface using hydroxyapatite

and B-tricalcium phosphate ceramics and replacement mechanism of B-

tricalcium phosphate in bone, Tissue & Cell 35, 427-440.

3) Shinn-Jyh DING. (2006). Preparation and Properties of Chitosan/Calcium

Phosphate Composites for Bone Repair, Dental Materials Journal

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4) Jue-Yeon Lee , Sung-Heon Nam , Su-Yeon Im , Yoon-Jeong Park ,Yong-Moo

Lee, Yang-Jo Seol, Chong-Pyoung Chung, Seung-Jin Lee. (2002). Enhanced

bone formation by controlled growth factor delivery from chitosan-based

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as scaffolds for bone regeneration, ITBM-RBM 26, 212-217.

6) Huipin Yuan, J. D. de Bruijn, Yubao Li, Jianqing Feng, Zongjian Yang, K. de

Groot, Xingdong Zhang. (2001). Bone formation induced by calcium phosphate

ceramics in soft tissue of dogs: a comparative study between porous -TCP and

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7) Stephen M. Warren, MD, Kenton D. Fong, MD, Randall P. Nacamuli, MD,

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vitro structural changes in porous HA/ -TCP scaffolds in simulated body fluid,

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chemically precipitated apatitic precursor into B-tricalcium phosphate upon

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Chemistry and Application of Chitin and its Derivatives, PTChit, X, p.13-17.

20)Niekraszewicz A., Kucharska M, Wisniewska-Wrona M. (1998). Some Aspects

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21)Pighinelli L, Kucharska M, Brzoza-Malczewska K, Gruchała B. (2011).

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preparing same. Poland patent application P 393758.

22)SZ-Chiau Liou, San-Yuan Chen. (2002). Transformation mechanism of different

chemically precipitated apatitic precursor into -tricalcium phosphate upon

calcinations, Biomaterials 23, 4541-4547.

23)Brugnerotto, J.; Lizardi, J.; Goycoolea, F.M.; ArguÈelles-Monal, W.;

DesbrieÁres, J.; Rinaudo, M. (2001). An infrared investigation in relation with

chitin and chitosan characterization. Polymer, 42, 3569-3580.

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26) Council Directive 93/42/EEC of 14 June 1993 concerning medical devices (OJ

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to medical directives.

Chapter 4.

Preparation of Microcrystalline chitosan (MCCh)/tricalcium phosphate complex

with Hydroxyapatite in sponge form.

4.1. Introduction

In the last past years, much attention has been given to develop materials for

bone tissue regeneration such as polymer-based scaffolds containing bioactive

bioceramics, which have been under development in the last past years for hard tissue

regeneration and can be produced making bioceramics serving two purposes: (a)

making the scaffolds osteoconductive, and (b) reinforcing the scaffolds. With this

composite strategy, there are two approaches for making bioceramic-polymer composite

scaffolds [1, 2]:

(1) Incorporating bioceramic particles in the scaffold through a variety of

techniques;

(2) Coating a polymer scaffold with a thin layer of apatite through biomimetic

processes.

(3) To prepare a complex polymer/calcium phosphate

All strategies have been employed to develop usable scaffolds for bone tissue

engineering. One of the manufacturing process as a freeze-drying method was used at

the final stage to produce the porous structure. In production of polymer/apatite

scaffolds, same factors such as polymer solution concentration, porous type and size,

can be changed in the freeze-drying method. Same parameters, such as pressure,

temperature and time play a very important role in forming the scaffolds of desired

porous structures (pore geometry, pore size and size distribution, pore interconnectivity,

thickness of pore walls, etc.) and, hence, the mechanical performance [1,2,3].

The incidence of fractures in the natural bones increases rapidly with age. This is

partly due to extra osseous factors such as the impaired reflexes of the elderly, their

reduced proprioceptive efficiency, reduced cushioning by fat, and weakened

musculature, and by osseous factors such as the structural changes in the shape and size

of the bones like diseases and deterioration of the condition of the bone material itself.

The intrinsic bone changes can be subdivided into quantity and quality effects. Quantity

refers to a reduction in bone mass and an increased macroscopic porosity. Quality refers

to changes in the bone matrix material, which is related with chemical, compositional

and physical factors. In terms of mechanical effects, between 0 and 35 years of age,

human bone shows an increase in its mineral content, which benefits both the strength

and stiffness of the tissue. After 35, the elastic, ultimate and fracture properties of bone

tissue invariably deteriorate in men and women. The aging effects on cancellous and

trabeculae bone are nowadays primarily reduced into describing its architecture,

connectivity and level of porosity of the tissue [2, 3, 4,5].

The concepts of stiffness and strength are easy to comprehend. However,

toughness is less obvious and is here defined in terms of the amount of energy required

for fracture. This energy will be absorbed in three principal ways. Some energy is

absorbed prior to the generation of a major crack in the form of diffuse damage; this we

may call the “pre-fracture” toughness of the material, or its damage tolerance level [2,

7]. Some energy is required to start the final fracture crack, the rest is required to drive

the crack through the tissue in order to break the material in two (or more) pieces; this

being the conventionally defined “fracture” toughness of the material. Toughness is of

primary importance, especially in relation to fractures [2,3].

It has been shown that in older bone, the fatigue strength and two separate

toughness measurements (like the work of fracture and the energy absorbed in impact)

are considerably reduced. Age, as a separate variable, was able to explain well the trend

for work of fracture and impact. The bone behaves like a composite material. It is likely

that aging is a more general deteriorative process, whose increase affects reducing the

biochemical and biomechanical properties of bone in a number of ways [2].

Modern composite materials, on the other hand, have a positive relationship

between strength and toughness; the tougher materials are also stronger. Composites

benefit by having a heterogeneous structure and an ability to accept widespread and

regenerate microstructural damage without breaking. The intracortical porosity reflects

an imbalance between the rates of new bone deposition and bone resorption as well as

bone formation. Bulk density and mineral weight ratio values are the result of the

combined effects of maturation of individual elements and also their relative fractions.

In vivo fatigue microcracks give an appreciation of the loading history of the material;

these cracks accumulate in life as a result of everyday loading activities and may also

have a harmful effect on the residual properties of the bone material itself [2, 3, 5].

The size of individual cracks stayed well within limits, probably as a result of an

active remodelling process. In the worst of cases, the crack length simply doubled in

size between 35 and 92 years of age (note that the cracks at 35 are at most up to 500 m

long, while at 92, there are a few up to 1 mm long). However, their number dramatically

increases with age, especially for the cracks of small lengths truly related with the

mechanical properties [2, 3, 5]. There are basically two major options for biodegradable

and biocompatible scaffold: ceramic and polymer as defined by Vert. The ceramic,

usually made of calcium-phosphate, may be delicate to use in such demanding

mechanical environment as they present a brittle behavior. The polymers are ductile but

may not have enough mechanical properties to withstand the load. If the polymer

scaffolds have enhanced mechanical properties, they may be an ideal material for bone

substitute. Bone is a Nano-structure of HAp crystals and organic matrix. Because rigid

crystals such as HAp crystals cannot dissipate much energy, the organic matrix must be

involved in this process. Definitions given by Vert [4, 5]:

Biodegradable are solid polymeric materials and devices which break down due

to macromolecular degradation with dispersion in vivo but no proof for the elimination

from the body (this definition excludes environmental, fungi or bacterial degradation).

Biodegradable polymeric systems or devices can be attacked by biological elements so

that the integrity of the system and in some cases but not necessarily, of the

macromolecules themselves, gives fragments or other degradation by-products. Such

fragments can move away from their site of action but not necessarily from the body.

Bioresorbable are solid polymeric materials and devices which show bulk

degradation and further resorb in vivo; i.e. polymers which are eliminated through

natural pathways either because of simple filtration of degradation by-products or after

their metabolization. Bioresorption is thus a concept which reflects total elimination of

the initial foreign material and of bulk degradation by-products (low molecular weight

compounds) with no residual side elects. The use of the word ‘bioresorbable’ assumes

that elimination is shown conclusively. Bioerodible are solid polymeric materials or

devices, which show surface degradation and further, resorb in vivo.

Bioerosion is thus a concept, too, which reflects total elimination of the initial

foreign material and of surface degradation by-products (low molecular weight

compounds) with no residual side effects. Bioabsorbable are solid polymeric materials

or devices, which can dissolve in body fluids without any polymer chain cleavage or

molecular mass decrease. For example, it is the case of slow dissolution of water-

soluble implants in body fluids. A bioabsorbable polymer can be bioresorbable if the

dispersed macromolecules are excreted.

The incorporation of a tricalcium phosphate (ß-TCP), hydroxyapatite (HAp) and

basic salts into a polymer matrix produces a hybrid/composite material. These inorganic

fillers allow tailoring the desired degradation and resorption kinetics of the polymer

matrix. A composite material would also improve biocompatibility and hard tissue

integration in a way that ceramic particles, which are embedded into the polymer

matrix, allow for increased initial fast spread of serum proteins compared to the more

hydrophobic polymer surface [5, 6, 7].

The similarity of synthetic hydroxyapatite [Ca10(PO4)6(OH)2; HAp and ß-TCP

(Ca3(PO4)2) ] to bone mineral has led to the extensive use of HAp and/or ß-TCP as a

bone grafting material in hard tissue implants. Pores in apatite composite scaffolds has

been introduced clinically for applications such as spinal fusions, bone tumors,

fractures, and in the replacement of failed or loose joint prostheses. The development of

pores in apatite composite scaffolds stemmed from the results of studies highlighting

the role of porosity in acting as a promoter for mechanical fixation to the surrounding

tissue by providing a template for bone ingrowth. Furthermore, it has been suggested

that the quality of bone surrounding porous implants is superior to that around dense

implants. There is an increasing trend towards engineering bone grafts with

interconnected porosity between the macropores of the scaffolds. Pore interconnections

with size 30-100 m act as pathways between the macropores to favor cellular and

vascular penetration assuring bone ingrowth into the pores. Porosity is defined as

micrometer-sized porosity within the structure of the implant, and found that strut

porosity increases bone ingrowth, mineral apposition rates, and bone organization [3, 5,

10, 11, 12].

The scaffold or three-dimensional (3-D) construct provides the necessary

support for cells to proliferate and maintain their differentiated function, and its

architecture the ultimate shape of the new bone and cartilage. Skeletal tissue is usually

organized into 3-D structures in the body. For the repair and regeneration and/or

generation of new hard and ductile tissue, scaffolds need to have a high elastic modulus

in order to be retained in the space they were designated for; and also to provide the

tissue with adequate space and nutrient flow for growth. Therefore, one of the basic

problems from a scaffold design is that to achieve significant mechanical properties and

sufficiently high interatomic and intermolecular bonding and also allows for hydrolytic

attack and degradation [4, 5, 6].

Another point is that the porosity has to be also focused in the diffusion of

nutrients, gas exchange, and elimination of by-products into the 3-D scaffold. Although,

an interconnected macropore structure of 300-500 m enhances the diffusion rates to

and/from the center of a scaffold, transportation of the nutrients is not sufficient for

large scaffold volumes [5, 6]. The properties of bone in health and disease attract much

attention, with an ever greater proportion of the population in need for medical devices

for hard tissue regeneration and/or replacement. With this, pressure is put on health and

welfare systems of all countries. Aging, diseases, fractures, and so on, when combined

with other skeletal complications, as a demineralization, contribute and improve the

suitability and development in the hard tissue engineering. The aim of this study is to

prepare a quantitative and qualitative composite using the MCCh/ß-TCP complex with

another commercial calcium phosphate (HAp), studying chemical characterization and

mechanical properties performance in sponge form.

4.2. Materials

1) MCCh/ß-TCP complex characterized by: water retention value (WRV) = 560%,

polymer content = 3.25 %, Moisture content 12.06%, pH=7.40.

2) Tricalcium phosphate (ß-TCP), (Ca3(PO4)2) - Sigma Aldrich Lab., Germany.

3) Hydroxyapatite (Ca5(PO4)3OH) - Sigma Aldrich Lab., Germany.

4) Plasticizer -Glycerol (C3H8O3) 99%, pure p.a., Sigma-Aldrich, Germany.

5) Hydrochloridric acid, 37.8% p.a., manufactured by Fluka, Germany

6) Ethanol - C2H5OH, Fluka, Germany.

4.3. Methods

4.3.1. Preparation of composite in sponge form

The preparation of composites in sponge form using freeze-dried method was

made using ALFA 1-4 dryer made by Christ Co in the temperature range from (-25) to

10 0C and vacuum from 0.1 to 0.53 mbar during 20 to 24 hours depending on size of

the charge. Drying accomplished that way resulted in the preparation of sponges with a

smooth surface without defects. The freeze-dried method used for sponge preparation

is also showed in the scheme in Figure 1.

Figure 1. Scheme of freeze-dried method used to prepare composites in sponge form.

4.3.2. Powder complex preparation

The composition for powder preparation was MCCh/ß-TCP complex

suspension (1 wt-%) /HAp (1 wt-%) /glycerol (1 wt-%) composite content 4% was with

freeze-dried method in lab, ALFA 1-4 dryer made by Christ Co in the temperature range

from (-25) to 10 0C and vacuum from 0.1 to 0.53 mbar during 20 to 24 hours depending

upon size of the charge. In the Figure 2, there is a schematic illustration of the method

to obtain a composite powder.

Figure 2. Scheme of preparation of composite powder used to prepare composites insponge form.

Composite

MCCh/ß-TCP complex (1 p.w)

HAp (1 p.w)

Glycerol (1 p.w)

Composite Powder

MCCh - 26.7 %

ß-TCP - 6.7 %

HAp - 33.3 %

Glycerol - 33.3 %

Freeze drying method

sponge

Teflon pin

Freeze-dried plate

Water and ethanol

Water and ethanol

Lyophilizing

Chamber

Surface A

Surface B

4.3.3. SEM study

The HAp particle morphology and distribution of the calcium phosphates in the

polymer matrix in a sponge and film forms were observed using a scanning electron

microscopy (SEM) - FEI Quanta 200, USA.

4.3.4. Infrared Spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a non-destructive technique

that was used to identify the functional groups through their chemical bonds, which

generate a spectrum of infrared bands characteristic of each connection type. The

infrared analysis was performed on a range from 500 to 4000 cm-1, resolution 4.0 cm-1

with a Spectrum Genesis Series. Samples were prepared for analysis with KBr, in the

form of tablets, to verify the presence of functional characteristic groups in the ß-TCP:

P-O, O-H, P-O-H, H-O-H and for MCCh material the functional groups: NH2, Amide I,

amide II. The FTIR study of the ß-TCP and the MCCh/ ß-TCP complex are showed in

chapter 3.

4.3.5. Determination of Ca and P in commercial HAp and ß-TCP powders and in

the composites.

The aim of this study is to determine and compare the quantity of Ca and P

remaining after film and sponge preparation of the composites, by ICP-OES, Germany,

through a method of microwave digestion according to SOP2.5L126. Quantification of

Calcium and Phosphorus was made by (DIN EN ISO 11885). This analysis was a

cooperation made between Institute of Biopolymers and Chemical Fibres (IBWCh) in

Lodz, Poland, and Thuringian Institute of Textile and Plastics Research (TITK),

Rudolstadt, Germany).

4.3.6. Determination of particles size of commercial HAp powder

The technique used was by laser particle size analyser by Sympatec Hellos

H1330, type BF (sympatec GmbH, Clausthal, Germany), to determine HAp range size

of particles and distribution. This analysis was a cooperation made between Institute of

Biopolymers and Chemical Fibres (IBWCh) in Lodz, Poland, and Thuringian Institute

of Textile and Plastics Research (TITK), Rudolstadt, Germany).

4.3.7. WAXS- Diffraction of ß-TCP and HAp powder

Two calcium phosphates in form of powder have been investigated by means of

WAXS-diffraction in transmission mode. Both powders, HAp and ß-TCP, were simply

placed between two polypropylene films for running the WAXS-scan in transmission

mode. The following parameters were adjusted: X-ray radiation - CuK -doublet, non-

monochromatic -portion suppressed by Ni-filter, mode of operation - Transmission,

anode Voltage - 40kV, tube current - 40mA, Steo width - 0,0250, scan rate - 5 sec/step,

aperture slit - 1mm, anti-diffusion slit - 1mm, detector slit - 1mm. This analyse was a

cooperation between Institute of Biopolymers and Chemical Fibres (IBWCh) in Lodz,

Poland, and Thuringian Institute of Textile and Plastics Research (TITK), Rudolstadt,

Germany).

4.3.8. Mechanical properties of composites

The mechanical properties of the composites in sponge and film forms were

determined in the Accredited Laboratory of Metrology at IBWCh (certificate No. AB

388) by using dynamometer Instron (type 5544) in accordance with the following

standards (Polish Standard PN-EN-ISO 527-1., Polish Standard PN-EN-ISO 527-3,

Polish Standard PN-ISO 4593).


4.4.1. Elaboration of the quantitative and qualitative of composites in sponge form

The main objective of this study was the elaboration of qualitative and

quantitative composition of MCCh/ß-TCP complex with HAp showed in Table 1, to

find the best concentration of complex resulting in the preparation of sponges with a

smooth surface without defects, the samples A, B, C the glycerol content is related

with wt-% complex content (MCCh/ß-TCP complex) and wt-% PC content.

Table 1. Quantitative and qualitative composite preparation in sponge form

Symbol ofsamples Concentration Composition wt-% %

A 5.0%Complex

PC Glyc.

(1.0)(4.0)(2.5)

MCCh - 32.00% ß-TCP - 8.00% HAp - 26.66%

Glycerol - 33.33%

B 8.0%Complex

PC Glyc.

(1.0)(4.0)(2.5)

MCCh - 32.00% ß-TCP - 8.00% HAp - 26.66%

Glycerol - 33.33%

C 10.0%Complex

PC Glyc.

(1.0)(4.0)(2.5)

MCCh - 32.00% ß-TCP - 8.00% HAp - 26.66%

Glycerol - 33.33%

* (MCCh/ß-TCP) complex,

** PC-Powder composite- (MCCh/ß-TCP complex (1.0 wt-%) / HAp (1.0 wt-%) / Glycerol (1.0 wt-%).

The decision to prepare the powder composite with HAp and mix with

the complex suspension to finally obtain sponges was made to minimize the

agglomeration and size of clusters formation. It was notice that preparation of the

complex suspension by mechanical mixing process using HAp powder was a challenge,

considering that the small particle size in a liquid suspension tends to move rapidly and

randomly, which is explained by micro Brownian movement, due to the impact between

the particles themselves, or with molecules of the suspension, or also against the surface

of the container, facilitating agglomeration, making the mixture more difficult and, at

the same time, a challenge. As a consequence, there is increase of the mechanical

degradation of the polymer. The small particles of the powder absorb more water and

reduce their free energy and charge distribution on surface, and typically the very small

particles tend to agglomerate due to the increased intensity of the attractive forces over

the repulsive forces, due to charge distribution on the surfaces, considering the cationic

nature of the HAp and microcrystalline chitosan and anionic nature of the ß-TCP also

responsible for high electrostatically bonding that reflects in the structure and suggests a

ceramic composite formation by the two calcium phosphates involved. This

phenomenon presents a remarkable influence on the rheological behaviour of

suspensions, as specific surface areas are highly conducive to cluster formation.

When the HAp powder is directly mixed in the complex suspension, it is noticed

that the smoothest surface of composites began to be disturbed with incorporation and

increasing amount of HAp powder, gradually resulting in a rough surface, which was

not smooth anymore. This phenomenon did not happen while the following steps

weren’t completed:

A) Preparation of the composite suspension by mechanical mixing ((MCCh/ß-TCP

complex (1.0 wt-%) / HAp (1.0 wt-%) / Glycerol (1.0 wt-%)).

B) Use the suspension to prepare sponges by freeze-dried method.

C) Crushing the sponge composite to a powder.

D) Mix the powder composite into complex (MCCh/ß-TCP), suspension by

mechanical mixing.

E) The final result of this mixture was a suspension used to obtain the final

composite sponges by freeze-dried method.

Another problem was to find the best concentration for the samples to obtain the

smoothest surface without defects, considering the composite samples in Table 1. It was

noticed that the concentration at 5 and 8% was difficult to prepare sponges. The way to

solve this problem was to increase the composite concentration by 10%, keeping the

same preparation method. In this 10% concentration, the composite showed a smooth

surface without defects, thus making continuous studies possible.

4.4.2. Composites with different ratios of ethanol to prepare sponge form

The set of samples in Table 2 shows the qualitative and quantitative analyses of

sample preparation, resulting in the sponges with a smooth surface without defects.

Table 2. Quantitative analysis of composite preparation with ethanol in sponge form

Symbolof

samplesContent of

compositionEthanolcontent

Composition wt-% Materials (%)

A1 10.0% 0%Complex

PC Glyc.

(1.0)(4.0)(2.5)

MCCh - 32.00% ß-TCP - 8.00% HAp - 26,.66%

Glycerol - 33.33%

B1 10.0% 1%Complex

PC Glyc.

(1.0)(4.0)(2.5)

MCCh - 32.00% ß-TCP - 8.00% HAp - 26.66%

Glycerol - 33.33%

C1 10.0% 2%Complex

PC Glyc.

(1.0)(4.0)(2.5)

MCCh - 32.00% ß-TCP - 8.00% HAp - 26.66%

Glycerol - 33.33%

D1 10.0% 5%Complex

PC Glyc.

(1.0)(4.0)(2.5)

MCCh - 32.00% ß-TCP - 8.00% HAp - 26.66%

Glycerol - 33.33%

* (MCCh/ß-TCP) complex,

** PC-Powder complex - (MCCh/ß-TCP complex (1.0 wt-%) / HAp (1.0 wt-%) / Glycerol (1.0 wt-%)

The glycerol used in samples A1, B1, C1 and D1 is related with wt-% complex

content (MCCh/ß-TCP complex) and wt-% of PC content. The different ratios of

ethanol are related only with the content of composition (10%) to study the viability to

increase the number and shape of pores by freeze-dried method.

4.4.3. FTIR study of the commercial HAp

The main peaks of energy vibration mode identified in the ß-TCP powder and

MCCh/ ß-TCP complex are showed in chapter 3.

Table 3 shows the main peaks of energy vibration mode identified in the

commercial HAp (Figure 3), the characteristic functional groups of orthophosphate,

hydroxyl, phosphate and probably pyrophosphate, the latter one in trace amount,

characteristic of HAp material. The presence of carbonate was also observed in the HAp

material as well as in ß-TCP (Chapter 3), which suggests that in some commercial HAp

the CaO and Ca (0H)2 also are used to get an ideal stoichiometric relation between Ca /

P in the material [25,26, 27,33].

Table 3. Localization of peaks of energy vibration of commercial HAp in transmission

FTIR.

Figure 3. Transmission FTIR spectrum of energy vibration of commercial HAp

In the transmission FTIR, spectra of the commercial HAp powder are observed

in Figure 3 at 839 (cm1), corresponding to deformation modes of phosphate groups (O-

P-H) bonds. The antisymmetric and symmetric stretching of OH- group are observed

3570 cm-1 and 3464 cm-1, respectively, referring the way they stretch and also

characteristic of the material in water adsorption. The band observed at 962 cm-1 of low

intensity corresponds to non-degenerate symmetric stretching of P-O bonds of


antisymmetric stretching

symmetric stretching

1040, 1093, 962 P-O of PO4

antisymmetric, symmetricstretching

3570, 3464 O-H

Antisymmetric stretching 876, 1456 CO of C-O


Antisymmetric stretching 839 P-O-H

Tra

nsm

itta

nce

%

phosphate groups. The bands 1040 and 1093 (cm-1) represent the asymmetric stretch

modes, respectively, the P-O bonds of phosphate groups. There is the presence of water

in the starting material for the existence of peaks associated in the regions of 1590, 1635

(cm-1) and the peaks around 3400 cm-1, in fair agreement with the literature [13, 25, 26].

4.4.4. FTIR study of the composites

The Figure 4 and 5 show the peaks of energy vibration of the selected

composites with MCCh/ß-TCP complex with HAp. The peaks of composite preparation

according with Table 2 with different ratios of ethanol confirm the presence of the

functional groups of amino groups of microcrystalline chitosan (NH2) at 1539 cm-1,

Amide I at 1648 cm-1, amide II at 1557 cm-1, the OH- group peak 3434 cm-1 and then N-

H peak 3298 and 3500 cm-1, referring to the way they stretch. The main peaks of energy

vibration at 1026 and 1086 cm-1 identified the C-O skeletal vibration stretching of

saccharide structure, also in fair agreement of FTIR study of MCCh/ß-TCP complex

showed in chapter 3 and literature [14, 15, 36]. The characteristic functional groups of

HAp, between 840 to 1100 cm-1, also are showed in Figure 4 and 5. The presence of

ethanol in the final sponge didn’t show a significant variation in the FTIR spectrum of

composite, suggesting no interaction between ethanol, polymer and calcium phosphates

involved.

Figure 4. Absorbance FTIR spectrum of energy vibration of the composite sample A1

Figure 5. Absorbance FTIR spectrum of energy vibration of the composite sample B1

The composites with HAp are characterized by the presence of mainly

coordinate bonds between the phosphate and calcium ions and the amide and hydroxide

groups of the microcrystalline chitosan and by the forming intra and intermolecular

hydrogen bonds between amino and hydroxide groups of the chitosan chains, with a

very high energy making the structure of the composite durable resulting in an excellent

stability of temperature, pH compared to known forms of chitosan and derivatives.

Sample A1

Sample B1

Another advantage of the method to obtain the composite with HAp is the

simple procedure providing unique properties like high content bound of Ca and P ions,

high water retention value, and two different calcium phosphates with high bioactivity.

4.4.5. WAXS-Diffraction investigation of ß-TCP and HAp powder

The aim of this study is to confirm the crystalline and amorphous phases of the

commercial HAp and ß-TCP powders. The crystalline phase of powders is directed

related with calcium phosphate dissolution and less time for new tissue stimulation and

formation. Crystalline HAp is considered to be the final, stable product in the

precipitation of calcium and phosphate ions from neutral or basic solutions like in the

physiological environmental. The possible role that amorphous calcium phosphate may

play as a precursor to HAp in biological calcification places it in the mainstream of

calcium phosphate chemistry [23, 26]. However, the WAXS-diffraction of the calcium

phosphate powders showed the intensity of peak at 32,5o and the intensities of peaks at

26o and 40o showing a characteristic peaks of HAp, Figure 6, in red color. Regarding

the peak values observed, the structure is hydroxyapatite, and no different new crystal

phase was observed. The increased sharpness and intensities and decreased broadness of

mentioned peaks can be explained by the increase in crystal sizes. The ß-TCP was

found to be completely amorphous.

Figure 6. WAXS-scan in transmission mode for investigation of commercial ß-TCP

and HAp in powder form.

Material applications for engineering tissue regeneration, the relatively high

solubility of amorphous calcium phosphate in aqueous environments make suitable or a

good choice as a mineralizing agent. When compounded with appropriate polymeric

material, amorphous calcium phosphate bioactivity may be particularly useful in

enhancing the performance of composites in physiological environmental [23, 26].

4.4.6. Analysis of the particles size and morphology of the HAp powder

The aim of this study was to determine the particle size and morphology of the

commercial HAp in form of powder. The ß-TCP particles size and morphology study

was showed in Chapter 3. Like in the ß-TCP procedure previously analyzed, HAp

powder was dried with vacuum at 105oC for 24h to remove moisture. The study of

particles morphology of HAp is showed in the Figure 7.

Figure 7. SEM photo at 2000x of the commercial HAp powder.

Notice that the morphologies of the HAp powder differ in shape when compared

with ß-TCP powder, being less spherical. The employed calcium phosphates, HAp and

ß-TCP, showed a great ability to agglomeration and cluster formation, explained by a

non-homogeneous charge distribution on the surfaces and high ability for water

adsorption. Typically the very small particles tend to agglomerate due to the increased

intensity of the attractive forces over the repulsive forces, due to charge distribution on

the surfaces and between the calcium phosphates. The analysis showed HAp grain size

fits in the range 2.0-7.0 m with average particle size of 3.16 m. The particles size

distribution of the commercial HAp is showed in the Figure 8.

Figure 8. Grain size and distribution of commercial HAp powder

In Table 4, it is the particles size of commercial HAp; the specified surface area

covered by HAp is 3.41 +04 cm2/g. Also it was found around 10% of the HAp particles

used in nano size, which also indicates a faster and easier release of calcium and

phosphate ions in physiological environmental. In Table 5, it is showed the particles

size of commercial HAp and ß-TCP following the literature.

Table 4. Particles size of commercial HAp.

POWDERS HAp

Particles size (90%), 8.81 m



Superficial area (cm2/g) 3.41 e+04

Table 5. Particles size of commercial HAp and ß-TCP [6, 7, 8, 12, 21].

POWDERS HAp ß-TCP

Particles size (90%), ( m) 7.37 9.77

Particles size (50%), ( m) 2.83 2.67

Particles size (10%), ( m) 0.80 0.60

Superficial area (cm2/g) 45.7 + 0.2 45.7 + 0.1

A great number of materials have been used for bone tissue engineering

including polymers, bioglass and a variety of calcium phosphate ceramics, and the

performance of the biocomposite also reflects in the method of preparation, particles

size, shape and ratio of inorganic part in the polymer matrix [6,7,8, 12, 21].

4.4.7. SEM study of the composites in sponge form.

The objective of the investigation was to study the HAp, suitability and

distribution in the MCCh/ß-TCP complex in the sponge form preparations.

The literature shows that scaffolds should possess an interconnected and diffuse

porosity (usually over 90%), if cell adhesion, ingrown and reorganization are to be

sustained in vitro, and room for neovascularisation has to be provided in vivo; pore

interconnections influence the diffusion of nutrients to the cells. Because excessive pore

size means decreased internal surface area, a compromise is necessary: for instance, for

regenerating bone tissue in vitro, some authors preferred pore size 200-400 m, while

others used scaffolds with 500 m nominal pore size. When pore diameter is too small,

pore occlusion prevents cellular penetration into the scaffold: as a consequence, pore

size 75-100 m resulted in ingrown non-mineralized osteoid tissue, while even smaller

pores were penetrated only by fibrous tissue. Because highly porous materials have

limited mechanical strength, the void volume must be tuned to allow for the

accommodation of a large number of cells and the preservation of the structural strength

particularly in load-bearing tissues. A proper porosity may improve mechanical

interlocking between the scaffold and the surrounding host tissue, providing necessary

mechanical stability at this critical interface. Scaffold surface properties such as

morphology, hydrophilicity, zeta-potential and surface energy influence cell adhesion,

migration, phenotype maintenance and intracellular signaling in vitro, as well as cell

recruitment at the tissue / scaffold interface in vivo [6, 7, 19, 37].

All composites of MCCh/ß-TCP complex with HAp in sponge form showed a

well-shaped 3-dimensional structure with high interconnected porous, suggesting a

good biological environment to cell attachment and proliferation, providing the passage

of nutrient flow through micro and macro interconnected porous. Those samples can be

used in future as a base for scaffolds production. The HAp powder aggregates well and

showed a homogenous distribution in all MCCh/ ß-TCP complex matrix that could be

explained also by the lyophilization process reducing the time for agglomeration and

cluster formation to obtain sponges and the mixing process to obtain the suspension. All

samples were obtained with smooth surface without defects, the pore morphology

structure showed more round shape, also affected by the glycerol used as a plasticizer

increasing the mixing process in the preparation of the suspension and directed related

with the homogenous interconnected pores formation.

The literature shows that more round shape porous, great number of porous and

smaller size decrease the chance for inflammatory reactions [9, 10, 14, 38].

There is a possibility to improve the number and shape of pores in the structure

of sponges, changing parameters in the freeze-dried method such as temperature,

pressure, time and adding ethanol for a faster removal of water in the samples and

method of liophylization showed in Figure 1. Figures 9-20 show the structure on surface

and cross-section of sponges according with Table 2.

x90 magnification x200 magnification x1000 magnification

Figure 9. SEM photographies cross-section of sample A 1, sponge conc. 10%.


Figure 10. SEM photographies surface A, of sample A 1, sponge conc. 10%.


Figure 11. SEM photographies surface B, of sample A 1, sponge conc.10%.

x90 magnification x200 magnification x1000 magnification

Figure 12. SEM photographies cross-section of sample B 1, sponge conc. 10% and 1%

of ethanol.


Figure 13. SEM photographies surface A, of sample B 1, sponge conc. 10% and 1% of

ethanol.


Figure 14. SEM photographies surface B, of sample B 1, sponge conc. 10% and 1% of

ethanol.


x1000 magnification

Figure 15. SEM photographies cross-section of sample C 1, sponge conc. 10% and 2%

of ethanol.

x200 magnification

x1000 magnification

Figure 16. SEM photographies surface A, of sample C 1, sponge conc. 10% and 2% of

ethanol.

x200 magnification

x1000 magnification

Figure 17. SEM photographies surface B, of sample C 1, sponge conc. 10% and 2% of

ethanol.

Figure 18. SEM photographies cross-section of sample D 1, sponge conc. 10% and 5% o


Figure 19. SEM photographies surface A, of sample D 1, sponge conc. 10% and 5% of

ethanol.

x200 magnification

x1000 magnification

Figure 20. SEM photographies surface B, of sample D 1, sponge conc. 10% and 5% of

ethanol.

The SEM observation of the composite sponge showed that the sample B 1 with

1 % of ethanol, showed in Figure 12, presents great network porosity with more

homogenous shape and size of pores and clusters. The estimate pore size ranges from 20

to 150 m were fabricated, suggesting that ethanol increases the evaporation of water

speed and directed related with the porosity. In general, the micro and macrostructure is

controlled by varying the polymer material, polymer concentration, solvents, processing

parameters (temperature, pressure and time), it is user and technique sensitive and the

processing parameters have to be well controlled.

4.4.8. The physical-mechanical tests of composite in sponge form.

From a biomechanical and clinical point of view, the hard tissue-

engineered implant should allow also for a mechanically stable fixation as well as

biological, chemical and physical properties on the host tissue. A great number of

fabrication technologies have been applied to process biodegradable and bioresorbable

materials for tissue engineering into 3-D polymeric scaffolds of high porosity and

surface area. The conventional techniques for scaffold fabrication include fibre bonding,

solvent casting, particulate leaching, membrane lamination and melt molding and

freeze-drying method applied in this research. The mechanical properties of bone or

scaffolds depend largely on the humidity, load application mode, load application

direction, age, materials involved, the kind of bone, and so on. While increasing level of

bone mineralization, strength increases [37]. Scaffolds for bone tissue engineering in

form of sponge must have a highly porous and interconnected pore structure to ensure a

biological environment conducive to cell attachment and proliferation as well as tissue

growth, in addition to provide the passage of nutrient flow [14, 24, 25]. Table 6 shows

the mechanical properties of composite sponges prepared according with Table 2.

Table 6. Mechanical properties of the sponge preparations with complex in paste formand powder complex.

Parameters

Samples

ethanolcontent

composition Tensile strength(MPa)

Elongationbreak (%)

ElasticModulus(MPa)

A1 0.0% Complex (1.0 wt-%)PC (4.0 wt-%)Glyc. (2.5 wt-%)

0.008 4.47 (cv 17.6% ) 0.005

B1 1.0% Complex (1.0 wt-%)PC (4.0 wt-%)Glyc. (2.5 wt-%)

0.006 5.42 (cv 66.5% ) 0.005

C1 2.0% Complex (1.0 wt-%)PC (4.0 wt-%)Glyc. (2.5 wt-%)

0.006 4.47 (cv114.5%) 0.005

D1 5.0% MCCh (1.0 wt-%)PC (4.0 wt-%)Glyc. (2.5 wt-%)

0.005 5.00 (cv 33.4%) 0.005

c.var. - Coefficient of variation, PC-Powder complex- (MCCh/ß-TCP complex (1.0 p.w) / HAp (1,0 p.w) /Glycerol (1.0 p.w)), complex (MCCh/ß-TCP)

Based on the results presented in Table 5, it has been found that addition of 1%

ethanol to MCCh/PC/Glycerol composite does not affect significantly the mechanical

properties of sponge, on the other hand, it showed a increase in the homogenous

structure with higher porosity directed related with no variation of tensile strength and

higher elongation at break. The MCCh/ß-TCP complex with HAp samples can be

summarized: the addition of PC powder to complex causes decrease of tensile strength

by the addition of calcium phosphate, but, on other hand, it could improve the

elongation at break by addition of glycerol, which could certainly alter the physical and

mechanical properties by enhancing the mobility of the polymer chains. According with

literature, the mechanical property tests have a glycerol and HAp influence in the

preparations. The presence of glycerol in causing significant differences in tensile

strength and elongation at break, without glycerol, presented greater tensile strength and

lower elongation. Glycerol interfered with MCCh chains, decreasing intermolecular

attraction and increasing polymer mobility which facilitate elongation [6, 19, 21, 38].

The literature also shows that the deacetylation degree was also a significant

factor on the mechanical properties. In the absence of additives, chitosan with lower

deacetylation (60.9% DD) presented higher tensile strength and higher elongation than

citosan of 96% DD. Significant interaction was observed only between glycerol and

deacetylation degree. Thus, glycerol effect was more intense on chitosan with lower DD

than on those with high DD [6].

4.4.9. Determination of Ca and P content in composites

The aim of this analysis is to determine the content of Ca and P that remains

after sponges preparation. This relation is also mentioned in the literature that minerals

in study are the most important elements for hard tissue regeneration activity and are

directly related with calcium phosphate dissolution and less time for new tissue

stimulation [7, 8, 9, 10, 12]. Table 7 shows the proportion of the Ca and P in

composites.

Table 7. Weight proportion of Ca and P in the raw material and composites.

The results showed a relation between Ca and P of complex and composite

approximately 2/1 wt-%, respectively, which shows a high content of Ca and P in all

samples.

4.5. Conclusions

The FTIR analysis of the composites showed that, in both cases, the components

(MCCh, ß-TCP and HAP) existed in the samples. The presence of ethanol in the final

sponge composite didn’t show a significant variation in the FTIR spectrum of

composite, suggesting a no interaction between ethanol, polymer and calcium

phosphates involved.

The WAXS-diffraction of the calcium phosphate powders showed the

characteristic crystalline peaks of commercial HAp, the ß-TCP was found to be

completely amorphous. The particles analysis showed HAp grain size fits in the range

2.0 - 7.0 m with average particles size 3.16 m with a highly tendency to agglomerate

and water absorption.

Symbol ofsample

Proportion of Ca and P

composition Ca(ppm)

P(ppm)

HAp powder 447200 186500ß-TCP powder 425400 199000MCCh 108 186MCCh/ß-TCPcomplex

53600 28200

(Sample A1) Complex (1.0 wt-%)PC (4.0 wt-%)Glyc. (2.5 wt-%)

183000 80800

All composites of MCCh/ß-TCP complex with HAp in sponge form showed a

well-shaped 3-dimensional structure with high interconnected porous, suggesting a

good biological environment to cells attachment and proliferation, providing the

passage of nutrient flow thought the micro and macro interconnected porous. Those

samples can be used in future as a base for scaffolds production.

Addition of 1% ethanol to MCCh/PC/Glycerol composite does not affect

significantly the mechanical properties of sponge, in another hand showed a increase in

the homogenous structure with higher porosity related with no variation of tensile

strength and higher elongation at break. The MCCh/ß-TCP complex with HAp samples

can be summarized: the addition of PC powder in to complex causes decrease of tensile

strength by the addition of calcium phosphate but in another hand could enhancing the

elongation at break by addition of glycerol could certain alter physical and mechanical

properties by enhancing the mobility of the polymer chains. The relation between Ca

and P approximately 2/1 wt-% respectively, that shows a high content of Ca and P in all

samples.

ACKNOWLEDGEMENTS

The research and work presented in this study were conducted with financial

support from the European Community's Seventh Framework Programme (“Marie Curie

Initial Training Network), FP7/2007-2013.

The particle size measurements, X-ray diffraction and quantification of calcium

and phosphate content were conducted using equipment provided by the TITK-

Thuringian Institute of Textile and Plastics Research in Rudolstadt, Germany.

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Membrane Prepared by Freeze-Gelation Method, Trends Biomater. Artif. Organs, 22

(1).

36. Henryk Pospieszny, Wojciech Folkman. (2004). Factors modyfying a biological

activity of chitin derivatives. ed. Progress on Chemistry and Application of Chitin and

its Derivatives, PTChit. X, p.07-12.

37. Qiaoling Hu*, Baoqiang Li, Mang Wang, Jiacong Shen. (2004). Preparation and

characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in

situ hybridization: a potential material as internal fixation of bone fracture, Biomaterials

25, 779-785.

38. Young Rho-Jae, Liisa Kuhn-Spearing, Peter Zioupos. (1998). Mechanical properties

and the hierarchical structure of bone, Medical Engineering & Physics 20, 92-102.

Chapter 5

Preparation of chitosan with nano calcium phosphates composite in fibre form.

5.1. Introduction

Fibres made of chitosan are widely used in new generation biomaterials. The

formation of chitosan fibres is a rather simple process usually accomplished by wet

spinning from a polymer solution. A variety of fibrous chitosan materials are available,

ranging from fine microfibrids [1] to very strong fibres and pseudo-dry-spun fibres [2]

and multifilament chitosan yarns [3,4]. Wet spinning from a solution of the polymer

enables modifications by adding various functional substances to the solution, notably

nanoparticles, carbon nanotubes (CNT) [5-7], nanosilver [8], calcium phosphates [9],

calcium sulphite [10] and a number of polymers such as fibroin and keratin proteins

[11,12]. Chitosan lends itself to use in biomaterials thanks to properties such as its

biodegradability, biosorption and ability to accelerate wound healing.

To confer functionality upon chitosan fibres, they are frequently coated with

other biomaterials such as collagen or calcium phosphates [13-17]. Following coating,

they are used to reinforce composite implants containing hydroxyapatites (HAp) [18].

Calcium phosphates are known for their compatibility, osteoconductivity and

easy absorption by the human body, which makes them suitable for the preparation of

composite dental implants [19] and, in combination with chitosan, artificial bones

[20,21]. Chitosan multifilament yarn, which is used in the construction of textile

scaffolds or as culture medium for cell growth, requires the addition of calcium

phosphates like HAp or ß-TCP and has been successfully used in medical applications

such as artificial bones, cartilage and glues for hard tissue engineering [3,13-15,20-23].

Calcium phosphates are not easy to dissolve in chitosan solutions; dissolution

depends on their chemical composition, crystallinity and dissolving conditions. For this

reason HAp nanoparticles can be used in the preparation of chitosan gels [24] or can be

introduced into the fibre matrix. Calcium phosphates HAp and ß-TCP have different

dissolving properties, and hence their release from composites or chitosan fibres may

differ. For this reason they are usually employed in a mixture to maintain a favorable

calcium-to-phosphorus ratio of 1.67 [25].

The aim of this study was to prepare novel chitosan fibres containing

nanoparticles of calcium phosphates for medical use, particularly for textile scaffolds

and cell growth. We also investigated how nanoparticles of ß-TCP, HAp and of the

complex HAp/ ß-TCP affect the rheology of the chitosan spinning solution, spinning

conditions and mechanical properties of the resulting HAp/ ß-TCP-modified fibres.

5.2. Materials

Tricalcium phosphate (ß-TCP) (Ca3(PO4)2), Hydroxyapatite (Ca10(PO4)6OH)2, supplied

by Sigma Aldrich, Germany.

Glycerol as a plasticizer (C3H8O3), 99% pure, Sigma Aldrich, Germany.

Hydrochloric acid 37.8% pure, manufactured by Fluka, Germany.

Acetic acid 80% and NaOH made by POCh S.A., Gliwice, Poland.

Chitosan derived from the northern shrimp (Pandalus borealis), supplied by Primex Co.,

Norway. Characteristics properties of chitosan such as Viscometric average molecular

mass [kD] 342; Moisture content [%] 5.58; Water retention value [%] 43.5; Dynamic

viscosity, 1 % chitosan in 1% acetic acid at 20 °C[CP] 63.1; Degree of deacetylation

[%] 83.2; Ash content [%] 0,40; Nitrogen content [%] 6.84.

5.3. Methods

5.3.1. Methods of manufacture composite chitosan fibres

The composite chitosan fibres production method is according to the invention,

polish patent application number. P. 393022, 2010 (Wawro, D.; Pighinelli, L.;

St plewski, W.) presents as follows: a homogeneous aqueous acid solution of 40 - 70

°C temperature, containing 0,1 - 10 wt% of hydroxyapatite and/or tricalcium phosphate

nano-particles, and 0,1 - 3 wt% of chitosan is entered, during mixing, into the spinning

solution of 20 - 55 °C temperature, containing chitosan of average molecular weight of

150 000 - 700 000, deacetylation degree of 80 - 95 %, poly-dispersion degree between 2

- 5, and concentration of 2 - 7 wt% in acetic acid aqueous solution of 1 - 3 wt%

concentration, containing 0,1 - 15 wt% of glycerine in relation to chitosan. Then, after

venting, the obtained spinning solution is being embossed through multi-hole spinning

nozzle into the coagulation bath of 25 - 45 °C temperature, which is a sodium hydroxide

aqueous solution of 1 - 5 wt% concentration. Obtained fibres are being stretched from

15 to 67 % in sodium hydroxide aqueous solution of 0,1 - 0,5 % concentration in

temperature of 80 - 95 °C. Then they are rinsed in water, finished in bath of 30 - 75 °C

temperature containing hydro-alcoholic emulsion with surfactants, and they are being

dried in temperature of 40 °C.

5.3.2. Preparation of Chitosan Spinning Solution Containing HAp, ß-TCP and

HAp/ß-TCP Nanoparticles

A 5.0 wt% weight chitosan solution (A) was prepared in aqueous 3.0 wt%

weight acetic acid. The dissolution of chitosan took 60 minutes, during which time

glycerol was introduced to the solution to a volume of 10%. After filtration and

deaeration at ambient temperature, the chitosan solution was blended with 2.0 wt%

chitosan solution containing ß-TCP particles (solution B), a chitosan solution containing

HAp (solution C) or a mixture of chitosan solutions containing HAp/ß-TCP

nanoparticles (solution B/C). Solution B was an aqueous 2.0 wt% solution of chitosan in

0.9 wt% solution of hydrochloric acid with a 2.0 wt% weight content of ß-TCP

nanoparticles. Solution C was a diluted aqueous 2.5 wt% chitosan solution in 1.5 wt%

acetic acid containing HAp particles at a concentration of 2.0 wt%.

5.3.3. Wet Spinning of Chitosan Fibres Containing HAp, ß-TCP and HAp/ ß -TCP

Nanoparticles

Chitosan fibres modified with HAp, ß-TCP and HAp/ß-TCP nanoparticles were

prepared by wet spinning on a pilot line equipped with a spinning head holding a

rhodium/platinum spinneret with 150 holes, each having a diameter of 0.08 mm. The

spinning solution is 35 °C, the same as a coagulation bath (aqueous 3.0 wt% sodium

hydroxide at 35 °C). The spun fibres were washed first in a water bath at 40 °C then in a

water-ethanol (60% v/v) bath and were then dried without tension in a loose bundle.

The spinning speed for the chitosan fibres modified with HAp/ß-TCP nanoparticles was

31 m/min.

5.3.4. Analytical Methods

The rheological properties of the chitosan solutions were measured using a

digital viscometer Brookfield model RV DV-II+, with the Rheocalc V3.1-1 programat

20, 25, 30, 35 and 40 °C. A Biolar ZPO polarizing microscope equipped with an

advanced image analysis system (MultiScan V. 14.02) was used to prepare images of

the aqueous solution of chitosan containing HAp and ß-TCP particles.

Images of the cross-section and surface of the fibres were obtained using a

scanning electron microscope SEM/ESEM, Quanta 200 (W), FEI Co., USA.

The size of the nanoparticles of HAp, ß-TCP and HAp/ß-TCP in chitosan solution was

measured using a ZETASIZER 2000 (Malvern Instruments) apparatus.

The amount of chitosan in the spinning solutions was determined using the

gravimetric method. A film was formed from a weighed amount of the solution by

evaporation of water and drying at 60 °C. This was then coagulated in an aqueous

solution of 3.0 wt% sodium hydroxide, rinsed and dried to constant weight at 105 °C.

The water retention value (WRV) was determined according to Standard

ISO/FDIS 23714. The mechanical properties of the chitosan fibres were tested

according to Standards PN-ISO-1973:1997 and PN-EN ISO 5079:1999 in an air-

conditioned room at 65 ± 4% relative humidity and 20 ± 2 °C.

Spectrophotometric spectra in the infrared range were prepared using FTIR

apparatus produced by Unicam Co. equipped with the control program Winfirst ATI

Mattson. Samples were prepared in the form of pressed cubes in potassium bromide

(KBr Aldrich Co.).

Samples of the fibres were mineralized at 575 °C in 6 M HCl to determine the

calcium content, which was measured in the mineralized residue by flame atomic

absorption spectrometry (FAAS) at a wavelength of 422.7 nm. A SCAN-1 f-my Thermo

Jarrell Ash atomic absorption spectrometer was used for the analysis.

The ash content in the chitosan fibres was estimated according to standard EN

ISO3451-1.


5.4.1. Preparation of Chitosan Solutions Containing -TCP, HAp and HAp/ -TCP

A 5.16 wt% aqueous chitosan solution (A) was prepared in 3.0 wt% acetic acid. The

solution was blended with a chitosan solution containing -TCP, HAp, or a mixture of

solutions containing HAp/ -TCP nanoparticles in the appropriate proportions. Table 1

presents the properties and the composition and some properties of these solutions. The

5.16 wt% chitosan solution (A) had a temperature of 52 °C and a high dynamic

viscosity of 19000 Pa. The dissolution of chitosan and the blending of the particular

solutions caused no problems. Admixing of the solution with calcium phosphate

resulted in a decrease in the concentration of the acid and chitosan and in the dynamic

viscosity. Small particles of calcium phosphate ( -TCP) were found in the chitosan

solutions upon microscopic observation. A clear, stabile chitosan solution suitable for

spinning was obtained by blending solutions A and B (code MCT 6).

Table 1. Some properties of chitosan solutions containing hydroxyapatite (HAp) and

tricalcium phosphate ( -TCP) particles.

Solutioncode

Percentage ofsolution used,

%

Concentration ofDynamic

viscosity/temp.-TCP HAp Acetic acid Chitosan

A B C wt% wt% wt% wt% Pa/°CChit 58 100 - - - - 3.00 5.16 19000/52MCT 6 83.3 16.7 - 0.333 - 2.75 4.50 7500/49MCT 7 83.3 - 16.7 - 0.330 2.75 4.63 9250/49MCT 8 71.4 14.3 14.3 0.286 0.283 2.36 4.46 4500/52MCT 11 62.5 25.0 12.5 0.707 0.252 2.10 4.01 1750/51

A chitosan solution (A) is 5.0 wt% chitosan was prepared in aqueous 3.0 wt%weight acetic acid; Solution B was an aqueous 2.0 wt% solution of chitosan in 0.9wt% solution of hydrochloric acid with a 2.0 wt% weight content of -TCPnanoparticles; Solution C was a diluted aqueous 2.5 wt% chitosan solution in 1.5wt% acetic acid containing HAp particles at a concentration of 2.0 wt%.

Comparison of microscopic images of the chitosan solutions denoted Chit 58 (A) and

MCT 6 indicated that the introduction of the chitosan solution containing -TCP

nanoparticles reduced the number of undissolved particles in the MCT 6 solution.

Hydroxyapatite is less soluble than -TCP; this was apparent in the course of

preparing solution C, when only a small amount of HAp dissolved while the majority

appeared as an opalescent suspension. Blending of the HAp-containing chitosan

solution with the chitosan spinning solution (A) only slightly improved the solubility of

HAp in the chitosan acetate solution. Hence, the chitosan acetate solution denoted MCT

7 contained HAp particles with a diameter of up to a few micrometres. Though not

negating the solution’s spinnability, this feature may negatively influence the quality of

the prepared calcium-containing fibre. The reverse was the case when chitosan acetate

solution was mixed with chitosan B and C solutions containing HAp and -TCP

(solution B contained 0.9 wt% hydrochloric acid). The result was the complete

dissolution of the hydroxyapatite and a clear solution denoted MCT 11.

It is likely that mixing of the chitosan solution containing HAp with the chitosan

solution containing -TCP in hydrochloric acid at a ratio of 2:1 provided adequate

conditions for the dissolution of HAp. The dissolution of HAp in chitosan solution is the

subject of further investigations and of a patent application [26]. The mechanism of

salts and amino acids upon the dissolution of HAp has been reported previously [27].

Mixing of solutions B and C in the ratio 1:1 did not cause HAp to dissolve (MCT 8).

Microscopic analysis of the MCT 7 and MCT 11 chitosan acetate solutions

confirmed the presence of HAp particles in the MCT 7 solution, while the B/C mixture

(MCT 11) clearly contained HAp/ -TCP nanoparticles. The microscopic image of the

MCT 11 solution was optically pure. The chitosan solution (B) containing -TCP

nanoparticles was also clear and optically pure. Hydroxyapatite added to chitosan acetate

solution (C) produced a suspension with only some of it going into solution.

Agglomerates with a size of 1500 nm that are present in the spinning solution may

negatively influence the mechanical properties of the spun fibres. The situation was

slightly improved when mixing the chitosan B/C solution with the chitosan A solution

in a 1:1 ratio, which resulted in a chitosan solution (MCT 8) with fewer HAp particles.

Another solution of the B/C mixture was prepared with more of the -TCP particle-

containing chitosan solution (2:1). During the blending of the HAp and -TCP particle-

containing chitosan solutions, the HAp particles dissolved immediately, resulting in a

clear solution (MCT 11). Comparison of the images of the chitosan solutions MCT 7

and MCT 11 revealed that mixing the HAp- and -TCP-containing chitosan solutions

had a beneficial effect by limiting the size of the nanoparticles within the range of 12.8-

58.0 nm in MCT 11. Figure 1(a-c), presents the particle size distribution in the chitosan

solutions containing HAp or -TCP and HAp and -TCP.

Figure 1. (a) Particle size distribution of -TCP in chitosan solution in hydrochloric

acid (solution B); (b) HAp particles in chitosan acetate solution (solution C); (c) The

HAp/ -TCP blend in chitosan (solution B/C).

(a)

(b)

(c)

Table 2 shows selected parameters of the solutions analyzed by means of the

ZetaSizer analyzer. The results presented in Table 2 and Figure 1 confirm. The smallest

nanoparticles found in the B/C mixture were much smaller than those in chitosan

solutions B and C before blending. As can be seen in the Figure 2 the pictures of the

solutions B, C and the blend solution B/C.

Figure 2. The cup number 1 shows the blend solution B/C (2:1), the cup number

2 shows the solution B and cup number 3 shows solution C.

Table 2. Selected parameters of chitosan solutions containing HAp, -TCP and HAp/ -

TCP.

Chitosan solutionRange of particle

sizeSize of fraction with

highest volume contentPercentage of

volumePotential Zeta

nm nm % mVSolution B 28.9-164.9 65.2 11.5 43.0 ± 2.3Solution C 417.3-1495 745.4 34.2 45.3 ± 2.3Blend of chitosansolutions B/Cin 2:1 ratio

12.8-58 22.9 19 52.9 ± 4.0

The chitosan solutions characterized in Table 2 were prepared for the spinning of

chitosan fibres. Addition of the calcium phosphate nanoparticles to the chitosan

solutions resulted in a slight drop in the chitosan content and concentration of acetic

acid, an insignificant increase in the hydrochloric acid content in the blended mixture,

and a radical decrease in the apparent dynamic viscosity. The Zeta potential showed

greater stability for the solution MCT-11 (solution A + B/C(2:1)) in this ratio than for

other solutions.

5.4.2. Rheology of Chitosan Solutions Modified with HAp/ -TCP Nanoparticles

The rheology of chitosan solution MCT 11 modified with HAp/ -TCP nanoparticles

was examined at 20, 25, 30, 35 and 40 °C. The impact of shearing speed and

temperature upon the dynamic viscosity of the chitosan solution modified with calcium

phosphate is presented in Figure 3. The flow curves were drawn for the rise and fall in

shearing speed.

Figure 3. Dependence of the apparent dynamic viscosity on the shearing rate and

temperature of the acetate chitosan solution modified with HAp/ -TCP (MCT 11).

It can be inferred from the curves that chitosan modified with HAp/ -TCP

nanoparticles has the attributes of a non-newtonian fluid. In the examined temperature

range, the viscosity of the solution decreased with increasing shearing speed. The flow

curves overlapped with each other both at increasing and decreasing shearing speed.

Chitosan solutions thus showed the features of a pseudo-plastic fluid (they are diluted by

shearing) typical of polymer solutions. An increase in temperature from 20 to 25 °C

distinctly reduced the dynamic viscosity. Further increases in temperature and shearing

speed caused much greater decreases in viscosity. Given that fibres are spun from

solutions of chitosan acetate and that the spinning process takes a relatively long time,

the stability of solutions as confirmed by the rheological examinations is of particular

importance.

5.4.3. Investigation into the Spinning of Chitosan Fibres Modified with HAp, -TCP and HAp/ -TCP

To allow comparison, regular chitosan fibres and those modified with calcium

phosphate were spun under the same conditions at a spinning speed of 31.0 m/min and a

draw ratio of 34%. The fibres were spun into a coagulation bath containing aqueous

3.0% NaOH without ethanol. The spinning process of both regular chitosan fibres and

those modified with -TCP, HAp and HAp/ -TCP was stable in the adopted conditions

with no disturbance in the spinneret zone. The addition of calcium phosphates to the

spinning solution had a beneficial effect on spinning stability, with fewer breaks in the

elementary filaments.

5.4.4. Mechanical Properties of Chitosan Fibres Modified with HAp, -TCP and

HAp/ -TCP

The mechanical properties of chitosan fibres modified with HAp/ -TCP are

presented in Table 3. The impact of the calcium phosphate on the mechanical properties

of the fibres varied depending on the type and amount of additive used. The addition of

-TCP had little effect on tenacity (MCT 6) while the same amount of HAp reduced

both the tenacity and elongation (MCT 7). The large amount of HAp agglomerates in

the fibres may be the underlying cause. Blending of the chitosan solutions with HAp

and -TCP led to an improvement in the solution with higher amounts of calcium

phosphates (some of the HAp particles dissolved), with a slight increase in fibre

tenacity (MCT 8). Doubling of the amount of chitosan solution B (containing -TCP) in

the mixture led to a homogenous solution of HAp/ -TCP (MCT 11) nanoparticles along

with the highest level of calcium phosphates, and resulted in fibres with the same

tenacity as that of regular chitosan fibres.

Table 3. Impact of HAp, -TCP and HAp/ -TCP concentration in the chitosan

solution on the mechanical properties of fibres.

Parameter Chit 58 MCT 6 MCT 7 MCT 8 MCT 11

Linear density dtex 4.39 4.48 5.14 5.41 4.16

Coefficient of variability of linear density % 1.25 2.48 1.93 3.57 1.64

Confidence interval of linear density % ±1.55 ±3.08 ±2.40 ±4.43 ±2.04

Breaking force cN 3.61 3.51 2.46 2.91 3.35

Coefficient of variability of breaking force

(conditioned)% 14.6 14.0 32.2 28.6 19.5

Confidence interval of breaking force % ±6.02 ±5.77 ±13.3 ±11.8 ±8.03

Tenacity (cond) cN/tex 8.22 7.83 4.79 5.38 8.05

Elongation at break (cond) % 17.0 22.0 9.9 11.0 12.0

Breaking force (wet) cN 2.80 2.21 1.78 2.48 2.25

Coefficient of variability of breaking force

(wet)% 36.8 31.9 49.9 33.4 18.3

Tenacity (wet) cN/tex 6.38 4.93 3.46 4.58 6.86

Elongation at break (wet) % 7.8 7.3 7.8 6.1 8.00

When dried in a loose bundle, chitosan fibres modified with HAp/ -TCP (MCT 11)

did not stick to each other; a feature that meant a finishing agent was not required. An

advantage of chitosan fibres containing HAp/ -TCP (MCT 11) nanoparticles is their

high tenacity and low coefficient of variability of breaking force in wet conditions in

comparison with other fibres.

5.4.5. FTIR Examination of Chitosan Fibres Modified with HAp, -TCP and

HAp/ -TCP Nanoparticles

The main peaks in energy vibration identified in the -TCP and HAp powders are

shown in Figure 4. The functional groups of orthophosphate (PO43−), hydroxyl (-OH)

and phosphate (HPO42−), the latter one in trace amounts, are characteristics of apatite

materials. Trace amounts of (CO32−) groups were observed at 1428 cm−1, for both -

TCP and HAp, indicating that in some commercial calcium phosphate powders, CaO

and Ca(OH)2 are added to achieve the ideal stoichiometric proportion between Ca/P

[23,25,28-30]. In the Figure 4 show the FTIR spectrum of the commercial calcium

phosphate.

Figure 4. FTIR spectrum of the commercial HAp and -TCP.

0

0,5

1

1,5

2

2,5

3

5001000150020002500300035004000

Absorbance

Wavenumber cm-1

-TCP

HAp

The absence of bands at 460 and 740 cm−1 and an isolated band at 600 cm−1,

characteristic of -TCP, indicate that the starting material was composed of -TCP only.

This calcium phosphate is easily identified by a broad band at 900-1200 cm−1, and the

presence of a peak at 724 cm−1, characteristic of the symmetric mode of (P-O-P), the

distortion of P-O. The peak at 1211 cm−1 is characteristic of the non-degenerate

deformation of hydrogen groups (H-OPO3, O-PO3, HPO42−), which may reflect the

interaction with water molecules in the structure [23,29,31,32-34]. Figure 3 shows the

spectrum of HAp with a peak at 839 cm−1 that corresponds to the deformation modes of

the phosphate group (O-P-H) bonds, which are associated with the energy levels and the

rotational type of (O-H) bond. The (-OH) group peaks are also apparent at 3570 cm−1

and 3464 cm−1, reflecting the way they stretch.

The low intensity peak visible at 962 cm−1 corresponds to the non-degenerate

symmetric stretching of P-O bonds in the phosphate groups. The peak at 1040 and 1093

cm−1 represent the asymmetric stretch modes of the P-O bonds in the phosphate groups

[23,31-34]. Figure 5 shows the FTIR spectra of the MCT 7 HAp composite. spectrum

exhibits characteristic bands such as: a strong and broad peak between 1032 cm−1 and

1086 cm−1 and 1160 cm−1 that reflects the skeletal vibrations stretch of the saccharide

structure and a peak at 1559, 1543 cm−1 attributed to the free primary amino group

(NH2), amide I (1637 cm−1) indicating that chitosan used in this research is partially

deacetylated (83.2%), amide II (1559 cm−1) and amide III (1319 cm−1). The anti-

symmetric stretch bridge C-O-C (1160 cm−1), stretch N-H (3298, 3500 cm−1), stretch O-

H (3445 cm−1) and the main peaks of energy vibration (1032, 1086 cm−1) identified the

chitosan. The peak at 1243 cm−1 represents the free amino groups and the C2 position of

glycosamine [23,25,27,30-32,35]. The main peaks of energy vibration identified in HAp

are characteristic of the functional groups of orthophosphate (PO43−) at 1100 cm−1,

hydroxyl (-OH) at 3700 and 2600 cm−1 and phosphate (HPO42−) at 1000 cm−1.

Figure 5. FTIR spectra of chitosan fibres (Chit 58) and modified -TCP (MCT 6),

and those modified with HAp (MCT 7), HAp/ -TCP (1:1) (MCT 8) and HAp/ -

TCP (1:2) (MCT 11).

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

5001000150020002500300035004000

Absorbance

Wavenumber, cm-1

MCT7CHIT 58MCT 6MCT 8MCT 11

The band in the region from 1590 to 1635 cm−1 and the peaks around 3400 cm−1,

corresponding to the (-OH) stretching absorption band, suggests the presence of water

molecules in the sample [19,25,27,31,36].The chemical interactions between the

inorganic and organic constituents in the composite probably take place via ionic

bonding between (Ca2+) and phosphate groups and the amino group of chitosan,

confirming the positive charge of chitosan and the negative charge of -TCP

[21,23,25,30,31,35].

All band features were similar to those described by Brugnerotto et al. [21,37] and

were present in all samples investigated, showing that all have basically the same

functional groups. The exact calcium phosphate phase formed under acidic conditions is

influenced by the specific anions present. In some cases, unusual phases may be formed

under acidic conditions, such as CaCl2·Ca(H2PO4)2·2H2O at low pH or in the presence

of HCl. The pH of the chitosan acetate solution was typically around 4 to 5. In this

regime, HAp is expected to show good solubility and may exhibit phase instability

[30,35].

High turbidity was observed in solutions containing HAp, which indicates a strong

adsorption interaction between chitosan and the HAp surface. Chitosan has a relatively

high zeta potential and can enhance the stability of HAp [30,35].

5.4.6. Morphology and Chemistry of Chitosan Fibres Modified with HAp, -TCP

and HAp/ -TCP Nanoparticles

The WRV of wet-spun regular- and HAp- and -TCP modified chitosan fibres

ranged from 150% to 331% depending on the calcium phosphate content.

MCT 8 had the highest WRV of 331%, which may be attributed to the high content

of apatite agglomerates capable of forming water-retaining pores. The amount of

calcium phosphate in the chitosan spinning solutions was estimated according to the

calcium and ash content (Table 4).

Table 4. Water retention value (WRV) content of calcium and ash in HAp/ -TCP

modified chitosan fibres.

Fibre codeWRV Ash Calcium content

% % g/kgChit 58 158 0.1 0.14MCT 6 163 0.4 0.35MCT 7 154 3.2 8.45MCT 8 331 4.8 9.95MCT 11 210 4.8 14.35

Cross-sections of selected HAp/ -TCP-modified chitosan fibres are illustrated in

SEM images in Figure 6.

Figure 6. (a,b) SEM images of the surface and cross-section of chitosan fibres

modified with HAp/ -TCP (MCT 8); (c,d) chitosan fibres modified with HAp/ -

TCP nanoparticles (MCT 11) and (e) picture of the final fibres.

(a) (b)

(c) (d)

(e)

The amount and size of HAp/ -TCP particles has a significant influence on the shape

of the fibre cross-section. In MCT 11 it is oval and uniform with an undeveloped brim.

The surface is characteristic of chitosan fibres with distinct recesses and grooves. The

white spots that can be seen in the fibre cross-sections in Figure 6 (a,b) represent

HAp/ -TCP agglomerates. In contrast, chitosan fibres spun from solutions containing

HAp/ -TCP nanoparticles have a pure cross-section with no traces of anything other

than the fibre-forming polymer substances (Figures 6 (c,d)).

As can be seen in the pictures represented below the clear chitosan/ -TCP/HAp

solution with nanoparticles of the respective calcium phosphates

Figure 7. Solution with chitosan and nano calcium phosphates composite used to

prepare fibre (solution MCT-11).

Figure 8. Wet-Spinning process using the solution with chitosan and nano calcium

phosphates composite used to prepare fibre (solution MCT-11).

5.5. Conclusions

The presence of HAp/ß-TCP nanoparticles in the solution of chitosan had a

beneficial effect on the production of the modified chitosan fibres. It allowed a smooth

run in the spinning process at a speed of 31 m/min. The addition of the HAp or ß-TCP

particles resulted in a decrease in the tenacity of the modified chitosan fibres. Only the

complex of chitosan with HAp/ ß-TCP nanoparticles had the same tenacity as regular

chitosan fibres. Chitosan fibres were prepared with a calcium content of 14.35 g/kg and

an ash content of 4.8%. The HAp/ ß-TCP-modified chitosan fibres had higher WRV

values than the regular ones. Their more hydrophilic character may contribute to the

higher susceptibility of fibres to enzymatic degradation.

When we compare the HAp/ ß-TCP-modified chitosan fibres with the regular

chitosan fibres (Chit 58) we found similar mechanical properties with higher content of

calcium, which is the main mineral responsible for hard tissue regeneration. The

nanoparticles of ceramic calcium phosphates in the modified chitosan fibres could

probably improve the speed of the release of minerals.

This resea rch also shows a method to obtain ceramic nanoparticles in chitosan

solution from commercial calcium phosphates.

The resulted Composite chitosan fibres have a Polish Patent Application No

393022 related with the composite chitosan fibres production method, according to the

invention, presents as follows: a homogeneous aqueous acid solution of 40 - 70 °C

temperature, containing 0,1 - 10 wt% of hydroxyapatite and/or tricalcium phosphate

nano-particles, and 0,1 - 3 wt% of chitosan is entered, during mixing, into the spinning

solution of 20 - 55 °C temperature, containing chitosan of average molecular weight of

150 000 - 700 000 Da, deacetylation degree of 80 - 95 %, poly-dispersion degree

between 2 - 5, and concentration of 2 - 7 wt% in acetic acid aqueous solution of 1 - 3

wt% concentration, containing 0,1 - 15 wt% of glycerine in relation to chitosan. Then,

after venting, the obtained spinning solution is being embossed through multi-hole

spinning nozzle into the coagulation bath of 25 - 45 °C temperature, which is a sodium

hydroxide aqueous solution of 1 - 5 wt% concentration. Obtained fibres are being

stretched from 15 to 67 % in sodium hydroxide aqueous solution of 0,1 - 0,5 %

concentration in temperature of 80 - 95 °C. Then they are rinsed in water, finished in

bath of 30 - 75 °C temperature containing hydro-alcoholic emulsion with surfactants,

and they are being dried in temperature of 40 °C.

Acknowledgments

This research was funded by the Ministry of Science and Higher Education in 2009-

2012 as research project No. N N508 445636.

The authors received funding from the European Community’s Seventh Framework

Program [FP7/2007-2013] under grant agreement no. PITN-GA-2008-214015.

Analysis of the HAp and -TCP particle size in the chitosan solutions was carried out

using equipment provided by the Institute of Polymer and Dye Technology of the

Technical University, Lodz, Poland.

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Chapter 6

Degradation and bioactivity of the MCCh/ -TCP complex

6.1. Introduction

The recent developments in the area of artificial bone materials involve

ceramics, which are bio-inert like alumina and zirconia, resorbable like a tri-calcium

phosphate, and bioactive like a hydroxyapatite. Ceramic applications in hard tissue

regeneration and replacement are well documented. The main fields of their applications

are the replacements of hips, knees, teeth, tendons, and ligaments and repair for

periodontal disease, maxillofacial reconstruction, augmentation and stabilisation of the

jaw bone, spinal fusion, and bone repair after tumour surgery [1-3].

Polysaccharides, such as chitosan and its derivatives like microcrystalline

chitosan (MCCh), have some excellent properties for medical applications: non-toxicity

(monomer residues are not harmful to health); water solubility or high swelling ability

after simple chemical modification; stability to pH variations; biocompatibility;

antibacterial, antifungal and antiviral activity; high adhesiveness; non-toxicity; and

extensive chemical reactivity. Moreover, these materials evidence a strong ability to

create hydrogen and ionic bonds and bio-stimulation of natural resistance by controlling

and improving bioactivity. These properties make chitosan a good candidate for the

preparation and modification of modern generation of scaffolds for tissue regeneration

[2,4-8,17-20]. Much attention has been given to different materials or composites

including organic and inorganic materials that can be used as a base material for

scaffold devices, as modification tools for currently used biomedical devices that

improve hard and soft tissue regeneration and/or reinforcement efficacy. Additionally,

they are applicable as a tissue formation precursor in regenerative therapy in the field of

periodontics, orthopaedics, cancer, plastic surgery, and veterinary applications [9-13].

The degradation behaviour of chitosan and dissolution of calcium phosphates in

physiological environmental, plays a crucial role in the long-term performance of a

tissue engineered cell/material construction. The degradation kinetics may affect many

cellular processes, including cell growth, tissue regeneration, and host response, the

polymer in case of microcrystalline chitosan also stimulates the forming of monocytes

and inhibits the growth of bacteria and fungi thus lowering the risk of wound

inflammation. Some characteristics of the scaffold material such as porosity, particle

size and particle shape were reported to have a significant influence on the

inflammatory response and reparative bone formation. Irregularly shaped, sharp-edged

particles prompted higher inflammatory response than round particles of the same size

[8,9,10,15-17]. The solution parameters of initial pH, ionic concentration and

temperature have a large effect on the rate of scaffold dissolution and even type of

calcium phosphate precipitated. Ionic concentration, and therefore pH, will obviously

change with time as dissolution progresses and this will in turn affect the dissolution

rate. If pH rises above a critical value, cytoxicity will occur [22]. This study compares

the hydrolytic and enzymatic biodegradation, bioactivity, structure and mechanical

properties in sponge form of microcrystalline chitosan and microcrystalline chitosan/ß-

TCP complex composites, the last one according with earlier investigation in the

formulation and method to prepare a new complex which is described in Polish patent

application P 393758, 2011, that can be potentially useful in the field of bone tissue

engineering.

6.2. Experimental Section

6.2.1. Materials

The following materials were used:

Microcrystalline chitosan (MCCh paste) - average molecular weight (Mw) = 330

kD, deacetylation degree (DD) = 82%, ash content = 0.7%, water retention value

(WRV) = 598%, polymer content = 2.79%, pH = 7.38.

MCCh/ß-TCP complex paste - water retention value (WRV) = 560%, complex

content = 3.76%, content of MCCh: 3.008% and ß -TCP: 0.752%, pH = 7.40.

Tri-calcium orthophosphate (ß-TCP) powder, (Ca3(PO4)2) – Sigma Aldrich Lab.,

Germany, with 425400 ppm Ca and 199000 ppm P.

Hydroxyapatite powder (Ca10(PO4)6(OH)2 – Sigma Aldrich Lab., Germany, with

447200 ppm Ca and 186500 ppm P.

Plasticiser - Glycerol (C3H8O3), 99%, pure p.a., Sigma-Aldrich, Germany.

Phosphate buffer – pH = 7.40

Lysozyme – muramidase from chicken protein, EC 3.2.1.17, by Merck Co. with

an activity of 50000 U/mg.

6.3. Methods

6.3.1. Preparation of the MCCh/ß-TCP complex

The MCCh/ß-TCP complex was prepared according to Poland Patent Application P

393758 [21].

6.3.2. Preparation of the composites in sponge form

Sponges were prepared via a freeze-drying method using an ALFA 1-4 dryer

made by Christ Co. in the temperature range from -25 to 10°C with a vacuum ranging

from 0.1 to 0.53 mbar for 20 to 24 hours, depending upon the size of the charge. Freeze-

drying resulted in the preparation of sponges with a smooth surface that was free of

defects. The compositions of the samples (MCCh and the MCCh/ß-TCP complex

sponges) are shown in Table 1.

6.3.3. SEM study of the composites in sponge form

The pores morphology, their size and distribution in the polymer sponge were

observed using a scanning electron microscope (SEM, FEI Quanta 200, USA).

6.3.4. Mechanical properties

The mechanical properties of the composites in sponge form were determined in

the Accredited Laboratory of Metrology at Institute of Biopolymers and Chemical

Fibers (IBWCh) (certificate No. AB 388) using a dynamometer Instron (type 5544) in

accordance with the following standards: Polish Standard PN-EN-ISO 527-1, Polish

Standard PN-EN-ISO 527-3, and Polish Standard PN-ISO 4593.

6.3.5. Assessment of the degradability of the composites

Tested composites were put into a phosphate buffer at pH = 7.41 and bath

module 1:300 w/w and sterilised in an autoclave at 121°C for 25 minutes. Then,

samples were dried under vacuum for 15 minutes. The susceptibility of the bio-

composites to biodegradation was assessed in the buffer solution with the addition of

lysozyme in amount of 200 µg/cm3. The test was conducted in an incubator at 37°C

under static conditions according to standard PN-81C-06504 ”preparation of buffer

solutions”. Bio-composites in the form of sponges were removed from bath after 20, 40,

and 60 days. Next, the samples were filtered in a Buchner funnel, washed with distilled

water at 50°C, poured over 70% ethanol, filtered after 5 minutes, and dried under

vacuum at 70°C until a constant mass. The estimation of the hydrolytic and enzymatic

degradation progress of the tested preparations was based on the change of the pH,

measurements of the concentration of aminosaccharides (products of degradation) in the

bath, and the mass loss of the samples.

6.3.6. Bioactivity

A quantity of 0.4 g of tested samples and 0.4 g of reference samples (cotton)

were put into separate vessels and sterilised in an autoclave at 121°C for 25 minutes.

After sterilisation and drying, all samples were inoculated with a suspension of bacteria

for the determination of antibacterial activity using a quantitative test according to

standard JIS L 1902:2002. Escherichia coli (ATCC 11229) and Staphylococcus aureus

(ATCC 6538) were used as testing microorganisms.

6.4. Results and Discussion

The Table 1 showed the composition of samples prepared, named SMC for

microcrystalline chitosan and SMC-TCP for (microcrystalline chitosan/ß-TCP

complex), to compare the biodegradation, bioactivity, structure and mechanical

properties in form of sponges.

Table 1. Samples composition.

Sample symbol Dry sample composition *[wt%]

SMC MCCh:66.7 Glycerol: 33.3

SMC-TCP MCCh: 53.4ß-TCP: 13.3

Glycerol: 33.3

* moisture not taken into account

The glycerol was used as a plastisizer for a better manufacture of the samples

increasing the mixing process in the preparation of the suspension and directed related

with the homogenous interconnected pore morphology structure showing, smooth

surface without defects. Notice the addition of glycerol increase the number and round

shape of pores in freeze-dried method in all samples.

6.4.1. Biodegradation mass loss

The assessment of hydrolytic and enzymatic degradation process of the SMC

and SMC-TCP in sponge form, was estimated in the course of the degradation: pH,

percentage of mass loss of the samples (calculated on the microcrystalline chitosan

contained in the biocomposite) and concentration of aminosugars (products of

degradation contained in the citric-phosphate buffer). Considering it that the

preparations only assist in the joining of bones and their role in the organism is short

lasting, the testing time was limited to 60 days. The results are showed in Table 2 a,b.

Table 2a, showed the hydrolytic degradation process by immersion the sample

into the buffer solution of pH 7.4 at 37°C. Notice that mass loss of SMC-TCP complex

after 60 days, gives evidence that samples are very susceptible for degradation. The

mass loss of SMC-TCP complex is slightly above 16 % and SMC is around 15 % with

forming of reductive aminosugars around 1.3 % and 2.0% respectively.

Table 2a. Percentage mass loss and saccharification before and after hydrolyticdegradation

Time in days /Sample

Polymer contentin sample before

degradation[g]

Polymer content insample afterdegradation

[g]

Mass loss[%]

Saccharification[%]

pH

0 / SMC 0.2273 0.1937 0 0 7.500 / SMC-TCP 0.3132 0.2842 0 0 7.39

60 / SMC 0.2237 0.1885 15.70 1.99 7.4760 / SMC-TCP 0.3373 0.2828 16.15 1.31 7.44

The results of enzymatic degradation of biocomposites are estimated by the

same parameters and compiled in Table 2 b. As can be seen the biocomposites are also

susceptible to enzymatic degradation after 60 days whose intensity depends on the

applied lysozyme concentration, at 200 µg/cm3, the mass loss of the SMC-TCP around

18 % and SMC biocomposite is around 20 % with higher concentration of aminosugars

as a result of degradation amounted 7.09 % and 10.68 % respectively. This result

suggest that in SMC-TCP complex sample, the calcium phosphate (ß-TCP) shields a

little the polymer against the lysozyme by crosslinking and probably ionic bonds

between ion phosphate and the amino group of polymer affecting the time and amount

of degradation in the microcrystalline chitosan.

Table 2b. Percentage mass loss and saccharification before and after enzymaticdegradation

Time in days /Sample

Polymer contentin sample before

degradation[g]

Polymer content insample afterdegradation

[g]

Mass loss[%]

Saccharification[%]

pH

0 / SMC 0.2273 0.1937 0 0 7.500 / SMC-TCP 0.3132 0.2842 0 0 7.39

60 / SMC 0.1878 0.1507 19.75 10.68 7.4760 / SMC-TCP 0.3159 0.2594 17.90 7.09 7.47

6.4.2. SEM study of the composites in sponge form

In this section, the structure and morphology of the samples before and after the

biodegradation process are presented according to the preparation conditions listed in

Table 1. Figures 1 a and b are SEM pictures of SMC before the degradation process,

and Figure 2 c is the digital picture of sample SMC.

Figure 1. SEM pictures of SMC before degradation process, a-200X, b-1000X, c-

digital picture.

The Figures 2 a and b are SEM pictures of the SMC-TCP before the degradation

process, and Figure 2 c is the digital picture of sample SMC-TCP.

Figure 2. SEM pictures of SMC-TCP complex before degradation process, a-200X, b-

1000X, c- digital picture.

The Figures 3 a and b are SEM pictures of SMC after 60 days of the hydrolytic

degradation process, and Figure 3 c is a digital picture of sample 60 / SMC.

Figure 3. SEM pictures of SMC after 60 days of hydrolytic degradation process, a-

200X, b-1000X, c- digital picture.

The Figures 4 a and b are SEM pictures of the of SMC-TCP complex after 60

days of the hydrolytic degradation process, and Figure 4 c is a digital picture of sample

60 / SMC-TCP.

Figure 4. SEM pictures of SMC-TCP complex after 60 days of hydrolytic degradation

process, a-200X, b-1000X, c- digital picture.

The Figures 5 a and b are SEM pictures of the SMC after 60 days of the

enzymatic degradation (concentration of lysozyme - 200 µg/cm3) and Figure 5 c is a

digital picture of sample 60 / SMC.

Figure 5. SEM pictures of the SMC after 60 days of enzymatic degradation process, a-

200X, b-1000X, c- digital picture.

The Figures 6 a and b are SEM pictures of SMC-TCP complex after 60 days of

the enzymatic degradation (concentration of lysozyme - 200 µg/cm3) and Figure 6 c is

the digital picture of sample 60 / SMC-TCP complex.

Figure 6. SEM pictures of SMC-TCP complex after 60 days of enzymatic degradation

process, a-200X, b-1000X, c- digital picture.

The morphology of the microcrystalline chitosan and complex sponges changed

progressively with time. In the case of the complex, the sample contained pores that

became more compact over time.

Comparing the samples before and after degradation process indicates that under

the action of the enzyme at 200 µg/cm3 concentration the surface structure of the sponge

undergoes changes with the forming of new and interconnected wider pores. The

complex structure reveals a homogenous distribution of micro and nanosized particles

and clusters formation of ß-TCP in the polymer matrix. The white colour of the sample

in the digital picture results from the calcium phosphate particles in the complex.

6.4.3. Mechanical properties of the sponges

The assessment of mechanical properties of the SMC and the SMC-TCP

complex in sponge form are presented in (Tables 3 a, b) in respect of strength,

elongation and elastic modulus, qualities that are crucial for implants in orthopedical

surgery. The addition of glycerol interfered with MCCh chains, decreasing

intermolecular attraction and increasing polymer chains mobility maintaining the

homogenous porous network structure which facilitates elongation, in case of complex

with -TCP had little effect on improving the elongation at break in another hand a little

decrease in tensile strength, but showing the good interaction (ionic and covalent

bonding) between the calcium phosphate and the MCCh according with the literature

[21]. After biocomposite materials were affected by 60 days of enzymatic and

hydrolytic degradation, similar behaviour of mechanical properties was observed, with

lower values of tensile strength, elastic modulus and with higher elongation at break due

the process. The SMC-TCP preparation feature by best mechanical parameters qualifies

for further investigations. In the case of enzymatic and hydrolytic degradation, similar

behaviour was observed, with lower values of tensile strength, elastic modulus with

higher elongation at break due the process

Table 3a. Mechanical properties of sponges before and after hydrolytic degradation

Parameters

Samples Degradationtime

[days]

Composition Tensilestrength[MPa]

Elongation atbreak [%]

Elastic modulus[MPa]

SMC 0 MCCh: 66.7 Glycerol: 33.3

0.1720 1.53 1.400

SMC-TCP 0 MCCh: 53.4ß-TCP: 13.3

Glycerol: 33.3

0.0150 2.41 1.000


0.0091 3.19 0.010


Glycerol: 33.3

0.0148 5.65 0.005

Table 3b. Mechanical properties of sponges before and after enzymaticdegradation

Parameters

Samples Degradationtime

[days]

Composition Tensilestrength[MPa]

Elongation atbreak [%]

Elastic modulus[MPa]


0.172 1.53 1.40


Glycerol: 33.3

0.015 2.41 1.00


0.0073 1.88 0.005


Glycerol: 33.3

0.0055 3.14 0.015

6.4.4. Bioactivity

The results of the bioactivity investigations are presented in (Table 4a,b), the

following test was estimated in the course of antibacterial activity (bacteriostatic and

bactericidal activity) using a quantitative test according to standard JIS L 1902:2002

with Escherichia coli and Staphylococcus aureus. Both samples SMC and SMC-TCP

complex showed a bacteriostatic activity and a bactericidal activity against Escherichia

coli, showing the SMC higher activity than SMC-TCP complex, suggesting more free

amino groups from SMC and lower free amino groups from complex that probably are

bonded with the phosphate groups of the calcium phosphate reducing the activity.

In the case of Staphylococcus aureus, SMC and SMC-TCP showed a

bacteriostatic activity and not bactericidal activity was found.

Table 4a. Determination of antibacterial activity (Escherichia coli)

Sample Time

[h]

Number ofliving bacteria[cfu/sample]

Confidence interval[cfu/sample]

Bacteriostaticactivity

Bactericidalactivity

Control 0 1.1x105 9.1 x104 – 1.4 x105 0 0

Control 24 1.4 x108 1.2 x108 – 1.7 x108 0 0

SMC24 6.5 x102 2.6 x102 – 1.3 x103 5.3 2.2

SMC-TCP24 6.7 x106 4.8 x105 – 1.4 x107 1.3 1.8

Table 4b. Determination of antibacterial activity (Staphylococcus aureus)

Sample Time

[h]

Number ofliving bacteria[cfu/sample]

Confidenceinterval

[cfu/sample]

Bacteriostaticactivity

Bactericidalactivity

Control 0 2.7x104 2.3 x104 – 3.1 x104 0 0

Control 24 7.4 x106 4.3 x106 – 1.1 x107 0 0

SMC24 6.8 x105 4.4 x104 – 1.8 x106 1.1 -1.4

SMC-TCP24 3.3 x106 1.2 x106 – 5.2 x106 0.4 -2.1

6.5. Conclusions

The investigation resulted compare two different microcrystalline chitosans

biocomposites designed for bone reconstruction and regeneration.

The biocomposite sponges keep a homogeneous and interconnected pore

network structure after 60 days of biodegradation processes, with a reduction in the size

of the pores and the clusters formation in case of SMC-TCP.

The SMC and SMC-TCP in sponges form are susceptible to hydrolytic and

enzymatic degradation process.

The hydrolytic degradation by immersion the sample into the buffer solution of

pH 7.4 at 37°C showed the mass loss of SMC-TCP complex is slightly above 16 % and

SMC is around 15 % with forming of reductive aminosugars around 1.3 % and 2.0%

respectively.

The enzymatic degradation in the presence of lysozyme, under the action of the

enzyme at a 200 µg/cm3 concentration, the mass loss of the SMC-TCP around 18 % and

SMC biocomposite is around 20 % with higher concentration of aminosugars as a result

of degradation amounted 7.09 % and 10.68 % respectively.

The mechanical properties after 60 days of enzymatic and hydrolytic

degradation, similar behaviour was observed, with lower values of tensile strength,

elastic modulus and with higher elongation at break showing that can be processed in

elastic forms which have the potential to be used as fillers in hard tissue regeneration.

Both samples SMC and SMC-TCP complex showed a bacteriostatic activity and

a bactericidal activity against Escherichia coli. In the case of Staphylococcus aureus,

SMC-TCP complex and SMC showed only a bacteriostatic activity.

Acknowledgements

The research and work presented in this study were conducted with financial

support from the European Community's Seventh Framework Programme (“Marie Curie

Initial Training Network), FP7/2007-2013.

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Poland patent application P 393758.

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Conclusions

Bone in healthy and diseased tissue attract much attention in the last decades, a

revolution in orthopaedics occurred which has led to a remarkable improvement of

quality of life for millions patients who needs those medical devices for hard tissue

regeneration and/or replacement. Significant results have been obtained and published

in the last past years in form of articles and patents showing the efforts are being made

to develop methods and materials in artificial tissues, including bones, cartilage, nerve,

blood vessels, and skin to restore the functions of damaged tissues in musculoskeletal

disorder. The material chemistry and the biochemical technology have progressed from

use of biomaterials like natural polymers (chitosan and its derivatives) and calcium

phosphates like HAp and -TCP to repair and/or replace wounded tissues to the

implantable scaffolds with excellent results.

One significant result of the research was reported in article published in 2011

and in Patent Application P. 393758 in Poland, about new method to obtain a

microcrystalline chitosan /tri-calcium orthophosphate complex showing the calcium

phosphate particles were uniformly dispersed in the amino-glucose macromolecule

aggregation (coagulation process) and no precipitation of the calcium phosphate was

noticed in the samples showing the good stability of the suspension. The study showed

the feasibility of freeze drying method in the preparation of the MCCh/ ß-TCP complex

in sponge form, resulting in a 3-dimensional interconected porous network structure that

can be used in future as a base for scaffolds production. The complex in sponge form is

susceptible to hydrolytic and enzymatic degradation with good mechanical properties

after 60 days of degradation, showing also a bacteriostatic activity and a bactericidal

activity against Escherichia coli and Staphylococcus aureus.

Another important result was publishing in 2010 in another Patent Application P.

393022 in Poland, resulting a article published in 2011 about new method of

manufacture nanocomposite chitosan fibres, showing a better mechanical properties in

wet conditions than regular chitosan fibres with a lower cost process, because no

finishing agent in the process was required. Their more hydrophilic character may

contribute to the higher susceptibility of fibers to enzymatic degradation. This research

also shows a method to obtain ceramic nanoparticles in chitosan solution from

commercial calcium phosphates.

The challenge of hard tissue engineering continue the development of suitable

material bone scaffold with sufficient interconnected porosity and mechanical strength

to allow cell adhesion, migration, growth and proliferation resulting in good integration

with surrounding tissues. These concepts will combine the understanding about

developments of materials, manufacturing technologies and surgical techniques creating

a new generation of scaffolds for skeletal reconstruction used in the regenerative

medicine.

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