Size effect of calcium phosphate coated with poly-DL-lactide-co-glycolide on healing processes in bone reconstruction

Nenad L. Ignjatovic,1 Zorica R. Ajdukovic,2 Vojin P. Savic,3 Dragan P. Uskokovic1

1Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade, Serbia2Faculty of Medicine, Nis, Clinic of Stomatology, Department of Prosthodontics, Nis, Serbia3Faculty of Medicine, Nis, Institute of Biomedical Research, Nis, Serbia

Received 21 January 2009; accepted 23 December 2009

Published online 8 April 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31630

Abstract: In this article, synthesis and application of calcium

phosphate/poly-DL-lactide-co-glycolide (CP/PLGA) composite

biomaterial in particulate form, in which each CP granule/

particle is coated with PLGA, are described. Two types of the

particulate material having different particle sizes were syn-

thesized: one with an average particle diameter between 150

and 250 lm (micron-sized particles, MPs) and the other with

an average particle diameter smaller than 50 nm (nanopar-

ticles, NPs). A comparative in vivo analysis was done by

reconstructing defects in osteoporotic alveolar bones using

both composites. The material, CP granules/particles covered

with polymer, was characterized using X-ray structural analy-

sis, scanning electron microscopy, and atomic force micros-

copy. Changes in reparatory functions of tissues affected by

osteoporosis were examined in mice in vivo, using these

two kinds of composite materials, with and without autolo-

gous plasma. Having defined the target segment, histomor-

phometric parameters—bone area fraction, area, and mean

density—were determined. The best results in the regenera-

tion and recuperation of alveolar bone damaged by osteopo-

rosis were achieved with the implantation of a mixture of

nanoparticulate CP/PLGA composite and autologous plasma.

After the implantation of microparticulate CP/PLGA, in the

form of granules, mixed with autologous plasma, into an ar-

tificial defect in alveolar bone, new bone formation was also

observed, although its formation rate was slower. VC 2010

Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater

94B:108–117, 2010.

Key Words: calcium phosphate/poly-DL-lactide-co-glycolide

(CP/PLGA), microparticles, nanoparticles, autologous plasma,

alveolar bone

INTRODUCTION

Each year millions of people are faced with numerous risksinvolved with bone tissue damage, caused by trauma or path-ological processes, which demand reconstruction and curing.Reconstruction carried out using allogenic materials containselements of risk such as infections or inadequate immunologicresponses. On the other hand, the use of synthetic materialsdecreases the risk maximally, thus increasing the success rateof reconstruction. Synthetic calcium hydroxyapatite (HAp), aswell as calcium phosphates (CP), belongs to the class of bio-compatible and bioactive materials that have been success-fully used for these purposes.1,2 Bone mineral is poorly crys-talline HAp. HAp particles in bone tissue are in the form ofplatelets, 2–7 nm thick, 5–20 nm long, and 10–80 nm wide.3

The key feature of particulate materials is their reducedsize. In that respect, it is necessary to establish the thresh-old size for considering a system to be a particulate one. Inthe literature, this issue is not a matter of universal consent.We will adopt the distinction according to which micron(lm)-sized systems (Ms) are those within the 1–1000 lmrange, whereas the nano-sized particle systems (NPs) arethose with particle size below 100 nm.4

Ceramic or composite materials in their particulate formare primarily used as fillers in bone engineering.5,6 Becausethey cannot adsorb plasma proteins, necessary for cell adhe-sion on their surface, such materials have to be coveredwith substances able to ensure cell–matrix adhesion.7 Toachieve cell adhesion on the material, plasma proteins haveto be bound to its surface first.8 Cells are not in directcontact with the surface of the material but are indirectlyconnected with it via proteins of the extracellular matrix.9

Bioresorptive polymers, such as PLGA, enable cell adhesion,the production of extracellular matrix and organization ofcytoskeleton.10 Investigations carried out so far confirmedsuccessful application of composite biomaterials based onHAp or CP with bioresorptive polymer (poly-L-lactide, PLLA;or poly-DL-lactide-co-glycolide, PLGA) in the reconstructionof bone defects.5,11–13 The practice of coating CP particleswith PLGA ensures adhesion of osteoprogenitor cells with thefinal aim of accelerating differentiation and osteogenesis.6,14

Coating of HAp or CA with PLGA resulted in better migra-tion and adhesion of the bone marrow stromal stem cellin bone tissue reconstruction.15,16 Microparticulate HApcoated with PLGA was also successfully used as controlled

Correspondence to: D. P. Uskokovic; e-mail: uskok@itn.sanu.ac.yu

Contract grant sponsor: Ministry of Science and Technological Development of the Republic of Serbia; contract grant numbers: 142006, 145068

108 VC 2010 WILEY PERIODICALS, INC.

drug release systems and biomaterial for bone tissuereconstruction.17

In recent years, nanotechnology has made a significantheadway in diagnostics and therapy of numerous humandiseases.18 Application of nanoparticulate HAp in bonereconstruction proved its superior biological properties.19

HAp and CP in the form of nanoparticles (NPs) with largespecific surface area and ultrafine structure are very similarto biological apatites, enabling thereby adequate adhesionand promoting interaction of cells with their surfaces.20

Composite biomaterials in the form of NPs may have signifi-cant advantages over those in micro- or submicro-particu-late form.21–23 NPs of HAp-based composites may haveimproved applicative characteristics, such as easy manipula-tion, close contact with the surrounding tissue, fast resorp-tion, and so on.24,25 HAp NPs in combination with silk fi-broin ensure good starting cell adhesion and promoteosteogenetic differentiation.26

This article discusses the possibility of synthesis ofhybrid micro- and nanoparticulate CP coated with PLGA.The possibility of improving reparatory properties of CP/PLGA composite biomaterial by decreasing particle sizefrom micro to nano dimensions was also examined. To doso, micro- and nanoparticulate CP/PLGA composite biomate-rials, with and without autologous plasma, used in thereconstruction of alveolar bones damaged by osteoporosisin rats, were comparatively analyzed. Osteoporotic modelwas used because bone affected by osteoporosis has lowerdensity, poor mechanical properties and is highly brittle,which can result in fracture induced by minimal force.16,27

The effect of the composite’s particle size on the phenome-nology and dynamics of alveolar bone reconstruction wasstudied. Histopathological and histomorphometric evalua-tion of the influence of the particle size on the regenerationof restored defects in rats’ osteoporotic alveolar bones wasalso carried out.

MATERIALS AND METHODS

Preparation of composite biomaterialsA CP gel was prepared by precipitation of calcium nitrateand ammonium phosphate in an alkaline medium. Poly-DL-lactide-co-glicolide (50:50) (Sigma Chemical Company, USA)was used as a polymer component. PVA with 98% hydroli-zation degree was used.

Procedure 1The gel was dried, granulated at room temperature and cal-cined at 1100�C for 6 h. Granules of CP were added intocompletely dissolved polymer, in an amount of 80 mass%.The suspension was mixed at 30 rpm, and then methanolwas added. PVA (0.02%) was subsequently added into thesuspension (PLGA/PVA ¼ 10/1). After the solvent evapora-tion, the granules were dried at room temperature for 24 h.Microparticles of CP/PLGA composite biomaterial, 150–200lm in size, were sieved and sterilized using c rays (25 kGy)before use.5

Procedure 2CP gel was added into completely dissolved polymer, in anamount of 80 mass%. The suspension was mixed at 1200rpm, and then methanol was added. PVA (0.02%) was subse-quently added into the suspension (PLGA/PVA ¼ 10/1). Afterthe solvent evaporation, the particles were dried at roomtemperature for 24 h. The particles of CP/PLGA compositebiomaterial were sterilized using c rays (25 kGy) before use.Mass ratio of CP/PLGA composite in both cases was 80/20.

Characterization of composite biomaterialsX-ray structural (XRD) analyses were carried out using anEnraf Nonius FR590 diffractometer. Microstructural charac-terization was done by AFM (Thermo Microscopes, Autop-robe CP Research) and SEM (JEOL JSM 5300).

In vivo researchThe research was carried out on female Sprague-Dawleysyngenic type rats, age 6–8 weeks, a period long enough forsexual maturity and full bone mineralization. The animalswere divided into two groups:

1. Experimental group (A) (72 animals).2. Control group (Co) (36 animals).

Osteoporosis was induced by glycocorticoids in the ani-mals of the group A. Glycocorticoids were administered toanimals intramuscularly during 12 weeks, alternately meth-ylprednisolone succinate sodium (Lemod-Solu, Hemofarm,Vrsac, Serbia), a dose of 2 mg/kg of corporal weight, anddexamethasone-sodium-phosphate (Dexason, ICN Gelenika,Belgrade, Serbia), a dose of 0.2 mg/kg. Twelve weeks afterthe treatment, the animals were prepared for interventionapplying diazepam (Bensedin, ICN Galenika, Belgrade, Ser-bia) and were anesthetized by ketamine hydrochloride USP(Ketalar, Rotexmedica Gmbh, Trittau, Germany). Defects1.6 mm in diameter and 1.8 mm deep were made in theregion between the medial line and the foramen mentale ofthe left side of osteoporotic mandible in animals by a ster-ile steel borer. The composite was implanted into as-madedefects.

The group A was divided into four subgroups:

• A1 (18 animals)—the composite obtained by procedure 1was implanted into a defect artificially made in osteopor-otic mandible.

• A2 (18 animals)—the composite, obtained by procedure 1,mixed with autologous plasma was implanted into an arti-ficial defect made in osteoporotic mandible. Blood takenfrom animal’s heart was centrifuged at 1500 rpm, and theseparated plasma was mixed with microcomposite par-ticles at a ratio 1:1. The as-prepared mixture wasimplanted into the defects in osteoporotic mandibles.

• A3 (18 animals)—the composite obtained by procedure 2was implanted into a defect artificially made in osteopor-otic mandible. The composite was mixed with physiologi-cal solution (0.9% NaCl solution) at a ratio 2:1. Theobtained nanocomposite paste was implanted into osteo-porotic mandibles of rats.

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• A4 (18 animals)—the composite, obtained by procedure 2,mixed with autologous plasma was implanted into adefect artificially made in osteoporotic mandible. Bloodtaken from animal’s heart was centrifuged at 1500 rpm,and the separated plasma was mixed with composite NPsat a ratio 1:2. The as-prepared nanocomposite material inthe form of paste was implanted into mandibles damagedby osteoporosis.

The control group B was kept without therapy in normalday-night rhythm and dietary regime.

Animals of both groups were killed at 6 and 12 weeksafter the implantation. Assessment of the regeneration ofrestored defects in rats’ osteoporotic alveolar bone was car-ried out by histopathological and histomorphometric analy-sis. Samples were taken from the lower jawbones, regionbetween the medial line and the foramen mentale (where adefect was artificially made and implantation performed).Bone samples were cut in the vestibule-oral direction. Thesamples were washed in physiological solution (0.9% NaClsolution), fixed in 10% formalin and then decalcified chemi-

cally. Chemical decalcification was performed in 15% nitricacid solution. After decalcification, the bone tissue wasdehydrated in alcohol, embedded in paraplast, cut andstained. The obtained histological sections 2–4 lm thickwere stained by a routine hematoxylin and eosin (HE) stain-ing method and histopathologically examined using a Lucia3.2 G (Laboratory Imaging, Prague, Czech Republic) systemfor image analysis, NU-2 microscope (Carl Zeiss, Jena, Ger-many). Having defined the target segment, histomorphomet-ric parameters, bone area fraction (the Haversian canal den-sity in newly formed bone structure of the studied bone),area (area occupied by new bone seen in view field, lm2),and mean bone density (mean value of the optical densityof the studied part of bone given in arbitrary units, a.u.)were determined. Statistical analysis of the obtained resultswas made using t-test.

RESULTS

Powder characterizationX-ray diffraction technique was used to characterize the sam-ples of both composites. Identification of phases in investi-gated samples was done by comparing the obtained diffrac-tion data with the standard ones from the JCPDS database.28

The obtained XRD results are presented in Figure 1.Figure 2a shows CP/PLGA micro-composite particles,

while single particles can be seen in Figure 2(b). It is evi-dent from Figure 2(a,b) that the average particle size wasbetween 150 and 250 lm, which means that this compositewas a microparticulate one (Ms).4 Porous and rough surfaceof CP particles covered with PLGA is shown in Figure 2(c).Pores of approximately 1 lm can be noticed on the particlesurface.

Figure 3(a) shows a 2D AFM image of a composite ag-glomerate, obtained according to procedure 2, the detail ofwhich is presented in Figure 3(b). Average composite parti-cle size was found to be 37.9 nm. A 3D AFM image of thedetail is shown in Figure 3(c). CP/PLGA NPs had relativelysmooth surface, their surface roughness was less than a fewnanometers, and they were not porous [Figure 3(c)].

FIGURE 1. XRD analysis of Ms and NPs CP/PLGA composite

biomaterials.

FIGURE 2. SEM micrographs of Ms CP/PLGA: (a) group of granules, (b) single granule, and (c) granule surface.

110 IGNJATOVIC ET AL. SIZE EFFECT OF CP COATED WITH PLGA

In vivo researchFigure 4 shows the healing of the artificial defect in an ex-perimental group (A1) of animals 6, 12, and 24 weeks afterthe implantation of CP/PLGA microparticles. Areas filledwith remains of biocomposite microparticles sporadicallyinterspersed with cells and fibres can be observed on Figure4(a). Well-vascularized granulated tissue and the formationof nonlamellar primitive bone were perceived. Areas inwhich the implanted material was fully resorbed were alsoevident. Twelve weeks after the implantation of the biocom-posite, remodeling processes were not evident [Figure 4(b)],while after 24 weeks they were pronounced [Figure 4(c)].

Figure 5 shows the healing of the artificial defect in anexperimental group (A2) of animals 6, 12, and 24 weeks af-ter the implantation of CP/PLGA microparticles mixed withautologous plasma. The growth of young bone tissue andthe formation of Haversian canals within the implanted ma-terial were somewhat more pronounced compared to theprevious group. Focal points of connective tissue residuewithin the implanted material were rare 6 weeks after theimplantation, but areas filled with remaining CP/PLGAmicroparticles covered with polymer were visible [Figure5(a)]. On Figure 5(b), areas in which the biocomposite wasalmost fully resorbed can be observed. Pronounced forma-tion of new bone with numerous Haversian canals was per-

ceived. Cement lines and a bone structure more regularthan that in the previous subgroup were present. New boneformation was more evident 24 weeks after the implanta-tion [Figure 5(c)]. On the preparations, the formation ofnew bone was evident on the periphery of CP/PLGA com-posite (on the contact surface between the bone and theimplanted material) and inside the pores distributed on thedefect surface.

Significantly better results in the process of regenera-tion, reparation, and recuperation of alveolar bone weak-ened by osteoporosis were achieved after the implantationCP/PLGA NPs. Histological preparations of the defect artifi-cially made in the experimental group of animals 6, 12, and24 weeks after the implantation CP/PLGA NPs are pre-sented in Figure 6. Figure 6(a) shows the implanted mate-rial intermingled with young bone tissue and the formationof Haversian system. Focal points of connective tissue resi-due within the implanted material were present in largeamount, and the traces of implanted material residue werealso noticeable. Figure 6(b) shows evident growth of newbone tissue and the formation of Haversian system withinthe matrix. Dense cement lines and vigorous osteogenesis,as well as a more regular bone structure were perceived.

Twenty-four weeks after the implantation of the nano-composite [Figure 6(c)], osteoclasts were less active and

FIGURE 3. AFM image of NPs CP/PLGA: (a) agglomerate, (b) 2D, and (c) 3D. [Color figure can be viewed in the online issue, which is available

at www.interscience.wiley.com.]

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small cavities were formed in the surrounding bone tissue.The cavities were filled with mononuclear cells of connec-tive tissue. There were numerous new blood vessels innewly formed bone tissue.

Histological preparations presented in Figure 7 illustratethe healing of the defects artificially made in healthy mandi-ble of animals 6, 12, and 24 weeks after the implantation of

FIGURE 4. Healing of artificial defect of the experimental group of ani-

mals. (a) 6 weeks, (b) 12 weeks, and (c) 24 weeks after Ms CP/PLGA

implantation. ( , composite biomaterials; , new bone tissue).

[Color figure can be viewed in the online issue, which is available at

www.interscience.wiley.com.]

FIGURE 5. Healing of artificial defect of the experimental group of ani-

mals. (a) 6 weeks, (b) 12 weeks, and (c) 24 weeks after implantation of Ms

CP/PLGA mixed with autologous plasma. [Color figure can be viewed in

the online issue, which is available at www.interscience.wiley.com.]

112 IGNJATOVIC ET AL. SIZE EFFECT OF CP COATED WITH PLGA

NPs CP/PLGA composite with autologous plasma. Rare focalpoints with connective tissue residue within the implantedmaterial can be noticed on Figure 7(a). Dense cement lines

and signs of an evident reparatory process can be observed.Vigorous growth of new bone tissue and the formation ofHaversian system within the implanted material are evident.

FIGURE 6. Healing of artificial defect of the experimental group of ani-

mals. (a) 6 weeks, (b) 12 weeks, and (c) 24 weeks after implantation of

NPs CP/PLGA composite. [Color figure can be viewed in the online

issue, which is available at www.interscience.wiley.com.]

FIGURE 7. Healing of artificial defect of the experimental group of ani-

mals. (a) 6 weeks, (b) 12 weeks, and (c) 24 weeks after the implanta-

tion of NPs CP/PLGA composite with autologous plasma. [Color

figure can be viewed in the online issue, which is available at www.

interscience.wiley.com.]

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Figure 7(b) shows numerous points at which complete inte-gration between new bone tissue and the implanted mate-rial occurred. These preparations show the pronouncedgrowth of new bone tissue and the formation of Haversiansystem within the implanted material. Newly formed bonetissue resembled mature bone. Twenty-four weeks later[Figure 7(c)], we noticed a large amount of mature bonemarrow cells with blood cells and a bone cell network,which was progressively replaced by lamellar bone and, fur-ther, by mature cortical bone

Histomorphometric analyses of alveolar bones of rats 6,12, and 24 weeks after the implantation of the compositeare presented in Figure 8.

DISCUSSION

Diffraction spectra of microparticulate and nanoparticulatecomposite are shown in Figure 1. All spectra had a part thatis characteristic of HAp (JCPDS file No. 09-432) and of b-TCP (JCPDS file No. 09-169).28,29

As shown in Figure 1, microparticles of composite bio-materials were highly crystalline. The most intense peaks at31.8� [2 1 1], 32.2� [1 1 2], 32.9� [3 0 0], and 49.5� [2 1 3]belonged to highly crystalline calcium HAp, whereas thoseat 2y ¼ 31� [0 2 10] and 34.4� [2 2 0] belonged to b-tri cal-cium phosphate (b-TCP). Thereby, this CP can be calledbiphasic calcium phosphate (BCP). XRD patterns showed nopeaks for PLGA, because the polymer is amorphous, andthis is in accordance with XRD studies of PLGA publishedby other authors.30

XRD patterns of NPs indicate poorly crystalline HAp(Figure 1). This may be the same as the calcium-deficientapatite with non-stoichiometric Ca/P ratios lower than 1.67observed by other authors.31 This result was expected sincethe CP gel was synthesized via precipitation at 100�C andpH ¼ 12 (procedures 1 and 2); CP synthesis at this temper-ature yields poorly crystalline CP.32

The microstructure of microparticulate and nanoparticu-late composite biomaterial was analyzed by SEM and AFM.Figure 2 shows the topology of the surface of microparticu-late composite biomaterial prepared according to procedure1. CP granules were coated with the polymer, and their av-erage diameter was 150–250 lm. The solvent–nonsolventprocedure ensured that CP was covered with PLGA poly-mer.33 Such a microstructured surface can be an ideal ma-trix for the adhesion of human cells after the implantation.In vitro tests carried out so far on this matrix planted withcell cultures MRC5 and L929 showed significantly low cyto-toxicity (10 and 23%), which indicates a high degree of bio-compatibility of this type of material.7

Figure 3(a) shows a 2D AFM image of nanoparticulatecomposite biomaterial obtained according to procedure 2.The use of CP gel resulted in the synthesis of CP/PLGA com-posite biomaterial in the form of nanosized spherical par-ticles. CP particles were coated with the polymer and theiraverage diameter was 38 nm [Figure 3(b)]. CP/PLGA in thisform was used for the synthesis of an injectable paste thatcan be applied in the reconstruction of small-scale bonedamages. Figure 3(c) shows a 3D AFM image of CP/PLGA

composite biomaterial shown in Figure 3(b). An analysis ofFigure 3(b) revealed particle diameter below 50 nm. Conse-quently, the composite obtained via procedure 2 was

FIGURE 8. Histomorphometric analysis of alveolar bones of rats. (a) 6

weeks, (b) 12 weeks, and (c) 24 weeks after composite implantation

(n.s., nonsignificant).

114 IGNJATOVIC ET AL. SIZE EFFECT OF CP COATED WITH PLGA

defined as nanoparticulate.4 The polymer was evenly dis-tributed along the surface of CP particles, which was con-firmed by electron microscopy and FTIR analysis in a previ-ous study carried out by the authors of this article.33

On histological preparations of alveolar bone, wheremicroparticulate CP/PLGA was implanted, newly formed,primitive, bone could be seen (Figure 4). Six weeks afterthe implantation, there were areas filled with microparti-culate CP/PLGA residue intermingled with fibrin anderythrocytes. In this space, temporarily occupied byextracellular matrix rich in fibrin, accumulation and activa-tion of thrombocytes take place.34 Thrombocytes releasegrowth factors and chemokines, which attract neutrophillicgranulocytes, monocytes, and macrophages.35 Extracellularmatrix was gradually replaced by well-vascularized granu-lation tissue, and the formation of primitive, nonlamellarbone was noticeable [Figure 4(a)]. Twelve weeks after theimplantation [Figure 4(b)], spaces filled with microparticu-late composite residue were still present. However, thesigns of polymer resorption were also evident. Primitivebone, formed as a trabecular meshwork, was graduallyreplaced, during remodeling, with a lamellar one. The for-mation of new bone was involved in the remodelling pro-cess of alveolar bone damaged by osteoporosis.36 Twenty-four weeks after the implantation of microparticulate CP/PLGA [Figure 4(c)], the regeneration process within thetissue surrounding the implanted material was moreadvanced compared to the 12th week, but very small areasof low alveolar bone density could still be noticed. A zoneof newly formed bone tissue can be observed in the lowerleft corner of Figure 4(c). In the central part of the samefigure, the penetration of the connective tissue into theimplanted material can be perceived. In the upper rightcorner, we can see that the implanted material was com-pletely replaced by the connective tissue, which was alsoconfirmed by high cell density.

Six weeks after the implantation of microparticulate bio-composite mixed with autologous plasma [Figure 5(a)], wecould notice connective tissue in the implanted material andthe initial stages of osteogenesis. Twelve weeks after the im-plantation of microparticulate biocomposite [Figure 5(b)],we could notice areas in which the implanted material wasresorbed and active osteogenesis was taking place. Theseareas were distinguished by a greater number of Haversiancanals and dense cement lines. More pronounced osteogene-sis in rats belonging to this subgroup (where a mixture ofthe composite and autologous plasma was implanted) com-pared to that in the previous subgroup was probably aresult of a joint action of the microparticles and compositeporosity37 and the growth factors present in autologousplasma, which can significantly improve cell activation andproliferation, as well as new bone tissue formation.36–38

New bone formation was even more advanced 24 weeksafter the implantation [Figure 5(c)] compared to that observedin the 12th week. On histological preparations [Figure 5(c)]new blood vessels and lamellar bone in the process of ossi-fication could be noticed in areas previously occupied bythe implanted composite.

In the case of nanoparticulate composite [Figure 6(a)],osteogenesis was more pronounced than that taking placein the microparticulate composite implant during the sameperiod of time [Figure 4(a)]. Six weeks after the implanta-tion, focal points with connective tissue residue within theimplanted material were present to a large extent, as wellas the traces of the implanted material. Small size of theseparticles (less than 50 nm) enables better contact and ad-herence of macrophages, facilitating growth factor produc-tion, and activation of cells—osteogenesis.20 After the poly-mer resorption, during hemostasis, this space is filled withextracellular fibrin-rich matrix, which accumulates andreleases granules containing growth-factors and chemo-kines, which attract granulocytes, monocytes, and macro-phages with proteolytic properties and have a role in angio-genesis and transport of osteoprogenitor cells, as noticedearlier.39,40 The growth of new bone tissue within theimplanted material and the formation of Haversian canalswere, however, more advanced 12 weeks after the implanta-tion. On histological preparations made in this subgroup,cement lines with deposited calcium salts indicating higherdegree of osteogenesis and a more regular bone structurecould be noticed [Figure 6(b)]. The given facts speak infavour of a pronounced reparatory osteogenesis. Twenty-four weeks after the implantation of the nanocomposite,less intense osteoclastic activity [Figure 6(c)] accompaniedby the formation of very small cavities in the mature bonecould be noticed. The cavities were filled with mononuclearcells of connective tissue. In the surrounding area, we couldobserve numerous new blood vessels within the newlyformed bone, which confirmed a positive effect of theimplanted nanocomposite on the angiogenesis and osteo-genesis processes.

Certainly, the best results in the process of regenerationand recuperation of alveolar bone affected by osteoporosiswere achieved after the implantation of nanoparticulate CP/PLGA composite mixed with autologous plasma. The growthof new bone tissue within the implanted material and theformation of Haversian canals were more evident comparedto the previous subgroups. Focal points with connective tis-sue residue within the implanted material were rare already6 weeks after the implantation [Figure 7(a)]. Cement lineswere denser, indicating an advanced reparatory process.The growth of new bone tissue within the implanted mate-rial and the formation of Haversian canals were more pro-nounced 12 weeks after the implantation. The integration ofnew bone tissue was complete in many places [Figure 7(b)].Nanocomposite (NPs CP/PLGA) coated with autologousplasma proteins (opsonization) yielded better resultsbecause it induced stimulation of macrophages and produc-tion of growth factors. Chemokines present in autologousplasma promoted migration of macrophages to the placesfilled with NPs.38,41 Twenty-four weeks after the implanta-tion [Figure 7(c)] we could notice a large quantity of maturebone cells, blood cells, and a bone cell network, progres-sively replaced by lamellar bone and, further, by maturecortical bone. As a result, new bone tissue was formed, fora relatively short time, and very soon it was interwoven

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with collagen fibres yielding mature collagen connective tis-sue—the reparation was entering its final stage.36,42 Theintegration of the new with the existing bone tissue wascomplete in many places, so the boundary between themwas barely visible. The given facts speak in favor of a vigor-ous reparatory osteogenesis after the implantation of thenanocomposite.43

Morphometric parameters [Figure 8(a–c)] are very wellsuited to the description of the histogram of the examinedtypes of implant. Mean density is an important histomor-phometric parameter. A small increase in optical density ofalveolar part of mandible in rats, already 6 weeks after theimplantation of the composite, was found especially in thecase of experimental subgroups A3 and A4, with implantedNPs CP/PLGA and the mixture with autologous plasma,respectively, in contrast to the control group (Co). This con-firmed much faster osteogenesis and the formation of newbone tissue with significantly higher optical density thanthat in defects left to heal spontaneously (Co).

According to Figure 8(b), there was a statistically signifi-cant difference in the studied histomorphometric parame-ters, such as the Haversian canal density on the bone sur-face observed in the view field between experimentalsubgroups A1 and A2 (p < 0.001), as well as the controlgroup Co, which indicated good osteogenesis twelve weeksafter the implantation of microparticulate composite bioma-terial. Statistically significant difference in area fractionbetween subgroups A3 and A4 (p < 0.001), where NPs CP/PLGA composite was implanted, and the control group (Co)was also noticed. There was a statistically significant differ-ence in histomorphometric parameters (area, mean density)between subgroups A3 and A4 and the control group (Co).The histomorphometric parameters confirmed more advancedbone integration and osteogenesis in alveolar bones of the ex-perimental group of animals where bone defects had beenfilled with NPs CP/PLGA mixed with autologous plasma, thanwith other subgroups, which is in accordance with histopath-ological findings.

A histomorphometric analysis of the rat mandibular al-veolar bone 24 weeks after the implantation of the nano-composite [Figure 8(c)] revealed a statistically significantdifference between the Haversian canal density within allexperimental groups—A groups (p < 0.001), and that in thecontrol group (Co). Twenty-four weeks after the implanta-tion, a deviation from mean bone density value obtained inthe control group could also be observed in all experimentalgroups (p < 0.001).

CONCLUSION

The best results in the process of regeneration and recupera-tion of alveolar bone weakened by osteoporosis wereachieved after the implantation of nanoparticulate CP/PLGAcomposite mixed with autologous plasma owing to the pres-ence of growth factors and chemokines, which attract cellswith pronounced proteolytic properties and active role inangiogenesis and transport of osteoprogenitor cells, provideconditions for enhanced cell proliferation, differentiation andnew, nonlamellar bone formation, which in the process of in-

tensive remodeling, becomes lamellar and then cortical withvery regular structure. The osteogenesis in rats belonging tothis group is more pronounced compared to that in thegroup in which microparticulate CP/PLGA was implanted,which is probably a result of a combined effect of the localinflammatory process, minimal interstices between the localbone and well-compressed nanoparticulate composite paste,sensitivity and average diameter of spherical NPs coated withpolymer matrix, which facilitates adhesion of osteoprogenitorcells, and together with growth factors, induces vigorous cellproliferation, differentiation, and osteogenesis, that is, newbone formation.

The implantation of microparticulate CP/PLGA, in theform of granules, mixed with autologous plasma into an artifi-cial defect in alveolar bone also led to the formation of newbone, but this process was less pronounced. Histomorphomet-ric analysis of the alveolar part of mandible in rats, wherenanoparticulate CP/PLGA composite mixed with autologousplasma was implanted, revealed the highest values of the stud-ied parameters: area fraction (the density of Haversian canalswithin the newly formed bone structure), area (area occupiedby new bone in the view field), and mean density (mean valueof the optical density of the studied part of the bone).

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ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JULY 2010 VOL 94B, ISSUE 1 117