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Science of Advanced MaterialsVol. 6, pp. 1–9, 2014

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Magnetic and Morphological Properties ofFerrofluid-Impregnated Hydroxyapatite/Collagen ScaffoldsAlberto Riminucci1,∗, Chiara Dionigi1, Chiara Pernechele2, Giulia De Pasquale2, Tilde De Caro3,Gabriel Maria Ingo3, Francesco Mezzadri4, Nathalie Bock1, 5, Massimo Solzi2,Giuseppina Padeletti3, Monica Sandri6, Anna Tampieri6 , and V. Alek Dediu1

1Consiglio Nazionale Delle Ricerche-Istituto per lo Studio Dei Materiali Nanostrutturati (CNR-ISMN), Via Gobetti 101,40129 Bologna, Italy2Università di Parma, Dipartimento di Fisica e Scienze della Terra, Parma, Italy3Consiglio Nazionale delle Ricerche-Istituto per lo Studio dei Materiali Nanostrutturati (CNR-ISMN), Rome, Italy4Università di Parma, Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica e Chimica Fisica, Parma, Italy5Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia6Consiglio Nazionale delle Ricerche-Istituto di Scienza e Tecnologia dei Materiali Ceramici, Faenza, Italy

ABSTRACT

In this article we present the morphological and magnetic characterization of ferrofluid-impregnated biomimeticscaffolds made of hydroxyapatite and collagen used for bone reconstruction. We describe an innovative andsimple impregnation process by which the ferrofluid is firmly adsorbed onto the hydroxyapatite/collagen scaf-folds. The process confers sufficient magnetization to attract potential magnetic carriers, which may be used totransport bioactive agents that favour bone regeneration. The crystalline structure of the magnetite contained inthe ferrofluid is preserved and its quantity, estimated from the weight gain due to the impregnation process, isconsistent with that obtained from energy dispersive X-ray spectroscopy. The magnetization, measured with asuperconducting quantum interference device, is uniform throughout the scaffolds, demonstrating the efficiencyof the impregnation process. The field emission gun scanning electron microscopy characterization demonstratesthat the process does not alter the morphology of the hydroxyapatite/collagen scaffolds, which is essential forthe preservation of their bioactivity and consequently for their effectiveness in promoting bone formation.

KEYWORDS:

1. INTRODUCTIONThe need for repairing osteochondral defects hasbecome a significant part of the health burden, due tothe increased incidence of degenerative osteochondralconditions1 caused by the growing median age of the worldpopulation. In particular, degenerative processes, such asarthritis and osteoporosis, are a major cause of disabilityfor the elderly. Regenerative medicine seeks to limit suchprocesses, as well as to repair damage caused by trau-matic events or tumour resection. Osteochondral tissue iscomplex and heterogeneous and has a graded composi-tion that comprises different extracellular components, celltypes and cell densities at various levels; it exhibits both

∗Author to whom correspondence should be addressed.Email: a.riminucci@bo.ismn.cnr.itReceived: xx xx xxxxAccepted: xx xx xxxx

cartilage and bone features.2 While autografts and allo-grafts have been used for treating osteochondral defectsin the past, they have several shortcomings, such as lim-ited supply, risk of disease transmission and immuno-logical rejection. Hence, an effective approach to tacklethis problem is the use of synthetic scaffolds.3–5 Scaffoldsare made of porous materials that provide an appropri-ate structure for cell loading and mimic tissues via phys-ical and chemical cues. Synthetic scaffolds technologiessuch as additive manufacturing have sought to recreate thecomplex architecture of osteochondral tissue6 by devel-oping graded matrices made of collagen, which is themost abundant component in this tissue, or made of sim-ilar hydrogels, such as alginate.7 However, these struc-tures lack hydroxyapatite (HA), the mineral component ofosteochondral tissue, which can be incorporated by wetchemical techniques.8 Composite HA/collagen scaffoldsbest reflect true osteochondral tissue when provided with

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a graded composition, with more collagen where the scaf-fold is in contact with cartilage, and more HA where thescaffold is in contact with the bone part of osteochon-dral tissue.9 Such scaffolds can be purchased from FIN-CERAMICA Biomedical Solutions S.p.A., Faenza, in Italyand have been validated as successful candidates for osteo-chondral repair. These bioactive scaffolds exert a stimulat-ing action on the surrounding healthy tissue that promotestissue regeneration within them. They can have variousproportions of HA and collagen depending on the type ofosteochondral tissue to be repaired.One way of improving the bioactivity of scaffolds is

to use bioactive molecules in the surroundings of theregenerating tissue, directing specific events to take placeduring the repair process. For instance, a crucial stepin tissue regeneration is the formation of blood vessels,i.e., angiogenesis, within the scaffolds, in order to sup-ply cells, nutrients, oxygen and waste exchange through-out the scaffolds.10 Angiogenesis is triggered by varioussignalling molecules such as vascular endothelial growthfactor (VEGF) and platelet-derived growth factor (PDGF),involved in angiogenic sprouting and capillary stabiliza-tion, necessary for optimal vascularisation.27 In order forbone to form and mature effectively, these potent angio-genic stimuli need to be present throughout the scaffold andduring the whole healing process.28 This can be achieved byusing a highly porous magnetic scaffold,11�29 that acts as amagnet when an external magnetic field is applied to it. In asufficiently high and uniform external magnetic field, themagnetic scaffold can attract functionalized (for examplewith VEGF) magnetic nanoparticles (MNPs). Once VEGFis delivered, it is available for cellular uptake in the scaf-fold surroundings. Reloading can take place by providingfresh, functionalized MNPs, while free MNPs are safelyeliminated from the bloodstream. The validity of this work-ing principle was confirmed by earlier studies: a permanent

Table I. Summary of the properties of the magnetic scaffolds that can be found in literature.

Type of scaffold Composition Porosity (%vol) MS (emu/g)

Composite HA/Coll+magnetite nanoparticles11 87–89 15 (calculated)HA/Coll+4 wt% magnetite nanoparticles12

Fe-doped foam-like carbon nanotubes/HA13 1.3HA nanocrystals on polylactic acid and doped with �-Fe2O3

14 0.06

Bioactive ceramics HA doped with Fe15 4HA dip-coated in colloidal suspension of magnetite nanoparticles16 1HA merged with magnetite nanoparticles17 2.5

Bioactive glasses Ca, P, Si, Fe18 6Ca, P, Si, Fe19 86.4 1.7Ca, P, Si, Fe20 83 1

Biological polymers Magnetite/chitosan/poly(vinyl alcohol) membranes21 83.9–85.1 3Chitosan coprecipitated with magnetite (5 wt%) magnetite22 4 (from magnetite concentration)

Synthetic polymers Fe-doped HA encased in polycaprolactone23 1Magnetite nanoparticles encased in polycaprolactone24 0.3Magnetite nanoparticles encased in poly(lactic-co-glycolic acid)25 10�-Fe2O3 nanoparticles coated with HA and encased in poly(vinyl alcohol)26 7

magnet was used to drive VEGF-functionalized MNPs intoa 90% HA/10% magnetite magnetic scaffold. The resultsshowed greater bone regeneration after 16 weeks comparedto a control experiment.31

For angiogenic and magnetic purposes, a high poros-ity and a high saturation magnetization (MS) are desirable.Table I summarizes the data available on the compo-sition, porosity and MS of current magnetic scaffoldsfound in the literature. Previous works on magnetic scaf-folds made of organic/inorganic HA/collagen compositesreport a maximum MS of 15 emu/g11�12 and a maxi-mum porosity of 89%; the MS value was not measuredbut extrapolated from the magnetization at lower fields.Several other magnetic composite scaffolds, chemicallyless biomimetic, can be found in literature, with a maxi-mum magnetization of 1.3 emu/g.13�14 The alternatives toorganic/inorganic composite materials are bioactive inor-ganic materials (bioactive ceramics and bioactive glasses)and biological or synthetic polymers.32 Bioactive ceram-ics and bioactive glasses often have a similar compositionto that of the mineral phase of natural bone and possessfavourable bioactive properties. For this type of magneticscaffolds the maximum reported MS was 6 emu/g andthe maximum reported porosity was 86.4%.15–20 Scaffoldsmade from biological polymers show a maximum MS of∼ 4 emu/g as deduced from a reported magnetite con-centration of 5 wt%, and a porosity of 85.1%;21�22 thesescaffolds provide appropriate signalling to cells. Syntheticpolymers offer a more versatile alternative and can beused for the fabrication of scaffolds with controllable 3Dstructures.33 A MS up to 10 emu/g has been reported forthis class of magnetic scaffolds.23–26

In this work we describe the fabrication process ofmagnetic scaffolds by impregnation of 70% hydroxyap-atite/30% collagen (hereafter referred to as HA/Coll) scaf-folds with a ferrofluid. Table II shows the mechanical

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Table II. Mechanical properties of HA-based scaffolds and of naturalhuman bone.

Young’s Flexural Compressivemodulus (GPa) strength (MPa) strength (MPa)

HA/Coll 0.5112 0.45∗9

Magnetic HA/Coll 0.6412

HA 1�1÷1�230 27÷3430

Human trabecular bone 12÷2230 0�7÷1530

Human cortical bone 10÷2630 131÷20630

Note. ∗Estimated.

properties of the HA/Coll scaffolds, in comparison withother HA-based scaffolds and with natural human bone.The HA/Coll scaffolds are not designed to be load-bearing,as opposed to pure HA scaffolds, and they have reducedmechanical properties compared to both pure HA scaf-folds and natural bone. Nevertheless, of all the magneticscaffolds presented in the literature, the HA/Coll compos-ite ones used here possess the best biomimetic properties,as extensively proven already.9�11�34 Conversely, ferrofluidsare made of an aqueous colloidal suspension of MNPs35

and can provide high magnetic moments at low costsand low cytotoxicity. The HA/Coll scaffolds impregnatedwith ferrofluid were tested in vivo (at a concentration of2.5 mg/ml) in rabbits.29 The histological and histomorpho-metric results of that study showed that magnetic scaffoldswere histocompatible, promoted bone healing, and werewell osteointegrated in the host bone without causing anysignificant adverse reactions. Impregnation is a simple andversatile technique which can potentially be used in a clin-ical setting to produce tailored magnetic scaffolds. Theobjective of this work is to study this technique and topresent a thorough morphological and magnetic character-ization of the magnetized HA/Collscaffolds. The controlon the amount of magnetization was obtained by carryingout several cycles of impregnation. They led to a uniformand strong magnetization throughout the sample, whichis essential to attract magnetic carriers. We also showthat the morphology of the scaffolds was not degraded bythe process, which is critical for the preservation of theirbiomimetic and bioactive properties.36

2. EXPERIMENTAL DETAILS2.1. MaterialsFor the fabrication of the magnetic scaffolds, westarted from composite HA/Coll scaffolds provided byFinceramica, Italy. These scaffolds mimic the microporos-ity and macroporosity of trabecular bone, as describedelsewhere.8 In order for this study to have sufficient sta-tistical significance we carried out experiments on 4 sam-ples, whose initial physical properties are summarized inTable III. The magnetization of the HA/Coll scaffolds wascarried out using the ferrofluid FluidMag-DP 200 nm,50 mg/ml MNPs concentration, purchased from Chemicell.

Table III. Physical characteristics of the pristine scaffolds used in thisstudy. The porosity was 93–95% for all scaffolds.

Sample A B C D

Initial weight (mg) 91�6 77�4 92�1 75�7Initial diameter (mm) 13�5 13�1 13�6 13�5Initial height (mm) 5�65 5 6�4 5�8

The ferrofluid will be referred to as FF in the remainderof this work.

2.2. Magnetic CharacterizationDuring the impregnation process, the magnetization ofthe scaffold was measured with a YSZ 01C�02C suscep-tometer (Sartorius Mechatronics, Italy S.r.l.) by applyinga magnetic field of 34 Oe. The in depth magnetic char-acterization of samples A and B was carried out with aQuantum Design MPMS Superconducting Quantum Inter-ference Device (SQUID) at room temperature.

2.3. Impregnation ProcessIn the impregnation process each scaffold was soaked inFF, as described in detail in Ref. [11]. Briefly, the pris-tine dry scaffold was placed in a plastic sample holder and1 ml aqueous solution of 50% v/v FF was poured on it tosoak it. The wet scaffold was then frozen by keeping it forat least 2 h at − 18 �C; subsequently the ice was sublimedat room temperature at a pressure lower than 0.2 mbar forat least 12 h. Upon drying, the magnetization of the scaf-fold was measured with the susceptometer by applying amagnetic field of 34 Oe and the weight was measured witha Gibertini E50S analytical balance. After this, the scaffoldwas placed in distilled water and washed for 20 minutesin a 100 W sonicator. The scaffold was then frozen andvacuum dried again as described above. The magnetizationand the weight were measured and compared to the previ-ous values to assess how much magnetic material was lostduring the rinsing. To achieve a greater magnetization, thisprocedure was repeated 3 times in total. Table IV summa-rizes some of the physical characteristics of the impreg-nated scaffolds used for this study.

2.4. Morphological CharacterizationMacro- and micromorphological observations were con-ducted on sample C by a high brilliance LEO 1530 fieldemission scanning electron microscope (FESEM) appara-tus equipped with an energy dispersive X-ray spectrome-ter (EDS) model INCA 450 and a back scattered electrondetector. The images were recorded both in the secondaryelectron image (InLens) and in the back scattered image(AsB) modes at acceleration voltages of 15 kV.

2.5. Thermogravimetric AnalysisThe thermogravimetric analysis (TGA) on MNPs, obtainedfrom drying the FF in air at 50 �C, was performed in air

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Table IV. Physical characteristics of the impregnated scaffolds. Samples A and B also underwent a SQUID characterization, whose results are reportedon the last column. C was used for the morphological characterization with a FESEM. In terms of porosity and MS , our scaffolds surpass all thosedescribed in literature, which are reported in Table I.

Final weight Final diameter Final height Volume Weight increase Final porosity SaturationSample (mg) (mm) (mm) loss (%) mg (%) (%) magnetization (emu/g)

A 130.2 11.4 4�95 37% 38.6 (42%) 90% 23.3B 111.8 11.1 4�3 38% 34.4 (44%) 89% 20.3C 136.3 11.8 5�6 34% 44.4 (48%) 91%D 113.3 11.7 5 35% 37.6 (50%) 91%

ramping up the temperature to 1000 �C (temperature rampof 5 �C/minute) with A STA1500 system equipped with asimultaneous thermal analyzer.

2.6. X-Ray Diffraction CharacterizationThe X-ray Diffraction (XRD) characterisation of both fer-rofluid and fully magnetized scaffolds was carried outwith a Thermo ARL X’tra diffractometer, equipped with aCuK� radiation source and a Si(Li) Thermo Electron solidstate detector allowing the exclusion of the fluorescencesignal of iron.

3. RESULTS AND DISCUSSION3.1. Impregnation ProcessIn order to characterize the impregnation process, theweight increase of the scaffolds after each round of mag-netization with the FF was monitored. Special care wastaken to exclude the effect of humidity, since the pristinescaffolds were stored in ambient humidity before use andthe sublimation process eliminated this ambient moisture.The inset in Figure 1(a) shows how the weight of a typicalscaffold increased over time after it was taken out of thesublimation chamber. A fitting of the weight increase as afunction of time was obtained with a curve of the type w=a+b ∗e−t/t1 +c ∗e−t/t2 , where w is the weight, a, b and care weight fitting parameters and t1 and t2 are time fittingparameters; the two exponential trends indicated that twodifferent rehydration processes were taking place simulta-neously. These two processes had approximately the samemagnitude (b � c � 0�005 g) but different time constants(t1 � 600 s and t2 � 20000 s). While a deeper understand-ing of the rehydration processes is beyond the scope of thisarticle, it is important to note that the difference betweent1 and t2 provides an ample time window in which weightscan be measured reproducibly. The weights reported inFigure 1(a) were taken at t � 1000 s, which is large com-pared to t1 and small compared to t2. This means that att � 1000 s the first hydration process was almost completeand that the second one was negligible.Figure 1(a) shows the average weight increase of sam-

ples A, B, C and D from their pristine state (left-most datapoints) up to the third impregnation (3 on the abscissa).The weight increase after each impregnation is similarwhile the loss of weight after each washing is around

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Fig. 1. Effect of each impregnation round. Black symbols indicate theincrease before washing the samples and the grey ones indicate the samequantity measured after washing. (a) average weight increase after eachimpregnation (average of samples A–D). After each impregnation theweight increase is similar. The loss of weight after washing increasesafter the first impregnation, indicating that the MNPs are more looselyattached. The inset shows the weight increase due to rehydration of afreeze-dried pristine HA/Coll scaffold. The process has two time con-stants. This provides a time window in which the weight can be mea-sured reproducibly. (b) average increase in magnetization at 34 Oe aftereach impregnation. The magnetization increase is greatest after the firstimpregnation; the effect of washing has no general trend. The inset showsa TGA curve, indicating that the fraction of volatile or combustible mate-rials in the MNPs is 9%. This is the sum of both water (2.5%) and theorganic component (6.5%), while the remaining 92±1% is made of ironoxide.

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2 mg after the first impregnation. This may be due to thedecreasing number of adhesion sites available to the MNPsin the scaffolds after each impregnation. The impregnationprocess caused the scaffolds to shrink, as it can be seenby comparing the diameter and height of the pristine scaf-folds in Table III to those of the impregnated scaffolds inTable IV; this in turn decreased the porosity from 93–95%for the pristine scaffolds to about 89–91%, as reported inTable IV. The shrinking was attributed to the interactionof the collagen fibres with the aqueous FF suspension.37

The average increase in magnetization after eachimpregnation is shown in Figure 1(b) at an applied fieldof 34 Oe. The magnetization increases less after thefirst impregnation because of the increased interactionbetween the MNPs inside the scaffolds after each impreg-nation round. Indeed, the initial susceptibility of MNPs,which is approximately proportional to the magnetizationat 34 Oe in our case, decreased with increasing inter-particle interaction.38�39

3.2. Thermogravimetric Analysis of the FFThe thermogravimetric analysis (TGA) of the FF (inset inFig. 1(b)), dried in ambient conditions, was carried outin order to determine what weight fraction of the MNPsin the dried FF was constituted by the magnetic phase. Thewater adsorbed on the MNPs evaporated at 150 �C andcorresponded to 2.5% of the total weight loss. Since thehydration dynamics of the FF was not known, this valueis considered as an uncertainty on the weight gain in theinset to Figure 1(a). The exothermic process at 300 �Cindicated the combustion of the organic fraction, whichamounted to 6.5% of the FF. As a result, 92± 1% of theweight increase reported in Figure 1(a) was attributed tothe inorganic component of MNPs (iron oxide).

3.3. Magnetic PropertiesThe magnetization curves of magnetic scaffolds A andB were measured with a SQUID magnetometer; thecurve for A, which is representative of sample B too,is shown in Figure 2. The curves show no appreciablehysteresis, which is consistent with the prevalent super-paramagnetism of the FF MNPs. The saturation magneti-zation MS is 23.3 emu/g for sample A (� = 0�258 g/cm3,V = 0�505 cm3, MS = 6�01 emu/cm3) and 20.3 emu/gfor sample B (� = 0�269 g/cm3, V = 0.416 cm3, MS =5�46 emu/cm3). Following the working principle proposedby Bock et al.11 the magnetization has to be sufficientlyhigh to be able to drive MNPs towards the scaffold whenan external uniform magnetic field is applied. The forcethat is exerted by scaffolds with the MS of sample A canbe easily estimated: in general, the force �F exerted by amagnetic field �B on a MNP with a magnetic moment �� isgiven by:40

�F = � �� · �� �B (1)

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Fig. 2. SQUID magnetization measurements on sample A, with an esti-mate of the force that it can exert on a 150 nm diameter magnetitenanoparticle. The solid line in the inset shows, as a function of the dis-tance from the surface, the force that a spherical scaffold, with a mag-netization equal to the saturation magnetization of A, would exert ona 150 nm diameter magnetite nanoparticle. The horizontal dashed lineshows the weight of the nanoparticle.

According to Eq. (1) the external uniform magnetic fieldcannot contribute to the force, as opposed to the space-varying magnetic field generated by the scaffold. As thedetails of the magnetic field generated by the scaffolddepend on its shape, we consider for simplicity a uni-formly magnetized spherical scaffold, whose magneticfield is given by:

�B = �0/�4× �3� �MV · �r · �r− r2 �MV �/r5 (2)

As an example we take a magnetically saturated, 150 nmdiameter cluster of superparamagnetic magnetite nanopar-ticles (M = 60 emu/g41 � 310 emu/cm3); its magneticmoment will be aligned with the applied magnetic field.The attractive force, along the axis defined by �B and thecentre of the scaffold, as a function of the distance fromthe surface of the scaffold is shown by the solid line inthe inset in Figure 2; the dashed line indicates the weightof one MNP. This value is easily overcome, which provesthat the scaffolds are effective in attracting functionalizedMNPs for repeated delivery of growth factors.

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Besides the strength of the magnetic force, anotherimportant measure of the effectiveness of the impregnationprocess is the uniformity of MS throughout the scaffold.In order to study how the MNPs distributed inside the scaf-folds, several similarly-sized pieces were cut from differ-ent parts of sample A to measure their magnetic moments.Figure 3(a) shows the MS of parts of sample A taken fromdifferent places, as mapped by the inset. Within the errorbars, which are due to the uncertainty in the weight, allthe parts have approximately the same MS . This meansthat impregnation is an effective and predictable way tohomogenously magnetize scaffolds.

3.4. Crystalline Structure of the ScaffoldsThe preservation of the crystalline structure of the mag-netite was checked by XRD. Figure 3(b) shows the spectrafor a sample of desiccated FF (top panel) and of sam-ple A (bottom panel). The spectrum on the top panel con-firms that the FF is made of a single phase, constitutedby magnetite MNPs, with a mean size of approximately12 nm as determined by using the Scherrer equation.42 Themagnetite peaks appear also in the spectrum from sam-ple A, confirming that the MNPs are not altered by theimpregnation process. Besides the broad contribution ofthe amorphous ceramic support and the weak reflectionsdue to hydroxyapatite, this spectrum displays the presenceof magnetite nanoparticles of the same size, suggestingthat the nanoparticles are included into the ceramic matrixwithout any structural modification.

3.5. Morphological and Elemental CharacterizationThe effect of the impregnation process on morphology isan essential point to address since morphology is one ofthe key properties of biomimetic and bioactive materials.43

In particular, for bone regeneration, macro-porosity is nec-essary for the vascularisation of scaffolds, so that new bonecan be produced and matured.44 At the same time, micro-porosity affects the adhesion of osteoblasts, bone cells,45

which is critical to successful bone deposition.46

To check that the impregnation procedure does notaffect the morphological properties of the scaffolds, weused FESEM. With this technique sample C was imagedin both the InLens and the AsB modes.47 The latter issensitive to the elemental composition: brighter areas cor-respond to elements with greater atomic number. In theAsB images the MNPs, which contain the heavy elementFe, appear brighter than areas that contain only HA/Coll,which is composed of lighter elements (C, H, P, O, Ca,N, S). With this technique it is therefore possible to gaina precise understanding of where the MNPs attached.Figure 4 shows micrographs of sample C (first two

columns) and of a pristine HA/Coll scaffold (third col-umn), taken with 4 different magnifications, increasingfrom top to bottom row. From left to right we show,with the same magnification, the InLens picture of C, the

(a)

(b)

Fig. 3. (a) MS of different parts of sample A as shown in the schematicdiagram in the panel. Within the experimental error on the weight mea-surements, the magnetization was approximately the same for all parts.(b) XRD spectra of the desiccated FF used for the impregnation (toppanel) and of sample A (bottom panel). The dashed lines highlight thepeaks corresponding to magnetite. The fact that they are preserved afterthe impregnation confirms the validity of our magnetizing approach.

AsB picture of the same area and a InLens picture ofthe blank scaffold for comparison. In the lower magnifi-cation pictures on the top row, the blank scaffold in 4(c)presents filamentary collagen structures covered by HA.9

As demonstrated by 4(a) and (b) taken at the same magni-fication, this macro-porosity, which allows the growth ofblood vessels, was preserved in the magnetized scaffold.The AsB image in 4(b) shows that the MNPs aggregatein clusters about 1 �m across (two examples are indicatedby the white arrows).At intermediate magnifications, shown on the 2nd and

3rd rows, the 1 �m Fe-rich clusters are more obvious(as highlighted by the AsB pictures in 4(e) and (h)). Thestrands that form the filaments are also visible.

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(a) 10 µm

(d) 1 µm

(g) 1 µm

(j) 100 nm

(f)

(i)

(l)

(b) (c)

(e)

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Fig. 4. SEM micrographs of C and of a pristine HA/Coll scaffold. From left to right, the first column is composed of secondary electrons images(InLens mode) of C, the second column contains micrographs taken with back scattered electrons (AsB mode) from the same area, and the thirdcontains InLens images of a pristine HA/Coll scaffold. In the AsB images on the second column, brighter areas correspond to elements with greateratomic number, Fe in this case. The arrows in (b) point to two such areas. In the InLens image (j), the dashed line enclosures show areas composed offiner grains, which correspond to the crystals that make up the MNPs. This is confirmed by the fact that the corresponding areas (which are shifted dueto a small drift) are brighter in the AsB mode image shown in (k). The solid line enclosure shows areas with bigger, HA crystals which are identicalto the ones found on the blank scaffold shown (l). The morphology of the magnetized sample C and that of the pristine HA/Coll scaffold is similar atall the magnifications shown, which confirms that the impregnation procedure does not affect the micro and macro porosity of the pristine scaffolds.This is essential for the preservation of the biomimetic and bioactive properties of the scaffold.

Upon looking at the InLens micrograph at the highestmagnification in 4(j), it is possible to distinguish crystalgrains of two different mean sizes. More specifically, finercrystals can be seen on the bottom left corner of the pictureand across the top (dashed line enclosures), while biggercrystals run across the picture from top left to bottom right(solid line enclosure). The AsB picture in 4(k) shows thatthe finer grains correspond to Fe-rich areas, thus suggest-ing that they belong to a MNP cluster; this is corroboratedby FESEM images on the dried FF (not shown), whichshow similar crystallites. The presence of MNPs was con-firmed in similar scaffolds by electron diffraction;12 crys-talline magnetite grains and amorphous HA ones could bedistinguished, in agreement with the XRD study reportedin the bottom panel of Figure 3(b). In Figure 4(l), on anequal magnification, the blank scaffold shows only the big-ger grains and no finer grains are present. The shape ofthe bigger grains in the magnetized scaffold is the sameto that of those in the blank scaffold and is similar to that

reported for the crystallization of HA on collagen fibres.48

This indicates that the bigger grains are made of HA.In order to confirm the amount of Fe present in the scaf-

fold an EDS analysis was also performed. Five randomareas (∼ 300× 200 �m2 on average) were sampled anda Fe content of 32± 5 w% was detected. The magnetitecontent of the impregnated scaffolds could be estimatedfrom the latter quantity, from the weight increase of thescaffold due to the impregnation and from the measuredmagnetic moment. Table V shows a comparison betweenthe values obtained using a MS of 60 emu/g for magnetite,considering that in the weight increase measurements inFigure 1(a), TGA characterization of the FF indicates thatonly 92±1% of the increase can be attributed to magnetite.The weight increase-based and EDS-based estimates inthe table agree reasonably well with each other, while theresults from the SQUID give a greater value. This can bedue to the difficulty in defining the MS of the magnetitenanoparticles, which is strongly dependent on the nanopar-ticle size.41

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Table V. Concentration of MNPs in the scaffold, as calculated fromsaturation magnetization, from energy dispersive X-ray spectroscopy andfrom the weight increase (the values are mean± standard deviation, 6repeats for SQUID data, 5 repeats for EDS data and 4 repeats for weightincrease data).

Measurement method Quantity of magnetite(samples considered) (mg/cm3)

SQUID-measured saturation 95±5magnetization (A, B)

EDS(C) 70±11Weight increase (A, B, C, D) 64±2

4. CONCLUSIONSThe magnetic and morphological properties of novel FF-impregnated HA/Coll scaffolds were investigated. Themethod of impregnation proved to be effective in uni-formly magnetizing the scaffolds, while preserving themorphology at the macro and micro-scale, and to be sim-ple enough to be potentially carried out in a clinical set-ting. The crystalline structure of the magnetite particlesused in the magnetization process was preserved and themagnetic scaffolds showed a magnetization that was suffi-ciently high to be effective in potentially attracting MNPs.The high magnetization obtained with biocompatible FFsand the intrinsic scaffold properties-high interconnectedporosity, 70%/30% hydroxyapatite/collagen composition-are superior to any of the scaffolds that can be found inthe literature. This unique combination makes the mag-netic scaffolds a highly innovative material for enhancedbone tissue regeneration by targeted delivery of bioactivefactors under a magnetic field.

Acknowledgments: The authors would like to thankFederico Bona and Mauro Paoletti for their invaluabletechnical contribution. Alberto Riminucci, Chiara Dionigi,Nathalie Bock, Massimo Solzi, Anna Tampieri and V. AlekDediu acknowledge financial support for this work from byFP7 EU project NMP3-LA-2008-214685 “MAGISTER.”

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