Mineralization, Biodegradation, and Drug Release Behavior of Gelatin

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Mineralization, Biodegradation, and Drug Release Behavior of Gelatin/Apatite Composite Microspheres for Bone Regeneration Sander C. G. Leeuwenburgh,*,† Junichiro Jo,‡ Huanan Wang,† Masaya Yamamoto,‡ John A. Jansen,† and Yasuhiko Tabata‡ Department of Biomaterials, Radboud University Nijmegen Medical Center, Philips van Leydenlaan 25, 6525 EX Nijmegen, The Netherlands, and Department of Biomaterials, Kyoto University, 53 Kawara-cho Shoguin, Sakyo-ku, Kyoto 606-8507, Japan Received June 10, 2010; Revised Manuscript Received July 29, 2010

Gelatin microspheres are well-known for their capacity to release growth factors in a controlled manner, but gelatin microspheres do not calcify in the absence of so-called bioactive substances that induce deposition of calcium phosphate (CaP) bone mineral. This study has investigated if CaP nanocrystals can be incorporated into gelatin microspheres to render these inert microspheres bioactive without compromising the drug releasing properties of gelatin microspheres. Incorporation of CaP nanocrystals into gelatin microspheres resulted into reduced biodegradation and drug release rates, whereas their calcifying capacity increased strongly compared to inert gelatin microspheres. The reduced drug release rate was correlated to the reduced degradation rate as caused by a physical cross-linking effect of CaP nanocrystals dispersed in the gelatin matrix. Consequently, these composite microspheres combine beneficial drug-releasing properties of organic gelatin with the calcifying capacity of a dispersed CaP phase.

1. Introduction Although synthetic options for bone-replacement are being researched for more than four decades, the only currently available treatment option that effectively fulfils all requirements needed for the replacement of bone tissue still involves the use of patient’s own (autograft) or donor (allograft) bone. The use of auto- or allografts, however, comes along with major drawbacks such as increasing donor shortages, limited bone volume, risk of disease transfer, and severe pain complications at the site of harvesting, as well as the need for additional surgery at the site of bone harvest, which is accompanied by severe pain complications and donor site morbidity.1 In view of the aging population, the above-mentioned problems will become increasingly evident, stressing the need for synthetic alternatives to human bone. Typical examples of synthetic bone fillers include calcium phosphate (CaP) ceramics2 and polymer/CaP composites3 because CaPs have strong chemical resemblance to the mineral phase of bone and teeth. Generally, however, the clinical success of these synthetic bone fillers is inferior to auto- or allografts for several reasons. First of all, the degradation rate of sintered CaP ceramics does not match with the rate of bone remodelling, which allows these CaP ceramics to be considered as static, permanent implants that hardly participate in the dynamic process of bone remodelling.4 Second, pure CaP ceramics cannot be molded into the desired defect shape when applied as granules due to the intrinsic brittleness of this class of materials. From a clinical point of view, injectable or moldable biomaterials for reconstruction of osseous defects offers several clinical and economical advantages as compared to solid, prefabricated implants.5 Using flowable materials, complete filling of the defect site can be established by means of minimally invasive * To whom correspondence should be addressed. Tel.: +31 (0)24 3667305. Fax: +31 (0)24 3614657. E-mail: [email protected]. † Radboud University Nijmegen Medical Center. ‡ Kyoto University.

techniques, thus, avoiding gaps that can lead to fibrous encapsulation or scar formation. Although injectable CaP cements have been developed to overcome this handling problem, these cements are also poorly degradable despite the fact that they are not sintered. Third, attempts to add signaling features to CaP ceramics and composites to increase their functionality were not successful in the past, because control over release and activity of incorporated biomolecules is generally poor due to the very strong interaction between proteins and CaPs.6 The current study hypothesizes that the above-mentioned drawbacks of a purely ceramic approach toward bone regeneration can be overcome by preparing bone fillers based on gelatinapatite microspheres. In that way, the controllable biodegradability, strong capacity for drug release, and injectability/ moldability of gelatin microspheres can be combined with the excellent osteoconductive properties of CaP ceramics. Gelatin has been extensively studied for pharmaceutical and medical purposes, and its biosafety has been proven through its long clinical usage in several pharmaceutical and medical applications.7 The biodegradation of gelatin can be tailored by controlling the cross-linking density using a wide variety of chemical and physical cross-linking techniques.8 Numerous studies have revealed that gelatin-derived scaffolds and microspheres are highly suitable for use as drug delivery vehicles for the controlled release of osteogenic and angiogenic growth factors.9,10 This can be understood from the physiological situation where growth factors are stored in the extracellular matrix (ECM), which consists mainly of fibrillar collagens like type I collagen as well as proteoglycans. Kanematsu et al. have shown that type I collagen can act as a reservoir of growth factors such as basic fibroblast growth factor (bFGF) by complexation and incorporation of bFGF molecules into the collagen fibers, thereby protecting bFGF from premature release by proteolysis until its release following triggering by environmental stimuli.11 From that perspective, incorporation of growth

10.1021/bm1006344  2010 American Chemical Society Published on Web 08/30/2010

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factors into and subsequent release from gelatin, which can be classified as denatured collagen, resembles the human physiological situation where growth factors are released from the ECM by orchestrated proteinase activity.11 With respect to clinical handling of bone fillers, one of the main practical advantages of gelatin is the ease by which it can be processed into various shapes such as porous sponges and microspheres.9,10 More specifically, microspheres with controlled size and shape can be injected into cavities with uniform packing using appropriate carrier liquids. Regarding drug delivery, Tabata et al. have shown in several in vivo studies that growth factorloaded microspheres are able to induce osteogenesis and angiogenesis.10,12-15 To this end, growth factors were loaded into cross-linked gelatin microspheres of opposite charge, resulting in the formation of polyion complexes between growth factors such as basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP-2) and gelatin of either bovine or porcine origin. Specifically, bFGF is a potent growth factor that promotes angiogenesis and proliferation of mesenchymal cells.15 The polyion complex between gelatin and bFGF was shown to be stable enough to bind bFGF to the organic matrix, thereby preserving the biological activity of bFGF until its release from the gelatin matrix by proteolytic degradation.9 This sustained release profile was shown to be far more effective than single-dose application of growth factors.9 Preparation of gelatin microspheres containing apatitic nanoparticles has been reported in literature,16,17 but none of both studies studied the functional properties of these composite microspheres so their physicochemical and biological behavior is still largely unknown. Therefore, the aim of the current study was to investigate if gelatin microspheres can be made bioactive by incorporation of CaP nanoparticles without compromising their capacity for drug release. More specifically, the biodegradation rate, the calcifying capacity and the drug releasing properties of gelatin/apatite microspheres in vitro has been studied in more detail. To this end, gelatin/apatite composite microspheres were loaded with radiolabeled bFGF and soaked in proteolytic simulated body fluid (SBF) for 4 weeks to characterize drug release, mineral deposition, and biodegradation of the microspheres simultaneously.

2. Experimental Section 2.1. Microsphere Preparation. Gelatin microspheres were prepared using a water-in-oil emulsion method adapted from Tabata et al.10 Briefly, 2 g of acidic gelatin type B (gelB, from bovine bone, isoelectric point 4.9, Nitta Gelatin, Osaka, Japan) was dissolved in 18 mL of distilled water at 40 °C (corresponding to 10 wt % gelatin concentration) and added dropwise into 600 mL of olive oil in a three-neck roundbottom flask. This mixture was stirred at 400 rpm for 15 min using an upperstirrer and chilled to below 10 °C for 30 min. After chilling, 200 mL of chilled acetone (4 °C) was added while stirring was continued for another 15 min. Microspheres were collected using filtration (Whatman, 90 mm filter paper grade 2), passed through sieves between 20-32 µm, and finally cross-linked dehydrothermally for 48 h at 160 °C. Gelatin/apatite composite microspheres were prepared using the water-in-oil emulsion method as described above except for the fact that premade aqueous suspensions of apatitic needle-shaped nanoparticles were used as water phase instead of CaP-free distilled water. An established neutralization reaction between calcium hydroxide and phosphoric acid was employed to prepare these suspensions.18,19 Briefly, a phosphoric acid solution (Wako, Kyoto, Japan) was added dropwise to a suspension of calcium hydroxide (Sigma, St. Louis, MO, ACS grade) at a stoichiometric ratio of 1.67 at room temperature. The apatitic suspensions were prepared in such way to yield final gelatin/apatite

Leeuwenburgh et al. weight ratios of 60:40 (gelB/CaP40%, 22.5 mL of 885 mM M H3PO4 added to 22.5 mL of 1476 mM Ca(OH)2) and 90:10 (gelB/CaP10%, 22.5 mL of 148 mM H3PO4 added to 22.5 mL of 246 mM of Ca(OH)2), respectively. After mixing the reactants the pH values of the suspensions were about 11-12. Subsequently, the suspensions were aged for 15-18 h and finally adjusted to a neutral pH value of 7.4 using HCl. Subsequently, 2 g of gelatin was dissolved in 18 mL of the apatitic suspensions (heated at 40 °C to allow for dissolution of the gelatin) and microspheres were prepared as described above for CaP-free microspheres. All microspheres were characterized using light microscopy (LM) and scanning electron microscopy (SEM, Hitachi, Tokyo, Japan), as well as energy dispersive spectroscopy (EDS, EDAX, Tilburg, The Netherlands) and X-ray diffraction (XRD, Panalytical PW3710, Almelo, The Netherlands). To determine fold swelling ratios, 20 mg of microspheres (n ) 5) were swollen in excess PBS at 37 °C overnight, patted dry with tissue to remove surface water, and weighed (Ws), lyophilized, and weighed again (Wd). Fold swelling ratios were calculated as (Ws - Wd)/Wd, while water content percentages were calculated as (100 × (Ws - Wd))/Ws. Additionally, fold swelling ratios and water content were also calculated based on the gelatin content only by compensating for the amount of nonswelling CaP phase. Dry apatite powders were obtained by washing and filtering (Whatman Japan, Tokyo, Japan, 90 mm filter paper grade 2) the apatitic suspensions as described above, drying at 37 °C overnight and crushing using a pestle and mortar. 2.2. Radioiodination of bFGF. Human recombinant basic fibroblast growth factor (bFGF, isoelectric point 9.6, Kaken Pharmaceutical Ltd. Tokyo, Japan) was radioiodinated using the chloramine-T method of Greenwood et al.20 Briefly, 5 µL Na125I (Perkin-Elmer) was added to a solution of 200 µL bFGF (0.5 mg/mL in 5 mM glutamic acid, 2.5 wt % glycine, 0.5% sucrose and 0.01 wt % Tween 80 (pH 4.5). This was followed by the addition of 100 µL of chloramine-T (Wako, Kyoto, Japan, 0.2 mg/mL) in 0.5 M potassium phosphate-buffered solution (pH 7.5) containing 0.5 M NaCl. After agitating the mixture for 2 min, the reaction was terminated by adding 100 µL of PBS containing 0.4 mg of sodium metabisulfate (Wako, Kyoto, Japan) and vortexing for another 2 min. Subsequently, purification was achieved by gel filtration through a Sephadex PD-10 column (Amersham Biosciences, Pittsburgh, PA) to remove free 125I from the solution. The commercially available bicinchoninic acid (BCA, Pierce Biotechnologies, Rockford, IL) total protein assay was used to determine the concentration of bFGF. 2.3. In Vitro Drug Release and Bioactivity Studies. Microspheres (gelB, gelB-10%CaP, gelB-40%CaP) or apatite powder were loaded with radiolabeled 125I-bFGF using diffusional loading; 20 µL of a 20 µg/mL 125I-bFGF solution was dripped onto 2 mg of sample overnight at 4 °C, yielding 400 ng of bFGF per sample. Subsequently, these drugloaded samples were incubated in proteolytic simulated body fluid (n ) 3, 1 mL SBF per sample, 400 ng/mL bacterial collagenase 1A (Sigma, St. Louis, MO), 0.001 w/v% sodium azide (Wako, Kyoto, Japan) to prevent bacterial contamination) up to 28 days to investigate the bioactivity as well as the drug release properties simultaneously. Simulated body fluid was used as buffer medium for this degradation study instead of PBS because this fluid allows for simultaneous evaluation of the calcification behavior in vitro.21 The capacity to nucleate CaP formation under in vitro conditions is often interpreted as a first indication for the possible bone-bonding capacity (“bioactivity”) of novel bone-substituting biomaterials.21 Because gelatin is digested enzymatically, addition of bacterial collagenase the SBF also allowed for simultaneous monitoring of in vitro degradation of gelatin/ apatite microspheres. The SBF was refreshed after 1, 3, 7, 14, 21, and 28 days after centrifugation at 10 krpm for 5 min to prevent loss of microparticles during SBF removal. Release of bFGF was quantified by monitoring the radioactivity of the supernatant using a gamma ray counter (ARC-301B, Aloka, Tokyo, Japan) and accounting for radioactive decay. The calcium concentration and protein concentration in the supernatant were also measured using commercially available kits (ortho-cresolphtalein (OCPC, Sigma, St. Louis, MO) for the calcium

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Table 1. Particle Size, Fold Swelling Ratio, and Water Content of Experimental Groups group

primary particle size (µm)

fold swelling

gelatin-based fold swelling

water content (%)

gelatin-based water content (%)

gelB gelB/CaP10% gelB/CaP40%

12.6 ( 3.1 15.6 ( 5.9 15.9 ( 4.5

10.9 ( 0.8 9.3 ( 1.0 6.6 ( 0.5

10.9 ( 0.8 10.4 ( 1.1 11.6 ( 0.9

91.5 ( 0.6 90.2 ( 1.0 86.8 ( 0.9

91.5 ( 0.6 91.2 ( 0.9 92.1 ( 0.6

concentration and BCA for the protein concentration) to evaluate the bioactivity (i.e., ability to induce CaP deposition onto the microspheres) and gelatin degradation simultaneously (n ) 3). In a separate study, all microspheres and powders were soaked in proteolytic SBF under similar conditions but without adsorbing 125I-bFGF onto the microspheres. After 7, 14, and 28 days of soaking, aliquots of all samples were flash-frozen in liquid nitrogen, freeze-dried, and analyzed using ATR-FTIR (Perkin-Elmer, Spectrum One, Groningen, The Netherlands) and SEM to study the molecular structure and morphology of the various microspheres after soaking in SBF. 2.4. Statistics. All data were analyzed using Student’s t-test and expressed as mean ( standard deviation. A value of P < 0.05 was accepted as statistically significant.

3. Results 3.1. Microsphere Properties. Table 1 summarizes the average microsphere size (as determined using light microscopy for 100 microspheres) as well as the fold swelling ratio and corresponding water content upon equilibrium swelling. With increasing CaP content, the average primary particle size increased, whereas the fold swelling ratio and water content decreased, revealing that incorporation of CaP nanocrystals into gelatin microspheres reduced the amount of water uptake by the microspheres. Based on the gelatin content only, however, no change in swelling behavior was observed upon incorporation of CaP into the microspheres. Figure 1 displays the corresponding scanning electron micrographs of the three types of microspheres. Pure gelatin microspheres were smooth, whereas their surfaces roughened and their shapes became more irregular with increased amounts of CaP incorporation. Light microscopy of swollen microspheres revealed that the opacity of the microspheres increased due to the light-scattering effect of incorporated CaP nanocrystals. The CaP phase was equally distributed throughout the microspheres without the

Figure 1. Scanning electron micrographs of pure gelatin B (gelB: a and d), gelB-10%CaP (b and e), and gelB-40%CaP microspheres (c and f) at low (a-c) and high (d-f) magnifications.

appearance of dark, clustered CaP aggregates (Figure 2). Elemental analysis of gelB-40%CaP microspheres by means of EDS revealed that the microspheres consisted mainly of C and O (derived from the organic matrix), as well as Ca and P (related to the inorganic CaP crystals; Figure 3). These CaP crystals were shown to match to a crystalline apatitic phase as revealed by main reflection peaks at 25.9 and 32.1° 2θ (Figure 4). Corresponding ATR-FTIR analyses confirmed the apatitic nature of the mineral phase in the microspheres as characterized by typical apatitic phosphate absorptions at 560, 600, 963, and 1030 cm-1 (Figure 5). As expected, apatite peaks decreased and gelatin amide I and II peaks (located at 1636 and 1535 cm-1) increased with increasing amount of CaP incorporation. 3.2. Drug Release. Figure 6 shows the release of bFGF from gelB (diamonds), gelB-10%CaP (triangles), and gelB-40%CaP (squares) microspheres, and CaP (circles) into proteolytic SBF. After day 1, a burst release was observed for all gelatincontaining microspheres, whereas this burst effect was absent

Figure 3. EDS spectrum of gelB-40%CaP microspheres.

Figure 4. XRD pattern of gelB-40%CaP microspheres.

Figure 2. Light micrographs of pure gelB (a), gelB-10%CaP (b), and gelB-40%CaP microspheres (c) in swollen state.

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Figure 7. Cumulative Ca deposition onto gelB ((), gelB-10%CaP (9), and gelB-40%CaP (2) microspheres and CaP powder (b) upon soaking in proteolytic SBF at 37 °C.

Figure 5. ATR-FTIR spectra of gelB (top), gelB-10%CaP (middle), and gelB-40%CaP (bottom) microspheres.

Figure 8. Total protein release of gelB ((), gelB-10%CaP (9), and gelB-40%CaP (2) microspheres and CaP powder (b) upon soaking in proteolytic SBF at 37 °C.

Figure 6. In vitro profiles of bFGF release from gelB ((), gelB10%CaP (9), and gelB-40%CaP (2) microspheres and CaP powder (b) upon soaking in proteolytic SBF at 37 °C.

for pure CaP. Release of bFGF from pure CaP proceeded at a lower rate following zero-order kinetics. Compared to CaP-free microspheres, the burst release for CaP-containing microspheres was significantly higher. Between days 1 and 7, release rates from CaP-containing microspheres were considerably lower than CaP-free gelatin microspheres and even comparable to pure CaP. From day 7 onward, the difference in cumulative release between pure gelatin versus gelatin-apatite composite microspheres decreased gradually due to slightly higher release rates for CaP-containing microspheres at prolonged soaking times. 3.3. Calcification and Biodegradation. From Figure 7, it can be clearly concluded that pure gelatin microspheres are not bioactive in vitro because CaP did not nucleate onto these

microspheres. All CaP-containing groups, however, displayed a continuous deposition of CaP, while the amount of deposited CaP increased with increasing CaP incorporation (during microsphere preparation). The corresponding extent of degradation of gelatin microspheres is displayed in Figure 8. Gelatin microspheres degraded almost completely within 7 days of soaking (initial amount of gelatin equal to 2 mg), whereas no protein was released from pure CaP as was expected. Incorporation of CaP into the gelatin microspheres reduced the degradation rate of gelatin, as reflected by the strongly reduced total protein release from both gelB-10%CaP and gelB-40%CaP composite microspheres. This observation was confirmed by SEM analysis of the various microspheres after 7 (left), 14 (middle), and 28 (right) days of soaking, as shown in Figure 9a,b (gelB); c-e (gelB10%CaP); and f-h (gelB-40%CaP). The spherical shape of the original gelB microspheres was hardly recognizable after 7 days (Figure 9a), while all spherical features disappeared at 14 days (Figure 9b). After 28 days, no gelatin remnants could be retrieved from the supernatant, indicating that gelatin degradation was completed. In contrast to pure gelatin microspheres,



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I and II peaks at 1634 and 1535 cm-1 decreased within the first two weeks of soaking, whereas apatitic phosphate absorptions at 562, 600, and 1025 cm-1 displayed a continuous increase in intensity with increasing soaking time.

4. Discussion

Figure 9. Scanning electron micrographs of gelB microspheres after 7 (a) and 14 (b) days of soaking (no gelatin was left after 28 days), gelB-10%CaP after 7 (c), 14 (d), and 28 (e) days of soaking, and gelB-40%CaP after 7 (f), 14 (g), and 28 (h) days of soaking in proteolytic SBF at 37 °C.

Figure 10. ATR-FTIR spectra of gelB-10%CaP microspheres after 0, 7, 14, and 28 days of soaking in proteolytic SBF.

gelB-10%CaP microspheres still revealed remnants of the original spherical shape after 7 days albeit in a fused network of porous microspheres (Figure 9c). After 14 (Figure 9d) and especially 28 days (Figure 9e), gelB-10%CaP microspheres were gradually replaced by a granular powder of irregular morphology. GelB-40%CaP microspheres were clearly recognizable after 7 days without any fusion between the microspheres, as observed for gelB-10%CaP (Figure 9f). After 14 days, large globular particles were still visible for these gelB-40%CaP (Figure 9g), but after 28 days the morphology of the remnants had become entirely granular (Figure 9h). The corresponding ATR-FTIR spectra confirmed the gradual transformation of gelatin-apatite composite microspheres into apatitic CaP powder due to degradation of the gelatin phase and simultaneous apatite precipitation from the SBF. As an example of this chemical transformation, Figure 10 shows the infrared spectra of gelB-10%CaP microspheres before and after soaking for 7, 14, and 28 days. The intensity of gelatin amide

The aim of the current study was to investigate the functional properties of gelatin/apatite composite microspheres to assess their potential use in bone-filling formulations. To this end, the effect of CaP incorporation into gelatin microspheres has been studied in vitro by soaking microspheres loaded with bFGF in proteolytic SBF and monitoring CaP deposition, drug release rates, and protein degradation, as well as the physicochemical characteristics of the microsphere remnants. Generally, the average primary microsphere size was smaller than the sieve aperture size due to occasional clustering of primary particles into small aggregates of 20-32 µm, but excessive aggregation was not observed for neither CaP-free nor CaP-containing microspheres. Incorporation of CaP nanocrystals into gelatin microspheres slightly increased their average primary particle size and decreased their capacity to swell in aqueous solutions due to the introduction of a nonswelling CaP phase into the water-in-oil emulsion. Although the microspheres lost their sphericity upon CaP incorporation, the CaP phase was distributed homogeneously throughout the gelatin matrix as observed using light microscopy (Figure 2). These results are in line with observations made by Kim et al.16 who used TEM analysis of resin-embedded microspheres to show that apatitic nanoparticles were homogeneously distributed throughout the gelatin matrix. The distortion of the spherical shape and increased surface roughness of the composite microspheres are in line with results obtained by Nukavarapu et al.,22 who observed similar trends for polyphosphazene/nanohydroxyapatite composite microspheres. However, these polyphosphaze/hydroxyapatite composite microspheres did not form beyond 30 wt % of apatite loading,22 while gelatin/apatite microspheres have been prepared with maximum mineral contents of up to 20 wt %.16,17 This study, on the contrary, indicates that gelatin microspheres can be loaded with apatite nanocrystals up to a higher percentage of 40 wt %. The water content of 91.5% for pure gelatin microspheres corresponded well with water contents of about 90-93% for gelatin microspheres or sponges crosslinked chemically with different concentrations of glutaraldehyde.10,23 Consequently, it can be concluded that dehydrothermal cross-linking can be used to prepare gelatin microspheres with similar water uptake as chemically cross-linked microspheres, thereby avoiding the use of potentially cytotoxic cross-linkers such as glutaraldehyde. After 4 weeks of soaking, cumulative release of bFGF from pure gelatin microspheres reached up to 80% while total cumulative release percentages of 65-90% (VEGF), 25-100% (BMP-2), and 80% (TGF-β) were reported in literature for different growth factors.23-25 Although the type of gelatin (gelatin B), microsphere fold swelling, collagenase concentration, and isoelectric points of these growth factors (bFGF ) 9.6, VEGF ) 8.6, BMP-2 ) 8.5, and TGF-β ) 9.5) were comparable for all study setups, the resultant release profiles differed considerably. The amount of burst release after one day of soaking, for instance, ranged from about 10% for BMP-2 to 26% for bFGF, 35-60% for VEGF, and 40% for TGF-β, stressing again that release characteristics cannot be explained merely in terms of single parameters such as the isoelectric point or cross-linking density. Tabata et al. observed a burst release

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Figure 11. Light micrographs of un-cross-linked gelB (a), gelB-10%CaP (b), and gelB-40%CaP (c) microspheres after soaking in PBS at 37 °C for 7 days. The dark spots were not material remnants but caused by fixed spots on the microscope lens.

of 30% bFGF due to release of loosely bound bFGF after 1 day of soaking glutaraldehyde-cross-linked gelatin microspheres in collagenase-free PBS,10 which is in agreement with the burst release of 26% observed in the current study. Consequently, the value of 26% release was suggested to be caused mainly by release of loosely bound bFGF. Incorporation of CaP into the gelatin microspheres resulted into a higher burst release (32-34%) than pure gelatin microsphere (26%), which can be explained by the lower water uptake for CaP-containing microspheres. Based on the observed fold swelling ratios (Table 1), it can be calculated that pure gelatin microspheres fully absorbed the total amount of 20 µL of bFGF solution, whereas the composite microspheres did not absorb the entire volume of bFGF-containing solution (93 and 66% for gelB-10%CaP and gelB-40%CaP, respectively). Consequently, this free bFGF did not bind to gelatin and was released instantaneously upon the addition of proteolytic SBF. On the contrary, CaP displayed a very strong affinity for bFGF as reflected by the absence of a burst effect. Protein adsorption onto hydroxyapatite has been shown to be driven mainly by electrostatic interactions between charged -COO- and -NH3+ protein functional groups and oppositely charged ions at the ceramic surface, which account for the total interaction energy for more than 95%.26 Compared to gelatin, which is a polyampholyte having only a limited number of charged amino acid residues along the polymer chain, the number of charged groups and their charge density for ionic solids such as hydroxyapatite is much higher, which explains the strong affinity of proteins such as bFGF for CaP ceramics6 that complicate controlled release of drugs from CaPs. After the initial burst phase, release from CaP-containing microspheres proceeded at a slower rate than from pure gelatin microspheres and reached rates that were as low as bFGF release from pure CaP. Seda Tigh et al.27 also observed an increase in bFGF retention upon incorporation of CaP into chitosan scaffolds. Still, the release profiles of gelB-10%CaP and gelB40%CaP were almost similar. This observation points to the fact that protein release rates from composite microspheres was not just determined by the ratio between organic and inorganic components but influenced by two simultaneously occurring processes, that is, the competing processes of calcification by CaP precipitation and enzymatic degradation of gelatin. Characterization by means of SEM and ATR-FTIR indeed showed a gradual transformation of both composite microspheres into a mixture of nonspherical gelatin remnants and abundant mineral deposition (Figures 9 and 10). Although the scanning electron micrographs of both composite microspheres revealed qualitative differences between the degradation process and the rate of gelB10%CaP and gelB-40%CaP microspheres (Figure 9c-e vs f-h), quantitative differences in protein release between both types of composite microspheres were only marginal (Figure 8). Therefore, it was concluded that release of bFGF was mainly controlled by enzymatic degradation of the gelatin phase because the release and degradation rates of both composite groups were comparable, although the amount of CaP incorporation and subsequent calcification differed considerably.

Regarding this calcification, it was observed that the composite microspheres strongly calcified by attracting abundant precipitation of apatitic mineral. This tendency increased with increasing CaP content, indicating that introduction of only minor amounts of CaP into gelatin microspheres can turn inert, bioinactive gelatin microspheres into highly bioactive microspheres in vitro without compromising their capacity to release proteins. Compared to the amount of apatite deposition onto purely ceramic hydroxyapatite powders after 4 weeks of soaking in SBF (0.85 mg hydroxyapatite as calculated from the amount of Ca deposition), the deposited amount of mineral onto composite microspheres was considerable (0.78 and 0.63 mg for gelB-40%CaP and gelB-10%CaP, respectively), indicating that calcification was not limited to the microsphere surface but occurred throughout the entire interior of the microspheres by influx of calcium and phosphate ions toward the embedded CaP nanocrystals. This mineralization behavior from inside out for swelling hydrogel microspheres can be considered as a significant advantage over conventional composites based on nonswelling polymers. The capacity of cross-linked gelatin to swell considerably in aqueous solutions allows diffusion of calcium and phosphate ions into the interior of water-swollen gelatin/ apatite microspheres, whereas nonswelling polymer/ceramic composites can only be calcified from outside by CaP deposition onto the outer surface. The incorporation of CaP into the microspheres, on the other hand, reduced the degradation rate of gelatin considerably. To test if CaP nanocrystals can decrease the degradation rate of the gelatin microspheres by forming electrostatic interactions between gelatin chains and CaP nanocrystals, CaP-free and both CaP-containing microspheres were soaked into PBS buffer at 37 °C for 7 days without any dehydrothermal cross-linking treatment. Figure 11 shows the resultant light micrographs, which reveal that CaP nanocrystals indeed were able to reduce gelatin dissolution. As expected, un-cross-linked gelatin microspheres were completely dissolved because gelatin dissolves in warm aqueous solutions (Figure 11a), but remnants of composite microspheres were still clearly recognizable after 7 days (Figure 11b,c). Zeta-potential measurements (at pH of 7.4) of the CaP nanoparticle surface charge revealed that neutral CaP nanoparticles (0 mV) became slightly charged upon soaking in dilute solutions of positively charged gelatin type A or negatively charged gelatin type B, yielding ζ-potentials of CaP nanoparticles of +0.9 and -1.3 mV, respectively (measurements performed at pH value of 7.4; unpublished results). This confirms again that hydroxyapatite has a strong adsorption affinity for proteins such as gelatin. Consequently, it can be concluded that the delayed degradation and release of bFGF from composite microspheres was caused in part by electrostatic interactions between CaP nanocrystals and charged amino acid sequences present on gelatin chains. Still, the marginal differences in terms of gelatin degradation and bFGF release for both composite microspheres containing either 10 or 40% of CaP cannot be attributed solely to the stabilizing effect of CaP nanocrystals. Kremer et al. have shown that hydroxyapatite crystals can induce autolytic degradation


and inactivation of matrix metalloproteinases such as MMP1 (interstitial collagenase) and MMP-3 (stromelysin 1).28 Similarly, the introduction of hydroxyapatite nanocrystals into gelatin microspheres might have caused conformational changes to matrix metalloproteinases upon binding to hydroxyapatite crystals, thereby reducing the proteolytic activity of the collagenase, as employed in the current study as well as the corresponding gelatin degradation rate.

5. Conclusion This study has investigated the functional properties of gelatin-apatite composite microspheres for potential use as injectable bone substitute. To this end, CaP-free versus CaPcontaining microspheres were loaded with radiolabeled basic fibroblast growth factor (bFGF) and soaked in proteolytic simulated body fluid (SBF) up to 4 weeks to allow for simultaneous monitoring of their degradation, calcification, and drug release properties. Incorporation of CaP nanocrystals into gelatin microspheres using a simple water-in-oil emulsification method yielded microspheres with reduced biodegradation and drug release rates, whereas their calcifying capacity increased strongly compared to inert CaP-free gelatin microspheres. The simultaneous processes of gelatin degradation and mineral deposition resulted into transformation of the original microspheres into a mixture of featureless gelatin remnants and abundant mineral deposition within 4 weeks of soaking. The reduced drug release rate was correlated to the reduced degradation rate as caused by stabilizing electrostatic interactions between CaP nanocrystals and gelatin chains in the composite matrix phase. By preparing gelatin microspheres containing CaP nanocrystals, gelatin microspheres can be rendered bioactive in vitro without compromising their capacity to release drugs in a controlled manner. Acknowledgment. S.C.G.L. was supported by a VENI fellowship and Grant No. 10700 from the Dutch Technology Foundation STW, Applied Science Division of NWO, and the Technology Program of the Ministry of Economic Affairs.

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