Biomacromolecules 2010, 11, 1470–1479
Three-Dimensional Mineralization of Dense Nanofibrillar Collagen-Bioglass Hybrid Scaffolds Benedetto Marelli,† Chiara E. Ghezzi,† Jake E. Barralet,‡ Aldo R. Boccaccini,§ and Showan N. Nazhat*,† Department of Mining and Materials Engineering, Faculty of Dentistry, McGill University, Montre´al, QC, Canada, H3A 2B2, and Department of Materials Science and Engineering, University of Erlangen-Nuremberg, 91058 Erlangen, Germany Received January 28, 2010; Revised Manuscript Received April 10, 2010
Scaffolds for bone tissue engineering must meet a number of requirements such as biocompatibility, osteoconductivity, osteoinductivity, biodegradability, and appropriate biomechanical properties. A combination of type I collagen and 45S5 Bioglass may meet these requirements, however, little has been demonstrated on the effect of Bioglass on the potential of the collagen nanofibrillar three-dimensional mineralization and its influence on the structural and mechanical properties of the scaffolds. In this work, rapidly fabricated dense collagen-Bioglass hybrid scaffolds were assessed for their potential for immediate implantation. Hybrid scaffolds were conditioned, in vitro, in simulated body fluid (SBF) for up to 14 days and assessed in terms of changes in structural, chemical, and mechanical properties. MicroCT and SEM analyses showed a homogeneous distribution of Bioglass particles in the as-made hybrids. Mineralization was detected at day 1 in SBF, while ATR-FTIR microscopy and XRD revealed the presence of hydroxyl-carbonated apatite on the surface and within the two hybrid scaffolds at days 7 and 14. FTIR and SEM confirmed that the triple helical structure and typical banding pattern of fibrillar collagen was maintained as a function of time in SBF. Principal component analysis executed on ATR-FTIR microscopy revealed that the mineralization extent was a function of both Bioglass content and conditioning time in SBF. Tensile mechanical analysis showed an increase in the elastic modulus and a corresponding decrease in strain at ultimate tensile strength (UTS) as imparted by mineralization of scaffolds as a function of time in SBF and Bioglass content. Change in UTS was affected by Bioglass content. These results suggested the achievement of a hybrid matrix potentially suitable for bone tissue engineering.
Introduction Scaffolds for bone tissue engineering (BTE) should be biocompatible, osteoconductive, osteoinductive, and biodegradable and should possess appropriate biomechanical properties prior to the regeneration of the tissue.1 The fulfillment of all these requirements may result in the direct implantation of scaffolds in vivo to lead to in situ tissue regeneration.2 This is an alternative approach to the in vitro tissue regeneration path, which requires a higher number of operations and, thus, has a longer preparation time preimplantation and a potentially longer healing time for the patient. BTE scaffolds can be made from synthetic or natural polymers.3 While the former is used due to its easier process ability, the latter is the more attractive option due to its similarities with the extracellular matrix (ECM) and its biological performance.4 Naturally derived type I collagen is the most abundant protein of the human body and the main organic component of bone.5 It has a long history as a material for biomedical applications due to its biocompatibility, biodegradability, biological properties, and natural role in tissue formation. Although collagen can be processed in films, sponges, or matrices, the fibrillar scaffold is the most biomimetic form due to its close similarity to the fibrillar ECM.6 However, in vitro reconstituted collagen gel scaffolds present low mechanical * To whom correspondence should be addressed. E-mail: [email protected]
. † Department of Mining and Materials Engineering, McGill University. ‡ Faculty of Dentistry, McGill University. § University of Erlangen-Nuremberg.
properties and low fibrillar density, relative to the native tissues, due to their highly hydrated nature (>99% fluid).6 To overcome this, Brown et al.7 developed a processing technique based on plastic compression (PC) to rapidly fabricate collagen matrices that mimic the ECM fibrillar density, microstructure, and biological properties.8 The mechanical properties of the matrices are significantly enhanced via the controlled increase in nanofibrillar density to greater than 10% in weight.9 Bitar et al. assessed the suitability of the PC dense collagen scaffold for BTE purposes. Osteoid-like dense collagen scaffolds seeded with human osteosarcome cells (ATCC HOS TE85) were capable of maintaining cell viability and function for up to 10 days, with preservation of the scaffold original mechanical properties.10 Furthermore, Buxton et al. cultivated for 14 days primary preosteoblasts in the presence of osteogenic supplements resulting in their mineralization.8 More recently, Pedraza et al. assessed in vitro cellular biomineralization of PC collagen scaffolds with MC3T3-E1 murine calvarial osteoblasts cultivated up to 7 weeks.11 Moreover, Nazhat et al. developed a dense collagen-phosphate glass fiber composite through PC to controllably incorporate aligned microchannels through the scaffold once the glass fibers degraded.12 Bioactive ceramics and glasses are another important category of materials used in BTE and more generally in bone replacement.3 Both are considered biomimetic and bioactive because they stimulate the formation, precipitation, and deposition of calcium phosphates from physiological solution and can result in enhanced bone-matrix interface strength.13 Bioglass 45S5 is osteoinductive and osteoconductive and has been found to
10.1021/bm1001087 2010 American Chemical Society Published on Web 05/05/2010
Mineralization of Collagen-Bioglass Scaffolds
Biomacromolecules, Vol. 11, No. 6, 2010
Table 1. Details of the Various Scaffolds Investigateda
material DC DC-B 60-40 DC-B 40-60
original original equivalent collagen equivalent Bioglass collagen Bioglass wt% collagen wt% Bioglass post vol% in the vol% in the concentration concentration weight loss post freeze-drying freeze-drying scaffold scaffold (mg/mL) (mg/mL) due to PC (%) (%) (%) (%) (%) 1.68 1.68 1.68
0 1.12 2.52
97.86 ( 0.15 95.51 ( 0.43 93.72 ( 0.85
11.27 ( 0.31 5.71 ( 0.44 3.50 ( 0.48
0 3.80 ( 0.34 5.25 ( 0.78
8.27 ( 0.24 4.22 ( 0.40 2.60 ( 0.37
0 1.47 ( 0.14 2.03 ( 0.31
a The Bioglass weight percentage value was calculated after freeze drying using the assumption that no collagen or Bioglass were lost during PC. This hypothesis has been verified by comparing the weight of the freeze-dried residue with the sum of the original collagen and Bioglass content in each gel (data not shown). Equivalent collagen and Bioglass volume percentages were calculated using the rule of mixtures and implementing density values of 1.41 and 2.7 g/cm3, respectively.
up-regulate the gene expression that controls osteogenesis and production of growth factors.14 Its network forming SiO2 content of 45 wt % exhibits a high bioactivity index (region A of the subdivision made by Hench and co-workers15). It bonds to both soft and hard tissues.16 To combine the advantages and minimize the drawbacks intrinsicineachmaterialchoice,thedevelopmentofpolymer-ceramic hybrids has been proposed17 of which type I collagen scaffolds incorporated with Bioglass particles is one approach. While attempts have been made to implant a hydrogel composed of collagen and Bioglass particles,18 until recently this remained an isolated study.19,20 More significantly, however, little has been demonstrated on the potential effect of Bioglass on the collagen type I network in terms of accelerating the mineralization in three-dimension and, in turn, the effect of mineralization on the structural and mechanical properties of the biomimetic nanofibrillar-based scaffolds. Therefore, the aim of this study was to investigate the effect of Bioglass on the mineralization extent of dense nanofibrillar collagen gel scaffolds when conditioned in simulated body fluid (SBF). Microcomputed tomography (microCT) and scanning electron microscopy (SEM) were used to characterize the scaffold morphology, as made, and during conditioning, with a particular focus on the calcium phosphate distribution within the gel scaffolds. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and microscopy and X-ray diffraction (XRD) were used to investigate the nucleation and growth of calcium phosphate on the surface and within the scaffolds and to assess the effect of the mineralization on the protein structure (triple helix and banding pattern). Furthermore, mechanical tensile analysis was performed to investigate the changes of the mechanical properties due to the biomineralization of the scaffolds.
Materials and Methods Preparation of Dense Collagen and Dense Collagen-Bioglass Hybrid Scaffolds. Dense collagen scaffolds (DC) were prepared using the PC technique, as described previously.7 In brief, 1.6 mL of rat tail tendons type I collagen (2.11 mg/mL, in 0.6% acetic acid; First Link Ltd., West Midlands, U.K.) was neutralized with 0.4 mL of 10 times concentrated Dulbecco modified Eagle medium (10× DMEM; Sigma Aldrich, Canada) and 37 µm of 5 M NaOH (Fisher Scientific, Canada). The solution was then cast in a circular mold (L ) 12 mm) and incubated at 37 °C for 25 min. The gel was then gently removed from the casting chamber and compressed using 1k Nm2- for 5 min in combination with blotting. Dense collagen-Bioglass (DC-B) hybrid scaffolds were produced at two relative 45S5 Bioglass contents: collagen-Bioglass 60-40 and 40-60 dry weight % (DC-B 60-40 and DC-B 40-60, respectively). These dense collagen-Bioglass concentrations were chosen based on literature findings.21 Bioglass particles of 5 µm diameter were incorporated by their addition to the 10× DMEM-5 M NaOH solution and then followed by sonication
(Ultrasonic Bath Model #2210, Branson, Danbury, CT) for 3 min before placing the collagen in the final mixture. By assuming that no collagen or Bioglass was lost during PC, the weight percent (wt%) of collagen and Bioglass in the scaffolds after PC was calculated by weighing the scaffolds before and after freezedrying (BenchTop K, VirTis, Canada; n ) 5). Table 1 summarizes the details of the hybrid scaffolds. The efficacy of PC was evaluated by measuring the weight loss. This hypothesis has been verified by comparing the freeze-dried residue weight with the sum of the original collagen and Bioglass content in each scaffold. The equivalent volume percentages (vol%) of collagen and Bioglass in the scaffold were obtained using the density values of 1.41 and 2.7 g/cm3 for collagen22 and Bioglass,23 respectively. Mineralization of Dense Collagen-Bioglass Scaffolds in SBF. Kokubo’s SBF24 was used to investigate the mineralization potential of DC and DC-B hybrid scaffolds. A comparison was made with dense collagen gels incorporated with alumina and phosphate-based glass particulates (in molar %: 50 P2O5, 40 CaO, 10 Fe2O3; Supporting Information, Figures 1S and 2S, respectively). To standardize the bioactivity investigation through SBF, samples (n ) 5) were conditioned in 50 mL of SBF at 37 °C and were assessed at days 1, 3, 7, and 14. SBF was prepared fresh and changed at two-day intervals. Morphological Characterization. DC and DC-B scaffolds, as made, and at days 3, 7, and 14 in SBF, were morphologically characterized through microCT and SEM. For microCT analysis, the samples were frozen with liquid nitrogen for 3 min and then freeze-dried for 36 h at -105 °C and 16 mTorr (BenchTop K freeze-dryer). The microCT analysis was performed with a SkyScan 1172 (SkyScan, Kontich, Belgium). Samples were analyzed through a 360° flat-field corrected scan at 40 kV and 250 µA, with a step size of 0.28°, a resolution of 3.5 µm, a large camera pixel, and no filter. The volumetric reconstruction (NRecon software, SkyScan) was set with a beam hardening correction of 10, a ring artifact correction of 20 and an “auto” misalignment correction. The 2D analysis (software CTAn, SkyScan) was carried out using a grayscale intensity range of 20-255 (8 bit images) to remove background noise. For each sample, thickness and volume measurement were taken using the proper functions in CTAn software. The collagen phase was set among an intensity range of 20-45 (data obtained from CT analysis of the as made DC scaffold), while the Bioglass and mineral phase was set over 45. This criterion allowed the 3D reconstruction and the visualization of the different phases using CTVol software, Skyscan. The collagen was highlighted in light gray, while the denser phase was highlighted in orange. For SEM, scaffolds were fixed with a 4% glutaraldehyde 0.1 M sodium cacodylate solution overnight at 4 °C. The samples were then washed with deionized distilled water and dried at 4 °C through a graded series of ethanol solutions. To maintain collagen triple helical structure, samples were subsequently dried with a Ladd critical point dryer (Ladd Research Industries, Burlington, VT). Samples were then sputter-coated with Au/Pd (Hummer VI Sputter Coater, Ladd Research Industries). The SEM analysis was performed with a S-4700 Field Emission-STEM (Hitachi, Japan) at 2 kV and 10 µA.
Biomacromolecules, Vol. 11, No. 6, 2010
Chemical Characterizations. Chemical characterizations were carried out with ATR-FTIR spectroscopy and microscopy and complemented by XRD. Samples were freeze-dried (see above) and analyzed on their outer surface as well as through their cross section. For ATR-FTIR spectroscopy, a Spectrum 400 (Perkin-Elmer, Waltham, MA) equipped with a single bounce ZnSe diamond-coated ATR crystal was used. A resolution of 2 cm-1, an infrared range of 4000-650 cm-1, and 64 scans per sample were used. The collected spectra were corrected with a linear baseline and normalized (absorbance of amide I at 1643 cm-1 ) 1.5) using Spectrum software (PerkinElmer, Waltham, MA). ATR-FTIR microscopy (Spotlight 400, Perkin-Elmer, Waltham, MA) was used to scan the cross section of the DC-B scaffolds and to investigate the presence and the distribution of Bioglass and calcium phosphates post SBF conditioning. The samples were carefully cut in approximately 1 mm wide strips with a width (using a microscopy sample preparation kit, Perkin-Elmer) and mounted on the sample holder so as to allow the sample cross section to be scanned. An area of 250 × 200 µm2 was scanned with a spatial resolution (spot size) of 1.25 µm2 and a resolution of 8 cm-1 at 4 scans/pixel. The images collected were postprocessed with principal component analysis (PCA) to highlight the presence of Bioglass (PO43- and Si-O-Si peak at 1000-1010 cm-1 and Si-O nonbounding oxygen peak at 905-920 cm-1) and calcium phosphate (PO43- V3 peak at 1017 cm-1). Furthermore, ATR-FTIR microscopy was used to carry out a semiquantitative analysis of the scaffold mineralization. The average PO43- V3 peak over amide I peak ratio (absorbance at 1017 cm-1/absorbance at 1643 cm-1) was calculated through five different images per hybrid scaffold (DC, DC-B 60-40 and 40-60 at days 7 and 14 in SBF). XRD spectra of all materials were recorded with a Bruker D8 Discover from 6 to 60° 2θ at 40 kV, 20 mA. Two frames of 30° were recorded for 15 min and then merged during the data post processing. Sample surfaces and cross sections were analyzed with two different pinhole sizes of 0.5 and 0.05 mm, respectively. The XRD spectra was analyzed with EVA software (Bruker) correcting the baseline (threshold ) 0.750). The phase composition was determined by comparing acquired spectra with peaks identified in the International Centre for Diffraction Data (ICDD) database. Characterization of the Mechanical Properties. Tensile mechanical analysis was carried out on spiral assemblies of all materials, as made.7 While the analysis of DC scaffolds was maintained in spiral form, strips of the hybrid scaffolds were analyzed to assess the potential effect of mineralization due to SBF conditioning on their mechanical properties. This was carried out to accommodate gripping restrictions. All the specimens were cast by gelling 4 mL collagen solutions into rectangular molds of 18 × 43 mm2 bottom surface area. For the spirals, after compression, the rectangular sheets were rolled along the long axis and halved to give cylindrical shaped specimens of 1.5 ( 0.1 mm diameter. For the strips, the compressed sheets were halved along the short axis and then folded once along the long axis to give specimens of 4.5 ( 0.1 width and 21.5 ( 0.5 mm length. Specimen thickness was sample dependent and was 200 ( 50 µm. All dimensional measurements were verified by microCT. As made, specimens were then characterized after 30 min of preconditioning in DMEM. The effect of SBF conditioning on the mechanical properties was assessed at days 7 and 14. Mechanical characterization was achieved on three repeat specimens using an ElectroForce Biodynamic Test Instrument 5160 (Bose Corp., MN) with a 15 N load cell and ad hoc modified grips. The system was used in displacement control and a rate of 0.01 mm s-1 was applied. During the test, SBF was added in a dropwise manner to maintain specimen hydration. Raw data were then post processed using Excel 2007 software (Microsoft Corp, WA). Statistical Analysis. Data of the ATR-FTIR microscopy semiquantitative analysis and mechanical tests were statistically compared with two way ANOVA tests using a two-way Anova tool (significance level ) 0.05 and Bonferroni means comparison) and Origin Pro v.8 software (OriginLab, MA).
Marelli et al.
Results Morphological Characterization. Table 1 gives the details of the various materials used in this study. Weight loss due to PC decreased with increasing Bioglass content. This decrease of PC efficacy led to a decrease in collagen fibrillar collagen density in the scaffolds. Figure 1 compares the macro- and micro- morphology of the DC and DC-B hybrid scaffolds. The morphologies of DC-B 60-40 and DC-B 40-60 were comparable, thus, only the analysis performed on DC-B 40-60 is presented. The morphological and structural characterizations of DC-B 60-40 are presented in Supporting Information (Figure S3). The 2D analysis of the samples revealed a thickness of 34 ( 12, 61 ( 17, and 65 ( 16 µm for DC, DC-B 60-40, and DC-B 40-60, respectively. Figure 1a and b are microCT 3D reconstructions of DC and DC-B 40-60, respectively. The 3D reconstruction of DC gave a homogeneous image and SEM confirmed the randomly arranged collagen nanofibrils exhibiting the typical banding pattern (Figure 1c,e,g). The orange zones (more dense) in Figure 1b represent areas with a higher presence of Bioglass. Figure 1d shows the Bioglass distribution at higher resolution, while Figure 1f and h show the detail of the surface of two Bioglass particles, indicating no specific interactions with the collagen fibrillar matrix. Morphological changes in DC-B scaffolds with conditioning time in SBF are illustrated in Figure 2. The 3D microCT reconstructions [Figure 2a (i, ii, iii)] of DC-B 40-60 as-made and at days 7 and 14 in SBF showed an increase in thickness with conditioning time, which were quantified by 2D analysis [Figure 2a (iv, v, vi)]. Moreover, the microCT images indicated the presence of a denser phase along their cross section (prevalence of orange zones in the middle of the reconstructed image). At day 14 in SBF, the presence of micropores was also indicated with a pore size ranging from 5 to 10 µm [black spot in Figure 2a (vi)]. SEM micrographs of DC-B hybrids at day 3 conditioning in SBF showed the nucleation of crystals [Figure 2b (i)]. Moreover, the surface of the Bioglass became rougher, suggesting surface reactions. The presence and growth of crystals on the surface of the hybrid scaffolds were confirmed by SEM micrographs at days 7 and 14 days in SBF [Figure 2b (ii and iii, respectively)]. In addition, SEM micrographs [Figure 2b (iv)] of the cross section of the hybrid scaffolds at day 14 confirmed the presence of these crystals within the bulk as well as open pores of similar range in size as indicated by microCT. A lower extent of crystal growth was found in DC at day 14 in SBF [Figure 2b (vi)]. Chemical Characterization. Qualitatively, both hybrid scaffolds presented similar results, therefore, only the results of DC-B 40-60 samples are given (Figure 3), except for the semiquantitative analysis illustrated in Figure 3e. ATR-FTIR spectroscopy on as-made DC-B scaffolds confirmed the presence of Bioglass within the collagen matrix (Figure 3a). FTIR spectra of DC-B scaffolds differed slightly from that of Bioglass alone. Bioglass exhibited a carbonate V3 peak at 1454 cm-1 and four peaks in the region between 1200 and 700 cm-1. Because the attribution of the peaks in this region is difficult due to the overlap of the absorbance of Si-O-Si and PO43- groups, the peak at 998 cm-1 can be attributed to both phosphate and silicate.25 Moreover, the peak at 906 cm-1 could be due to the vibration of Si-O nonbonding oxygen (NBO).26 While a weak peak at 867 cm-1 was present, its attribution is difficult due to double absorbance of CO32- V2 and HPO42- ions at this wavelength.27 Finally, the δ Si-O-Si shaped the weak absorbance peak at 740 cm-1.26 In DC-B scaffolds, all three Amide
Mineralization of Collagen-Bioglass Scaffolds
Biomacromolecules, Vol. 11, No. 6, 2010
Figure 1. Morphological analysis of the as-made DC and DC-B 40-60. MicroCT 3D reconstruction of (a) DC and (b) DC-B 40-60. Areas of higher Bioglass content (higher density) are highlighted in orange. SEM micrographs of (c, e, g) DC at increasing magnifications and of (d, f, h) DC-B 40-60 at increasing magnification.
peaks that are typical of the collagen protein are visible: amide I, II, and III peaks at 1643, 1550, and 1243 cm-1, respectively.28 The carbonate V3 peak of the Bioglass component of the hybrid scaffold was summed to the carbonate peak of the collagen side chains. The peak at 998 cm-1 registered in the spectra of Bioglass alone shifted to 1010 cm-1. Furthermore, the peak at 906 cm-1 showed a blue shift to 920 cm-1, which may indicate a lower amount of NBO26 and the formation of Si-OH bonds29 due to the interactions between Bioglass and water. Bioglass alone had a peak at 740 cm-1, which was absent in DC-B scaffolds. Two new peaks at 730 and 700 cm-1 were formed. As a function of conditioning time in SBF, the spectra of DC-B scaffolds showed an increase in the absorbance of the phosphate peaks (V3 at 1078 and 1017 cm-1 and V1 at 961 cm-1) and of the carbonate peaks (V3 at 1454 cm-1 and V2 at 873 cm-1; Figure 3b).27 A time-dependent increase of the Si-O-Si peaks absorbance (V1 at 1100 cm-1 and δ at 803 cm-1) was also observed.26 X-ray diffraction spectra of DC-B 40-60 showed the passage from an amorphous to a more crystalline scaffold with increasing
conditioning time in SBF (Figure 3c). The amorphous Bioglass structure presented a broad peak between 28 and 34° 2θ. Another broad peak between 10 and 25° is visible in the DC-B as-made scaffold, which corresponds to the unordered components of the collagen nanofibrils.30 With increasing time in SBF, however, the intensity of the main peaks of hydroxyapatite (HA) became more apparent. In particular, at day 1, the 002 reflection at 25.80°, the main 211 reflection at 31.68°, and the 300 reflection at 32.80° appeared [ICDD 9-432]. A weak reflection at 31°, typical of β-tricalcium phosphate (β-TCP), was also visible [ICCD 9-169]. However, this reflection disappeared by day 3 in SBF, suggesting the conversion of β-TCP to HA. At day 14 in SBF, the majority of the reflections of HA were identifiable (Figure 3d). ATR-FTIR microscopy semiquantitative analysis (Figure 3 e) confirmed that the ratio of phosphate V3 to amide I peaks (1017/1643 cm-1) had increased with conditioning time in SBF in all materials. In as made DC scaffold, the ratio was measured at 0.35 ( 0.08 (data not shown), which increased to 0.44 ( 0.06 and 0.61 ( 0.09 at days 7 and 14 in SBF, respectively. In
Biomacromolecules, Vol. 11, No. 6, 2010
Marelli et al.
Figure 2. Morphological analysis of DC-B 40-60 scaffolds at different time points in SBF. (a) MicroCT 3D reconstruction at days (i) 0, (ii) 7, and (iii) 14. Orange indicates higher density regions. In the as-made material they represent the presence of Bioglass, while at days 7 and 14 in SBF, they represent zones of mineral formation. (iv, v, vi) microCT scan of DC-B 40-60, as made, and at days 7 and 14 in SBF, respectively. The lighter the gray, the higher the density. 2D analysis also permitted the evaluation of scaffold expansion through increase in the crosssectional thickness (t). (b) SEM micrographs of DC-B 40-60 hybrid scaffolds and DC. Images i, ii, and iii refer to days 3, 7, and 14 in SBF, respectively, and iv and v refer to SEM micrographs of the cross section of DC-B 40-60 at day 14 in SBF, confirming the presence of pores (dimension = 5-10 µm). (vi) SEM micrograph of the DC after 14 days in SBF.
the hybrid scaffolds, the ratio significantly increased as a function of both Bioglass content and time in SBF. Moreover, the interaction between time in SBF and Bioglass concentration was to be considered significant (p < 0.05). The Bonferroni means comparison revealed that the average ratio values were relatively statistically significantly different (p < 0.05). Cross-sectional investigations of DC-B 40-60 hybrid scaffolds, as-made and at days 7 and 14 in SBF, are presented in Figure 4. In the PCA of the as-made DC-B 40-60 (image in Figure 4a), Bioglass is highlighted in light gray, while collagen matrix is indicated by the darker regions. Spectrum C presented a higher absorbance in the 1200-800 cm-1 region due to the vibrations of Si-O-Si and phosphate groups present in Bioglass. PCA of DC-B scaffolds at days 7 and 14 in SBF highlighted the presence of calcium phosphate in light gray (Figure 4c,e). The lighter the gray, the higher the absorbance of PO43- ions, as shown in the corresponding illustrative spectra in Figure 4c and e, respectively. Moreover, the black dots present in the DC-B 40-60 image at day 14 in SBF (Figure 4e) are due to the loose contact between ATR crystal and sample
surface, probably caused by the presence of pores [Figure 2b (v)]. Cross-sectional XRD analysis of the hybrids confirmed the presence of HA within the matrix. Mechanical Analysis. The results of the tensile tests are illustrated in Figure 5. Representative stress-strain curves for DC, DC-B 60-40, and DC-B 40-60 scaffolds as-made and at days 7 and 14 in SBF are shown in Figure 5a, b, and c, respectively. In all curves, three regions were identified: a nonlinear stress-strain response at low strain (toe region), a linear region, where the elastic modulus was calculated, and a failure region, where the ultimate tensile strength (UTS) was identified. The elastic modulus increased with conditioning time in SBF and increasing content of Bioglass (Figure 5d). In the as made hybrid scaffolds, Bioglass incorporation resulted in an increase in UTS when compared to DC alone. However, there was no statistical difference (p > 0.05) between the strain at UTS of all as-made materials. With conditioning time in SBF, both the UTS (except for DC) and strain at UTS were found to decrease (Figure 5e and f, respectively).
Mineralization of Collagen-Bioglass Scaffolds
Biomacromolecules, Vol. 11, No. 6, 2010
Figure 3. Chemical characterization of the surface of DC-B 40-60 at different time points in SBF. (a) ATR-FTIR spectroscopy of the as made DC-B 40-60, DC, and Bioglass and (b) ATR-FTIR spectroscopy of DC-B 40-60 at days 1, 3, 7, and 14 in SBF. Longer conditioning time in SBF led to an increase in the phosphate v3 peak at 1017 cm-1, which is typical of hydroxyapatite. (c) XRD of DC-B 40-60 at days 1, 3, 7, and 14 in SBF. Longer conditioning time in SBF led to greater crystallinity. (d) XRD spectrum of DC-B 40-60 at day 14 in SBF and its match with diffraction data of hydroxyapatite. All hydroxyapatite major peaks matched the mineralized hybrids. (e) Semiquantitative analysis (ratio of phosphate v3 peak at 1017 cm-1 to the amide I peak at 1643 cm-1) in DC and DC-B 60-40 and 40-60 at days 7 and 14 in SBF via ATR-FTIR microscopy. The ratio was calculated as the average of the mean value of three images (200 × 200 µm, spatial resolution ) 1.25 µm2) and a total of 96000 spectra per sample. As from two-way ANOVA test results, the population means of time in SBF and Bioglass concentration were statistically different. Bonferroni means comparison revealed that the average ratio values were relatively significant statistically different (p < 0.05).
Discussion Current tissue engineering approaches involve the conversion of a cell-seeded synthetic scaffold into a tissue-like structure through the cellular production of ECM. ECM biomineralization is an essential additional step that is required in the tissue engineering of bone. These cell-reliant processes are often slow, requiring weeks in culture, and the biochemical and mechanical factors in inducing optimal outcomes are not fully defined. In this study, the incorporation of 45S5 Bioglass particles into rapidly fabricated dense fibrillar type I collagen matrices is hypothesized to enhance the 3D biomineralization of the scaffolds. Dense, biomimetic collagen scaffolds produced
through plastic compression exhibit similar characteristics to the ECM in terms of their nanofibrillar structures and bulk density levels. Moreover, the mechanical properties of these scaffolds have been reported to be similar to those of an osteoid, a nonmineralized bone matrix.8 The potential of this 3D environment as a bone tissue engineering scaffold has previously been assessed in terms of its ability to be remodelled by cells, its induction of osteoblastic differentiation and its subsequent mineralization.8,10,11 This work investigated the mineralization potential of these scaffolds through an acellular approach via the incorporation of Bioglass, and was assessed, in vitro, by conditioning in SBF.24 Although the use of SBF to test
Biomacromolecules, Vol. 11, No. 6, 2010
Marelli et al.
Figure 4. Chemical characterization of a cross-section of DC-B 40-60 as-made and at days 7 and 14 in SBF. Left column: ATR-FTIR microscopy spectra refer of the relative area shown in the image insert of (a) the scaffolds as-made and (c and e) at days 7 and 14 in SBF, respectively. The lighter the gray, the higher the presence of Bioglass (in as-made scaffolds) and hydroxyapatite (in conditioned scaffolds). Right column: XRD pattern of the cross sections of the scaffolds (b) as-made, (d) day 7, and (f) day 14 in SBF.
bioactivity was recently questioned by Bohner and Lemaitre,31 in this study, Kokubo SBF24 was used and strictly reported to allow for the correlation of its findings with others in literature. It was used to prove the enhanced bioactivity of dense collagen-Bioglass hybrid gel compared to dense collagen alone and when incorporated with either alumina or phosphate-based glass (Supporting Information). A successful outcome of this investigation may expedite the production of readily implantable scaffolds. The rapid implantation of cell incorporated dense collagen scaffold has been carried out by Mudera et al (2007).32 Scaffolds were implanted in an in vivo nursery site for potential applications in tendon regeneration. Bioglass particles were homogeneously incorporated within the DC scaffolds without affecting collagen fibril formation, as confirmed by the presence of the typical collagen banding pattern at high SEM images,33 and by the amide I absorbance
peak value at 1643 cm-1 (ATR-FTIR spectra) that is typical of a nondenatured collagen exhibiting the triple helical structure. The incorporation of Bioglass resulted in a more hydrated scaffold due to its hydrophilic nature and reduced the weight loss due to plastic compression. There was an increase in the initial tensile strength of the scaffolds as a result of the particulate inclusions suggesting that the integrity of collagen fibrillar matrix was maintained in dense collagen-Bioglass scaffolds. The strain at UTS value was maintained while the elastic modulus underwent a statistically significant increase with Bioglass content (EDC ) 2.02 ( 0.04, EDC-B 60-40 ) 2.11 ( 0.12, EDC-B 60-40 ) 2.24 ( 0.07). Conditioning the hybrid scaffolds in SBF confirmed their potential for rapid biomineralization. In contrast, the incorporation of alumina and phosphate based glass did not demonstrate any mineralization (Supporting Information, Figures 1S and 2S).
Mineralization of Collagen-Bioglass Scaffolds
Biomacromolecules, Vol. 11, No. 6, 2010
Figure 5. Tensile mechanical analysis of DC and hybrid scaffolds. Representative curves of the stress-strain relationship for DC, DC-B 60-40, and DC-B 40-60 gels as-made (a), at day 7 (b), and at day 14 in SBF (c), respectively. (d) Elastic modulus, (e) strain at ultimate tensile strength (UTS), and (f) UTS for the different materials. Two-way ANOVA statistical analysis showed that the elastic modulus, the strain at UTS and UTS population means of time in SBF and Bioglass concentration were statistically different. (The relative Bonferroni means comparison is highlighted as follows: Op > 0.05, (p > 0.05, *p > 0.05, bp > 0.05, 9p > 0.05, +p > 0.05.)
SEM revealed the presence of crystals as early as in day 3 in SBF, and ATR-FTIR spectroscopy and XRD confirmed these crystals to be calcium phosphate based. MicroCT 3D reconstruction of the hybrids revealed the formation and growth of a denser phase on the surface and within the scaffolds when conditioned for longer time periods in SBF. The nucleation and growth of calcium phosphate crystals within the collagen nanofibrils caused a gradual expansion of the scaffolds, an effect that was not registered for the DC alone. ATR-FTIR spectra of mineralized scaffolds demonstrated that the shape and the value of the phosphate V3 peak at 1017 cm-1 was compatible with the presence of hydroxyapatite in the scaffolds. The broad XRD peaks, confirmed a low crystalline form of hydroxyapatite34 that is typical of the precipitated form and resembling biological hydroxyapatite.35 Moreover, the presence of the carbonate V2 peak at 873 cm-1 suggested hydroxyl-carbonated apatite (HCA).36 Besides, the increase of the Si-O-Si absorbance peaks (V1 at 1100 cm-1 and δ at 803 cm-1) indicated the presence of SiO2 layers deposited on collagen fibrils. Particularly, the Si-O-Si δ vibration is typical of siloxane methyl rocking mode (Si-CH3)2. ATR-FTIR microscopy allowed for the detailed assessment of the scaffolds’ cross sections. At later time points in SBF, HCA was confirmed as the composition of mineralized phase within the scaffolds. Moreover, semiquantitative analysis on ATR microscopy images of the hybrid scaffolds revealed a 4-fold increase in the ratio of phosphate V3 to amide I as a function of time and Bioglass content. In comparison, DC scaffolds provided a slight but statistically significant increase in this ratio. It has previously been shown that the rate of HCA formation on 45S5 Bioglass depends on its concentration in SBF solution.37 Furthermore, Bioglass incorporated in synthetic polymers, such as poly(DL-lactide) foams, induced HA nucleation, and growth after one week of conditioning in SBF38 or after two weeks of immersion in PBS.39 Analysis of the hybrid scaffolds at day 14 in SBF indicated the presence of pores of similar dimensions to that of Bioglass particles. The presence of Bioglass particles was confirmed by SEM and detected by FTIR up to day 3 in SBF (phosphate V3 peak distorted by the presence of Si-O-Si bonds), but not at
days 7 and 14. The increase in Si-O-Si V1 absorbencies with time in SBF, on the other hand, suggested the formation of glassy Me+Si2O5 (where Me+ ) K+, Na+)40 as a consequence of Bioglass degradation. The presence of open pores inside the so-formed mineralized matrix may be important in terms of facilitating the metabolite and catabolite diffusion through the dense matrix. As expected, the mineralization of the neat dense collagen scaffold resulted in an increase in its modulus as has been previously reported.8 This was accompanied by a significant decrease in the strain at UTS. However, there was no significant change in the UTS of these scaffolds as a function of time in SBF. This effect was in contrast to DC-B 60-40 and DC-B 40-60 scaffolds, which resulted in a significant reduction in this parameter as a function of time in SBF. The upturn of the UTS of the scaffold with a higher Bioglass at day 14 in SBF content is interesting and is hypothesized to be due to the mineralization extent of this material to be high enough for the HCA crystals to interact with each other, limiting the damage to collagen matrix. The respective significant increase and decrease in the elastic modulus and strain at UTS in these hybrid scaffolds with time in SBF confirmed the ductile-brittle transition in these scaffolds with the continual growth of HCA. In this study, the well-characterized surface dissolution behavior of Bioglass led to the release of Si-O-Si and Si-OH groups and resulted in the subsequent migration and deposition of Ca2+ and PO43- ions. This enhanced the nucleation and growth of HCA within the dense collagen matrix without altering the triple helical structure of the protein. It is known that the mineralization of collagen is one of the main prerequisites to developing successful collagen-based scaffolds for bone tissue engineering.41 Nevertheless, efforts conducted to understand and reproduce, in vitro, the biomineralization process and, in particular, the acellular realization of biomineralized collagen scaffolds that mimic the chemical, biological and mechanical properties of bone seem to be far from being fulfilled.42 Moreover, the in vitro imitation of the unique in vivo mineralization process is currently not fully understood.43 While a number of scenarios have been previously attempted in the acellular induction of collagen mineralization, the
Biomacromolecules, Vol. 11, No. 6, 2010
outcomes have been variable. These approaches include the alternate soaking of collagen structures in the form of films, matrices, sponges, as well as scaffolds in calcium and phosphate solutions,44 or the direct titration of calcium-phosphate solutions on the collagen scaffold.45 Both of these approaches could compromise the structure of collagen as they require environments outside physiological boundaries. Alternatively, SBF has been used to induce hydroxyapatite nucleation and growth on phosphorilated collagen46 and collagen sponges.47 In either way, the use of collagen sponges or chemically treated collagen may lead to similar collagen denaturation processes. In addition, the use of non collagenous proteins such as alkaline phosphatase and bone sialoprotein that are involved in the ECM biomineralization steps have also been pursued to enhance the mineralization of collagen matrices. However, the collagen structures used were 2D matrices48,49 or 3D collagen matrices undergoing physicochemical process such as methanol/water dry or UV irradiation.50 More recently, a three component material of silicacollagen-calcium phosphate cement was developed.51 The composite resulted in a bioactive scaffold with modifiable mechanical properties. Although the silication of collagen is a promising technique in terms of enhancing its bioactivity, the effects on collagen structures, suprastructure and biological properties needs further investigations. In this scenario, the use of 45S5 Bioglass as a biocompatible, osteoinductive, and osteoconductive mineralization enhancer for dense collagen gels is a promising and unexplored strategy. Furthermore, the plastic compression of highly hydrated collagen gels allowed the investigation of a unique dense fibrillar collagen gel which mimics the fibrillar and hydrated nature of the ECM and is distinct from other forms of collagen. The combination of the plastic compression of collagen and Bioglass resulted in the biomineralization of the scaffolds without altering the natural and physiological structure of the protein. Although it has been demonstrated that Bioglass 45S5 does not activate macrophages either in vivo or in vitro52 and thus should not instigate or enhance inflammatory responses at the site of glass insertion, further investigations concerning the macrophage response are a prerequisite to assess the use of the DC-B hybrids for in vivo bone tissue engineering.
Conclusion In this study, the combination of dense nanofibrillar collagen scaffolds and Bioglass 45S5 was successfully used to produce scaffolds for potential bone tissue engineering purposes. The plastic compression technique resulted in the rapid production of the hybrid scaffolds, while the presence of 45S5 Bioglass within dense collagenous “osteoid-like” environment resulted in an accelerated three-dimensional mineralization of the matrix when exposed to an in vitro physiological environment. Hybrid scaffolds biomineralized as early as day 1 in SBF. The nucleation and growth of calcium phosphates were characteristic of low crystalline hydroxyl-carbonated apatite. Semiquantitatively, the mineralization extent significantly increased with conditioning time in SBF without altering both the collagen triple helix and fibrillar nature. Tensilemechanicalanalysisontheasmadedensecollagen-Bioglass specimen revealed mechanical properties similar to the viscoelastic dense collagen scaffold. In spite of this, at days 7 and 14 conditioning in SBF, it was possible to relate the progression of mineralization with changes in the scaffolds mechanical properties. DC-B mineralized scaffolds demonstrated a ductilebrittle transition behavior through an increase in the elastic
Marelli et al.
modulus and a corresponding decrease in the strain at UTS. This increase in the mechanical properties was found to be dependent on Bioglass content and conditioning time in SBF. The results suggested the formation of a scaffold that is suitable for bone tissue engineering purposes. Acknowledgment. The funding of the Canadian Natural Sciences and Engineering Research Council for Strategic Research Grant No. 350725-07 (with BioSyntech as partner), Discovery Grant No. RGPIN 341235-2007, and the Canadian Foundation for Innovation, Leaders Opportunity Funds Project No. 13054 are gratefully acknowledged. Funding for Benedetto Marelli is also supported by the Werner Groupe Fellowship and McGill Engineering Doctoral Award. Funding for Chiara E. Ghezzi is also supported by Showan Nazhat’s Hatch Faculty Fellowship and McGill Principal’s Graduate Fellowship. Supporting Information Available. Investigations of the bioactivityofdensecollagen-alumina-anddensecollagen-phosphatebased glass hybrid gels in SBF. A brief description of the experiment rationale, materials and methods, results, and discussion (Figures 1S and 2S). Biomineralization of DC-B 60-40 hybrid gels at day 14 in SBF. Figure 3S shows the morphological and structural biomineralization of DC-B 60-40 gels at day 14 in SBF. The interested reader should access this information to compare the effects of the incorporation of other microparticles on the bioactivity of dense collagen gels and to compare the effect of different Bioglass concentration on the carbonated hydroxyapatite nucleation and growth. This material is available free of charge via the Internet at http://pubs.acs.org.
References and Notes (1) Mohamad Yunos, D.; Bretcanu, O.; Boccaccini, A. R. Polymerbioceramic composites for tissue engineering scaffolds. J. Mater. Sci. 2008, 43 (13), 4433–4442. (2) Zdrahala, R. J.; drahala, I. J. In vivo tissue engineering: Part I. Concept genesis and guidelines for its realization. J. Biomater. Appl. 1999, 14 (2), 192–209. (3) Rezwan, K.; Chen, Q. Z.; Blaker, J. J.; Boccaccini, A. R. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 2006, 27 (18), 3413–3431. (4) Mano, J. F.; Silva, G. A.; Azevedo, H. S.; Malafaya, P. B.; Sousa, R. A.; Silva, S. S.; Boesel, L. F.; Oliveira, J. M.; Santos, T. C.; Marques, A. P.; Neves, N. M.; Reis, R. L. Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends. J. R. Soc. Interface 2007, 4 (17), 999–1030. (5) Fratzl, P.; Gupta, H. S.; Paschalis, E. P.; Roschger, P. Structure and mechanical quality of the collagen-mineral nano-composite in bone. J. Mater. Chem. 2004, 14 (14), 2215–2123. (6) Lee, C. H.; Singla, A.; Lee, Y. Biomedical applications of collagen. Int. J. Pharm. 2001, 221 (1-2), 1–22. (7) Brown, R. A.; Wiseman, M.; Chuo, C. B.; Cheema, U.; Nazhat, S. N. Ultrarapid engineering of biomimetic materials and tissues: Fabrication of nano- and microstructures by plastic compression. AdV. Funct. Mater. 2005, 15 (11), 1762–1770. (8) Buxton, P. G.; Bitar, M.; Gellynck, K.; Parkar, M.; Brown, R. A.; Young, A. M.; Knowles, J. C.; Nazhat, S. N. Dense collagen matrix accelerates osteogenic differentiation and rescues the apoptotic response to MMP inhibition. Bone 2008, 43 (2), 377–385. (9) Abou Neel, E. A.; Cheema, U.; Knowles, J. C.; Brown, R. A.; Nazhat, S. N. Use of multiple unconfined compression for control of collagen gel scaffold density and mechanical properties. Soft Mater. 2006, 2, 986–992. (10) Bitar, M.; Salih, V.; Brown, R. A.; Nazhat, S. N. Effect of multiple unconfined compression on cellular dense collagen scaffolds for bone tissue engineering. J. Mater. Sci.: Mater. Med. 2007, 18 (2), 237– 244. (11) Pedraza, C. E.; Marelli, B.; Chicatun, F.; McKee, M. D.; Nazhat, S. N. An in vitro assessment of a cell-containing collagenous extracellular matrix-like scaffold for bone tissue engineering. Tissue Eng., Part A 2010, 16, 781-793.
Mineralization of Collagen-Bioglass Scaffolds (12) Nazhat, S. N.; Abou Neel, E. A.; Kidane, A.; Ahmed, I.; Hope, C.; Kershaw, M.; Lee, P. D.; Stride, E.; Saffari, N.; Knowles, J. C.; Brown, R. A. Controlled microchannelling in dense collagen scaffolds by soluble phosphate glass fibers. Biomacromolecules 2006, 8 (2), 543– 551. (13) Khan, Y.; Yaszemski, M. J.; Mikos, A. G.; Laurencin, C. T. Tissue engineering of bone: Material and matrix considerations. J. Bone Joint Surg. Am. 2008, 90 (Supplement_1), 36–42. (14) Xynos, I. D.; Edgar, A. J.; Buttery, L. D. K.; Hench, L. L.; Polak, J. M. Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II mRNA expression and protein synthesis. Biochem. Biophys. Res. Commun. 2000, 276 (2), 461–465. (15) Hench, L. BioceramicssFrom concept to clinic. J. Am. Ceram. Soc. 1991, 74 (7), 1487–1510. (16) Hench, L. L. Bioceramics. J. Am. Ceram. Soc. 1998, 81 (7), 1705– 1728. (17) Chen, Q.; Roether, J. A.; Boccaccini, A. R., Tissue engineering scaffolds from bioactive glass and composite materials. In Topics in Tissue Engineering; Ashammakhi, N., Reis, R., Chiellini, F., Eds.; 2008; Vol. 4, pp 3-5. (18) Pohunkova`, H.; Adam, M. Reactivity and the fate of some composite bioimplants based on collagen in connective tissue. Biomaterials 1995, 16 (1), 67–71. (19) Eglin, D.; Maalheem, S.; Livage, J.; Coradin, T. In vitro apatite forming ability of type I collagen hydrogels containing bioactive glass and silica sol-gel particles. J. Mater. Sci.: Mater. Med. 2006, 17 (2), 161– 167. (20) Andrade, A. L.; Andrade, S. P.; Domingues, R. Z. In vivo performance of a sol-gel glass-coated collagen. J. Biomed. Mater. Res., Part B: Appl. Biomater. 2006, 79B (1), 122–128. (21) Kim, H.-W.; Song, J.-H.; Kim, H.-E. Bioactive glass nanofiber-collagen nanocomposite as a novel bone regeneration matrix. J. Biomed. Mater. Res., Part A 2006, 79A (3), 698–705. (22) Noda, H. Partial specific volume of collagen. J. Biochem. 1972, 71 (4), 699–703. (23) Chen, Q. Z.; Boccaccini, A. R. Poly(D,L-lactic acid)-coated 45S5 Bioglass-based scaffolds: Processing and characterization. J. Biomed. Mater. Res., Part A 2006, 77A (3), 445–457. (24) Kokubo, T.; Takadama, H. How useful is SBF in predicting in vivo bone bioactivity. Biomaterials 2006, 27 (15), 2907–2915. (25) Rehman, I.; Knowles, J. C.; Bonfield, W. Analysis of in vitro reaction layers formed on Bioglass using thin-film X-ray diffraction and ATRFTIR microspectroscopy. J. Biomed. Mater. Res. 1998, 41 (1), 162– 166. (26) Cerruti, M.; Greenspan, D.; Powers, K. Effect of pH and ionic strength on the reactivity of Bioglass 45S5. Biomaterials 2005, 26 (14), 1665– 1674. (27) Koutsopoulos, S. Synthesis and characterization of hydroxyapatite crystals: A review study on the analytical methods. J. Biomed. Mater. Res. 2002, 62 (4), 600–612. (28) Doyle, B. B.; Bendit, E. G.; Blout, E. R. Infrared spectroscopy of collagen and collagen-like polypeptides. Biopolymers 1975, 14 (5), 937–957. (29) Shirosaki, Y.; Kubo, M.; Takashima, S.; Tsuru, K.; Hayakawa, S.; Osaka, A. In vitro apatite formation on organic polymers modified with a silane coupling reagent. J. R. Soc. Interface 2005, 2 (4), 335– 340. (30) Wu, B.; Mu, C.; Zhang, G.; Lin, W. Effects of Cr3+ on the structure of collagen fiber. Langmuir 2009, 25 (19), 11905–11910. (31) Bohner, M.; Lemaitre, J. Can bioactivity be tested in vitro with SBF solution. Biomaterials 2009, 30 (12), 2175–2179. (32) Mudera, V.; Morgan, M.; Cheema, U.; Nazhat, S. N.; Brown, R. A. Ultra-rapid engineered collagen constructs tested in an in vivo nursery site. J. Tissue Eng. Regener. Med. 2007, 1 (3), 192–198.
Biomacromolecules, Vol. 11, No. 6, 2010
(33) Sander, E. A.; Barocas, V. H. Biomimetic collagen tissues: Collagenous tissue engineering and other applications. In Collagen - Structure and Mechanics; Fratzl, P., Ed.; Springer: Postdam, Germany, 2008; p 477. (34) Preve´y, P. X-ray diffraction characterization of crystallinity and phase composition in plasma-sprayed hydroxyapatite coatings. J. Thermal Spray Technol. 2000, 9 (3), 369–376. (35) LeGeros, R. Z. Calcium Phosphates in Oral Biology and Medicine; S Karger: Basel, Switzerland, 1991; Vol. 15, pp 8-9. (36) Antonakos, A.; Liarokapis, E.; Leventouri, T. Micro-Raman and FTIR studies of synthetic and natural apatites. Biomaterials 2007, 28 (19), 3043–3054. (37) Jones, J. R.; Sepulveda, P.; Hench, L. L. Dose-dependent behavior of bioactive glass dissolution. J. Biomed. Mater. Res. 2001, 58 (6), 720– 726. (38) Gough, J. E.; Arumugam, M.; Blaker, J.; Boccaccini, A. R. Bioglasscoated poly(DL-lactide) foams for tissue engineering scaffolds. Materialwiss. Werkstofftech. 2003, 34 (7), 654–661. (39) Kazarian, S. G.; Andrew Chan, K. L.; Maquet, V.; Boccaccini, A. R. Characterisation of bioactive and resorbable polylactide/Bioglass composites by FTIR spectroscopic imaging. Biomaterials 2004, 25 (18), 3931–3938. (40) Sitarz, M.; Mozgawa, W.; Handke, M. Rings in the structure of silicate glasses. J. Mol. Struct. 1999, 511-512, 281–285. (41) Wiesmann, H.-P.; Luttenberg, B.; Meyer, U., Tissue Engineering of Bone. In Handbook of Biomineralization - Medical and Clinical Aspects; Epple, M., Baeuerlein, E., Eds.; Wiley-VCH: Weinheim, Germany, 2007; pp 145-156. (42) Wahl, D. A.; Czernuszka, J. T. Collagen-hydroxyapatite composites for hard tissue repair. Eur. Cells Mater. 2006, 11, 43–56. (43) Gower, L. B. Biomimetic model systems for investigating the amorphous precursor pathway and its role in biomineralization. Chem. ReV. 2008, 108 (11), 4551–4627. (44) Go`es, J. C.; Figueiro`, S. D.; Oliveira, A. M.; Macedo, A. A. M.; Silva, C. C.; Ricardo, N. M. P. S.; Sombra, A. S. B. Apatite coating on anionic and native collagen films by an alternate soaking process. Acta Biomater. 2007, 3 (5), 773–778. (45) Kikuchi, M.; Ikoma, T.; Itoh, S.; Matsumoto, H. N.; Koyama, Y.; Takakuda, K.; Shinomiya, K.; Tanaka, J. Biomimetic synthesis of bone-like nanocomposites using the self-organization mechanism of hydroxyapatite and collagen. Compos. Sci. Technol. 2004, 64 (6), 819– 825. (46) Li, X.; Chang, J. Preparation of bone-like apatite-collagen nanocomposites by a biomimetic process with phosphorylated collagen. J. Biomed. Mater. Res., Part A 2008, 85A (2), 293–300. (47) Jalota, S.; Bhaduri, S. B.; Tas, A. C. Using a synthetic body fluid (SBF) solution of 27 mM HCO3- to make bone substitutes more osteointegrative. Mater. Sci. Eng., C 2008, 28 (1), 129–140. (48) Berendsen, A. D.; Smit, T. H.; Hoeben, K. A.; Walboomers, X. F.; Bronckers, A. L. J. J.; Everts, V. Alkaline phosphatase-induced mineral deposition to anchor collagen fibrils to a solid surface. Biomaterials 2007, 28 (24), 3530–3536. (49) Baht, G. S.; Hunter, G. K.; Goldberg, H. A. Bone sialoproteincollagen interaction promotes hydroxyapatite nucleation. Matrix Biol. 2008, 27 (7), 600–608. (50) Yamauchi, K.; Goda, T.; Takeuchi, N.; Einaga, H.; Tanabe, T. Preparation of collagen/calcium phosphate multilayer sheet using enzymatic mineralization. Biomaterials 2004, 25 (24), 5481–5489. (51) Heinemann, S.; Heinemann, C.; Bernhardt, R.; Reinstorf, A.; Nies, B.; Meyer, M.; Worch, H.; Hanke, T. Bioactive silica-collagen composite xerogels modified by calcium phosphate phases with adjustable mechanical properties for bone replacement. Acta Biomater. 2009, 5 (6), 1979–1990. (52) Silver, I. A.; Erecinska, M. Interactions of osteoblastic and other cells with bioactive glasses and silica in vitro and in vivo. Materialwiss. Werkstofftech. 2003, 34 (12), 1069–1075.