Article pubs.acs.org/Langmuir
Preparation of a Partially Calcified Gelatin Membrane as a Model for a Soft-to-Hard Tissue Interface Meital Aviv-Gavriel, Nissim Garti, and Helga Füredi-Milhofer* Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel ABSTRACT: Cartilage and/or bone tissue engineering is a very challenging area in modern medicine. Since cartilage is an avascular tissue with limited capacity for self-repair, using scaffolds provides a promising option for the repair of severe cartilage damage caused by trauma, agerelated degeneration, and/or diseases. Our aim in this study was to design a model for a functional biomedical membrane to form the interface between a cartilage-forming scaffold and bone. To realize such a membrane gelatin gels containing calcium or phosphate ions were exposed from one side to a solution of the other constituent ion (i.e., a sodium phosphate solution was allowed to diffuse into a calcium-containing gel and vice versa). The partially calcified gels were analyzed by XRD, ATR-FTIR spectra, E-SEM, and EDX. Thus, we confirmed the existence of a gradient of crystals, with a dense top layer, extending several micrometers into the gel. XRD spectra and Ca/P atomic ratios confirmed the existence of calcium deficient apatites. The effect of different experimental parameters on the calcification process within the gelatin membranes has been elucidated. It was shown that increasing the gelatin concentration from 5 wt % to 10 wt % retards calcification. A similar effect was observed when glycerol, which is frequently used as plasticizer, was added to the system. With increasing calcium concentration within the organic matrix, the quantity and density of calcium phosphate crystals over/within the gel increased. The possible explanations for the above phenomena are discussed.
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INTRODUCTION Tissue engineering is an emerging field of regenerative medicine, which aims to repair or restore tissues and organs affected by chronic diseases, age-linked degeneration, congenital deformity, and/or trauma. For this purpose, synthetic functional materials are used as scaffolds that contain cells meant to generate specific tissues.1,2 Interface tissue engineering is a special area, which focuses on regenerating or repairing diseased or damaged zones between different tissue types.3 Tissue interfaces, such as ligament-tobone, tendon-to-bone, and cartilage-to-bone, exhibit anisotropic structural properties, which gradually vary from one tissue to another. Therefore, biomimetic scaffolds with graded properties are needed in order to engineer interface tissues. Such scaffolds should be three-dimensional (3D) and should exhibit a gradient in composition, structure, and mechanical features, among other functional properties, in order to mimic those of the native interface zones. The interfacial tissue reconstruction between soft and hard tissues, such as cartilage and bone, is particularly challenging because cartilage is an avascular tissue with limited capacity for self-repair. Cartilage regeneration may be achieved by organic− inorganic composite scaffold materials which exhibit gradients in composition and structure and can interface with both the mineralized (bone) and nonmineralized (cartilage) tissue. To date, for cartilage tissue engineering, two types of scaffolds have been used experimentally: solid-type scaffolds and hydrogels. Hydrogels have advantages because they can provide a 3D environment for the organization of cartilage forming cells.4 When used for these applications, they should © 2012 American Chemical Society
be biodegradable, biocompatible, and have mechanical and structural properties similar to cartilage. Gelatin was chosen in this work as a model biopolymer to form an interface membrane between a scaffold, containing cartilage forming cells5,6 and bone. It is a natural polymer which has a wide range of medical applications, because it is biodegradable and biocompatible. In addition, gelatin displays several features that are attractive in the biomedical field: it has not shown antigenicity under physiological conditions, it is completely resorbable in vivo, and its physicochemical properties can be suitably modulated.7 Gelatin is derived from the parent protein collagen by thermal denaturation or by its physical and/or chemical degradation. The network structure of gelatin gels and their physical properties are mainly influenced by the source and the conditions of extraction. There are two main types of gelatin depending on the treatment of collagen. Type A is extracted from collagen by acid treatment, while type B is extracted by alkaline treatment. Gelatin based-films are hygroscopic materials, which are very sensitive to environmental conditions, such as temperature and relative humidity.8 In order to modify some of their functional and physical properties, plasticizers such as glycerol and sorbitol are usually added to the system. Gelatin−hydroxyapatite composites have been prepared for use as bone cements, bone fillers, and/or scaffold materials. Received: September 13, 2012 Revised: November 12, 2012 Published: December 11, 2012 683
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The instrumental analyses were performed from both the upper and lower parts of calcified samples. Treatment of data. Atom % of Ca, P, and C as obtained from EDX spectra were used to calculate C/Ca and C/P atomic ratios, which are an indication of the concentration of crystals on the surface of the organic matrix.
Several studies of gelatin calcification have been described in the past decades. Some of the studies involve the preparation of the gel and hydroxyapatite crystals separately and mixing them together.9,10 Other studies, based on diffusion systems, involve in situ precipitation of the inorganic component within the gel. One-dimensional single diffusion11,12 and double diffusion13−15 systems have been described. Recently, in our laboratory a gelatin membrane with a gradient of calcium phosphate crystals has been prepared.16 Here, we describe the influence of different experimental parameters on the process of calcification and the characteristics of the ensuing membrane.
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RESULTS Preparation and Characterization of the Partially Calcified Membrane. When thin films of gelatin gels, containing calcium or phosphate ions, were exposed from the top of the gel to a phosphate or calcium solution, respectively, the upper parts of the gels became visibly turbid after several hours of diffusion. The lower parts of the membranes stayed transparent throughout the whole reaction time. The two layers were easily distinguished by the naked eye already during the preparation of the membrane. This pointed to a way to obtain films of partially calcified hydrogels. The upper part of the gel contained a crystalline layer, while the bottom part consisted only of the organic phase as confirmed by ATR-FTIR (Figure 1) and XRD (not shown). The crystalline matter appeared as
EXPERIMENTAL SECTION
Materials. Analytical-grade chemicals and ultrapure water (UPW; Milli Q-plus from Barnsted system) were used for all experiments. Calcium chloride and sodium phosphate stock solutions were prepared from CaCl2·2H2O and Na2HPO4 (both from Merck), which were dried overnight in a desiccator over silica-gel before weighing and dissolving in UPW (phosphate solution) or Tris buffer (calcium solution). Type A (300 Bloom) and B (250 Bloom) gelatin powder were purchased from Sigma−Aldrich and used as received. Sample Preparation. The composition of the solvent for gelatin solution was Tris buffer pH 8 containing the required concentration of calcium ([Ca] = 0.02, 0.03, or 0.05 mol dm−3) or phosphate ions ([PO4] = 0.019 mol dm−3) and 0−50% of glycerol (the actual concentrations of each additive pertain to the solvent before addition of gelatin and are stated in the Results section). Five or ten grams of gelatin powder per 100 mL of solvent were then dissolved under constant stirring and subsequently allowed to swell at room temperature for 4−5 h. After swelling, the gelatin gel was dissolved in a water bath shaker (60 rpm, 45 °C) during 30 min and 4 mL aliquots of the final solution were transferred into Petri dishes (diameter = 3.5 cm). The samples were then left to gel overnight in a dust-free drybox at room temperature. Gelatin gels (thickness approx 0.5 cm, surface area 9.62 cm2) containing different amounts of the inner electrolyte: system I (0.02− 0.05 mol dm−3 calcium ions) and/or system II (0.019 mol dm−3 phosphate ions) were prepared as described above and were exposed from the upper side to 3 mL of a solution of the respective outer electrolyte (phosphate for calcium containing and calcium for phosphate containing gels). The concentration of the outer electrolyte was adjusted to obtain an initial Ca/P molar ratio of 1.5 or 1.6. If not otherwise stated, the systems were kept for 48 h in a water bath shaker at 25 °C to allow diffusion of the counterions into the gel. The thickness of the gels did not change significantly during this procedure. After 48 h, the gels were washed with UPW and dried for specified times at room temperature under a flow of filtered air. The conditions and time of aging are given for each experiment in the Results section. Methods and Instrumentation. Calcified and noncalcified gels were visualized by environmental electron microscopy (E-SEM) and subjected to electron-dispersive X-ray (EDX) surface and spot analysis (E-SEM with EDX attachment from Quanta 200, FEI, Eindhoven, The Netherlands) and to ATR-FTIR analysis on an Alpha model spectrometer equipped with a single reflection diamond ATR sampling module (Bruker Optics GmbH, Ettlingen, Germany). The spectra were recorded with 25 scans, at 25 °C; a spectral resolution of 2 cm−1 was obtained. Calcified samples were, in addition, characterized by X-ray powder diffraction patterns, obtained by a D8 Advance diffractometer (Bruker AXS, Karlsruhe, Germany) with a goniometer radius 217.5 mm, Göbel Mirror parallel-beam optics, 2° Sollers slits, and 0.2 mm receiving slit. XRD patterns within the range 4° to 54° 2θ were recorded at room temperature using Cu Kα radiation (λ = 1.5418 Å) with the following measurement conditions: tube voltage of 40 kV, tube current of 40 mA, step-scan mode with a step size of 0.02° 2θ, and counting time of 1 s/step.
Figure 1. ATR-FTIR spectra of the upper (a) and bottom (b) layers of a partially calcified gelatin gel (pH 8.0; 0.03 mol dm−3 calcium; outer electrolyte 0.019 mol dm−3 phosphate). Arrows show phosphate bands: 1116.5 cm−1 and 1026.7 cm−1 due to the presence of CO32−, HPO42−, and crystal imperfections in non-stoichiometric apatites, 595.9 cm−1 and 553.2 cm−1triply degenerated bending modes, ν4a, ν4c of O−P−O bonds; assignments after references 17−19.
globular aggregates above and within the upper layer of the gel (Figure 2A). Their Ca/P atomic ratio, obtained from EDX spectra, was 1.46 ± 0.04 (average from 5 samples). The ATRFTIR spectra obtained from our samples typically exhibit the characteristic vibrational absorption band amide I of the protein at 1634−1637 cm−1, as well as a broad band between 3000 and 3500 cm−1 that can be attributed to the OH stretching band of water molecules (Figure 1). The spectrum obtained from the top layer displays, in addition, the characteristic vibrational phosphate bands (Figure 1A). The strong bands at 553.2 and 595.9 cm−1 correspond to a triply degenerated bending mode of the O−P−O bonds of the phosphate group, while peaks appearing at 1026.7 and 1116.5 cm−1 are due to the presence of CO32−, HPO42−, and crystal imperfections in non-stoichiometric apatites.17−19 The observed phosphate bands and the Ca/P atomic ratio indicate that the crystals are calciumdeficient hydroxyapatite (CDHA), which is similar to the main inorganic component of bone and dentin. Likewise, in the XRD pattern taken from the upper part of a calcified gelatin sample the main peaks characteristic of apatite are discernible (Table 1), while the pattern of the bottom part of the sample indicates a completely amorphous substance (not shown). In order to examine the deeper layers of the system, a cross section of the sample was examined by E-SEM (Figure 3) and EDX (Figure.4). Four layers of the gel: top, next to top (i.e., the immediate layer below the top), center, and bottom layers are shown. Both the E-SEM images and the changing intensities of the EDX peaks characteristic of Ca and P, respectively, indicate 684
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Figure 4. EDX spectra of different layers within the partially calcified gelatin gel, shown in Figure 3A−D (pH 8.0; inner electrolyte 0.019 mol dm−3 phosphate; outer electrolyte 0.03 mol dm−3 calcium). C/Ca atomic ratios: (A) 3.64; (B) 25.69; (C) 83.38; (D) ∞.
Figure 2. Scanning electron micrographs of calcium phosphate precipitates formed within/upon the upper layers of 5 wt % gelatin gels observed after 5 days of aging: (A) pH 8.0; 0.03 mol dm−3 calcium; outer electrolyte 0.019 mol dm−3 phosphate; Ca/P atomic ratio = 1.46 ± 0.04; (B) pH 8.0; 0.019 mol dm−3 phosphate; outer electrolyte 0.03 mol dm−3 calcium; Ca/P atomic ratio = 1.81 ± 0.07.
a concentration gradient of crystals, decreasing with increasing distance from the upper layer of the gel. The increasing C/Ca atomic ratios (as calculated from EDX data; see caption of Figure 4) give a quantitative estimate of this trend. In the bottom part of the gel, there was no evidence at all of the inorganic phase. Influence of Different Parameters on the Calcification of Gelatin Gels. Exchange of the Inner and Outer Electrolytes. There are considerable differences between the two diffusion systems with different inner electrolytes, calcium or phosphate ions (system I and system II as defined in the Experimental Section). E-SEM images taken from the top of system I after 5 days of aging show high density of crystalline aggregates with quite homogeneous dispersion over the surface of the gel (Figure 2A), while significantly fewer aggregates were formed in system II (Figure 2B). Moreover, the latter crystal aggregates appeared smaller and the Ca/P atomic ratio was considerably higher (Ca/P = 1.81 ± 0.07) compared to the crystals grown in system I, (Ca/P = 1.46 ± 0.04). When systems II were allowed to age for another three weeks, the quantity of the crystal aggregates increased and the Ca/P atomic ratio decreased to 1.53 ± 0.03, which is in the range of calcium-deficient hydroxyapatite. From these observations, it can be inferred that the crystallization process was slower when phosphate, rather than calcium ions constituted the inner electrolyte. In system II, the initial precipitate was poorly crystalline or amorphous, and perfected itself only during longer aging periods. Because of the obvious advantages of system I, the effect of changes in experimental parameters was investigated on this system. Gelatin Concentration. The influence of increased concentration of gelatin on the calcification process is shown in Figure 5. In contrast to samples with 5 wt % of gelatin in which crystallization was almost complete after 5 days of aging (Figure 2A), on the surface of gelatin membranes containing 10 wt % of gelatin, after the same period there was almost no evidence of inorganic crystals (Figure 5A). After 17 days of aging, just a small amount of crystalline matter was found also over/within the latter membranes (Figure 5B). A semiquantitative estimate of changes of the concentration of crystalline matter within the upper layers of the gel can be
Table 1. Characteristic XRD Peaks from the Top Layer of Calcified Gelatin, Compared to the Nearest Corresponding Literature Values19 for Hydroxyapatite (HA) d (Å) (as measured)
d (Å) HAP
Miller index of the corresponding reflection
3.435 2.809 2.772 2.263 1.947 1.721
3.440 2.814 2.778 2.262 1.943 1.722
(002) (211) (112) (310) (222) (004), (411)
Figure 3. Scanning electron micrographs of a cross section through a calcified gel, showing the gradient of crystals (pH 8.0; inner electrolyte 0.019 mol dm−3 phosphate; outer electrolyte 0.03 mol dm−3 calcium): (A) top view; (B)−(D) side views: (B) top (approximately 7−8 μm) and next to top, (C) center, (D) bottom of the gel.
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Figure 5. Scanning electron micrographs of calcium phosphate precipitates within 10 wt % gelatin gels showing the influence of the time of aging: 5 days (A) and 17 days (B). Conditions of precipitation: pH 8.0, 0.03 mol dm−3 calcium; diffusion of 0.019 mol dm−3 phosphate.
obtained from the respective C/Ca and C/P atomic ratios, calculated from EDX spectra. For 10 wt % of gelatin, these values decreased by about 10 times in the period between 5 and 17 days of aging of the gel, indicating a relative increase of the crystalline matter. Calcium Concentration within the Gelatin Membrane. In order to demonstrate how the calcium concentration within the gel matrix affects the calcification process, three different concentrations of calcium ions within the gelatin gels were examined: 0.02 mol dm−3, 0.03 mol dm−3, and 0.05 mol dm−3. The other parameters of all three systems were identical. The scanning electron micrographs display the density of the crystals as a function of the calcium ion concentrations and a quantitative estimate has been obtained from EDX spectra (Figure 6 and Table 2). It is seen that, with increasing calcium concentration within the gel, C/Ca and C/P atomic ratios decrease, indicating an increasing amount of the crystals relative to the organic matrix. Addition of Glycerol to the Gelatin Membrane. The effect of the addition of glycerol (0−50%) is shown in Figures 7−9. From the E-SEM images and corresponding EDX spectra taken from the upper parts of the gels, it is obvious that adding glycerol to the gelatin membranes significantly slows down calcification (Figure 7). The C/Ca and C/P atomic ratios increase almost linearly with increasing glycerol concentration (Figure 8), indicating a corresponding decrease in the amount of crystals within the gel, while the Ca/P ratio varies only slightly around 1.35 (Figure 8, inset). The effect of addition of 50 wt % glycerol on the morphology of the crystal aggregates is shown in Figure 9. These composites appeared quite different from those obtained without glycerol (Figure 7A), as large aggregates of calcium phosphate precipitate appeared in isolated areas over the gel instead of a relatively homogeneous distribution at zero and low glycerol concentrations (Figure 7). The appearance of the large crystal aggregates indicates that fewer sites for calcium phosphate nucleation are available, since most of the nucleation sites have been occupied by glycerol molecules.
Figure 6. Scanning electron micrographs of calcium phosphate precipitates formed within/upon the upper layers of gelatin gels containing different concentrations of calcium ions: (A) 0.02 mol dm−3, (B) 0.03 mol dm−3, (C) 0.05 mol dm−3 (pH 8.0; phosphate diffusion, initial Ca/P atomic ratio 1.6).
Table 2. C/Ca, C/P, and Ca/P Atomic Ratios Obtained from Partially Calcified Gelatin Gels Containing Different Concentrations of Calcium Ionsa
a
Ca concentration (mol dm−3)
C/Ca
C/P
Ca/P
0.02 0.03 0.05
4.55 ± 0.90 2.77 ± 0.18 3.20 ± 0.32
6.62 ± 1.19 4.02 ± 0.20 4.31 ± 0.36
1.46 ± 0.04 1.46 ± 0.04 1.36 ± 0.03
Each value is the mean of 5 determinations.
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DISCUSSION In this work, we demonstrated a way to obtain a hydrogel membrane with a gradient of apatite crystals, which could be used as interface between a chondrocyte containing scaffold and bone. As model biopolymer, gelatin gel was chosen because of its similarity to collagen from which it is obtained by degradation. A gradient of calcium phosphate crystals within the membrane (calcification gradient) was obtained by a simple diffusion system, when gels containing calcium or phosphate ions were exposed from one side to a solution of the other constituent ion. XRD, ATR-FTIR spectra, E-SEM, and EDX have confirmed the existence of a concentration gradient of crystals within the gel, with a dense top layer extending several micrometers into the gel (Figures 1−4). Such a gradient is reminiscent of the phenomenon of periodic precipitation known as Liesegang’s rings, which for the gelatin/calcium phosphate system were investigated by Devik.12 Moreover, EDX and ATR-FTIR experimental results have shown that, despite the fact that the calcium or phosphate ions dissolved 686
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Figure 9. Scanning electron micrographs (different magnifications) of a calcium phosphate precipitate formed within/upon the upper layer of a 5 wt % gelatin gel containing 50 wt % glycerol (pH 8.0; 0.03 mol dm−3 calcium; outer electrolyte 0.019 mol dm−3 phosphate).
Figure 7. Scanning electron micrographs and the corresponding EDX spectra of calcium phosphate precipitates formed within/upon the upper layers of 5 wt % gelatin gels containing glycerol: (A) control, 0 wt %; (B) 10 wt %; (C) 30 wt % (pH 8.0; 0.03 mol dm−3 calcium; outer electrolyte 0.019 mol dm−3 phosphate).
Figure 8. C/P (■), C/Ca (◆), and Ca/P (▲) atomic ratios of calcium phosphate precipitates formed within/upon the upper layers of 5 wt % gelatin gels as a function of glycerol concentration (wt %) (pH 8.0; 0.03 mol dm−3 calcium; outer electrolyte 0.019 mol dm−3 phosphate). Each value is the mean of 5 determinations and is reported with its standard deviation (see bars). Inset: Ca/P atomic ratio vs glycerol concentration (wt %).
Figure 10. Schematic presentation of a possible mechanism of the formation of a gradient of calcium phosphate crystals within a gelatin gel membrane. In this example, Ca2+ ions and HPO42− ions are the inner and outer electrolyte, respectively. Scanning electron micrographs represent the top (A), center (B), and bottom (C) of the gel.
homogeneously within the gelatin matrix during gel preparation, there was no evidence of the inner electrolyte (calcium or phosphate ions) in the bottom part of the gel after the diffusion process was completed (Figure 4D). The basis of understanding the mechanism of the formation of a calcification gradient within the gelatin membranes is the diffusion process of calcium and phosphate ions within the gel (Figure 10). At our experimental conditions (pH = 8.0), the gelatin strands have negatively charged sites. If calcium ions are the inner electrolyte, they are positioned close to these sites and serve as nucleation centers upon the addition of phosphate ions (template crystallization). Crystallization commences in the upper parts of the gel, which in the process is depleted of free
calcium ions, and as a consequence, a concentration gradient is formed within the gel, inducing diffusion of calcium ions toward the top (Figure 10). The formation of calcium-deficient apatites is a relatively slow process which probably occurs via amorphous calcium phosphate formation as shown in solution systems.20−22 Therefore, crystal perfection (via recrystallization and phase transformation) proceeds long after phosphate addition has been terminated. 687
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gradient of in situ grown calcium phosphate crystals, is to be used as an interface between a cartilage forming cells containing scaffold and bone. As a model, gelatin gels have been calcified and the essential parameters effecting the amount and quality of the crystals have been examined. Calcification may be optimized by (i) using calcium ions as the internal and phosphate ions as the external component, (ii) increasing the concentration of one or both constituent ions, or (iii) increasing the time of diffusion and/or crystal ripening and other adjustments. The method is simple and cost-effective and could be easily adapted to calcify any other hydrogel that is convenient from a medical standpoint. The above research is of broader significance because it can be modified to develop organic−inorganic nanocomposite biomedical membranes for guided tissue regeneration using other types of hydrogels and repairing other soft connective tissues, such as muscles and/or nerves, which are in contact with bone.
When phosphate is the inner electrolyte, the mechanism is similar but the phosphate ions within the gel are randomly distributed, because they are of the same charge as the gelatin sites and therefore repelled by them. The result is random precipitation of poorly crystalline calcium phosphate (Figure 2B) with high Ca/P atomic ratio because of adsorbed calcium ions. Only after extended periods of time does the amorphous precipitate transform into a crystalline phase, with Ca/P atomic ratios in the range characteristic of calcium-deficient hydroxyapatite (CDHA). Since the crystallization rate is diffusion-controlled, it is also affected by the different diffusion coefficients, i.e., for 10 wt % gelatin gels Dcalcium (6.0 × 10−6) > Dphosphate (3.9 × 10−6 cm2/ s).15 This is an additional explanation for the slower crystallization process, when phosphate is the inner electrolyte. The observations are consistent with the results of Pokric and Pucar,13 who have shown that in double diffusion systems the positions of the first formed precipitates are shifted toward the phosphate side of the gel columns with increasing gel concentrations, even though the initial concentrations of the precipitating components were equal. Because of the obvious advantages of the system in which calcium was the inner electrolyte, the influence of different parameters on the calcification process was investigated on this system. The following observations were made: • Increasing gelatin concentration from 5 to 10 wt % caused a significant decrease in the rate of crystallization as well as in the total amount of crystals (Figure 5). The effect is most probably due to the impaired mobility of the constituent ions with increasing gelatin concentration; • With increasing calcium concentration within the organic matrix, the quantity of calcium phosphate crystals within/upon the gel increases, as shown by SEM pictures (Figure 6) and by decreasing C/Ca and C/P atomic ratios (Table 2). The effect is due to the formation of more nucleation sites within the gel. • Addition of glycerol to the gelatin membrane has the most significant effect on the calcification process within this system. Figure 7 shows that this additive inhibits calcium phosphate crystallization, most probably by competing with calcium ions for the hydrophilic sites in the gelatin gel. An interesting phenomenon confirming this assumption is the formation of large crystal aggregates in the presence of high glycerol concentrations (Figure 9), indicating the lack of nucleation sites within/upon the gel. Glycerol is frequently used as plasticizer for gelatin, altering some physical properties of the gel.8,23 Thus, the additive may play an important role for a partially calcified gelatin membrane by giving it the required flexibility necessary for temporary cartilage replacing tissue. Therefore, the above effect, which increases with increasing gelatin and glycerol concentration, respectively (Figures 7−9), has to be taken into account.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 972-2-6585885. Fax: 972-2-6528250. E-mail: helga@vms. huji.ac.il. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS It is a pleasure to thank Professor Dr. Avner Yayon, Prochon Biotech Ltd, and Dr Boaz Amit, Procore Ltd, Ness Ziona, Israel for their interest in this work and helpful discussions. The financial support granted by the Ministry of Industry, Trade and Labor of Israel, supplemented by Prochon, Biotech Ltd, as well as the partial financial support by the German-Israeli Foundation (GIF) are gratefully acknowledged.
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REFERENCES
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SUMMARY In this study, we have demonstrated that, by carefully adjusting experimental parameters, it is possible to obtain partially calcified hydrogel membranes for cartilage and/or bone regeneration. A simple method for the preparation of such a functional biomedical membrane has been devised. The membrane, which is based on a hydrogel with a concentration 688
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