Characterization and deposition behavior of silk hydrogels soaked in simulated body fluid

Characterization and deposition behavior of silk hydrogels soaked in simulated body fluid

Materials Science and Engineering C 32 (2012) 945–952 Contents lists available at SciVerse ScienceDirect Materials Science and Engineering C journal...

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Materials Science and Engineering C 32 (2012) 945–952

Contents lists available at SciVerse ScienceDirect

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Characterization and deposition behavior of silk hydrogels soaked in simulated body fluid Catalin Zaharia a,⁎, Mihaela-Ramona Tudora a, Izabela-Cristina Stancu a, Bianca Galateanu b, Adriana Lungu a, Corneliu Cincu a a b

University Politehnica of Bucharest, Department of Bioresources and Polymer Science, Bucharest 010072, Romania Department of Biochemistry and Molecular Biology, University of Bucharest, Romania

a r t i c l e

i n f o

Article history: Received 29 March 2011 Received in revised form 23 December 2011 Accepted 2 February 2012 Available online 12 February 2012 Keywords: Silk fibroin Polyacrylamide Mineralization Hydrogel Cytotoxicity

a b s t r a c t This paper reports the mineralization ability of semi-interpenetrating networks composed of regenerated silk fibroin and polyacrylamide hydrogels soaked in simulated body fluid (SBF1x). Hydrogels were prepared by polymerization of acrylamide and N,N′-methylenebisacrylamide in silk fibroin solution with a redox pair as initiator. The incorporation of the fibroin within the polyacrylamide matrix was proved by FTIR–ATR spectroscopy. Swelling measurements in saline solution were performed to evaluate the behavior of these hydrogels having various compositions. Mineralization assays in SBF1x solution revealed the presence of apatite-like crystals onto the surface of the silk fibroin/polyacrylamide hydrogels. Cytotoxicity test by extract method revealed that these hydrogels with various contents in silk fibroin have not developed cytotoxic effects on human fibroblast cultures which increases the possibility of their use in biomedical applications. Mechanical compressive tests revealed good strengths for silk fibroin/polyacrylamide hydrogels. In this way, organic–inorganic hybrids analogous to bone structure can be produced under biomimetic conditions and could be further used in biomedical applications. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Various polymeric scaffolds have been used as substitutes for the extracellular matrix, which is rapidly becoming the most challenging experimental approach for regenerating the native structural and functional properties of living tissue [1–3]. These materials may consist of natural macromolecules and synthetic polymers, providing an adhesive substrate that can serve as a 3D physical support matrix for cell culture and tissue regeneration. Natural bone is a composite in which inorganic apatite nanocrystals are deposited on organic collagen fibers woven into a three-dimensional structure. An interesting task is to mimic bone structure in the design of novel bone-repairing materials. Kokubo and his colleagues proposed a biomimetic process that utilizes a reaction between bioactive glass and a simulated body fluid (SBF) [4–6]. This process has attracted much attention because a bone-like apatite layer can be coated onto organic substrates. In this process, an organic substrate is initially placed in the neighborhood of bioactive glass in SBF that has ion concentrations nearly equal to those of human extracellular fluid. Apatite deposition is initiated by the

⁎ Corresponding author. E-mail address: [email protected] (C. Zaharia). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.02.018

heterogeneous nucleation of apatite induced by dissolved silicate and calcium ions arising from the bioactive glass. The dissolved silicate ions provide a heterogeneous nucleation site for apatite on the substrate, and dissolved calcium ions increase the degree of supersaturation of the fluid with respect to the apatite. Once the apatite nuclei are formed on the substrate, they can spontaneously grow by consuming calcium and phosphate ions from the surrounding fluid [5,7–9]. In the production of an apatite–organic polymer hybrid through such a biomimetic route, the polymer should act as a substrate with heterogeneous nucleation sites for apatite deposition [10–12]. It also should be flexible enough for the desired mechanical performance of the hybrid. Hydrogels can be regarded as three dimensional hydrophilic polymer networks that swell, but do not dissolve, when brought into contact with water [13–20]. Semi-interpenetrating polymer networks (semi-IPNs) are defined as compounds in which one or more polymers are cross-linked. Cross-linked polymers exhibiting high equilibrium swelling in water or aqueous solutions can be based exclusively on macromolecules with a high hydrophilicity and flexibility [16,18,20]. Silks, the unique proteins of silkworm fibers, are high-molecularweight block copolymers consisting of a heavy (~370 kDa) and a light (~ 26 kDa) chain with varying amphiphilicity linked by a single disulfide bond [1,21–27]. Bombyx mori silk is the most characterized silkworm silk. As a fibrous protein, silk fibroin


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(SF) fibers from B. mori have been used as biomedical suture material for a long time, because of their outstanding mechanical properties, in contrast to the catalytic and molecular recognition functions of globular proteins. Also, researchers have investigated fibroin as one of the promising resources of biotechnology and biomedical materials due to its other unique properties including excellent biocompatibility, favorable oxygen permeability and outstanding biodegradability, and the degradation product can be readily absorbed by the body with minimal inflammatory reaction, etc [6,22,28–32]. Silk hydrogels have been thoroughly studied for potential biotechnological applications due to their mechanical properties, biocompatibility, controllable degradation rates, and self-assembly into β-sheet networks [13,33,34]. To further improve the properties of hydrogel, silk fibroin has been blended with various other polymers which includes both synthetic molecules polyvinyl alcohol as well as natural macromolecules like gelatin, collagen etc. The major disadvantages of the silk fibroin hydrogels are the poor mechanical properties and swelling behavior, which are very important parameters in biomedical applications. To overcome this major disadvantage, semi-interpenetrating networks of silk fibroin and polyacrylamide hydrogels are designed. In this respect, semi-interpenetrating networks composed of regenerated silk fibroin (RSF) and polyacrylamide (PAA) were obtained and characterized for their ability to form apatite-like crystals in SBF solution. The hydrogels were obtained by polymerization of acrylamide and N,N′-methylenebis (acrylamide) in fibroin solution with a redox initiating system composed of potassium persulfate and triethanol amine. Next, the physico-chemical properties were evaluated by swelling behavior, morphological studies, and ATR–FTIR spectroscopy. The mineralization ability of the silk fibroin–polyacrylamide hydrogels in SBF1x was further investigated. The cytotoxicity of possible substances that could leach from the semi-IPNs was evaluated by direct microscopic observation and by monitoring cell viability and lysis. Compressive mechanical tests were also employed for silk fibroin/polyacrylamide hydrogels. 2. Experimental 2.1. Materials Cocoons of B. mori silkworm silk were kindly supplied by Marilena Constantinescu, Commercial Society SERICAROM SA (Bucharest, Romania). Acrylamide (AA) was purified by recrystallization from water and dried in air. N,N′-methylenebisacrylamide (MBA), potassium persulfate (KPS) and triethanol amine (TEA) were purchased from Sigma Aldrich, St-Quentin Fallavier, France. Lithium bromide (LiBr), sodium bicarbonate and sodium dodecyl sulfate (SDS) were provided by Alfa Aesar GmbH&Co KG, Germany and dialysis tubing cellulose membrane from Sigma. 2.2. Methods 2.2.1. Obtaining of silk fibroin solution B. mori silkworm cocoons were boiled for 30 min in an aqueous solution of 0.5% (w/v) NaHCO3 and SDS and then rinsed thoroughly with distilled water to extract the sericin protein. This operation was repeated three times to get the pure silk fibroin. The degummed silk fibroin was dried at 40 °C and atmospheric pressure. The extracted silk fibroin was then dissolved in a 9.5 M LiBr solution at 60 °C for 8 h, yielding a 10% (w/v) solution. This solution was dialyzed in distilled water using a dialysis tubing cellulose membrane (molecular weight cutoff, MWCO, 12.4 kDa) for 4 days (frequent water changes). The final concentration of the silk fibroin aqueous solution was 3 wt.%, which was determined by weighing the remaining solid after drying.

2.2.2. Obtaining of silk fibroin–polyacrylamide hydrogels Hydrogels of silk fibroin and polyacrylamide were obtained by free radical polymerization. Briefly aqueous solution of 15 wt.% AA having 1 wt.% N,N′-MBA was prepared and then mixed with regenerated silk fibroin (RSF) solution in various ratios. The blended solutions were placed in glass vials together with the redox initiating system composed of KPS and TEA. The reaction proceeded at 40 °C for 4 days. The prepared hydrogel matrices were purified by repeated extraction with ethanol/methanol solution and distilled water for 5 days. The water was changed every several hours to wash out the unreacted acrylamide monomer and cross-linking agent. The samples were then dried at room temperature to constant weight. 2.2.3. ATR–FTIR characterization The FT-IR spectra were taken on a Jasco 4200 spectrometer equipped with a Specac Golden Gate attenuated total reflectance (ATR) accessory, using a resolution of 4 cm − 1 and an accumulation of 60 spectra, in the 4000–600 cm − 1 wavenumber region. 2.2.4. Swelling measurements Determination of the swelling degree. Swelling behavior of the hydrogels was performed in saline solution at 37 °C. The weight changes of the hydrogels were recorded at regular time intervals during the course of swelling. The swelling degree of the hydrogels was determined according to the following equation: SD ¼ ððW t –W 0 Þ=W 0 Þ  100


where W and W0 denote the weight of the wet hydrogel at a predetermined time and the weight of the dry sample, respectively. The equilibrium swelling degrees (ESD) were measured until the weight of the swollen hydrogels was constant. At least three swelling measurements were performed for each hydrogel sample and the mean values were reported. Swelling kinetics. The dynamics of water sorption process was studied by monitoring the saline solution imbibed by the hydrogels at different time intervals. For diffusion kinetic analysis, the swelling results obtained were utilized only up to 60% of the swelling curves. The following equation was used: f ¼kt



where f is the fractional water uptake, k is a constant, t is swelling time and n the swelling coefficient that indicates whether diffusion or relaxation controls the swelling process. The fractional water content f is Mt/Mn where Mt is the mass of water in the hydrogel at time t, and Mn is the mass of the water at equilibrium. 2.2.5. Morphology of silk fibroin–polyacrylamide hydrogels Morphological information including internal structure was obtained through the scanning electron microscopy (SEM) analysis of the gold-coated hydrogels. Hydrogel samples were frozen at −50 °C followed by lyophilization. The analysis has been performed using a QUANTA INSPECT F SEM device equipped with a field emission gun (FEG) with a resolution of 1.2 nm and with an X-ray energy dispersive spectrometer (EDS). 2.2.6. Mineralization assay For mineralization assay, three samples of each hydrogel composition were incubated in synthetic body fluid (SBF1x) at pH =7.4, adjusted with tris(hydroxy-methyl) aminomethane (Tris) and hydrochloric acid (HCl), for 14 days, under sterile conditions, in containers with 45 mL of the incubation medium at 37 °C. The incubation medium was changed

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every 48 h. After incubation, the hydrogels were rinsed with distilled water to remove any traces of salts from the surface and dried at 40 °C for 24 h. The composition of SBF1x is presented below: Na+: 142.19 mM, Ca2 +: 2.49 mM, Mg2 +: 1.5 mM, HCO3−: 4.2 mM, Cl−: 141.54 mM, HPO42 −, 0.9 mM, SO42 −: 0.5 mM, K+: 4.85 mM. The presence of mineral crystals onto the surface of the hydrogels was evaluated by SEM analysis. The Ca/P molar ratio was investigated by EDS spectroscopy. XRD patterns for mineralized silk fibroin/ polyacrylamide hydrogels were obtained using a RIGAKU miniflex II diffractometer with CuKα radiation. 2.2.7. Biological tests Cell culture. Human dermal fibroblast cell line CCD-1070Sk provided by American Type Culture Collection was cultured in MEM (Sigma-Aldrich Co.) supplemented with 1% Antibiotic/Antimycotic (ABAM, Sigma-Aldrich Co.) and 10% Fetal Bovine Serum (FBS, Gibco). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, with the growth media changed every third day. Cytotoxicity tests on extracts. The hydrogels used in this study were sterilized by 12 h exposure to UV per side and then washed in culture medium for 24 h at 4 °C. A combination approach, MTT and lactate dehydrogenase (LDH) assays, was used to provide valuable information about cell viability and possible cytotoxic effects of the analyzed materials. These tests were performed in accordance with ISO 10993-5 (ISO 10993: Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity). Briefly, samples were immersed in culture medium and incubated in standard culture conditions. After 24 h, medium containing the cell culture extracts were tested undiluted. Fibroblasts cell type recommended by ISO procedure were seeded at a density of 1.2 × 104 cells/cm2 in 12-well plates and allowed to proliferate for 24 h. Then the culture media was changed with culture extracts the MEM containing the extracts, and incubated cells were allowed to proliferate for further 72 h at 37 °C in a 5% CO2 enriched atmosphere. Cells incubated with complete MEM and wells containing only complete MEM were used as controls. Experiments were performed in triplicates and statistically analyzed with GraphPrism software using one-way ANOVA with Bonferroni's multiple comparison tests. All results are expressed as means ± SD (standard deviation) and differences at p ≤ 0.05 were considered statistically significant. The cell morphology was examined by phase contrast microscopy. The MTT assay is based on the reduction of a tetrazolium salt solution — MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) to purple formazan by metabolic active cells. The precipitated formazan was then solubilized, and the concentration was determined by optical density at 550 nm. The result is a sensitive assay with a colorimetric signal proportional to the cell number. As an additional test, we also measured the leakage of lactate dehydrogenase (LDH) in the culture medium as a result of plasma membrane lysis with a cytotoxicity detection kit (TOX-7, Sigma-Aldrich Co.) according to the manufacturer's protocol. The optical density at 490 nm was measured in a microplate reader. 2.2.8. Mechanical tests Mechanical properties of silk fibroin/polyacrylamide hydrogels were evaluated by uniaxial compression on Universal Testing Machine (Instron 3382 2 kN cell force). The test speed of the crosshead was set at 1 mm/min. Data were collected for at least three specimens for each hydrogel composition with 0.5% accuracy of force measurement and position accuracy of 0.001 mm. Hydrogel disks (15 mm diameter and 4 mm height) with flat and parallel surfaces were prepared and were allowed to swell in saline water solution at 37 °C for 25 h before the test. Compressive strength and strain were determined for fully swollen hydrogel samples.


3. Results and discussion 3.1. Obtaining of silk fibroin/polyacrylamide hydrogels Silk fibroin/polyacrylamide hydrogels were obtained by polymerization reaction of acrylamide and methylenebisacrylamide in silk fibroin solution by a redox initiating system. The compositions of the semi-interpenetrating networks were as follows: 0/100, 10/90, 20/ 80, 30/70, 40/60, 50/50, 70/30, 80/20, and 90/10 in volumetric ratios. The polymerization and cross-linking reactions were almost complete after 4 days leading to three-dimensional polymeric networks where silk fibroin was physically entrapped. The last three compositions were gelly-like masses and were not furthermore studied due to low physical integrity. 3.2. ATR–FTIR characterization The results of the FTIR–ATR spectra gave us the specific absorbance wavelengths of the specific bonds which appeared in the silk fibroin/polyacrylamide hydrogels, confirming the structure of the new materials (Fig. 1). Typical peaks of silk fibroin are 1621, 1515 and 1231 cm− 1, characteristic for amide I (C_O stretching), amide II (NH deformation and C\N stretching) and amide III (C\N stretching and N\H deformation) [2]. In the case of polyacrylamide, there is a peak with two spikes assigned to N\H stretch and a strong C_O stretch having also two spikes (1649 and 1605 cm− 1). The FTIR analysis of the hydrogels confirmed the spectral modification as a function of composition. As expected, the presence of an increasing amount of silk fibroin within the hydrogel is associated with a shifting toward right of the signal from 3332 cm− 1 (polyacrylamide) to 3286 cm− 1 (RSF/PAA, 10/90) and 3289 cm − 1 (RSF/PAA, 50/50). The characteristic peak for OH and NH groups becomes broader as the concentration of silk fibroin increases in the hydrogel. Another significant spectral modification is present between 1700 and 1500 cm − 1 (amide I + II). Polycrylamide spectrum shows a peak with two spikes (1649 and 1605 cm − 1) characteristic for C_O vibrations from primary amide. As the silk fibroin concentration increases this peak with two spikes is maintained and the characteristic peak for amide II appears between1544 and 1533 cm − 1. This peak is clearly the contribution of the silk fibroin present in the hydrogel. This shifting of the amides is characteristic for β-sheet structure of silk fibroin within the hydrogel. The main conclusion is that the polyacrylamide plays a major role in the transformation of random conformation of silk fibroin to a more organized β-sheet structure. 3.3. Swelling measurements The most important property of a hydrogel is its ability to absorb and hold an amount of solvent in its network structure. The equilibrium swelling of a hydrogel is a result of the balance of osmotic forces determined by the affinity to the solvent and network elasticity. The two most important factors controlling the extent of swelling are the hydrophilicity of polymer chains and the cross-linking density. Hydrogel properties depend strongly on the degree of cross-linking, the chemical composition of the polymer chains, and the interactions of the network and surrounding liquid. Fig. 2 shows the water swelling behavior of the PAA and RSF/PAA hydrogels. As can be seen in Fig. 2, the water swelling occurred rapidly, reaching equilibrium of water uptake in about 24 h. The swelling degree increases with the decrease of the acrylamide content within the hydrogel: 50/50 composition showed a maximum swelling degree of 689%, 30/70 had a swelling degree of 574%, whereas the value of the swelling degree for pure polyacrylamide gel was 273%. This behavior could be explained by the higher pore diameter for 50/50 hydrogels as compared to pure PAA which exhibited lower pore diameter.


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Fig. 1. FTIR–ATR spectra for silk fibroin (RSF), polyacrylamide (PAA) and silk fibroin/polyacrylamide hydrogels (RSF/PAA).

Further, the swelling mechanism was evaluated by Eq. (1). Here, by plotting ln f versus ln t, we may calculate the swelling coefficient n as the slope of the linear graph. It is known that the swelling process could be controlled by a Fickian-type mechanism, by relaxation of the chain or by both mechanisms depending on composition. The values of n were below 0.5, which means a diffusion controlled process (Fickian mechanism). With the decrease of PAA content within

the hydrogel the value of n also decreases to 0.2 for 50/50 RSF/PAA hydrogel composition. 3.4. Morphology of silk fibroin/polyacrylamide hydrogels SEM analysis showed the porous RSF/PAA hydrogels for various compositions with close-ended pores. We may notice the shape of the pores which varies with hydrogel composition. At high polyacrylamide content the shape tends to be tubular with longitudinal orientation (Fig. 3). While the polyacrylamide content decreases the pores become more spherical. The decrease of the polyacrylamide content led to higher pores upon lyophilization due to the decrease of the cross-linking density within the network. Thus the water content increased with silk fibroin concentration leading finally to higher pores upon lyophilization process. 3.5. Mineralization assay

Fig. 2. Swelling degrees in saline solution at 37 °C for RSF/PAA hydrogels.

The mineralization capacity of the semi-interpenetrating RSF/PAA hydrogels was assessed through SEM analysis. All the hydrogels were uniformly covered with a mineral layer whose morphology strongly depended on hydrogels composition (Fig. 4). The mineral phase was composed of microglobule type elementary features. A higher content of silk fibroin within the hydrogel (30/70 and 50/50) led to a more

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Fig. 3. SEM microphotographs for hydrogel cross-section morphology revealing pores for RSF/PAA network with A) PAA; B) 20/80; C) 30/70; D) 50/50.

uniform cover of the surface with mineral phase, a higher number of microglobules and the decrease of the their size. The elementary features became needle-like structures embedded within 50/50 hydrogel. As shown in Fig. 4, the mineral phase emerged from the hydrogel surface. As the silk fibroin content decreased the morphology of the hydrogels changed. The features of the mineral phase became elementary platelets (in the case of pure polyacrylamide) and

the number of microglobules decreased with the increase in polyacrylamide content in the hydrogel. EDS analysis clearly identified Ca and P onto the surfaces of RSF/ PAA hydrogels. The Ca/P molar ratios ranged between 1.5 and 1.7 for all the hydrogels except pure polyacrylamide. In this case the value of Ca/P ratio (1.08) revealed other types of calcium phosphates onto PAA surface (probably brushite type crystals).

Fig. 4. SEM microphotographs showing the morphology of mineral deposits onto the surface for RSF/PAA hydrogels: A) PAA; B) 20/80; C) 30/70; D) 50/50.


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Fig. 7. Viability of cells cultured for 72 h in medium containing only MEM (control) and in MEM containing hydrogel extracts.

Fig. 5. XRD patters for RSF/PAA hydrogels incubated in SBF1x at 37 °C for 14 days.

Fig. 5 shows XRD patterns of silk fibroin hydrogels soaked in SBF1x for 14 days by comparison with a pure hydrogel before soaking. The diffraction peaks situated at 2 θ angles of 26.0 31.9, 39.5 and 49.6 in the RSF/PAA hydrogels soaked in SBF1x could be clearly seen. All these diffraction peaks are ascribed to hydroxyapatite deposited onto the surfaces of RSF/PAA hydrogels and their intensities increase with increase in fibroin content (the highest intensity for 50/50 composition).

The mineralization of RSF/PAA networks is intrinsically favored by the 3-D structure of the hydrogel and by the micro- and macroporosity induced by lyophilization. Nevertheless, it is clear that the presence of silk fibroin influences the mineralization of hydrogels as apatite-like crystals. 3.6. Cytotoxicity tests on extracts Fig. 5 shows phase contrast micrographs obtained after the examination of fibroblast cultures treated for 24, 48 and 72 h with RSF/ PAA:50/50, RSF/PAA:20/80 and RSF/PAA:10/90 extracts. Note that the cells retain their phenotypic appearance during treatment, cell proliferation occurs during the incubation, and cell density in cultures

Fig. 6. Phase contrast morphology of CCD-1070Sk cells at 24, 48 and 72 h post-seeding, grown in standard conditions (control) and in medium with RSF/PAA extracts (× 10).

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Table 1 Compressive properties for silk/polyacrylamide hydrogels as a function of composition. RSF/PAA hydrogel composition

Compressive strength, kPa

Compressive strain, %


151.3 ± 2.12 147 ± 9.19 117 ± 7.07 64 ± 1.41 62 ± 0.35

46.7 58.12 60.01 63.5 72.5

treated with hydrogels with a higher content of silk fibroin (RSF/PAA: 50/50, RSF/PAA: 20/80) is similar to that of the control experiment. MTT assay performed after 72 h of incubation of fibroblasts with hydrogel extracts shows decreased cell viability compared with control experiment with 7% (p b 0.01), 10.7% (p b 0.001) and 25.9% (p b 0.01) if the treatment with RSF/PAA 50/50, 20/80 and 10/90 respectively (Fig. 6). LDH release into the environment of the cytosol is a measure of plasma membrane lysis. Considering 100% release of LDH in cell culture for control, data presented in Fig. 7 shows that incubation of cells with extracts of hydrogels RSF/PAA increases cell lysis with 9.7% (p b 0.05), 14.5% (p b 0.01) and 21.4% (p b 0.001) with the decrease of the silk fibroin content: 50%, 20% and 10% respectively. Biological investigations have shown that gels RSF/PAA with high volumetric percentage content in silk fibroin did not show a significant in vitro cytotoxicity and could be used for developing biomedical applications. 3.7. Mechanical properties RSF/PAA hydrogels should have adequate mechanical properties to be used for load-bearing biomedical applications. Compressive tests were performed on fully hydrated hydrogels specimens. Results of compressive strength and compressive strain are shown in Table 1 for silk fibroin/polyacrylamide hydrogels with various compositions. Compressive strength of hydrogels shows a decreasing behavior with the increase in fibroin content. This trend is however normal as the hydrogels become less dense and more soft with the increase of fibroin content. At high polyacrylamide content the network is rigid and compact so more stress is required to fail. This mechanical behavior could be also correlated with the swelling ability and morphology of RSF/PAA hydrogels. As we can see from Fig. 2 the increase of fibroin content (50/50) leads to a high swelling degree (689%) so the water molecules act as a plasticizer thus decreasing the compressive strength of hydrogels (Fig. 8). Although the 50/50 hydrogel composition has the lowest strength of 62 kPa it undergoes the highest deformation (72.5% compressive strain) as compared to pure PAA

Fig. 9. Stress–strain curves for full swollen RSF/PAA hydrogels with various compositions.

hydrogel (151.33 kPa compressive strength at 46.7% compressive strain). Although it is clear that the mechanical properties of silk fibroin hydrogels were improved by the introduction of the crosslinked polyacrylamide matrix, the compressive strength could be further improved (Fig. 9). 4. Conclusions This work emphasizes the use of silk fibroin embedded in a 3-D polyacrylamide networks as mineralization scaffolds. Such silk fibroin hydrogels could be easily obtained and their properties could be manipulated by varying the composition. We have shown the good mineralization ability of the porous hydrogels incubated in SBF1x. The hydrogels were uniformly covered with a mineral layer whose morphology depended on their composition. A higher content of silk fibroin within the hydrogel (30/70, 50/50) led to a more uniform cover of the surface with apatite-like mineral phase. These findings support the proposition that novel organic–inorganic hybrids analogous to bone structure can be produced under biomimetic conditions utilizing hydrogels with fibroin content as a substrate for the deposition of bone-like apatite crystals. Acknowledgments This work was supported by CNCSIS-UEFISCSU, project number PNII-IDEI code 248/2010. References

Fig. 8. Assessment of LDH leakage from cells cultured for 72 h in medium containing only MEM (control) and in MEM containing hydrogel extracts.

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