Osteoblast: Osteoclast co-cultures on silk fibroin, chitosan and PLLA films

This study investigates the growth of a co-culture of osteoblasts and osteoclasts on four different types of degradable biomaterials with bone tissue engineering potential. Single or co-cultures of osteoblasts and osteoclasts (used at a ratio of 1:100 osteoblast:osteoclasts) were cultured on vapour stabilised silk fibroin, methanol stabilised silk fibroin, chitosan and poly (l lactic acid) (PLLA) films for 10 days. Osteoclast differentiation was determined by tartrate resistant acid phosphatase (TRAP) staining, total cell number by a picogreen DNA assay, cell morphology by scanning electron microscopy (SEM) and the material topography by atomic force microscopy (AFM). Samples were also monitored for degradation by differential scanning calorimetry (DSC) and fourier transform infrared (FTIR). Results demonstrated that vapour stabilised silk fibroin, methanol stabilised silk fibroin and chitosan all support the growth of osteoblasts and osteoclasts in both single and co-cultures. PLLA showed poor osteoclast differentiation in both single and co-cultures but supported osteoblast attachment and proliferation. Both silk fibroin materials showed sign of early degradation in the ten-day period, but very little change was seen in chitosan and PLLA samples. This study indicates that this novel co-culture approach for bone tissue engineering may be possible if scaffolds are created from silk fibroin or chitosan.     

In vivo degradation of three-dimensional silk fibroin scaffolds

Three-dimensional porous scaffolds prepared from regenerated silk fibroin using either an all-aqueous process or a process involving an organic solvent, hexafluoroisopropanol (HFIP), have shown promise in cell culture and tissue engineering applications. However, their biocompatibility and in vivo degradation have not been fully established. The present study was conducted to systematically investigate how processing method (aqueous vs. organic solvent) and processing variables (silk fibroin concentration and pore size) affect the short-term (up to 2 months) and long-term (up to 1 year) in vivo behavior of the protein scaffolds in both nude and Lewis rats. The samples were analyzed by histology for scaffold morphological changes and tissue ingrowth, and by real-time RT-PCR and immunohistochemistry for immune responses. Throughout the period of implantation, all scaffolds were well tolerated by the host animals and immune responses to the implants were mild. Most scaffolds prepared from the all-aqueous process degraded to completion between 2 and 6 months, while those prepared from organic solvent (hexafluoroisopropanol (HFIP)) process persisted beyond 1 year. Due to widespread cellular invasion throughout the scaffold, the degradation of aqueous-derived scaffolds appears to be more homogeneous than that of HFIP-derived scaffolds. In general and especially for the HFIP-derived scaffolds, a higher original silk fibroin concentration (e.g. 17%) and smaller pore size (e.g. 100-200microm) resulted in lower levels of tissue ingrowth and slower degradation. These results demonstrate that the in vivo behavior of the three-dimensional silk fibroin scaffolds is related to the morphological and structural features that resulted from different scaffold preparation processes. The insights gained in this study can serve as a guide for processing scenarios to match desired morphological and structural features and degradation time with tissue-specific applications.     

Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications

This study describes the developmental physicochemical properties of silk fibroin scaffolds derived from high-concentration aqueous silk fibroin solutions. The silk fibroin scaffolds were prepared with different initial concentrations (8, 10, 12 and 16%, in wt.%) and obtained by combining the salt-leaching and freeze-drying methodologies. The results indicated that the antiparallel β-pleated sheet (silk-II) conformation was present in the silk fibroin scaffolds. All the scaffolds possessed a macro/microporous structure. Homogeneous porosity distribution was achieved in all the groups of samples. As the silk fibroin concentration increased from 8 to 16%, the mean porosity decreased from 90.8 ± 0.9 to 79.8 ± 0.3% and the mean interconnectivity decreased from 97.4 ± 0.5 to 92.3 ± 1.3%. The mechanical properties of the scaffolds exhibited concentration dependence. The dry state compressive modulus increased from 0.81 ± 0.29 to 15.14 ± 1.70 MPa and the wet state dynamic storage modulus increased by around 20- to 30-fold at each testing frequency when the silk fibroin concentration increased from 8 to 16%. The water uptake ratio decreased with increasing silk fibroin concentration. The scaffolds present favorable stability as their structure integrity, morphology and mechanical properties were maintained after in vitro degradation for 30 days. Based on these results, the scaffolds developed in this study are proposed to be suitable for use in meniscus and cartilage tissue-engineered scaffolding.     

The effect of hyaluronic acid on silk fibroin conformation

The molecular conformation of silk fibroin drastically changes the physical properties of this biomaterial. Herein, we investigated the capacity of hyaluronic acid to modify the conformational transition of silk fibroin into its crystalline beta-sheet form. For this aim, matrices composed of these two polymers were prepared and studied. Instrumental analysis confirmed the presence of two intermixed phases: one of pure hyaluronic acid, and another consisting of a molecular dispersion of silk fibroin and hyaluronic acid. Studies performed with silk fibroin/hyaluronic acid matrices indicated that hyaluronic acid induces molecular transition of silk fibroin into a beta-sheet structure when incubated in water, and that it synergistically enhances beta-sheet formation together with methanol treatment. The enhancement of beta-sheet content observed for silk fibroin/hyaluronic acid matrices correlated with improved mechanical properties: blended matrices had higher compressive moduli and higher breaking strengths than pure silk fibroin matrices. These new properties, together with the capacity of silk fibroin/hyaluronic acid to form partially insoluble matrices without any treatment with organic solvents, make this blend composition an interesting material for biomedical applications.     

Poly(epsilon-caprolactone-co-D,L-lactide) /silk fibroin composite materials: preparation and characterization

Poly(epsilon-caprolactone-co-D,L-lactide) copolymers with 10, 30, and 50% by weight of silk particles (size range: 5-250 microm) derived from Bombyx mori were blended in acetone solution. After evaporation of the solvent, the morphology, thermal behavior, and mechanical properties of the composites were examined. The composites were transparent and the silk fibroin particles were homogeneously distributed within the composite structure. The particles appeared as bright reflected images under the optical microscope, suggesting that they were in a crystalline state. DSC thermograms of the composites revealed that the glass transition of the matrix was at ca. -18 degrees C. Degradation of the silk fibroin occurred beyond 270 degrees C. The decomposition temperatures and degradation rate decreased with increasing silk fibroin content as revealed by TGA analysis. FTIR spectra of the composites showed absorption bands at 1730 and 1088 cm(-1) for the copolymer and at 3273 and 1617 cm(-1) for the silk fibroin. Although the characteristic lines of poly(epsilon-caprolactone-co-D,L-lactide) were independent of filler concentration. the absorption bands of the beta-sheet form of the silk fibroin increased slightly due to the interaction of silk fibroin with the copolymer.     

Silk coatings on PLGA and alginate microspheres for protein delivery

Bombyx mori silk fibroin self-assembles on surfaces to form ultrathin nanoscale coatings based on our prior studies using layer-by-layer deposition techniques driven by hydrophobic interactions between silk fibroin protein molecules. In the present study, poly(lactic-co-glycolic acid) (PLGA) and alginate microspheres were used as substrates and coated with silk fibroin. The coatings were visualized by confocal laser scanning microscopy using fluorescein-labeled silk fibroin. On PLGA microspheres, the coating was approximately 1microm and discontinuous, reflecting the porous surface of these microspheres determined by SEM. In contrast, on alginate microspheres the coating was approximately 10microm thick and continuous. The silk fibroin penetrated into the alginate gel matrix. The silk coating on the PLGA microspheres delayed PLGA degradation. The silk coating on the alginate microspheres survived ethylenediamine tetraacetic acid (EDTA) treatment used to remove the Ca(2+)-cross-links in the alginate gels to solubilize the alginate. This suggests that alginate microspheres can be used as templates to form silk microcapsules. Horseradish peroxidase (HRP) and tetramethylrhodamine-conjugated bovine serum albumin (Rh-BSA) as model protein drugs were encapsulated in the PLGA and alginate microspheres with and without the silk fibroin coatings. Drug release was significantly retarded by the silk coatings when compared to uncoated microsphere controls, and was retarded further by methanol-treated silk coating when compared to silk water-based coatings on alginate microspheres. Silk coatings on PLGA and alginate microspheres provide mechanically stable shells as well as a diffusion barrier to the encapsulated protein drugs. This coating technique has potential for biosensor and drug delivery applications due to the aqueous process employed, the ability to control coating thickness and crystalline content, and the biocompatibility of the silk fibroin protein used in the process.     

Enzymatic degradation behavior of porous silk fibroin sheets

We investigated the degradation behavior of porous silk fibroin sheets by in vitro enzymatic experiments with alpha-chymotrypsin, collagenase IA, and protease XIV. With 1.0 U/ml protease XIV, 70% of a silk fibroin sheet was degraded within 15 days at 37 degrees C. When the fibroin sheet was exposed to collagenase IA, the amount of Silk II crystalline structure in the sheets decreased slightly, and a small amount of Silk I crystalline structure was formed. When protease XIV was used, almost all Silk II disappeared, but the crystallinity increased overall because the amount of Silk I increased. During digestion with protease XIV, the pore size of the fibroin sheets increased with increasing degradation time, until the sheets finally collapsed and became totally shapeless. The average molecular weight of the products after degradation with the three enzymes followed the order protease XIV < collagenase IA < alpha-chymotrypsin. More than 50% of the products resulting from degradation with protease XIV were free amino acids.     

Biodegradation behavior of silk fibroin membranes in repairing tympanic membrane perforations

Silk fibroin (SF) from silkworms has been widely studied as a biomaterial. The degradation behavior of silk biomaterials is important for medical applications, but few studies have examined long-term degradation behavior in vivo. In this study, we investigated the degradation behavior of SF membranes in vitro and in vivo. For the in vitro assay, we observed degradation of silk membranes in phosphate buffered saline, culture media, and an enzyme (proteinase K) solution. In the proteinase K solution, 80% of the silk membranes degraded within 10 days. Silk membranes exhibited no cytotoxicity toward L929 cells and rat tissues. To investigate the degradation of silk membranes in vivo, they were implanted subcutaneously in rats and harvested 19 months after surgery. Scanning electron microscopy imaging and histological analysis of silk membrane explants showed that they broke into several pieces after 16 months. Results show that silk membranes are biocompatible and display excellent long-term degradation behavior when used as biomaterials.     

Sonication-induced gelation of silk fibroin for cell encapsulation

Purified native silk fibroin forms beta-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of beta-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical cross-links responsible for gel stabilization. K(+) at physiological concentrations and low pH promoted gelation, which was not observed in the presence of Ca(2+). The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5-2 h. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol-gel transitions, such as for cell encapsulation.     

Poly(ε-caprolactone-co-D,L-lactide)/silk fibroin composite materials: Preparation and characterization

Poly(ε-caprolactone-co-D,L-lactide) copolymers with 10, 30, and 50% by weight of silk particles (size range: 5-250 μm) derived from Bombyx mori were blended in acetone solution. After evaporation of the solvent, the morphology, thermal behavior, and mechanical properties of the composites were examined. The composites were transparent and the silk fibroin particles were homogeneously distributed within the composite structure. The particles appeared as bright reflected images under the optical microscope, suggesting that they were in a crystalline state. DSC thermograms of the composites revealed that the glass transition of the matrix was at ca. -18°C. Degradation of the silk fibroin occurred beyond 270°C. The decomposition temperatures and degradation rate decreased with increasing silk fibroin content as revealed by TGA analysis. FTIR spectra of the composites showed absorption bands at 1730 and 1088 cm-1 for the copolymer and at 3273 and 1617 cm^-1 for the silk fibroin. Although the characteristic lines of poly(ε-caprolactone-coD,L-lactide) were independent of filler concentration, the absorption bands of the β-sheet form of the silk fibroin increased slightly due to the interaction of silk fibroin with the copolymer.     

Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends

Adult bone marrow derived mesenchymal stem cells are undifferentiated, multipotential cells and have the potential to differentiate into multiple lineages like bone, cartilage or fat. In this study, polyelectrolyte complex silk fibroin/chitosan blended porous scaffolds were fabricated and examined for its ability to support in vitro chondrogenesis of mesenchymal stem cells. Silk fibroin matrices provide suitable substrate for cell attachment and proliferation while chitosan are promising biomaterial for cartilage repair due to it’s structurally resemblance with glycosaminoglycans. We compared the formation of cartilaginous tissue in the silk fibroin/chitosan blended scaffolds with rat mesenchymal stem cells and cultured in vitro for 3 weeks. Additionally, pure silk fibroin scaffolds of non-mulberry silkworm, Antheraea mylitta and mulberry silkworm, Bombyx mori were also utilized for comparative studies. The constructs were analyzed for cell attachment, proliferation, differentiation, histological and immunohistochemical evaluations. Silk fibroin/chitosan blended scaffolds supported the cell attachment and proliferation as indicated by SEM observation, Confocal microscopy and metabolic activities. Alcian Blue and Safranin O histochemistry and expression of collagen II indicated the maintenance of chondrogenic phenotype in the constructs after 3 weeks of culture. Glycosaminoglycans and collagen accumulated in all the scaffolds and was highest in silk fibroin/chitosan blended scaffolds and pure silk fibroin scaffolds of A. mylitta. Chondrogenic differentiation of MSCs in the silk fibroin/chitosan and pure silk fibroin scaffolds was evident by real-time PCR analysis for cartilage-specific ECM gene markers. The results represent silk fibroin/chitosan blended 3D scaffolds as suitable scaffold for mesenchymal stem cells-based cartilage repair.     

丝素蛋白作为组织工程材料应用于角膜的研究进展

背景：丝素蛋白纤维材料具有透明性、结构可塑性、成分单一性、力学强韧性及生物相容性等特点。 目的：综述国内外丝素蛋白应用于角膜组织工程的研究进展。 方法：由第一作者在标题和摘要中以“silk fibroin, corneal, ocular”或“丝素,角膜”为检索词,检索1980至2011年PubMed及1990至2011年CNKI数据库中关于丝素蛋白角膜的文章。 结果与结论：从天然蚕丝中提取的高分子丝素蛋白,因其良好的生物相容性、独特的力学性能、光学透明性及降解速率可控性,既可以单独应用于角膜组织结构的重建,又可与其他组织材料联合应用,成为角膜组织工程学应用的理想材料。现已证明多种角膜细胞可在丝素纤维膜上良好生长,但体外培养的细胞应用于动物模型的相关研究较少;此外丝素蛋白材料植入角膜内对其产生何种影响的研究数据较缺乏,这些均是亟待解决的问题。     

丝素蛋白溶液诱导成骨分化及其性能评价

BACKGROUND: Silk fibroin, as a kind of high-performance biomaterial, has been widely used to construct scaffolds in bone tissue engineering. However, whether silk fibroin itself holds osteoinductive ability has not been reported yet. OBJECTIVE: To investigate the impact of different concentrations of silk fibroin solution on the proliferation and differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. METHODS: Silk fibroin and BMSCs were respectively isolated from silkworm cocoon and rat tibia, and were identified. Then, BMSCs were cultured in different concentrations of silk fibroin solution (0.01%, 0.05% and 0.1%), and the cell proliferation and the alkaline phosphatase activity were detected at different time points. RESULTS AND CONCLUSION: FTIR spectra of the sample extracted from silkworm cocoon showed distinct absorption peaks at 1 653 (amide I), 1 530.5 (amide II) and 1 212.3 cm-1 (amide III), which could be confirmed to be silk fibroin. Thus generated BMSCs showed long fusiform or astral morphology, positive for representative markers (CD29, CD44 and CD90) relating to mesenchymal stem cells, and could differentiate into osteocytes, chondrocytes and adipocytes under specific induction conditions, which further confirmed the extracted cells were BMSCs. Compared with the control group (without silk fibroin), 0.05% silk fibroin not only significantly promoted the cell adhesion, migration and proliferation, but also enhanced the alkaline phosphatase activity (P < 0.01). With the increasing of the silk fibroin concentrations, the osteodifferentiation capacity of the BMSCs was progressively improved within the range of 0-0.05% and then declined at 0.01% of silk fibroin solutions. These results suggest that silk fibroin can promote osteogenesis, thus providing scientific evidence for developing silk fibroin-based tissue-engineered scaffolds.     

蚕丝丝素纤维的制备及其与骨髓间充质干细胞相容性的研究

组织工程为韧带损伤修复提供了可行途径，但韧带修复对支架材料各方面性能要求都很高。在力学性能方面，不仅要求材料有一定的强度而且需要有良好的韧性。在满足力学性能的同时，支架材料还必须兼具优良的生物相容性。蚕丝作为一种天然生物蛋白质，由于其良好的力学性能显示了在组织工程方面应用的前景。但由于丝胶存在污染问题，因此脱胶成为蚕丝在医学领域应用的首要问题。本实验首先比较了三种脱胶试剂对蚕丝力学性质的影响，选择了影响最小的碳酸钠作为脱胶试剂，进而确定了碳酸钠脱胶的最佳条件为：试剂浓度0．40%，温度90℃，时间为1h。然后在脱胶后的丝紊纤维上种植了大鼠骨髓问充质干细胞（Rat bone marrow mesenchymal stem cells，rMSCs），通过扫描电镜（SEM）、荧光显微镜检测了丝素上细胞的生长情况，结果显示蚕丝具有良好的生物相容性，细胞亲和力。为蚕丝在韧带组织工程方面的进一步应用奠定了基础。     

In Vitro and In Vivo Release of Basic Fibroblast Growth Factor Using a Silk Fibroin Scaffold as Delivery Carrier

Two different solvents were used to prepare two types of silk fibroin scaffolds via the salt-leaching technique, i.e., hexafluoroisopropanol (HFIP) and water. The in vitro release study suggests that the opposite charge between the silk fibroin and basic fibroblast growth factor (bFGF) at physiological pH rendered them to form a complex, and the difference in the solvents used to produce the silk fibroin scaffold did not affect the affinity of silk fibroin to bFGF. However, a higher degradation rate of the aqueous-derived silk fibroin scaffolds provided faster in vitro release kinetics of the bFGF, as compared to the HFIP-derived scaffolds. From the in vivo studies, the use of silk fibroin scaffolds as the carrier matrix enabled the control of the in vivo release of bFGF in a sustained fashion over two weeks, while the majority of the bFGF disappeared within one day after the injection of the bFGF in soluble form. In addition, the in vivo release of bFGF from the silk fibroin scaffolds was not affected by the mode of processing due to their similar degradation behavior in vivo.     

Silk fibroin porous scaffolds by N2O foaming

Silk fibroin has acquired increasing interest for biomedical applications, and namely for the fabrication of scaffolds for tissue engineering, because of its highly positive biological interaction and the possibility to adapt the material to several application requirements by adopting different fabrication methods, in order to make films, sponges, fibers, nets or gels with predictable degradation times. For tissue engineering, in most cases porous scaffolds are required, in some cases possibly in situ forming and therefore fabricated in mild body-compatible conditions. In this work, we present a novel one-step method for the preparation of silk fibroin foams starting from water solutions and using low-pressure nitrous oxide gas as foaming agent. This foaming technique allows preparing fibroin porous scaffolds with easily tunable porosity, in mild processing conditions with the use of a relatively inert foaming agent saturating a fibroin water solution, that could be occasionally injected through a thin needle in the implantation site where expansion and foaming would occur. Optimal foaming processing conditions have been investigated, and the prepared foams have been characterized with Fourier Transform Infrared Spectroscopy (FTIR) compressive mechanical and rheological properties measurements, and by scanning electron microscopy and microCT.     

许旺细胞在不同孔径丝素蛋白支架上的生长

背景：细胞在生物支架上的生长行为受到支架表面形貌、润湿性、孔径及孔隙率等多种因素影响。 目的：观察许旺细胞在不同孔径丝素蛋白支架上的生长情况。 方法：制备大孔径50~60 µm、小孔径10~   20 µm两种多孔丝素材料。选用许旺细胞永生化细胞R3 [33-10ras3]为种子细胞,当细胞在培养瓶底形成致密单层时即可消化细胞并进行接种实验,将许旺细胞悬液种于不同形貌的多孔丝素材料表面。复合培养1周后,扫描电镜观察许旺细胞的生长形态及增殖等情况。 结果与结论：不同孔径丝素材料的表面可见许旺细胞生长情况不一。在10~20 µm孔径材料支架上,细胞浓度较低,细胞表现为特异的双极性形态,细胞与细胞之间或平行排列,或首尾相连成细胞链;细胞与细胞之间或平行排列,或首尾相连成细胞链;在50~ 60 µm孔径丝素材料支架上,细胞浓度较高,细胞多为球形,单个分散在多孔支架表面,或呈现成团成串葡萄样聚集在孔的底部,未延展成双极性形态,只有极少量生长在孔与孔之间嵴上的细胞呈双极样。说明多孔丝素蛋白支架的孔径对许旺细胞的黏附、生长有一定的影响,许旺细胞更适合生长在孔径略大于胞体直径的支架材料上。     

Lyophilized silk fibroin hydrogels for the sustained local delivery of therapeutic monoclonal antibodies

The development of sustained delivery systems compatible with protein therapeutics continues to be a significant unmet need. A lyophilized silk fibroin hydrogel matrix (lyogel) for the sustained release of pharmaceutically relevant monoclonal antibodies is described. Sonication of silk fibroin prior to antibody incorporation avoids exposing the antibody to the sol-gel transition inducing shear stress. Fourier Transform Infrared (FTIR) analysis showed no change in silk structural composition between hydrogel and lyogel or with increasing silk fibroin concentration. Antibody release from hydrogels occurred rapidly over 10 days regardless of silk concentration. Upon lyophilization, sustained antibody release was observed over 38 days from lyogels containing 6.2% (w/w) silk fibroin and above. In 3.2% (w/w) silk lyogels, antibody release was comparable to hydrogels. Swelling properties of lyogels followed a similar threshold behavior. Lyogels at 3.2% (w/w) silk recovered approximately 90% of their fluid mass upon rehydration, while approximately 50% fluid recovery was observed at 6.2% (w/w) silk and above. Antibody release was primarily governed by hydrophobic/hydrophilic silk-antibody interactions and secondarily altered by the hydration resistance of the lyogel. Hydration resistance was controlled by altering β-sheet (crystalline) density of the matrix. The antibody released from lyogels maintained biological activity. Silk lyogels offer an advantage as a delivery matrix over other hydrogel materials for the slow release of the loaded protein, making lyogels suitable for long-term sustained release applications.     

Tissue engineered bulking agent with adipose-derived stem cells and silk fibroin microspheres for the treatment of intrinsic urethral sphincter deficiency

In this study we developed a tissue engineered bulking agent that consisted of adipose-derived stem cells (ADSCs) and silk fibroin microspheres to treat stress urinary incontinence caused by severe intrinsic sphincter deficiency (ISD). ISD models were established by completely transection of the bilateral pudendal nerve (PNT) and confirmed by the decreased leak-point pressure (LPP) and increased lumen area of urethra. Injection of silk fibroin microspheres could recover LPP and lumen area at 4 weeks but its efficacy disappears at 8, 12 weeks. Moreover, it was exciting to find that tissue engineered bulking agent brought long-term efficacy (at 4, 8, 12 weeks post-injection) on the recovery of LPP and lumen area. Concomitantly with the function, tissue engineered bulking agent treated group also improved the urethral sphincter structure as exhibited by better tissue regeneration. The findings showed that silk fibroin microspheres alone could work effectively in short-term, while tissue engineered bulking agent that combined silk fibroin microspheres with ADSCs exhibited promising long-term efficacy. This study developed a new strategy of tissue engineered bulking agent for future ISD therapy.     

Silk fibroin/hyaluronan scaffolds for human mesenchymal stem cell culture in tissue engineering

The design of new bioactive scaffolds mimicking the physiologic environment present during tissue formation is an important frontier in biomaterials research. Herein, we evaluated scaffolds prepared from blends of two biopolymers: silk fibroin and hyaluronan. Our rationale was that such blends would allow the combination of silk fibroin's superior mechanical properties with the biological characteristics of hyaluronan. We prepared scaffolds with porous microstructures by freeze-drying aqueous solutions of silk fibroin and hyaluronan and subsequent incubation in methanol to induce water insolubility of silk fibroin. Hyaluronan acted as an efficient porogenic excipient for the silk fibroin scaffolding process, allowing the formation of microporous structures within the scaffolds under mild processing conditions. Mesenchymal stem cells were seeded on silk fibroin/hyaluronan scaffolds and cultured for three weeks. Histology of the constructs after cell culture showed enhanced cellular ingrowth into silk fibroin/hyaluronan scaffolds as compared to plain silk fibroin scaffolds. In the presence of tissue-inductive stimuli, in vitro stem cell culture on silk fibroin/hyaluronan scaffolds resulted in more efficient tissue formation when measured by glycosaminoglycan and type-I and type-III collagen gene expression, as compared to plain silk fibroin scaffolds. In conclusion, our data encourages further exploration of silk fibroin/hyaluronan scaffolds as biomimetic platform for mesenchymal stem cells in tissue engineering.