Bombyx mori silk fibroin membranes as potential substrata for epithelial constructs used in the management of ocular surface disorders

Membranes were prepared from fibroin, a protein isolated from the domesticated silkworm (Bombyx mori) silk, and evaluated as a potential substratum for corneal limbal epithelial cells. These membranes (i.e., B. mori silk fibroin [BMSF] membranes) were cast from dialyzed solutions of fibroin protein (4% w/v) dispensed into 35-mm-diameter culture dishes and dried at room temperature (23-24 degrees C). The resulting material was transparent, easy to handle, and supported levels of human limbal epithelial (HLE) cell growth comparable to that observed on tissue culture plastic. Remarkably, these results were obtained utilizing a commercial serum-free medium (CnT-20) designed for the ex vivo expansion of corneal epithelial progenitor cells. The potential benefits of serum proteins on this culture system were examined through addition of fetal bovine serum (FBS) either to fibroin stocks prior to membrane casting or by supplementation of the CnT-20 medium. Membranes cast in the presence of FBS displayed increasing opacity and induced little change in HLE growth. Supplementation of CnT-20 medium with FBS deterred cell growth on all substrata, including tissue culture plastic control substrata. The remarkable properties of BMSF membranes demonstrated under serum-free conditions warrant investigation of this material as a substratum in the creation of tissue-engineered constructs for the restoration of diseased or damaged ocular surface.     

Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes

Membranes prepared from a protein, fibroin, isolated from domesticated silkworm (Bombyx mori) silk, support the cultivation of human limbal epithelial (HLE) cells and thus display significant potential as biomaterials for ocular surface reconstruction. We presently extend this promising avenue of research by directly comparing the attachment, morphology and phenotype of primary HLE cell cultures grown on fibroin to that observed on donor amniotic membrane (AM), the current clinical standard substrate for HLE transplantation. Fibroin membranes measuring 6.3 ± 0.5 μm (mean ± sd) in thickness and permeable to FITC dextran of a molecular weight up to 70 kDa, were used. Attachment of HLE cells to fibroin was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. Nevertheless, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (K3/K12 expression) and displayed a comparable number and distribution of ΔNp63(+) progenitor cells to that seen in cultures grown on AM. These results support the suitability of membranes constructed from Bombyx mori silk fibroin as substrata for HLE cultivation and encourage progression to studies of efficacy in preclinical models.     

犬骨髓基质细胞和蚕丝丝素的体外生物相容性

目的 探讨体外培养的犬骨髓基质细胞(BMSCs)与蚕丝丝素材料的生物相容性,寻找BMSCs组织工程化神经的支架材料.方法 通过差速贴壁法体外分离、培养犬骨髓基质细胞,与丝素共培养后,通过光镜(经免疫荧光染色)、扫描电镜观察细胞在丝素上黏附和生长情况.利用丝素浸出液培养BMSCs后,通过透射电镜观察细胞内部超微结构,用四甲基偶氮唑盐(MTT)法检测丝素、羟基磷灰石、有机锡浸出液和普通IMDM完全培养基培养细胞12、24、48、72h和7d的细胞活力,每组重复12次.流式细胞术检测丝素浸出液培养BMSCs的细胞周期及表型,实验重复3次.结果 通过光镜、扫描电镜观察,发现BMSCs 紧紧黏附于丝素材料,并沿着丝素纤维延伸,黏附于丝素纤维的细胞呈圆形、椭圆形及呈梭形.与普通IMDM完全培养基培养的细胞相比,透射电镜下可见丝素浸出液培养后的BMSCs内部结构未见异常;MTT检测丝素和羟基磷灰石浸出液对骨髓基质细胞的活力无显著性影响(P＞0.05);流式细胞术检测丝素浸出液对骨髓基质细胞周期和表型无明显影响.结论 蚕丝丝素材料与犬BMSCs生物相容性好,且未见丝素对BMSCs有毒性作用,可作为BMSCs组织工程化神经的支架材料.     

Porous Silk Fibroin Film as a Transparent Carrier for Cultivated Corneal Epithelial Sheets

Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin β1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium.     

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.     

Regenerated silk fibroin scaffold and infrapatellar adipose stromal vascular fraction as feeder-layer: a new product for cartilage advanced therapy

Articular cartilage has limited repair and regeneration potential, and the scarcity of treatment modalities has motivated attempts to engineer cartilage tissue constructs. The use of chondrocytes in cartilage tissue engineering has been restricted by the limited availability of these cells, their intrinsic tendency to lose their phenotype during the expansion, as well as the difficulties during the first cell adhesion to the scaffold. Aim of this work was to evaluate the intra-articular adipose stromal vascular fraction attachment on silk fibroin scaffold to promote chondrocytes adhesion and proliferation. Physicochemical characterization has demonstrated that three-dimensionally organized silk fibroin scaffold is an ideal biopolymer for cartilage tissue engineering; it allows cell attachment, scaffold colonization, and physically cell holding in the area that must be repaired; the use of adipose-derived stem cells is a promising strategy to promote adhesion and proliferation of chondrocytes to the scaffold as an autologous human feeder layer.     

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.     

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.     

Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering

The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.     

Engineered silk fibroin protein 3D matrices for in vitro tumor model

3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs.     

Oriented nanofibrous silk as a natural scaffold for ocular epithelial regeneration

The aim of this study was to develop nanofibrous silk substrates for limbal stem cell expansion that can serve as a potential alternative substrate to replace human amniotic membrane. The human limbal stem cell was used to evaluate the biocompatibility of substrates (random and oriented nanofibrous mats, and human amniotic membrane) based on their phenotypic profile, viability, proliferation, and attachment ability. Biocompatibility results indicated that all substrates were highly biocompatible, as limbal stem cells could favorably attach and proliferate on the nanofibrous surfaces. Microscopic figures showed that the human limbal stem cells were firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the nanofibers and successfully formed a three-dimensional corneal epithelium, which was viable for 15 days. Immunocytochemistry and real-time PCR results revealed no change in the expression profile of limbal stem cells grown on nanofibrous substrates when compared to those grown on human amniotic membrane. In addition, electrospun nanofibrous silk substrates especially oriented mat provides not only a milieu supporting limbal stem cells expansion, but also serve as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to amniotic membrane.     

Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro

Silk-based materials have been used in the field of bone or ligament tissue engineering. In order to explore the feasibility of using purified silk fibroin to construct artificial nerve grafts, it is necessary to evaluate the biocompatibility of silk fibroin material with peripheral nerve tissues and cells. We cultured rat dorsal root ganglia (DRG) on the substrate made up of silk fibroin fibers and observed the cell outgrowth from DRG during culture by using light and electron microscopy coupled with immunocytochemistry. On the other hand, we cultured Schwann cells from rat sciatic nerves in the silk fibroin extract fluid and examined the changes of Schwann cells after different times of culture. The results of light microscopy, MTT test and cell cycle analysis showed that Schwann cells cultured in the silk fibroin extract fluid showed no significant difference in their morphology, cell viability and proliferation as compared to that in plain L15 medium. Furthermore, no significant difference was found in expression of the factors secreted by Schwann cells, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and S-100, between Schwann cells cultured in the silk fibroin extraction fluid and in plain L15 medium by the aid of immunocytochemistry, RT-PCR and Western analysis. Collectively, these data indicate that silk fibroin has good biocompatibility with DRG and is also beneficial to the survival of Schwann cells without exerting any significant cytotoxic effects on their phenotype or functions, thus providing an experimental foundation for the development of silk fibroin as a candidate material for nerve tissue engineering applications.     

Aspects of the in vitro bioactivity of hydraulic calcium (alumino)silicate cement

Silk fibroin-based biomaterials have recently found increasing applications in the tissue-engineering field including the generation of artificial nerve guides for peripheral nerve repair. The aim of this study was to investigate the suitability of silk fibroin as a candidate biomaterial for central nervous system (CNS) therapy. We found that substrates made up of silk fibroin fibers supported the survival and growth of the attached hippocampal neurons by using morphological observation. We also cultured the hippocampal neurons in silk fibroin extract for different times, and observed no significant difference occurring in their morphology, cell viability for these cultured hippocampal neurons as compared to those cultured in plain neuronal culture medium. Moreover, immunocytochemistry, RT-PCR, and Western blot analysis revealed that no significant difference was found in mRNA or protein levels of the growth-associated molecules, such as brain-derived neurotrophic factor, growth-associated protein-43, neurofilament, nerve growth factor, and nerve growth factor-receptor P75, between the hippocampal neurons cultured in the silk fibroin extract and in plain neuronal culture medium. Taken together, all the results demonstrate that silk fibroin has good biocompatibility with primarily cultured hippocampal neurons without any significant cytotoxic effects on their cell phenotype and functions, suggesting a potential possible use of silk fibroin for preparing the tissue-engineered nerve guides or drug delivery vehicles to treat CNS injuries or diseases.     

Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells

Silk fibroin scaffolds were studied as a new biomaterial option for tissue-engineered cartilage-like tissue. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on silk, collagen, and crosslinked collagen scaffolds and cultured for 21 days in serum-free chondrogenic medium. Cells proliferated more rapidly on the silk fibroin scaffolds than on the collagen matrices. The total content of glycosaminoglycan deposition was three times higher on silk as compared to collagen scaffolds. Glycosaminoglycan deposition coincided with overexpression of collagen type II and aggrecan genes. Cartilage-like tissue was homogeneously distributed throughout the entire silk scaffolds, while on the collagen and crosslinked collagen systems tissue formation was restricted to the outer rim, leaving a doughnut appearance. Round or angular-shaped cells resided in deep lacunae in the silk systems and stained positively for collagen type II. The aggregate modulus of the tissue-engineered cartilage constructs was more than 2-fold higher than that of the unseeded silk scaffold controls. These results suggest that silk fibroin scaffolds are suitable biomaterial substrates for autologous cartilage tissue engineering in serum-free medium and enable mechanical improvements along with compositional features suitable for durable implants to generate or regenerate cartilage.     

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.     

再生多孔丝素膜创面局部运用激活不同部位T细胞的作用

背景：丝素蛋白是从蚕丝中提取的天然高分子纤维蛋白,其具有良好的理化特性及生物相容性。而目前国内外有关再生多孔丝素膜植入体内后对T淋巴细胞的激活作用研究未见报道。 目的：观察再生多孔丝素膜植入大鼠局部创面后对外周血单个核细胞、脾脏及胸腺中T淋巴细胞的激活作用。 方法：切除SD大鼠背部皮肤建立2 cm×2 cm创面,随机分为2组：实验组覆盖经预处理的再生多孔丝素膜,将已切除皮肤的表皮盖在丝素膜上进行缝合,对照组仅覆盖自身表皮层。于术后第3,14,28,56,90天观察创面愈合及丝素膜的残留情况,采用双色免疫荧光及流式细胞术分析大鼠外周血、脾脏及胸腺中CD3⁺CD25⁺/ CD3⁺T细胞百分率。 结果与结论：植入早期,两组均可见局部、轻微的炎症细胞浸润,主要为淋巴细胞;至28 d后,炎症细胞逐渐减少,并且在整个动态观察过程中,两组结果一致。实验组外周血中活化T细胞在14 d之前呈一过性升高,随后开始下降,至28 d以后,活化T细胞水平趋于稳定,与对照组无明显差异(P > 0.05);脾脏与胸腺T细胞均有少量活化,随后即开始下降并趋于稳定,胸腺中CD3⁺CD25⁺/ CD3⁺T细胞的阳性率较脾脏微高,与对照组均无明显差异(P > 0.05)。说明再生多孔丝素膜对T淋巴细胞的激发作用较小,引发机体细胞免疫应答的能力较弱。     

固相基质表面von Willebrand Factor抗体的固定化研究

以丝素蛋白膜和聚乳酸膜为基质,采用辉光放电等离子体处理技术和共价交联的方法对vonWillebrand factor(vWf)抗体进行了固定化研究。利用抗体过剩法和酶联免疫实验对固定化效率进行了评价,固定化抗体的活性采用体外凝血时间(APTT,TT和PT)测定进行检测。结果显示固定化效率最高可以达到23.88%,酶联免疫实验均显示阳性,APTT、TT项有明显延长,有的还超出了仪器的测量范围,由此说明,通过这种方法可以有效地对vWf抗体进行共价固定化。本研究拓宽了抗体固定化技术的应用范围,为血管性假血友病的早期快速诊断仪器或试剂盒的研制打下技术基础,同时为其它凝血因子抗体相关的生物材料研究与开发提供一种新思路。     

Silk-based biomaterials

Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined silkworm silk fibers and films suggest that the core silk fibroin fibers exhibit comparable biocompatibility in vitro and in vivo with other commonly used biomaterials such as polylactic acid and collagen. Furthermore, the unique mechanical properties of the silk fibers, the diversity of side chain chemistries for 'decoration' with growth and adhesion factors, and the ability to genetically tailor the protein provide additional rationale for the exploration of this family of fibrous proteins for biomaterial applications. For example, in designing scaffolds for tissue engineering these properties are particularly relevant and recent results with bone and ligament formation in vitro support the potential role for this biomaterial in future applications. To date, studies with silks to address biomaterial and matrix scaffold needs have focused on silkworm silk. With the diversity of silk-like fibrous proteins from spiders and insects, a range of native or bioengineered variants can be expected for application to a diverse set of clinical needs.     

Human corneal endothelial cell growth on a silk fibroin membrane

Tissue engineering of the cornea could overcome shortages of donor corneas for transplantation and improve quality. Our aim was to grow an endothelial layer on a substratum suitable for transplant. Silkworm (Bombyx mori) fibroin was prepared as 5 μm thick transparent membranes. The B4G12 cell line was used to assess attachment and growth of human corneal endothelial cells on fibroin and compare this with a reference substratum of tissue-culture plastic. To see if cell attachment and proliferation could be improved, we assessed coatings of collagen IV, FNC Coating Mix(?) and a chondroitin sulphate-laminin mixture. All the coatings improved the final mean cell count, but consistently higher cell densities were achieved on a tissue-culture plastic rather than fibroin substratum. Collagen-coated substrata were the best of both groups and collagen-coated fibroin was comparable to uncoated tissue-culture plastic. Only fibroin with collagen coating achieved cell confluency. Primary human corneal endothelial cells were then grown using a sphere-forming technique and when seeded onto collagen-coated fibroin they grew to confluency with polygonal morphology. We report the first successful growth of primary human corneal endothelial cells on coated fibroin as a step in evaluating fibroin as a substratum for the transplantation of tissue-constructs for endothelial keratoplasty.