Evaluation of silk sericin as a biomaterial: In vitro growth of human corneal limbal epithelial cells on Bombyx mori sericin membranes

Sericin and fibroin are the two major proteins in the silk fibre produced by the domesticated silkworm, Bombyx mori. Fibroin has been extensively investigated as a biomaterial. We have previously shown that fibroin can function successfully as a substratum for growing cells of the eye. Sericin has been so far neglected as a biomaterial because of suspected allergenic activity. However, this misconception has now been dispelled, and sericin’s biocompatibility is currently indisputable. Aiming at promoting sericin as a possible substratum for the growth of corneal cells in order to make tissue-engineered constructs for the restoration of the ocular surface, in this study we investigated the attachment and growth in vitro of human corneal limbal epithelial cells (HLECs) on sericin-based membranes. Sericin was isolated and regenerated from the silkworm cocoons by an aqueous procedure, manufactured into membranes, and characterized (mechanical properties, structural analysis, contact angles). Primary cell cultures from two donors were established in serum-supplemented media in the presence of murine feeder cells. Membranes made of sericin and fibroin-sericin blends were assessed in vitro as substrata for HLECs in a serum-free medium, in a cell attachment assay and in a 3-day cell growth experiment. While the mechanical characteristics of sericin were found to be inferior to those of fibroin, its ability to enhance the attachment of HLECs was significantly superior to fibroin, as revealed by the PicoGreen® assay. Evidence was also obtained that cells can grow and differentiate on these substrata.

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.

Fabrication of a corneal-limbal tissue substitute using silk fibroin

Fibroin extracted from silkworm cocoon silk provides an intriguing and potentially important biomaterial for corneal reconstruction. In the present chapter we outline our methods for producing a composite of two fibroin-based materials that supports the co-cultivation of human limbal epithelial (HLE) cells and human limbal stromal (HLS) cells. The resulting tissue substitute consists of a stratified epithelium overlying a three-dimensional arrangement of extracellular matrix components (principally ‘degummed’ fibroin fibers) and mesenchymal stromal cells. This tissue substitute is currently being evaluated as a tool for reconstructing the corneal limbus and corneal epithelium.

Incorporation of exogenous RGD peptide and inter-species blending as strategies for enhancing human corneal limbal epithelial cell growth on Bombyx mori silk fibroin membranes

While fibroin isolated from the cocoons of domesticated silkworm Bombyx mori supports growth of human corneal limbal epithelial (HLE) cells, the mechanism of cell attachment remains unclear. In the present study we sought to enhance the attachment of HLE cells to membranes of Bombyx mori silk fibroin (BMSF) through surface functionalization with an arginine-glycine-aspartic acid (RGD)-containing peptide. Moreover, we have examined the response of HLE cells to BMSF when blended with the fibroin produced by a wild silkworm, Antheraea pernyi, which is known to contain RGD sequences within its primary structure. A procedure to isolate A. pernyi silk fibroin (APSF) from the cocoons was established, and blends of the two fibroins were prepared at five different BMSF/APSF ratios. In another experiment, BMSF surface was modified by binding chemically the GRGDSPC peptide using a water-soluble carbodiimide. Primary HLE were grown in the absence of serum on membranes made of BMSF, APSF, and their blends, as well as on RGD-modified BMSF. There was no statistically significant enhancing effect on the cell attachment due to the RGD presence. This suggests that the adhesion through RGD ligands may have a complex mechanism, and the investigated strategies are of limited value unless the factors contributing to this mechanism become better known.

Assessment of freestanding membranes prepared from Antheraea pernyi silk fibroin as a potential vehicle for corneal epithelial cell transplantation

Freestanding membranes created from Bombyx mori silk fibroin (BMSF) offer a potential vehicle for corneal cell transplantation since they are transparent and support the growth of human corneal epithelial cells (HCE). Fibroin derived from the wild silkworm Antheraea pernyi (APSF) might provide a superior material by virtue of containing putative cell- attachment sites that are absent from BMSF. Thus we have investigated the feasibility of producing transparent, freestanding membranes from APSF and have analysed the behaviour of HCE cells on this material. No significant differences in cell numbers or phenotype were observed in short term HCE cell cultures established on either fibroin. Production of transparent freestanding APSF membranes, however, proved to be problematic as cast solutions of APSF were more prone to becoming opaque, displayed significantly lower permeability and were more brittle than BMSF-membranes. Cultures of HCE cells established on either membrane developed a normal stratified morphology with cytokeratin pair 3/12 being immuno-localized to the superficial layers. We conclude that while it is feasible to produce transparent freestanding membranes from APSF, the technical difficulties associated with this biomaterial, along with an absence of enhanced cell growth, currently favours the continued development of BMSF as a preferred vehicle for corneal cell transplantation. Nevertheless, it remains possible that refinement of techniques for processing APSF might yet lead to improvements in the handling properties and performance of this material.

Treatment of silk fibroin with poly(ethylene glycol) for the enhancement of corneal epithelial cell growth

A silk protein, fibroin, was isolated from the cocoons of the domesticated silkworm (Bombyx mori) and cast into membranes to serve as freestanding templates for tissue-engineered corneal cell constructs to be used in ocular surface reconstruction. In this study, we sought to enhance the attachment and proliferation of corneal epithelial cells by increasing the permeability of the fibroin membranes and the topographic roughness of their surface. By mixing the fibroin solution with poly(ethylene glycol) (PEG) of molecular weight 300 Da, membranes were produced with increased permeability and with topographic patterns generated on their surface. In order to enhance their mechanical stability, some PEG-treated membranes were also crosslinked with genipin. The resulting membranes were thoroughly characterized and compared to the non-treated membranes. The PEG-treated membranes were similar in tensile strength to the non-treated ones, but their elastic modulus was higher and elongation lower, indicating enhanced rigidity. The crosslinking with genipin did not induce a significant improvement in mechanical properties. In cultures of a human-derived corneal epithelial cell line (HCE-T), the PEG treatment of the substratum did not improve the attachment of cells and it enhanced only slightly the cell proliferation in the longer term. Likewise, primary cultures of human limbal epithelial cells grew equally well on both non-treated and PEG-treated membranes, and the stratification of cultures was consistently improved in the presence of an underlying culture of irradiated 3T3 feeder cells, irrespectively of PEG-treatment. Nevertheless, the cultures grown on the PEG-treated membranes in the presence of feeder cells did display a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. We concluded that while the treatment with PEG had a significant effect on some structural properties of the B. mori silk fibroin (BMSF) membranes, there were minimal gains in the performance of these materials as a substratum for corneal epithelial cell growth. The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes.

The osteogenic properties of CaP/silk composite scaffolds

The rationale for the present study was to develop porous CaP/silk composite scaffolds with a CaP-phase distribution and pore architecture better suited to facilitate osteogenic properties of human bone mesenchymal stromal cells (BMSCs) and in vivo bone formation abilities. This was achieved by first preparing CaP/silk hybrid powders which were then incorporated into silk to obtain uniform CaP/silk composite scaffolds, by means of a freeze-drying method. The composition, microstructure and mechanical properties of the CaP/silk composite scaffolds were ascertained by X-ray diffraction (XRD), Fourier transform infrared spectra (FTIR), scanning electron microscope (SEM) and a universal mechanical testing machine. BMSCs were cultured in these scaffolds and cell proliferation analyzed by confocal microscopy and MTS assay. Alkaline phosphatase (ALP) activity and osteogenic gene expression were assayed to determine if osteogenic differentiation had taken place. A calvarial defect model in SCID mice was used to determine the in vivo bone forming ability of the hybrid CaP/silk scaffolds. Our results showed that incorporating the hybrid CaP/silk powders into silk scaffolds improved both pore structure architecture and distribution of CaP powders in the composite scaffolds. By incorporating the CaP phase into silk scaffolds in vitro osteogenic differentiation of BMSCs was enhanced and there was increased in vivo cancellous bone formation. Here we report a method with which to prepare Ca/P composite scaffolds with a pore structure and Ca/P distribution better suited to facilitate BMSC differentiation and bone formation.

Structure–property relationships of silk-modified mesoporous bioglass scaffolds

Porous mesopore-bioglass (MBG) scaffolds have been proposed as a new class of bone regeneration materials due to their apatite-formation and drug-delivery properties; however, the material’s inherent brittleness and high degradation and surface instability are major disadvantages, which compromise its mechanical strength and cytocompatibility as a biological scaffold. Silk, on the other hand, is a native biomaterial and is well characterized with respect to biocompatibility and tensile strength. In this study we set out to investigate what effects blending silk with MBG had on the physiochemical, drug-delivery and biological properties of MBG scaffolds with a view to bone tissue engineering applications. Transmission electron microscopy (TEM), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were the methods used to analyze the inner microstructure, pore size and morphology, and composition of MBG scaffolds, before and after addition of silk. The effect of silk modification on the mechanical property of MBG scaffolds was determined by testing the compressive strength of the scaffolds and also compressive strength after degradation over time. The drug-delivery potential was evaluated by the release of dexamethasone (DEX) from the scaffolds. Finally, the cytocompatibility of silk-modified scaffolds was investigated by the attachment, morphology, proliferation, differentiation and bone-relative gene expression of bone marrow stromal cells (BMSCs). The results showed that silk modification improved the uniformity and continuity of pore network of MBG scaffolds, and maintained high porosity (94%) and large-pore size (200–400 mm). There was a significant improvement in mechanical strength, mechanical stability, and control of burst release of DEX in silkmodified MBG scaffolds. Silk modification also appeared to provide a better environment for BMSC attachment, spreading, proliferation, and osteogenic differentiation on MBG scaffolds.

The silk road project : dance and embodiment in the age of motion capture

The Silk Road Project was a practice-based research project investigating the potential of motion capture technology to inform perceptions of embodiment in dance performance. The project created a multi-disciplinary collaborative performance event using dance performance and real-time motion capture at Deakin University’s Deakin Motion Lab. Several new technological advances in producing real-time motion capture performance were produced, along with a performance event that examined the aesthetic interplay between a dancer’s movement and the precise mappings of its trajectories created by motion capture and real-time motion graphic visualisations.

The effects of pore architecture in silk fibroin scaffolds on the growth and differentiation of mesenchymal stem cells expressing BMP7

The pore architecture of scaffolds is known to play a critical role in tissue engineering as it provides the vital framework for seeded cells to organize into a functioning tissue. In this report we have investigated the effects of different concentrations of silk fibroin protein on three-dimensional (3D) scaffold pore microstructure. Four pore size ranges of silk fibroin scaffolds were made by the freeze drying technique, with the pore sizes ranging from 50 to 300 lm. The pore sizes of the scaffolds decreased as the concentration of fibroin protein increased. Human bone marrow mesenchymal stromal cells (BMSC) transfected with the BMP7 gene were cultured in these scaffolds. A cell viability colorimetric assay, alkaline phosphatase assay and reverse transcription-polymerase chain reaction were performed to analyze the effect of pore size on cell growth, the secretion of extracellular matrix (ECM) and osteogenic differentiation. Cell migration in 3D scaffolds was confirmed by confocal microscopy. Calvarial defects in SCID mice were used to determine the bone forming ability of the silk fibroin scaffolds incorporating BMSC expressing BMP7. The results showed that BMSC expressing BMP7 preferred a pore size between 100 and 300 lm in silk fibroin protein fabricated scaffolds, with better cell proliferation and ECM production. Furthermore, in vivo transplantation of the silk fibroin scaffolds combined with BMSC expressing BMP7 induced new bone formation. This study has shown that an optimized pore architecture of silk fibroin scaffolds can modulate the bioactivity of BMP7-transfected BMSC in bone formation.

The Silk Road project

The Silk Road Project was a practice-based research project investigating the potential of motion capture technology to inform perceptions of embodiment in dance performance. The project created a multi-disciplinary collaborative performance event using dance performance and real-time motion capture at Deakin University’s Deakin Motion Lab. Performances at Deakin University, December 2007.

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.

Mesoporous bioglass/silk composite scaffolds for bone tissue engineering

In the past 20 years, mesoporous materials have been attracted great attention due to their significant feature of large surface area, ordered mesoporous structure, tunable pore size and volume, and well-defined surface property. They have many potential applications, such as catalysis, adsorption/separation, biomedicine, etc. [1]. Recently, the studies of the applications of mesoporous materials have been expanded into the field of biomaterials science. A new class of bioactive glass, referred to as mesoporous bioactive glass (MBG), was first developed in 2004. This material has a highly ordered mesopore channel structure with a pore size ranging from 5–20 nm [1]. Compared to non-mesopore bioactive glass (BG), MBG possesses a more optimal surface area, pore volume and improved in vitro apatite mineralization in simulated body fluids [1,2]. Vallet-Regí et al. has systematically investigated the in vitro apatite formation of different types of mesoporous materials, and they demonstrated that an apatite-like layer can be formed on the surfaces of Mobil Composition of Matters (MCM)-48, hexagonal mesoporous silica (SBA-15), phosphorous-doped MCM-41, bioglass-containing MCM-41 and ordered mesoporous MBG, allowing their use in biomedical engineering for tissue regeneration [2-4]. Chang et al. has found that MBG particles can be used for a bioactive drug-delivery system [5,6]. Our study has shown that MBG powders, when incorporated into a poly (lactide-co-glycolide) (PLGA) film, significantly enhance the apatite-mineralization ability and cell response of PLGA films. compared to BG [7]. These studies suggest that MBG is a very promising bioactive material with respect to bone regeneration. It is known that for bone defect repair, tissue engineering represents an optional method by creating three-dimensional (3D) porous scaffolds which will have more advantages than powders or granules as 3D scaffolds will provide an interconnected macroporous network to allow cell migration, nutrient delivery, bone ingrowth, and eventually vascularization [8]. For this reason, we try to apply MBG for bone tissue engineering by developing MBG scaffolds. However, one of the main disadvantages of MBG scaffolds is their low mechanical strength and high brittleness; the other issue is that they have very quick degradation, which leads to an unstable surface for bone cell growth limiting their applications. Silk fibroin, as a new family of native biomaterials, has been widely studied for bone and cartilage repair applications in the form of pure silk or its composite scaffolds [9-14]. Compared to traditional synthetic polymer materials, such as PLGA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the chief advantage of silk fibroin is its water-soluble nature, which eliminates the need for organic solvents, that tend to be highly cytotoxic in the process of scaffold preparation [15]. Other advantages of silk scaffolds are their excellent mechanical properties, controllable biodegradability and cytocompatibility [15-17]. However, for the purposes of bone tissue engineering, the osteoconductivity of pure silk scaffolds is suboptimal. It is expected that combining MBG with silk to produce MBG/silk composite scaffolds would greatly improve their physiochemical and osteogenic properties for bone tissue engineering application. Therefore, in this chapter, we will introduce the research development of MBG/silk scaffolds for bone tissue engineering.