Development of silk-based scaffolds for tissue engineering of bone from human adipose-derived stem cells.

Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and its demonstrated ability to support bone formation both in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASCs), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol (HFIP)), pore sizes (250-500 µm vs. 500-1000 µm) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes, with decellularized trabecular bone (DCB) included as a "gold standard". The scaffolds were seeded with hASCs and cultured for 7 weeks in osteogenic medium. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400-600 µm porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes, as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2 and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation.

Materials fabrication from Bombyx mori silk fibroin.

Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery.

The effect of manipulation of silk scaffold fabrication parameters on matrix performance in a murine model of bladder augmentation.

Autologous gastrointestinal segments are utilized as the primary option for bladder reconstructive procedures despite their inherent morbidity and significant complication rate. Multi-laminate biomaterials derived from Bombyx mori silk fibroin and prepared from a gel spinning process may serve as a superior alternative for bladder tissue engineering due to their robust mechanical properties, biocompatibility, and processing plasticity. In the present study, we sought to determine the impact of variations in winding (axial slew rate: 2 and 40 mm/s) and post-winding (methanol and lyophilization) fabrication parameters on the in vivo performance of gel spun silk scaffolds in a murine model of bladder augmentation. Three silk matrix groups with distinct structural and mechanical properties were investigated following 10 weeks of implantation including our original prototype previously shown to support bladder regeneration, Group 1 (2 mm/s, methanol) as well as Group 2 (40 mm/s, methanol) and Group 3 (40 mm/s, lyophilization) configurations. Non surgical animals were assessed in parallel as controls. Quantification of residual scaffold area demonstrated that while Group 1 and 2 scaffolds were largely intact, processing parameters utilized for Group 3 led to significantly higher degrees of scaffold degradation in comparison to Group 1. Histological (hematoxylin and eosin, masson's trichrome) and immunohistochemical (IHC) analyses showed comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within the original defect site throughout all matrix groups similar to controls. Parallel evaluations demonstrated transitional urothelial formation with prominent uroplakin and p63 protein expression supported by Group 1 and 3 scaffolds, while Group 2 variants supported a thin, immature epithelium composed primarily of uroplakin-negative, p63-positive basal cells. Voided stain on paper analysis revealed similar voiding patterns between all matrix groups; however Group 2 animals displayed substantially lower voided volumes with increased frequency in comparison to controls. In addition, cystometric assessments revealed all matrix groups supported comparable degrees of bladder compliance similar to control levels. The results of this study demonstrate that selective alterations in winding and post-winding fabrication parameters can enhance the degradation rate of gel spun silk scaffolds in vivo while preserving their ability to support bladder tissue regeneration and function.

Mechanical improvements to reinforced porous silk scaffolds.

Load-bearing porous biodegradable scaffolds are required to engineer functional tissues such as bone. Mechanical improvements to porogen leached scaffolds prepared from silk proteins were systematically studied through the addition of silk particles in combination with silk solution concentration, exploiting interfacial compatibility between the two components. Solvent solutions of silk up to 32 w/v % were successfully prepared in hexafluoroisopropanol (HFIP) for the study. The mechanical properties of the reinforced silk scaffolds correlated to the material density and matched by a power law relationship, independent of the ratio of silk particles to matrix. These results were similar to the relationships previously shown for cancellous bone. From these data we conclude that the increased mechanical properties were due to a densification effect and not due to the inclusion of stiffer silk particles into the softer silk matrix. A continuous interface between the silk matrix and the silk particles, as well as homogeneous distribution of the silk particles within the matrix was observed. Furthermore, we note that the roughness of the pore walls was controllable by varying the ratio of the particles matrix, providing a route to control topography. The rate of proteolytic hydrolysis of the scaffolds decreased with increase in mass of silk used in the matrix and with increasing silk particle content.

Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds.

We describe a composite hydroxyapatite (HA)-silk fibroin scaffold designed to induce and support the formation of mineralized bone matrix by human mesenchymal stem cells (hMSCs) in the absence of osteogenic growth factors. Porous three-dimensional silk scaffolds were extensively used in our previous work for bone tissue engineering and showed excellent biodegradability and biocompatibility. However, silk is not an osteogenic material and has a compressive stiffness significantly lower than that of native bone. In the present study, we explored the incorporation of silk sponge matrices with HA (bone mineral) micro-particles to generate highly osteogenic composite scaffolds capable of inducing the in vitro formation of tissue-engineered bone. Different amounts of HA were embedded in silk sponges at volume fractions of 0%, 1.6%, 3.1% and 4.6% to enhance the osteoconductive activity and mechanical properties of the scaffolds. The cultivation of hMSCs in the silk/HA composite scaffolds under perfusion conditions resulted in the formation of bone-like structures and an increase in the equilibrium Young's modulus (up to 4-fold or 8-fold over 5 or 10 weeks of cultivation, respectively) in a manner that correlated with the initial HA content. The enhancement in mechanical properties was associated with the development of the structural connectivity of engineered bone matrix. Collectively, the data suggest two mechanisms by which the incorporated HA enhanced the formation of tissue engineered bone: through osteoconductivity of the material leading to increased bone matrix production, and by providing nucleation sites for new mineral resulting in the connectivity of trabecular-like architecture.

Ingrowth of human mesenchymal stem cells into porous silk particle reinforced silk composite scaffolds: An in vitro study.

Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous three-dimensional (3-D) silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (non-reinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to 6 weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microcomputer tomography (µCT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per µg of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1, and 1:2, respectively. In addition, µCT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for non-reinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3-D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes.

Simple modular bioreactors for tissue engineering: a system for characterization of oxygen gradients, human mesenchymal stem cell differentiation, and prevascularization.

Large-scale tissue engineering is limited by nutrient perfusion and mass transport limitations, especially oxygen diffusion, which restrict construct development to smaller than clinically relevant dimensions and limit the ability for in vivo integration. The goal of this work was to develop a modular approach to tissue engineering, where scaffold and tissue size, transport issues, and surgical implantation in vivo are considered from the outset. Human mesenchymal stem cells (hMSCs) were used as the model cell type, as their differentiation has been studied for several different cell lineages and often with conflicting results. Changes in the expression profiles of hMSCs differentiated under varied oxygen tensions are presented, demonstrating tissue-specific oxygen requirements for both adipogenic (20% O2) and chondrogenic (5% O2) differentiation. Oxygen and nutrient transport were enhanced by developing a bioreactor system for perfusing hMSC-seeded collagen gels using porous silk tubes, resulting in enhanced oxygen transport and cell viability within the gels. These systems are simple to use and scaled for versatility, to allow for the systematic study of relationships between cell content, oxygen, and cell function. The data may be combined with oxygen transport modeling to derive minimally sized modular units for construction of clinically relevant tissue-engineered constructs, a generic strategy that may be employed for vascularized target tissues.

Three-dimensional culture alters primary cardiac cell phenotype.

The directed formation of complex three-dimensional (3D) tissue architecture is a fundamental goal in tissue engineering and regenerative medicine. The growth of cells in 3D structures is expected to influence cellular phenotype and function, especially relative cell distribution, expression profiles, and responsiveness to exogenous signals; however, relatively few studies have been carried out to examine the effects of 3D reaggregation on cells from critical target organs, like the heart. Accordingly, we cultured primary cardiac ventricular cells in a 3D model system using a serum-free medium to test the hypothesis that expression profiles, multicellular organizational pathways, tissue maturation markers, and responsiveness to hormone stimulation were significantly altered in stable cell populations grown in 3D versus 2D culture. We found that distinct multi-cellular structures formed in 3D in conjunction with changes in mRNA expression profile, up-regulation of endothelial cell migratory pathways, decreases in the expression of fetal genes (Nppa and Ankrd1), and increased sensitivity to tri-iodothyronine stimulation when compared to parallel 2D cultures comprising the same cell populations. These results indicate that the culture of primary cardiac cells in 3D aggregates leads to physiologically relevant alterations in component cell phenotype consistent with cardiac ventricular tissue formation and maturation.

Culture on electrospun polyurethane scaffolds decreases atrial natriuretic peptide expression by cardiomyocytes in vitro.

The function of the mammalian heart depends on the functional alignment of cardiomyocytes, and controlling cell alignment is an important consideration in biomaterial design for cardiac tissue engineering and research. The physical cues that guide functional cell alignment in vitro and the impact of substrate-imposed alignment on cell phenotype, however, are only partially understood. In this report, primary cardiac ventricular cells were grown on electrospun, biodegradable polyurethane (ES-PU) with either aligned or unaligned microfibers. ES-PU scaffolds supported high-density cultures and cell subpopulations remained intact over two weeks in culture. ES-PU cultures contained electrically-coupled cardiomyocytes with connexin-43 localized to points of cell:cell contact. Multi-cellular organization correlated with microfiber orientation and aligned materials yielded highly oriented cardiomyocyte groupings. Atrial natriuretic peptide, a molecular marker that shows decreasing expression during ventricular cell maturation, was significantly lower in cultures grown on ES-PU scaffolds than in those grown on tissue culture polystyrene. Cells grown on aligned ES-PU had significantly lower steady state levels of ANP and constitutively released less ANP over time indicating that scaffold-imposed cell organization resulted in a shift in cell phenotype to a more mature state. We conclude that the physical organization of microfibers in ES-PU scaffolds impacts both multi-cellular architecture and cardiac cell phenotype in vitro.

Seeding bioreactor-produced embryonic stem cell-derived cardiomyocytes on different porous, degradable, polyurethane scaffolds reveals the effect of scaffold architecture on cell morphology.

A successful regenerative therapy to treat damage incurred after an ischemic event in the heart will require an integrated approach including methods for appropriate revascularization of the infarct site, mechanical recovery of damaged tissue, and electrophysiological coupling with native cells. Cardiomyocytes are the ideal cell type for heart regeneration because of their inherent electrical and physiological properties, and cardiomyocytes derived from embryonic stem cells (ESCs) represent an attractive option for tissue-engineering therapies. An important step in developing tissue engineering-based approaches to cardiac cell therapy is understanding how scaffold architecture affects cell behavior. In this work, we generated large numbers of ESC-derived cardiomyocytes in bioreactors and seeded them on porous, 3-dimensional scaffolds prepared using 2 different techniques: electrospinning and thermally induced phase separation (TIPS). The effect of material macro-architecture on the adhesion, viability, and morphology of the seeded cells was determined. On the electrospun scaffolds, cells were elongated in shape, a morphology typical of cultured ESC-derived cardiomyocytes, whereas on scaffolds fabricated using TIPS, the cells retained a rounded morphology. Despite these gross phenotypic and physiological differences, sarcomeric myosin and connexin 43 expression was evident, and contracting cells were observed on both scaffold types, suggesting that morphological changes induced by material macrostructure do not directly correlate to functional differences.

Characterization of biodegradable polyurethane microfibers for tissue engineering.

A polyurethane designed to be biodegradable via hydrolysis and enzyme-mediated chain cleavage, has been investigated for its use as a temporary scaffold in tissue-engineering applications. The phase-segregated nature of the polyurethane imparts elastomeric properties that are attractive for soft tissue engineering. This polyurethane has been electrospun in order to create scaffolds that incorporate several biomimetic features including small fiber diameter, large void volume, and an interconnected porous network. Material properties were evaluated via gel-permeation chromatography, differential scanning calorimetry and Raman spectroscopy before and after processing. Analysis by gel-permeation chromatography showed that the molecular weights were similar, indicating that the bulk of the polymer chains were not degraded during processing. Thermal analysis revealed that the glass transition temperature did not shift and Raman spectra of the bulk polyurethane film compared to the electrospun mat were identical, confirming that the conformation of the polymer was unaffected by the shear and electric field used in the electrospinning process. In addition, field emission scanning electron microscopy revealed that the morphology of the electrospun mats had a broad fiber diameter distribution, and mechanical analysis showed that the mats had an ultimate tensile stress of 1.33 MPa and ultimate tensile strain of 78.6%. The degradation profile was investigated in the presence of chymotrypsin. These results were compared to a previous study of thin films of this polyurethane, and it was found that the increase of surface area aided the surface-mediated erosion of the material. It is believed that an electrospun matrix of this biodegradable polyurethane shows promise for use in soft tissue engineering and regenerative medicine applications.