Swati Midha

Silk fibroin as biomaterial for bone tissue engineering

UNLABELLED Silk fibroin (SF) is a fibrous protein which is produced mainly by silkworms and spiders. Its unique mechanical properties, tunable biodegradation rate and the ability to support the differentiation of mesenchymal stem cells along the osteogenic lineage, have made SF a favorable scaffold material for bone tissue engineering. SF can be processed into various scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified, which provides an impressive toolbox and allows SF scaffolds to be tailored to specific applications. This review discusses and summarizes recent advancements in processing SF, focusing on different fabrication and functionalization methods and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current challenges, uncertainties and gaps in knowledge are highlighted. STATEMENT OF SIGNIFICANCE Silk fibroin is a natural biomaterial with remarkable biomedical and mechanical properties which make it favorable for a broad range of bone tissue engineering applications. It can be processed into different scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified which provides a unique toolbox and allows silk fibroin scaffolds to be tailored to specific applications. This review discusses and summarizes recent advancements in processing silk fibroin, focusing on different fabrication and functionalization methods and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current challenges, uncertainties and gaps in knowledge are highlighted.

Preconditioned 70S30C bioactive glass foams promote osteogenesis in vivo.

Bioactive glass scaffolds (70S30C; 70% SiO2 and 30% CaO) produced by a sol-gel foaming process are thought to be suitable matrices for bone tissue regeneration. Previous in vitro data showed bone matrix production and active remodelling in the presence of osteogenic cells. Here we report their ability to act as scaffolds for in vivo bone regeneration in a rat tibial defect model, but only when preconditioned. Pretreatment methods (dry, pre-wetted or preconditioned without blood) for the 70S30C scaffolds were compared against commercial synthetic bone grafts (NovaBone® and Actifuse®). Poor bone ingrowth was found for both dry and wetted sol-gel foams, associated with rapid increase in pH within the scaffolds. Bone ingrowth was quantified through histology and novel micro-CT image analysis. The percentage bone ingrowth into dry, wetted and preconditioned 70S30C scaffolds at 11 weeks were 10±1%, 21±2% and 39±4%, respectively. Only the preconditioned sample showed above 60% material-bone contact, which was similar to that in NovaBone and Actifuse. Unlike the commercial products, preconditioned 70S30C scaffolds degraded and were replaced with new bone. The results suggest that bioactive glass compositions should be redesigned if sol-gel scaffolds are to be used without preconditioning to avoid excess calcium release.

Bioactive glass foam scaffolds are remodelled by osteoclasts and support the formation of mineralized matrix and vascular networks in vitro.

Remodelling of scaffolds and new bone formation is critical for effective bone regeneration. Herein is reported the first demonstration of resorption pits due to osteoclast activity on the surface of sol-gel bioactive glass foam scaffolds. Bioactive glass foam scaffolds are known to have osteogenic potential and suitable pore networks for bone regeneration. Degradation of the scaffolds is known to be initially solution mediated, but for effective bone regeneration, remodelling of the scaffold by osteoclasts and vascularisation of the scaffold is necessary. The culture of C7 macrophages on a bioactive glass scaffold induces the cells to differentiate into (TRAP(+ve) ) osteoclasts. They then form distinctive resorption pits within 3 weeks, while MC3T3-E1 pre-osteoblasts deposit mineralized osteoid on their surfaces in co-culture. The scaffolds are of the 70S30C (70 mol% SiO2 , 30 mol% CaO) composition, with modal pore and interconnect diameters of 373 μm and 172 μm respectively (quantified by X-ray micro-tomography and 3D image analysis). The release of soluble silica and calcium ions from 70S30C scaffolds induces an increase in osteoblast numbers as determined via the MTT assay. Scaffolds also support growth of endothelial cells on their surface and tube formation (characteristic of functional microvasculature) following 4 days in culture. This data supports the hypothesis that 70S30C bioactive glass scaffolds promote the differentiation of the 3 main cell types involved in vascularized bone regeneration.

Effect of visco-elastic silk–chitosan microcomposite scaffolds on matrix deposition and biomechanical functionality for cartilage tissue engineering

Commonly used polymer‐based scaffolds often lack visco‐elastic properties to serve as a replacement for cartilage tissue. This study explores the effect of reinforcement of silk matrix with chitosan microparticles to create a visco‐elastic matrix that could support the redifferentiation of expanded chondrocytes. Goat chondrocytes produced collagen type II and glycosaminoglycan (GAG)‐enriched matrix on all the scaffolds (silk:chitosan 1:1, 1:2 and 2:1). The control group of silk‐only constructs suffered from leaching out of GAG molecules into the medium. Chitosan‐reinforced scaffolds retained a statistically significant (p < 0.02) higher amount of GAG, which in turn significantly increased (p < 0.005) the aggregate modulus (as compared to silk‐only controls) of the construct akin to that of native tissue. Furthermore, the microcomposite constructs demonstrated highly pronounced hysteresis at 4% strain up to 400 cycles, mimicking the visco‐elastic properties of native cartilage tissue. These results demonstrated a step towards optimizing the design of biomaterial scaffolds used for cartilage tissue engineering. Copyright

Elucidation of differential mineralisation on native and regenerated silk matrices

Bone mineralisation is a well-orchestrated procedure triggered by a protein-based template inducing the nucleation of hydroxyapatite (HA) nanocrystals on the matrix. In an attempt to fabricate superior nanocomposites from silk fibroin, textile braided structures made of natively spun fibres of Bombyx mori silkworm were compared against regenerated fibroin (lyophilized and films) underpinning the influence of intrinsic properties of fibroin matrices on HA nucleation. We found that native braids could bind Ca(2+) ions through electrostatic attraction, which initiated the nucleation and deposition of HA, as evidenced by discrete shift in amide peaks via ATR-FTIR. This phenomenon also suggests the involvement of amide linkages in promoting HA nucleation on fibroin. Moreover, CaCl2-SBF immersion of native braids resulted in preferential growth of HA along the c-axis, forming needle-like nanocrystals and possessing Ca/P ratio comparable to commercial HA. Though regenerated lyophilized matrix also witnessed prominent peak shift in amide linkages, HA growth was restricted to (211) plane only, albeit at a significantly lower intensity than braids. Regenerated films, on the other hand, provided no crystallographic evidence of HA deposition within 7days of SBF immersion. The present work sheds light on the primary fibroin structure of B. mori which probably plays a crucial role in regulating template-induced biomineralisation on the matrix. We also found that intrinsic material properties such as surface roughness, geometry, specific surface area, tortuosity and secondary conformation exert influence in modulating the extent of mineralisation. Thus our work generates useful insights and warrants future studies to further investigate the potential of bone mimetic, silk/mineral nanocomposite matrices for orthopaedic applications.

Strategies for faster detachment of corneal cell sheet using micropatterned thermoresponsive matrices

The development of transplantable cell sheets of functional keratocytes embedded within an aligned collagen type I matrix is a viable approach for constructing a bioequivalent of corneal stroma. Thermoresponsive materials based on poly(N-isopropylacrylamide) (PolyNIPA) have been utilized to recover carrier-free corneal cell sheets by inducing temperature changes. In this study, we employed direct-write assembly (DWA) to develop microperiodic parallel patterns of silk-PolyNIPA and gelatin-PolyNIPA. Semi-interpenetrating networks of PolyNIPA hybrids (with silk/gelatin) exhibited temperature-responsive nature and thereby have potential use in cell sheet engineering. Silk-PolyNIPA and gelatin-PolyNIPA hybrids demonstrated a hydrophobic surface at 37 °C (i.e. above their lower critical solution temperature) with a contact angle of 59°± 0.3° and 55°± 3°, respectively, whereas the surface roughness of silk-PolyNIPA was double that of gelatin-PolyNIPA. The reduction of temperature to 20 °C resulted in a decrease in the value of surface roughness and water contact angle for both hybrids. All four parallel patterned substrates guided corneal cell alignment along the direction of the patterns. Collagen type-I and aggrecan gene expression was higher when the cells were grown over the gelatin-PolyNIPA matrix after 3 weeks of culture when compared to silk-PolyNIPA. In addition, a significantly higher metabolic activity as well as enhanced vinculin expression of keratocytes on the gelatin-PolyNIPA matrix indicated the improved cytocompability compared to the silk, gelatin and silk-PolyNIPA matrices. Interestingly, the detachment of keratocytes cell sheet was achieved from the silk-PolyNIPA and gelatin-PolyNIPA planar films only within 10 min and 30 min, respectively, but the patterns could not yield intact sheet recovery. Hence, we conclude that while gelatin-PolyNIPA hybrids with parallel patterns fabricated using DWA will benefit from the application of cellular alignment, some optimization in the pattern parameters may be required for rapid sheet recovery from such substrates. Understanding the keratocytes responses to such hybrid biomaterials suggests viable options to develop a corneal stromal bioequivalent.

Preservation of biomacromolecular composition and ultrastructure of a decellularized cornea using a perfusion bioreactor

An attempt has been made to formulate a new method of corneal decellularization using a direct perfusion system through the cornea. Here, we compared the direct perfusion method to some commonly used decellularization strategies including chemical methods; non-ionic detergent TRITON X-100 and ionic detergent; sodium dodecyl sulphate (SDS) based orbital shaker method and physical methods of liquid nitrogen and freeze–thaw to decellularize a goat cornea. Histochemical evaluation and biochemical estimation highlighted that liquid nitrogen, freeze–thaw and TRITON-based orbital shaker methods resulted in incomplete removal of resident cells from the native cornea. On the contrary, direct perfusion of the cornea using TRITON and SDS completely removed all the cells from the cornea while preserving the ultrastructure of the extracellular matrix at a steady flow rate of 10 μl min−1. Raman and ATR-FTIR spectra indicated the relative abundance of the α-helical conformation of collagen type I in the perfused cornea while a β-sheet conformation was predominantly observed in other treatment methods. FACS was used to determine the cell death modality in different methods of decellularization. In the direct perfusion system, 13.1% higher apoptotic cells, the preferred route of cell death, were observed in the cornea compared to orbital shaker-based methods. Further, feasibility studies conducted for 7 days to investigate the recellularization potential of the perfused decellularized matrix demonstrated a well attached viable population of seeded corneal stromal cells. In summary, we demonstrated that the direct perfusion method for decellularization of a cornea using 0.1% TRITON detergent at 10 μl min−1 is an optimal strategy for efficiently removing the resident corneal cells while maintaining the ultrastructure of the corneal matrix intact and therefore could serve as an excellent source for corneal transplantation.

Silk‐Based Bioinks for 3D Bioprinting

3D bioprinting field is making remarkable progress; however, the development of critical sized engineered tissue construct is still a farfetched goal. Silk fibroin offers a promising choice for bioink material. Nature has imparted several unique structural features in silk protein to ensure spinnability by silkworms or spider. Researchers have modified the structure-property relationship by reverse engineering to further improve shear thinning behavior, high printability, cytocompatible gelation, and high structural fidelity. In this review, it is attempted to summarize the recent advancements made in the field of 3D bioprinting in context of two major sources of silk fibroin: silkworm silk and spider silk (native and recombinant). The challenges faced by current approaches in processing silk bioinks, cellular signaling pathways modulated by silk chemistry and secondary conformations, gaps in knowledge, and future directions acquired for pushing the field further toward clinic are further elaborated.

Antioxidant and antibacterial hydroxyapatite-based biocomposite for orthopedic applications

Post-implantation, vicinity acquired oxidative stress and bacterial infections lead to apoptosis with eventual bone-resorption and implant failure, respectively. Thus, in order to combat aforementioned complications, present research aims in utilizing antioxidant ceria (CeO2) and antibacterial silver (Ag) reinforced hydroxyapatite (HA) composite with enhanced mechanical and cytocompatible properties. Highly dense (>90%) spark plasma sintered HA-based composites elicits enhanced elastic modulus (121-133 GPa) in comparison to that of HA. The antioxidant activity is quantified using ceria alone, wherein HA-ceria and HA-ceria-Ag pellets exhibits ~36 and 30% antioxidant activity, respectively, accrediting ceria as a scavenger of reactive oxygen species, which was corroborated with the % Ce3+ change quantified by X-ray photoelectron spectroscopy. The HA-Ag pellet shows antibacterial efficacy of ~61% for E. coli and ~53% for S. aureus, while a reduction of ~59% for E. coli and ~50% for S. aureus is observed for HA-ceria-2.5Ag pellet, affirming Ag reinforcement as an established bactericidal agent. The enhanced hydrophobicity on all the HA-based composites affords a high protein adsorption (24 h incubation). Further, elevated hFOB cell count (~6.7 times for HA-ceria-Ag on day 7) with filopodial extensions (60-150 μm) and matrix-like deposition reflect cell-substrate intimacy. Thus, synergistic antioxidant ceria and antibacterial Ag reinforcement with enhanced mechanical integrity can potentially serve as cytocompatible porous bone scaffolds or bioactive coatings on femoral stems.