Biofabrication of the osteochondral unit and its applications: Current and future directions for 3D bioprinting.

Multiple prevalent diseases, such as osteoarthritis (OA), for which there is no cure or full understanding, affect the osteochondral unit; a complex interface tissue whose architecture, mechanical nature and physiological characteristics are still yet to be successfully reproduced in vitro. Although there have been multiple tissue engineering-based approaches to recapitulate the three dimensional (3D) structural complexity of the osteochondral unit, there are various aspects that still need to be improved. This review presents the different pre-requisites necessary to develop a human osteochondral unit construct and focuses on 3D bioprinting as a promising manufacturing technique. Examples of 3D bioprinted osteochondral tissues are reviewed, focusing on the most used bioinks, chosen cell types and growth factors. Further information regarding the applications of these 3D bioprinted tissues in the fields of disease modelling, drug testing and implantation is presented. Finally, special attention is given to the limitations that currently hold back these 3D bioprinted tissues from being used as models to investigate diseases such as OA. Information regarding improvements needed in bioink development, bioreactor use, vascularisation and inclusion of additional tissues to further complete an OA disease model, are presented. Overall, this review gives an overview of the evolution in 3D bioprinting of the osteochondral unit and its applications, as well as further illustrating limitations and improvements that could be performed explicitly for disease modelling.

Three-Dimensional Engineered Peripheral Nerve: Toward a New Era of Patient-Specific Nerve Repair Solutions.

Reconstruction of peripheral nerve injuries (PNIs) with substance loss remains challenging because of limited treatment solutions and unsatisfactory patient outcomes. Currently, nerve autografting is the first-line management choice for bridging critical-sized nerve defects. The procedure, however, is often complicated by donor site morbidity and paucity of nerve tissue, raising a quest for better alternatives. The application of other treatment surrogates, such as nerve guides, remains questionable, and it is inefficient in irreducible nerve gaps. More importantly, these strategies lack customization for personalized patient therapy, which is a significant drawback of these nerve repair options. This negatively impacts the fascicle-to-fascicle regeneration process, critical to restoring the physiological axonal pathway of the disrupted nerve. Recently, the use of additive manufacturing (AM) technologies has offered major advancements to the bioengineering solutions for PNI therapy. These techniques aim at reinstating the native nerve fascicle pathway using biomimetic approaches, thereby augmenting end-organ innervation. AM-based approaches, such as three-dimensional (3D) bioprinting, are capable of biofabricating 3D-engineered nerve graft scaffolds in a patient-specific manner with high precision. Moreover, realistic in vitro models of peripheral nerve tissues that represent the physiologically and functionally relevant environment of human organs could also be developed. However, the technology is still nascent and faces major translational hurdles. In this review, we spotlighted the clinical burden of PNIs and most up-to-date treatment to address nerve gaps. Next, a summarized illustration of the nerve ultrastructure that guides research solutions is discussed. This is followed by a contrast of the existing bioengineering strategies used to repair peripheral nerve discontinuities. In addition, we elaborated on the most recent advances in 3D printing and biofabrication applications in peripheral nerve modeling and engineering. Finally, the major challenges that limit the evolution of the field along with their possible solutions are also critically analyzed. Impact statement Complex nerve injuries, including critical-sized gaps (>3 cm loss of substance), gaps involving nerve bifurcations, and those associated with ischemic environments, are difficult to manage. A biomimetic, personalized peripheral nerve tissue surrogate to address these injuries is lacking. The peripheral nerve repair market currently represents a multi-billion-dollar industry that is projected to expand. Given the clinical and economical dilemmas posed by this medical condition, it is crucial to devise novel and effective nerve substitutes. In this review article, we discuss progress in three-dimensional printing technologies, including biofabrication and nerve computer-aided design modeling, toward achieving a patient-specific and biomimetic nerve repair solution.

Design and In Vivo Testing of Novel Single-Stage Tendon Graft Using Polyurethane Nanocomposite Polymer for Tendon Reconstruction.

Severe trauma, failure of prior surgical repair, delayed presentation and excessive scarring around the flexor tendon bed often necessitate a two-stage surgical reconstruction, where a silicone spacer is used in the first stage to recreate the fibro-osseous tunnel through which the tendon graft can glide in the second stage. This staged procedure involves great commitment on the part of both patient and surgeon, over the course of several months, involving a prolonged period of rehabilitation that can be quite disruptive to the patient's life and work. Reducing this from a two-stage into a single-stage procedure, therefore, has the potential to reduce rehabilitation time and cost, expedite return to work, and improve outcomes. To address this, we developed polyurethane (PU) nanocomposite, as an engineered tendon sheath, for treatment of delayed flexor tendon division as a single-stage procedure. The clinically conformant tubular grafts were tested for their efficacy in the peroneus tertius tendon of 6 Mule sheep for 3 months. Semi-quantitative histological assessment was carried out by analysing four descriptive layers: tendon, tendon/polymer sheath interface, polymer sheath, and polymer sheath/surrounding tissue. Four (out of 6) of the implanted PU nanocomposites showed moderate to substantial healing of the injured tendons, with minimal adhesion after repair, ensuring good gliding movement. No statistical differences were observed in tendon repair based on intra-regional variation in the explanted grafts, indicating homogeneity in tendon repair. Overall, the PU nanocomposite bears morphological stability and functionality for tendon repair, in single-stage surgical reconstruction, demonstrating promising evidence for clinical translation.

Rapid tumor inhibition via magnetic hyperthermia regulated by caspase 3 with time-dependent clearance of iron oxide nanoparticles.

Among conventional cancer therapies, radio-frequency magnetic hyperthermia (MHT) has widely been investigated for use with magnetic nanoparticles (MNPs). However, the majority of in vivo biodistribution studies have tested very low MNP dosages (equivalent to magnetic resonance imaging (MRI) applications) to check for clearance rate; which is far below the clinical dose of MHT. Due to this poor validation in preclinical scenarios, quite a few MNPs already in clinical use were later discontinued, on grounds of unexpected clinical outcomes in terms of inflammation, and prolonged clearance in vivo. By exploiting an economical method of synthesis, we have developed chitosan-coated Fe3O4 nanoparticles with high heating efficiency performance. Their anti-tumor response was evaluated in an ectopic tumor model of C6 glioblastoma by MHT. The intratumoral injection of MNPs on days 1 and 7 resulted in rapid tumor inhibition rate of 69.4% within 8 days, with complete inhibition within 32 days, and no recurrence recorded over a 5-month follow-up. Notably, the MNP-mediated MHT therapy achieved the highest degree of therapeutic efficacy required for complete tumor ablation by combining controlled temperature range (<44 °C), reduced MNP dosage; much lower than in most reported studies, and AMF parameters (time of exposure and frequency) within the clinical safety limit. Periodic body weight measurements confirmed negligible adverse side effects in rats. The anti-tumor activity was validated by severe apoptosis (TUNEL, cleaved Caspase-3), reduced proliferation (Ki 67) and disrupted vasculature (CD 31) in the Fe3O4-MHT-treated group. Real-time gene expression of pro-inflammatory cytokines (IL-6, TNF-α, IL-1α, IL-1ß) confirmed the intratumoral activation of IL-6, suggesting the role of immunomodulation in triggering the adaptive immune response for faster tumor regression in the treated group. In addition, the biodistribution and clearance rate of MNPs monitored using ICP-OES confirmed their time-dependent biodegradation via excretion (urine, feces), phagocytosis (liver) and circulatory system (blood), with negligible deposition in other major organs (kidney, heart, lungs). Although we could not show complete clearance of our MNPs within the time frame tested, future studies should focus on combining MHT with immunotherapy, and target tumors at a much-reduced iron dose, consequently improving in vivo clearance rate, and hence overcoming the limitations of MHT in clinical therapy.

Tissue-specific mesenchymal stem cell-dependent osteogenesis in highly porous chitosan-based bone analogs.

Among conventional fabrication techniques, freeze-drying process has widely been investigated for polymeric implants. However, the understanding of the stem cell progenitor-dependent cell functionality modulation and quantitative analysis of early osseointegration of highly porous scaffolds have not been explored. Here, we developed a novel, highly porous, multimaterial composite, chitosan/hydroxyapatite/polycaprolactone (CHT/HA/PCL). The in vitro studies have been performed using mesenchymal stem cells (MSCs) from three tissue sources: human bone marrow-derived MSCs (BM-MSCs), adipose-derived MSCs (AD-MSCs), and Wharton's jelly-derived MSCs (WJ-MSCs). Although cell attachment and metabolic activity [3-4,5-dimethylthiazol-2yl-(2,5 diphenyl-2H-tetrazoliumbromide) assay] were ore enhanced in WJ-MSC-laden CHT/HA/PCL composites, scanning electron microscopy, real-time gene expression (alkaline phosphatase [ALP], collagen type I [Col I], osteocalcin [OCN], and bone morphogenetic protein 4 [BMP-4]), and immunostaining (COL I, ß-CATENIN, OCN, and SCLEROSTIN [SOST]) demonstrated pronounced osteogenesis with terminal differentiation on BM-MSC-laden CHT/HA/PCL composites only. The enhanced cell functionality on CHT/HA/PCL composites was explained in terms of interplay among the surface properties and the optimal source of MSCs. In addition, osteogenesis in rat tibial model over 6 weeks confirmed a better ratio of bone volume to the total volume for BM-MSC-laden composites over scaffold-only and defect-only groups. The clinically conformant combination of 3D porous architecture with pore sizes varying in the range of 20 to 200 µm together with controlled in vitro degradation and early osseointegration establish the potential of CHT/HA/PCL composite as a potential cancellous bone analog.

Functional Skin Grafts: Where Biomaterials Meet Stem Cells.

Skin tissue engineering has attained several clinical milestones making remarkable progress over the past decades. Skin is inhabited by a plethora of cells spatiotemporally arranged in a 3-dimensional (3D) matrix, creating a complex microenvironment of cell-matrix interactions. This complexity makes it difficult to mimic the native skin structure using conventional tissue engineering approaches. With the advent of newer fabrication strategies, the field is evolving rapidly. However, there is still a long way before an artificial skin substitute can fully mimic the functions and anatomical hierarchy of native human skin. The current focus of skin tissue engineers is primarily to develop a 3D construct that maintains the functionality of cultured cells in a guided manner over a period of time. While several natural and synthetic biopolymers have been translated, only partial clinical success is attained so far. Key challenges include the hierarchical complexity of skin anatomy; compositional mismatch in terms of material properties (stiffness, roughness, wettability) and degradation rate; biological complications like varied cell numbers, cell types, matrix gradients in each layer, varied immune responses, and varied methods of fabrication. In addition, with newer biomaterials being adopted for fabricating patient-specific skin substitutes, issues related to escalating processing costs, scalability, and stability of the constructs under in vivo conditions have raised some concerns. This review provides an overview of the field of skin regenerative medicine, existing clinical therapies, and limitations of the current techniques. We have further elaborated on the upcoming tissue engineering strategies that may serve as promising alternatives for generating functional skin substitutes, the pros and cons associated with each technique, and scope of their translational potential in the treatment of chronic skin ailments.

Advances in three-dimensional bioprinting of bone: Progress and challenges.

Several attempts have been made to engineer a viable three-dimensional (3D) bone tissue equivalent using conventional tissue engineering strategies, but with limited clinical success. Using 3D bioprinting technology, scientists have developed functional prototypes of clinically relevant and mechanically robust bone with a functional bone marrow. Although the field is in its infancy, it has shown immense potential in the field of bone tissue engineering by re-establishing the 3D dynamic micro-environment of the native bone. Inspite of their in vitro success, maintaining the viability and differentiation potential of such cell-laden constructs overtime, and their subsequent preclinical testing in terms of stability, mechanical loading, immune responses, and osseointegrative potential still needs to be explored. Progress is slow due to several challenges such as but not limited to the choice of ink used for cell encapsulation, optimal cell source, bioprinting method suitable for replicating the heterogeneous tissues and organs, and so on. Here, we summarize the recent advancements in bioprinting of bone, their limitations, challenges, and strategies for future improvisations. The generated knowledge will provide deep insights on our current understanding of the cellular interactions with the hydrogel matrices and help to unravel new methodologies for facilitating precisely regulated stem cell behaviour.

Silk fibroin-bioactive glass based advanced biomaterials: towards patient-specific bone grafts.

A major challenge in bone tissue engineering is to develop patient-specific, defect-site specific grafts capable of triggering specific cell signaling pathways. We could programmably fabricate the 3D printed bone constructs via direct ink writing of silk-gelatin-bioactive glass (SF-G-BG) hybrids using two different compositions of melt-derived bioactive glasses (with and without strontium) and compared against commercial 45S5 Bioglass®. Physico-chemical characterization revealed that released ions from bioactive glasses inhibited the conformational change of Bombyx mori silk fibroin protein (from random coil to ß-sheet conformation), affecting printability of the SF-G-BG ink. In-depth molecular investigations showed that strontium containing SF-G-BG constructs demonstrated superior osteogenic differentiation of mesenchymal stem cells (TVA-BMSCs) over 21 days towards osteoblastic (marked by upregulated expression of runt related transcription factor, alkaline phosphatase, osteopontin, osteonectin, integrin bone sialoprotein, osteocalcin) and osteocytic (marked by podoplanin, dentin matrix acidic phosphoprotein, sclerostin) phenotype compared to other BG compositions and silk-gelatin alone. Moreover, ionic release from bioactive glasses in the silk-gelatin ink triggered the activation of signaling pathways (BMP-2, BMP-4 and IHH), which are critical in regulating bone formation in vivo. Overall, the presence of strontium containing bioactive glass in silk-gelatin matrices provided appropriate cues in regulating the development of custom-made 3D in vitro human bone constructs.

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.

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.

Differential Regulation of Hedgehog and Parathyroid Signaling in Mulberry and Nonmulberry Silk Fibroin Textile Braids.

Even after several decades of research, the most optimal source of silk for promoting osteogenesis in situ is still a subject of debate. A major gap in existing knowledge is role of underlying signaling mechanisms in both the mulberry and nonmulberry silk species that leads to the development of differential levels of osteogenesis. In our previous study, we elucidated the role of Wnt/ß-catenin signaling for promoting superior osteogenic differentiation in nonmulberry silk braids in the presence of TGF-ß and pro-osteogenic supplements. Here, we provide a comparative osteogenic analysis of the two most popular silk species (mulberry and nonmulberry silk), in the form of silk braids prepared from natively spun fibers, by conducting detailed gene expression profiling using 25 different osteogenic markers, followed by further validation by immunohistochemistry. Our study provides novel insights into the direct regulatory role of nonmulberry silk fibroin braids on hedgehog and parathyroid signaling pathways in controlling osteogenic differentiation of cultured human fetal osteoblasts (hFOBs), a phenomenon not very evident in the mulberry silk textile braids. Although both silk braids enabled adequate cellular attachment, proliferation, and extracellular collagen matrix formation, superior expression of osteogenic markers (ALP, VDR, Runx2), matrix proteins (Col1A2, OPN), and signaling molecules (GLI1, GLI2, Shh) with characteristic terminal osteocytic phenotype could only be observed in nonmulberry silk. Therefore, our study provided detailed insights into the development of engineered bone to be a prospective tissue equivalent with potential to provide the essential instructive elements for activating physiological pathways of bone differentiation. Such engineered constructs have potential for use as an in vitro model for drug testing and as scaffolds for bone regeneration strategies.

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 © 2015 John Wiley & Sons, Ltd.

Nonmulberry Silk Braids Direct Terminal Osteocytic Differentiation through Activation of Wnt-Signaling.

Silk polymers can regulate osteogenesis by mimicking some features of the extracellular matrix of bone and facilitate mineralized deposition on their surface by cultured osteoprogenitors. However, terminal differentiation of these mineralizing osteoblasts into osteocytic phenotypes has not yet been demonstrated on silk. Therefore, in this study we test the hypothesis that flat braids of natively (nonregenerated) spun nonmulberry silk A. mylitta, possessing mechanical stiffness in the range of trabecular bone, can regulate osteocyte differentiation within their 3D microenvironment. We seeded human preosteoblasts onto these braids and cultured them under varied temperatures (33.5 and 39 °C), soluble factors (dexamethasone, ascorbic acid, and ß-glycerophosphate), and cytokine (TGF-ß1). After 1 week, cell dendrites were conspicuously evident, confirming osteocyte differentiation, especially, in the presence of osteogenic factors and TGF-ß1 expressing all characteristic osteocyte markers (podoplanin, DMP-1, and sclerostin). A. mylitta silk braids alone were sufficient to induce this differentiation, albeit only transiently. Therefore, we believe that the combinatorial effect of A. mylitta silk (surface chemistry, braid rigidity, and topography), osteogenic differentiation factors, and TGF-ß1 were critical in stabilizing the mature osteocytic phenotype. Interestingly, Wnt signaling promoted osteocytic differentiation as evidenced by the upregulated expression of ß-catenin in the presence of osteogenic factors and growth factor. This study highlights the role of nonmulberry silk braids in regulating stable osteocytic differentiation. Future studies could benefit from this understanding of the signaling mechanisms associated with silk-based matrices in order to develop 3D in vitro bone model systems.

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.

Osteogenic signaling on silk-based matrices.

Bone tissue engineering has mainly focused on generating 3D grafts to repair bone defects. However, the underlying signaling mechanisms responsible for development of such 3D bone equivalents have largely been ignored. Here we describe the crucial aspects of embryonic osteogenesis and bone development including cell sources and general signaling cascades that guide mesenchymal progenitors towards osteogenic lineage. Drawing from the knowledge of developmental biology, we then review how silk biomaterial can regulate osteogenic signaling by focusing on the expression of cell surface markers, functional genomic information (mRNA) of stem cells cultured on silk matrices. In an attempt to recapitulate exact in vivo microenvironment of osteogenesis, role of scaffold architecture and material chemistry in regulating cellular differentiation is elaborated. The generated knowledge will not only improve our understanding of cell-material interactions but reveal newer strategies beyond a conventional tissue engineering paradigm and open new prospects for developing silk-based therapies against clinically relevant bone disorders.

Regulation of Chondrogenesis and Hypertrophy in Silk Fibroin-Gelatin-Based 3D Bioprinted Constructs.

To date, the development of phenotypically stable, functionally equivalent engineered cartilage tissue constructs remains elusive. This study explored chondrogenic differentiation and suppression of hypertrophic differentiation in tyrosinase cross-linked silk-gelatin bioink using different cell modalities (dispersed, aggregates) for chondrocytes and mesenchymal progenitor cells (hMSCs) compared against the "gold standard" hMSC spheroids. Chondrogenic differentiation of hMSC spheroids (without silk-gelatin) showed a constant increase in hypertrophy over 21 days (gradual upregulated expression of COL10A1, MMP13). On the contrary, hMSC-laden constructs (both dispersed and aggregates) in bioink showed upregulated hypoxia (HIF1A) which positively regulated the expression of chondrogenic markers (aggrecan, COMP1) over chondrocyte-laden constructs. The gelatin component in the bioink induced MMP2 activity, which degraded the synthesized matrix, creating a pericellular zone for the accumulation of growth factors and newly synthesized matrices. We believe that the combinatorial effect of these accumulated factors as well as the hypoxia-regulated HDAC4 pathway played a pivotal role in stabilizing the chondrogenic phenotype of differentiated hMSCs along with suppressed hypertrophy. Therefore, the results suggest that tyrosinase cross-linked silk-gelatin bioink offers a suitable material composition for 3D bioprinting of cartilage constructs. Further standardization is warranted to investigate the biological mechanisms minimizing hypertrophic differentiation of hMSC/chondrocytes toward development of improved cartilage constructs.

Silk fibroin as biomaterial for bone tissue engineering.

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.

Nonmulberry Silk Fibroin Scaffold Shows Superior Osteoconductivity Than Mulberry Silk Fibroin in Calvarial Bone Regeneration.

Recent years have witnessed the advancement of silk biomaterials in bone tissue engineering, although clinical application of the same is still in its infancy. In this study, the potential of pure nonmulberry Antheraea mylitta (Am) fibroin scaffold, without preloading with bone precursor cells, to repair calvarial bone defect in a rat model is explored and compared with its mulberry counterpart Bombyx mori (Bm) silk fibroin. After 3 months of implantation, Am scaffold culminates in a completely ossified regeneration with a progressive increase in mineralization at the implanted site. On the other hand, the Bm scaffold fails to repair the damaged bone, presumably due to its low osteoconductivity and early degradation. The deposition of bone matrix on scaffolds is evaluated by scanning electron and atomic force microscopy. These results are corroborated by in vitro studies of enzymatic degradation, colony formation, and secondary conformational features of the scaffold materials. The greater biocompatibility and mineralization in pure nonmulberry fibroin scaffolds warrants the use of these scaffolds as an "ideal bone graft" biomaterial for effective repair of critical size defects.

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.

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.