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Silks are appealing materials for numerous biomedical applications involving drug delivery, tissue engineering, or implantable devices, because of their tunable mechanical properties and wide range of physical structures. In addition to the functionalities needed for specific clinical applications, a key factor necessary for clinical success for any implanted material is appropriate interactions with the body in vivo . This review summarizes our current understanding of the in vivo biological responses to silks, including degradation, the immune and inflammatory response, and tissue remodeling with particular attention to vascularization. While we focus in this review on silkworm silk fibroin protein due to the large quantity of in vivo data thanks to its widespread use in medical materials and consumer products, spider silk information is also included if available. Silk proteins are degraded in the body on a time course that is dependent on the method of silk fabrication and can range from hours to years. Silk protein typically induces a mild inflammatory response that decreases within a few weeks of implantation. The response involves recruitment and activation of macrophages and may include activation of a mild foreign body response with the formation of multinuclear giant cells, depending on the material format and location of implantation. The number of immune cells present decreases with time and granulation tissue, if formed, is replaced by endogenous, not fibrous, tissue. Importantly, silk materials have not been demonstrated to induce mineralization, except when used in calcified tissues. Due to its ability to be degraded, silk can be remodeled in the body allowing for vascularization and tissue ingrowth with eventual complete replacement by native tissue. The degree of remodeling, tissue ingrowth, or other specific cell behaviors can be modulated with addition of growth or other signaling factors. Silk can also be combined with numerous other materials including proteins, synthetic polymers, and ceramics to enhance its characteristics for a particular function. Overall, the diverse array of silk materials shows excellent bioresponses in vivo with low immunogenicity and the ability to be remodeled and replaced by native tissue making it suitable for numerous clinical applications. [ABSTRACT FROM AUTHOR]

Abstract: The development of biomaterials that mimic the physiological binding of growth factors to the extracellular matrix (ECM) is an appealing strategy for advanced growth factor delivery systems. In vivo, fibroblast growth factor 2 (FGF-2) binds to the sulfated glycosaminoglycan heparan sulfate, which is a major component of the ECM. Therefore, we tested whether silk fibroin (SF) decorated with a sulfonated moiety could mimic the natural ECM environment and lead to advanced delivery of this heparin-binding growth factor. Using a diazonium coupling reaction, modified SF derivatives containing approximately 20, 40, 55 and 70 sulfonic acid groups per SF molecule were obtained. Films of the SF derivative decorated with 70 sulfonic acid groups per SF molecule resulted in a 2-fold increase in FGF-2 binding as compared to native SF. More than 99% of bound FGF-2 could be retained on all SF derivatives. However, protection of FGF-2 potency was only achieved with at least 40 sulfonic acid groups per SF molecule, as observed by reduced metabolic activity and enhanced levels of phosphorylated extracellular signal-regulated kinases (pERK1/2) in cultured human mesenchymal stem cells (hMSCs). This study introduces a first step towards the development of an ECM-mimicking biomaterial for sustained, non-covalent binding, controlled delivery and preserved potency of biomolecules. [Copyright &y& Elsevier]

Abstract: A simple chemical modification method using diazonium coupling chemistry was developed to tailor the structure and hydrophilicity of silk fibroin protein. The extent of modification using several aniline derivatives was characterized using UV–vis and 1H NMR spectroscopies, and the resulting protein structure was analyzed with ATR-FTIR spectroscopy. Introduction of hydrophobic functional groups facilitated rapid conversion of the protein from a random coil to a β-sheet structure, while addition of hydrophilic groups inhibited this process. hMSCs were grown on these modified silks to assess the biocompatibility of these materials. The hydrophilicity of the silk derivatives was found to affect the growth rate and morphology, but hMSCs were able to attach, proliferate and differentiate into an osteogenic lineage on all of the silk derivatives. [Copyright &y& Elsevier]

In this study, silk fibroin and hyaluronic acid (HA) were enzymatically crosslinked to form biocompatible composite hydrogels with tunable mechanical properties similar to that of native tissues. The formation of di-tyrosine crosslinks between silk fibroin proteins via horseradish peroxidase has resulted in a highly elastic hydrogel but exhibits time-dependent stiffening related to silk self-assembly and crystallization. Utilizing the same method of crosslinking, tyramine-substituted HA forms hydrophilic and bioactive hydrogels that tend to have limited mechanics and degrade rapidly. To address the limitations of these singular component scaffolds, HA was covalently crosslinked with silk, forming a composite hydrogel that exhibited both mechanical integrity and hydrophilicity. The composite hydrogels were assessed using unconfined compression and infrared spectroscopy to reveal of the physical properties over time in relation to polymer concentration. In addition, the hydrogels were characterized by enzymatic degradation and for cytotoxicity. Results showed that increasing HA concentration, decreased gelation time, increased degradation rate, and reduced changes that were observed over time in mechanics, water retention, and crystallization. These hydrogel composites provide a biologically relevant system with controllable temporal stiffening and elasticity, thus offering enhanced tunable scaffolds for short or long term applications in tissue engineering. [ABSTRACT FROM AUTHOR]

Silk fibers spun by silkworms and spiders exhibit exceptional mechanical properties with a unique combination of strength, extensibility and toughness. In contrast, the mechanical properties of regenerated silk materials can be tuned through control of the fabrication process. Here we introduce a biomimetic, all-aqueous process, to obtain bulk regenerated silk-based materials for the fabrication of functionalized orthopedic devices. The silk materials generated in the process replicate the nano-scale structure of natural silk fibers and possess excellent mechanical properties. The biomimetic materials demonstrate excellent machinability, providing a path towards the fabrication of a new family of resorbable orthopedic devices where organic solvents are avoided, thus allowing functionalization with bioactive molecules to promote bone remodeling and integration. [ABSTRACT FROM AUTHOR]

Recombinant spider silk materials with antimicrobial peptides are a promising new class of drug-free antimicrobial materials capable of preventing surgical site infections (SSI), but their potential to impede infections is unclear. Herein, we aimed to unravel the biological and inflammatory potential of bioengineered spider silk materials to prevent SSI using an infection animal model. Silk-like fibers made of silk fibroin and spider silk proteins with antimicrobial peptides (6mer-HNP1) held improved stiffness (2.9 GPa) and had a slow biodegradation profile while inhibiting bacterial adherence in vitro by 5-log and 6-log reduction on Methicillin-Resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), respectively. In vivo studies showed that fibers with 6mer-HNP1 elicited a short-term low to mild local inflammatory response, similar to implanted commercial sutures. In the presence of a bacterial infection, the mediators related to infection and inflammation were downregulated suggesting that the fibers maintained a low but active response to bacterial infection. Thus, the presence of 6mer-HNP1 helped the host maintain an active response to bacterial infection, impairing the development of an acute infection. Our findings further support the use of bioengineered spider silk proteins to develop drug-free antimicrobial sutures capable to impair SSI. [ABSTRACT FROM AUTHOR]

3D printing has experienced swift growth for biological applications in the field of regenerative medicine and tissue engineering. Essential features of bioprinting include determining the appropriate bioink, printing speed mechanics, and print resolution while also maintaining cytocompatibility. However, the scarcity of bioinks that provide printing and print properties and cell support remains a limitation. Silk Fibroin (SF) displays exceptional features and versatility for inks and shows the potential to print complex structures with tunable mechanical properties, degradation rates, and cytocompatibility. Here we summarize recent advances and needs with the use of SF protein from Bombyx mori silkworm as a bioink, including crosslinking methods for extrusion bioprinting using SF and the maintenance of cell viability during and post bioprinting. Additionally, we discuss how encapsulated cells within these SF-based 3D bioprinted constructs are differentiated into various lineages such as skin, cartilage, and bone to expedite tissue regeneration. We then shift the focus towards SF-based 3D printing applications, including magnetically decorated hydrogels, in situ bioprinting, and a next-generation 4D bioprinting approach. Future perspectives on improvements in printing strategies and the use of multicomponent bioinks to improve print fidelity are also discussed. [ABSTRACT FROM AUTHOR]

Silk fibroin (SF) is a natural polymer widely used and studied for diverse applications in the biomedical field. Recently, genetically modified silks, particularly fluorescent SF fibers, were reported to have been produced from transgenic silkworms. However, they are currently limited to textile manufacturing. To expand the use of transgenic silkworms for biomedical applications, a solution form of fluorescent SF needed to be developed. Here, we describe a novel method of preparing a fluorescent SF solution and demonstrate long-term fluorescent function up to one year after subcutaneous insertion. We also show that fluorescent SF labeled p53 antibodies clearly identify HeLa cells, indicating the applicability of fluorescent SF to cancer detection and bio-imaging. Furthermore, we demonstrate the intraoperative use of fluorescent SF in an animal model to detect a small esophageal perforation (0.5 mm). This study suggests how fluorescent SF biomaterials can be applied in biotechnology and clinical medicine. [ABSTRACT FROM AUTHOR]

A silk fibroin (silk) hydrogel was prepared by using diglycidyl ether (BDDE), a chemical crosslinker commonly used to generate Food and Drug Administration (FDA)-approved hyaluronic acid (HA) medical products. The silk/BDDE hydrogels exhibited high elasticity (compressive modulus of 166 ± 15.0 kPa), anti-fatigue properties, and stable structure and mechanical strength in aqueous solution. Chemical crosslinking was conducted in a high concentration (9.3 M) of lithium bromide (LiBr) solution, a salt that is commonly used to dissolve degummed silk fibers during silk solubilization. The unfolded and extended structure of silk molecules with these reaction conditions, as well as the unique ionic environment provided by LiBr facilitated a high degree of crosslinking in the hydrogel. Similar hydrogels were not obtained when the silk was dissolved in other silk fiber-dissolving reagents (e.g., Ajisawa's, formic acid (FA)/LiBr, FA/CaCl 2 solutions), likely because partially folded silk structures and the ionic conditions with these reagents were less favorable for the crosslinking reaction. Based on these findings, silk/BDDE hydrogel spheres were prepared using an oil/water (o/w) emulsification method and biocompatibility and biodegradation were evaluated in vivo , along with other silk gel control systems (e.g., enzyme-catalyzed di-tyrosine and pulverized silk/BDDE gel particles with irregular shapes). Histological and immunohistochemical analyses demonstrated that the silk/BDDE hydrogel spheres were biocompatible and served as a bio-lubricant to treat osteoarthritis (OA). The intra-articular injection of the gel spheres reduced pain as measured with OA rats, reduced cartilage damage and resisted the digestive environment in the articular cavity for extended time frames (>4 weeks), suggesting utility for pain relief and sustained drug release for future OA treatments. [ABSTRACT FROM AUTHOR]

Despite the promise for stem cell-based tissue engineering for regenerative therapy, slow and insufficient vascularization of large tissue constructs negatively impacts the survival and function of these transplanted cells. A combination of channeled porous silk scaffolds and prevascularization with endothelial cells was investigated to test the ability of this tissue engineering strategy to support rapid and extensive vascularization process. We report that hollow channels promote in vitro prevascularization by facilitating endothelial cell growth, VEGF secretion, and capillary-like tube formation. When implanted in vivo , the pre-established vascular networks in the hollow channel scaffolds anastomose with host vessels and exhibit accelerated vascular infiltration throughout the whole tissue construct, which provides timely and sufficient nutrients to ensure the survival of the transplanted stem cells. This tissue engineering strategy can promote the effective application of stem cell-based regeneration to improve future clinical applications. [ABSTRACT FROM AUTHOR]

Surgical management of long-gap esophageal defects with autologous gastrointestinal tissues is frequently associated with adverse complications including organ dysmotility, dysphagia, and donor site morbidity. In order to develop alternative graft options, bi-layer silk fibroin (SF) scaffolds were investigated for their potential to support functional tissue regeneration in a rodent model of esophageal repair. Onlay esophagoplasty was performed with SF matrices (N = 40) in adult rats for up to 2 m of implantation. Parallel groups consisted of animals implanted with small intestinal submucosa (SIS) scaffolds (N = 22) or sham controls receiving esophagotomy alone (N = 20). Sham controls exhibited a 100% survival rate while rats implanted with SF and SIS scaffolds displayed respective survival rates of 93% and 91% prior to scheduled euthanasia. Animals in each experimental group were capable of solid food consumption following a 3 d post-op liquid diet and demonstrated similar degrees of weight gain throughout the study period. End-point μ-computed tomography at 2 m post-op revealed no evidence of contrast extravasation, fistulas, strictures, or diverticula in any of the implant groups. Ex vivo tissue bath studies demonstrated that reconstructed esophageal conduits supported by both SF and SIS scaffolds displayed contractile responses to carbachol, KCl and electrical field stimulation while isoproterenol produced tissue relaxation. Histological (Masson's trichrome and hematoxylin and eosin) and immunohistochemical (IHC) evaluations demonstrated both implant groups produced de novo formation of skeletal and smooth muscle bundles positive for contractile protein expression [fast myosin heavy chain (MY32) and α-smooth muscle actin (α-SMA)] within the graft site. However, SF matrices promoted a significant 4-fold increase in MY32+ skeletal muscle and a 2-fold gain in α-SMA+ smooth muscle in comparison to the SIS cohort as determined by histomorphometric analyses. A stratified squamous, keratinized epithelium expressing cytokeratin 5 and involucrin proteins was also present at 2 m post-op in all experimental groups. De novo innervation and vascularization were evident in all regenerated tissues indicated by the presence of synaptophysin (SYP38)+ boutons and vessels lined with CD31 expressing endothelial cells. In respect to SIS, the SF group supported a significant 4-fold increase in the density of SYP38+ boutons within the implant region. Evaluation of host tissue responses revealed that SIS matrices elicited chronic inflammatory reactions and severe fibrosis throughout the neotissues, in contrast to SF scaffolds. The results of this study demonstrate that bi-layer SF scaffolds represent promising biomaterials for onlay esophagoplasty, capable of producing superior regenerative outcomes in comparison to conventional SIS scaffolds. [ABSTRACT FROM AUTHOR]

A silk-protein based reservoir rod was developed for zero-order and long-term sustained drug delivery applications. Silk reservoir rod formulations were processed in three steps. First, a regenerated silk fibroin solution, rich in random-coil content was transformed into a tubular silk film with controllable dimensions, uniform film morphology and a structure rich in silk II , β -sheet content via “film-spinning.” Second, the drug powder was loaded into swollen silk tubes followed by tube end clamping. Last, clamped silk tube ends were sealed completely via dip coating. Anastrozole, an FDA approved active ingredient for the treatment of breast cancer, was used as a model drug to investigate viability of the silk reservoir rod technology for sustained drug delivery. The in vitro and in vivo pharmacokinetic data (in a female Sprague–Dawley rat model) analyzed via liquid chromatography-tandem mass spectroscopy indicated zero-order release for 91 days. Both in vitro and in vivo anastrozole release rates could be controlled simply by varying silk rod dimensions. The swelling behavior of silk films and zero-order anastrozole release kinetics indicated practically immediate film hydration and formation of a linear anastrozole concentration gradient along the silk film thickness. The dependence of anastrozole release rate on the overall silk rod dimensions was in good agreement with an essentially diffusion-controlled sustained release from a reservoir cylindrical geometry. In vivo results highlighted a strong in vitro - in vivo pharmacokinetic correlation and a desirable biocompatibility profile of silk reservoir rods. During a 6-month implantation in rats, the apparent silk molecular weight values decreased gradually, while rod dry mass and β -sheet crystal content values remained essentially constant, providing a suitable timeframe for controlled, long-term sustained delivery applications. Overall, the silk reservoir rod may be a viable candidate for sustained delivery of breast cancer therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: Adverse side-effects associated with enterocystoplasty for neurogenic bladder reconstruction have spawned the need for the development of alternative graft substitutes. Bi-layer silk fibroin (SF) scaffolds and small intestinal submucosa (SIS) matrices were investigated for their ability to support bladder tissue regeneration and function in a rat model of spinal cord injury (SCI). Bladder augmentation was performed with each scaffold configuration in SCI animals for 10 wk of implantation and compared to non-augmented control groups (normal and SCI alone). Animals subjected to SCI alone exhibited a 72% survival rate (13/18) while SCI rats receiving SIS and bi-layer SF scaffolds displayed respective survival rates of 83% (10/12) and 75% (9/12) over the course of the study period. Histological (Masson's trichrome analysis) and immunohistochemical (IHC) evaluations demonstrated both implant groups supported de novo formation of smooth muscle layers with contractile protein expression [α-smooth muscle actin (α-SMA) and SM22α] as well as maturation of multi-layer urothelia expressing cytokeratin (CK) and uroplakin 3A proteins. Histomorphometric analysis revealed bi-layer SF and SIS scaffolds respectively reconstituted 64% and 56% of the level of α-SMA+ smooth muscle bundles present in SCI-alone controls, while similar degrees of CK+ urothelium across all experimental groups were detected. Parallel evaluations showed similar degrees of vascular area and synaptophysin+ boutons in all regenerated tissues compared to SCI-alone controls. In addition, improvements in certain urodynamic parameters in SCI animals, such as decreased peak intravesical pressure, following implantation with both matrix configurations were also observed. The data presented in this study detail the ability of acellular SIS and bi-layer SF scaffolds to support formation of innervated, vascularized smooth muscle and urothelial tissues in a neurogenic bladder model. [Copyright &y& Elsevier]

Abstract: Blended polymers are used extensively in many critical medical conditions as components of permanently implanted devices. Hybrid protein polymers containing recombinant human tropoelastin and silk fibroin have favorable characteristics as implantable scaffolds in terms of mechanical and biological properties. A firefly luciferase transgenic mouse model was used to monitor real-time IL-1β production localized to the site of biomaterial implantation, to observe the acute immune response (up to 5 days) to these materials. Significantly reduced levels of IL-1β were observed in silk/tropoelastin implants compared to control silk only implants at 1, 2 and 3 days post-surgery. Subsequently, mice (n = 9) were euthanized at 10 days (10D) and 3 weeks (3W) post-surgery to assess inflammatory cell infiltration and collagen deposition, using histopathology and immunohistochemistry. Compared to control silk only implants, fewer total inflammatory cells were found in silk/tropoelastin (∼29% at 10D and ∼47% at 3W). Also fewer ingrowth cells (∼42% at 10D and ∼63% at 3W) were observed within the silk/tropoelastin implants compared to silk only. Lower IL-6 (∼52%) and MMP-2 (∼84%) (pro-inflammatory) were also detected for silk/tropoelastin at 10 days. After 3 weeks implantation, reduced neovascularization (vWF ∼43%), fewer proliferating cells (Ki67 ∼58% and PCNA ∼41%), macrophages (F4/80 ∼64%), lower IL-10 (∼47%) and MMP-9 (∼55%) were also observed in silk/tropoelastin materials compared to silk only. Together, these results suggest that incorporation of tropoelastin improves on the established biocompatibility of silk fibroin, uniquely measured here as a reduced foreign body inflammatory response. [Copyright &y& Elsevier]

Abstract: Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a porcine model of augmentation cystoplasty. Two bi-layer matrix configurations were fabricated by solvent-casting/salt leaching either alone (Group 1) or in combination with silk film casting (Group 2) to yield porous foams buttressed by heterogeneous surface pore occlusions or homogenous silk films, respectively. Bladder augmentation was performed with each scaffold group (6 × 6 cm2) in juvenile Yorkshire swine for 3 m of implantation. Augmented animals exhibited high rates of survival (Group 1: 5/6, 83%; Group 2: 4/4, 100%) and voluntary voiding over the course of the study period. Urodynamic evaluations demonstrated mean increases in bladder capacity over pre-operative levels (Group 1: 277%; Group 2: 153%) which exceeded nonsurgical control gains (144%) encountered due to animal growth. Similarly, elevations in bladder compliance were substantially higher in augmented animals from baseline (Group 1: 357%; Group 2: 147%) in comparison to controls (41%). Gross tissue evaluations revealed that both matrix configurations supported extensive de novo tissue formation throughout the entire original implantation site which exhibited ultimate tensile strength similar to nonsurgical counterparts. Histological and immunohistochemical analyses showed that both implant groups promoted comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent cytokeratin, uroplakin, and p63 protein expression in both matrix groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Ex vivo organ bath studies demonstrated that regenerated tissues supported by both silk matrices displayed contractile responses to carbachol, α,β-methylene-ATP, KCl, and electrical field stimulation similar to controls. Our data detail the ability of acellular silk scaffolds to support regeneration of innervated, vascularized smooth muscle and urothelial tissues within 3 m with structural, mechanical, and functional properties comparable to native tissue in a porcine model of bladder repair. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Purified native silk fibroin forms β-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of β-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical cross-links responsible for gel stabilization. K+ at physiological concentrations and low pH promoted gelation, which was not observed in the presence of Ca2+. The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5–2h. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol–gel transitions, such as for cell encapsulation. [Copyright &y& Elsevier]

Abstract: Currently available synthetic grafts demonstrate moderate success at the macrovascular level, but fail at the microvascular scale (<6mm inner diameter). We report on the development of silk fibroin microtubes for blood vessel repair with several advantages over existing scaffold materials/designs. These microtubes were prepared by dipping straight lengths of stainless steel wire into aqueous silk fibroin, where the addition of poly(ethylene oxide) (PEO) enabled control of microtube porosity. The microtube properties were characterized in terms of pore size, burst strength, protein permeability, enzymatic degradation, and cell migration. Low porosity microtubes demonstrated superior mechanical properties in terms of higher burst pressures, but displayed poor protein permeability; whereas higher porosity tubes had lower burst strengths but increased permeability and enhanced protein transport. The microtubes also exhibited cellular barrier functions as low porosity tubes prevented outward migration of GFP-transduced HUVECs, while the high porosity microtubes allowed a few cells per tube to migrate outward during perfusion. When combined with the biocompatible and suturability features of silk fibroin, these results suggest that silk microtubes, either implanted directly or preseeded with cells, are an attractive biomaterial for microvascular grafts. [Copyright &y& Elsevier]

Abstract: Natural bone consists of cortical and trabecular morphologies, the latter having variable pore sizes. This study aims at engineering different bone-like structures using scaffolds with small pores (112–224μm) in diameter on one side and large pores (400–500μm) on the other, while keeping scaffold porosities constant among groups. We hypothesized that tissue engineered bone-like structure resulting from silk fibroin (SF) implants is pre-determined by the scaffolds’ geometry. To test this hypothesis, SF scaffolds with different pore diameters were prepared and seeded with human mesenchymal stem cells (hMSC). As compared to static seeding, dynamic cell seeding in spinner flasks resulted in equal cell viability and proliferation, and better cell distribution throughout the scaffold as visualized by histology and confocal microscopy, and was, therefore, selected for subsequent differentiation studies. Differentiation of hMSC in osteogenic cell culture medium in spinner flasks for 3 and 5 weeks resulted in increased alkaline phosphatase activity and calcium deposition when compared to control medium. Micro-computed tomography (μCT) detailed the pore structures of the newly formed tissue and suggested that the structure of tissue-engineered bone was controlled by the underlying scaffold geometry. [Copyright &y& Elsevier]

Traditional bone fixation devices made from inert metal alloys provide structural strength for bone repair but are limited in their ability to actively promote bone healing. Although several naturally derived bioactive materials have been developed to promote ossification in bone defects, it is difficult to translate small-scale benchtop fabrication of these materials to high-output manufacturing. Standard industrial molding processes, such as injection and compression molding, have typically been limited to use with synthetic polymers since most biopolymers cannot withstand the harsh processing conditions involved in these techniques. Here we demonstrate injection and compression molding of a bioceramic composite comprised of hydroxyapatite (HA) and silk fibroin (SF) from Bombyx mori silkworm cocoons. Both the molding behavior of the HA-SF slurry and final scaffold mechanics can be controlled by modulating SF protein molecular weight, SF content, and powder-to-liquid ratio. HA-SF composites with up to 20 weight percent SF were successfully molded into stable three-dimensional structures using high pressure molding techniques. The unique durability of silk fibroin enables application of common molding techniques to fabricate composite silk-ceramic scaffolds. This work demonstrates the potential to move bone tissue engineering one step closer to large-scale manufacturing of natural protein-based resorbable bone grafts and fixation devices. [ABSTRACT FROM AUTHOR]