Dry Surface Treatments of Silk Biomaterials and Their Utility in Biomedical Applications

Silk-based materials are widely used in biomaterial and tissue engineering applications due to their cytocompatibility and tunable mechanical and biodegradation properties. Aqueous-based processing techniques have enabled the fabrication of silk into a broad range of material formats, making it a highly versatile material platform across multiple industries. Utilizing the full potential of silk in biomedical applications frequently requires modification of silk's surface properties. Dry surface modification techniques, including irradiation and plasma treatment, offer an alternative to the conventional wet chemistry strategies to modify the physical and chemical properties of silk materials without compromising their bulk properties. While dry surface modification techniques are more prevalent in textiles and sterilization applications, the range of modifications available and resultant changes to silk materials all point to the utility of dry surface modification for the development of new, functional silk biomaterials. Dry surface treatment affects the surface chemistry, secondary structure, molecular weight, topography, surface energy, and mechanical properties of silk materials. This Review describes and critically evaluates the effect of physical dry surface modification techniques, including irradiation and plasma processes, on silk materials and discusses their utility in biomedical applications, including recent examples of modulation of cell/protein interactions on silk biomaterials, in vivo performance of implanted biomaterials, and applications in material biofunctionalization and lithographic surface patterning approaches

The biomedical use of silk: past, present, future

Humans have long appreciated silk for its lustrous appeal and remarkable physical properties, yet as the mysteries of silk are unraveled, it becomes clear that this outstanding biopolymer is more than a high-tech fiber. This progress report provides a critical but detailed insight into the biomedical use of silk. This journey begins with a historical perspective of silk and its uses, including the long-standing desire to reverse engineer silk. Selected silk structure-function relationships are then examined to appreciate past and current silk challenges. From this, biocompatibility and biodegradation are reviewed with a specific focus of silk performance in humans. The current clinical uses of silk (e.g., sutures, surgical meshes, and fabrics) are discussed, as well as clinical trials (e.g., wound healing, tissue engineering) and emerging biomedical applications of silk across selected formats, such as silk solution, films, scaffolds, electrospun materials, hydrogels, and particles. The journey finishes with a look at the roadmap of next-generation recombinant silks, especially the development pipeline of this new industry for clinical use

Rapid Photocrosslinking of Silk Hydrogels with High Cell Density and Enhanced Shape Fidelity

Silk fibroin hydrogels crosslinked through di-tyrosine bonds are clear, elastomeric constructs with immense potential in regenerative medicine applications. In this study, demonstrated is a new visible light-mediated photoredox system for di-tyrosine bond formation in silk fibroin that overcomes major limitations of current conventional enzymatic-based crosslinking. This photomediated system rapidly crosslinks silk fibroin (80%). The photocrosslinked silk hydrogels present more stable mechanical properties which do not undergo spontaneous transition to stiff, β-sheet-rich networks typically seen for enzymatically crosslinked systems. These hydrogels also support long-term culture of human articular chondrocytes, with excellent cartilage tissue formation. This system also facilitates the first demonstration of biofabrication of silk fibroin constructs in the absence of chemical modification of the protein structure or rheological additives. Cell-laden constructs with complex, ordered, graduated architectures, and high resolution (40 µm) are fabricated using the photocrosslinking system, which cannot be achieved using the enzymatic crosslinking system. Taken together, this work demonstrates the immense potential of a new crosslinking approach for fabrication of elastomeric silk hydrogels with applications in biofabrication and tissue regeneration

Silk fibroin photo-lyogels containing microchannels as a biomaterial platform for: In situ tissue engineering

The biophysical properties of biomaterials are key to directing the biological responses and biomaterial integration and function in in situ tissue engineering approaches. We present silk photo-lyogels, a biomaterial format fabricated using a new combinatorial approach involving photo-initiated crosslinking of silk fibroin via di-tyrosine bonds followed by lyophilization to generate 3D, porous lyogels showing physical properties distinct to those of lyophilized silk sponges or silk hydrogels. This fabrication approach allowed introduction of microchannels into 3D constructs via biofabrication approaches involving silk crosslinking around an array of 3D printed photocurable resin pillars to generate parallel channels or around a 3D printed sacrificial thermosensitive gel to generate interconnected channels in a rapid manner and without the need for chemical modification of silk fibroin. The presence of interconnected microchannels significantly improved migration of endothelial cells into 3D photo-lyogels in vitro, and tissue infiltration, photo-lyogel integration, and vascularization when implanted in vivo in a mouse subcutaneous model. Taken together, these findings demonstrate the feasibility and utility of a new combinatorial fabrication approach for generation of silk biomaterials that support cell interactions and implant integration for in situ tissue engineering approaches

Bone tissue engineering using 3D silk scaffolds and human dental pulp stromal cells epigenetic reprogrammed with the selective histone deacetylase inhibitor MI192.

Epigenetics plays a critical role in regulating mesenchymal stem cells’ (MSCs) fate for tissue repair and regeneration. There is increasing evidence that the inhibition of histone deacetylase (HDAC) isoform 3 can enhance MSC osteogenesis. This study investigated the potential of using a selective HDAC2 and 3 inhibitor, MI192, to promote human dental pulp stromal cells (hDPSCs) bone-like tissue formation in vitro and in vivo within porous Bombyx Mori silk scaffolds. Both 2 and 5 wt% silk scaffolds were fabricated and characterised. The 5 wt% scaffolds possess thicker internal lamellae, reduced scaffold swelling and degradation rates, whilst increased compressive modulus in comparison to the 2 wt% silk scaffold. MI192 pre-treatment of hDPSCs on 5 wt% silk scaffold significantly enhanced hDPSCs alkaline phosphatase activity (ALP). The expression of osteoblast-related genes (RUNX2, ALP, Col1a, OCN) was significantly upregulated in the MI192 pre-treated cells. Histological analysis confirmed that the MI192 pre-treated hDPSCs-silk scaffold constructs promoted bone extracellular matrix (ALP, Col1a, OCN) deposition and mineralisation compared to the untreated group. Following 6 weeks of subcutaneous implantation in nude mice, the MI192 pre-treated hDPSCs-silk scaffold constructs enhanced the vascularisation and extracellular matrix mineralisation compared to untreated control. In conclusion, these findings demonstrate the potential of using epigenetic reprogramming and silk scaffolds to promote hDPSCs bone formation efficacy, which provides evidence for clinical translation of this technology for bone augmentation

Towards engineering heart tissues from bioprinted cardiac spheroids.

Currentin vivoandin vitromodels fail to accurately recapitulate the human heart microenvironment for biomedical applications. This study explores the use of cardiac spheroids (CSs) to biofabricate advancedin vitromodels of the human heart. CSs were created from human cardiac myocytes, fibroblasts and endothelial cells (ECs), mixed within optimal alginate/gelatin hydrogels and then bioprinted on a microelectrode plate for drug testing. Bioprinted CSs maintained their structure and viability for at least 30 d after printing. Vascular endothelial growth factor (VEGF) promoted EC branching from CSs within hydrogels. Alginate/gelatin-based hydrogels enabled spheroids fusion, which was further facilitated by addition of VEGF. Bioprinted CSs contracted spontaneously and under stimulation, allowing to record contractile and electrical signals on the microelectrode plates for industrial applications. Taken together, our findings indicate that bioprinted CSs can be used to biofabricate human heart tissues for long termin vitrotesting. This has the potential to be used to study biochemical, physiological and pharmacological features of human heart tissue

Release of insulin from PLGA-alginate dressing stimulates regenerative healing of burn wounds in rats

Burn wound healing involves a complex set of overlapping processes in an environment conducive to ischemia, inflammation, and infection costing $7.5 billion/year in the US alone, in addition to the morbidity and mortality that occur when the burns are extensive. We previously showed that insulin, when topically applied to skin excision wounds, accelerates re-epithelialization, and stimulates angiogenesis. More recently, we developed an alginate sponge dressing (ASD) containing insulin encapsulated in PLGA microparticles that provides a sustained release of bioactive insulin for >20days in a moist and protective environment. We hypothesized that insulin-containing ASD accelerates burn healing and stimulates a more regenerative, less scarring, healing. Using a heat-induced burn injury in rats, we show that burns treated with dressings containing 0.04mg insulin/cm2, every three days for 9 days, have faster closure, faster rate of disintegration of dead tissue, and decreased oxidative stress.In addition, in insulin-treated wounds the pattern of neutrophil inflammatory response suggests faster clearing of the burn dead tissue. We also observe faster resolution of the pro-inflammatory macrophages. We also found that insulin stimulates collagen deposition and maturation with the fibers organized more like a basket weave (normal skin) than aligned and crosslinked (scar tissue). In summary , application of ASD-containing insulin-loaded PLGA particles on burns every three days stimulates faster and more regenerative healing. These results suggest insulin as a potential therapeutic agent in burn healing and, because of its long history of safe use in humans, insulin could become one of the treatments of choice when repair and regeneration are critical for proper tissue function.This work was supported by the National Natural Science Fund of China [grant numbers 81170761 and 81270909 (to Y.L.)]; the Natural Sciences and Engineering Research Council of Canada [grant numbers 204794-2011 (to M.H.) and private donor (to M.M.-G.)]