Michael L. Lovett

Materials fabrication from Bombyx mori silk fibroin

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

Tubular silk scaffolds for small diameter vascular grafts.

Vascular surgeries such as coronary artery bypass require small diameter vascular grafts with properties that are not available at this time. Approaches using synthetic biomaterials have been not completely successful in producing non-thrombogenic grafts with inner diameters less than 6 mm, and there is a need for new biomaterials and graft designs. We propose silk fibroin as a microvascular graft material and describe tubular silk scaffolds that demonstrate improved properties over existing vascular graft materials. Silk tubes produced using an aqueous gel spinning technique were first assessed in vitro in terms of thrombogenicity (thrombin and fibrinogen adsorption, platelet adhesion) and vascular cell responses (endothelial and smooth muscle cell attachment and proliferation) in comparison with polytetrafluoroethylene (PTFE), a synthetic material most frequently used for vascular grafts. Silk tubes were then implanted into the abdominal aortas of Sprague-Dawley rats. At time points of 2 weeks and 4 weeks post implantation, tissue outcomes were assessed through gross observation (acute thrombosis, patency) and histological staining (H&E, Factor VIII, smooth muscle actin). Over the 4-week time period, we observed graft patency and endothelial cell lining of the lumen surfaces. These results demonstrate the feasibility of using silk fibroin as a vascular graft material and some advantages of silk tubes over the currently used synthetic grafts.

Gel spinning of silk tubes for tissue engineering

Tubular vessels for tissue engineering are typically fabricated using a molding, dipping, or electrospinning technique. While these techniques provide some control over inner and outer diameters of the tube, they lack the ability to align the polymers or fibers of interest throughout the tube. This is an important aspect of biomaterial composite structure and function for mechanical and biological impact of tissue outcomes. We present a novel aqueous process system to spin tubes from biopolymers and proteins such as silk fibroin. Using silk as an example, this method of winding an aqueous solution around a reciprocating rotating mandrel offers substantial improvement in the control of the tube properties, specifically with regard to winding pattern, tube porosity, and composite features. Silk tube properties are further controlled via different post-spinning processing mechanisms such as methanol treatment, air-drying, and lyophilization. This approach to tubular scaffold manufacture offers numerous tissue engineering applications such as complex composite biomaterial matrices, blood vessel grafts and nerve guides, among others.

Silk-Based Biomaterials for Sustained Drug Delivery

Silk presents a rare combination of desirable properties for sustained drug delivery, including aqueous-based purification and processing options without chemical cross-linkers, compatibility with common sterilization methods, controllable and surface-mediated biodegradation into non-inflammatory by-products, biocompatibility, utility in drug stabilization, and robust mechanical properties. A versatile silk-based toolkit is currently available for sustained drug delivery formulations of small molecule through macromolecular drugs, with a promise to mitigate several drawbacks associated with other degradable sustained delivery technologies in the market. Silk-based formulations utilize silks well-defined nano- through microscale structural hierarchy, stimuli-responsive self-assembly pathways and crystal polymorphism, as well as sequence and genetic modification options towards targeted pharmaceutical outcomes. Furthermore, by manipulating the interactions between silk and drug molecules, near-zero order sustained release may be achieved through diffusion- and degradation-based release mechanisms. Because of these desirable properties, there has been increasing industrial interest in silk-based drug delivery systems currently at various stages of the developmental pipeline from pre-clinical to FDA-approved products. Here, we discuss the unique aspects of silk technology as a sustained drug delivery platform and highlight the current state of the art in silk-based drug delivery. We also offer a potential early development pathway for silk-based sustained delivery products.

Evaluation of gel spun silk-based biomaterials in a murine model of bladder augmentation.

Currently, gastrointestinal segments are considered the gold standard for bladder reconstructive procedures. However, significant complications including chronic urinary tract infection, metabolic abnormalities, urinary stone formation, bowel dysfunction, and secondary malignancies are associated with this approach. Biomaterials derived from silk fibroin may represent a superior alternative due their robust mechanical properties, biodegradable features, and processing plasticity. In the present study, we evaluated the efficacy of a gel spun silk-based matrix for bladder augmentation in a murine model. Over the course of 70 d implantation period, H&E and Massons trichrome (MTS) analysis revealed that silk matrices were capable of supporting both urothelial and smooth muscle regeneration at the defect site. Prominent uroplakin and contractile protein expression (α-actin, calponin, and SM22α) was evident by immunohistochemical analysis demonstrating maturation of the reconstituted bladder wall compartments. Gel spun silk matrices also elicited a minimal acute inflammatory reaction following 70 d of bladder integration, in contrast to parallel assessments of small intestinal submucosa (SIS) and poly-glycolic acid (PGA) matrices which routinely promoted evidence of fibrosis and chronic inflammatory responses. Voided stain on paper analysis revealed that silk augmented animals displayed similar voiding patterns in comparison to non surgical controls by 42 d of implantation. In addition, cystometric evaluations of augmented bladders at 70 d post-op demonstrated that silk scaffolds supported significant increases in bladder capacity and voided volume while maintaining similar degrees of compliance relative to the control group. These results provide evidence for the utility of gel spun silk-based matrices for functional bladder tissue engineering applications.

Bone Marrow Osteoblastic Niche: A New Model to Study Physiological Regulation of Megakaryopoiesis

Background The mechanism by which megakaryocytes (Mks) proliferate, differentiate, and release platelets into circulation are not well understood. Growing evidence indicates that a complex regulatory mechanism, involving cellular interactions, composition of the extracellular matrix and physical parameters such as oxygen tension, may contribute to the quiescent or permissive microenvironment related to Mk differentiation and maturation within the bone marrow. Methodology/Principal Findings Differentiating human mesenchymal stem cells (hMSCs) into osteoblasts (hOSTs), we established an in vitro model for the osteoblastic niche. We demonstrated for the first time that the combination of HSCs, Mks and hypoxia sustain and promote bone formation by increasing type I collagen release from hOSTs and enhancing its fibrillar organization, as revealed by second harmonic generation microscopy. Through co-culture, we demonstrated that direct cell-cell contact modulates Mk maturation and differentiation. In particular we showed that low oxygen tension and direct interaction of hematopoietic stem cells (HSCs) with hOSTs inhibits Mk maturation and proplatelet formation (PPF). This regulatory mechanism was dependent on the fibrillar structure of type I collagen released by hOSTs and on the resulting engagement of the alpha2beta1 integrin. In contrast, normoxic conditions and the direct interaction of HSCs with undifferentiated hMSCs promoted Mk maturation and PPF, through a mechanism involving the VCAM-1 pathway. Conclusions/Significance By combining cellular, physical and biochemical parameters, we mimicked an in vitro model of the osteoblastic niche that provides a physiological quiescent microenvironment where Mk differentiation and PPF are prevented. These findings serve as an important step in developing suitable in vitro systems to use for the study and manipulation of Mk differentiation and maturation in both normal and diseased states.

Three-Dimensional System for the In Vitro Study of Megakaryocytes and Functional Platelet Production Using Silk-Based Vascular Tubes

Platelets are specialized cells produced by megakaryocytes in the bone marrow that represent the first defense against hemorrhage, yet they also play a pathological role in thrombosis, inflammation, and cancer. Millions of platelet transfusions are conducted each year, and the supply of this blood component is limited. There are many diseases where platelet production or function is impaired with severe consequences for patients. With such clinical need, new insight into the formation of platelets would have a major impact on patients and healthcare. We developed an innovative 3D system to study platelet production that represents the first spatial reconstruction of the bone marrow environment. In this system human megakaryocytes were able to migrate toward the vascular niche, extend proplatelets, and release functional platelets into vascular tubes. The combination of different bone marrow components and the compliance of silk-based vascular tubes makes this model a unique tool for the study of platelet formation and production for use in healthcare needs.

Silk Fibroin Conduits: A Cellular and Functional Assessment of Peripheral Nerve Repair

Silk fibroin conduits were designed with appropriate porosity for peripheral nerve repair. The aim of this work was to use these conduits to examine cell inflammatory responses and functional recovery in a sciatic nerve defect model. A total of 45 randomized Lewis rats were used to create an 8-mm defect bridged by a silk guide, commercial collagen guide, or an autograft. After 1, 4, and 8 weeks, macrophage recruitment, percentage of newly formed collagen, number of myelinated axons, and gastrocnemius muscle mass were evaluated. Following 8 weeks, ED1+ cells in autograft and silk conduits decreased to <1% and 17% of week 1 values, respectively. Collagen formation revealed no difference for all measured time points, suggesting a similar foreign body response. Myelinated axon counts within the silk guide revealed a greater number of proximal spouts and distal connections than collagen guides. Gastrocnemius weights demonstrated a 27% decrease between silk and autografts after 8 weeks. This study demonstrates that, in addition to tailorable degradation rates, our silk conduits possess a favorable immunogenicity and remyelination capacity for nerve repair.

Role of electrospun fibre diameter and corresponding specific surface area (SSA) on cell attachment

In order to develop scaffolds for tissue regeneration applications, it is important to develop an understanding of the kinetics of cell attachment as a function of scaffold geometry. In the present study, we investigated how the specific surface area of electrospun scaffolds affected cell attachment and spreading. Number of cells attached to the scaffold was measured by the relative intensity of a metabolic dye (MTS) and cell spreading was analysed for individual cells by measuring the area of projected F‐actin cytoskeleton. We varied the fibre diameter to obtain a specific surface area distribution in the range 2.24–18.79 µm−1. In addition, we had one case where the scaffolds had beads in them and therefore had non‐uniform fibres. For each of these different geometries, we varied the cell‐seeding density (0.5–1 × 105) and the serum concentration (0–12%) over the first 8 h in an electrospun polycaprolactone NIH 3T3 fibroblast system. Cells on beaded scaffolds showed the lowest attachment and almost no F‐actin spreading in all experiments indicating uniform fibre diameter is essential for electrospun scaffolds. For the uniform fibre scaffolds, cell attachment was a function of scaffold specific surface area (SSA) (18.79–2.24 µm−1) and followed two distinct trends: when scaffold SSA was < 7.13 µm−1, cell adhesion rate remained largely unchanged; however, for SSA > 7.13 µm−1 there was a significant increase in cellular attachment rate with increasing SSA. This indicated that nanofibrous scaffolds increased cellular adhesion compared to microfibrous scaffolds. This phenomenon is true for serum concentrations of 7.5% and higher. For 5% and lower serum concentration, cell attachment is low and higher SSA fails to make a significant improvement in cell attachment. When cell attachment was investigated at a single‐cell level by measuring the projected actin area, a similar trend was noted where the effect of higher SSA led to higher projected area for cells at 8 h. These results indicate that uniform electrospun scaffolds with SSA provide a faster cell attachment compared to lower SSA and beaded scaffolds. These results indicate that continuous electrospun nanofibrous scaffolds may be a good substrate for rapid tissue regeneration. Copyright

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

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