Academic literature on the topic 'Biomaterials, regenerative medicine, carbohydrates, proteins'

Elastin like polypeptides (ELPs) are a class of naturally derived biomaterials that are non-immunogenic, genetically encodable, and biocompatible making them ideal for a variety of biomedical applications, ranging from drug delivery to tissue engineering. Also, ELPs undergo temperature-mediated inverse phase transitioning, which allows them to be purified in a relatively simple manner from bacterial expression hosts. Being able to genetically encode ELPs allows for the incorporation of bioactive peptides and functionalization of ELPs. This work utilizes ELPs for regenerative medicine and drug delivery. The goal of the first study was to synthesize a biologically active epidermal growth factor-ELP (EGF-ELP) fusion protein that could aid in the treatment of chronic wounds. EGF plays a crucial role in wound healing by inducing epithelial cell proliferation and migration, and fibroblast proliferation. The use of exogenous EGF has seen success in the treatment of acute wounds, but has seen relatively minimal success in chronic wounds because the method of delivery does not protect exogenous EGF from degradation, or prevent it from diffusing away from the application site. We created an EGF-ELP fusion protein to combat these issues. As demonstrated through the proliferation of human skin fibroblasts in vitro, the EGF-ELP may be able to aid in the treatment of chronic wounds. Furthermore, the ability of the EGF-ELP to self-assemble near physiological temperatures could allow for the formation of drug depots at the wound site and minimize diffusion, increasing the bioavailability of EGF and enhancing tissue regeneration. The objective of the second study was to create an injectable hydrogel platform that does not require conjugation of functional moieties for crosslinking or biological activity. Hydrogels are three-dimensional polymer networks that are able to absorb water and biological fluids without dissolving. Their high water content gives them physical properties similar to soft tissues, making them useful as scaffolds for cell migration and drug delivery vehicles. Injectable hydrogels that crosslink in situ are particularly useful because they can form to the shape of the defect, providing a near perfect fit. However, many hydrogel platforms cannot be crosslinked in situ because cytotoxic crosslinking reagents are required. Additionally, hydrogels typically require the chemical conjugation of crosslinking domains and bioactive peptides to the polymer backbone, adding more steps and time required for hydrogel production. We devised an injectable hydrogel platform that can be synthesized in a single step using photoreactive ELPs as the polymer backbone. Leucine auxotrophic Eshcherichia coli expressed ELPs containing photoleucine, a leucine analog and photoreactive diazirine crosslinker, which is substituted for leucine periodically throughout the ELP sequence. Upon exposure to ultraviolet radiation (~370 nm), photoleucine is able to form covalent crosslinks with amino acid side chains, forming a polymer network for hydrogel formation. Additionally, recombinant growth factors and morphogens can be encoded into the ELP sequence providing a simple method of hydrogel functionalization for regenerative medicine applications. The potential for this platform was demonstrated through in vivo crosslinking of photoreactive ELPs in the expression hosts. Though the production of the photoreactive ELP was not as forthright as originally assumed. The substitution of noncanonical amino acids typically requires the auxotrophic expression hosts to be starved of the amino acid that they are auxotrophic for. A noncanonical analog of said amino acid can then be supplemented into expression media, maximizing incorporation. In this investigation, it was found the addition of photoleucine alone inhibited photoreactive ELP expression. ELP expression only occurred in the presence of photoleucine if valine or leucine was also present in the media. Furthermore, valine was found to aid the production of ELPs as much as leucine. It was postulated the bacterial translational machinery might need to be altered for optimal ELP expression.

Volumetric muscle loss (VML) typically results from traumatic incidents; such as those presented from combat missions, where soft-tissue extremity injuries account for approximately 63% of diagnoses. These injuries lead to a devastating loss of function due to the complete destruction of large amounts of tissue and its native basement membrane, removing important biochemical cues such as hepatocyte growth factor (HGF), which initiates endogenous muscle regeneration by recruiting progenitor cells. Clinical strategies to treat these injuries consist of autologous tissue transfer techniques, requiring large amounts of healthy donor tissue and extensive surgical procedures that can result in donor site morbidity and limited functional recovery. As such, there is a clinical need for an off-the-shelf, bioactive scaffold that directs patient’s cells to align and differentiate into muscle tissue in situ. In this thesis, we developed fibrin microthreads, scaffolds composed of aligned fibrin material that direct cell alignment along the longitudinal axis of the microthread structure, with specific structural and biochemical properties to recreate structural cues lost in VML injuries. We hypothesized that fibrin microthreads with an increased resistance to proteolytic degradation and loaded with HGF would enhance the functional, mechanical regeneration of skeletal muscle tissue in a VML injury. We developed a crosslinking strategy to increase fibrin microthread resistance to enzymatic degradation, and increased their tensile strength and stiffness two- to three-fold. This crosslinking strategy enhanced the adsorption of HGF, facilitated its rapid release from microthreads for 2 to 3 days, and increased the chemotactic response of myoblasts twofold in 2D and 3D assays. Finally, we implanted HGF-loaded, crosslinked (EDCn-HGF) microthreads into a mouse model of VML to evaluate tissue regeneration and functional recovery. Fourteen days post-injury, we observed more muscle ingrowth along EDCn-HGF microthreads than untreated controls, suggesting that released HGF recruited additional progenitor cells to the injury site. Sixty days post-injury, EDCn-HGF microthreads guided mature, organized muscle to replace the microthreads in the wound site. Further, EDCn-HGF microthreads restored the contractile mechanical strength of the tissue to pre-injured values. In summary, we designed fibrin microthreads that recapitulate regenerative cues lost in VML injuries and enhance the functional regeneration of skeletal muscle.

Biomimetic materials that recapitulate the complex mechanical and biochemical cues in load-bearing tissues are of significant interest in regenerative medicine and tissue engineering applications. Several investigators have endeavored to not only emulate the mechanical properties of the vasculature, but to also mimic the biologic responsiveness of the blood vessel in creating vascular substitutes. Previous studies in our lab generated the elastin-like protein polymer LysB10, which was designed with the capability of physical and chemical crosslinks, and was shown to display a range of elastomeric properties that more closely matched those of the native artery. While extensive validation of the mechanical properties of elastin-mimetic polymers has demonstrated their functionality in a number of tissue engineering applications, limited cell growth on the surfaces of the polymers has motivated further optimization for biological interaction. Recent biologically-inspired surface strategies have focused on functionalizing material surfaces with extracellular matrix molecules and bioactive motifs in order to encourage integrin-mediated cellular responses that trigger precise intracellular signaling processes, while limiting nonspecific biomaterial interactions. Consequently, this dissertation addresses three approaches to modulating cellular behavior on elastin-mimetic analogs with the goal of promoting vascular wall healing and tissue regeneration: genetic engineering of elastin-like protein polymers (ELPs) with cell-binding domains, biofunctionalization of elastin-like protein polymers via chemoselective ligation of bioactive ligands, and incorporation of matrix protein fibronectin for engineering of cell-seeded multilamellar collagen-reinforced elastin-like constructs. The synthesis of recombinant elastin-like protein polymers that integrate biologic functions of the extracellular matrix provides a novel design strategy for generating clinically durable vascular substitutes. Ultimately, the synthesis of model protein networks provides new insights into the relationship between molecular architecture, biomimetic ligand presentation, and associated cellular responses at the cell-material interface. Understanding how each of these design parameters affects cell response will contribute significantly to the rational engineering of bioactive materials. Potential applications for polymer blends with enhanced mechanical and biological properties include surface coatings on vascular grafts and stents, as well as composite materials for tissue engineered scaffolds and vascular substitutes.

Challenges in treating injuries, together with an increased need for repair of damaged tissues and organs, have made regenerative medicine a major research area today. Biomaterials such as silk fibroin (SF) have proven to be excellent tissue scaffolds possessing properties essential in tissue engineering such as biocompatibility, biodegradability and exceptional mechanical properties. SF nanofibres are especially attractive due to their large surface-to-volume ratio and high porosity which is beneficial in regenerative medicine. However, to design biomaterial scaffolds, chemical and physical properties of SF have to be sufficiently known. The thesis aims to contribute to knowledge by characterizing silk fibroin from the African wild silkworm species Gonometa rufobrunnae, Gonometa postica, Argema mimosae, Epiphora bahuniae and Anaphe panda. Moreover, the feasibility of producing nanofibrous biomaterial scaffolds from these fibroins is explored. The chemical composition of degummed fibres was investigated using Capillary electrophoresis whilst Infrared (IR) and Raman spectroscopic techniques were utilized to determine structural characteristics of the fibroin. In addition, thermal behaviour and mechanical properties of the fibroins were also investigated. Nanofibres were fabricated via electrospinning. The effects of solution concentration, voltage, polymer flow rate and tip to collector distance were studied to give optimum electrospinning conditions. IR spectroscopy was also utilized to observe the conformational structure of the degummed and electrospun fibres whilst scanning electron microscopy (SEM) provided information on the size and morphology of the fibres. The use of the nanofibres as biomaterials was evaluated using cytotoxicity tests. Results showed that glycine, alanine and serine constituted over 70% of the amino acid composition of all the fibroins. Gonometa fibroin had more glycine than alanine whilst the opposite was true for Argema mimosae, Epiphora bahuniae and Anaphe panda fibroin. The abundance of basic amino acids in Gonometa rufobrunnae, Gonometa postica, Argema mimosae and Epiphora bahuniae fibroin makes them prime candidates for cell and tissue culture. The amino acid composition of the fibroins influenced secondary structure as the β-sheet structure. Anaphe panda, Argema mimosae and Epiphora bahuniae silks was made up of mostly alanine-alanine (Ala-Ala)n polypeptides whilst Gonometa fibroin had an interesting mixture of both glycine-alanine (Gly-Ala)n and (Ala-Ala)n units. The unique structures impacted the mechanical and thermal properties of the fibroins. Production of Gonometa nanofibres was mainly dependent on fibroin solution concentration. A minimum of 27 % w/v was needed to produce defect free nanofibres. Diameters of the electrospun fibres produced ranged from 300 to 2500 nm. IR spectroscopy data highlighted that the β-sheet conformation of degummed fibroin was degraded during the formation of the nanofibres rendering them water soluble. It was however possible to regenerate the β-sheet structure in the nanofibres by exposing them to various solvents. Cytotoxicity tests using Sulforhodamine B (SRB) assay demonstrated that the nanofibres were not toxic to cells, a major prerequisite for use as a biomaterial. This thesis successfully provides useful data in an area that has been minimally explored. Results suggest that SF from African silkworm species offers diversity in properties and are therefore attractive for use as biomaterials, especially in cell and tissue engineering. As far as we could determine, we are the first to extend the use of fibroin from African silk species by producing Gonometa SF nanofibres that are of potential use as biomaterial scaffolds.<br>Chemistry<br>D. Phil. (Chemisty)