Non-Covalently Associated Streptavidin Multi-Arm Nanohubs Exhibit Mechanical and Thermal Stability in Cross-Linked Protein-Network Materials.

Constructing protein-network materials that exhibit physicochemical and mechanical properties of individual protein constituents requires molecular cross-linkers with specificity and stability. A well-known example involves specific chemical fusion of a four-arm polyethylene glycol (tetra-PEG) to desired proteins with secondary cross-linkers. However, it is necessary to investigate tetra-PEG-like biomolecular cross-linkers that are genetically fused to the proteins, simplifying synthesis by removing additional conjugation and purification steps. Non-covalently, self-associating, streptavidin homotetramer is a viable, biomolecular alternative to tetra-PEG. Here, a multi-arm streptavidin design is characterized as a protein-network material platform using various secondary, biomolecular cross-linkers, such as high-affinity physical (i.e., non-covalent), transient physical, spontaneous chemical (i.e., covalent), or stimuli-induced chemical cross-linkers. Stimuli-induced, chemical cross-linkers fused to multi-arm streptavidin nanohubs provide sufficient diffusion prior to initiating permanent covalent bonds, allowing proper characterization of streptavidin nanohubs. Surprisingly, non-covalently associated streptavidin nanohubs exhibit extreme stability, which translates into material properties that resemble hydrogels formed by chemical bonds even at high temperatures. Therefore, this study not only establishes that the streptavidin nanohub is an ideal multi-arm biopolymer precursor but also provides valuable guidance for designing self-assembling nanostructured molecular networks that can properly harness the extraordinary properties of protein-based building blocks.

Engineering skeletal muscle: Building complexity to achieve functionality.

Volumetric muscle loss (VML) VML is defined as the loss of a critical mass of skeletal muscle that overwhelms the muscle's natural healing mechanisms, leaving patients with permanent functional deficits and deformity. The treatment of these defects is complex, as skeletal muscle is a composite structure that relies closely on the action of supporting tissues such as tendons, vasculature, nerves, and bone. The gold standard of treatment for VML injuries, an autologous muscle flap transfer, suffers from many shortcomings but nevertheless remains the best clinically available avenue to restore function. This review will consider the use of composite tissue engineered constructs, with multiple components that act together to replicate the function of an intact muscle, as an alternative to autologous muscle flaps. We will discuss recent advances in the field of tissue engineering that enable skeletal muscle constructs to more closely reproduce the functionality of an autologous muscle flap by incorporating vasculature, promoting innervation, and reconstructing the muscle-tendon boundary. Additionally, our understanding of the cellular composition of skeletal muscle has evolved to recognize the importance of a diverse variety of cell types in muscle regeneration, including fibro/adipogenic progenitors and immune cells like macrophages and regulatory T cells. We will address recent advances in our understanding of how these cell types interact with, and can be incorporated into, implanted tissue engineered constructs.

Cytoskeleton-inspired artificial protein design to enhance polymer network elasticity.

Reducing topological network defects to enhance elasticity in polymeric materials remains a grand challenge. Efforts to control network topology, primarily focused on crosslinking junctions, continue to underperform compared to theoretical estimations from idealized networks using affine and phantom network theories. Here, artificial protein technology was adapted for the design of polymer-network hydrogels with precisely defined coil-like and rod-like strands to observe the impact of strand rigidity on the mechanical properties of polymeric materials. Cytoskeleton-inspired polymer-network hydrogels incorporated with rod-like protein strands nearly tripled the gel shear elastic modulus and relaxation time compared to coil-like protein strands, indicating an enhanced effective crosslinking density. Furthermore, asymmetric rod-coil protein designs in network strands with an optimal rod:coil ratio improved the hydrogel relaxation time, enhancing the stability of physical macromolecular associations by modulating crosslinker mobility. The careful design of strand rigidity presents a new direction to reduce topological defects for optimizing polymeric materials.

Recent advances toward understanding the role of transplanted stem cells in tissue-engineered regeneration of musculoskeletal tissues.

Stem cell-based tissue engineering is poised to revolutionize the treatment of musculoskeletal injuries. However, in order to overcome scientific, practical, and regulatory obstacles and optimize therapeutic strategies, it is essential to better understand the mechanisms underlying the pro-regenerative effects of stem cells. There has been an attempted paradigm shift within the last decade to think of transplanted stem cells as "medicinal" therapies that orchestrate healing on the basis of their secretome and immunomodulatory profiles rather than acting as bona fide stem cells that proliferate, differentiate, and directly produce matrix to form de novo tissues. Yet the majority of current bone and skeletal muscle tissue engineering strategies are still premised on a direct contribution of stem cells as building blocks to tissue regeneration. Our review of the recent literature finds that researchers continue to focus on the quantification of de novo bone/skeletal muscle tissue following treatment and few studies aim to address this mechanistic conundrum directly. The dichotomy of thought is reflected in the diversity of new advances ranging from in situ three-dimensional bioprinting to a focus on exosomes and extracellular vesicles. However, recent findings elucidating the role of the immune system in tissue regeneration combined with novel imaging platform technologies will have a profound impact on our future understanding of how stem cells promote healing following biomaterial-mediated delivery to defect sites.