Volumetric muscle loss (VML) is a debilitating injury which results in full or partial loss of function. Current clinical options utilize tissue grafts and bracing to restore function. Tissue graft implantation oftentimes leads to serious complications, some of which end in graft rejection and thereby necessitate further surgeries and procedures. Polymeric scaffolds show promise as scaffolding systems due to their mechanical properties and overall degradation profiles. Scaffolds need appropriate mechanical properties, 10-60 kPa modulus, and overall degradation times, five days to two weeks, to initiate tissue regeneration. Poly(β-amino ester)s (PBAE), a class of synthetic polymers, act as a safe biocompatible material with overall degradation times that are suitable for healing; however, due to harmful ultraviolet light (UV) irradiation from common crosslinking methods, these scaffold systems cannot be synthesized in vivo. This research presents the development and characterization of blue light (BL) crosslinked PBAEs. BL PBAEs showed vastly higher swelling ratios, 300-400% increase; decreased mechanical strength, an average decrease of 877 kPa in compressive modulus and 431 kPa in tensile modulus; and prolonged degradation patterns, 22% average mass retention. BL PBAEs show mechanical properties and degradation profiles that could be used as a skeletal muscle scaffolds.

This study investigated the effect of bone marrow derived mesenchymal stem cells (MSCs) in a PEGylated fibrin gel (PEG) seeded into a decellularized extracellular matrix (ECM) on recovery of skeletal muscle following a volumetric muscle loss (VML) injury. Six to nine month old male Sprague-Dawley rats were used in this study. Approximately one-third of the skeletal muscle mass of the lateral gastrocnemius (LGAS) was removed from the LGAS, which was immediately replaced with an acellular ECM of the same dimensions. Seven days after injury, animals were injected with one of four solutions: saline (SAL), MSCs (MSC), PEGylated fibrin hydrogel (PEG), or MSCs in PEG (PEG+MSC). Maximal isometric tetanic tension (Po) of the LGAS was assessed fifty-six days after VML injury, followed by histological evaluation. VML injury resulted in a functional impairment of the LGAS capable of producing 76.1± 4.9% of the force generated in the non-injured contralateral LGAS. Tetanic tension of the PEG+MSC treated group was significantly higher compared to all other treatment groups (p < 0.05), although specific tension (N/cm2) in the PEG+MSC group (79.7±4.0%) was only significantly higher compared to SAL (58.2±3.0) and PEG (64.0±2.1%) treated groups (p < 0.05). However, LGAS mass was significantly higher in the PEG+MSC group compared to all other groups (p < 0.05). These findings suggest the combination of the PEG+MSC did not lead to a significant increase in muscle function compared to MSC treatment alone, and demonstrates the importance of MSCs in skeletal muscle regeneration in VML injury models. However, as evident by the significant increase in LGAS mass, PEG+MSC treatment may lead to histological differences not evaluated in this study. Gross morphology of the repaired gastrocnemius was indistinguishable from the contralateral control. / text

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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.

Skeletal muscle injuries and muscle degenerative diseases pose significant challenges to the healthcare. Surgical interventions are restricted due to tissue availability, donor site morbidity and alterations to tissue biomechanics. Current cell-based therapies are hindered by low survival and long-term engraftment for the transplanted cells due to the lack of appropriate supportive microenvironment (cell niche) in the injured muscle. Therefore, there is a critical need for developing strategies that provide cellular and structural support in the regeneration of functional muscle. In the present work, a bioengineered cell niche mimicking the native skeletal muscle microenvironment has been developed for skeletal muscle regenerative engineering. It is hypothesized that the bioengineered scaffolds with appropriate structural and cell instructive properties will support myoblast alignment and function, as well as promote the myogenic responses in clinically relevant skeletal muscle injuries. The current work utilized a three-pronged approach to design biomaterial scaffolds to aid in skeletal muscle regeneration. In the first part, aligned poly(lactide-co-glycolide) (PLGA) fiber scaffolds mimicking the oriented muscle fiber microenvironment with fiber diameters of 335±154 nm (nanoscale), 1352±225 nm (microscale) and 3013±531 nm (microscale) were fabricated and characterized. Myoblasts were found to respond to fiber diameter as observed from the differences in cell alignment, cell elongation, cell spreading area, proliferation and differentiation. <i>In vivo</i> study demonstrated the potential of using microscale fiber scaffolds to improve myogenic potential in the <i>mdx</i> mouse model. In the second part, we designed, synthesized, and characterized an implantable glycosaminoglycan-based composite hydrogel consisting of hyaluronic acid, chondroitin sulfate and polyethylene glycol (HA-CS) with tailored structural and mechanical properties for skeletal muscle regeneration applications. We demonstrated that HA-CS hydrogels provided a suitable microenvironment for <i>in vitro</i> myoblast proliferation and differentiation. Furthermore, <i>in vivo</i> studies using a volumetric muscle loss model in the mouse quadriceps showed that HA-CS hydrogels integrated with the surrounding host tissue and facilitated <i>de novo</i> myofiber generation, angiogenesis, nerve innervation and minimized scar tissue formation. In the third part, we investigated the effects of PC12 secreted signaling factors in modulating C2C12 myoblast behavior. We showed that PC12 conditioned media modulated myoblast proliferation and differentiation in both 2D culture and 3D aligned electrospun fiber scaffold system in a dose dependent manner. We also demonstrated the biomimetic HA-CS hydrogel system enabled 3D encapsulation of PC12 cells secreting signaling factors and promoted survival and proliferation of myoblasts in co-culture. Further proteomics analysis identified a total of 2088 protein/peptides from the secretome of the encapsulated PC12 cells and revealed the biological role and overlapping functions of nerve secreted proteins for skeletal muscle regeneration, potentially through regulating myoblast behavior, nerve function, and angiogenesis. These set of experiments not only provide critical insight on exploiting the interactions between muscle cells and their microenvironment, but they also open new avenues for developing advanced bioengineered scaffolds for regenerative engineering of skeletal muscle tissues.<br>

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