Volumetric Muscle Loss Repair Research Articles

Bioactive Nb2C MXene-Functionalized Hydrogel with Microenvironment Remodeling and Enhanced Neurogenesis to Promote Skeletal Muscle Regeneration and Functional Restoration.

The complete structure-functional repair of volumetric muscle loss (VML) remains a giant challenge and biomedical hydrogels to remodel microenvironment and enhance neurogenesis have appeared to be a promising direction. However, the current hydrogels for VML repair hardly achieve these two goals simultaneously due to their insufficient functionality and the challenge in high-cost of bioactive factors. In this study, a facile strategy using Nb2C MXene-functionalized hydrogel (OPTN) as a bioactive scaffold is proposed to promote VML repair with skeletal muscle regeneration and functional restoration. In vitro experiments show that OPTN scaffold can effectively scavenge reactive oxygen species (ROS), guide macrophages polarization toward M2 phenotype, and resist bacterial infection, providing a favorable microenvironment for myoblasts proliferation as well as the endothelial cells proliferation, migration, and tube formation. More importantly, OPTN scaffold with electroactive feature remarkably boosts myoblasts differentiation and mesenchymal stem cells neural differentiation. Animal experiments further confirm that OPTN scaffold can achieve a prominent structure-functional VML repair by attenuating ROS levels, alleviating inflammation, reducing fibrosis, and facilitating angiogenesis, newborn myotube formation, and neurogenesis. Collectively, this study provides a highly promising and effective strategy for the structure-functional VML repair through designing bioactive multifunctional hydrogel with microenvironment remodeling and enhanced neurogenesis.

Biodegradable conductive IPN in situ cryogels with anisotropic microchannels and sequential delivery of dual-growth factors for skeletal muscle regeneration

Biodegradable and anisotropic cryogels that simulate conductivity and ECM orientation structure of skeletal muscle, and release multiple growth factors, are expected for in situ skeletal muscle tissue engineering. Herein, biodegradable, conductive and anisotropic interpenetrating network (IPN) in situ cryogels are fabricated through Schiff base/acylhydrazone crosslinking via combining unidirectional freezing and cyclic freeze-thaw processes. The cryogels have good anisotropic mechanical properties and oriented microchannel structure, and induce the oriented alignment of myoblasts. The introduction of aniline tetramer enhances the mechanical properties and conductivity of the cryogels. The conductive cryogels significantly improve the proliferation and myogenic differentiation of C2C12 cells during 3D culture. The sequential delivery of insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) can induce migration of human umbilical vein endothelial cells (HUVECs), proliferation of human skin myofibroblasts (HMFB), and myogenic differentiation of C2C12 cells in vitro. In particular, conductive and anisotropic cryogels with dual-growth factors can significantly improve the repair efficiency of volumetric muscle loss (VML) in vivo. This study provides a new strategy to fabricate anisotropic and conductive IPN in situ cryogel biomimetic scaffolds that can encapsulate dual-growth factors and deliver them in time sequence, which significantly promote the efficient repair of VML via an in situ tissue engineering approach.

Impact of Passaging Primary Skeletal Muscle Cell Isolates on the Engineering of Skeletal Muscle.

Volumetric muscle loss (VML) is a clinical state that results in impaired skeletal muscle function. Engineered skeletal muscle can serve as a treatment for VML. Currently, large biopsies are required to achieve the cells necessary for the fabrication of engineered muscle, leading to donor-site morbidity. Amplification of cell numbers using cell passaging may increase the usefulness of a single muscle biopsy for engineering muscle tissue. In this study, we evaluated the impact of passaging cells obtained from donor muscle tissue by analyzing characteristics of in vitro cellular growth and tissue-engineered skeletal muscle unit (SMU) structure and function. Human skeletal muscle cell isolates from three separate donors (P0-Control) were compared with cells passaged once (P1), twice (P2), or three times (P3) by monitoring SMU force production and determining muscle content and structure using immunohistochemistry. Data indicated that passaging decreased the number of satellite cells and increased the population doubling time. P1 SMUs had slightly greater contractile force and P2 SMUs showed statistically significant greater force production compared with P0 SMUs with no change in SMU muscle content. In conclusion, human skeletal muscle cells can be passaged twice without negatively impacting SMU muscle content or contractile function, providing the opportunity to potentially create larger SMUs from smaller biopsies, thereby producing clinically relevant sized grafts to aid in VML repair.

Bioactive Nanofiber-Hydrogel Composite Regulates Regenerative Microenvironment for Skeletal Muscle Regeneration after Volumetric Muscle Loss.

Volumetric muscle loss (VML) is a severe form of muscle trauma that exceeds the regenerative capacity of skeletal muscle tissue, leading to substantial functional impairment. The abnormal immune response and excessive reactive oxygen species (ROS) accumulation hinder muscle regeneration following VML. Here, an interfacial cross-linked hydrogel-poly(ε-caprolactone) nanofiber composite, that incorporates both biophysical and biochemical cues to modulate the immune and ROS microenvironment for enhanced VML repair, is engineered. The interfacial cross-linking is achieved through a Michael addition between catechol and thiol groups. The resultant composite exhibits enhanced mechanical strength without sacrificing porosity. Moreover, it mitigates oxidative stress and promotes macrophage polarization toward a pro-regenerative phenotype, both in vitro and in a mouse VML model. 4 weeks post-implantation, mice implanted with the composite show improved grip strength and walking performance, along with increased muscle fiber diameter, enhanced angiogenesis, and more nerve innervation compared to control mice. Collectively, these results suggest that the interfacial cross-linked nanofiber-hydrogel composite could serve as a cell-free and drug-free strategy for augmenting muscle regeneration by modulating the oxidative stress and immune microenvironment at the VML site.

Biocompatible and stretchable piezoelectric elastomer based on lactic acid for synchronous repairing and function monitoring of volumetric muscle loss

The treatment of volumetric muscle loss (VML) and the real-time monitoring of damaged muscle function have long posed challenges for clinicians. The novel in-situ electric stimuli therapy using piezoelectric materials for VML repairing has attracted numerous attentions. However, the reciprocating motion of the muscle requires the piezoelectric materials with both high piezoelectricity and stretchability. In this study, a biocompatible piezoelectric elastomer (VMLRE) was synthesized through the copolymerization of several bio-based diacids and diols. By controlling the chemical compositions and chain structure, the VMLRE possess elastic modulus accurately matching with that of muscle tissue, allowing the reciprocal deformation during muscle movements to realize function recovery evaluating. The backbone of VMLRE is enriched with CO dipoles to achieve excellent piezoelectric properties, which enable converting mechanical stimuli generated by physical activity into electrical energy, providing real-time and in situ electrical stimulation. The electric stimulation promotes myoblast proliferation and differentiation by potentially activating calcium/calmodulin signaling pathways, facilitating cell migration, and enhancing neovascularization. Consequently, the rate of VML repair is significantly improved. Our innovative piezoelectric elastomer enables simultaneous repair and real-time biofeedback monitoring of VML, offering a novel strategy for the treatment of volumetric muscle loss.

Local IL-10 delivery modulates the immune response and enhances repair of volumetric muscle loss muscle injury

This study was designed to test the hypothesis that in addition to repairing the architectural and cellular cues via regenerative medicine, the delivery of immune cues (immunotherapy) may be needed to enhance regeneration following volumetric muscle loss (VML) injury. We identified IL-10 signaling as a promising immunotherapeutic target. To explore the impact of targeting IL-10 signaling, tibialis anterior (TA) VML injuries were created and then treated in rats using autologous minced muscle (MM). Animals received either recombinant rat IL-10 or phosphate buffered saline (PBS) controls injections at the site of VML repair beginning 7 days post injury (DPI) and continuing every other day (4 injections total) until 14 DPI. At 56 DPI (study endpoint), significant improvements to TA contractile torque (82% of uninjured values & 170% of PBS values), TA mass, and myofiber size in response to IL-10 treatment were detected. Whole transcriptome analysis at 14 DPI revealed activation of IL-10 signaling, muscle hypertrophy, and lymphocytes signaling pathways. Expression of ST2, a regulatory T (Treg) cell receptor, was dramatically increased at the VML repair site in response to IL-10 treatment when compared to PBS controls. The findings suggest that the positive effect of delayed IL-10 delivery might be due to immuno-suppressive Treg cell recruitment.

3D printing a biocompatible elastomer for modeling muscle regeneration after volumetric muscle loss

Volumetric muscle loss (VML) injuries due to trauma, tumor ablation, or other degenerative muscle diseases are debilitating and currently have limited options for self-repair. Advancements in 3D printing allow for the rapid fabrication of biocompatible scaffolds with designer patterns. However, the materials chosen are often stiff or brittle, which is not optimal for muscle tissue engineering. This study utilized a photopolymerizable biocompatible elastomer – poly (glycerol sebacate) acrylate (PGSA) – to develop an in vitro model of muscle regeneration and proliferation into an acellular scaffold after VML injury. Mechanical properties of the scaffold were tuned by controlling light intensity during the 3D printing process to match the specific tension of skeletal muscle. The effect of both geometric (channel sizes between 300 and 600 μm) and biologic (decellularized muscle extracellular matrix (dECM)) cues on muscle progenitor cell infiltration, proliferation, organization, and maturation was evaluated in vitro using a near-infrared fluorescent protein (iRFP) transfected cell line to assess cells in the 3D scaffold. Larger channel sizes and dECM coating were found to enhance cell proliferation and maturation, while no discernable effect on cell alignment was observed. In addition, a pilot experiment was carried out to evaluate the regenerative capacity of this scaffold in vivo after a VML injury. Overall, this platform demonstrates a simple model to study muscle progenitor recruitment and differentiation into acellular scaffolds after VML repair.

Injectable conductive micro-cryogel as a muscle stem cell carrier improves myogenic proliferation, differentiation and in situ skeletal muscle regeneration

Volumetric muscle loss (VML) results in the impediment of skeletal muscle function, and there were still great challenges in cell delivery approach with the minimally invasive operation to repair muscle defects. To deliver cells to the VML defects site efficiently, the injectable conductive porous nanocomposite microcryogels based on gelatin (GT) and reduced graphene oxide (rGO) were designed and prepared. The microcryogels were loaded with myoblasts to form an injectable cell delivery system and show the ability to protect cells during injection. Conductive microcryogel with 4 mg/mL rGO (GT/rGO4) enhanced C2C12 cell proliferation and myogenic differentiation during 3D culture compared with pure gelatin microcryogel. In a mice VML model, injection of microcryogel loaded with muscle-derived stem cells into the injury site significantly improved the generation of new muscle fibers and blood vessels, and anti-inflammatory properties. The results show that injectable biodegradable conductive microcryogel can be used as muscle stem cell carriers with the potential to maintain cell activity and deliver cells to defective sites, thereby in situ enhancing skeletal muscle regeneration. Statement of significanceVolumetric muscle loss overwhelms the regenerative capacity of skeletal muscle, which results in severe damage to muscle tissues. In the treatment of volumetric muscle loss, conductive niche and muscle stem cells are needed to alleviate excessive scar formation and inflammation to improve muscle regeneration. Injectable gelatin/reduced graphene oxide based nanocomposite microcryogel can enhance the differentiation of seeded muscle stem cells. The improved repair of volumetric muscle loss was achieved via reducing collagen deposition and inflammation in the injected region through the microcryogel cell-delivery therapy, suggesting great potential of the injectable microcryogel as a cell carrier in soft tissue repair.

A myogenic niche with a proper mechanical stress environment improves abdominal wall muscle repair by modulating immunity and preventing fibrosis

Volumetric muscle loss (VML) healing is often complicated by fibrosis, which impairs muscle regeneration and function. Adjusting mechanical stress in the repair environment may modulate immunity and reduce fibrosis. In this study, we aimed to create a biomaterial with suitable tension conditions and bidirectional tissue-inducing abilities to prevent fibrosis thus promote muscle regeneration and induce aponeurosis-like structures to restore muscle force transmission. A protocol was developed to manufacture decellularized muscle aponeurosis (D-MA) patches with an intact extracellular matrix (ECM) and low cytotoxicity. D-MA optimized the mechanical stress distribution in muscle injury sites and decreased the number of proinflammatory macrophages and myofibroblasts, thereby attenuating muscle fibrosis. Muscle and aponeurosis ECM environments had different microstructures and mechanical properties, which specifically enhanced stem cell differentiation into muscle-like cells on muscle ECM and tenocyte-like cells on aponeurosis ECM in vitro. Four weeks after orthotopic implantation, the biphasic muscle-aponeurosis-like tissue was successfully regenerated by the D-MA scaffold. The regenerated muscle fibers in D-MA were more abundant than those in the fibrotic decellularized muscle (D-M) scaffold. D-MA can be used to repair abdominal defects, which significantly improves the repair outcomes. Our results suggest D-MA as a promising material for VML repair.

Nandrolone supplementation does not improve functional recovery in an aged animal model of volumetric muscle loss injury.

Aging hinders the effectiveness of regenerative medicine strategies targeting the repair of volumetric muscle loss (VML) injury. Anabolic steroids have been shown to improve several factors which contribute to the age-related decline in muscle's regenerative capacity. In this study, the impact of exogenous nandrolone decanoate (ND) administration on the effectiveness of a VML regenerative repair strategy was explored using an aged animal model. Unilateral tibialis anterior VML injuries were repaired in 18-month-aged animal models (male Fischer 344 rat) using decellularized human skeletal muscle scaffolds supplemented with autologous minced muscle. The contralateral limb was left untreated/uninjured. Following repair, ND(+) or a carrier control (ND-) was delivered via weekly injection for a period of 8weeks. At 8weeks, muscle isometric torque, gene expression, and tissue structure were assessed. ND(+) treatment did not improve contractile torque recovery following VML repair when compared to carrier only ND(-) injection controls. Peak isometric torque in the ND(+) VML repair group remained significantly below contralateral uninjured control values (4.69±1.18vs. 7.46±1.53N mm/kg) and was statistically indistinguishable from carrier only ND(-) VML repair controls (4.47±1.18N mm/kg). Gene expression for key myogenic genes (Pax7, MyoD, MyoG, IGF-1) were not significantly elevated in response to ND injection, suggesting continued age related myogenic impairment even in the presence of ND(+) treatment. ND injection did reduce the histological appearance of fibrosis at the site of VML repair, and increased expression of the collagen III gene, suggesting some positive effects on repair site matrix regulation. Overall, the results presented in this study suggest that a decline in regenerative capacity with aging may present an obstacle to regenerative medicine strategies targeting VML injury and that the delivery of anabolic stimuli via ND administration was unable to overcome this decline.

Photoreactive Hydrogel Stiffness Influences Volumetric Muscle Loss Repair.

Volumetric muscle loss (VML) injuries are characterized by permanent loss of muscle mass, structure, and function. Hydrogel biomaterials provide an attractive platform for skeletal muscle tissue engineering due to the ability to easily modulate their biophysical and biochemical properties to match a range of tissue characteristics. In this work, we successfully developed a mechanically tunable hyaluronic acid (HA) hydrogel system to investigate the influence of hydrogel stiffness on VML repair. HA was functionalized with photoreactive norbornene groups to create hydrogel networks that rapidly crosslink through thiol-ene click chemistry with tailored mechanics. Mechanical properties were controlled by modulating the amount of matrix metalloproteinase-degradable peptide crosslinker to produce hydrogels with increasing elastic moduli of 1.1 ± 0.002, 3.0 ± 0.002, and 10.6 ± 0.006 kPa, mimicking a relevant range of developing and mature muscle stiffnesses. Functional muscle recovery was assessed following implantation of the HA hydrogels by in situ photopolymerization into rat latissimus dorsi (LD) VML defects at 12 and 24 weeks postinjury. After 12 weeks, muscles treated with medium stiffness (3.0 kPa) hydrogels produced maximum isometric forces most similar to contralateral healthy LD muscles. This trend persisted at 24 weeks postinjury, suggestive of sustained functional recovery. Histological analysis revealed a significantly larger zone of regeneration with more de novo muscle fibers following implantation of medium stiffness hydrogels in VML-injured muscles compared to other experimental groups. Lower (low and medium) stiffness hydrogels also appeared to attenuate the chronic inflammatory response characteristic of VML injuries, displaying similar levels of macrophage infiltration and polarization to healthy muscle. Together these findings illustrate the importance of hydrogel mechanical properties in supporting functional repair of VML injuries. Impact statement This report defines the role hydrogel mechanical properties play in the repair of volumetric muscle loss (VML) injuries. We show that an intermediate hydrogel stiffness (3 kPa) more compliant than adult muscle tissue facilitated improved and sustained regenerative outcomes up to 24 weeks postinjury in a rat latissimus dorsi model of VML. Muscles treated with 3 kPa hydrogels showed enhanced myogenesis and attenuation of the chronic inflammatory response characteristic of VML injuries. These results should help guide the future design of hydrogels for skeletal muscle tissue engineering and regeneration.

Human Skeletal Muscle Units for the Repair of Volumetric Muscle Loss

Human Skeletal Muscle Units for the Repair of Volumetric Muscle Loss

Replace and repair: Biomimetic bioprinting for effective muscle engineering.

The debilitating effects of muscle damage, either through ischemic injury or volumetric muscle loss (VML), can have significant impacts on patients, and yet there are few effective treatments. This challenge arises when function is degraded due to significant amounts of skeletal muscle loss, beyond the regenerative ability of endogenous repair mechanisms. Currently available surgical interventions for VML are quite invasive and cannot typically restore function adequately. In response to this, many new bioengineering studies implicate 3D bioprinting as a viable option. Bioprinting for VML repair includes three distinct phases: printing and seeding, growth and maturation, and implantation and application. Although this 3D bioprinting technology has existed for several decades, the advent of more advanced and novel printing techniques has brought us closer to clinical applications. Recent studies have overcome previous limitations in diffusion distance with novel microchannel construct architectures and improved myotubule alignment with highly biomimetic nanostructures. These structures may also enhance angiogenic and nervous ingrowth post-implantation, though further research to improve these parameters has been limited. Inclusion of neural cells has also shown to improve myoblast maturation and development of neuromuscular junctions, bringing us one step closer to functional, implantable skeletal muscle constructs. Given the current state of skeletal muscle 3D bioprinting, the most pressing future avenues of research include furthering our understanding of the physical and biochemical mechanisms of myotube development and expanding our control over macroscopic and microscopic construct structures. Further to this, current investigation needs to be expanded from immunocompromised rodent and murine myoblast models to more clinically applicable human cell lines as we move closer to viable therapeutic implementation.

COX-2 inhibition does not alter wound healing outcomes of a volumetric muscle loss injury treated with a biologic scaffold.

Volumetric muscle loss (VML) injuries are characterized by a heightened immune response that alters canonical wound healing outcomes and results in a chronic reduction of both total myofiber number and functional capacity. Preclinical and clinical investigations aimed at repairing VML injuries have largely utilized biologic scaffolds (BSs) as a potential therapeutic intervention. BSs rely on the recruitment of a myriad of host-derived immune, stem, and stromal cells to induce a wound healing response that has been routinely characterized as largely fibrous matrix deposition and limited myofiber regeneration at the site of the defect. While the mechanisms by which this occurs are not fully elucidated, the role of the immune response and modulation thereof is believed to be critical to BS-mediated wound healing outcomes. Given the known roles of nonsteroidal anti-inflammatory drugs (NSAIDs) on cyclooxygenase (COX) signaling and of COX signaling on macrophage polarization and myogenesis, it is plausible that prescription of NSAIDs could alter BS-mediated VML repair. To study the effect of COX-2 inhibition on BS-mediated repair of VML, an established rat model of VML was acutely treated with BSs with and without adjunct administration of mavacoxib, a COX-2-specific inhibitor. Evaluation of the function of the affected musculature as well as the tissue-level histomorphology, evaluated at 14 and 90 days postoperatively, suggested that COX-2 inhibition does not alter the baseline repair outcomes of the BS therapy. These studies provide useful information to clinicians regarding the postoperative prescription of NSAIDS in concert with a BS therapy in the treatment of VML.

The Effects of Engineered Skeletal Muscle on Volumetric Muscle Loss in The Tibialis Anterior Of Rat After Three Months In Vivo.

Volumetric muscle loss (VML) is traumatic, degenerative, or surgical loss of skeletal muscle that exceeds the regenerative capacity of the remaining muscle, thus resulting in impaired muscle function. In humans, the loss of 30% or more mass of any one muscle will result in permanent structural and functional loss. Current VML repair treatments are limited by donor site morbidity and graft tissue availability, necessitating alternative muscle graft sources. To address this need, our lab has fabricated tissue-engineered skeletal muscle units (SMUs) for implantation into a 30 % VML model in the tibialis anterior (TA) muscle of rat. Previous results showed that after 28 days in vivo, muscle with a 30% VML repaired with our SMUs produced significantly more force than muscle with acute VML. But repair with our SMU did not fully restore muscle force production to that of native muscle. Thus, we hypothesized that more time for in vivo tissue regeneration would allow for greater force recovery. Therefore, the purpose of this study was to examine the long-term (3-month) effects of our SMUs on a 30% VML repair. We also assessed the effects of reinnervation by redirecting a branch of the peroneal nerve to the repair site. Thirty-nine, 2-month old female F344 rats were separated into a nonsurgical control group (n=5) and four surgical experimental groups (VML Only, n=5; VML+Nerve Redirect, n=6; VML+SMU, n=5; VML+SMU+ Nerve Redirect, n=8). Experimental rats were allowed a 3-month recovery period post-surgery before undergoing in situ force testing of the surgical (left) TA. The left TA of the control animals also underwent in situ force testing. Finally, the surgical (left) and contralateral (right) TAs of the experimental animals, as well as the left TA of the control animals, were explanted for histological analysis. Results for specific force showed that while all groups recovered specific forces similar to that of native muscle, the two SMU groups had significantly higher specific forces, on average, compared to the uninjured control group. Histological staining showed small muscle fibers in the repair site in animals that received an SMU. The average cross-sectional area of the native fibers just outside the area of repair (or the equivalent area in control animals) was not significantly different between groups, indicating that hypertrophy of remaining fibers did not contribute to the recovery of force following the VML. Our results suggest that following a 30% VML of the TA muscle, all surgical groups were able to recover TA mass, maximum tetanic and specific force production. Thus, creating a 30% VML in the TA in a rat model is not enough a sufficient VML to produce the sustained VML seen in humans following similar 30% loss of muscle volume.

Pre-Clinical Cell Therapeutic Approaches for Repair of Volumetric Muscle Loss

Extensive damage to skeletal muscle tissue due to volumetric muscle loss (VML) is beyond the inherent regenerative capacity of the body, and results in permanent functional debilitation. Current clinical treatments fail to fully restore native muscle function. Recently, cell-based therapies have emerged as a promising approach to promote skeletal muscle regeneration following injury and/or disease. Stem cell populations, such as muscle stem cells, mesenchymal stem cells and induced pluripotent stem cells (iPSCs), have shown a promising capacity for muscle differentiation. Support cells, such as endothelial cells, nerve cells or immune cells, play a pivotal role in providing paracrine signaling cues for myogenesis, along with modulating the processes of inflammation, angiogenesis and innervation. The efficacy of cell therapies relies on the provision of instructive microenvironmental cues and appropriate intercellular interactions. This review describes the recent developments of cell-based therapies for the treatment of VML, with a focus on preclinical testing and future trends in the field.

Long-Term Evaluation of Functional Outcomes Following Rat Volumetric Muscle Loss Injury and Repair.

Volumetric muscle loss (VML) injuries, by definition, exceed the endogenous repair capacity of skeletal muscle resulting in permanent structural and functional deficits. VML injuries present a significant burden for both civilian and military medicine. Despite progress, there is still considerable room for therapeutic improvement. In this regard, tissue-engineered constructs show promise for VML repair, as they provide an opportunity to introduce both scaffolding and cellular components. We have pioneered the development of a tissue-engineered muscle repair (TEMR) technology created by seeding muscle progenitor cells onto a porcine-derived bladder acellular matrix followed by cyclic stretch preconditioning before implantation. Our work to date has demonstrated significant functional repair (60-90% functional recovery) in progressively larger rodent models of VML injury following TEMR implantation. Notwithstanding this success, TEMR implantation in cylindrically shaped VML injuries in the tibialis anterior (TA) muscle was associated with more variable functional outcomes than has been observed in sheet-like muscles such as the latissimus dorsi. In fact, previous observations documented a dichotomy of responses following TEMR implantation in a rodent TA VML injury model; with an ≈61% functional improvement observed in fewer than half (46%) of TEMR-implanted animals at 12 weeks postinjury. This current study builds directly from those observations as we modified the geometry of both the VML injury and the TEMR construct to determine if improved matching of the implanted TEMR construct to the surgically created VML injury resulted in increased functional recovery posttreatment. Following these modifications, we observed a comparable degree of functional improvement in a larger proportion of animals (≈67%) that was durable up to 24 weeks post-TEMR implantation. Moreover, in ≈25% of all TEMR-implanted animals, functional recovery was virtually complete (TEMR max responders), and furthermore, the functional recovery in all 67% of responding animals was accompanied by the presence of native-like muscle properties within the repaired TA muscle, including fiber cross-sectional area, fiber type, vascularization, and innervation. This study emphasizes the importance of tuning the application of tissue engineering technology platforms to the specific requirements of diverse VML injuries to improve functional outcomes. Impact Statement This report confirms and extends previous observations with our implantable tissue-engineered technology platform for repair of volumetric muscle loss (VML) injuries. Based on our prior work, we addressed factors hypothesized to be responsible for significant outcome variability following treatment of VML injuries in a rat tibialis anterior model. Through customization of the muscle repair technology to a specific VML injury, we were able to significantly increase the frequency at which functional recovery occurred, and furthermore, demonstrate durability out to 6 months. In addition, the enhanced biomimetic qualities of repaired muscle tissue were associated with the most robust functional outcomes.

Encapsulation of bone marrow-MSCs in PRP-derived fibrin microbeads and preliminary evaluation in a volumetric muscle loss injury rat model: modular muscle tissue engineering

Repair of volumetric muscle loss (VML) injuries is a complicated endeavour which necessitates the collaborative use of different regenerative approaches and technologies. Herein is proposed the development of fibrin-based microbeads (FMs) alone or as a bone marrow mesenchymal stem cell (MSC) encapsulation matrix for modular muscle engineering. FMs were generated through the ionotropic gelation of alginate and fibrinogen obtained from the platelet-rich plasma of whole blood, and then removing the alginate by citrate treatment. FMs were first characterized by FT-IR, SEM and water uptake tests. Then, the stability of FMs and the mitochondrial dehydrogenase activity of the MSCs encapsulated in FMs were evaluated under in vitro culture conditions. Eventually, the regenerative capacity of the cell-devoid and MSCs-encapsulated FMs was evaluated in a rat VML injury model involving 8 × 4×4 mm3-size bilateral defects in the biceps femoris muscles. The histochemical, immunohistochemical and semi-quantitative histomorphological scoring results retrieved at 30, 60 and 180 days demonstrated that the cell-devoid FMs supported muscle regeneration to a great extent. Moreover, MSCs-encapsulated FMs were more effective in shortening the regeneration period of the injured tissue of the rat VML, resulting in good myofibre orientation, while the Sham group resulted in incomplete repair with fibrotic scar tissue formations.

A Porcine Urinary Bladder Matrix Does Not Recapitulate the Spatiotemporal Macrophage Response of Muscle Regeneration after Volumetric Muscle Loss Injury

Volumetric muscle loss (VML) results in irrecoverable loss of muscle tissue making its repair challenging. VML repair with acellular extracellular matrix (ECM) scaffolds devoid of exogenous cells has shown improved muscle function, but limited de novo muscle fiber regeneration. On the other hand, studies using minced autologous and free autologous muscle grafts have reported appreciable muscle regeneration. This raises the fundamental question whether an acellular ECM scaffold can orchestrate the spatiotemporal cellular events necessary for appreciable muscle fiber regeneration. This study compares the macrophage and angiogenic responses including the remodeling outcomes of a commercially available porcine urinary bladder matrix, MatriStem™, and autologous muscle grafts. The early heightened and protracted M1 response of the scaffold indicates that the scaffold does not recapitulate the spatiotemporal macrophage response of the autograft tissue. Additionally, the scaffold only supports limited de novo muscle fiber formation and regressing vessel density. Furthermore, scaffold remodeling is accompanied by increased presence of transforming growth factor and α-smooth muscle actin, which is consistent with remodeling of the scaffold into a fibrotic scar-like tissue. The limited muscle formation and scaffold-mediated fibrosis noted in this study corroborates the findings of recent studies that investigated acellular ECM scaffolds (devoid of myogenic cells) for VML repair. Taken together, acellular ECM scaffolds when used for VML repair will likely remodel into a fibrotic scar-like tissue and support limited de novo muscle fiber regeneration primarily in the proximity of the injured musculature. This is a work of the US Government and is not subject to copyright protection in the USA. Foreign copyrights may apply. Published by S. Karger AG, Basel

Porcine urinary bladder matrix does not generate appreciable de novo muscle tissue in rat models of volumetric muscle loss injury

Event Abstract Back to Event Porcine urinary bladder matrix does not generate appreciable de novo muscle tissue in rat models of volumetric muscle loss injury Amit Aurora1, Janet L. Roe1, Benjamin T. Corona1 and Thomas J. Walters1 1 US Army Institute of Surgical Research, Extremity Trauma Research and Regenerative Medicine, United States Background: The high prevalence of volumetric muscle loss (VML) among battlefield injuries has stimulated research efforts aimed at regenerating muscle de novo using tissue engineering approaches. Extracellular matrix (ECM) scaffolds, in particular, have been extensively used based on their ability to provide a bio-inductive platform that supports endogenous cell recruitment, proliferation, and differentiation, ultimately improving healing of the tissue[1][2]. Recently, surgeons have used commercially available porcine derived urinary bladder matrix (UBM) to repair lower extremity VML in a small cohort of patients[3][4]. The porcine UBM scaffold has clinically been used for surgical reinforcement of soft tissue where weakness exits and general wound management-indications for which it has been approved by the U.S. Food and Drug Administration. However, there is need to establish evidence that will support further clinical use of the scaffold for the repair of traumatic muscle injuries. The overarching objective of this study is to investigate the efficacy of the porcine UBM scaffold as a tissue engineered strategy for the repair of traumatic muscle injuries. Methods: A musculotendinous junction (MTJ) or volumetric muscle loss (VML) injury was created in Lewis rats. For the MTJ injury, the defect created either received no repair or was repaired with a 6-layer MatriStem Surgical Matrix PSMX (time points: 2, 4, or 8 weeks). For the VML injury, the defect created received no repair, repaired with autograft tissue or with a 3-layer MatriStem Surgical Matrix PSM (time points: 2, 8, or 16 weeks). The m-ECM was only investigated at 2 weeks. Upon finding that the porcine UBM scaffold partially restored the muscle function in VML injury at eight weeks; a second study was performed to determine if physical rehabilitation during an eight week period would further improve function. The porcine UBM repaired rats were given free access to running wheels beginning at one week or four weeks post - injury. In vivo function testing was done (MTJ injury: 4 weeks post-injury; VML injury: 8 and 16 weeks post-injury) followed by tissue harvest for histological analysis. Results and Discussion: The porcine UBM scaffold did not promote appreciable muscle tissue regeneration as demonstrated in two distinct models of extremity VML. The porcine UBM scaffold completely resorbed when used as an interpositional graft for MTJ repair. When used to augment a VML defect surrounded by muscle and fascia, the porcine UBM scaffold improves the contractile properties of the injured limb by 17%, but does so without significant muscle fiber regeneration compared to autograft that showed robust regeneration (A-C). Finally, inclusion of physical rehabilitation (i.e., voluntary wheel running) did not improve tissue remodeling or functional improvements observed with scaffold implantation alone. Conclusions: The scaffold may not be suitable for clinical repair of MTJ injuries. The porcine UBM scaffold when used for VML repair may hasten functional recovery not due to significant muscle regeneration, but through force transmission via scaffold mediated fibrosis. The study suggests that if appreciable muscle generation is to be realized, combinatorial approaches that incorporate myogenic cells, a myoconductive scaffold, and a vasculogenic accelerant need to be developed.