Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy.

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5' AMP-activated protein kinase (AMPKα1/α2/ß2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

Agent-based model provides insight into the mechanisms behind failed regeneration following volumetric muscle loss injury.

Skeletal muscle possesses a remarkable capacity for repair and regeneration following a variety of injuries. When successful, this highly orchestrated regenerative process requires the contribution of several muscle resident cell populations including satellite stem cells (SSCs), fibroblasts, macrophages and vascular cells. However, volumetric muscle loss injuries (VML) involve simultaneous destruction of multiple tissue components (e.g., as a result of battlefield injuries or vehicular accidents) and are so extensive that they exceed the intrinsic capability for scarless wound healing and result in permanent cosmetic and functional deficits. In this scenario, the regenerative process fails and is dominated by an unproductive inflammatory response and accompanying fibrosis. The failure of current regenerative therapeutics to completely restore functional muscle tissue is not surprising considering the incomplete understanding of the cellular mechanisms that drive the regeneration response in the setting of VML injury. To begin to address this profound knowledge gap, we developed an agent-based model to predict the tissue remodeling response following surgical creation of a VML injury. Once the model was able to recapitulate key aspects of the tissue remodeling response in the absence of repair, we validated the model by simulating the tissue remodeling response to VML injury following implantation of either a decellularized extracellular matrix scaffold or a minced muscle graft. The model suggested that the SSC microenvironment and absence of pro-differentiation SSC signals were the most important aspects of failed muscle regeneration in VML injuries. The major implication of this work is that agent-based models may provide a much-needed predictive tool to optimize the design of new therapies, and thereby, accelerate the clinical translation of regenerative therapeutics for VML injuries.

Adipose-derived stem cells delay muscle atrophy after peripheral nerve injury in the rodent model.

INTRODUCTION: Injuries to peripheral nerves cause distal muscle atrophy. The effects of adipose-derived stem cell (ASC) injections into a muscle after injury were examined. METHODS: A 1.5 cm defect in the rat sciatic nerve was created, resulting in gastrocnemius muscle atrophy. The nerve defect was repaired with autograft; DiR-labeled ASCs were injected into the gastrocnemius immediately postoperatively. Quantitation of gross musculature and muscle fiber area, cell survival, fibrosis, lipid deposition, inflammation, and reconstructive responses were investigated. RESULTS: ASCs were identified in the muscle at 6 weeks, where injections showed increased muscle mass percentage retained, larger average fiber area, and less overall lipid content accumulated throughout the musculature. Muscles having received ASCs showed increased presence of interlukin-10 and Ki67, and decreased inducible nitric oxide synthase (iNOS). DISCUSSION: This investigation is suggestive that an ASC injection into denervated muscle post-operatively is able to delay the onset of atrophy. Muscle Nerve 59:603-603, 2019.

The Potential of Combination Therapeutics for More Complete Repair of Volumetric Muscle Loss Injuries: The Role of Exogenous Growth Factors and/or Progenitor Cells in Implantable Skeletal Muscle Tissue Engineering Technologies.

Despite the robust regenerative capacity of skeletal muscle, there are a variety of congenital and acquired conditions in which the volume of skeletal muscle loss results in major permanent functional and cosmetic deficits. These latter injuries are referred to as volumetric muscle loss (VML) injuries or VML-like conditions, and they are characterized by the simultaneous absence of multiple tissue components (i.e., nerves, vessels, muscles, satellite cells, and matrix). There are currently no effective treatment options. Regenerative medicine/tissue engineering technologies hold great potential for repair of these otherwise irrecoverable VML injuries. In this regard, three-dimensional scaffolds have been used to deliver sustained amounts of growth factors into a variety of injury models, to modulate host cell recruitment and extracellular matrix remodeling. However, this is a nascent field of research, and more complete functional improvements require more precise control of the spatiotemporal distribution of critical growth factors over a physiologically relevant range. This is especially true for VML injuries where incorporation of a cellular component into the scaffolds might provide not only a source of new tissue formation but also additional signals for host cell migration, recruitment, and survival. To this end, we review the major features of muscle repair and regeneration for largely recoverable injuries, and then discuss recent cell- and/or growth factor-based approaches to repair the more profound and irreversible VML and VML-like injuries. The underlying supposition is that more rationale incorporation of exogenous growth factors and/or cellular components will be required to optimize the regenerative capacity of implantable therapeutics for VML repair.

The pharmacology of regenerative medicine.

Regenerative medicine is a rapidly evolving multidisciplinary, translational research enterprise whose explicit purpose is to advance technologies for the repair and replacement of damaged cells, tissues, and organs. Scientific progress in the field has been steady and expectations for its robust clinical application continue to rise. The major thesis of this review is that the pharmacological sciences will contribute critically to the accelerated translational progress and clinical utility of regenerative medicine technologies. In 2007, we coined the phrase "regenerative pharmacology" to describe the enormous possibilities that could occur at the interface between pharmacology, regenerative medicine, and tissue engineering. The operational definition of regenerative pharmacology is "the application of pharmacological sciences to accelerate, optimize, and characterize (either in vitro or in vivo) the development, maturation, and function of bioengineered and regenerating tissues." As such, regenerative pharmacology seeks to cure disease through restoration of tissue/organ function. This strategy is distinct from standard pharmacotherapy, which is often limited to the amelioration of symptoms. Our goal here is to get pharmacologists more involved in this field of research by exposing them to the tools, opportunities, challenges, and interdisciplinary expertise that will be required to ensure awareness and galvanize involvement. To this end, we illustrate ways in which the pharmacological sciences can drive future innovations in regenerative medicine and tissue engineering and thus help to revolutionize the discovery of curative therapeutics. Hopefully, the broad foundational knowledge provided herein will spark sustained conversations among experts in diverse fields of scientific research to the benefit of all.

Age-dependent changes cooperatively impact skeletal muscle regeneration after compartment syndrome injury.

Declining skeletal muscle function, due to injury and aging (sarcopenia), results in a significantly decreased quality of life and is a major cause of disability in the United States. Studies examining recovery from muscle injury in models of older animals principally used insults that primarily affect only the myofibers without affecting the muscle tissue microenvironment. This type of injury does not adequately represent the full extent of tissue damage observed in older humans, which encompasses injury not only to the muscle fibers, but also to the surrounding tissue components, such as the vasculature and nerves. Previously, we described a novel rat model of compression-induced muscle injury that results in multicomponent injury to the muscle and adequately mimics compartment syndrome injuries seen in patients. Herein, we characterized tissue regeneration in young, adult, and aged rats after compartment syndrome injury. We observed significant differences between the regeneration process in the different aged rats that involved muscle function, tissue anatomical features, neovascularization, and innervation. Compared to young rats, adult rats had delayed functional recovery, whereas the aged rats were deficient in their regenerative capacity. Age-dependent changes in both the ability to restore the contractile apparatus and myogenesis are important, and must be taken into consideration when designing therapies for the treatment of muscle injury.

Age-related alterations in regeneration of the urinary bladder after subtotal cystectomy.

Prior work documented that surgical removal of approximately 70% of the bladder (subtotal cystectomy) in 12-week-old female rats induced complete functional regeneration of the bladder within 8 weeks. To determine whether animal age affects bladder regeneration, female F344 rats aged 12 weeks (young) and 12 months (old) underwent subtotal cystectomy, and then were evaluated from 1 to 26 weeks after subtotal cystectomy. At 26 weeks after subtotal cystectomy, bladder capacity in young animals was indistinguishable from that in age-matched controls, but bladder capacity in old animals was only approximately 56% of that in age-matched controls. There was no detectable difference in residual volume among treatment groups, but the diminished regeneration in old animals was associated with a corresponding increase in the ratio of residual volume to micturition volume. The majority of old animals exhibited evidence of chronic kidney damage after subtotal cystectomy. Maximal contraction of bladder strips to electrical field stimulation, as well as activation with carbachol, phenylephrine, and KCl, were lower in old than in young animals at 26 weeks after subtotal cystectomy. Immunostaining with proliferating cell nuclear antigen and Von Willebrand factor revealed delayed and/or diminished proliferative and angiogenic responses, respectively, in old animals. These results confirm prior work and suggest that multiple mechanisms may contribute to an age-related decline in the regenerative capacity of the bladder.

Optimized Biomanufacturing for Treatment of Volumetric Muscle Loss Enables Physiomimetic Recovery.

Volumetric muscle loss (VML) injuries are defined by loss of sufficient skeletal muscle to produce persistent deficits in muscle form and function, with devastating lifelong consequences to both soldiers and civilians. There are currently no satisfactory treatments for VML injuries. The work described herein details the implementation of a fully enclosed bioreactor environment (FEBE) system that efficiently interfaces with our existing automated bioprinting and advanced biomanufacturing methods for cell deposition on sheet-based scaffolds for our previously described tissue-engineered muscle repair (TEMR) technology platform. Briefly, the TEMR technology consists of a porcine bladder acellular matrix seeded with skeletal muscle progenitor cells and preconditioned via 10% uniaxial cyclic stretch in a bioreactor. Overall, TEMR implantation in an established rat tibialis anterior (TA) VML injury model can result in 60 to ∼90% functional recovery. However, our original study documented >50% failure rate. That is, more than half of the implanted TEMR constructs produced no functional improvement beyond no treatment/repair. The high failure rate was attributed to the untoward mechanical disruption of TEMR during surgical implantation. In a follow-up study, adjustments were made to the geometry of both the VML injury and the TEMR construct, and the "nonresponder" group was reduced from over half the TEMR-treated animals to just 33%. Nonetheless, additional improvement is needed for clinical applicability. The main objectives of the current study were twofold: (1) explore the use of advanced biomanufacturing methods (i.e., FEBE bioreactor) to further improve TEMR reliability (i.e., increase functional response rate), (2) determine if previously established bioprinting methods, when coupled to the customized FEBE system would further improve the rate, magnitude or amplitude of functional outcomes following TEMR implantation in the same rat TA VML injury model. The current study demonstrates the unequivocal benefits of a customized bioreactor system that reduces manipulation of TEMR during cell seeding and maturation via bioprinting while simultaneously maximizing TEMR stability throughout the biofabrication process. This new biomanufacturing strategy not only accelerated the rate of functional recovery, but also eliminated all TEMR failures. In addition, implementation of bioprinting resulted in more physiomimetic skeletal muscle characteristics of repaired muscle tissue.

Freeze-dried porous collagen scaffolds for the repair of volumetric muscle loss injuries.

Volumetric muscle loss (VML) injuries are characterized by the traumatic loss of skeletal muscle resulting in permanent damage to both tissue architecture and electrical excitability. To address this challenge, we previously developed a 3D aligned collagen-glycosaminoglycan (CG) scaffold platform that supported in vitro myotube alignment and maturation. In this work, we assessed the ability of CG scaffolds to facilitate functional muscle recovery in a rat tibialis anterior (TA) model of VML. Functional muscle recovery was assessed following implantation of either non-conductive CG or electrically conductive CG-polypyrrole (PPy) scaffolds at 4, 8, and 12 weeks post-injury by in vivo electrical stimulation of the peroneal nerve. After 12 weeks, scaffold-treated muscles produced maximum isometric torque that was significantly greater than non-treated tissues. Histological analysis further supported these reparative outcomes with evidence of regenerating muscle fibers at the material-tissue interface in scaffold-treated tissues that was not observed in non-repaired muscles. Scaffold-treated muscles possessed higher numbers of M1 and M2 macrophages at the injury while conductive CG-PPy scaffold-treated muscles showed significantly higher levels of neovascularization as indicated by the presence of pericytes and endothelial cells, suggesting a persistent wound repair response not observed in non-treated tissues. Finally, only tissues treated with non-conductive CG scaffolds displayed neurofilament staining similar to native muscle, further corroborating isometric contraction data. Together, these findings show that CG scaffolds can facilitate improved skeletal muscle function and endogenous cellular repair, highlighting their potential use as therapeutics for VML injuries.

Compression-induced muscle injury in rats that mimics compartment syndrome in humans.

Compartment syndrome (CS) is a serious complication arising from a variety of extremity injuries and resultant swelling within the fascicles of the muscle tissue. The current standard of care for CS is fasciotomy, which relieves the intracompartmental pressure of CS but inflicts further tissue damage. The development of new techniques to treat CS include angiogenic therapy, antifibrosis treatments, and stem cell therapy, all which aim to enhance tissue regeneration and functional recovery. Current rodent models of skeletal muscle injury do not accurately mimic the complex physiological tissue damage found in CS in human patients, and large-animal models of CS cannot be used as an experimental model of human cell therapy because of the lack of immunocompromised animals. We developed a rat model of CS that mimics the sequelae of the human condition. Compression of the hindlimb of rats using neonatal blood pressure cuffs maintaining 120 to 140 mmHg for 3 hours resulted in considerable muscular, vascular, and neural damage. Histological and functional analyses documented the initial degeneration and subsequent regeneration of the muscle tissue over time. The complex muscular, vascular, and neural injury observed in this model provides an ideal platform for testing cellular, biological, and pharmacological agents for the restoration of muscle volume and function.

Impact of partial urethral obstruction on bladder function: time-dependent changes and functional correlates of altered expression of Ca²âº signaling regulators.

In animal models of partial urethral obstruction (PUO), altered smooth muscle function/contractility may be linked to changes in molecules that regulate calcium signaling/sensitization. PUO was created in male rats, and urodynamic studies were conducted 2 and 6 wk post-PUO. Cystometric recordings were analyzed for the presence or absence of nonvoiding contractions [i.e., detrusor overactivity (DO)]. RT-PCR and Western blots were performed on a subpopulation of rats to study the relationship between the expression of RhoA, L-type Ca(2+) channels, Rho kinase-1, Rho kinase-2, inositol 1,4,5-trisphosphate, ryanodine receptor, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and protein kinase C (PKC)-potentiated phosphatase inhibitor of 17 kDa, and urodynamic findings in the same animal. Animals displayed DO at 2 (38%) and 6 wk (43%) post-PUO, increases were seen in in vivo pressures at 2 wk, and residual volume at 6 wk. Statistical analysis of RT-PCR and Western blot data at 2 wk, during the compensatory phase of detrusor hypertrophy, documented that expression of molecules that regulate calcium signaling and sensitization was consistently lower in obstructed rats without DO than those with DO or control rats. Among rats with DO at 2 wk, linear regression analysis revealed positive correlations between in vivo pressures and protein and mRNA expression of several regulatory molecules. At 6 wk, in the presence of overt signs of bladder decompensation, no clear or consistent alterations in expression of these same targets were observed at the protein level. These data extend prior work to suggest that molecular profiling of key regulatory molecules during the progression of PUO-mediated bladder dysfunction may shed new light on potential biomarkers and/or therapeutic targets.

Bioengineered vascular access maintains structural integrity in response to arteriovenous flow and repeated needle puncture.

OBJECTIVE: Tissue-engineered blood vessels (TEBV) have been proposed as an alternative to prosthetic grafts for dialysis access. However, arteriovenous (AV) grafts must withstand extreme flow rates and frequent needle trauma. In a proof-of-concept study, we sought to determine whether scaffold-based TEBV could withstand the hemodynamic and mechanical challenges of chronic dialysis access. METHODS: TEBV were constructed using decellularized arterial scaffolds seeded with autologous ovine endothelial cells (EC) derived from circulating endothelial progenitor cells (EPC) using a novel high-affinity capture approach. Seeded scaffolds were preconditioned to arterial pressure and flow in a bioreactor for 2 weeks prior to implantation to create carotid artery to jugular vein AV grafts in each animal. TEBV were healed for 1 month before initiating percutaneous needle puncture 3 days/week. TEBV wall geometry and patency were monitored using duplex imaging and were either explanted for histologic analysis at 2 months (n = 5) or followed for up to 6 months until venous outflow stenosis threatened AV graft patency (n = 6). RESULTS: Despite high flow, TEBV maintained stable geometry with only modest wall dilation (under 6%) by 4 months after implantation. Needle access was well tolerated with a single puncture site complication, a small pseudoaneurysm, occurring in the late group. Time-to-hemostasis at puncture sites averaged 4 ± 2 minutes. Histologic analysis at 2 months demonstrated repopulation of the outer TEBV wall by host cells and healing of needle punctures by cellular ingrowth and new matrix deposition along the tract. TEBV followed beyond 2 months showed stable wall geometry but, consistent with the primary mode of clinical AV graft failure, all TEBV eventually developed venous anastomotic stenosis (mean, 4.4 ± 0.9 months; range, 3.3-5.6 months postimplantation; n = 6). CONCLUSIONS: This pilot study supports the concept of creating dialysis access from scaffold-based autologous TEBV. Engineered AV grafts were created within a clinically relevant time frame and demonstrated stable wall geometry despite high flow and repeated puncture. Cellular ingrowth and puncture site healing may improve wall durability, but venous outflow stenosis remains the primary mode of TEBV graft failure in the ovine model.

Comprehensive dynamic and kinematic analysis of the rodent hindlimb during over ground walking.

The rat hindlimb is a frequently utilized pre-clinical model system to evaluate injuries and pathologies impacting the hindlimbs. These studies have demonstrated the translational potential of this model but have typically focused on the force generating capacity of target muscles as the primary evaluative outcome. Historically, human studies investigating extremity injuries and pathologies have utilized biomechanical analysis to better understand the impact of injury and extent of recovery. In this study, we expand that full biomechanical workup to a rat model in order to characterize the spatiotemporal parameters, ground reaction forces, 3-D joint kinematics, 3-D joint kinetics, and energetics of gait in healthy rats. We report data on each of these metrics that meets or exceeds the standards set by the current literature and are the first to report on all these metrics in a single set of animals. The methodology and findings presented in this study have significant implications for the development and clinical application of the improved regenerative therapeutics and rehabilitative therapies required for durable and complete functional recovery from extremity traumas, as well as other musculoskeletal pathologies.

Controlling scaffold conductivity and pore size to direct myogenic cell alignment and differentiation.

Skeletal muscle's combination of three-dimensional (3D) anisotropy and electrical excitability is critical for enabling normal movement. We previously developed a 3D aligned collagen scaffold incorporating conductive polypyrrole (PPy) particles to recapitulate these key muscle properties and showed that the scaffold facilitated enhanced myotube maturation compared with nonconductive controls. To further optimize this scaffold design, this work assessed the influence of conductive polymer incorporation and scaffold pore architecture on myogenic cell behavior. Conductive PPy and poly(3,4-ethylenedioxythiophene) (PEDOT) particles were synthesized and mixed into a suspension of type I collagen and chondroitin sulfate prior to directional freeze-drying to produce anisotropic scaffolds. Energy dispersive spectroscopy revealed homogenous distribution of conductive PEDOT particles throughout the scaffolds that resulted in a threefold increase in electrical conductivity while supporting similar myoblast metabolic activity compared to nonconductive scaffolds. Control of freezing temperature enabled fabrication of PEDOT-doped scaffolds with a range of pore diameters from 98 to 238 µm. Myoblasts conformed to the anisotropic contact guidance cues independent of pore size to display longitudinal cytoskeletal alignment. The increased specific surface area of the smaller pore scaffolds helped rescue the initial decrease in myoblast metabolic activity observed in larger pore conductive scaffolds while also promoting modestly increased expression levels of the myogenic marker myosin heavy chain (MHC) and gene expression of myoblast determination protein (MyoD). However, cell infiltration to the center of the scaffolds was marginally reduced compared with larger pore variants. Together these data underscore the potential of aligned and PEDOT-doped collagen scaffolds for promoting myogenic cell organization and differentiation.

Administration of particulate oxygen generators improves skeletal muscle contractile function after ischemia-reperfusion injury in the rat hindlimb.

Extended tourniquet application, often associated with battlefield extremity trauma, can lead to severe ischemia-reperfusion (I/R) injury in skeletal muscle. Particulate oxygen generators (POGs) can be directly injected into tissue to supply oxygen to attenuate the effects of I/R injury in muscle. The goal of this study was to investigate the efficacy of a sodium percarbonate (SPO)-based POG formulation in reducing ischemic damage in a rat hindlimb during tourniquet application. Male Lewis rats were anesthetized and underwent tourniquet application for 3 h at a pressure of 300 mmHg. Shortly after tourniquet inflation, animals received intramuscular injections of either 0.2 mg/mL SPO with catalase (n = 6) or 2.0 mg/mL SPO with catalase (n = 6) directly into the tibialis anterior (TA) muscle. An additional Tourniquet-Only group (n = 12) received no intervention. Functional recovery was monitored by in vivo contractile testing of the hindlimb at 1, 2, and 4 wk after injury. By the 4 wk time point, the Low-Dose POG group continued to show improved functional recovery (85% of baseline) compared with the Tourniquet-Only (48%) and High-Dose POG (56%) groups. In short, the low-dose POG formulation appeared, at least in part, to mitigate the impact of ischemic tissue injury, thus improving contractile function after tourniquet application. Functional improvement correlated with maintenance of larger muscle fiber cross-sectional area and the presence of fewer fibers containing centrally located nuclei. As such, POGs represent a potentially attractive therapeutic solution for addressing I/R injuries associated with extremity trauma.NEW & NOTEWORTHY Skeletal muscle contraction was evaluated in the same animals at multiple time points up to 4 wk after injury, following administration of particulate oxygen generators (POGs) in a clinically relevant rat hindlimb model of tourniquet-induced ischemia. The observed POG-mediated improvement of muscle function over time confirms and extends previous studies to further document the potential clinical applications of POGs. Of particular significance in austere environments, this technology can be applied in the absence of an intact circulation.

Assembled Cell-Decorated Collagen (AC-DC) Fiber Bioprinted Implants with Musculoskeletal Tissue Properties Promote Functional Recovery in Volumetric Muscle Loss.

Musculoskeletal tissue injuries, including volumetric muscle loss (VML), are commonplace and often lead to permanent disability and deformation. Addressing this healthcare need, an advanced biomanufacturing platform, assembled cell-decorated collagen (AC-DC) bioprinting, is invented to rapidly and reproducibly create living biomaterial implants, using clinically relevant cells and strong, microfluidic wet-extruded collagen microfibers. Quantitative analysis shows that the directionality and distribution of cells throughout AC-DC implants mimic native musculoskeletal tissue. AC-DC bioprinted implants further approximate or exceed the strength and stiffness of human musculoskeletal tissue and exceed collagen hydrogel tensile properties by orders of magnitude. In vivo, AC-DC implants are assessed in a critically sized muscle injury in the hindlimb, with limb torque generation potential measured over 12 weeks. Both acellular and cellular implants promote functional recovery compared to the unrepaired group, with AC-DC implants containing therapeutic muscle progenitor cells promoting the highest degree of recovery. Histological analysis and automated image processing of explanted muscle cross-sections reveal increased total muscle fiber count, median muscle fiber size, and increased cellularization for injuries repaired with cellularized implants. These studies introduce an advanced bioprinting method for generating musculoskeletal tissue analogs with near-native biological and biomechanical properties with the potential to repair myriad challenging musculoskeletal injuries.

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.

Vascular smooth muscle enhances functionality of tissue-engineered blood vessels in vivo.

OBJECTIVES: There is significant room for improvement in the development of tissue-engineered blood vessels (TEBVs) for vascular reconstruction. Most commonly, TEBVs are seeded with endothelial cells (ECs) only. This provides an antithrombogenic surface but suboptimal physiologic characteristics compared with native arteries, due to lack of smooth muscle cells (SMCs) in the vessel media. Although SMCs are critical in vessel architecture and function throughout the vascular tree, few studies have incorporated SMCs in TEBVs implanted in vivo. As such, the goal of the present study was to evaluate the effect of SMC coseeding with ECs on TEBV maturation, structure, and function after prolonged in vivo maturation. METHODS: Dual-seeded TEBVs (dsTEBVs) were created by coseeding autologous ECs derived from circulating progenitor cells and SMCs from artery explants onto the lumen and outer surface of extracellular matrix scaffolds, respectively. Control vessels were seeded with ECs alone (ecTEBV). All vessels were preconditioned to pulsatile flow for 10 to 14 days in a bioreactor, implanted as arterial interposition grafts in sheep, and allowed to heal and adapt in vivo for 4 months before ex vivo physiologic testing and histologic analysis. RESULTS: All implants were patent at 4 months. There were no structural failures, aneurysms, or infectious complications. The dsTEBVs exhibited a greater degree of wall maturation, characterized by higher medial cellularity (P = .01) and greater percentage of α-actin (P = .005) and SMC-specific muscle myosin heavy chain (P = .005) staining compared with ecTEBVs. Contractile responses to phenylephrine and serotonin were significantly greater in isolated rings of dsTEBVs than those observed in ecTEBVs (P = .01). CONCLUSIONS: To our knowledge, this is the first study that demonstrates enhanced in vivo wall maturation and contractile function of TEBVs coseeded with autologous SMCs and ECs compared with EC seeding alone. These data suggest a coseeding strategy can be accomplished in a clinically relevant timeframe (typically 6 weeks) and may provide advantages for arterial reconstruction compared with vessels engineered only with endothelium.

Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways.

OBJECTIVE: • To investigate the role that oxidative stress plays in the development of diabetic cystopathy. MATERIALS AND METHODS: • Comparative gene expression in the bladder of non-diabetic and streptozotocin (STZ)-induced 2-month- old diabetic rats was carried out using microarray analysis. • Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S-transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. • The activity of protein degradation pathways was assessed using Western blot analysis. RESULTS: • Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ-induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P = 1.27 × 10(-10)). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. • It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. • This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up-regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0-fold, respectively, and the apoptosis inducing protein, BAX, was up-regulated by 6.76 ± 0.76-fold. CONCLUSION: • Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function.