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.

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.

Endothelial alpha globin is a nitrite reductase.

Resistance artery vasodilation in response to hypoxia is essential for matching tissue oxygen and demand. In hypoxia, erythrocytic hemoglobin tetramers produce nitric oxide through nitrite reduction. We hypothesized that the alpha subunit of hemoglobin expressed in endothelium also facilitates nitrite reduction proximal to smooth muscle. Here, we create two mouse strains to test this: an endothelial-specific alpha globin knockout (EC Hba1Δ/Δ) and another with an alpha globin allele mutated to prevent alpha globin's inhibitory interaction with endothelial nitric oxide synthase (Hba1WT/Δ36-39). The EC Hba1Δ/Δ mice had significantly decreased exercise capacity and intracellular nitrite consumption in hypoxic conditions, an effect absent in Hba1WT/Δ36-39 mice. Hypoxia-induced vasodilation is significantly decreased in arteries from EC Hba1Δ/Δ, but not Hba1WT/Δ36-39 mice. Hypoxia also does not lower blood pressure in EC Hba1Δ/Δ mice. We conclude the presence of alpha globin in resistance artery endothelium acts as a nitrite reductase providing local nitric oxide in response to hypoxia.

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.

Perfusable cell-laden micropatterned hydrogels for delivery of spatiotemporal vascular-like cues to tissues.

The integration of vasculature at physiological scales within 3D cultures of cell-laden hydrogels for the delivery of spatiotemporal mass transport, chemical and mechanical cues, is a stepping-stone towards building in vitro tissue models that recapitulate in vivo cues. To address this challenge, we present a versatile method to micropattern adjoining hydrogel shells with a perfusable channel or lumen core, for enabling facile integration with fluidic control systems, on one hand, and to cell-laden biomaterial interfaces, on the other hand. This microfluidic imprint lithography methodology utilizes the high tolerance and reversible nature of the bond alignment process to lithographically position multiple layers of imprints within a microfluidic device for sequential filling and patterning of hydrogel lumen structures with single or multiple shells. Through fluidic interfacing of the structures, the ability to deliver physiologically relevant mechanical cues for recapitulating cyclical stretch on the hydrogel shell and shear stress on endothelial cells in the lumen are validated. We envision application of this platform for recapitulation of the bio-functionality and topology of micro-vasculatures, alongside the ability to deliver transport and mechanical cues, as needed for 3D culture to construct in vitro tissue models.

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.

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.

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.

Gene Therapy for Overactive Bladder: A Review of BK-Channel α-Subunit Gene Transfer.

A need exists for local (ie, bladder-specific) interventions to treat overactive bladder (OAB) with low risk of unwanted postprocedural outcomes. Gene therapy targeted to leverage endogenous physiology in bladder cells may assist in restoring normal cell and organ function. Herein, we review the potential promise of gene therapy for treating OAB, focusing on gene transfer of URO-902, a non-viral naked plasmid DNA expressing the big potassium (BK) channel. We searched PubMed for articles concerning functional aspects of the BK channel and its potential use for gene transfer as local OAB treatment. Results from preclinical, phase 1, and phase 2 studies of URO-902 for erectile dysfunction and phase 1 studies of URO-902 for OAB are included. The BK channel has been extensively studied; however, URO-902 is the first gene therapy used in clinical trials directed toward treating OAB via the BK channel. In both URO-902 studies, there were no serious adverse events considered treatment related and no adverse events leading to early withdrawal. Both studies included secondary efficacy endpoints with promising results suggesting improvement in OAB symptoms, and quality of life, with use of URO-902 versus placebo. Gene therapy involving the BK channel, such as gene transfer with URO-902, has demonstrated promising safety and efficacy results in women with OAB. Findings warrant further investigation of the use of URO-902 for OAB 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.

Aligned and electrically conductive 3D collagen scaffolds for skeletal muscle tissue engineering.

Skeletal muscle is characterized by its three-dimensional (3D) anisotropic architecture composed of highly aligned and electrically-excitable muscle fibers that enable normal movement. Biomaterial-based tissue engineering approaches to repair skeletal muscle are limited due to difficulties combining 3D structural alignment (to guide cell/matrix organization) and electrical conductivity (to enable electrically-excitable myotube assembly and maturation). In this work we successfully produced aligned and electrically conductive 3D collagen scaffolds using a freeze-drying approach. Conductive polypyrrole (PPy) nanoparticles were synthesized and directly mixed into a suspension of type I collagen and chondroitin sulfate followed by directional lyophilization. Scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and confocal microscopy showed that directional solidification resulted in scaffolds with longitudinally aligned pores with homogeneously-distributed PPy content. Chronopotentiometry verified that PPy incorporation resulted in a five-fold increase in conductivity compared to non-PPy-containing collagen scaffolds without detrimentally affecting myoblast metabolic activity. Furthermore, the aligned scaffold microstructure provided contact guidance cues that directed myoblast growth and organization. Incorporation of PPy also promoted enhanced myotube formation and maturation as measured by myosin heavy chain (MHC) expression and number of nuclei per myotube. Together these data suggest that aligned and electrically conductive 3D collagen scaffolds could be useful for skeletal muscle tissue engineering.

Semisynthetic Hyaluronic Acid-Based Hydrogel Promotes Recovery of the Injured Tibialis Anterior Skeletal Muscle Form and Function.

Volumetric muscle loss (VML) injuries are characterized by a degree of tissue loss that exceeds the endogenous regenerative capacity of muscle, resulting in permanent structural and functional deficits. Such injuries are a consequence of trauma, as well as a host of congenital and acquired diseases and disorders. Despite significant preclinical research with diverse biomaterials, as well as early clinical studies with implantation of decellularized extracellular matrices, there are still significant barriers to more complete restoration of muscle form and function following repair of VML injuries. In fact, identification of novel biomaterials with more advantageous regenerative profiles is a critical limitation to the development of improved therapeutics. As a first step in this direction, we evaluated a novel semisynthetic hyaluronic acid-based (HyA) hydrogel that embodies material features more favorable for robust muscle regeneration. This HyA-based hydrogel is composed of an acrylate-modified HyA (AcHyA) macromer, an AcHyA macromer conjugated with the bsp-RGD(15) peptide sequence to enhance cell adhesion, a high-molecular-weight heparin to sequester growth factors, and a matrix metalloproteinase-cleavable cross-linker to allow for cell-dependent remodeling. In a well-established, clinically relevant rat tibialis anterior VML injury model, we report observations of robust functional recovery, accompanied by volume reconstitution, muscle regeneration, and native-like vascularization following implantation of the HyA-based hydrogel at the site of injury. These findings have important implications for the development and clinical application of the improved biomaterials that will be required for stable and complete functional recovery from diverse VML injuries.

Adipose Stem Cells Enhance Nerve Regeneration and Muscle Function in a Peroneal Nerve Ablation Model.

Severe peripheral nerve injuries have devastating consequences on the quality of life in affected patients, and they represent a significant unmet medical need. Destruction of nerve fibers results in denervation of targeted muscles, which, subsequently, undergo progressive atrophy and loss of function. Timely restoration of neural innervation to muscle fibers is crucial to the preservation of muscle homeostasis and function. The goal of this study was to evaluate the impact of addition of adipose stem cells (ASCs) to polycaprolactone (PCL) nerve conduit guides on peripheral nerve repair and functional muscle recovery in the setting of a critical size nerve defect. To this end, peripheral nerve injury was created by surgically ablating 6 mm of the common peroneal nerve in a rat model. A PCL nerve guide, filled with ASCs and/or poloxamer hydrogel, was sutured to the nerve ends. Negative and positive controls included nerve ablation only (no repair), and reversed polarity autograft nerve implant, respectively. Tibialis anterior (TA) muscle function was assessed at 4, 8, and 12 weeks postinjury, and nerve and muscle tissue was retrieved at the 12-week terminal time point. Inclusion of ASCs in the PCL nerve guide elicited statistically significant time-dependent increases in functional recovery (contraction) after denervation; ∼25% higher than observed in acellular (poloxamer-filled) implants and indistinguishable from autograft implants, respectively, at 12 weeks postinjury (p < 0.05, n = 7-8 in each group). Analysis of single muscle fiber cross-sectional area (CSA) revealed that ASC-based treatment of nerve injury provided a better recapitulation of the overall distribution of muscle fiber CSAs observed in the contralateral TA muscle of uninjured limbs. In addition, the presence of ASCs was associated with improved features of re-innervation distal to the defect, with respect to neurofilament and S100 (Schwann cell marker) expression. In conclusion, these initial studies indicate significant benefits of inclusion of ASCs to the rate and magnitude of both peripheral nerve regeneration and functional recovery of muscle contraction, to levels equivalent to autograft implantation. These findings have important implications to improved nerve repair, and they provide input for future work directed to restoration of nerve and muscle function after polytraumatic injury. Impact Statement This works explores the application of adipose stem cells (ASCs) for peripheral nerve regeneration in a rat model. Herein, we demonstrate that the addition of ASCs in poloxamer-filled PCL nerve guide conduits impacts nerve regeneration and recovery of muscle function, to levels equivalent to autograft implantation, which is considered to be the current gold standard treatment. This study builds on the importance of a timely restoration of innervation to muscle fibers for preservation of muscle homeostasis, and it will provide input for future work aiming at restoring nerve and muscle function after polytraumatic injury.

Taking the Next Steps in Regenerative Rehabilitation: Establishment of a New Interdisciplinary Field.

The growing field of regenerative rehabilitation has great potential to improve clinical outcomes for individuals with disabilities. However, the science to elucidate the specific biological underpinnings of regenerative rehabilitation-based approaches is still in its infancy and critical questions regarding clinical translation and implementation still exist. In a recent roundtable discussion from International Consortium for Regenerative Rehabilitation stakeholders, key challenges to progress in the field were identified. The goal of this article is to summarize those discussions and to initiate a broader discussion among clinicians and scientists across the fields of regenerative medicine and rehabilitation science to ultimately progress regenerative rehabilitation from an emerging field to an established interdisciplinary one. Strategies and case studies from consortium institutions-including interdisciplinary research centers, formalized courses, degree programs, international symposia, and collaborative grants-are presented. We propose that these strategic directions have the potential to engage and train clinical practitioners and basic scientists, transform clinical practice, and, ultimately, optimize patient outcomes.

Evaluating the safety and potential activity of URO-902 (hMaxi-K) gene transfer by intravesical instillation or direct injection into the bladder wall in female participants with idiopathic (non-neurogenic) overactive bladder syndrome and detrusor overactivity from two double-blind, imbalanced, placebo-controlled randomized phase 1 trials.

AIMS: Two phase 1 trials were performed in healthy women with the overactive bladder (OAB) syndrome and urodynamically demonstrated detrusor overactivity (DO), with the aim to demonstrate the safety and potential efficacy of URO-902, which comprises a gene therapy plasmid vector expressing the human big potassium channel α subunit. METHODS: ION-02 (intravesical instillation) and ION-03 (direct injection) were double-blind, placebo-controlled, multicenter studies without overlap in enrollment between studies. Active doses were administered and evaluated sequentially (lowest dose first) for safety. ION-02 participants received either 5000 µg or 10 000 µg URO-902, or placebo. ION-03 participants received either 16 000 or 24 000 µg URO-902, or placebo, injected directly into the bladder wall using cystoscopy. Primary outcome variables were safety parameters occurring subsequent to URO-902 administration; secondary efficacy variables also were evaluated. RESULTS: Among the safety outcomes, there were no dose-limiting toxicities or significant adverse events (AEs) preventing dose escalation during either trial, and no participants withdrew due to AEs. For efficacy, in ION-02 (N = 21), involuntary detrusor contractions on urodynamics at 24 weeks in patients receiving URO-902 (P < .0508 vs placebo) and mean urgency incontinence episodes in the 5000 µg group (P = .0812 vs placebo) each showed a downward trend. In ION-03 (N = 13), significant reduction versus placebo in urgency episodes (16 000 µg, P = .036; 24 000 µg, P = .046) and number of voids (16 000 µg, -2.16, P = .044; 24 000 µg, -2.73, P = .047) were observed 1 week after injection. CONCLUSION: Promising safety and efficacy results in these preliminary phase 1 studies suggest gene transfer may be a promising therapy for OAB/DO, warranting further investigation.

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.

Analysis and Modeling of Rat Gait Biomechanical Deficits in Response to Volumetric Muscle Loss Injury.

There is currently a substantial volume of research underway to develop more effective approaches for the regeneration of functional muscle tissue as treatment for volumetric muscle loss (VML) injury, but few studies have evaluated the relationship between injury and the biomechanics required for normal function. To address this knowledge gap, the goal of this study was to develop a novel method to quantify the changes in gait of rats with tibialis anterior (TA) VML injuries. This method should be sensitive enough to identify biomechanical and kinematic changes in response to injury as well as during recovery. Control rats and rats with surgically-created VML injuries were affixed with motion capture markers on the bony landmarks of the back and hindlimb and were recorded walking on a treadmill both prior to and post-surgery. Data collected from the motion capture system was exported for post-hoc analysis in OpenSim and Matlab. In vivo force testing indicated that the VML injury was associated with a significant deficit in force generation ability. Analysis of joint kinematics showed significant differences at all three post-surgical timepoints and gait cycle phase shifting, indicating augmented gait biomechanics in response to VML injury. In conclusion, this method identifies and quantifies key differences in the gait biomechanics and joint kinematics of rats with VML injuries and allows for analysis of the response to injury and recovery. The comprehensive nature of this method opens the door for future studies into dynamics and musculoskeletal control of injured gait that can inform the development of regenerative technologies focused on the functional metrics that are most relevant to recovery from VML injury.

In Silico and In Vivo Studies Detect Functional Repair Mechanisms in a Volumetric Muscle Loss Injury.

IMPACT STATEMENT: Despite medical advances, volumetric muscle loss (VML) injuries to craniofacial muscles represent an unmet clinical need. We report an implantable tissue-engineered construct that leads to substantial tissue regeneration and functional recovery in a preclinical model of VML injury that is dimensionally relevant to unilateral cleft lip repair, and a series of corresponding computational models that provide biomechanical insight into mechanism(s) responsible for the VML-induced functional deficits and recovery following tissue-engineered muscle repair implantation. This unique combined approach represents a critical first step toward establishing a crucial biomechanical basis for the development of efficacious regenerative technologies, considering the spectrum of VML injuries.