Highly Aligned Ternary Nanofiber Matrices Loaded with MXene Expedite Regeneration of Volumetric Muscle Loss

Current therapeutic approaches for volumetric muscle loss(VML)face challenges due to limited graft availability and insufficient bioactivities.To overcome these limitations,tissue-engineered scaffolds have emerged as a promising alternative.In this study,we developed aligned ternary nanofibrous matrices comprised of poly(lactide-co-ε-caprolactone)integrated with collagen and Ti_(3)C_(2)T_(x)MXene nanoparticles(NPs)(PCM matrices),and explored their myogenic potential for skeletal muscle tissue regeneration.The PCM matrices demonstrated favorable physicochemical properties,including structural uniformity,alignment,microporosity,and hydrophilicity.In vitro assays revealed that the PCM matrices promoted cellular behaviors and myogenic differentiation of C2C12 myoblasts.Moreover,in vivo experiments demonstrated enhanced muscle remodeling and recovery in mice treated with PCM matrices following VML injury.Mechanistic insights from next-generation sequencing revealed that MXene NPs facilitated protein and ion availability within PCM matrices,leading to elevated intracellular Ca^(2+)levels in myoblasts through the activation of inducible nitric oxide synthase(i NOS)and serum/glucocorticoid regulated kinase 1(SGK1),ultimately promoting myogenic differentiation via the m TOR-AKT pathway.Additionally,upregulated i NOS and increased NO–contributed to myoblast proliferation and fiber fusion,thereby facilitating overall myoblast maturation.These findings underscore the potential of MXene NPs loaded within highly aligned matrices as therapeutic agents to promote skeletal muscle tissue recovery.

Harnessing the synergy of perfusable muscle flap matrix and adipose-derived stem cells for prevascularization and macrophage polarization to reconstruct volumetric muscle loss

Muscle flaps must have a strong vascular network to support a large tissue volume and ensure successful engraftment.We developed porcine stomach musculofascial flap matrix(PDSF)comprising extracellular matrix(ECM)and intact vasculature.PDSF had a dominant vascular pedicle,microcirculatory vessels,a nerve network,well-retained 3-dimensional(3D)nanofibrous ECM structures,and no allo-or xenoantigenicity.In-depth proteomic analysis demonstrated that PDSF was composed of core matrisome proteins(e.g.,collagens,glycoproteins,proteoglycans,and ECM regulators)that,as shown by Gene Ontology term enrichment analysis,are functionally related to musculofascial biological processes.Moreover,PDSF􀀀human adipose-derived stem cell(hASC)synergy not only induced monocytes towards IL-10􀀀producing M2 macrophage polarization through the enhancement of hASCs’paracrine effect but also promoted the proliferation and interconnection of both human skeletal muscle myoblasts(HSMMs)and human umbilical vein endothelial cells(HUVECs)in static triculture conditions.Furthermore,PDSF was successfully prevascularized through a dynamic perfusion coculture of hASCs and HUVECs,which integrated with PDSF and induced the maturation of vascular networks in vitro.In a xenotransplantation model,PDSF demonstrated myoconductive and immunomodulatory properties associated with the predominance of M2 macrophages and regulatory T cells.In a volumetric muscle loss(VML)model,prevascularized PDSF augmented neovascularization and constructive remodeling,which was characterized by the predominant infiltration of M2 macrophages and significant musculofascial tissue formation.These results indicate that hASCs’integration with PDSF enhances the cells’dual function in immunomodulation and angiogenesis.Owing in part to this PDSF-hASC synergy,our platform shows promise for vascularized muscle flap engineering for VML reconstruction.

Biomimetic glycosaminoglycan-based scaffolds improve skeletal muscle regeneration in a Murine volumetric muscle loss model

Volumetric muscle loss(VML)injuries characterized by critical loss of skeletal muscle tissues result in severe functional impairment.Current treatments involving use of muscle grafts are limited by tissue availability and donor site morbidity.In this study,we designed and synthesized an implantable glycosaminoglycan-based hydrogel system consisting of thiolated hyaluronic acid(HA)and thiolated chondroitin sulfate(CS)cross-linked with poly(ethylene glycol)diacrylate to promote skeletal muscle regeneration of VML injuries in mice.The HA-CS hydrogels were optimized with suitable biophysical properties by fine-tuning degree of thiol group substitution to support C2C12 myoblast proliferation,myogenic differentiation and expression of myogenic markers MyoD,MyoG and MYH8.Furthermore,in vivo studies using a murine quadriceps VML model demonstrated that the HA-CS hydrogels supported integration of implants with the surrounding host tissue and facilitated migration of Pax7+satellite cells,de novo myofiber formation,angiogenesis,and innervation with minimized scar tissue formation during 4-week implantation.The hydrogel-treated and autograft-treated mice showed similar functional improvements in treadmill performance as early as 1-week post-implantation compared to the untreated groups.Taken together,our results demonstrate the promise of HA-CS hydrogels as regenerative engineering matrices to accelerate healing of skeletal muscle injuries.

In vivo measurement of NADH fluorescence lifetime in skeletal muscle via -ber-coupled time-correlated single photon counting

Nicotinamide adenine dinucleotide(NADH)is a cofactor that serves to shuttle electrons during metabolic processes such as glycolysis,the tricarboxylic acid cycle,and oxidative phosphorylation(OXPHOS).NADH is autofluorescent,and itsfluorescence lifetime can be used to infer metabolic dynamics in living cells.Fiber-coupled time-correlated single photon counting(TCSPC)equipped with an implantable needle probe can be used to measure NADH lifetime in vivo,enabling investigation of changing metabolic demand during muscle contraction or tissue regeneration.This study illustrates a proof of concept for point-based,minimally-invasive NADHfluorescence lifetime measurement in vivo.Volumetric muscle loss(VML)injuries were created in the left tibialis anterior(TA)muscle of male Sprague Dawley rats.NADH lifetime measurements were collected before,during,and after a 30 s tetanic contraction in the injured and uninjured TA muscles,which was subsequently-t to a biexponential decay model to yield a metric of NADH utilization(cytoplasmic vs protein-bound NADH,the A11/A22 ratio).On average,this ratio was higher during and after contraction in uninjured muscle compared to muscle at rest,suggesting higher levels of free NADH in contracting and recovering muscle,indicating increased rates of glycolysis.In injured muscle,this ratio was higher than uninjured muscle overall but decreased over time,which is consistent with current knowledge of inflammatory response to injury,suggesting tissue regeneration has occurred.These data suggest that-ber-coupled TCSPC has the potential to measure changes in NADH binding in vivo in a minimally invasive manner that requires further investigation.