S100b treatment overcomes RAGE signaling deficits in myoblasts on advanced glycation end-product cross-linked collagen and promotes myogenic differentiation.

Advanced glycation end-products (AGEs) stochastically accrue in skeletal muscle and on collagen over an individual's lifespan, stiffening the muscle and modifying the stem cell (MuSC) microenvironment while promoting proinflammatory, antiregenerative signaling via the receptor for advanced glycation end-products (RAGEs). In the present study, a novel in vitro model was developed of this phenomenon by cross linking a 3-D collagen scaffold with AGEs and investigating how myoblasts responded to such an environment. Briefly, collagen scaffolds were incubated with d-ribose (0, 25, 40, 100, or 250 mM) for 5 days at 37°C. C2C12 immortalized mouse myoblasts were grown on the scaffolds for 6 days in growth conditions for proliferation, and 12 days for differentiation and fusion. Human primary myoblasts were also used to confirm the C2C12 data. AGEs aberrantly extended the DNA production stage of C2C12s (but not in human primary myoblasts) which is known to delay differentiation in myogenesis, and this effect was prevented by RAGE inhibition. Furthermore, the differentiation and fusion of myoblasts were disrupted by AGEs, which were associated with reductions in integrins and suppression of RAGE. The addition of S100b (RAGE agonist) recovered the differentiation and fusion of myoblasts, and the addition of RAGE inhibitors (FPS-ZM1 and Azeliragon) inhibited the differentiation and fusion of myoblasts. Our results provide novel insights into the role of the AGE-RAGE axis in skeletal muscle aging, and future work is warranted on the potential application of S100b as a proregenerative factor in aged skeletal muscle.NEW & NOTEWORTHY Collagen cross-linked by advanced glycation end-products (AGEs) induced myoblast proliferation but prevented differentiation, myotube formation, and RAGE upregulation. RAGE inhibition occluded AGE-induced myoblast proliferation, while the delivery of S100b, a RAGE ligand, recovered fusion deficits.

Specific MicroRNAs Found in Extracellular Matrix Vesicles Regulate Proliferation and Differentiation in Growth Plate Chondrocytes.

Matrix vesicles (MVs) are extracellular organelles produced by growth plate cartilage cells in a zone-specific manner. MVs are similar in size to exosomes, but they are tethered to the extracellular matrix (ECM) via integrins. Originally associated with matrix calcification, studies now show that they contain matrix processing enzymes and microRNA that are specific to their zone of maturation. MVs produced by costochondral cartilage resting zone (RC) chondrocytes are enriched in microRNA 503 whereas those produced by growth zone (GC) chondrocytes are enriched in microRNA 122. MVs are packaged by chondrocytes under hormonal and factor regulation and release of their contents into the ECM is also under hormonal control, suggesting that their microRNA might have a regulatory role in growth plate proliferation and maturation. To test this, we selected a subset of these enriched microRNAs and transfected synthetic mimics back into RC and GC cells. Transfecting growth plate chondrocytes with select microRNA produced a broad range of phenotypic responses indicating that MV-based microRNAs are involved in the regulation of these cells. Specifically, microRNA 122 drives both RC and GC cells toward a proliferative phenotype, stabilizes the matrix and inhibits differentiation whereas microRNA 22 exerts control over regulatory factor production. This study demonstrates the strong regulatory capability possessed by unique MV enriched microRNAs on growth plate chondrocytes and their potential for use as therapeutic agents.

Genetic variability affects the response of skeletal muscle to disuse.

OBJECTIVE: To examine whether genetic variability plays a role in skeletal muscle response to disuse. METHODS: We examined skeletal muscle response to disuse in five different strains of mice: CAST/EiJ, NOD/ShiLtJ, NZO/HILtJ, 129S1/SvImJ and A/J. Mice had one limb immobilized by a cast for three weeks. RESULTS: Response to immobilization was dependent on the strain of mice. Skeletal muscle mass/body weight was decreased by immobilization in all strains except 1291/SvImJ. Immobilization decreased absolute skeletal muscle mass in quadriceps and gastrocnemius in NOD/ShiltJ and NZO/HILtJ mice. Three weeks of immobilization resulted in an increase in quadriceps levels of atrogenes in CAST/EiJ. Immobilization resulted in an increase in quadriceps and gastrocnemius levels of Myh4 in CAST/EiJ. A similar trend was observed for Myh7 in gastrocnemius muscle. Immobilization resulted in a decrease of the p-p70S6K1/total p706SK1 ratio in quadriceps of NOD/ShiLtJ mice and the gastrocnemius of A/J mice. Immobilization did not affect the p-4EBP1/total 4EBP1 ratio in quadriceps of any of the strains examined. However, the p-4EBP1/total 4EBP1 ratio in gastrocnemius was greater in immobilized, relative to control, limbs in CAST/EiJ mice. CONCLUSION: Genetic variability affects the response of skeletal muscle to disuse.

Advanced Glycation End Products Are Retained in Decellularized Muscle Matrix Derived from Aged Skeletal Muscle.

Decellularized tissues are biocompatible materials that engraft well, but the age of their source has not been explored for clinical translation. Advanced glycation end products (AGEs) are chemical cross-links that accrue on skeletal muscle collagen in old age, stiffening the matrix and increasing inflammation. Whether decellularized biomaterials derived from aged muscle would suffer from increased AGE collagen cross-links is unknown. We characterized gastrocnemii of 1-, 2-, and 20-month-old C57BL/6J mice before and after decellularization to determine age-dependent changes to collagen stiffness and AGE cross-linking. Total and soluble collagen was measured to assess if age-dependent increases in collagen and cross-linking persisted in decellularized muscle matrix (DMM). Stiffness of aged DMM was determined using atomic force microscopy. AGE levels and the effect of an AGE cross-link breaker, ALT-711, were tested in DMM samples. Our results show that age-dependent increases in collagen amount, cross-linking, and general stiffness were observed in DMM. Notably, we measured increased AGE-specific cross-links within old muscle, and observed that old DMM retained AGE cross-links using ALT-711 to reduce AGE levels. In conclusion, deleterious age-dependent modifications to collagen are present in DMM from old muscle, implying that age matters when sourcing skeletal muscle extracellular matrix as a biomaterial.

Integrin-α7 signaling regulates connexin 43, M-cadherin, and myoblast fusion.

Regenerative medicine treatments for severe skeletal muscle injuries are limited, resulting in persistent functional deficits. Clinical options include neglecting the wound with the expectation that fibrosis will develop or using an autologous muscle graft with minimal functional improvement. A regenerative matrix can be used, but muscle fiber development on these matrices remains a challenge in vivo. Here, we explored the fundamental mechanisms that mediate cell-substrate signaling and its effect on cell-cell communication during myoblast fusion and tube formation to improve outcomes following implantation of matrices used to stimulate muscle regeneration. We previously reported that integrin-α7 was increased on anisotropic biomaterials, suggesting a role for α7ß1 signaling in myoblast communication via connexin 43 and M-cadherin. Our results demonstrated that α7 silencing blocked expression of myogenic differentiation factor 1 (Myod), myogenin (Myog), myogenic factor 6 (Myf6), myosin heavy chain type 1 (Myh1), and transmembrane protein 8c (Tmem8c), indicating that myoblast fusion was inhibited. Expression of α5 and M-cadherin decreased but ß1 and connexin 43 increased. We examined protein production and observed reduced extracellular-signal regulated kinase 1/2 (ERK) in α7-silenced cells that correlated with upregulation of connexin 43 and M-cadherin, suggesting a compensatory pathway. These results indicate that α7 signaling plays a critical role in ex vivo fusion and implicates a relationship with connexin 43 and M-cadherin.

Reduced osseointegration in disuse and denervation rat models results from impaired cellular responses to multiscale microstructured titanium surfaces.

Immobilization-induced skeletal unloading results in muscle atrophy and rapid bone loss, thereby increasing the risk of falling and the need for implant therapy in patients with extended bed rest or neuromuscular injuries. Skeletal unloading causes bone loss by altering bone growth and resorption, suggesting that implant performance might be affected. To test this, we focused on early events in implant osseointegration. We used the rat sciatic neurectomy-induced disuse model under two different settings. In Study 1, 16 Sprague Dawley rats (SD) were separated into control, sham operated+cast immobilization, and sciatic neurectomy+casting groups; titanium implants with multiscale microtextured topography and hydrophilic chemistry (modSLA) were inserted in the distal femoral metaphysis. Neurectomy surgeries and casting were performed at the same surgical setting as implant placement; rats were euthanized 4 weeks post-implantation. In Study 2, we established the unloaded condition before implantation. A total of 12 SD rats were divided into control and sciatic+femoral neurectomy groups. A total of 24 days after sciatic and femoral neurectomy surgery, rats received implants. Study 2 rats were euthanized at 4 weeks post-implantation. MicroCT and histomorphometry showed that trabecular bone and osseointegration were reduced when disuse was established before implantation. Osteoblasts isolated from Study 1 sciatic neurectomy tibial bones exhibited impaired differentiation on modSLA culture disks, revealing a possible mechanism responsible for the decreased osseointegration observed in the Study 2 rats. This study addressed the importance of considering the mechanical unloading and muscle function history before implant insertion and suggests that implant performance was reduced due to poor cellular ability to regenerate.

Osseointegration of Titanium Implants in a Botox-Induced Muscle Paralysis Rat Model Is Sensitive to Surface Topography and Semaphorin 3A Treatment.

Reduced skeletal loading associated with many conditions, such as neuromuscular injuries, can lead to bone fragility and may threaten the success of implant therapy. Our group has developed a botulinum toxin A (botox) injection model to imitate disease-reduced skeletal loading and reported that botox dramatically impaired the bone formation and osseointegration of titanium implants. Semaphorin 3A (sema3A) is an osteoprotective factor that increases bone formation and inhibits bone resorption, indicating its potential therapeutic role in improving osseointegration in vivo. We first evaluated the sema3A effect on whole bone morphology following botox injections by delivering sema3A via injection. We then evaluated the sema3A effect on the osseointegration of titanium implants with two different surface topographies by delivering sema3A to cortical bone defect sites prepared for implant insertion and above the implants after insertion using a copper-free click hydrogel that polymerizes rapidly in situ. Implants had hydrophobic smooth surfaces (PT) or multiscale biomimetic micro/nano topography (SLAnano). Sema3A rescued the botox-impaired bone formation. Furthermore, biomimetic Ti implants improved the bone-to-implant contact (BIC) and mechanical properties of the integrated bone in the botox-treated rats, which sema3A enhanced. This study demonstrated the value of biomimetic approaches combining multiscale topography and biologics in improving the clinical outcomes of implant therapy.

Human Adipose-Derived Stromal Cells Delivered on Decellularized Muscle Improve Muscle Regeneration and Regulate RAGE and P38 MAPK.

Volumetric muscle loss (VML) is the acute loss of muscle mass due to trauma. Such injuries occur primarily in the extremities and are debilitating, as there is no clinical treatment to restore muscle function. Pro-inflammatory advanced glycation end-products (AGEs) and the soluble receptor for advanced glycation end-products (RAGE) are known to increase in acute trauma patient's serum and are correlated with increased injury severity. However, it is unclear whether AGEs and RAGE increase in muscle post-trauma. To test this, we used decellularized muscle matrix (DMM), a pro-myogenic, non-immunogenic extracellular matrix biomaterial derived from skeletal muscle. We delivered adipose-derived stromal cells (ASCs) and primary myoblasts to support myogenesis and immunomodulation (N = 8 rats/group). DMM non-seeded and seeded grafts were compared to empty defect and sham controls. Then, 56 days after surgery muscle force was assessed, histology characterized, and protein levels for AGEs, RAGE, p38 MAPK, and myosin heavy chains were measured. Overall, our data showed improved muscle regeneration in ASC-treated injury sites and a regulation of RAGE and p38 MAPK signaling, while myoblast-treated injuries resulted in minor improvements. Taken together, these results suggested that ASCs combined with DMM provides a pro-myogenic microenvironment with immunomodulatory capabilities and indicates further exploration of RAGE signaling in VML.

Prophylactic administration of miR-451 inhibitor decreases osteoarthritis severity in rats.

Transfection of chondrocytes with microRNA-451(miR-451), present in growth zone cartilage of the growth plate, upregulates production of enzymes association with extracellular matrix degradation. miR-451 is also present in articular cartilage and exacerbates IL-1ß effects in articular chondrocytes. Moreover, when osteoarthritis (OA) was induced in Sprague Dawley rats via bilateral anterior cruciate ligament transection (ACLT), miR-451 expression was increased in OA cartilage compared to control, suggesting its inhibition might be used to prevent or treat OA. To examine the prophylactic and therapeutic potential of inhibiting miR-451, we evaluated treatment with miR-451 power inhibitor (451-PI) at the onset of joint trauma and treatment after OA had developed. The prophylactic animal cohort received twice-weekly intra-articular injections of either 451-PI or a negative control (NC-PI) beginning on post-surgical day 3. OA was allowed to develop for 24 days in the therapeutic cohort before beginning injections. All rats were killed on day 45. Micro-CT, histomorphometrics, OARSI scoring, and muscle force testing were performed on samples. 451-PI mitigated OA progression compared to NC-PI limbs in the prophylactic cohort based on histomorphometric analysis and OARSI scoring, but no differences were detected by micro-CT. 451-PI treatment beginning 24 days post-surgery was not able to reduce OA severity. Prophylactic administration of 451-PI mitigates OA progression in a post-trauma ACLT rat model supporting its potential to prevent OA development following an ACLT injury clinically.

Advanced Glycation End-Products in Skeletal Muscle Aging.

Advanced age causes skeletal muscle to undergo deleterious changes including muscle atrophy, fast-to-slow muscle fiber transition, and an increase in collagenous material that culminates in the age-dependent muscle wasting disease known as sarcopenia. Advanced glycation end-products (AGEs) non-enzymatically accumulate on the muscular collagens in old age via the Maillard reaction, potentiating the accumulation of intramuscular collagen and stiffening the microenvironment through collagen cross-linking. This review contextualizes known aspects of skeletal muscle extracellular matrix (ECM) aging, especially the role of collagens and AGE cross-linking, and underpins the motor nerve's role in this aging process. Specific directions for future research are also discussed, with the understudied role of AGEs in skeletal muscle aging highlighted. Despite more than a half century of research, the role that intramuscular collagen aggregation and cross-linking plays in sarcopenia is well accepted yet not well integrated with current knowledge of AGE's effects on muscle physiology. Furthermore, the possible impact that motor nerve aging has on intramuscular cross-linking and muscular AGE levels is posited.

Differential Effects of Neurectomy and Botox-induced Muscle Paralysis on Bone Phenotype and Titanium Implant Osseointegration.

Metabolic bone is highly innervated by both sensory and sympathetic nerves. In addition to skeletal development, neural regulation participates in local bone remodeling, which is important for successful osseointegration of titanium implants. Neurectomy is a model used to investigate the lack of neural function on bone homeostasis, but the relative impacts of direct denervation to bone or denervation-induced muscle paralysis are less well defined. To investigate this difference, we used two nerve intervention models, sciatic and femoral neurectomy (SFN) v. botox-induced muscle paralysis (BTX) and assessed the resulting femoral bone phenotype and Ti implant osseointegration. Male Sprague Dawley rats (19) were randomly divided into three groups: implant control (n = 5), SFN (n = 7), and BTX (n = 7). Ti implants (microrough/hydrophilic [modSLA], Institut Straumann AG) were placed in the distal metaphysis of each femur on day 24 post-SFN or BTX. Bone and muscle were examined on day 28 after implant insertion. Both nerve intervention models impaired osseointegration. MicroCT and histology indicated that both models had reduced trabecular bone formation. Only BTX reduced cortical bone formation and increased cortical bone porosity. BTX resulted in more bone loss characterized by the least trabecular and cortical bone, as well as osseointegration. Osteoblasts isolated from the tibia exhibited a model-specific phenotype when they were grown on Ti substrates in vitro. Neurectomy caused more severe muscle atrophy than botox injection. These results indicate that neural regulation directly modulates bone formation and osseointegration. Muscle paralysis modulated the effects of loss of neural inputs into bone, supporting the hypothesis that mechanical loading of bone is a factor in achieving successful osseointegration. The different effects of botox and neurectomy on bone phenotype indicated that the sensory and sympathetic nerves had a role in the osseointegration process.

RNU (Foxn1 RNU-Nude) Rats Demonstrate an Improved Ability to Regenerate Muscle in a Volumetric Muscle Injury Compared to Sprague Dawley Rats.

Products developed for skeletal muscle regeneration frequently incorporate allogeneic and xenogeneic materials to elicit a regenerative response to heal skeletal muscle wounds. To avoid graft rejection in preclinical studies, immunodeficient rodents are used. Whether the immunodeficiency alters the host response to the material in skeletal muscle has not been studied. In this study, we hypothesized that an allogeneic acellular skeletal muscle grafts implanted in an immunodeficient rat (RNU, Foxn1-deficient) would exhibit better new muscle fiber formation compared to grafts implanted in immunocompetent Sprague Dawley (SD) rats. Decellularized SD skeletal muscle matrix (DMM) was implanted in the gastrocnemius (N = 8 rats/group). 56 days after surgery, animal gait was examined and animals were euthanized. Muscle force was assessed and fiber number as well as immune cell infiltrate was measured by histomorphometry and immunohistochemistry. Animal gait and percent recovery of muscle force were unchanged in both groups, but newly regenerated muscle fibers increased in RNU rats. Macrophage staining for CD68 was higher in RNU rats than in SD rats. These data show differences in muscle regeneration between animal models using the same biomaterial treatment, but these differences could not be ascribed to the immune response. Overall, our data provide awareness that more studies are needed to understand how host responses to biomaterials differ based on the animal model used.

Randomized, Placebo-Controlled Analysis of the Knee Synovial Environment Following Platelet-Rich Plasma Treatment for Knee Osteoarthritis.

BACKGROUND: Platelet-rich-plasma (PRP) is used to treat knee osteoarthritis; however, mechanistic evidence of PRP effectiveness for pain relief is limited. OBJECTIVE: To assess molecular biomarkers and mesenchymal stem cells (MSCs) in synovial fluid during PRP treatment of the osteoarthritic knee joint. DESIGN: Single blinded, randomized, placebo controlled pilot study. SETTING: Veterans Affairs Medical Center. PARTICIPANTS: Seventeen participants with mild to moderate knee osteoarthritis were randomized in a 2:1 placebo-controlled ratio, receiving PRP or saline (placebo) intra-articular injection into the knee joint. METHODS: Knee synovial fluid was analyzed before the respective injections and again 10 days following injection. Participants were followed up to 12 months completing visual analog scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaires at intervals over that period. MAIN OUTCOME MEASURES: The effects of PRP on synovial protein and MSC gene expression levels were measured by multiplex enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. RESULTS: Novel biomarkers including levels of interleukin (IL)-5, IL-6, IL-10, and tumor necrosis factor-α were measured in synovial fluid 10 days after PRP treatment. Altered gene expression profiles in MSCs from patients treated with PRP were observed for matrix metalloproteinases and inflammatory markers (IL-6, IL-8, CCL2, TNF-α). A2M protease was significantly increased following PRP treatment (P = .005). WOMAC scores declined for up to 3 months from baseline levels and remained low at 6 and 12 months in the PRP group. In contrast, WOMAC scores for patients receiving the saline injection were relatively unchanged for up to 12 months. CONCLUSIONS: We report significant changes for the biomarker A2M (P = .005) as well as differences in expression of cellular markers and postulate that PRP modulates the local knee synovial environment by altering the inflammatory milieu, matrix degradation, and angiogenic growth factors. The PRP treatment group had less pain and stiffness and improved function scores.

Regulation of extracellular matrix vesicles via rapid responses to steroid hormones during endochondral bone formation.

Endochondral bone formation is a precise and highly ordered process whose exact regulatory framework is still being elucidated. Multiple regulatory pathways are known to be involved. In some cases, regulation impacts gene expression, resulting in changes in chondrocyte phenotypic expression and extracellular matrix synthesis. Rapid regulatory mechanisms are also involved, resulting in release of enzymes, factors and micro RNAs stored in extracellular matrisomes called matrix vesicles. Vitamin D metabolites modulate endochondral development via both genomic and rapid membrane-associated signaling pathways. 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] acts through the vitamin D receptor (VDR) and a membrane associated receptor, protein disulfide isomerase A3 (PDIA3). 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] affects primarily chondrocytes in the resting zone (RC) of the growth plate, whereas 1α,25(OH)2D3 affects cells in the prehypertrophic and upper hypertrophic cell zones (GC). This includes genomically directing the cells to produce matrix vesicles with zone specific characteristics. In addition, vitamin D metabolites produced by the cells interact directly with the matrix vesicle membrane via rapid signal transduction pathways, modulating their activity in the matrix. The matrix vesicle payload is able to rapidly impact the extracellular matrix via matrix processing enzymes as well as providing a feedback mechanism to the cells themselves via the contained micro RNAs.

Polymer scaffold architecture is a key determinant in mast cell inflammatory and angiogenic responses.

Implanted polymer scaffolds can induce inflammation leading to the foreign body response (FBR), fibrosis, and implant failure. Thus, it is important to understand how immune cells interact with scaffolds to mitigate inflammation and promote a regenerative response. We previously demonstrated that macrophage phenotype is modulated by fiber and pore diameters of an electrospun scaffold. However, it is unclear if this effect is consistent among other innate immune cells. Mast cells are inflammatory sentinels that play a vital role in the FBR of implanted biomaterials, as well as angiogenesis. We determined if altering electrospun scaffold architecture modulates mast cell responses, with the goal of promoting regenerative cell-scaffold interactions. Polydioxanone (PDO) scaffolds were made from 60 mg/mL or 140 mg/mL PDO solutions, yielding structures with divergent fiber and pore diameters. Mouse mast cells plated on these scaffolds were activated with IL-33 or lipopolysaccharide (LPS). Relative to the 60 mg/mL scaffold, 140 mg/mL scaffolds yielded less IL-6 and TNF, and greater VEGF secretion. Pores >4-6 µm elicited less IL-6 and TNF secretion. IL-33-induced VEGF regulation was more complex, showing effects of both pore size and fiber diameter. These data indicate parameters that can predict mast cell responses to scaffolds, informing biomaterial design to increase wound healing and diminish implant rejection. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 884-892, 2019.

Suture-reinforced electrospun polydioxanone-elastin small-diameter tubes for use in vascular tissue engineering: a feasibility study.

This study characterizes the cross-linking of electrospun elastin and the mechanical properties of suture-reinforced 1.5mm internal diameter electrospun tubes composed of blended polydioxanone (PDO) and soluble elastin. Several tube configurations were tested to assess the effects of reinforcement on tube mechanical properties. Between the electrospun layers of each double-layered prosthetic, zero, one or two 6-0 sutures were wound, maintaining 1mm spacing with a pitch of 9 degrees . Single-layered tubes without suture were also examined. Samples were cross-linked and tested for compliance and burst strength. Compliance decreased significantly (p <0.05) and burst strength significantly increased (p <0.01) with reinforcement. Uncross-linked tubes were also tested to determine the effects of cross-linking. Results demonstrated that cross-linking significantly decreases burst strength (p <0.01), while decreases in compliance for cross-linked tubes were not significant. Cross-linked suture-reinforced PDO-elastin tubes had burst pressures more than 10 times greater than normal systolic pressures and exhibited a range of compliance values, including those matching native artery. These tubes display many characteristics of the "ideal" small-diameter graft, having mechanical properties that can be tailored to match those desired in vascular replacement applications.

Platelet-rich plasma and alignment enhance myogenin via ERK mitogen activated protein kinase signaling.

Volumetric muscle loss is debilitating and involves extensive rehabilitation. One approach to accelerate healing, rehabilitation, and muscle function is to repair damaged skeletal muscle using regenerative medicine strategies. In sports medicine and orthopedics, a common clinical approach is to treat minor to severe musculoskeletal injuries with platelet-rich plasma (PRP) injections. While these types of treatments have become commonplace, there are limited data demonstrating their effectiveness. The goal of this study was to determine the effect of PRP on myoblast gene expression and protein production when incorporated into a polymer fiber. To test this, we generated extracellular matrix mimicking scaffolds using aligned polydioxanone (PDO) fibers containing lyophilized PRP (SmartPReP® 2, Harvest Technologies Corporation, Plymouth, MA). Scaffolds with PRP caused a dose-dependent increase in myogenin and myosin heavy chain but did not affect myogenic differentiation factor-1 (MyoD). Integrin α7ß1D decreased and α5ß1A did not change in response to PRP scaffolds. ERK inhibition decreased myogenin and increased Myod on the PDO-PRP scaffolds. Taken together, these data suggest that alignment and PRP produce a substrate-dependent, ERK-dependent, and dose-dependent effect on myogenic differentiation.

MicroRNA Contents in Matrix Vesicles Produced by Growth Plate Chondrocytes are Cell Maturation Dependent.

Chondrocytes at different maturation states in the growth plate produce matrix vesicles (MVs), membrane organelles found in the extracellular matrix, with a wide range of contents, such as matrix processing enzymes and receptors for hormones. We have shown that MVs harvested from growth zone (GC) chondrocyte cultures contain abundant small RNAs, including miRNAs. Here, we determined whether RNA also exists in MVs produced by less mature resting zone (RC) chondrocytes and, if so, whether it differs from the RNA in MVs produced by GC cells. Our results showed that RNA, small RNA specifically, was present in RC-MVs, and it was well-protected from RNase by the phospholipid membrane. A group of miRNAs was enriched in RC-MVs compared RC-cells, suggesting that miRNAs are selectively packaged into MVs. High throughput array and RNA sequencing showed that ~39% miRNAs were differentially expressed between RC-MVs and GC-MVs. Individual RT-qPCR also confirmed that miR-122-5p and miR-150-5p were expressed at significantly higher levels in RC-MVs compared to GC-MVs. This study showed that growth plate chondrocytes at different differentiation stages produce different MVs with different miRNA contents, further supporting extracellular vesicle miRNAs play a role as "matrisomes" that mediate the cell-cell communication in cartilage and bone development.

Decellularized Muscle Supports New Muscle Fibers and Improves Function Following Volumetric Injury.

Current strategies to treat volumetric muscle loss use primarily pedicle or free muscle transfers, but these grafts fail to adequately regenerate functional tissue. Decellularized soft tissue grafts possess physical and chemical cues to promote muscle regeneration, suggesting their potential for use in large muscle defects. In this study, we developed a decellularized muscle matrix (DMM) graft using rat gastrocnemius. Anisotropy and chemical components of the extracellular matrix were retained, including laminin, fibronectin, and collagen. We compared the ability of DMM, autologous muscle grafts (clinical standard), and type I collagen plugs (negative control) to support muscle regeneration. DMM supported regeneration over a 56-day period in 1 × 1 cm and 1.5 × 1 cm gastrocnemius defects in rats. Muscle function tests demonstrated improved muscle recovery in rats with DMM grafts when compared to collagen. Histological sections were assessed using morphometrics and immunostaining. DMM supported muscle regeneration with less fibrosis and more de novo neuromuscular receptors than either autograft or collagen. Overall, our results indicate that DMM may be used as a muscle replacement graft based on its ability to improve muscle function recovery, promote muscle regeneration, and support new neuromuscular junctions.

Galectin-1 promotes an M2 macrophage response to polydioxanone scaffolds.

Regulating soft tissue repair to prevent fibrosis and promote regeneration is central to creating a microenvironment conducive to soft tissue development. Macrophages play an important role in this process. The macrophage response can be modulated using biomaterials, altering cytokine and growth factor secretion to promote regeneration. Electrospun polydioxanone (PDO) fiber scaffolds promoted an M2 phenotype when macrophages were cultured on large diameter, highly porous scaffolds, but an M1 phenotype on smaller diameter fibers. In this study, we investigated whether incorporation of galectin-1, an immunosuppressive protein that enhances muscle regeneration, could promote the M2 response. Galectin-1 was incorporated into large and small fiber PDO scaffolds during electrospinning. Galectin-1 incorporation increased arginase-1 and reduced iNOS and IL-6 production in mouse bone-marrow derived macrophages compared with PDO alone for both scaffold types. Inhibition of ERK mitogen-activated protein kinase did not alter galectin-1 effects on arginase-1 and iNOS expression, but reversed IL-6 suppression, indicating that IL-6 is mediated by a different mechanism. Our results suggest that galectin-1 can be used to modulate macrophage commitment to a pro-regenerative M2 phenotype, which may positively impact tissue regeneration when using small diameter PDO scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2562-2571, 2017.