Introduction Volumetric muscle loss (VML) is a significant clinical challenge that severely compromises patients' motor function and often results in irreversible disability. While conventional hydrogels have been explored for VML repair, their inability to address peripheral nerve denervation has limited functional recovery. Objectives The objective of this study was to develop a conductive double-crosslinking hydrogel (PPY@SF/GelMA) by integrating polypyrrole (PPY) into gelatin methacryloyl (GelMA) and silk fibroin (SF), aiming to simultaneously promote myotube formation and nerve re-innervation. Methods The micro-architecture, compressive strength, rheological properties, swelling behavior, and conductivity of the PPY@SF/GelMA hydrogel were assessed. The influence of the conductive hydrogel on in vitro myogenic differentiation of C2C12 myoblast cells and angiogenic differentiation of endothelial cells was evaluated. The in vivo biodegradation and biocompatibility of the conductive hydrogel were assessed through subcutaneous implantation in the dorsal region of C57BL mice. The regenerative potential of the conductive hydrogel for skeletal muscle and peripheral nerve repair was investigated using a mouse tibialis anterior VML model. Results Compared to pure GelMA or SF hydrogels, the PPY@SF/GelMA composite exhibited superior mechanical resilience, tunable swelling kinetics, exceptional biocompatibility, and enhanced electrical conductivity. In vitro experiments using C2C12 murine myoblasts demonstrated that the PPY@SF/GelMA hydrogel markedly upregulated myogenic differentiation markers (e.g., Mhc, Myog, and MyoD) and promoted the formation of multinucleated myotubes. Additionally, the conductive hydrogel exhibited pro-angiogenic potential by enhancing endothelial cell differentiation, as evidenced by new formation of endothelial tubes. In vivo, histopathological analysis showed no signs of toxicity from the implanted conductive hydrogel. PPY@SF/GelMA implantation facilitated the regeneration of aligned muscle fibers, reduced fibrotic collagen deposition, and accelerated neovascularization. Importantly, the conductive hydrogel successfully promoted peripheral nerve re-innervation by restoring neuromuscular junctions. RNA-seq analysis further revealed the involvement of the phosphoinositide 3-kinase (PI3K) signaling pathway in the newly regenerated muscle treated with PPY@SF/GelMA. Conclusions Our findings demonstrate PPY@SF/GelMA as a promising therapeutic scaffold to facilitate both myogenic and neurogenic regeneration after VML injuries, offering a translatable strategy for complex musculoskeletal repair. The translational potential of this article The failure of skeletal muscle regeneration following VML injuries is primarily attributed to the loss of peripheral nerve innervation. The present investigation has demonstrated that the conductive PPY@SF/GelMA hydrogel effectively promoted myofiber maturation and restored neuromuscular junctions, thereby promoting the re-innervation of peripheral nerves in newly regenerated skeletal muscle. Our objective is to translate this conductive hydrogel into a viable clinical strategy for patients requiring the repair of severe muscle damage.

The functional regeneration of skeletal muscle following traumatic injury-induced volumetric muscle loss (VML) remains a significant challenge in orthopedic clinics. Myoblast mitochondrial dysfunction at the VML defect site hinders the formation of mature myotubes by disrupting energy metabolism and redox homeostasis. Here we demonstrate that activation of mitochondrial Sirtuin 3 (SIRT3) by honokiol (HKL) enhances skeletal muscle regeneration in a mouse model of cardiotoxin-induced acute injury and promotes functional restoration in a rat VML model. HKL treatment dose-dependently increased mitochondrial respiratory chain activity, activated mitochondrial antioxidant defense mechanisms, and enhanced the myogenic differentiation of myoblast cells via the SIRT3-mediated pathway. Conversely, silencing Sirt3 abrogated the protective effects of HKL on mitochondrial function and redox homeostasis. To evaluate the therapeutic potential for VML injuries, we developed a reactive oxygen species (ROS)-responsive hydrogel based on hyaluronic acid methacrylate (HAMA) by incorporating phenylboronic acid (PBA) with HKL (HAMA-PBA@HKL) through the formation of boronic ester bonds. When applied to rat tibialis anterior defects, the HAMA-PBA@HKL hydrogel significantly enhanced myofiber formation, improved vascularization, restored neuromuscular junction innervation, and recovered muscle contractile performance, while effectively inhibiting fibrotic tissue formation. Single-cell RNA sequencing analysis of the newly regenerated muscle demonstrated that the implantation of the HAMA-PBA@HKL hydrogel significantly increased the proportions of myonuclei cells, satellite cells, endothelial cells, and Schwann cells, while reducing the presence of macrophages, monocytes, and T lymphocytes. Transcriptomic profiling analysis confirmed that treatment with the HAMA-PBA@HKL hydrogel up-regulated genes associated with myoblast differentiation, oxidative phosphorylation, and mitochondrial fusion, while down-regulating gene expression related to immune response, fibroblast proliferation, and collagen deposition. Collectively, these findings highlight the translational potential of the HAMA-PBA@HKL hydrogel that targets mitochondrial SIRT3 in promoting functional skeletal muscle regeneration following severe traumatic injuries.

Intervertebral disc degeneration (IVDD) is a leading cause of spinal disorders, affecting millions globally, particularly the aging population. Current treatments, however, fail to fully restore disc structure and function, highlighting the need for regenerative therapies. This study aims to construct an antioxidant artificial nucleus pulposus (NP) by incorporating cuttlefish ink nanoparticles (CINPs) into GelMA microspheres, thereby enhancing nucleus pulposus cell (NPC) viability and extracellular matrix (ECM) synthesis. Oxidative stress is a key driver of disc degeneration. CINPs, rich in proline and fucose, significantly enhanced the antioxidant capacity of NPCs, as evidenced by reduced intracellular reactive oxygen species (ROS) levels and activation of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (NRF2/HO-1) pathway in our study. In vitro experiments demonstrated that GelMA@CINPs microspheres significantly enhanced NPC antioxidant capacity and promoted ECM secretion. Implantation of these microspheres into intervertebral discs (IVDs) of rats following discectomy validated their therapeutic efficacy in promoting NP tissue regeneration. In this experiment, the introduction of CINPs facilitates a dual antioxidant mechanism, comprising chemical (e.g., free radical scavenging by eumelanin via HAT/SET mechanisms) and biological (activation of the NRF2/HO-1 pathway) components. This synergistic approach directly addresses oxidative stress, a critical driver of intervertebral disc degeneration (IVDD) progression. This research introduces a novel strategy for improving cell-material interactions in tissue engineering, which enhances the potential for constructing an artificial NP and effectively treating IVDD.

Nucleotomy, a surgical procedure employed to address nucleus pulposus (NP) herniation, frequently results in defects within the annulus fibrosus (AF). Owing to the inherently limited self-repair ability of AF tissue, untreated lesions may precipitate reherniation of the NP, thereby accelerating the degeneration of the intervertebral disc. In this study, we introduce a novel composite scaffold comprising hydrogel-reinforced electrospun nanofibers, engineered to mimic the heterogeneous microarchitecture of the native AF matrix while offering improved mechanical support. A mesh-like polycaprolactone (PCL)/gelatin methacryloyl (GelMA) hybrid electrospun nanofibrous membrane was designed to replicate the AF outer region, while a hydrogel mixture of fucoidan methacryloyl (FuMA) and GelMA was sprayed to the membrane surface to emulate the inner region. The integrated FuMA/GelMA hydrogel and PCL/GelMA nanofiber layer enhanced the proliferation of AF cells and markedly improved the synthesis of AF matrix components. Importantly, even under oxidative stress induced by hydrogen peroxide, this hydrogel-reinforced nanofibrous composite effectively maintained the mitochondrial functions of AF cells, thereby supporting their energy metabolism and matrix anabolism. Further molecular experiments unraveled that this composite protected AF cells from oxidative damage through activation of the NRF2-mediated antioxidant enzymes, such as heme oxygenase 1. In a rat caudal disc box defect model, the implantation of a hydrogel-reinforced nanofibrous composite promoted the regeneration of AF tissue, preserved the hydration of NP tissue, and inhibited intervertebral disc degeneration. This approach represents a promising strategy for the repair of nucleotomy-induced AF damage, thereby offering potential therapeutic benefits for patients experiencing intervertebral disc degeneration.

The degeneration of intervertebral discs (IVD) remains a significant challenge in regenerative medicine. This study introduces novel hydrogel microspheres (GelMA-FCD-GA) designed to mimic the native extracellular matrix (ECM) of nucleus pulposus cells (NPCs). These microspheres integrate Gelatin Methacrylate (GelMA) with Fucoidan (FCD) and are further modified with aldehyde groups (GA) to establish a dynamic viscoelastic scaffold capable of transducing mechanical signals and promoting cellular functions. In vitro, GelMA-FCD-GA microspheres enhanced mitochondrial function and antioxidant capacity of NPCs, with increased expression of respiratory chain factors and reduced reactive oxygen species. In vivo, the transplantation of NPC-laden GelMAFCD-GA microspheres into Rat caudal IVDs demonstrated significant regenerative effects, as evidenced by improved MRI signals, restored disc height, and favorable histological outcomes compared to controls. This innovative approach presents a significant advancement in IVDD treatment, combining the mechanical benefits of bioactive materials with the bioactive properties of fucoidan. The dual-network design supports cell adhesion and growth and dynamically adapts to the physiological environment, offering a robust platform for regenerative medicine applications.

Background: The treatment of bone defects in the context of osteoporosis encounters numerous challenges. In the osteoporotic microenvironment, bone resorption outweighs bone formation, impeding the self-repair of bone defect areas. Furthermore, the deterioration of osteogenesis-angiogenesis coupling function at the defect sites and excessive inflammatory responses further complicate the treatment of bone defects. Hence, an improved approach is urgently needed to enhance the treatment of osteoporotic bone defects. Methods: Our efficient strategy has developed a multi-scale biomimetic fusion alendronate sodium cerium ion hydrogel scaffold, integrating 3D-printed tricalcium phosphate (TCP) scaffolds, collagen-methacrylate (COMA) hydrogel, and nanoparticles of alendronate sodium cerium ions. In vitro, we intervened osteoporosis rat derived bone marrow stromal cells (BMSCs) with the extract of TCP-H-Alendronate sodium cerium ion nanoparticles (ACNP) scaffold and detected the osteogenesis-related indicators through alkaline phosphatase (ALP) enzymatic activity staining, alizarin red staining, Western Blot, RT-qPCR and immunofluorescence staining to evaluate the osteogenic differentiation effect of TCP-H-ACNP scaffold. Through transcriptome sequencing, we explored the mechanism of TCP-H-ACNP scaffold affecting osteogenic differentiation of osteoporotic BMSCs. We intervened human umbilical vein endothelial cells (HUVECs) with the extract of TCP-H-ACNP scaffold and evaluated the angiogenic effect of TCP-H-ACNP scaffold through tube formation assay and cell scratch assay. In vivo, we established a distal femoral bone defect model in osteoporotic rats and evaluated the therapeutic effect in vivo through Mirco CT, Hematoxylin and Eosin (H&E) stainin, Masson staining and immunohistochemical staining. Results: The results demonstrated that in vitro, TCP-H-ACNP scaffolds could promote osteogenic differentiation of osteoporotic BMSCs from rats and angiogenesis of HUVECs. In vivo, TCP-H-ACNP scaffolds could promote bone regeneration and repair of distal femoral bone defects in osteoporotic rats and improve local angiogenesis. Mechanistically, TCP-H-ACNP scaffolds could directly promote osteogenic differentiation of osteoporotic BMSCs from rats through the Wnt signaling pathway, and indirectly promote osteogenic differentiation by influencing Ca ion transport and improving mitochondrial function. Conclusion: We create a hydrogel scaffold that not only offers adequate mechanical support but also possesses a favorable microenvironment for cell growth and contains biological factors promoting osteogenic and angiogenic differentiation. The translational potential of this paper: This application represents a pioneering aspect of multi-scale biomimetic hydrogel scaffolds in addressing osteoporotic bone defects, providing a novel direction for the treatment of osteoporotic bone defects.

Cell transplantation for nucleus pulposus (NP) regeneration represents a promising strategy for intervertebral disc degeneration (IVDD). Nonetheless, the hostile microenvironment within the degenerated intervertebral discs, characterized by redox imbalance and elevated mechanical pressure, poses risks of low cell survival and inadequate cell colonization for efficient NP regeneration. To address these challenges, we developed a biomimetic, esterase-responsive composite hydrogel microsphere (GHKM) for cell delivery, consisting of gelatin methacrylate (GelMA) mixed with HAMA-KGN, a conjugate of hyaluronic acid methacrylate (HAMA) and the small heterocyclic molecule kartogenin (KGN) via ester bonds. GHKM mimic the NP extracellular matrix (ECM), providing essential adhesion and mechanical support for cell proliferation, while facilitating cellular adaptation to the adverse microenvironment through the esterase-responsive release of KGN. Furthermore, GHKM exhibit favorable biocompatibility and promote or protect ECM synthesis by nucleus pulposus cells (NPCs) under both normal and inflammatory conditions. Transcriptomic sequencing analysis indicates a correlation between enhanced ECM synthesis and enrichment of antioxidant-related pathways. Subsequent cellular biological studies reveal that GHKM can also reduce reactive oxygen species production within the inflammatory milieu. The underlying mechanism of its protective effect on matrix metabolism may involve the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and the upregulation of downstream antioxidant enzymes. In vivo implantation of NPCs-laden GHKM into rat tail nuclectomy models for 4 and 8 weeks preserved disc height, structure, and biological function, with histological analysis confirming NP regeneration. These findings present GHKM as a promising, synergistic transplantation strategy for NP regeneration in IVDD. STATEMENT OF SIGNIFICANCE: This study introduces an esterase-responsive gelatin methacrylate/hyaluronic acid methacrylate-kartogenin composite hydrogel microsphere (GHKM) system, aimed at mimicing the extracellular matrix (ECM) of the nucleus pulposus (NP) to address the pressing challenge of intervertebral disc degeneration (IVDD). These microspheres offer an innovative solution for cell transplantation therapy by simultaneously addressing two critical barriers: the harsh microenvironment of the degenerated disc and the need for sustained therapeutic effects. GHKM provide mechanical support, enhance cell survival, and adapt dynamically to adverse conditions through esterase-responsive release of kartogenin (KGN), a multifunctional molecule with chondrogenic, anti-inflammatory, and antioxidative properties. This study will not only interest researchers focused on regenerative medicine and biomaterials but also inspire new directions for tackling complex degenerative diseases.

Degeneration of the nucleus pulposus (NP) is the primary contributor to lumbar intervertebral disc (IVD) disorders. While clinical discectomy is frequently employed to address advanced stages of IVD degeneration, this surgical intervention compromises the structural integrity of NP tissue, leading to a substantial decrease in the IVD's load-bearing capacity and hindering long-term pain relief. Selenoprotein is crucial in modulating mitochondrial redox homeostasis within NP cells, suggesting that selenium-based antioxidative stress therapy may be a promising approach for NP regeneration. In this study, we developed a selenium-functionalized hydrogel microsphere (SeNPs@GelMA) consisting of gelatin methacrylate (GelMA) and selenium nanoparticles (SeNPs). The SeNPs@GelMA microspheres serve as a vehicle for cell-based NP regeneration, supporting both cell delivery and antioxidative activity. In vitro experiments indicated that SeNPs@GelMA microspheres possess favorable cytocompatibility. Nucleus pulposus cells (NPCs) cultured on SeNPs@GelMA microspheres exhibited a marked enhancement in matrix synthesis, including aggrecan and type II collagen, alongside a significant suppression of matrix-degrading enzymes, even in an interleukin-1β-induced inflammatory environment. Notably, SeNPs@GelMA microspheres preserved mitochondrial redox homeostasis through the activation of glutathione peroxidase 1 (GPX1), thereby maintaining mitochondrial integrity and enhancing energy metabolism. When implanted into rat IVDs following discectomy, NPC-loaded SeNPs@GelMA microspheres facilitated NP regeneration and effectively restored biomechanical function, as evidenced by a substantial increase in IVD disc height and water content at the defect site. These findings indicate that the integration of SeNPs@GelMA microspheres with cell therapy constitutes a promising approach for NP regeneration, offering potential therapeutic benefits for patients undergoing discectomy.

Extracellular matrix (ECM) derived from mesenchymal stem cells regulates antioxidant properties and bone metabolism by providing a favorable extracellular microenvironment. However, its functional role and molecular mechanism in mitochondrial function regulation and aged bone regeneration remain insufficiently elucidated. This proteomic analysis has revealed a greater abundance of proteins supporting mitochondrial function in the young ECM (Y-ECM) secreted by young bone marrow-derived mesenchymal stem cells (BMMSCs) compared to the aged ECM (A-ECM). Further studies demonstrate that Y-ECM significantly rejuvenates mitochondrial energy metabolism in adult BMMSCs (A-BMMSCs) through the promotion of mitochondrial respiratory functions and amelioration of oxidative stress. A-BMMSCs cultured on Y-ECM exhibited enhanced multi-lineage differentiation potentials in vitro and ectopic bone formation in vivo. Mechanistically, silencing of silent information regulator type 3 (SIRT3) gene abolished the protective impact of Y-ECM on A-BMMSCs. Notably, a novel composite biomaterial combining hyaluronic acid methacrylate hydrogel microspheres with Y-ECM is developed, which yielded substantial improvements in the healing of bone defects in an aged rat model. Collectively, these findings underscore the pivotal role of Y-ECM in maintaining mitochondrial redox homeostasis and present a promising therapeutic strategy for the repair of aged bone defects.

Intervertebral disc degeneration (IVDD) is a common cause of debilitating spinal conditions, necessitating regenerative therapies to restore tissue function. This study explores the potential of enhancing nucleus pulposus cell (NPC) viability and extracellular matrix (ECM) synthesis through surface modification of GelMA microspheres with His-Ala-Val (HAV) peptides. The HAV peptides, mimicking N-cadherin's adhesive properties, aim to promote cell-cell interactions akin to NPCs' native environment. In vitro studies demonstrated enhanced ECM secretion by NPCs cultured on HAV-functionalized GelMA microspheres, suggesting a potential for improved regenerative capacity. The microspheres promoted NP tissue regeneration when implanted in rat tail IVDs post-discectomy, indicating their therapeutic efficacy in vivo. This research provides insights into novel strategies for enhancing cell-material interactions in tissue engineering applications to mitigate IVDD.

Volumetric muscle loss (VML) is a severe condition caused by extensive damage to muscle tissue that exceeds the body's intrinsic regenerative capacity, resulting in significant functional deficits and long-term disability. Current clinical treatments, including autologous muscle grafts and physical therapy, often fall short due to donor site morbidity, limited functional recovery, and an inability to fully restore muscle structure and function. Polysaccharides as natural macromolecules are characterized by inherent biocompatibility, degradability, and tunable mechanical properties, making them highly suitable for tissue engineering applications. This comprehensive review examines the innovative application of polysaccharide-based biomaterials in VML repair, emphasizing their translational potential to regenerative treatment outcomes. Polysaccharide-based biomaterials can significantly enhance muscle regeneration by providing structural support, modulating immune responses to minimize fibrosis, and promoting new vascular and nerve formation, which are essential for functional muscle recovery. More importantly, these materials serve as effective carriers for cell therapy and the delivery of bioactive molecules, thereby enhancing regenerative efficacy. Despite challenges such as standardizing preparation protocols, ensuring consistent biocompatibility, and optimizing degradation rates, polysaccharide-based biomaterials hold great promise for advancing skeletal muscle regeneration and offer a potential breakthrough in treating VML. Statement of Significance Volumetric muscle loss (VML) is a severe clinical condition characterized by extensive muscle tissue damage that exceeds the body's natural regenerative capacity. Polysaccharide-based biomaterials have been extensively applied in tissue repair due to their favorable biological properties. They offer a promising therapeutic strategy for VML, for which currently available long-term treatments are limited in both safety and efficacy. However, a comprehensive review detailing the interaction mechanisms between polysaccharide-based biomaterials and muscle tissue regeneration remains lacking. This article focuses on the interactions and highlights recent evidence on the capacity of polysaccharide promoting myogenesis, modulating immune responses, restoring neural innervation, stimulating angiogenesis, and enhancing antioxidant activity. This review article provides insights into the future design and application of polysaccharide-based biomaterials for skeletal muscle regeneration after acute injuries.

The heightened activity of osteoclasts and diminished function of osteoblasts observed in individuals with osteoporosis present additional complexities in the management of critical bone defects. Consequently, an improved approach is urgently required to enhance bone regeneration in patients with osteoporosis. Our effective strategy involves the integration of hydrogels with spinning membrane technology, and accurately replicates the composition and structure of the native extracellular matrix found in periosteum tissues. Cerium, as a substitute for conventional protein growth factors, facilitates osteogenic differentiation and angiogenesis in stem cells, concurrently impeding bone resorption. In this study, we successfully synthesized a biomimetic electrospun hydrogel periosteum, grafted cerium ions onto the membrane via non-covalently bound phosphate, and achieved cyclic amplification of bone regeneration. This approach represents a pioneering and efficient utilization of tissue-engineered bionic periosteum to address osteoporotic bone defects.

Osteoporotic bone defects, a severe complication of osteoporosis, are distinguished by a delayed bone healing process and poor repair quality. While bone marrow-derived mesenchymal stem cells (BMMSCs) are the primary origin of bone-forming osteoblasts, their mitochondrial function is impaired, leading to inadequate bone regeneration in osteoporotic patients. Melatonin is well-known for its antioxidant properties and regulation on bone metabolism. The present study postulated that melatonin has the potential to enhance the repair of osteoporotic bone defects by restoring the mitochondrial function of BMMSCs. In vitro administration of melatonin at varying concentrations (0.01, 1, and 100 mu M) demonstrated a significant dose-dependent improvement in the mitochondrial function of BMMSCs obtained from ovariectomized rats (OVX-BMMSCs), as indicated by an elevation in mitochondrial membrane potential, adenosine triphosphate synthesis and expression of mitochondrial respiratory chain factors. Melatonin reduced the level of mitochondrial superoxide by activating the silent information regulator type 1 (SIRT1) and its downstream antioxidant enzymes, particularly superoxide dismutase 2 (SOD2). The protective effects of melatonin were found to be nullified upon silencing of Sirt1 or Sod2, underscoring the crucial role of the SIRT1-SOD2 axis in the melatonin-induced enhancement of mitochondrial energy metabolism in OVX-BMMSCs. To achieve a sustained and localized release of melatonin, silk fibroin scaffolds loaded with melatonin (SF@MT) were fabricated. The study involved the surgical creation of bilateral femur defects in OVX rats, followed by the implantation of SF@MT scaffolds. The results indicated that the application of melatonin partially restored the mitochondrial energy metabolism and osteogenic differentiation of OVX-BMMSCs by reinstating mitochondrial redox homeostasis. These findings suggest that the localized administration of melatonin through bone implants holds potential as a therapeutic approach for addressing osteoporotic bone defects.

Intervertebral disc degeneration (IVDD) is a prevalent musculoskeletal disorder that involves the excessive accumulation of reactive oxygen species (ROS), resulting in mitochondrial dysfunction and matrix metabolism imbalance in nucleus pulposus cells (NPCs). Selenium, an indispensable trace element, plays a crucial role in maintaining mitochondrial redox homeostasis by being incorporated into antioxidant selenoproteins as selenocysteine. In this study, we employed a straightforward synthesis method to produce selenium nanoparticles (SeNPs) with consistent size and distribution, and evaluated their potential protective effects in ameliorating IVDD. In a simulated inflammatory environment induced by interleukin-1beta (IL-1β) in vitro, SeNPs demonstrated a protective effect on the matrix synthesis capacity of NPCs through the up-regulation of aggrecan and type II collagen, while concurrently suppressing the expression of matrix degradation enzymes including MMP13 and ADAMTS5. Additionally, SeNPs preserved mitochondrial integrity and restored impaired mitochondrial energy metabolism by activating glutathione peroxidase1 (GPX1) to rebalance redox homeostasis. In a rat lumbar disc model induced by puncture, the local administration of SeNPs preserved the hydration of nucleus pulposus tissue, promoted matrix deposition, and effectively mitigated the progression of IVDD. Our results indicate that the enhancement of GPX1 by SeNPs may offer a promising therapeutic approach for IVDD by restoring mitochondrial function and redox homeostasis.

IntroductionThe cartilage endplate (CEP) plays a crucial role as both a mechanical barrier and nutrient channel for the intervertebral disc, but it is vulnerable to excessive axial loading. We modified the Ilizarov external fixator and applied it to the CEP of the rat tail to impose diurnal, controllable excess axial loading. The objective was to measure morphological changes in the CEP when subjected to loading during the aging process.MethodsTwo Kirschner wires were, respectively, inserted into the center of the eighth and ninth coccygeal vertebrae (Co8/9) of rat (n = 54) to apply axial loading to the CEP. A remote control device was used to establish the diurnal loading schedule. At the end of 4, 8, and 12-week periods, the Co8/9 CEPs in each group were analyzed using MRI, histological staining, and immunohistochemical staining techniques.ResultsThe novel Ilizarov model that we modified successfully induced degeneration of the rat coccygeal CEP. MRI analysis revealed significant degenerative changes in the loaded Co8/9 CEP, including decreased signal intensity and the formation of Schmorl’s nodes at 8 and 12 weeks. Histological examination showed progressive CEP degeneration (CEPD), characterized by decreased microporosity, thinning, and structural irregularities. Immunohistochemical analysis demonstrated a significant reduction in Aggrecan and Collagen II expression in the CEP and nucleus pulposus over time. Control and sham groups maintained normal CEP structure and composition throughout the study period.ConclusionExcessive axial loading induced CEPD in the rat tail, primarily characterized by the formation of Schmorl’s nodes and a reduction in CEP microporosity in this study. Our modified Ilizarov rat tail compression model, featuring stable and controllable axial loading capabilities, provided an alternative experimental paradigm for further investigation into CEPD.

Volumetric muscle loss (VML) is a condition that results in the extensive loss of 20 % or more of skeletal muscle due to trauma or tumor ablation, leading to severe functional impairment and permanent disability. The current surgical interventions have limited functional regeneration of skeletal muscle due to the compromised self-repair mechanism. Melatonin has been reported to protect skeletal muscle from exercise-induced oxidative damage and holds great potential to treat muscle diseases. In this study, we hypothesize that melatonin can enhance myoblast differentiation and promote effective recovery of skeletal muscle following VML. In vitro administration of melatonin resulted in a significant enhancement of myogenesis in C2C12 myoblast cells, as evidenced by the upregulation of myogenic marker genes in a dose-dependent manner. Further experiments revealed that silent information of regulator type 3 (SIRT3) played a critical role in the melatonin-enhanced myoblast differentiation through enhancement of mitochondrial energy metabolism and activation of mitochondrial antioxidant enzymes such as superoxide dismutase 2 (SOD2). Silencing of Sirt3 completely abrogated the protective effect of melatonin on the mitochondrial function of myoblasts, evidenced by the increased reactive oxygen species, decreased adenosine triphosphate production, and down-regulated myoblast-specific marker gene expression. In order to attain a protracted and consistent release, liposome-encapsuled melatonin was integrated into gelatin methacryloyl hydrogel (GelMA-Lipo@MT). The implantation of GelMA-Lipo@MT into a tibialis anterior muscle defect in a VML model effectively stimulated the formation of myofibers and new blood vessels in situ, while concurrently inhibiting fibrotic collagen deposition. The findings of this study indicate that the incorporation of melatonin with GelMA hydrogel has facilitated the de novo vascularized skeletal muscle regeneration by augmenting mitochondrial energy metabolism. This represents a promising approach for the development of skeletal muscle tissue engineering, which could be utilized for the treatment of VML and other severe muscle injuries.

The repair of critical-sized bone defects poses a significant challenge due to the absence of periosteum, which plays a crucial role in coordinating the processes of osteogenesis and vascularization during bone healing. Herein, we hypothesized that melatonin-encapsuled silk Fibronin electrospun nanofibers (SF@MT) could provide intrinsic induction of both osteogenesis and angiogenesis, thereby promoting vascularized bone regeneration. The sustained release of melatonin from the SF@MT nanofibers resulted in favorable biocompatibility and superior osteogenic induction of bone marrow mesenchymal stem cells (BMMSCs). Interestingly, melatonin promoted the migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a BMMSC-dependent manner, potentially through the upregulation of vascular endothelial growth factor (VEGFA) expression in SF@MT-cultured BMMSCs. SF@MT nanofibers enhanced the BMMSC-mediated angiogenesis by activating the PI3K/Akt signaling pathway. In vivo experiments indicated that the implantation of SF@MT nanofibers into rat critical-sized calvarial defects significantly enhances the production of bone matrix and the development of new blood vessels, leading to an accelerated process of vascularized bone regeneration. Consequently, the utilization of melatonin-encapsulated silk Fibronin electrospun nanofibers shows great promise as a potential solution for artificial periosteum, with the potential to regulate the coupling of osteogenesis and angiogenesis in critical-sized bone defect repair.