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Abstract Rotator cuff tendinopathy is the most common tendinopathy type with the worst prognosis. Conventional treatments often elicit heterogeneous drug responses due to the diversity of tendinopathy. Hence, this study attempted a classification of 126 diseased tendons into three distinct subtypes with opposite pathogenic mechanisms based on transcriptomic and clinical features. The hypoxic atrophic subtype with white appearance (Hw) exhibits downregulated neovascularization pathways. The inflammatory proliferative subtype with white appearance (Iw) shows a moderate upregulation of inflammatory characteristics. The inflammatory proliferative subtype with red appearance (Ir) exhibits the highest levels of upregulated neovascularization and inflammatory pathways, along with severe joint dysfunction. We then established research models, including subtype-specific simulations in animal models and clinical data analysis. These revealed that glucocorticoid, a controversial commonly used drug, was only effective in treating the Ir subtype. Hence, the tendinopathy subtypes elucidated in this study have significant implications for developing precision treatment of tendinopathy.

Abstract The anterior cruciate ligament is anchored to the femur and tibia via specialized interfaces known as entheses. These play a critical role in ligament homeostasis and joint stability by transferring forces, varying in magnitude and direction between structurally and functionally dissimilar tissues. However, the precise structural and mechanical characteristics underlying the femoral and tibial entheses and their intricate interplay remain elusive. In this study, two thin-graduated mineralization regions in the femoral enthesis (~21 μm) and tibial enthesis (~14 μm) are identified, both exhibiting distinct biomolecular compositions and mineral assembly patterns. Notably, the femoral enthesis interface exhibits progressively maturing hydroxyapatites, whereas the mineral at the tibial enthesis interface region transitions from amorphous calcium phosphate to hydroxyapatites with increasing crystallinity. Proteomics results reveal that Matrix Gla protein uniquely enriched at the tibial enthesis interface, may stabilize amorphous calcium phosphate, while C-type lectin domain containing 11 A, enriched at the femoral enthesis interface, could facilitate the interface mineralization. Moreover, the finite element analysis indicates that the femoral enthesis model exhibited higher resistance to shearing, whereas the tibial enthesis model contributes to tensile resistance, suggesting that the discrepancy in biomolecular expression and the corresponding mineral assembly heterogeneities collectively contribute to the superior mechanical properties of both the femoral enthesis and tibial enthesis models. These findings provide novel perspectives on the structure-function relationships of anterior cruciate ligament entheses, paving the way for improved management of anterior cruciate ligament injury and regeneration.

Although natural polymers have been widely used in constructing bone scaffolds, it still remains challenging to fabricate natural polymer-derived bone scaffolds with biomimetic mechanical properties as well as outstanding osteogenic properties for large-size and weight-bearing bone defects regeneration. Herein, an “organic-inorganic assembly” strategy is developed to construct silk fibroin (SF)-based bone scaffolds with the aforementioned merits. After secondary structure reshuffling, the 3.3-fold increment of β-sheet structures in SF hydrogel resulted in a 100-fold improvement of mineral-assembly efficacy via influencing the ion adsorption process and providing templates for mineral growth. Notably, abundant minerals were deposited within the hydrogel and also on the surface, which indicated entire mineral-assembly, which ensured the biomimetic mechanical properties of the digital light processing 3D printed SF hydrogel scaffolds with haversian-mimicking structure. In vitro experiments proved that the assembly between the mineral and SF results in rapid adhesion and enhanced osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. In vivo experiments further proved that the mineral-assembled SF hydrogel scaffold could significantly enhance integration and bone regeneration at the weight-bearing site within one month. This SF-based “organic-inorganic assembly” strategy sheds light on constructing cell-free, growth factor-free and natural polymer-derived bone scaffolds with biomimetic 3D structure, mechanical properties and excellent osteogenic properties.

Abstract The knee joint has long been considered a closed system. The pathological effects of joint diseases on distant organs have not been investigated. Herein, our clinical data showed that post-traumatic joint damage, combined with joint bleeding (hemarthrosis), exhibits a worse liver function compared with healthy control. With mouse model, hemarthrosis induces both cartilage degeneration and remote liver damage. Next, we found that hemarthrosis induces the upregulation in ratio and differentiation towards Th17 cells of CD4+ T cells in peripheral blood and spleen. Deletion of CD4+ T cells reverses hemarthrosis-induced liver damage. Degeneration of cartilage matrix induced by hemarthrosis upregulates serological type II collagen (COL II), which activates CD4+ T cells. Systemic application of a COL II antibody blocks the activation. Furthermore, bulk RNAseq and single-cell qPCR analysis revealed that the cartilage Akt pathway is inhibited by blood treatment. Intra-articular application of Akt activator blocks the cartilage degeneration and thus protects against the liver impairment in mouse and pig models. Taken together, our study revealed a pathological joint–liver axis mediated by matrikine-activated CD4+ T cells, which refreshes the organ-crosstalk axis and provides a new treatment target for hemarthrosis-related disease. Intra-articular bleeding induces cartilage degradation through down-reulation of cartilage Akt pathway. During this process, the soluble COL II released from the damaged cartilage can activate peripheral CD4+ T cells, differention into Th17 cells and secretion of IL-17, which consequently induces liver impairment. Intra-articular application of sc79 (inhibitor of Akt pathway) can prevent the cartilage damage as well as its peripheral influences.

Abstract Despite orientationally variant tears of the meniscus, suture repair is the current clinical gold treatment. However, inaccessible tears in company with re-tears susceptibility remain unresolved. To extend meniscal repair tools from the perspective of adhesion and regeneration, we design a dual functional biologic-released bioadhesive (S-PIL10) comprised of methacrylated silk fibroin crosslinked with phenylboronic acid-ionic liquid loading with growth factor TGF-β1, which integrates chemo-mechanical restoration with inner meniscal regeneration. Supramolecular interactions of β-sheets and hydrogen bonds richened by phenylboronic acid-ionic liquid (PIL) result in enhanced wet adhesion, swelling resistance, and anti-fatigue capabilities, compared to neat silk fibroin gel. Besides, elimination of reactive oxygen species (ROS) by S-PIL10 further fortifies localized meniscus tear repair by affecting inflammatory microenvironment with dynamic borate ester bonds, and S-PIL10 continuously releases TGF-β1 for cell recruitment and bridging of defect edge. In vivo rabbit models functionally evidence the seamless and dense reconstruction of torn meniscus, verifying that the concept of meniscus adhesive is feasible and providing a promising revolutionary strategy for preclinical research to repair meniscus tears.

Objective: It is a common clinical phenomenon that blood infiltrates into the injured tendon caused by sports injuries, accidental injuries, and surgery. However, the role of blood infiltration into the injured tendon has not been investigated. Methods: A blood-induced rat model was established and the impact of blood infiltration on inflammation and HO of the injured tendon was assessed. Cell adhesion, viability, apoptosis, and gene expression were measured to evaluate the effect of blood treatment on tendon stem/progenitor cells (TSPCs). Then RNA-seq was used to assess transcriptomic changes in tendons in a blood infiltration environment. At last, the small molecule drug PI3K inhibitor LY294002 was used for in vivo and in vitro HO treatment. Results: Blood caused acute inflammation in the short term and more severe HO in the long term. Then we found that blood treatment increased cell apoptosis and decreased cell adhesion and tenonic gene expression of TSPCs. Furthermore, blood treatment promoted osteochondrogenic differentiation of TSPCs. Next, we used RNA-seq to find that the PI3K/AKT signaling pathway was activated in blood-treated tendon tissues. By inhibiting PI3K with a small molecule drug LY294002, the expression of osteochondrogenic genes was markedly downregulated while the expression of tenonic genes was significantly upregulated. At last, we also found that LY294002 treatment significantly reduced the tendon HO in the rat blood-induced model. Conclusion: Our findings indicate that the upregulated PI3K/AKT signaling pathway is implicated in the aggravation of tendon HO. Therefore, inhibitors targeting the PI3K/AKT pathway would be a promising approach to treat blood-induced tendon HO.

Spinal cord injury (SCI) is a severe central nerve diseases which lacks effective of treatment. Oxidative stress is a major pathological factor for SCI. It may induce mitochondrial damage, which further lead to ferroptosis and apoptosis in neural cells during SCI. Thus, targeting oxidative stress may be the key strategy for SCI treatment. In this study, we carried out screening experiment in the natural product library against oxidative stress in neural cells. It was identified that idebenone (IDE) may potently suppress oxidative stress induced cell death in neural cells. Furthermore, we successfully developed a multifunctional nanovesicles, which we named IDE@PPNs, by encapsulating IDE within proanthocyanidin polyphenol nanovesicles (PPNs). IDE@PPNs exhibited lysosomal escape capability and mitochondrial ROS responsiveness; meanwhile, they may suppress ferroptosis and apoptosis in neural cells. In vivo study demonstrated that IDE@PPNs may promote motor function recovery in rats and suppress ferroptosis and apoptosis in neurons. It may also show high biocompatibility in in vivo study. In summary, we fabricated a novel natural product loaded ROS responsive nanovesicles, IDE@PPNs may have the great potential for SCI therapy.

Dynamic regulation of cell-extracellular matrix (ECM)-material interactions is crucial for various biomedical applications. In this study, a light-activated molecular switch for the modulation of cell attachment/detachment behaviors was established on monolayer graphene (Gr)/n-type Silicon substrates (Gr/Si). Initiated by light illumination at the Gr/Si interface, pre-adsorbed proteins (bovine serum albumin, ECM proteins collagen-1, and fibronectin) underwent protonation to achieve negative charge transfer to Gr films (n-doping) through π-π interactions. This n-doping process stimulated the conformational switches of ECM proteins. The structural alterations in these ECM interactors significantly reduced the specificity of the cell surface receptor-ligand interaction (e.g., integrin recognition), leading to dynamic regulation of cell adhesion and eventual cell detachment. RNA-sequencing results revealed that the detached bone marrow mesenchymal stromal cell sheets from the Gr/Si system manifested regulated immunoregulatory properties and enhanced osteogenic differentiation, implying their potential application in bone tissue regeneration. This work not only provides a fast and feasible method for controllable cells/cell sheets harvesting but also gives new insights into the understanding of cell-ECM-material communications.

Inflammatory bowel disease (IBD) is a chronic, immune-mediated inflammatory disease characterized by the destruction of the structure and function of the intestinal epithelial barrier. Due to the poor remission effect and severe adverse events associated with current clinical medications, IBD remains an incurable disease. Here, we demonstrated a novel treatment strategy with high safety and effective inflammation remission via tissue-adhesive molecular coating. The molecular coating is composed of o-nitrobenzaldehyde (NB)-modified Gelatin (GelNB), which can strongly bond with –NH2 on the intestinal surface of tissue to form a thin biophysical barrier. We found that this molecular coating was able to stay on the surface of the intestine for long periods of time, effectively protecting the damaged intestinal epithelium from irritations of external intestinal metabolites and harmful flora. In addition, our results showed that this coating not only provided a beneficial environment for cell migration and proliferation to promote intestinal repair and regeneration, but also achieved a better outcome of IBD by reducing intestinal inflammation. Moreover, the in vivo experiments showed that the GelNB was better than the classic clinical medication—mesalazine. Therefore, our molecular coating showed potential as a promising strategy for the prevention and treatment of IBD.

Autologous mosaicplasty is a common approach used to treat osteochondral defects in clinical practice. Gap integration between host and transplanted plugs requires bone tissue reservation and hyaline cartilage regeneration without uneven surface, graft necrosis and sclerosis. However, poor gap integration is a serious concern, which eventually leads to deterioration of joint function. To deal with such complications, this study has developed a strategy to effectively enhance integration of the gap region following mosaicplasty by applying injectable bioactive supramolecular nanofiber-enabled gelatin methacryloyl (GelMA) hydrogel (BSN-GelMA). A rabbit osteochondral defect model demonstrated that BSN-GelMA achieved seamless osteochondral healing in the gap region between plugs of osteochondral defects following mosaicplasty, as early as six weeks. Moreover, the International Cartilage Repair Society score, histology score, glycosaminoglycan content, subchondral bone volume, and collagen II expression were observed to be the highest in the gap region of BSN-GelMA treated group. This improved outcome was due to bio-interactive materials, which acted as tissue fillers to bridge the gap, prevent cartilage degeneration, and promote graft survival and migration of bone marrow mesenchymal stem cells by releasing bioactive supramolecular nanofibers from the GelMA hydrogel. This study provides a powerful and applicable approach to improve gap integration after autologous mosaicplasty. It is also a promising off-the-shelf bioactive material for cell-free in situ tissue regeneration.

Nano-hydroxyapatite (nHAP) has been widely used in bone repair as an osteo-inductive and naturally-occurring material. However, the optimal applied form of nHAP and the underlying mechanisms involved remain unclear. Herein, to investigate into these, a range of corresponding models were designed, including three applied forms of nHAP (Free, Coating and 3D) that belong to two states (Free or fixed). The results indicate that when fixed nHAP was applied in the 3D form, optimal osteogenesis was induced in human bone marrow stem cells (hBMSCs) with increased bone volume via integrin α7 (ITGA7)-mediated upregulation of the PI3K-AKT signaling pathway, while contrary results were observed with free nHAP. Ectopic osteogenesis experiments in mice subcutaneous transplantation model further confirmed the different tendencies of ITGA7 expression and osteogenesis of hBMSCs in free and fixed states of nHAP. Our results revealed that the two states of nHAP play a different regulatory role in cell morphology and osteogenesis through the valve role of ITGA7, providing cues for better application of nanoparticles and a potential new molecular target in bone tissue engineering.

Abstract Cartilage damage affects millions of people worldwide. Tissue engineering strategies hold the promise to provide off‐the‐shelf cartilage analogs for tissue transplantation in cartilage repair. However, current strategies hardly generate sufficient grafts, as tissues cannot maintain size growth and cartilaginous phenotypes simultaneously. Herein, a step‐wise strategy is developed for fabricating expandable human macromass cartilage (macro‐cartilage) in a 3D condition by employing human polydactyly chondrocytes and a screen‐defined serum‐free customized culture (CC). CC‐induced chondrocytes demonstrate improved cell plasticity, expressing chondrogenic biomarkers after a 14.59‐times expansion. Crucially, CC‐chondrocytes form large‐size cartilage tissues with average diameters of 3.25 ± 0.05 mm, exhibiting abundant homogenous matrix and intact structure without a necrotic core. Compared with typical culture, the cell yield in CC increases 2.57 times, and the expression of cartilage marker collagen type II increases 4.70 times. Transcriptomics reveal that this step‐wise culture drives a proliferation‐to‐differentiation process through an intermediate plastic stage, and CC‐chondrocytes undergo a chondral lineage‐specific differentiation with an activated metabolism. Animal studies show that CC macro‐cartilage maintains a hyaline‐like cartilage phenotype in vivo and significantly promotes the healing of large cartilage defects. Overall, an efficient expansion of human macro‐cartilage with superior regenerative plasticity is achieved, providing a promising strategy for joint regeneration.

Critical-sized bone defects still present clinical challenges. Here the authors show that transplantation of neurotrophic supplement-incorporated hydrogel grafts promote full-thickness regeneration of the calvarium and perform scRNA-seq to reveal contributing stem/progenitor cells, notably a resident Msx1+ skeletal stem cell population.

Summary: The global prevalence and burden of musculoskeletal (MSK) disorders are immense. Advancements in next-generation sequencing (NGS) have generated vast amounts of data, accelerating the research of pathological mechanisms and the development of therapeutic approaches for MSK disorders. However, scattered datasets across various repositories complicate uniform analysis and comparison. Here, we introduce MSdb, a database for visualization and integrated analysis of next-generation sequencing data from human musculoskeletal system, along with manually curated patient phenotype data. MSdb provides various types of analysis, including sample-level browsing of metadata information, gene/miRNA expression, and single-cell RNA-seq dataset. In addition, MSdb also allows integrated analysis for cross-samples and cross-omics analysis, including customized differentially expressed gene/microRNA analysis, microRNA-gene network, scRNA-seq cross-sample/disease integration, and gene regulatory network analysis. Overall, systematic categorizing, standardized processing, and freely accessible knowledge features MSdb a valuable resource for MSK research community.

Abstract As a critical node for insulin/IGF signaling, insulin receptor substrate 1 (IRS-1) is essential for metabolic regulation. A long and unstructured C-terminal region of IRS-1 recruits downstream effectors for promoting insulin/IGF signals. However, the underlying molecular basis for this remains elusive. Here, we found that the C-terminus of IRS-1 undergoes liquid-liquid phase separation (LLPS). Both electrostatic and hydrophobic interactions were seen to drive IRS-1 LLPS. Self-association of IRS-1, which was mainly mediated by the 301–600 region, drives IRS-1 LLPS to form insulin/IGF-1 signalosomes. Moreover, tyrosine residues of YXXM motifs, which recruit downstream effectors, also contributed to IRS-1 self-association and LLPS. Impairment of IRS-1 LLPS attenuated its positive effects on insulin/IGF-1 signaling. The metabolic disease-associated G972R mutation impaired the self-association and LLPS of IRS-1. Our findings delineate a mechanism in which LLPS of IRS-1-mediated signalosomes serves as an organizing center for insulin/IGF-1 signaling and implicate the role of aberrant IRS-1 LLPS in metabolic diseases.

Abstract Articular cartilage damage is a universal health problem. Despite recent progress, chondrocyte dedifferentiation has severely compromised the clinical outcomes of cell-based cartilage regeneration. Loss-of-function changes are frequently observed in chondrocyte expansion and other pathological conditions, but the characteristics and intermediate molecular mechanisms remain unclear. In this study, we demonstrate a time-lapse atlas of chondrocyte dedifferentiation to provide molecular details and informative biomarkers associated with clinical chondrocyte evaluation. We performed various assays, such as single-cell RNA sequencing (scRNA-seq), live-cell metabolic assays, and assays for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), to develop a biphasic dedifferentiation model consisting of early and late dedifferentiation stages. Early-stage chondrocytes exhibited a glycolytic phenotype with increased expression of genes involved in metabolism and antioxidation, whereas late-stage chondrocytes exhibited ultrastructural changes involving mitochondrial damage and stress-associated chromatin remodeling. Using the chemical inhibitor BTB06584, we revealed that early and late dedifferentiated chondrocytes possessed distinct recovery potentials from functional phenotype loss. Notably, this two-stage transition was also validated in human chondrocytes. An image-based approach was established for clinical use to efficiently predict chondrocyte plasticity using stage-specific biomarkers. Overall, this study lays a foundation to improve the quality of chondrocytes in clinical use and provides deep insights into chondrocyte dedifferentiation.

Abstract Tendon heterotopic ossification (HO) is characterized by bone formation inside tendon tissue, which severely debilitates people in their daily life. Current therapies fail to promote functional tissue repair largely due to our limited understanding of HO pathogenesis. Here, we investigate the pathological mechanism and propose a potential treatment method for HO. Immunofluorescence assays showed that the Mohawk (MKX) expression level was decreased in human tendon HO tissue, coinciding with spontaneous HO and the upregulated expression of osteochondrogenic and angiogenic genes in the tendons of Mkx −/− mice. Single-cell RNA sequencing analyses of wild-type and Mkx −/− tendons identified three cell types and revealed the excessive activation of osteochondrogenic genes during the tenogenesis of Mkx −/− tendon cells. Single-cell analysis revealed that the gene expression program of angiogenesis, which is strongly associated with bone formation, was activated in all cell types during HO. Moreover, inhibition of angiogenesis by the small-molecule inhibitor BIBF1120 attenuated bone formation and angiogenesis in the Achilles tendons of both Mkx mutant mice and a rat traumatic model of HO. These findings provide new insights into the cellular mechanisms of tendon HO and highlight the inhibition of angiogenesis with BIBF1120 as a potential treatment strategy for HO.

Abstract Background Osteoarthritis (OA), a prevalent degenerative disease characterized by degradation of extracellular matrix (ECM), still lacks effective disease-modifying therapy. Mesenchymal stem cells (MSCs) transplantation has been regarded as the most promising approach for OA treatment while engrafting cells alone might not be adequate for effective regeneration. Genetic modification has been used to optimize MSC-based therapy; however, there are still significant limitations that prevent the clinical translation of this therapy including low efficacy and safety concerns. Recently, chemically modified mRNA (modRNA) represents a promising alternative for the gene-enhanced MSC therapy. In this regard, we hypothesized that adipose derived stem cells (ADSCs) engineered with modRNA encoding insulin-like growth factor 1 (IGF-1) were superior to native ADSCs on ameliorating OA development. Methods Mouse ADSCs were acquired from adipose tissue and transfected with modRNAs. First, the kinetics and efficacy of modRNA-mediated gene transfer in mouse ADSCs were analyzed in vitro. Next, we applied an indirect co-culture system to analyze the pro-anabolic potential of IGF-1 modRNA engineered ADSCs (named as IGF-1-ADSCs) on chondrocytes. Finally, we evaluated the cell retention and chondroprotective effect of IGF-1-ADSCs in vivo using fluorescent labeling, histology and immunohistochemistry. Results modRNA transfected mouse ADSCs with high efficiency (85 ± 5%) and the IGF-1 modRNA-transfected ADSCs facilitated burst-like production of bio-functional IGF-1 protein. In vitro, IGF-1-ADSCs induced increased anabolic markers expression of chondrocytes in inflammation environment compared to untreated ADSCs. In a murine OA model, histological and immunohistochemical analysis of knee joints harvested at 4 weeks and 8 weeks after OA induction suggested IGF-1-ADSCs had superior therapeutic effect over native ADSCs demonstrated by lower histological OARSI score and decreased loss of cartilage ECM. Conclusions These findings collectively supported the therapeutic potential of IGF-1-ADSCs for clinical OA management and cartilage repair.

Although aging is an increasingly severe healthy, economic, and social global problem, it is far from well-modeling aging due to the aging process’s complexity. To promote the aging modeling, here we did the quantitative measurement based on aging blood transcriptome. Specifically, the aging blood transcriptome landscape was constructed through ensemble modeling in a cohort of 505 people, and 1138 age-related genes were identified. To assess the aging rate in the linear dimension of aging, we constructed a simplified linear aging clock, which distinguished fast-aging and slow-aging populations and showed the differences in the composition of immune cells. Meanwhile, the non-linear dimension of aging revealed the transcriptome fluctuations with a crest around the age of 40 and showed that this crest came earlier and was more vigorous in the fast-aging population. Moreover, the aging clock was applied to evaluate the rejuvenation effect of molecules in vitro, such as Nicotinamide Mononucleotide (NMN) and Metformin. In sum, this study developed a de novo aging clock to evaluate age-dependent precise medicine by revealing its fluctuation nature based on comprehensively mining the aging blood transcriptome, promoting the development of personal aging monitoring and anti-aging therapies.

Corneal injuries will cause corneal surface diseases that may lead to blindness in millions of people worldwide. There is a tremendous need for biomaterials that can promote corneal regeneration with practical feasibility. Here we demonstrate a strategy of a protein coating for corneal injury regeneration. We synthesize an o-nitrosobenzaldehyde group (NB)-modified gelatin (GelNB), which could adhere directly to the corneal surface with covalent bonding to form a thin molecular coating. The molecular coating could avoid rapid clearance and provide a favorable environment for cell migration, thereby effectively accelerating corneal repair and regeneration. The histological structure of the regenerated cornea is more similar to the native cornea. This molecular coating can be used conveniently as an eye drop solution, which makes it a promising strategy for corneal regeneration.

Heterotopic ossification (HO) is the formation of pathological mature bone within extraskeletal soft tissues, and there are currently no reliable methods for removing these calcified plaques. Here, the authors demonstrate that chemically engineered osteoclasts coated with tetracycline can improve their targeting capacity to ectopic calcifications, which extends their bone resorption functions for the treatment of HO.

Abstract Background During the intervertebral disc (IVD) degeneration process, initial degenerative events occur at the extracellular matrix level, with the appearance of neoepitope peptides formed by the cleavage of aggrecan and collagen. This study aims to elucidate the spatial and temporal alterations of aggrecan and collagen neoepitope level during IVD degeneration. Methods Bovine caudal IVDs were cultured under four different conditions to mimic different degenerative situations. Samples cultured after 1‐ or 8‐days were collected for analysis. Human IVD samples were obtained from patients diagnosed with lumbar disc herniation (LDH) or adolescent idiopathic scoliosis (AIS). After immunohistochemical (IHC) staining of Aggrecanase Cleaved C‐terminus Aggrecan Neoepitope (NB100), MMP Cleaved C‐terminus Aggrecan Neoepitope (MMPCC), Collagen Type 1α1 1/4 fragment (C1α1) and Collagenase Cleaved Type I and II Collagen Neoepitope (C1,2C), staining optical density (OD)/area in extracellular matrix (OECM) and pericellular zone (OPCZ) were analyzed. Conditioned media of the bovine IVD was collected to measure protein level of inflammatory cytokines and C1,2C. Results For the bovine IVD sections, the aggrecan MMPCC neoepitope was accumulated in nucleus pulposus (NP) and cartilage endplate (EP) regions following mechanical overload in the one strike model after long‐term culture; as for the TNF‐α induced degeneration, the OECM and OPCZ of collagen C1,2C neoepitope was significantly increased in the outer AF region after long‐term culture; moreover, the C1,2C was only detected in conditioned medium from TNF‐α injection + Degenerative loading group after 8 days of culture. LDH patients showed higher MMPCC OECM in NP and higher C1,2C OECM in AF region compared with AIS patients. Conclusions In summary, aggrecan and collagen neoepitope profiles showed degeneration induction trigger‐ and region‐specific differences in the IVD organ culture models. Different IVD degeneration types are correlated with specific neoepitope expression profiles. These neoepitopes may be helpful as biomarkers of ECM degradation in early IVD degeneration and indicators of different degeneration phenotypes.

Abstract Chondrocyte differentiation is a critical process for endochondral ossification, which is responsible for long bone development and fracture repair. Considerable progress has been made in understanding the transcriptional control of chondrocyte differentiation; however, epigenetic regulation of chondrocyte differentiation remains to be further studied. NSD1 is a H3K36 (histone H3 at lysine 36) methyltransferase. Here, we showed that mice with Nsd1 deficiency in Prx1+ mesenchymal progenitors but not in Col2+ chondrocytes showed impaired skeletal growth and fracture healing accompanied by decreased chondrogenic differentiation. Via combined RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we identified sex determining region Y box 9 (Sox9), the key transcription factor of chondrogenic differentiation, as a functional target gene of NSD1. Mechanistically, NSD1 regulates Sox9 expression by modulating H3K36me1 and H3K36me2 levels in the Sox9 promoter region, constituting a novel epigenetic regulatory mechanism of chondrogenesis. Moreover, we found that NSD1 can directly activate the expression of hypoxia-inducible factor 1α (HIF1α), which plays a vital role in chondrogenic differentiation through its regulation of Sox9 expression. Collectively, the results of our study reveal crucial roles of NSD1 in regulating chondrogenic differentiation, skeletal growth, and fracture repair and expand our understanding of the function of epigenetic regulation in chondrogenesis and skeletal biology.

Cartilage defects are one of the most common symptoms of osteoarthritis (OA), a degenerative disease that affects millions of people world-wide and places a significant socio-economic burden on society. Hydrogels, which are a class of biomaterials that are elastic, and display smooth surfaces while exhibiting high water content, are promising candidates for cartilage regeneration. In recent years, various kinds of hydrogels have been developed and applied for the repair of cartilage defects in vitro or in vivo, some of which are hopeful to enter clinical trials. In this review, recent research findings and developments of hydrogels for cartilage defects repair are summarized. We discuss the principle of cartilage regeneration, and outline the requirements that have to be fulfilled for the deployment of hydrogels for medical applications. We also highlight the development of advanced hydrogels with tailored properties for different kinds of cartilage defects to meet the requirements of cartilage tissue engineering and precision medicine.

Abstract Adhesive patches are advanced but challenging alternatives to suture, especially in treating fragile internal organs. So far there is no suture‐free adhesive patch based on metabolizable poly(amino acid) materials with excellent mechanical strength as well as immunomodulation functionality. Here, a polyglutamic acid‐based elastic and tough adhesive patch modified by photosensitive groups on the surface to achieve robust light‐activated adhesion and sealing of flexible internal organs is explored. With the porous internal morphology and excellent biodegradability, the patches promote regeneration through a macrophage‐regulating microenvironment. Treated rabbits achieve rapid full‐thickness gastric regeneration with complete functional structure within 14 d, suggesting its robust tissue adhesion and repair‐promoting ability.

Abstract Nonuniform microstretching (NUMS) naturally occurs in real bone tissues in vivo, but its profound effects have not been identified yet. In order to explore the biological effects of NUMS and static stretch (uniform stretch [US]) on cells, a new “musical dish” device was developed. Musical signal was used to provide NUMS to cells. More stress fibers, arranging along the long axis of cells, were formed throughout the cells under NUMS, compared with US and untreated control group, although cell morphology did not show any alteration. Whole transcriptome sequencing revealed enhanced osteogenic differentiation of cells after NUMS treatment. Cells in the NUMS group showed a higher expression of bone‐related genes, while genes related to stemness and other lineages were down‐regulated. Our results give insights into the biological effects of NUMS and US on stem cell osteogenic differentiation, suggesting beneficial effects of micromechanical stimulus for osteogenesis. The newly developed device provides a basis for the development of NUMS derived rehabilitation technology to promote bone healing.

Objective: The magnitude of the therapeutic effects of intra-articular injection of platelet-rich plasma (PRP) on osteoarthritis (OA) is still under debate. The goal of this study that was a systematic review of randomised controlled trials ​of PRP injections for the treatment of OA was to elucidate the therapeutic efficacy of PRP. Methods: Electronic databases of PubMed, CENTRAL, EMBASE, EBSCO, ClinicalTrials.gov, and International Clinical Trials Registry Platform ​were searched from inception to June 2018 for RCTs that compared PRP injections to controls in patients with OA. A random-effects approach was used to compile data and subgroups according to trial size (large trials versus small trials), patient profile (age and gender), and PRP preparation method was performed. Results: Thirty trials met the inclusion criteria and were analysed. All results had unexplained statistical heterogeneity. Patients treated with PRP compared with control showed statistically relevant pain relief and function improvement at short term (standardised mean difference [SMD] ​= ​−0.62, 95% confidence interval [CI]: −0.98 to −0.27, P ​= ​0.0006, SMD ​= ​−0.74, 95% CI: −1.11 to 0.36, P ​= ​0.0001, respectively), medium term (SMD ​= ​−0.53, 95% CI: −0.83 to −0.23, P ​= ​0.0006, SMD ​= ​−0.50, 95% CI: −0.75 to −0.25, P ​= ​0.0006), and long term (SMD ​= ​−0.69, 95% CI: −1.08 to −0.30, P ​= ​0.0006, SMD ​= ​−0.68, 95% CI: −0.1.09 to −0.27, P ​= ​0.001, respectively). A subgroup analysis of the data from large trials and from trials composed of less than 50% female patients revealed that therapeutic effects of the treatment are insignificant. Conclusions: According to the currently available data, PRP injections are beneficial for pain relief and function improvement in patients with OA. This meta-analysis, however, demonstrated that the efficacy of PRP is related to sample size and gender composition. Thus, more randomised controlled trials of high quality and larger patient size, also including gender aspects, are required to understand this phenomenon. The translational potential of this article: The translation potential of this meta-analysis is that provided another perspective to analyse the treatment effect of PRP for OA. In future research, phenotypes subpopulation and gender difference of OA patient should be considered for PRP treatment.

Summary: Articular cartilage injury and degeneration causing pain and loss of quality-of-life has become a serious problem for increasingly aged populations. Given the poor self-renewal of adult human chondrocytes, alternative functional cell sources are needed. Direct reprogramming by small molecules potentially offers an oncogene-free and cost-effective approach to generate chondrocytes, but has yet to be investigated. Here, we directly reprogrammed mouse embryonic fibroblasts into PRG4+ chondrocytes using a 3D system with a chemical cocktail, VCRTc (valproic acid, CHIR98014, Repsox, TTNPB, and celecoxib). Using single-cell transcriptomics, we revealed the inhibition of fibroblast features and activation of chondrogenesis pathways in early reprograming, and the intermediate cellular process resembling cartilage development. The in vivo implantation of chemical-induced chondrocytes at defective articular surfaces promoted defect healing and rescued 63.4% of mechanical function loss. Our approach directly converts fibroblasts into functional cartilaginous cells, and also provides insights into potential pharmacological strategies for future cartilage regeneration. : In this article, Ouyang and colleagues develop a chemical-driven direct reprogramming of mouse embryonic fibroblasts into PRG4+ chondrocytes, with a screen-defined cocktail VCRTc. Their induced chondrocytes promote in vivo cartilage functional regeneration. The dynamical cellular phenotypes were analyzed by single-cell RNA sequencing, illustrating the inhibition of fibroblast features and activation of chondrogenesis pathways in early reprogramming. Keywords: chemical reprogramming, single-cell analysis, cartilage regeneration, drug discovery

The myotendinous junction (MTJ) is a complex and special anatomical area that connects muscles and tendons, and it is also the key to repairing tendons. Nevertheless, the anatomical structure and connection structure of MTJ, the cluster and distribution of cells, and which cells are involved in repairing the tissue are still unclear. Here, we analyzed the cell subtype distribution and function of human MTJ at single-cell level. We identified four main subtypes, including stem cell, muscle, tendon, and muscle-tendon progenitor cells (MTP). The MTP subpopulation, which remains the characteristics of stem cells and also expresses muscle and tendon marker genes simultaneously, may have the potential for bidirectional differentiation. We also found the muscle-tendon progenitor cells were distributed in the shape of a transparent goblet; muscle cells first connect to the MTP and then to the tendon. And after being transplanted in the MTJ injury model, MTP exhibited strong regenerative capability. Finally, we also demonstrated the importance of mTOR signaling for MTP maintenance by in vitro addition of rapamycin and in vivo validation using mTOR-ko mice. Our research conducted a comprehensive analysis of the heterogeneity of myotendinous junction, discovered a special cluster called MTP, provided new insights into the biological significance of myotendinous junction, and laid the foundation for future research on myotendinous junction regeneration and restoration.

Abstract Stem cell therapies are unsatisfactory due to poor cell survival and engraftment. Stem cell used for therapy must be properly “tuned” for a harsh in vivo environment. Herein, we report that transfer of exogenous mitochondria (mito) to adipose‐derived mesenchymal stem cells (ADSCs) can effectively boost their energy levels, enabling efficient cell engraftment. Importantly, the entire process of exogeneous mitochondrial endocytosis is captured by high‐content live‐cell imaging. Mitochondrial transfer leads to acutely enhanced bioenergetics, with nearly 17% of higher adenosine 5′‐triphosphate (ATP) levels in ADSCs treated with high mitochondrial dosage and further results in altered secretome profiles of ADSCs. Mitochondrial transfer also induced the expression of 334 mRNAs in ADSCs, which are mainly linked to signaling pathways associated with DNA replication and cell division. We hypothesize that increase in ATP and cyclin‐dependent kinase 1 and 2 expression might be responsible for promoting enhanced proliferation, migration, and differentiation of ADSCs in vitro. More importantly, mito‐transferred ADSCs display prolonged cell survival, engraftment and horizontal transfer of exogenous mitochondria to surrounding cells in a full‐thickness skin defect rat model with improved skin repair compared with nontreated ADSCs. These results demonstrate that intracellular mitochondrial transplantation is a promising strategy to engineer stem cells for tissue regeneration.

Abstract Meniscus‐derived stem cells (MeSCs) are a potential cell source for meniscus tissue engineering. The stark morphological and structural changes of meniscus tissue during development indicate the complexity of MeSCs at different tissue regions and stages of development. In this study, we characterized and compared postnatal rat meniscus tissue and MeSCs at different tissue regions and stages of development. We observed that the rat meniscus tissue exhibited marked changes in tissue morphology during development, with day 7 being the most representative time point of different developmental stages. All rat MeSCs displayed typical stem cell characteristics. Rat MeSCs derived from day 7 inner meniscus tissue exhibited the highest self‐renewal capacity, cell proliferation, differentiation potential toward various mesenchymal lineage and the highest expression levels of chondrogenic genes and proteins. Transplantation of rat MeSCs derived from day 7 inner meniscus tissue promoted neo‐tissue formation and effectively protected joint surface cartilage in vivo. Our results demonstrated for the first time that rat MeSCs are not necessarily better at earlier developmental stages, and that rat MeSCs derived from day 7 inner meniscus tissue may be a superior cell source for effective meniscus regeneration and articular cartilage protection. This information could make a significant contribution to human meniscus tissue engineering in the future. Stem Cells Translational Medicine 2019;8:1318&1329

Uncontrollable bleeding is a major problem in surgery and after trauma. Here the authors design a photo-reactive adhesive that mimics the composition of connective tissue and is able to stop high pressure bleeding within half a minute.

Summary: A low surface expression level of human leukocyte antigen class I (HLA-I) ensures that the mesenchymal stem cells (MSCs) escape from the allogeneic recipients' immunological surveillance. Here, we discovered that both transcriptional and synthesis levels of HLA-I in MSCs increased continuously after interferon (IFN)-γ treatment, whereas interestingly, their surface HLA-I expression was downregulated after reaching an HLA-I surface expression peak. Microarray data indicated that the post-transcriptional process plays an important role in the downregulation of surface HLA-I. Further studies identified that IFN-γ-treated MSCs accelerated HLA-I endocytosis through a clathrin-independent dynamin-dependent endocytosis pathway. Furthermore, cells that have self-downregulated surface HLA-I expression elicit a weaker immune response than they previously could. Thus uncovering the plasticity of MSCs in the regulation of HLA-I surface expression would reveal insights into the membrane transportation events leading to the maintenance of low surface HLA-I expression, providing more evidence for selecting and optimizing low-immunogenic MSCs to improve the therapeutic efficiency. : Biological Sciences; Immunology; Cell Biology; Stem Cells Research Subject Areas: Biological Sciences, Immunology, Cell Biology, Stem Cells Research

Bearing compression from adjacent joints, the articular cartilage is cumulatively pressured in daily life, thus making it prone to injuries; however, once damaged, the self-healing capacity of articular cartilage is limited owing to its low metabolic property. Autologous chondrocyte implantation, a three-step repairing technique for articular lesions, has received satisfactory short-term clinical outcomes, whereas its long-term effect remains controversial. Currently, improved stem-cell therapies and novel biomaterials have shed new lights on autologous chondrocyte implantation. We would, therefore, synthesize these optimization strategies in order of their presences in the three-step protocol, seeking to find and amplify synergic effects between these strategies. The translational potential of this article: Autologous chondrocytes implantation serves as an alternative for the treatment of articular cartilage lesions to avoid potentially detrimental effects of applying microfracture. The optimized ACI should improve the cost-effectiveness of repairing articular cartilage while circumventing latent complications like osteophyte. This article synthesized optimization strategies for ACI and provided appropriate applying approaches to maximize their synergic effects. It will be a pioneering trial for combinedly using stem cells and nanotechnology to regenerate cartilage. Keywords: Autologous chondrocyte implantation, Scaffold, Stem cell, Nanotechnology

Tendon injury commonly occurs during sports activity, which may cause interruption or rapid decline in athletic career. Tensile strength, as one aspect of tendon biomechanical properties, is the main parameter of tendon function. Tendon injury will induce an immune response and cause the loss of tensile strength. Regulation of mechanical forces during tendon healing also changes immune response to improve regeneration. Here, the effects of internal/external forces and immune response on tendon regeneration are reviewed. The interaction between immune response and internal/external forces during tendon regeneration is critically examined and compared, in relation to other tissues. In conclusion, it is essential to maintain a fine balance between internal/external forces and immune response, to optimize tendon functional regeneration.

Osteoarthritis (OA) is a leading cause of physical disability among aging populations, with no available drugs able to efficiently restore the balance between cartilage matrix synthesis and degradation. Also, OA has not been accurately classified into subpopulations, hindering the development toward personalized precision medicine.In the present study, we identified a subpopulation of OA patients displaying high activation level of epidermal growth factor receptor (EGFR). With Col2a1-creERT2; Egfrf/f mice, it was found that the activation of EGFR, indicated by EGFR phosphorylation (pEGFR), led to the destruction of joints. Excitingly, EGFR inhibition prohibited cartilage matrix degeneration and promoted cartilage regeneration. The Food and Drug Administration (FDA)-approved drug gefitinib could efficiently inhibit EGFR functions in OA joints and restore cartilage structure and function in the mouse model as well as the clinical case report.Overall, our findings suggested the concept of the EGFR activated OA subpopulation and illustrated the mechanism of EGFR signaling in regulating cartilage homeostasis. Gefitinib could be a promising disease-modifying drug for this OA subpopulation treatment. Keywords: Osteoarthritis, Disease subpopulation, Epidermal growth factor receptor, Gefitinib

Abstract Background Mesenchymal stem cell therapy for osteoarthritis (OA) has been widely investigated, but the mechanisms are still unclear. Exosomes that serve as carriers of genetic information have been implicated in many diseases and are known to participate in many physiological processes. Here, we investigate the therapeutic potential of exosomes from human embryonic stem cell-induced mesenchymal stem cells (ESC-MSCs) in alleviating osteoarthritis (OA). Methods Exosomes were harvested from conditioned culture media of ESC-MSCs by a sequential centrifugation process. Primary mouse chondrocytes treated with interleukin 1 beta (IL-1β) were used as an in vitro model to evaluate the effects of the conditioned medium with or without exosomes and titrated doses of isolated exosomes for 48 hours, prior to immunocytochemistry or western blot analysis. Destabilization of the medial meniscus (DMM) surgery was performed on the knee joints of C57BL/6 J mice as an OA model. This was followed by intra-articular injection of either ESC-MSCs or their exosomes. Cartilage destruction and matrix degradation were evaluated with histological staining and OARSI scores at the post-surgery 8 weeks. Results We found that intra-articular injection of ESC-MSCs alleviated cartilage destruction and matrix degradation in the DMM model. Further in vitro studies illustrated that this effect was exerted through ESC-MSC-derived exosomes. These exosomes maintained the chondrocyte phenotype by increasing collagen type II synthesis and decreasing ADAMTS5 expression in the presence of IL-1β. Immunocytochemistry revealed colocalization of the exosomes and collagen type II-positive chondrocytes. Subsequent intra-articular injection of exosomes derived from ESC-MSCs successfully impeded cartilage destruction in the DMM model. Conclusions The exosomes from ESC-MSCs exert a beneficial therapeutic effect on OA by balancing the synthesis and degradation of chondrocyte extracellular matrix (ECM), which in turn provides a new target for OA drug and drug-delivery system development.

The endometrial layer comprises luminal and glandular epithelia that both develop from the same simple layer of fetal uterine epithelium. Mechanisms of uterine epithelial progenitor self-renewal and differentiation are unclear. This study aims to systematically analyze the molecular and cellular mechanisms of uterine epithelial development by single-cell analysis. An integrated set of single-cell transcriptomic data of uterine epithelial progenitors and their differentiated progenies is provided. Additionally the unique molecular signatures of these cells, characterized by sequential upregulation of specific epigenetic and metabolic activities, and activation of unique signaling pathways and transcription factors, were also investigated. Finally a unique subpopulation of early progenitor, as well as differentiated luminal and glandular lineages, were identified. A complex cellular hierarchy of uterine epithelial development was thus delineated. Our study therefore systematically decoded molecular markers and a cellular program of uterine epithelial development that sheds light on uterine developmental biology.

Abstract One of the key problems hindering skin repair is the deficiency of dermal vascularization and difficulty of epidermis regeneration, which makes it challenging to fabricate scaffolds that can biologically fulfill the requirements for skin regeneration. To overcome this problem, three-dimensional printing was used to fabricate a gelatin-sulfonated silk composite scaffold that was incorporated with basic fibroblast growth factor 2 (FGF-2) through binding with a sulfonic acid group (SO3) (3DG-SF-SO3-FGF). The efficacy and mechanism by which the 3DG-SF-SO3-FGF scaffolds promote skin regeneration were investigated both within in vitro cell culture and in vivo with a full-thickness skin defect model. The histological results showed that the gelatin-sulfonated silk composite scaffolds promoted granulation, and that incorporation of FGF-2 significantly enhanced the regeneration of skin-like tissues after implantation in rat skin defects for 14 and 28 days. Further investigations demonstrated that 3DG-SF-SO3-FGF scaffolds might stimulate dermal vascularization. These findings thus suggest that incorporation of FGF-2 into the 3D printed scaffolds is a viable strategy for enhancing skin regeneration.

Tendon injuries are common and present a clinical challenge, as they often respond poorly to treatment and result in long‐term functional impairment. Inferior tendon healing responses are mainly attributed to insufficient or failed tenogenesis. The main objective of this study was to establish an efficient approach to induce tenogenesis of bone marrow‐derived mesenchymal stem cells (BMSCs), which are the most common seed cells in tendon tissue engineering. First, representative reported tenogenic growth factors were used as media supplementation to induce BMSC differentiation, and the expression of teno‐lineage transcription factors and matrix proteins was compared. We found that transforming growth factor (TGF)‐β1 significantly induced teno‐lineage‐specific gene scleraxis expression and collagen production. TGF‐β1 combined with connective tissue growth factor (CTGF) elevated tenomodulin and Egr1 expression at day 7. Hence, a stepwise tenogenic differentiation approach was established by first using TGF‐β1 stimulation, followed by combination with CTGF for another 7 days. Gene expression analysis showed that this stepwise protocol initiated and maintained highly efficient tenogenesis of BMSCs. Finally, regarding in situ rat patellar tendon repair, tendons treated with induced tenogenic BMSCs had better structural and mechanical properties than those of the control group, as evidenced by histological scoring, collagen I and tenomodulin immunohistochemical staining, and tendon mechanical testing. Collectively, these findings demonstrate a reliable and practical strategy of inducing tenogenesis of BMSCs for tendon regeneration and may enhance the effectiveness of cell therapy in treating tendon disorders. Significance The present study investigated the efficiency of representative tenogenic factors on mesenchymal stem cells' tenogenic differentiation and established an optimized stepwise tenogenic differentiation approach to commit tendon lineage differentiation for functional tissue regeneration. The reliable tenogenic differentiation approach for stem cells not only serves as a platform for further studies of underlying molecular mechanisms but also can be used to enhance cell therapy outcome in treating tendon disorders and develop novel therapeutics for tendon injury.