The study of functional heterogeneity in mesenchymal stem cells relies on the efficient isolation of specific subpopulations (e.g., podoplanin (PDPN)-positive cells). However, for most membrane protein targets, directly available commercial magnetic beads for sorting are limited. To address this, our laboratory has established and tested a standardized magnetic bead sorting protocol based on the indirect coupling principle of "phycoerythrin (PE)-labeled flow cytometry antibodies/anti-PE magnetic beads." Using PDPN-positive cells as a model target population, this protocol enriches a target cell fraction without relying on expensive flow cytometric cell sorters. Researchers can use compatible PE-labeled flow cytometry antibodies targeting accessible cell-surface proteins and combine them with commercially available anti-PE microbeads to establish a sorting workflow for selected targets after target-specific optimization. This approach avoids the lengthy lead times and high costs associated with customizing or ordering target-specific magnetic beads for each new target, making it particularly suitable for accessible surface markers that are rare or understudied. In the PDPN model system tested here, the protocol enabled enrichment of the target subpopulation with high post-sort PDPN positivity and preserved CCK-8-based proliferation/metabolic activity compared with unsorted MSCs.

Ossification of the posterior longitudinal ligament (OPLL) is a heterotopic ossification process of the posterior longitudinal ligament (PLL) that can compress the spinal cord and nerve roots, yet its cellular heterogeneity and pathogenesis remain unclear. We performed single-cell RNA sequencing (scRNA-seq) with integrative computational analyses and histological validation on 4,683 cells from surgically resected cervical OPLL lesions obtained from human patients. We identified 15 major cell subsets, including chondrocyte-lineage populations-progenitor/proliferative fibrocartilage chondrocytes (ProFCs), pre-hypertrophic chondrocytes (preHTCs), and hypertrophic chondrocytes (HTCs)-as well as osteoblasts, endothelial cells, and diverse immune cells. Pseudotime analysis suggested an inferred trajectory from cartilage progenitor-like cells through inflammatory/reactive and hypertrophic states toward osteogenic programs, with progressive activation of ossification, extracellular-matrix, and inflammation-related pathways. Cell–cell communication analyses showed coordinated immune–stromal crosstalk, with FGF signaling enriched in earlier pseudotime states and SPP1-CD44 signaling enriched in later states. SPP1 signals originated predominantly from HTCs, whereas macrophages were prominently involved in the broader inflammatory niche and immune-stromal communication within OPLL lesions. Consistently, SPP1/CD44-positive cells were enriched in OPLL tissues and further increased by IL-1β stimulation in vitro and in an Enpp1-driven OPLL mouse model. This exploratory single-cell atlas suggests that an inflammatory niche may couple chondrocyte hypertrophy with osteogenic activation and highlights candidate immune-stromal pathways for future biomarker and therapeutic exploration in OPLL. Single-cell transcriptomic profiling reveals cellular heterogeneity and the SPP1-CD44 signaling axis in OPLL

Traditional wound dressings have problems such as a lack of bioactive substances, poor shape adaptability and secondary damage, which make it difficult to meet clinical needs. Therefore, this study developed a new type of photocurable hydrogel preparation (Ferulic acid-modified gelatin, GelFA) to promote wound healing. GelFA can be solidified in situ under ultraviolet light to form a hydrogel. Inspired by the compatibility of classical drug pairs, it is loaded with Bletilla striata polysaccharide (BSP) and notoginsenoside R1 (R1), which improves the moisturizing performance of GelFA but also synergistically promotes wound repair. GelFA/BSP-R1 hydrogel has excellent environmental adaptability, can prevent wound infection. At the same time, the hydrogel has anti-inflammatory, antioxidant, hemostatic and other functional properties. The results of cell experiments showed that GelFA/BSP-R1 hydrogel had good biocompatibility and significantly promoted angiogenesis, which laid a solid foundation for tissue repair. In wound models of rats and Bama pigs, it accelerates tissue repair by reducing inflammation, promoting collagen deposition, and accelerating angiogenesis. Inspired by the above research results, based on the new photocuring material GelFA, this study further developed various dosage forms to expand its clinical application. In summary, this study provides a promising new treatment strategy for clinical tissue regeneration.

Osteoarthritis (OA) is one of the most common causes of physical disability among older people and its incidence increases with age. Removal of the senescent cells (SNCs) delays OA pathologies, but little is known about the heterogeneity of SNCs and their roles in OA pathogenesis. Here, we identify a subpopulation of senescent synovial cells and proposed a molecular mechanism governing pathogenic synovium-cartilage crosstalk in OA progression. Using single-cell RNA sequencing and synovial organoids, we demonstrate that RCAN1 + IL1α + senescent synovial fibroblasts, predominantly located in the lining layer of human OA synovium, exhibit proinflammatory phenotype, mitochondrial dysfunction, and promote cartilage degeneration. Mechanistically, RCAN1 stabilizes ATF4 mRNA and mediates saturated fatty acids (SFA) secretion from synovial fibroblasts, which could promote chondrocyte senescence and cartilage matrix degradation. Synovium-targeted delivery of anti-RCAN1 siRNA significantly ameliorated posttraumatic OA development in mice, reducing of SNC accumulation in synovium and increasing cartilage regeneration. Coculture experiments with human OA cartilage explants and synovial organoids confirm that RCAN1 silencing in synovial fibroblasts suppressess chondrocyte senescence and cartilage degradation. Our findings reveal a prodegenerative interaction between RCAN1 + IL1α + senescent synovial fibroblasts and chondrocytes mediated by secreted lipid in OA progression. Targeted RCAN1 knockdown in senescent synovium could be a new treatment strategy for restoring the joint homeostasis.

Background: Macrophages play a critical role in carotid plaque. Understanding the mechanisms of carotid plaque formation based on macrophage heterogeneity could provide valuable insights for clinical intervention. Methods: Single-cell transcriptome and bulk RNA-seq data of carotid plaque were obtained from public databases. Weighted gene correlation network analysis (WGCNA) identified gene modules linked to unstable plaques. Macrophage marker genes were intersected with module genes of WGCNA, followed by using randomForest and LASSO regression to pinpoint key genes. Quantitative real-time PCR (qRT-PCR) and Western blot were used to verify the regulation of key genes at the cellular level. The correlation between the key genes and inflammatory phenotypes was examined by single-sample gene set enrichment analysis (ssGSEA). Results: Single-cell clustering revealed major cellular subpopulations, with elevated macrophage infiltration in carotid plaque. Six key macrophage-associated genes (ADPGK, ATP6V1F, CX3CR1, MYO9B, RNF135, and SLC7A8) were discovered. The qRT-PCR results demonstrated upregulation of ADPGK, ATP6V1F, and RNF135 genes in vascular smooth muscle cells (VSMCs) treated with oxidized low-density lipoprotein (ox-LDL), except for CX3CR1, which was downregulated. Protein expression results showed that expressions of ADPGK, ATP6V1F, RNF135, and SLC7A8 were significantly elevated in the ox-LDL-VSMC group. In addition, most of the immune cells showed significant differences between the unstable arterial plaque group and the control group. Conclusion: This study discovered potential biomarkers that affected carotid plaque progression and macrophage regulation at the single-cell level, and examined their regulatory roles in immune regulation, programed cell death (PCD), and inflammatory factor modulation.

Aims Photothermal therapy shows potential for treating wound infection, but unstable agents and possible tissue damage limit its use. We aimed to develop a strategy that removes Methicillin-resistant Staphylococcus aureus (MRSA) and supports wound healing. Methods After confirming the stable photothermal activity of difluoroboron-curcumin (DF-Cur), we prepared a Mg2+-chelated microgel via microfluidics that co-delivers DF-Cur and glycyrrhizic acid (GA) within liposomes (termed GD Lip@Mg). And then its photothermal performance and in vitro and in vivo antibacterial ability were systematically examined. We evaluated the therapeutic efficacy of GD Lip@Mg in MRSA-infected full-thickness wounds in both rat and pig model, as well as exploring the underlying mechanisms. Results Proteomic analysis revealed that GA inhibits the bacterial stress-response chaperone HSP60, thereby directly sensitizing MRSA to DF-Cur-mediated photothermal killing. GD Lip@Mg plus 450nm laser reduced bacterial counts by >99.9%, increased re-epithelialization, collagen deposition and vessel density, and shifted macrophages from M1 to M2 without thermal damage. Transcriptomic data associated these effects with photothermal-induced up-regulation of nerve growth factor (NGF) and down-regulation of matrix metalloproteinases (MMPs). Conclusions GD Lip@Mg combined with laser decreases MRSA load and promotes wound healing, providing a translatable approach for infected wounds caused by resistant bacteria.

Osteoarthritis (OA) is one of the most common joint degenerative diseases without effective treatment, whose pathology is related to the local accumulation of senescent cells (SnCs). However, existing SnCs-scavenging drugs "senolytics" may lead to the exhaustion of stem and progenitor cells, impairing chondrocyte proliferation and cartilage regeneration. Here, ADAM19, a kind of endopeptidases from the ADAM (a disintegrin and metalloproteinase) family, is identified as a novel target for senescent chondrocyte rejuvenation. ADAM19 is elevated in senescent chondrocytes in both mice and human osteoarthritic joints, as well as in cellular senescence model in vitro. ADAM19 knockdown not only significantly attenuated senescent phenotype of chondrocytes, but also promoted cell proliferation and extracellular matrix synthesis. RNA sequencing revealed ADAM19 may regulate chondrocyte senescence mainly through the PI3K/AKT signal axis. In addition, a senescence-targeting small interfering RNA (siRNA) delivery system is developed for in vivo delivery of therapeutic siRNA. The complex selectively released ADAM19 siRNA in SnCs and performed high silencing effect on target gene. Furthermore, intra-articular (IA) injection of the complex once every two weeks in OA mice effectively reduced SnCs accumulation and promoted hyaline cartilage regeneration. This study provides a promising strategy for the development of regenerative RNA interference therapy.

INTRODUCTION:Heterotopic ossification of the tendon and ligament (HOTL) is a chronic progressive disease that is usually accompanied by thickening and ossification of ligaments and high osteogenic activity of the surrounding ligament tissue. However, the molecular mechanism of maintaining the cellular phenotype of HOTL remains unclear. MATERIALS AND METHODS:We first constructed a model of HOTL, Enpp1flox/flox/EIIa-Cre mice, a novel genetic mouse system. Imaging, histological, and cell-level analyses were performed to investigate the progressive ossification of the posterior longitudinal ligament, Achilles tendons, and degeneration joints caused by Enpp1 deficiency. RESULTS:The results indicate that Enpp1 deficiency led to markedly progressive heterotopic ossification (HO), especially spine, and Achilles tendons, and was associated with progressive degeneration of the knees. The bone mass was decreased in the long bone. Furthermore, fibroblasts from Enpp1flox/flox/EIIa-Cre mice had greater osteogenic differentiation potential following induction by osteogenesis, accompanied by enhanced hedgehog (Hh) signaling. In addition, fibroblast cells show senescence, and aggravation of the senescence phenotype by further osteogenic induction. CONCLUSION:Our study indicated that with increasing age, mutations in Enpp1 promote ectopic ossification of spinal ligaments and endochondral ossification in tendons and further aggravate knee degeneration by upregulating hedgehog signaling.

Cartilage Decellularized ExtraCellular Matrix (dECM) materials have shown promising cartilage regeneration capacity due to their chondrogenic bioactivity. However, the limited retention of ECM components and the reduced integrity of functional ECM molecules during traditional decellularization processes impair the biomimicry of these materials. The current study aims to fabricate biomimetic materials containing decellularized cartilage particles that have an intact molecular structure and native composition as biomaterial inks and hydrogels for cartilage repair. For this, we established a novel two-fraction decellularization strategy for the preparation of reconstituted dECM (rdECM) particles by mixing the two-fraction components, as well as a one-fraction decellularization strategy for the preparation of biomimetic dECM (bdECM) particles. Hyaluronic acid-tyramine (THA) hydrogels containing rdECM or bdECM particles were produced and characterized via rheological test, swelling and stability evaluation, and compression test. The results showed that our novel decellularization strategies preserved intact proteoglycans and collagen at a higher retention rate with adequate DNA removal compared to traditional methods of decellularization. The addition of rdECM or bdECM particles significantly increased the shear moduli of the THA bioinks while preserving their shear-thinning properties. bdECM particle-embedded THA hydrogels also achieved long-term stability with a swelling ratio of 70% and high retention of glycosaminoglycans and collagen after long-term incubation, while rdECM particle-embedded THA hydrogels showed unsatisfactory stability as self-standing biomaterials. Compared to pure THA hydrogels, the addition of bdECM particles significantly enhanced the compression moduli. In summary, our decellularization methods are successful in the retention of functional and intact cartilage components with high yield. Both rdECM and bdECM particles can be supplemented in THA bioinks for biomimetic cartilage 3D printing. Hydrogels with cartilage bdECM particles possess the functional structure and the natural composition of cartilage ECM, long-term stability, and enhanced mechanical properties, and are promising biomaterials for cartilage repair.

Lower limb microcirculatory ischemic disease is a vascular disorder primarily characterized by limb pain, gangrene, and potential amputation. It can be caused by various factors, such as hyperglycemia, atherosclerosis, and infection. Due to the extremely narrow luminal diameter in lower limb microcirculatory ischemic lesions, both surgical and medical interventions face challenges in achieving satisfactory therapeutic outcomes within the microvessels. Extracellular vesicles derived from mesenchymal stem cells (MSCs-EVs) exhibit promising potential in the treatment of microcirculation ischemic lesions due to their small size and ability to promote angiogenesis. After undergoing substantial losses during the process of EVs transportation, only a minimal fraction of EVs can effectively reach the site of microcirculatory lesions, thereby compromising the therapeutic efficacy for microcirculatory disorders. Herein, an ultrasound-responsive system utilizing 2-(dimethylamino)ethyl methacrylate-b-2-tetrahydropyranyl methacrylate (DMAEMA-b-THPMA) micelles to encapsulate MSCs-EVs has been successfully constructed, with the aim of achieving localized and targeted release of EVs at the site of microcirculatory lesions. The reversible addition-fragmentation chain transfer (RAFT) polymerization method facilitates the successful synthesis of diblock copolymers comprising monomer 2-(dimethylamino)ethyl methacrylate (DMAEMA) and monomer 2-tetrahydropyranyl methacrylate (THPMA). The DMAEMA-b-THPMA micelles exhibit a nanoscale structure, reliable biocompatibility, ultrasound responsiveness, and conspicuous protection of EVs. Furthermore, the implementation of low-energy-density ultrasound can enhance angiogenesis by upregulating the levels of the vascular endothelial growth factor (VEGF). In in vivo experiments, the ultrasound-responsive system of the DMAEMA-b-THPMA micelles and MSCs-EVs synergistically enhances therapeutic efficacy by promoting angiogenesis, improving vascular permeability, and optimizing vascular. In conclusion, this work demonstrates bioapplication of an ultrasound-responsive micellar nanosystem loaded with EVs for the treatment of lower limb microcirculatory ischemic disorders.

Basedon the excellent photothermal conversion performance of grapheneoxide (GO) and the shrinkage of thermoresponsive copolymer chain segments,a GO-polymer hybrid hydrogel carrier system was designed toload stem cell exosomes that have repair and nutritional functionson nerve cells and play a synergistic role in the proliferation andmigration of Schwann cells (SCs). The surface modification of hydroxypropylchitosan on GO was carried out based on the EDC reaction, and GO-hydroxypropylchitosan (GC) with well hydrophilicity and dispersion was obtained.The thermoresponsive copolymer poly(2-hydroxyethyl methacrylate-co-2-(2-methoxyethoxy) ethylmethacrylate-co-oligo(ethylene glycol) monomethyl ether methacrylate) (P(HEMA-co-OEGMA-co-MEO(2)MA), PHOM) wasprepared by atom transfer radical polymerization. After aldehyde modification,PHOM-CHO was cross-linked with GC through a dynamic Schiffbase bond to form a thermoresponsive hydrogel. Due to the near-infrared(NIR) photothermal conversion of GO and the thermoresponsive contractionof the hydrogel network, the hydrogel can achieve controlled releaseof the loaded exosomes. Cell experiments showed that the GO-thermoresponsivepolymer hybrid hydrogel had good biocompatibility. Further tests onthe promoting effect of the exosome-loaded hydrogel on Schwann cells(SCs) showed that the exosomes could be released in a controlled waythrough NIR irradiation, and the synergistic effect of exosomes andthe GO hybrid hydrogel promoted the proliferation and migration ofSCs.

Abstract Background Microvascular dysfunction is one of the most common pathological characteristics in Type 2 diabetes. Human mesenchymal stem cell-derived exosomes (hUCMSCs-Exo) have diverse functions in improving microcirculation; however, the molecular mechanism of hUCMSCs-Exo in regulating burn-induced inflammation is not well understood. Methods hUCMSCs-Exo were extracted by hypervelocity centrifugation method, and exosome morphology was observed by transmission electron microscopy, exosome diameter distribution was detected by particle size analysis, and exosome specific proteins were identified by Western blot.2. DB/DB mice were randomly divided into exosomes group and PBS group. Exosomes and PBS were injected into the tail vein, respectively, and the calf muscle tissue was taken 28 days later. 0.5% Evans blue fluorescence assessment microvascular permeability. The expression of CD31 was detected by immunofluorescence.The morphology and function of microvessels in muscle tissue of lower limbs was evaluated by transmission electron microscopy.3. TMT proteomics was used to detect the changes of differential protein expression in lower limb muscle tissues of the PBS group and the exosome group, and data analysis was performed to screen key signal molecules and their involved biological pathways. Key signal molecules CD105 were verified by Western blot. The expression of TGF-β1 in exosomes were evaluated by Western blot. Results Electron microscopy showed that hUCMSCs-Exo presented a uniform vesicle structure, and NTA showed that its diameter was about 160 nm. Western blot showed positive expression of specific proteins CD9, CD81 and TSG101 on exosomes.2. There is no significant change in blood glucose and body weight before and after the exosome treatment. The exosome group can significantly reduce the exudation of Evans blue. Compared with the PBS group. Meanwhile, CD31 immunofluorescence showed that the red fluorescence of exosome treatment was significantly increased, which was higher than that of PBS group. Transmission electron microscopy showed smooth capillary lumen and smooth and complete surface of endothelial cells in the exosome group, while narrow capillary lumen and fingerlike protrusion of endothelial cells in the PBS group.3.Quantitative analysis of TMT proteomics showed that there were 82 differential proteins, including 49 down-regulated proteins and 33 up-regulated proteins. Go enrichment analysis showed that the differential proteins were involved in molecular function, biological process, cell components,among which CD105 was one of the up-regulated proteins. Through literature search, CD105 was found to be related to endothelial cell proliferation. Therefore, this study verified the changes of CD105 in the exosome group, and it was used as the mechanism study of this study. 4. Western blot analysis showed that the expression of CD105 protein in lower limb muscle tissue of exosome group was significantly increased compared with that of PBS group. Based on the fact that CD105 is a component of the TGF-β1 receptor complex and exosomes are rich in growth factors and cytokines, this study further examined the expression of TGF-β1 in exosomes, and the results showed that exosomes had high expression of TGF-β1. Conclusion By improving the integrity of microvascular endothelial cells, hUCMSCs-Exo can improve the permeability of microvessels in diabetic lower muscle tissue, further promote the proliferation of lower limb muscle cells and inhibit the apoptosis of tissue cells. The mechanism may be associated with exosomes rich in TGF-β1, which is likely to promote endothelial cell proliferation and improve permeability through binding to the endothelial CD105/TβR-II receptor complex, while promoting angiogenesis and protecting skeletal muscle cells from apoptosis.

Objective: N6-methyladenosine (m6A) has been implicated in the progression of several diseases, and the role of epigenetic regulation in immunity is emerging, particularly for RNA m6A modification. However, it is unclear how m6A-related genes affect the immune microenvironment of ligamentum flavum hyperplasia (LFH). Therefore, we aimed to investigate the effect of m6A modification on the LFH immune microenvironment. Methods: The GSE113212 dataset was downloaded from the Gene Expression Omnibus (GEO) database. We systematically analyzed m6A regulators in eight patient samples and the corresponding clinical information of the samples. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA) and protein-protein interactions (PPIs) were used to explore the correlation of m6A clusters with the immune microenvironment in LFH. A least absolute shrinkage and selection operator (Lasso) regression was then used to further explore the m6A prognostic signature in LFH. The relative abundance of immune cell types was quantified using a single-sample Gene Set Enrichment Analysis (ssGSEA) algorithm. We explored the relationship between hub genes and small molecule drug sensitivity by clustering hub gene-based samples. In addition, Real-Time quantitative PCR (RT-qPCR) as well as western blotting (WB) were used to validate the gene expression of the differentially expressed genes. Results: A total of 1259 differentially expressed genes were identified, of which 471 were upregulated and 788 were downregulated. A total of three genes showed significant differences (METTL16, PCIF1, and FTO). According to the enrichment analysis, immune factors may play a key role in LFH. ssGSEA was used to cluster the immune infiltration score, construct the hub gene diagnosis model, and screen a total of 6 LFH immune-related prediction model genes. The predictive diagnostic model of LFH was further constructed, revealing that METTL16, PCIF1, FTO and ALKBH5 had superior diagnostic efficiency. RT-qPCR results showed that 6 genes (METTL16, PCIF1, POSTN, TNNC1, MMP1 and ACTA1; P < 0.05) exhibited expression consistent with the results of the bioinformatics analysis of the mRNA microarray. Up-regulated METTL16, PCIF1, and ALKBH5 levels in LFH were validated by western blotting. Conclusion: Diversity and complexity of LFH's immune microenvironment are influenced by M6A modification, and our study provides strong evidence for predicting the diagnosis and prognosis of LFH.

Background The single-cell platform provided revolutionary way to study cellular biology. Technologically, a sophistic protocol of isolating qualified single cells would be key to deliver to single-cell platform, which requires high cell viability, high cell yield and low content of cell aggregates or doublets. For musculoskeletal tissues, like bone, cartilage, nucleus pulposus and tendons, as well as their pathological state, which are tense and dense, it’s full of challenge to efficiently and rapidly prepare qualified single-cell suspension. Conventionally, enzymatic dissociation methods were wildly used but lack of quality control. In the present study, we designed the rapid cycling enzymatic processing method using tissue-specific enzyme cocktail to treat different human pathological musculoskeletal tissues, including degenerated nucleus pulposus (NP), ossifying posterior longitudinal ligament (OPLL) and knee articular cartilage (AC) with osteoarthritis aiming to rapidly and efficiently harvest qualified single-cell suspensions for single-cell RNA-sequencing (scRNA-seq). Results We harvested highly qualified single-cell suspensions from NP and OPLL with sufficient cell numbers and high cell viability using the rapid cycling enzymatic processing method, which significantly increased the cell viability compared with the conventional long-time continuous digestion group ( P < 0.05). Bioanalyzer trace showed expected cDNA size distribution of the scRNA-seq library and a clear separation of cellular barcodes from background partitions were verified by the barcode-rank plot after sequencing. T-SNE visualization revealed highly heterogeneous cell subsets in NP and OPLL. Unfortunately, we failed to obtain eligible samples from articular cartilage due to low cell viability and excessive cell aggregates and doublets. Conclusions In conclusion, using the rapid cycling enzymatic processing method, we provided thorough protocols for preparing single-cell suspensions from human musculoskeletal tissues, which was timesaving, efficient and protective to cell viability. The strategy would greatly guarantee the cell heterogeneity, which is critical for scRNA-seq data analysis. The protocol to treat human OA articular cartilage should be further improved.

Articular cartilage (AC) is an avascular and flexible connective tissue located on the bone surface in the diarthrodial joints. AC defects are common in the knees of young and physically active individuals. Because of the lack of suitable tissue-engineered artificial matrices, current therapies for AC defects, especially full-thickness AC defects and osteochondral interfaces, fail to replace or regenerate damaged cartilage adequately. With rapid research and development advancements in AC tissue engineering (ACTE), functionalized hydrogels have emerged as promising cartilage matrix substitutes because of their favorable biomechanical properties, water content, swelling ability, cytocompatibility, biodegradability, and lubricating behaviors. They can be rationally designed and conveniently tuned to simulate the extracellular matrix of cartilage. This article briefly introduces the composition, structure, and function of AC and its defects, followed by a comprehensive review of the exquisite (bio)design and (bio)fabrication of functionalized hydrogels for AC repair. Finally, we summarize the challenges encountered in functionalized hydrogel-based strategies for ACTE both in vivo and in vitro and the future directions for clinical translation.

The early postnatal limb developmental progression bridges embryonic and mature stages, and in many ways mirrors pathomechanisms of articular cartilage degeneration. However, compared with multitudinous research on embryonic limb development, the early postnatal stage seems relative unattractive. Here, a systematic work to portray the postnatal spatial-temporal developmental landscape and the interlinks with osteoarthritis was carried out by characterization of 19952 single cells from murine hindlimbs at 4 postnatal stages using unbiased single-cell RNA sequencing technique. By delineation of cell heterogeneity in each type of tissues, novel candidate progenitor subclusters marked by Cd34 and Ly6e were discovered both in articular cartilage and enthesis, and three cellular developmental branches marked by Col10a1, Spp1 and Tnni2 were reflected in growth plate. The spatial-temporal developmental patterns, key regulation mechanisms, and evolvement in osteoarthritis were intensively explored. Above all, these results expand horizons of biology of postnatal limb development and more importantly highlight the internal commonality with pathology of osteoarthritis, which would help leverage to develop novel therapies.

• A new method for preparing high-quality single-cell nuclear suspension of frozen spinal cord issue. • Tissue beating method breaks the colloidal scar and facilitates full grinding. • Two density gradient centrifugation were performed to remove impurities.

The early postnatal limb developmental progression bridges embryonic and mature stages and mirrors the pathological remodeling of articular cartilage. However, compared with multitudinous research on embryonic limb development, the early postnatal stage seems relatively unnoticed. Here, a systematic work to portray the postnatal limb developmental landscape was carried out by characterization of 19,952 single cells from murine hindlimbs at 4 postnatal stages using single-cell RNA sequencing technique. By delineation of cell heterogeneity, the candidate progenitor sub-clusters marked by Cd34 and Ly6e were discovered in articular cartilage and enthesis, and three cellular developmental branches marked by Col10a1, Spp1, and Tnni2 were reflected in growth plate. The representative transcriptomes and developmental patterns were intensively explored, and the key regulation mechanisms as well as evolvement in osteoarthritis were discussed. Above all, these results expand horizons of postnatal limb developmental biology and reach the interconnections between limb development, remodeling, and regeneration.

Spinal cord injury (SCI) often leads to sensory and motor dysfunction. Two major factors that hinder spinal cord repair are local inflammation and glial scar formation after SCI, and thus appropriate immunotherapy may alleviate damage. To characterize changes in gene expression that occur during SCI and thereby identify putative targets for immunotherapy, here we analyzed the dataset GSE5296 (containing one control group and six SCI groups at different timepoints) to identify differentially-expressed genes. Functional enrichment analysis was performed and a protein-protein interaction network was created to identify possible hub genes. Finally, we performed quantitative PCR to verify changes in gene expression. The CIBERSORT algorithm was used to analyze innate immune cell infiltration patterns. The dataset GSE162610 (containing one control group and three SCI groups at different timepoints) was analyzed to evaluate innate immune cell infiltration at the single-cell level. The dataset GSE151371 (containing one control group [n = 10] and an SCI group [n = 38]) was used to detect the expression of hub genes in the blood from SCI patients. Differentially-expressed innate immune-related genes at each timepoint were identified, and the functions and related signaling pathways of these genes were examined. Six hub genes were identified and verified. We then analyzed the expression characteristics of these hub genes and characteristics of innate immune infiltration in SCI; finally, we examined ligand expression in the context of the CCL signaling pathway and COMPLEMENT signaling pathway networks. This study reveals the characteristics of innate immune cell infiltration and temporal expression patterns of hub genes, and may aid in the development of immunotherapies for SCI.

Osteoarthritis (OA) is the most common joint disease worldwide; however, disease-modifying treatments are lacking because of the complicated pathological mechanisms. As a breakthrough, aberrant activation of transforming growth factor-β 1 (TGF-β1)in subchondral bone has been confirmed as an essential pathomechanism for OA progression, and has become a potential therapeutic target. In addition to R&D on neutralizing antibodies, small-molecule antagonists and chemical medicines, native antagonists of TGF-β1 could be exploited as another promising approach. Noggin (NOG) is an antagonist of bone morphogenetic proteins (BMPs) and was reported to effectively attenuate OA by protecting cartilage and preventing pathological subchondral bone remodeling. However, the underlying mechanisms have not been fully clarified. We first detected the distribution of NOG in knee joints of an OA mouse model, which showed upregulation at early stage of OA but downregulation later in the subchondral bone and no significant change in the articular cartilage. Furthermore, the interaction between NOG and TGF-β1 was verified, which in turn suppressed the downstream SMAD2/3 activity of TGF-β1. Moreover, the proliferation and chondrogenesis of mesenchymal stem cells (MSCs) were not significantly influenced by NOG. Taken together, the results showed that NOG antagonized TGF-β1 but did not repress MSC proliferation and chondrogenesis; thus, it seems promising for OA treatment.