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Inhibitory receptors (IRs) play an indispensable role in regulating T cell activation and expansion. This study is aimed at exploring the correlation between IRs and ankylosing spondylitis (AS). Bioinformatics analysis of two datasets (GSE25101 and GSE73754), including 68 AS cases and 36 healthy controls, demonstrated that “T cell receptor signaling pathway” was significantly enriched, and two IRs (CD112R and CD96) were downregulated in AS cases. Real-time Quantitative PCR Detecting System (qPCR) analysis confirmed the decreased expression of CD112R and CD96 in the peripheral blood of AS patients. Flow cytometry demonstrated that the frequency of CD96-positive cells among CD4 T cells in AS patients was significantly reduced and that expressed on the cells was also significantly lower than the healthy controls. In addition, the expression of CD96 was altered on human primary CD4 T cells extracted from 3 healthy volunteers and cocultured with allogeneic dendritic cells (DCs). Also, low expression of CD96 elevated the phosphorylation of ERK in CD4 T cells and increased the level of TNF-α, IL-23, IL-17A, IL-6, and IFN-γ in the cell culture supernatant. These results suggested that CD96 is crucial for the pathogenesis of AS and may be a potential target in the treatment of the disease.

Background: The study aimed to explore the efficacy of oblique lateral interbody fusion (OLIF) combined with Wiltse approach pedicle screw fixation in the treatment of single-level degenerative lumbar spondylolisthesis（DLS）.Methods: This is a retrospective study. The study enrolled 54 patients with single-level DLS who were treated at the Affiliated Jiangnan Hospital of Zhejiang Chinese Medical University from May 2018 to June 2020. (OLIF group) 24 cases operated using OLIF combined with Wiltse approach pedicle screw fixation, and (PLIF group) 30 cases operated by PLIF. The primary outcome measures were visual analog scale (VAS) pain scores, Oswestry dysfunction index (ODI), and the lower lumbar spine anterior convexity angle. The secondary outcome measures were operation time, intraoperative blood loss, postoperative drainage, hospitalized days, intervertebral space height, and postoperative complications. Results: All patients were followed up for 12-20 months, with a mean of 16.2 months. There were significant differences in VAS (2.63±0.58 vs 3.57±0.63, PConclusion: OLIF combined with the Wiltse approach pedicle screw fixation can achieve good short-term clinical results in the treatment of single-level DLS. This surgical approach is less invasive, promotes early functional recovery, shortens hospitalization time, and improves the quality of life.

BACKGROUND Many studies have confirmed that electroacupuncture can regulate the body's environment to treat a variety of diseases. However, there are few reports on the mechanism of electroacupuncture therapy for diseases involving skin injury. Transcriptome sequencing can reveal changes in gene expression within cells and the signaling pathways involved. In this study, we used transcriptome sequencing to study the molecular mechanisms by which electroacupuncture promotes the healing of skin lesions. METHODS A total of 10 SD rats were divided into two groups of 5: a control group and an electroacupuncture treatment group. The wound-healing area was compared between the two groups after 3 and 14 days. Then, mRNA sequencing and bioinformatics were used to analyze the changes in gene expression profiles in skin tissue after electroacupuncture stimulation. RESULTS (I) The wound area was significantly reduced after 3 and 14 days of electroacupuncture compared with the control group (P

Repair of large skin defects caused by burns, trauma, or tumor operations is a clinical challenge. Hair follicle stem cells (HFSCs) are involved in epithelialization of wounds, formation of new hair follicles and promote vascularization in the newly formed skin, and human acellular amniotic membrane (hAAM) is a promising scaffold for skin substitute. Here, we investigated the ability of rat HFSCs (rHFSCs) combined with an hAAM to repair full thickness skin defects in nude mice. The effect of the rHFSC-hAAM composite on the repair of skin defects in nude mice was assessed by hematoxylin and eosin staining, immunohistochemistry, and EdU-labeled cell tracking. Isolated and cultured rHFSCs had strong cloning and proliferation potentials. Immunofluorescence staining and flow cytometry assays showed that rHFSCs expressed high levels of integrin α6, CK15, p63, and Sox9. Cells cultured in hAAM showed flaky and cluster-like morphology and were able to adhere and grow effectively. After transplantation, the rHFSC-hAAM composite promoted wound healing in nude mice. Moreover, cells in the rHFSC-hAAM composite were directly involved in hair follicle formation and angiogenesis of tissue around the hair follicle. These results provide an experimental and theoretical basis for the clinical application of HFSCs in repair of human skin defects and a new approach for skin tissue engineering.

Spinal cord injury (SCI) is a catastrophic disease associated with damaged neurological structures and has become a significant social and economic burden for the health care system and patients’ families. The use of Chinese Herbal Medicine (CHM) to treat SCI has been increasing in recent years. This meta-analysis aimed to investigate the effectiveness of CHM for patients with SCI. Therefore, we included randomized controlled trials (RCTs) of CHM for SCI in seven databases. A total of 26 studies involving 1961 participants were included in this study. No serious heterogeneity or publication bias was observed across each study. The results showed that significant improvements of the American Spinal Injury Association (ASIA)-grading improvement rate ([Formula: see text], [Formula: see text]), clinical effective rate ([Formula: see text], [Formula: see text]), ASIA motor score ([Formula: see text], [Formula: see text]), ASIA sensory score (total) ([Formula: see text], [Formula: see text]), ASIA sensory score (light touch) ([Formula: see text], [Formula: see text]), ASIA sensory score (pinprick) ([Formula: see text], [Formula: see text]), and activities of daily living (ADL) score ([Formula: see text], [Formula: see text]) in CHM group compared with the control group. Among the CHM groups, Buyang Huanwu decoction was the most frequently prescribed herbal formula, while Astragalus membranaceus was the most commonly used single herb. In addition, there were no serious and permanent adverse effects in the two groups. The methodological quality of the most included RCTs was poor and the quality of evidence for the main outcomes was from very low to moderate according to the GRADE system. Current evidence suggests that CHM is an effective and safe treatment for SCI and could be treated as a complementary and alternative option with few side effects. However, considering the low quality, small size, and high risk of the studies identified in this meta-analysis, higher methodological quality, rigorously designed RCTs with large sample sizes are needed to confirm the results.

Ankylosing spondylitis (AS) is an autoimmune disease with unknown aetiology. To unravel the mechanisms mediating AS pathogenesis, we profiled peripheral blood mononuclear cells (PBMCs) from AS patients and healthy subjects using 10X single-cell RNA sequencing. The frequencies of immune cell subsets were evaluated by flow cytometry. NK cells were purified from PBMCs using isolation kit and were examined for gene expression by RT-qPCR. Plasma levels of cytolytic molecules were examined by enzyme-linked immunosorbent assay. Compared to healthy controls, AS patients showed a significant decrease in total NK cells as well as CD56

Background. Ankylosing spondylitis (AS) is a chronic disease in which the column is the main lesion. It is caused by a combination of genetic and environmental factors, mainly involving the axial skeleton, resulting in column rigidity and difficulty in movement, and there may be different degrees of eye, lung, cardiovascular, kidney, and other organ damage. Long-term treatment lacks in ankylosing spondylitis. Wenbu Zhibi granule (WZG) is a prescription handed down from the history of Chinese medicine for thousands of years, which is used to treat the pain of patients with AS and to prevent the further development of the disease. However, there is no scientific evidence based on clinical trials to evaluate the efficacy and safety of WZG for ankylosing spondylitis. Methods/Design. We will conduct a multicenter, randomized, double-blind, placebo-controlled trial to evaluate the efficacy and safety of the WZG in the treatment of AS. We will randomly assign 100 patients with active AS to two groups, treated for 16 weeks. The primary efficacy endpoint is the proportion of subjects who reached 40% improvement criteria proposed by Assessment of SpondyloArthritis International Society (ASAS40) at 16 weeks from baseline, the secondary efficacy endpoint includes ASAS20 response rate, ASAS partial remission response rate, 5/6 improvement criteria proposed by ASAS (ASAS5/6) response rate, and change in the Spondyloarthritis Research Consortium of Canada (SPARCC) MRI spine score, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Ankylosing Spondylitis Disease Activity Score (ASDAS), linear Bath Ankylosing Spondylitis Metrology Index (BASMI), ankylosing spondylitis quality of life (ASQoL). In addition, the time points will be set as baseline, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 24 weeks, and 48 weeks. Discussion. The results of this study will elucidate the efficacy and safety of WZG and provide an appropriate treatment option for patients with AS. Trial registration: ClinicalTrials.gov ID: https://clinicaltrials.gov/ct2/show/ChiCTR2000041010. (Chinese Clinical Trail Registry, Registered 16 December 2020, http://www.chictr.org.cn).

BACKGROUND: Induced pluripotent stem cells (iPSCs) can generate bone marrow mesenchymal stem cells (BMSCs) as seed cells for tissue-engineered bone to repair bone defects. In this study, we investigated the effects of hydroxyapatite-zirconia (HA/ZrO(2)) composites combined with iPSC-derived BMSCs as a bone substitute on repairing skull defects in rats. METHODS: Human urinary cells isolated from a healthy donor were reprogramed into iPSCs and then induced into BMSCs. Immunocytochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) were used to examine the characteristics of the induced MSCs. The iPSC-derived BMSCs were cultured on HA/ZrO(2) composites, and cytocompatibility of the composite was analyzed by cell counting kit-8 (CCK-8) assays, RT-PCR, and scanning electron microscopy. Then, HA/ZrO(2) combined with iPSC-derived expanded potential stem cells (EpSCs) were transplanted onto skull defects of rats. The effects of this composite on bone repair were evaluated by IHC. RESULTS: The results showed that MSCs induced from iPSCs displayed the phenotypes and property of normal BMSCs. After seeding on the HA/ZrO(2), iPSC-derived BMSCs had the ability to proliferate and differentiate into osteoblasts. After transplantation, iPSC-derived BMSCs on HA/ZrO(2) promoted construction of bone on rat skulls. CONCLUSIONS: These results indicated that transplantation of a HA/ZrO(2) combined with iPS-derived BMSCs is feasible to reconstruct bones and may be a substantial reference for iPSC-based therapy for bone defects.

Background Ankylosing spondylitis (AS) is a progressive rheumatic disease and studies reveal that the immune system is critical for the pathogenesis of AS. In the present study, various bioinformatics analysis methods were comprehensively applied, designed to identify potential key genes and inflammation states of AS. Methods The transcriptome profiles of GSE25101 and GSE73754 obtained from the Gene Expression Omnibus (GEO) database were merged for subsequent analyses. The differentially expressed genes (DEGs) were identified using the Bioconductor package Limma and threshold values. Functional enrichment and pathway enrichment analyses were performed using the clusterProfiler package and Gene Set Enrichment Analysis (GSEA). Next, protein–protein interaction (PPI) network of the identified DEGs was constructed by the online database, the Search Tool for the Retrieval of Interacting Genes (STRING), visualization and analysis were performed through Cytoscape software. Subsequently, we applied CIBERSORT algorithm to identify subpopulation proportions of immune cells in peripheral blood samples. Finally, we validated the hub genes with the GSE18781 dataset. Samples were collected from patients to validate gene and protein expression using qRT-PCR and ELISA. Results A total of 334 DEGs were identified, including 182 upregulated and 152 downregulated DEGs, between AS patients and normal human controls, which were primarily involved in immune response, autophagy, and natural killer cell-mediated cytotoxicity. The most prominent module and candidate biomarkers were identified from the PPI network. Biomarkers were selected for validation and their expressions were significantly decreased in peripheral blood samples which was consistent with transcriptome sequencing results. Nine genes with AUC > 0.70 were considered to be AS hub genes for ROC curve analysis, including GZMA, GZMK, PRF1, GNLY, NKG7, KLRB1, KLRD1, IL2RB and CD247. Furthermore, CIBERSORT results suggest that AS contained a higher proportion of CD8+ T cells, naive CD4+ T cells, neutrophils, and lower levels of gamma delta T cells compared with the normal controls. Conclusion In this study, we identified DEGs combined with their closely related biological functions and propose that granule-associated proteins and immune infiltration maybe involved in the progression of ankylosing spondylitis. These validated hub genes may provide new perspectives for understanding the molecular mechanisms of ankylosing spondylitis.

The role of autophagy in the transplantation of induced pluripotent stem cells (iPSCs)-derived neural stem cells (NSCs) to treat spinal cord injury (SCI) and neurogenic bladder was investigated in this study. NSCs derived from human iPSCs were identified by and immunofluorescence assay. To clarify the role of autophagy, iPSCs were treated with either an autophagy inducer (rapamycin), or an autophagy inhibitor (chloroquine). Cell Counting kit-8 (CCK-8), western blot and flow cytometry were used to detect the effect of autophagy on the viability and differentiation of iPSCs. Sixty Wistar rats were selected to establish the SCI model and treated with iPSCs-derived NSCs transplantation. The effect of autophagy on the bladder function of rats with different treatments was evaluated by Basso, Beattie, and Bresnahan (BBB) score, bladder function score, bladder weight measurement, Hematoxylin & Eosin (H&E) staining, and Masson staining. The results of in vitro experiment showed that rapamycin enhanced the cell activity of iPSCs, increased the number of nestin positive cells, up-regulated Beclin-1 and LC3BI/II expressions, and down-regulated p62 expression. And the results of in vivo experiment showed that rapamycin improved exercise ability and bladder function, partially restored bladder weight, and significantly reduced bladder tissue damage in SCI rats. However, chloroquine showed the opposite results. The differentiation of iPSCs into NSCs could be promoted by induced autophagy, while neurogenic bladder of SCI was restored by autophagy activation.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by erosive arthritis, which has not been thoroughly cured yet, and standardized treatment is helpful for alleviating clinical symptoms. Here, various bioinformatics analysis tools were comprehensively utilized, aiming to identify critical biomarkers and possible pathogenesis of RA. Three gene expression datasets profiled by microarray were obtained from GEO database. Dataset GSE55235 and GSE55457 were merged for subsequent analyses. We identified differentially expressed genes (DEGs) in RStudio with limma package, performing functional enrichment analysis based on GSEA software and clusterProfiler package. Next, protein-protein interaction (PPI) network was set up through STRING database and Cytoscape. Moreover, CIBERSORT website was used to assess the inflammatory state of RA. Finally, we validated the candidate hub genes with dataset GSE77298. As a result, we identified 106 DEGs (72 upregulated and 34 downregulated genes). Through GO, KEGG, and GSEA analysis, we found that DEGs were mainly involved in immune response and inflammatory signaling pathway. With the help of Cytoscape software and MCODE plug-in, the most prominent subnetwork was screened out, containing 14 genes and 45 edges. For ROC curve analysis, eight genes with AUC >0.80 were considered as hub genes of RA. In conclusion, compared with healthy controls, the DEGs and their closely related biological functions were analyzed, and we held that chemokines and immune cells infiltration promote the progression of rheumatoid arthritis. Targeting the eight biomarkers we identified may be useful for the diagnosis and treatment of rheumatoid arthritis.

Background ：To assess the short-term clinical outcomes of oblique lateral lumbar interbody fusion (OLIF) combined with multiple level fixation with the guidance of posterior "O" arm CT navigation for degenerative scoliosis. Methods ：A total of 15 patients (3 males and 12 females; average age: 62.2±5.3 years) with degenerative scoliosis were enrolled in our study. All patients were treated with OLIF combined with posterior "O" arm CT navigation and long segment (T 10 -L 5 ) internal fixation during Apr. 2016 and Dec. 2017. Collected perioperative data included surgery time, blood loss, excellent rate of intraoperative pedicle screw. Coronal Cobb angle, lumbar lordosis (LL), intervertebral space height, sagittal balance (SVA) were measured by plain radiograph. Accuracy of pedicle screw placement and intervertebral fusion was investigated with postoperative CT. Visual analogue scale (VAS) and Oswestry disability index (ODI) were used to evaluate the clinical efficacy of surgery. Results ： All patients were followed for 12-25 months (16.5±5.3 months).The OLIF operation time was (98.7±16.8) min, and the blood loss was (50.2±10.7) ml. The posterior "O" arm CT-navigated long segment internal fixation was (101.5 ± 23.4) min, and the blood loss was (63.1 ± 19.7) ml. The total operation time was (200.2±40.2) min, and the blood loss was (113.3±30.4) ml. Coronal Cobb angle, LL, intervertebral space height, SVA, VAS, ODI at the last follow-up were 5.5°± 3.1°, 39.3°± 9.1°, (10.9±1.2) mm, (25.1±10.9) mm, (1.3± 0.8)and(15.8±4.5)%, respectively, showing statistically significant differences ( P

BACKGROUND The sustained negative pressure created by vacuum sealing drainage (VSD) on exposed vascular wounds can result in blood vessel compression, embolism, or necrosis. The objective of this research was to explore the ability of an experimental vascular protective shield combined with VSD to protect exposed vessels of the lower limbs and accelerate wound repair. METHODS (I) The vascular protective shield was prepared; (II) the material was subjected to acute toxicity and hemolysis tests; (III) and 30 New Zealand rabbits were divided into three groups: the control, VSD-only, and combined shield-VSD groups (with ten rabbits in each group). The wound-healing rate, myocardial function, wound histopathology, expression of angiogenesis markers, and exposed vascular compression of these three groups were compared on day 7. RESULTS (I) The internal structure of the material was smooth; and (II) no toxicity or death was observed in mice of any group. The hemolysis rate in the combined shield-VSD group was very low. (III) The combined shield-VSD group showed a higher wound-healing rate, and higher levels of cluster of differentiation 31 (CD31), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF), than the other groups (P

Objective. This meta-analysis aimed to investigate the effectiveness of acupuncture therapy plus hyaluronic acid injection versus hyaluronic acid injection alone for patients with knee osteoarthritis. Methods. Relevant randomized controlled trials that compared the combined effect of acupuncture therapy and hyaluronic acid injection with hyaluronic acid injection alone for knee osteoarthritis patients were included. 10 studies were included in this meta-analysis, and the relative risk (RR) and weight mean difference (MD) with 95% CI for the Lysholm knee score (LKSS), visual analogue scale (VAS), and effective rate (ER) were evaluated by using RevMan 5.3 software. Besides, the bias assessment of the included studies was evaluated using the Cochrane risk of bias tool, and the GRADE (Grading of Recommendations, Assessment Development, and Evaluation) system was applied to assess the overall quality of the evidence. Results. A total of 10 studies involving 998 participants were included in this study. Compared to hyaluronic acid injection alone, the combined therapy significantly reduced pain on the visual analogue scale (VAS) and improved the ER and knee function on the Lysholm knee score (LKSS). Of these, the pooled LKSS (MD = 8.09, 95% CI = [7.02, 9.16], p<0.00001, 7 studies) and ER (RR = 1.23, 95% CI 1.15 to 1.31, p<0.00001, 8 studies) suggested that combination therapy yielded a significantly higher ER and improved the LKSS scores to a greater degree than hyaluronic acid injection alone in patients with KOA. The VAS (MD = −1.39, 95% CI = [−1.99, −0.79], p<0.00001, 7 studies) showed that the combined therapy significantly reduced pain than hyaluronic acid injection alone. The quality of evidence for the main outcomes was from very low to low according to the GRADE system. Conclusion. Current evidence suggests that acupuncture therapy combined with hyaluronic acid injection is more effective in alleviating pain, improving the ER and knee function compared with hyaluronic acid injection alone. However, considering the low quality, small size, and high risk of the studies identified in this meta-analysis, more higher methodological quality, rigorously designed randomized controlled trials with large sample sizes are needed to confirm the results.

Heredity is the major factor contributing to the susceptibility to ankylosing spondylitis(AS). Janus kinase 2 (JAK2) has been associated with AS. Urine-derived cells from an AS patient with JAK2 mutation were used to generate induced pluripotent stem cells (iPSCs) with five episomal iPSC reprogramming vectors (pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, pCXLE-EGFP and pCXWB-EBNA1). The iPSCs were pluripotent and will be valuable for research on the role and mechanism of JAK2 in the pathogenesis of AS.

Genomic editing to correct disease-causing mutations is a promising approach for the treatment of human diseases. As a simple and programmable nuclease-based genomic editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has substantially improved the ability to make precise changes in the human genome. Rapid development of CRISPR-based technologies in recent years has expanded its application scope and promoted CRISPR-based therapies in preclinical trails. Here, we review the application of the CRISPR system over the last 2 years; including its development and application in base editing, transcription modulation and epigenetic editing, genomic-scale screening, and cell and embryo therapy. Finally, the prospects and challenges related to application of CRISPR/Cas9 technologies are discussed.

Bone formation is linked with osteogenic differentiation of mesenchymal stem cells (MSCs) in the bone marrow. Microgravity in spaceflight is known to reduce bone formation. In this study, we used a real microgravity environment of the SJ-10 Recoverable Scientific Satellite to examine the effects of space microgravity on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). hMSCs were induced toward osteogenic differentiation for 2 and 7 d in a cell culture device mounted on the SJ-10 satellite. The satellite returned to Earth after going through space experiments in orbit for 12 d, and cell samples were harvested and analyzed for differentiation potentials. The results showed that space microgravity inhibited osteogenic differentiation and resulted in adipogenic differentiation, even under osteogenic induction conditions. Under space microgravity, the expression of 10 genes specific for osteogenesis decreased, including collagen family members, alkaline phosphatase ( ALP), and runt-related transcription factor 2 ( RUNX2), whereas the expression of 4 genes specific for adipogenesis increased, including adipsin ( CFD), leptin ( LEP), CCAAT/enhancer binding protein β ( CEBPB), and peroxisome proliferator-activated receptor-γ ( PPARG). In the analysis of signaling pathways specific for osteogenesis, we found that the expression and activity of RUNX2 was inhibited, expression of bone morphogenetic protein-2 ( BMP2) and activity of SMAD1/5/9 were decreased, and activity of focal adhesion kinase (FAK) and ERK-1/2 declined significantly under space microgravity. These data indicate that space microgravity plays a dual role by decreasing RUNX2 expression and activity through the BMP2/SMAD and integrin/FAK/ERK pathways. In addition, we found that space microgravity increased p38 MAPK and protein kinase B (AKT) activities, which are important for the promotion of adipogenic differentiation of hMSCs. Space microgravity significantly decreased the expression of Tribbles homolog 3 ( TRIB3), a repressor of adipogenic differentiation. Y15, a specific inhibitor of FAK activity, was used to inhibit the activity of FAK under normal gravity; Y15 decreased protein expression of TRIB3. Therefore, it appears that space microgravity decreased FAK activity and thereby reduced TRIB3 expression and derepressed AKT activity. Under space microgravity, the increase in p38 MAPK activity and the derepression of AKT activity seem to synchronously lead to the activation of the signaling pathway specifically promoting adipogenesis.-Zhang, C., Li, L., Jiang, Y., Wang, C., Geng, B., Wang, Y., Chen, J., Liu, F., Qiu, P., Zhai, G., Chen, P., Quan, R., Wang, J. Space microgravity drives transdifferentiation of human bone marrow-derived mesenchymal stem cells from osteogenesis to adipogenesis.

BACKGROUND: Stem cell transplantation has been increasingly used for spinal cord repair, and some achievements have been made. However, limited stem cell sources as well as immune rejection and ethical issues have restricted its wide application. Therefore, to achieve further breakthroughs regarding the application of stem cell transplantation to treat spinal cord injury (SCI), it is important to develop a stem cell line that can effectively avoid immune rejection and ethical issues. METHODS: Urine cells (UCs) were induced to differentiate into induced pluripotent stem cells (iPSCs), which then further differentiated into neural stem cells (NSCs). Relevant tests were performed, and three-dimensional (3D) printed scaffolds were prepared. Thirty C57BL/6 mice were divided into 5 groups based on a random number table: a sham group, an SCI group, an SCI + control group, an SCI + siNC group, and an SCI + siGAS5 group (n=6). The latter 4 groups replicated SCI models. Mice in the SCI + control group were transplanted with 3D scaffolds loaded with iPSC-derived NSCs (iPSd-NSCs). Mice in the SCI + siNC group and the SCI + siGAS5 group were transplanted with scaffolds loaded with iPSd-NSCs-siNC and 3D scaffolds loaded with iPSd-NSCs-siGAS5, respectively. Mice in the other groups were injected with the same amount of normal saline. Hematoxylin-eosin staining was used to observe the histopathology of the injured spinal cord, the Basso-Mouse Scale was used to assess the motor function of the hind limbs of the mice, and Western blot was used to detect the expression of apoptosis-related proteins after SCI. RESULTS: iPSd-NSCs were successfully induced and differentiated, and 3D printed heparin sulfate-collagen scaffolds were prepared, inside which a 3D loose porous structure was shown by electron microscopy. Morphological observations showed that iPSd-NSC transplantation improved SCI in mice, while GAS5 silencing inhibited the reparative effect of iPSd-NSC transplantation on SCI in mice. Western blot results indicated that iPSd-NSC transplantation significantly increased the expression level of B cell lymphoma/leukemia-2 (Bcl-2) (P

Background and Aims A host of studies investigated the associations between tumor necrosis factor alpha inducible protein 3 (TNFAIP3) gene rs2230926 and rs5029937 polymorphisms and rheumatoid arthritis (RA) susceptibility, but with conflicting findings. Therefore, we explored whether TNFAIP3 gene rs2230926 and rs5029937 polymorphisms are associated with RA by meta-analysis. Material and Methods We performed out a comprehensive literature search of PubMed, Elsevier, Embase, and CNKI databases to identify relevant studies. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the associations. Results Literature search identified 10 case-control studies involving 18,014 cases and 20,112 controls in this meta-analysis. Our data supported an association between TNFAIP3 gene rs2230926 and rs5029937 polymorphisms and RA risk. Stratification analysis of ethnicity indicated that rs5029937 polymorphism increased the risk of RA among Caucasians, while rs2230926 polymorphism increased the risk of RA among Asians and Caucasians. Conclusions In conclusion, this meta-analysis demonstrates that TNFAIP3 gene polymorphisms (rs2230926 and rs5029937) are associated with the increased risk of RA.

Within the vascular endothelial growth factor (VEGF) family of five subtypes, VEGF165 secreted by endothelial cells has been identified to be the most active and widely distributed factor that plays a vital role in courses of angiogenesis, vascularization and mesenchymal cell differentiation. Hair follicle stem cells (HFSCs) can be harvested from the bulge region of the outer root sheath of the hair follicle and are adult stem cells that have multi‐directional differentiation potential. Although the research on differentiation of stem cells (such as fat stem cells and bone marrow mesenchymal stem cells) to the endothelial cells has been extensive, but the various mechanisms and functional forms are unclear. In particular, study on HFSCs’ directional differentiation into vascular endothelial cells using VEGF165 has not been reported. In this study, VEGF165 was used as induction factor to induce the differentiation from HFSCs into vascular endothelial cells, and the results showed that Notch signalling pathway might affect the differentiation efficiency of vascular endothelial cells. In addition, the in vivo transplantation experiment provided that HFSCs could promote angiogenesis, and the main function is to accelerate host‐derived neovascularization. Therefore, HFSCs could be considered as an ideal cell source for vascular tissue engineering and cell transplantation in the treatment of ischaemic diseases.

Background Induced pluripotent stem cells (iPSCs) can generate epithelial stem cells (EpSCs) as seed cells for skin substitutes to repair skin defects. Here, we investigated the effects of a human acellular amniotic membrane (hAAM) combined with iPSC-derived CD200+/ITGA6+ EpSCs as a skin substitute on repairing skin defects in nude mice. Methods Human urinary cells isolated from a healthy donor were reprogrammed into iPSCs and then induced into CD200+/ITGA6+ epithelial stem cells. Immunocytochemistry and RT-PCR were used to examine the characteristics of the induced epithelial stem cells. iPSC-derived EpSCs were cultured on a hAAM, and cytocompatibility of the composite was analyzed by CCK8 assays and scanning electron microscopy. Then, hAAMs combined with iPSC-derived EpSCs were transplanted onto skin defects of mice. The effects of this composite on skin repair were evaluated by immunohistochemistry. Results The results showed that CD200+/ITGA6+ epithelial stem cells induced from iPSCs displayed the phenotypes of hair follicle stem cells. After seeding on the hAAM, iPSC-derived epithelial stem cells had the ability to proliferate. After transplantation, CD200+/ITGA6+ epithelial stem cells on the hAAM promoted the construction of hair follicles and interfollicular epidermis. Conclusions These results indicated that transplantation of a hAAM combined with iPS-derived EpSCs is feasible to reconstruct skin and skin appendages, and may be a substantial reference for iPSC-based therapy for skin defects.

Yttria-tetragonal zirconia polycrystal (Y-TZP) ceramic with hydroxyapatite (HA) coating has been recognized as one of the most common biomaterials. However, its lower interfacial bonding strength has restricted its clinical application. In this study, CaCO3, Ca3(PO4)2 (TCP), and Ag were added into the interface of the HA coating and Y-TZP substrate by a pressureless sintering process to increase interfacial bonding strength. The morphology and microstructure of the interlayers were analyzed by scanning electronic microscope. The results show that the comprehensive performances of HA/interlayer/ZrO2 biomaterials were much better than that of the sample without interlayer, and the shear and tensile strengths of the HA/CaCO3/ZrO2 biomaterial reached 15.19 and 16.88 MPa, respectively, which were higher than those of TCP and Ag. The CaZrO3 and carbonated apatite, formed by decomposed products of CaCO3 and HA reacting with ZrO2 at the interface under sintering, increased the mechanical properties of the...

In this study, we demonstrated the elongated needle therapy would improved functional recovery after Acute Spinal Cord Injury (ASCI). Fifty-four Sprague-Dawley rats were randomly divided into three groups: sham group (n=18), control group (n=18) and elongated needle therapy group (n=18). ASCI-induced injury was induced at T9–T11 in spinal cords of control and elongated needle therapy groups. The sham and elongated needle therapy group was treated with elongated needles (0.35*75) on the ST28 (Chibian) and BL54 (Water Channel points) once daily for 7 days with following therapeutic parameters: frequency of 2/100 Hz, current of 1 mA. Postoperative Basso, Beattie and Bresnahan locomotors rating scale scores were measured on days 1, 3, and 7 and on day 1, 3 and 7d following spinal cord tissues were collected for Enzyme Linked Immunosorbent Assay (ELISA) were used to detect Tumor Necrosis Factor α (TNF-α), Interleukin-6 (IL-6), Interleukin-1 (IL-1) and Nuclear transcription Factor κB (NF-κB) expression. As the time goes on, we found that elongated needle therapy promoted the functional recovery and the expression of TNF-α, IL-6, IL-1β and NF-κB. Compared with control group, elongated needle therapy can significantly promote locomotor recovery at 3 and 7d; and it can significantly decrease the expression of TNF-α, IL-6, IL-1β and NF-κB at 1,3 and 7d. Thus, our results suggest that elongated needle therapy can promoted functional recovery and Inhibition of inflammation as a potential mechanism.

Ethnopharmacological relevance Acupuncture is a key part of traditional Chinese medicine, shown to induce favorable neuroplasticity for injuries in the central and peripheral nervous systems. Recent studies report elongated needle therapy (ENT) with BL54 and ST28 may restore acute spinal cord injury (ASCI). However, the precise mechanism for this has not been elucidated. Aim of the study In our current study, we investigated the effects of ENT on inflammation and neuronal apoptosis induced by ASCI, and whether PI3K/Akt and MAPK/ERK signaling pathways are involved in the ENT restoration effect. Materials and methods Rat models of moderate SCI were established in accordance with the modified Allen's method and were treated with ENT continuously for 7 days. Spontaneous activities were evaluated by the Basso Beattie and Bresnahan locomotor scale. Levels of inflammatory cytokines, such as tumor necrosis factor alpha, interleukin-6, IL-1β, and nuclear factor kappa-β, were determined by enzyme-linked immunosorbent assay. Cell apoptosis was examined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The proportions of cells with positive Bcl-2 and Bax expression were determined by immunohistochemical assays, whilst the expression profiles of p-AKT and p-ERK in spinal cord tissues were evaluated by western blotting. Furthermore, the expression profiles of Cytochrome-C (Cyt-C) and caspase-3 in vivo were analyzed by reverse transcription polymerase chain reaction. The potential inhibitory effects downstream of the Akt and ERK signaling pathways were examined by administration of specific inhibitors LY294002 and PD98059 in vivo. Results As indicated by this study, inflammation as well as PI3K/Akt- and MAPK/ERK signaling pathway-mediated neuronal apoptosis were involved in the course of SCI in rats. The neuro-protective effect of ENT was associated with reduced Bax protein-positive neurons and increased Bcl-2 protein-positive neurons. ENT enhanced recovery of rat activities. Activation of p-Akt and p-ERK in the PI3K/Akt and MAPK/ERK signaling pathways, inhibited expression of the critical component Cyt-C. Cyt-C is required for the mitochondrial apoptosis pathway and cascade of caspase-3, which is involved in activation of neuronal apoptosis through down-regulation of Bax protein and up-regulation of Bcl-2, as determined by TUNEL. The administration of PI3K/Akt and MAPK/ERK signaling pathway specific inhibitors, LY294002 and PD98059, suppressed expression of both p-Akt and p-ERK. Conclusion ENT with BL54 and ST28 points can promote the recovery of ASCI. And the neuro-protective effect of ENT during the restoration of SCI may be associated with the suppression of both inflammation and activation of PI3K/Akt and MAPK/ERK signaling pathways, resulting from down-regulation of Bax protein, up-regulation of Bcl-2, and inhibition of the mitochondrial apoptosis pathway.

Dense biomaterial plays an important role in bone replacement. However, it fails to induce bone cell migration into graft material. In the present study, a novel bone graft substitute (BGS) consisting of porous gradient hydroxyapatite/zirconia composite (PGHC) and gelatin/chitosan slow-release hydrogel containing bone morphogenetic protein 2 and bone mesenchymal stem cells was designed and prepared to repair lumbar vertebral defects. The morphological characteristics of the BGS evaluated by a scanning electron microscope showed that it had a three-dimensional network structure with uniformly distributed chitosan microspheres on the surfaces of the graft material and the interior of the pores. Then, BGS (Group A), PGHC (Group B), or autologous bone (Group C) was implanted into lumbar vertebral body defects in a total of 24 healthy rhesus monkeys. After 8 and 16 weeks, anteroposterior and lateral radiographs of the lumbar spine, microcomputed tomography, histomorphometry, biomechanical testing, and biochemical testing for bone matrix markers, including Type I collagen, osteocalcin, osteopontin, basic fibroblast growth factor, alkaline phosphatase, and vascular endothelial growth factor, were performed to examine the reparative efficacy of the BGS and PGHC. The BGS displayed excellent ability to repair the lumbar vertebral defect in rhesus monkeys. Radiography, microcomputed tomography scanning, and histomorphological characterization showed that the newly formed bone volume in the interior of the pores in the BGS was significantly higher than in the PGHC. The results of biomechanical testing indicated that the vertebral body compression strength of the PGHC implant was lower than the other implants. Reverse-transcription polymerase chain reaction and western blot analyses showed that the expression of bone-related proteins in the BGS implant was significantly higher than in the PGHC implant. The BGS displayed reparative effects similar to autologous bone. Therefore, BGS use in vertebral bone defect repair appears promising.

This study compared bony fusion in autologous bone grafting with HA/ZrO2 graded composite in terms of mineral depositions, histological characteristics, and biomechanical properties of bonding interface. Twenty-four beagle dogs were established four places of bone defect in two adjacent lumbar vertebral bodies (L4 and L5) and were successively implanted with HA/ZrO2 graded composite (group A), HA/ZrO2 unilayer composite (group B), pure ZrO2 (group C), and pure HA (group D). After operation, lumbar vertebral specimens were respectively harvested per time at week 6, 12, and 16. Then, the bony fusion interface was evaluated by fluorescence microscope and computer image analysis system to measure mineral apposition rates (MAR). Histological analysis of specimen was used to determine bone bonding rates (BBR) and possible foreign body reactions associated with each groups. And interface bonding force between implant and autogenous bone was quantified with biomechanical push-out test. Compared with other groups,...

Department of Orthopedics, the second Affiliated Hospital, Medical College of Zhejiang University,ZhengJiang Province 310009, ChinaThe goals of bone tissue engineering are to apply biomaterial scaffolds with adhered cells, such as osteoblasts, bone marrowstromal stem cells, or chondrocytes, to repair, regenerate, and restore the functions of damaged bone tissue or to replace thosetissues with porous engineered biomaterials. Over recent decades, a diverse class of biomaterials has been applied in bone tissueengineering field. Porous hydroxyapatite bioceramic is currently receiving significant attention as a bone tissue engineeringsubstitute because of its biological characteristics, including biocompatibility, bioactivity, osteoconduction, and vasculogenesis.This biomaterial has a three-dimensional structure with interconnected spherical pores of uniform size, which encourages boneingrowth and achieves good integration of the material and the host bone over time. However, the compressive strength andelastic modulus of porous hydroxyapatite scaffolds generally weaken as the porosity increases, in both in vitro and in vivo testing.Zirconia can be used to toughen hydroxyapatite materials for bone repair and replacement because of its unique biomechanicalproperties, including compressive strength and fracture toughness. Additionally, a zirconia chitosan hybrid containing bonemorphogenetic protein-2 and mesenchymal stem cells derived from induced pluripotent stem cells has been used as a coatingmaterial adhered to surface of scaffolds to promote bone regeneration and repair. Here, we provide a succinct review of zirconiatoughened hydroxyapatite biomaterial scaffolds that incorporate bone morphogenetic protein-2 and mesenchymal stem cells forbone tissue engineering and describe the biomaterials that are currently being investigated based on the recent literature and ourown data.

The purpose of this study was to establish methods for isolation, culture, expansion, and characterization of rat hair follicle stem cells (rHFSCs). Hair follicles were harvested from 1-week-old Sprague-Dawley rats and digested with dispase and collagenase IV. The bulge of the hair follicle was dissected under a microscope and cultured in Dulbecco's modified Eagle's medium/F12 supplemented with KnockOut™ Serum Replacement serum substitute, penicillin-streptomycin, L-glutamine, non-essential amino acids, epidermal growth factor, basic fibroblast growth factor, polyhydric alcohol, and hydrocortisone. The rHFSCs were purified using adhesion to collagen IV. Cells were characterized by detecting marker genes with immunofluorescent staining and real-time polymerase chain reaction (PCR). The proliferation and vitality of rHFSCs at different passages were evaluated. The cultured rHFSCs showed typical cobblestone morphology with good adhesion and colony-forming ability. Expression of keratin 15, integrin α6, and integrin β1 were shown by immunocytochemistry staining. On day 1-2, the cells were in the latent phase. On day 5-6, the cells were in the logarithmic phase. Cell vitality gradually decreased from the 7th passage. Real-time PCR showed that the purified rHFSCs had good vitality and proliferative capacity and contained no keratinocytes. Highly purified rHFSCs can be obtained using tissue culture and adhesion to collagen IV. The cultured cells had good proliferative capacity and could therefore be a useful cell source for tissue-engineered hair follicles, vessels, and skin.

Osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs) is regulated by various factors, including bone morphogenetic proteins (BMPs), Notch, growth hormones and mitogen-activated protein kinases (MAPKs). Tribbles homolog 3 (TRIB3), a pseudokinase, plays an important role in cancer cells and adipocytes. However, TRIB3 function in osteogenic differentiation is unknown, although it is involved in regulating signaling pathways associated with osteogenic differentiation. Here, we found that TRIB3 was highly expressed during osteogenic differentiation in hBMSCs. Inhibition of focal adhesion kinase (FAK) or phosphatidylinositol 3-kinase (PI3K) resulted in a significant decrease in TRIB3 expression, and expression of TRIB3 was restored by increasing insulin-like growth factor-1 (IGF-1) via activating phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling. TRIB3 knock-down enhanced proliferation and decreased osteogenic differentiation at the middle stage of differentiation, and these effects were reversed by inhibiting the activation of extracellular signal-regulated kinase (ERK)-1/2. In conclusion, TRIB3 plays an important role in proliferation and osteogenic differentiation by regulating ERK1/2 activity at the middle stage of differentiation, and expression of TRIB3 is regulated by FAK in a PI3K/AKT-dependent manner.

Objective: To investigate the effect and significance of electroacupuncture (EA) on neuronal apoptosis and hindlimb motor and bladder functional improvement in rats with acute spinal cord injury (SCI). Methods: Sixty healthy Sprague Dawley rats were randomly assigned to sham, model, EA, and EA control groups (n = 15 each). EA group rats received EA treatment at Zhibian and Shuidao acupoints seven times daily, whereas EA control group rats received EA at two points, 0.5 cm away from Zhibian and Shuidao, respectively. Histomorphological changes in spinal cord tissue were examined using hematoxylin-eosin staining. Neuronal apoptosis was detected by TUNEL assay. Bcl-2, Bax, and Bad protein levels were detected using immunohistochemistry. Additionally, hindlimb motor function, residual urine volume and maximum bladder capacity were measured. Results: HE staining revealed a morphologically and structurally intact spinal cord in the EA group, and the tissue contained scattered blood cells without edema. In the EA control group, there were small morphological defects in the spinal cord, and the tissue contained fewer blood cells with local edema. Compared with the EA control and model groups, Bax and Bad levels were significantly decreased in the EA group and Bcl-2 expression was increased (P < 0.05). After SCI, hindlimb function scores, residual urine volume, and maximum bladder capacity in rats of the EA group significantly differed from those of the EA control group (P < 0.05). Conclusion: EA may induce SCI-induced improvements in hindlimb motor and bladder functions by affecting neuronal apoptosis and relevant gene expression changes.

The study investigate three kinds of interfacial patterns (convex, corrugated, grid-like) were designed so as to improve the interfacial bonding strengths of hydroxyapatite (HA)/ZrO2 biomaterials. The thicknesses of prepared HA coatings were 50–250 μm. Both shear and tensile bonding strengths of the composites with different interfacial patterns were improved compared with the tiled samples because of the different contact geometry. The analysis on the fracture surfaces by SEM and XRD confirmed that the adhesive modes of HA and ZrO2 were the combination of mechanical and physical bonding. The research is beneficial to the clinical application of HA for a layered composite.

Surgery is the preferred treatment for intertrochanteric fractures. This study aimed to analyze risk factors for predicting mortality after intertrochanteric hip fracture surgery by arthroplasty and internal fixation. We performed a retrospective analysis of 1,263 patients who received surgical treatment for intertrochanteric fractures from January 2005 to December 2010. The clinical and follow-up data from 491 eligible patients were collected and analyzed. The age, activity before surgery, activity after surgery, anesthesia, medical illness, and the time between fracture and surgery were significantly associated with the mortality as single factor. Multifactor analysis showed that the activity after surgery, medical illness, and the time between fracture and surgery were correlated with postoperative mortality. In conclusion, these data suggest that the age, activity after surgery, medical illness could affect postoperative mortality of patients of intertrochanteric fractures. These factors may be used to predict the postoperative mortality of intertrochanteric fractures.

To obtain rat hair follicle stem cells (rHFSCs) which can constantly and highly express vascular endothelial growth factor 165 (VEGF165), and to observe the expression of VEGF165 gene in rat HFSCs.The cirri skin of 1-week-old Sprague Dawley rat was harvested and digested by using combination of Dispase and type IV collagenases. The bulge was isolated under microscope. The rHFSCs were cultured by tissue block method. After purified by rapid adhering on collagen type IV, the growth curve of different generations rHFSCs was drawn. The cells were identified by immunofluorescence staining and real time quantitative PCR (RT-qPCR) analysis that tested the expression level of correlated genes. Lentivirus of pLV-internal ribosome entry site (IRES)-VEGF165-enhanced green fluorescent protein (EGFP) (experimental group) and pLV-IRES-EGFP empty vector (control group) was packaged by calcium transfected method and the rHFSCs were transfected. The green fluorescent protein expression was observed by inverted fluorescence microscope, and VEGF165 mRNA and protein expressions were detected using RT-PCR and Western blot.The rHFSCs which were isolated, cultured, and purified were like the "slabstone", and had strong adhesion ability and colony formation ability. The purified cells were in latent growth phase at 2-3 days; they were in exponential growth phase at 5-6 days. The expressions of cytokeration 15 (CK15), integrin alpha6, and integrin beta1 (markers of HFSCs) were positive by immunocytochemistry. The RT-qPCR analysis showed that CK15, CK19, integrin alpha6, and integrin beta1 expressed highly, but CD34 (a marker of epidermal stem cells) and CK10 (a marker of keratinocyte) expressed lowly. After 14 days, the transfection efficiency was up to 85.76% +/- 1.91%. RT-PCR analysis and Western blot showed that VEGF165 mRNA and protein expressions were positive in experimental group, and were negative in control group.The rHFSCs with high purity and strong proliferation ability can be obtained by using microscope combined with tissue cultivation and rapid cell adhesion on collagen type IV. The rHFSCs with high expression of VEGF165 can be successfully obtained by lentiviral transfection. This method provides good seeding cells for tissue engineering to construct artificial hair follicles, blood vessels, and skins.

Acute spinal cord injury is one of the most common and complicated diseases among human spinal injury. We aimed to explore the effect of point-through-point acupuncture therapy with elongated needles on acute spinal cord injury in rabbits and its possible mechanism. Adult rabbits were randomly divided into a model group, elongated needle therapy group, and blank group. Immunohistochemical staining showed that the protein levels of Fas and caspase-3 in the model group were significantly higher than those in the blank group at each time point (P<0.05) and significantly lower than those in the elongated needle therapy group on the 3rd and 5th days after operation (P<0.05). RT-PCR showed that Fas and caspase-3 mRNA levels in the model group and elongated needle therapy group were significantly higher than those in the blank group (P<0.05, 0.01). The mRNA levels of Fas and caspase-3 in the elongated needle therapy group were significantly lower than those in model group on the 3rd day (P<0.05, 0.01). Therefore, we confirmed that elongated needle therapy has an obvious effect on acute spinal cord injury in rabbits. Its mechanism is made possible by inhibiting the expression of the Fas→caspase-3 cascade, thereby inhibiting cell apoptosis after spinal cord injury.