Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

Nanofat lysate ameliorates pain and cartilage degradation of osteoarthritis through activation of TGF-ß-Smad2/3 signaling of chondrocytes.

Introduction: Nanofat is an effective cell therapy for osteoarthritis (OA). However, it has clinical limitations due to its short half-life. We developed Nanofat lysate (NFL) to overcome the defect of Nanofat and explore its anti-OA efficacy and mechanism. Methods: Monoiodoacetate (MIA) was employed to establish rat OA model. For pain assessment, paw withdrawal latency (PWL) and thermal withdrawal latency (TWL) were evaluated. Degeneration of cartilage was observed by histopathological and immunohistochemical examination. Primary chondrocytes were treated with TNF-α to establish the cellular model of OA. MTT, wound healing, and transwell assays were performed to assess effects of NFL on chondrocytes. RNA-seq, qPCR and Western blot assays were conducted to clarify the mechanism of NFL. Results and Discussion: The animal data showed that PWL and TWL values, Mankin's and OARSI scorings, and the Col2 expression in cartilage were significantly improved in the NFL-treated OA rats. The cellular data showed that NFL significantly improved the proliferation, wound healing, and migration of chondrocytes. The molecular data showed that NFL significantly restored the TNF-α-altered anabolic markers (Sox9, Col2 and ACAN) and catabolic markers (IL6 and Mmp13). The RNA-seq identified that TGF-ß-Smad2/3 signaling pathway mediated the efficacy of NFL, which was verified by qPCR and Western blot that NFL significantly restored the abnormal expressions of TGFßR2, phosphorylated-Smad2, phosphorylated-Smad2/3, Col2, Mmp13 and Mmp3. After long-term storage, NFL exerted similar effects as its fresh type, indicating its advantage of storability. In sum, NFL was developed as a new therapeutic approach and its anti-OA efficacy and mechanism that mediated by TGF-ß-Smad2/3 signaling was determined for the first time. Besides, the storability of NFL provided a substantial advantage than other living cell-based therapies.

Intra-articular injection of placental mesenchymal stromal cells ameliorates pain and cartilage anabolism/catabolism in knee osteoarthritis.

Background: Knee Osteoarthritis (kOA), the most common joint degenerative disorder, lacks effective therapeutics. Placenta-derived mesenchymal stromal cells (PMSCs) are effective in tissue repairing and generation, which have potential in treating kOA. This study aimed to determine the anti-kOA efficacy of PMSCs and to explore its action mode. Methods: Flow cytometry and three-line differentiation were performed for identification of PMSCs. In vivo, a rat kOA model established by anterior cruciate ligament transection (ACLT) surgery was used to evaluate the efficacy of PMSCs. Histopathological HE and SO staining with Osteoarthritis Research Society International scoring were conducted, and cartilage expressions of MMP13 and Col2 were measured by immunohistochemistry. Pain behavior parameters by mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL), were measured. In vitro, wound healing and cell immunofluorescence assays were conducted to detect the proliferation and migration ability of chondrocytes treated with PMSCs conditioned medium (PMSCs-CM). Quantitative real-time PCR (qRT-PCR) and Western blot (WB) assays were applied to explore the molecular action of PMSCs on chondrocytes. Results: The results of flow cytometry indicated that the surface markers of PMSCs (CD73 > 95%, CD90 > 95%, and CD34 < 2%) were consistent with the typical mesenchymal stromal cells. The in vivo data showed that PMSCs significantly reversed the kOA progression by protection of cartilage, regulation of anabolic (Col2) and catabolic (MMP13) expressions, and relief of pain symptoms. The in vitro data showed that PMSCs promoted chondrocyte proliferation and migration and significantly restored the IL-1ß-induced abnormal gene expressions of Col2, Mmp13, Adamts4, Adamts5 and Sox9 and also restored the abnormal protein expressions of Col2, Mmp13 and Sox9 of chondrocytes. The molecular actions of PMSCs on chondrocytes in nested co-culture way or in conditioned medium way were similar, confirming a paracrine-based mode of action. Conclusion: This study demonstrated PMSCs' anti-kOA efficacy and its paracrine-based action mode, providing novel knowledge of PMSCs and suggesting it as a promising cell therapy for treatment of kOA.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Ameliorate Skin Aging of Nude Mice Through Autophagy-Mediated Anti-Senescent Mechanism.

Skin aging is a currently irreversible process, affected by increased oxidative stress, activated cellular senescence, and lacked regeneration of the dermal layer. Mesenchymal stem cells (MSCs), such as human umbilical cord-derived MSCs (hucMSCs), have pro-regeneration and anti-aging potencies. To explore whether hucMSCs can be used to treat skin aging, this study employed skin-aging model of nude mice to conduct in vivo assays, including biochemical analysis of superoxide dismutase (SOD) and malondialdehyde (MDA), gross observation, histopathological observation, and immunohistochemical analysis. To clarify how hucMSCs work on skin aging, this study employed skin-aging model of human dermal fibroblasts (HDFs) to conduct in vitro assays by applying conditional medium of hucMSCs (CMM), including wound healing assay, senescence staining, flow cytometric oxidative detection, real time PCR, and western blot analysis. The in vivo data demonstrated that hucMSCs dose-dependently removed wrinkles, smoothed skin texture, and increased dermal thickness and collagen production of aged skin by reversing SOD and MDA levels and up-regulating Col-1 and VEGF expressions, indicating anti-oxidative and pro-regenerative effects against skin aging. The in vitro data revealed that hucMSCs significantly reversed the senescence of HDFs by promoting cell migration, inhibiting ROS production, and restoring the overexpressions of oxidative and senescent markers through paracrine mode of action, and the paracrine mechanism was mediated by the inhibition of autophagy. This study provided novel knowledge regarding the anti-aging efficacy and paracrine mechanism of hucMSCs on skin, making hucMSCs-based therapy a promising regime for skin aging treatment.

Intra-Articular Injection of Adipose-Derived Stem Cells Ameliorates Pain and Cartilage Anabolism/Catabolism in Osteoarthritis: Preclinical and Clinical Evidences.

Background: Osteoarthritis (OA) is the most common joint disorder, lacking disease-modifying treatments. Adipose-derived mesenchymal stem cells (ADSCs) are adult multipotent stromal cells obtained from fat tissue, which holds great potential in treating OA. This study aimed to evaluate the anti-OA efficacy of ADSCs from preclinical and clinical facets and explore the underlying mechanism of action. Methods: In vivo, a single dose of 5 × 105 ADSCs was injected into the knee joints of monoiodoacetate-induced OA rat model. The levels of metabolic and hypertrophic molecules (MMP13, Collagen II, Collagen X) of chondrocytes were measured by immunohistochemistry. In vitro, cell viability assay was conducted to detect the proliferation ability of chondrocytes treated with ADSCs conditioned medium (ADSCs-CM). Quantitative real-time polymerase chain reaction and Western blot assays were applied to explore the mechanism of action of ADSCs. Moreover, a retrospective analysis was conducted to determine the clinical efficacy and safety of ADSCs on OA patients. Results: The animal study showed that ADSCs significantly alleviated OA cartilage lesions in rats, as was confirmed by downregulation of the MMP13 and Collagen X and upregulation of the Collagen II. In vitro data showed that ADSCs-CM promoted the proliferation of chondrocytes, and significantly restored the IL-1ß-induced abnormal expressions of molecular markers IL-6, Aggrecan, MMP3, MMP13, Collagen II, Collagen X, ADAMTS5, ADAMTS9, SOX6, and SOX9 in chondrocytes. Such regulatory effects of ADSCs-CM on the proliferation and these anabolic, catabolic, and hypertrophic markers of chondrocytes suggested a paracrine-based mode of action of ADSCs. Furthermore, the clinical data showed that ADSCs reduced pain and repaired cartilage damage in OA patients, with no adverse events. Conclusion: This study demonstrated the anti-OA efficacy, safety, and a paracrine-based mechanism of ADSCs, providing a promising cell-based therapeutic option for OA treatment.

Fuzi decoction ameliorates pain and cartilage degeneration of osteoarthritic rats through PI3K-Akt signaling pathway and its clinical retrospective evidence.

BACKGROUND: Osteoarthritis (OA) is a difficult disease but the clinic lacks effective therapy. As a classic formula of traditional Chinese medicine (TCM), Fuzi decoction (FZD) has been clinically applied for treating OA-related syndromes, but its anti-OA efficacy and mechanism remain unclear. PURPOSE: To experimentally and clinically determine the anti-OA efficacy of FZD and clarify the underlying mechanism. METHODS: UPLC/MS/MS was applied to identify the main components of FZD. A monoiodoacetate (MIA)-induced OA rat model was employed to evaluate the in vivo efficacy of FZD against OA, by using pain behavior assessment, histopathological observation, and immunohistochemical analysis. Primary rat chondrocytes were isolated to determine the in vitro effects of FZD by using cell viability assay, wound healing assay, and real-time PCR (qPCR) analysis on anabolic/catabolic mRNA expressions. RNA sequencing (RNA-seq) and network pharmacology analysis were conducted and the overlapping data were used to predict the mechanism of FZD, followed by verification with qPCR and Western blot assays. Finally, a retrospective analysis was performed to confirm FZD's efficacy and safety in OA patients. RESULTS: The UPLC/MS/MS result showed that FZD contained atractylenolide I, benzoylhypaconitine, benzoylmesaconitine, benzoylaconitine, hypaconitine, mesaconitine, aconitine, lobetyolin, paeoniflorin, and pachymic acid. The in vivo data showed that FZD restored the cartilage degeneration in MIA-induced OA rats by ameliorating pain behavior parameters, recovering histopathological alterations, benefitting cartilage anabolism (up-regulating Col2 expression), and suppressing catabolism (down-regulating MMP13 and Col10 expressions). The in vitro data showed that FZD increased cell viability and wound healing capacity of chondrocytes, and restored the altered expressions of anabolic and catabolic genes of chondrocytes. The overlapping results of RNA-seq and network pharmacology analysis suggested that PI3K/Akt signaling mediated the anti-OA mechanism of FZD, which was verified by qPCR and Western blot experiments. Clinically, the anti-OA efficacy and safety of FZD were confirmed by the retrospective analysis on OA patients. CONCLUSION: The scientific innovation of this study was the determination of anti-OA efficacy of FZD by experimental and clinical evidence and the discovery of its mechanism by integrated RNA-seq, network pharmacology, and molecular experiments, which suggests FZD as a promising TCM agency for OA treatment.

Network Pharmacology and Experimental Validation to Reveal the Pharmacological Mechanisms of Liuwei Dihuang Decoction Against Intervertebral Disc Degeneration.

PURPOSE: To explore the pharmacological mechanisms of Liuwei Dihuang Decoction (LWDHD) against intervertebral disc (IVD) degeneration (IVDD) via network pharmacology analysis combined with experimental validation. METHODS: First, active ingredients and related targets of LWDHD, as well as related genes of IVDD, were collected from public databases. The protein-protein interaction (PPI) network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed to predict the core targets and pathways of LWDHD against IVDD. Secondly, the IVDD model of mice treated with LWDHD was selected to validate the major targets predicted by network pharmacology. RESULTS: By searching the intersection of the active ingredient targets and IVDD targets, a total of 110 targets matched the related targets of 30 active ingredients in LWDHD and IVDD were retrieved. PPI network analysis indicated that 17 targets, including Caspase-3, IL-1ß, P53, etc., were hub targets. GO and KEGG enrichment analyses showed that the apoptosis pathway was enriched by multiple targets and served as the target for in vivo experimental study validation. The results of animal experiments revealed that LWDHD administration not only restored the decrease in disc height and abnormal degradation of matrix metabolism in IVDD mice but also reversed the high expression of Bax, Caspase-3, IL-1ß, P53, and low expression of Bcl-2, thereby inhibiting the apoptosis of IVD tissue and ameliorating the progression of IVDD. CONCLUSION: Using a comprehensive network pharmacology approach, our findings predicted the active ingredients and potential targets of LWDHD intervention for IVDD, and some major target proteins involved in the predictive signaling pathway were validated experimentally, which gave us a new understanding of the pharmacological mechanism of LWDHD in treating IVDD at the comprehensive level.

A Novel Mutation of the KLK6 Gene in a Family With Knee Osteoarthritis.

To investigate the correlation between gene mutation and knee osteoarthritis (KOA), a whole-exome sequencing (WES) was applied to analyze blood samples of four KOA patients and two normal subjects in a family. Gene mutations were identified by gene-trapping and high-throughput sequencing analysis across the differences between the patients and normal subjects. The interactive gene network analysis on the retrieval of interacting genes (STRING) database and the KOA-related genes expression data sets was performed. A possibly detrimental and nonsynonymous mutation at the kallikrein-related peptidase 6 (KLK6) gene (rs201586262, c. C80A, P27H) was identified and attracted our attention. KLK6 belongs to the kallikrein family of serine proteases and its serum level is known as a prevalent biomarker in inflammatory and malignant diseases. KLK6 expresses in the extracellular compartment for matrix degradation, highlighting that KLK6 plays a role in the pathogenesis of KOA. By using the gene databases, the KOA-related genes were mined after de-duplication and IL6 was selected as the most relevant gene through interactive analysis of protein-protein interaction (PPI) network. The data suggested that KLK6 gene mutation and the related expression alteration of IL6 gene might determine the occurrence of hereditary KOA. The is the first study discovering the gene mutation of KLK6 as a factor of pathogenesis of KOA, especially the hereditary KOA.

Identification and validation of hub genes of synovial tissue for patients with osteoarthritis and rheumatoid arthritis.

BACKGROUND: Osteoarthritis (OA) and rheumatoid arthritis (RA) were two major joint diseases with similar clinical phenotypes. This study aimed to determine the mechanistic similarities and differences between OA and RA by integrated analysis of multiple gene expression data sets. METHODS: Microarray data sets of OA and RA were obtained from the Gene Expression Omnibus (GEO). By integrating multiple gene data sets, specific differentially expressed genes (DEGs) were identified. The Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and protein-protein interaction (PPI) network analysis of DEGs were conducted to determine hub genes and pathways. The "Cell Type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT)" algorithm was employed to evaluate the immune infiltration cells (IICs) profiles in OA and RA. Moreover, mouse models of RA and OA were established, and selected hub genes were verified in synovial tissues with quantitative polymerase chain reaction (qPCR). RESULTS: A total of 1116 DEGs were identified between OA and RA. GO functional enrichment analysis showed that DEGs were enriched in regulation of cell morphogenesis involved in differentiation, positive regulation of neuron differentiation, nuclear speck, RNA polymerase II transcription factor complex, protein serine/threonine kinase activity and proximal promoter sequence-specific DNA binding. KEGG pathway analysis showed that DEGs were enriched in EGFR tyrosine kinase inhibitor resistance, ubiquitin mediated proteolysis, FoxO signaling pathway and TGF-beta signaling pathway. Immune cell infiltration analysis identified 9 IICs with significantly different distributions between OA and RA samples. qPCR results showed that the expression levels of the hub genes (RPS6, RPS14, RPS25, RPL11, RPL27, SNRPE, EEF2 and RPL19) were significantly increased in OA samples compared to their counterparts in RA samples (P < 0.05). CONCLUSION: This large-scale gene analyses provided new insights for disease-associated genes, molecular mechanisms as well as IICs profiles in OA and RA, which may offer a new direction for distinguishing diagnosis and treatment between OA and RA.

A Systematic Review and Meta-Analysis of the SuperPATH Approach in Hip Arthroplasty.

OBJECTIVE: To compare the clinical and radiographic results of the supercapsular percutaneously assisted total hip (SuperPATH) approach and the conventional approach in hip arthroplasty. DESIGN: Based on a prepublished protocol (PROSPERO: CRD42020177717), we searched PubMed, Embase, and Cochrane for relevant literatures up to January 30, 2021. The methodological qualities were assessed using the guidelines provided by the Cochrane Collaboration for Systematic Reviews. Randomized- or fixed-effect models were used to calculate the weighted mean difference (WMD) or odds ratio (OR), respectively, for continuous and dichotomous variables. RESULTS: 6 articles were included in the study, and 526 patients were selected, which included 233 cases in the SuperPATH groups and 279 cases in the conventional groups, and 4 cases performed two surgeries in succession. The SuperPATH group demonstrated shorter incision length (WMD = -7.87, 95% CI (-10.05, -5.69), P < 0.00001), decreased blood transfusion rate (OR = 0.48, 95% CI (0.25, 0.89), P = 0.02), decreased visual analogue scale (VAS) (WMD = -0.40, 95% CI (-0.72, -0.08), P = 0.02), and higher Harris hip score (HHS) (WMD = 1.98, 95% CI (0.18, 3.77), P = 0.03) than the conventional group. However, there was no difference in VAS (P = 0.14) and HHS (P = 0.86) between the two groups 3 months later, nor in the acetabular abduction angle (P = 0.32) in either group. CONCLUSIONS: SuperPATH, as a minimally invasive approach with its reduced tissue damage, quick postoperative recovery, and early rehabilitation, demonstrates the short-term advantages of hip arthroplasty. As the evidences in favor of the SuperPATH technique were limited in a small number of studies and short duration of follow-up, more research is required to further analyze its long-term effect.

Pain relief and cartilage repair by Nanofat against osteoarthritis: preclinical and clinical evidence.

BACKGROUND: Osteoarthritis (OA) is the most common joint degenerative disorder, with little effective therapy to date. Nanofat is a cocktail of cells obtained from fat tissue, which possesses regenerative capacity and has a potential in treating OA. This study aimed to determine the anti-OA efficacy of Nanofat from basic and clinical aspects and explore its action mode. METHODS: Flow cytometry was performed to characterize Nanofat. A monoiodoacetate-induced OA rat model was employed for in vivo study. Cell viability and wound healing assays were conducted for in vitro study. Real-time PCR and Western blot assays were applied to explore the molecular action mode of Nanofat. Moreover, a retrospective analysis was conducted to determine the clinical efficacy and safety of Nanofat on knee OA patients. RESULTS: The in vivo results showed that Nanofat significantly attenuated pain symptoms and protected cartilage ECM (Col2) from damage, and its effects were not significantly differed with adipose tissue-derived stem cells (both P > 0.05). The in vitro results showed that Nanofat promoted the cell viability and migration of chondrocytes and significantly restored the IL-1ß-induced abnormal gene expressions of Col2, Aggrecan, Sox9, Adamts5, Mmp3, Mmp9 Mmp13, IL-6 and Col10 and protein expressions of Col2, MMP9, MMP13, and Sox9 of chondrocytes. The regulatory actions of Nanofat on these anabolic, catabolic, and hypertrophic molecules of chondrocytes were similar between two treatment routes: co-culture and conditioned medium, suggesting a paracrine-based mode of action of Nanofat. Moreover, the clinical data showed that Nanofat relieved pain and repaired damaged cartilage of OA patients, with no adverse events. CONCLUSION: In sum, this study demonstrated the anti-OA efficacy as well as a paracrine-based action mode of Nanofat, providing novel knowledge of Nanofat and suggesting it as a promising and practical cell therapy for clinical treatment of OA.

Identification of differentially expressed genes, signaling pathways and immune infiltration in rheumatoid arthritis by integrated bioinformatics analysis.

BACKGROUND: The disability rate associated with rheumatoid arthritis (RA) ranks high among inflammatory joint diseases. However, the cause and potential molecular events are as yet not clear. Here, we aimed to identify differentially expressed genes (DEGs), pathways and immune infiltration involved in RA utilizing integrated bioinformatics analysis and investigating potential molecular mechanisms. MATERIALS AND METHODS: The expression profiles of GSE55235, GSE55457, GSE55584 and GSE77298 were downloaded from the Gene Expression Omnibus database, which contained 76 synovial membrane samples, including 49 RA samples and 27 normal controls. The microarray datasets were consolidated and DEGs were acquired and further analyzed by bioinformatics techniques. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were performed using R (version 3.6.1) software, respectively. The protein-protein interaction (PPI) network of DEGs were developed utilizing the STRING database. Finally, the CIBERSORT was used to evaluate the infiltration of immune cells in RA. RESULTS: A total of 828 DEGs were recognized, with 758 up-regulated and 70 down-regulated. GO and KEGG pathway analyses demonstrated that these DEGs focused primarily on cytokine receptor activity and relevant signaling pathways. The 30 most firmly related genes among DEGs were identified from the PPI network. The principal component analysis showed that there was a significant difference between the two tissues in infiltration immune. CONCLUSION: This study shows that screening for DEGs, pathways and immune infiltration utilizing integrated bioinformatics analyses could aid in the comprehension of the molecular mechanisms involved in RA development. Besides, our study provides valuable data related to DEGs, pathways and immune infiltration of RA and may provide new insight into the understanding of molecular mechanisms.