Connective Tissue Research最新文献

Purpose: Non-weight bearing improves and immobilization worsens contracture induced by anterior cruciate ligament reconstruction (ACLR), but effect persistence after reloading and remobilization remains unclear, and the combined effects of these factors on ACLR-induced contracture are unknown. We aimed to determine 1) whether the effects of short-term (2-week) non-weight bearing or immobilization after ACLR on contracture would be sustained by reloading or remobilization during a 10-week observation period, and 2) how the combination of both interventions compared to the outcome of either alone.

Methods: We divided 88 ACL-reconstructed male rats into four groups: non-intervention, non-weight bearing, joint immobilization, and both interventions. Interventions were performed for 2 weeks, followed by rearing without intervention. Twelve untreated rats were used as controls. At 2, 4, and 12 weeks post-surgery, we assessed range of motion (ROM) and histological changes.

Results: ACLR resulted in persistent loss of ROM, accompanied by synovial shortening, capsule thickening, and osteophyte formation. Two weeks of non-weight bearing increased ROM and reduced osteophyte size, but the beneficial effects disappeared within 10 weeks after reloading. Two-week immobilization decreased ROM and facilitated synovial shortening. After remobilization, ROM partially recovered but remained below non-intervention levels at 12 weeks. When both interventions were combined, ROM was similar to immobilization alone.

Conclusions: The beneficial effects of 2-week non-weight bearing on contracture diminished within 10 weeks after reloading. The adverse effects of 2-week immobilization on contracture persisted after 10 weeks of remobilization. The effects of the combined use of both interventions on contracture were primarily determined by immobilization.

Objective: Osteoporosis, a skeletal ailment marked by bone metabolism imbalance and disruption of bone microarchitecture, Neferine, a bisbenzylisoquinoline alkaloid with diverse pharmacological activities, has received limited attention in the context of osteoporosis treatment.

Methods: We employed a bilateral ovariectomy (OVX) rat model to induce osteoporosis and subsequently administered Neferine treatment for four weeks following successful model establishment. Throughout the modeling and treatment phases, we closely monitored rat body weights. We assessed alterations in bone tissue microstructure through micro-CT, HE staining, and safranin O-fast green staining. Levels of bone formation and resorption markers in serum were evaluated using ELISA assay. Western blot analysis was employed to determine the expression levels of p38MAPK, p-p38MAPK, and bone formation-related genes in bone tissue. We isolated and cultured OVX rat BMSCs (OVX-BMSCs) and induced osteogenic differentiation while simultaneously introducing Neferine and the p38MAPK inhibitor SB203580 for intervention.

Results: Neferine treatment effectively curbed the rapid weight gain in OVX rats, ameliorated bone loss, and decreased serum levels of TRAP, CTX-I, PINP, and BALP. Most notably, Neferine promoted the expression of bone formation-related factors in bone tissue of OVX rats, while concurrently activating the p38MAPK signaling pathway. In in vitro experiments, Neferine facilitated the expression of bone formation-related factors in OVX-BMSCs, increased the osteogenic differentiation potential of OVX-BMSCs, and activated the p38MAPK signaling pathway. Nevertheless, SB203580 partially reversed Neferine's promotive effect.

Conclusion: Neferine can boost the osteoblastic differentiation of BMSCs and alleviate OVX-induced osteoporosis in rats by activating the p38MAPK signaling pathway.

Purpose/aim of study: During the development of the vertebrate skeleton, the progressive differentiation and maturation of chondrocytes from mesenchymal progenitors is precisely coordinated by multiple secreted factors and signaling pathways. The WNT signaling pathway has been demonstrated to play a major role in chondrogenesis. However, the identification of secreted factors that fine-tune WNT activity has remained elusive. Here, in this study, we have identified PI15 (peptidase inhibitor 15, protease Inhibitor 15, SugarCrisp), a member of the CAP (cysteine rich secretory proteins, antigen 5, and pathogenesis related 1 proteins) protein superfamily, as a novel secreted WNT antagonist dynamically upregulated during chondrocyte differentiation.

Materials and methods: ATDC5 cells, C3H10T1/2 micromass cultures and primary chondrocyte cells were used as in vitro models of chondrogenesis. PI15 levels were stably depleted or overexpressed by viral shRNA or expression vectors. Chondrogenesis was evaluated by qPCR gene expression analysis and Alcian blue staining. Protein interactions were determined by coimmunoprecipitation assays.

Results and conclusions: shRNA-mediated knockdown of PI15 in ATDC5 cells, C3H10T1/2 cells or primary chondrocytes inhibits chondrogenesis, whereas the overexpression of PI15 strongly enhances chondrogenic potential. Mechanistically, PI15 binds to the LRP6 WNT co-receptor and blocks WNT-induced LRP6 phosphorylation, thus repressing WNT-induced transcriptional activity and alleviating the inhibitory effect of WNT signaling on chondrogenesis. Altogether, our findings suggest that PI15 acts as a key regulator of chondrogenesis and unveils a mechanism through which chondrocyte-derived molecules can modulate WNT activity as differentiation proceeds, thereby creating a positive feedback loop that further drives differentiation.

Purpose: This study aimed to evaluate whether cilostazol (phosphodiesterase III inhibitor) could enhance the healing of Achilles tendon ruptures in rats.

Materials and methods: The Achilles tendons of 24 healthy male adult rats were incised and repaired. The rats were randomly allocated to cilostazol and control groups. The cilostazol group received daily intragastric administration of 50 mg/kg cilostazol for 28 days, while the control group did not receive any medication. The rats were sacrificed on the 30th day, and the Achilles tendon was evaluated for biomechanical properties, histopathological characteristics, and immunohistochemical analysis.

Results: All rats completed the experiment. The Movin sum score of the control group was significantly higher (p = 0.008) than that of the cilostazol group, with means of 11 ± 0.63 and 7.50 ± 1.15, respectively. Similarly, the mean Bonar score was significantly higher (p = 0.026) in the control group compared to the cilostazol group (8.33 ± 1.50 vs. 5.5 ± 0.54, respectively). Moreover, the Type I/Type III Collagen ratio was notably higher (p = 0.016) in the cilostazol group (52.2 ± 8.4) than in the control group (34.6 ± 10.2). The load to failure was substantially higher in the cilostazol group than in the control group (p = 0.034), suggesting that the tendons in the cilostazol group were stronger and exhibited greater resistance to failure.

Conclusions: The results of this study suggest that cilostazol treatment significantly improves the biomechanical and histopathological parameters of the healing Achilles tendon in rats. Cilostazol might be a valuable supplementary therapy in treating Achilles tendon ruptures in humans. Additional clinical studies are, however, required to verify these outcomes.

Purpose: Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells.

Materials and methods: Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted.

Results: Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis.

Conclusion: This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy.

Objective: The COL1A1 proximal promoter contains two GC-rich regions and two inverted CCAAT boxes. The transcription factors Sp1 and CBF bind to the GC sequence at -122 to -115 bp and the inverted CCAAT box at -101 to -96 bp, respectively, and stimulate COL1A1 transcriptional activity.

Methods: To further define the regulatory mechanisms controlling COL1A1 expression by Sp1 and CBF, we introduced 2, 4, 6, or 8 thymidine nucleotides (T-tracts) at position -111 bp of the COL1A1 gene promoter to increase the physical distance between these two binding sites and examined in vitro the transcriptional activities of the resulting constructs and their response to TGF-β1.`.

Results: Insertion of 2 or 4 nucleotides decreased COL1A1 promoter activity by up to 70%. Furthermore, the expected increase in COL1A1 transcription in response to TGF-β1 was abolished. Computer modeling of the modified DNA structure indicated that increasing the physical distance between the Sp1 and CBF binding sites introduces a rotational change in the DNA topology that disrupts the alignment of Sp1 and CBF binding sites and likely alters protein-protein interactions among these transcription factors or their associated co-activators.

Conclusion: The topology of the COL1A1 proximal promoter is crucial in determining the transcriptional activity of the gene and its response to the stimulatory effects of TGF-β1.

Background: Osteoarthritis (OA) is a degenerative joint disease that affects millions worldwide. Synovitis and macrophage polarization are important factors in the development of OA. However, the specific components of synovial fluid (SF) responsible for promoting macrophage polarization remain unclear.

Methods: Semi-quantitative antibody arrays were used to outline the proteome of SF. Differential expression analysis and GO/KEGG were performed on the obtained data. Immunohistochemistry and ELISA were used to investigate the relationship between SF S100A12 levels and synovitis levels in clinalclinical samples. In vitro cell experiments were conducted to investigate the effect of S100A12 on macrophage polarization. Public databases were utilized to predict and construct an S100A12-centered lncRNA-miRNA-mRNA competing endogenous RNA network, which was preliminarily validated using GEO datasets.

Results: The study outlines the protein profile in OA and non-OA SF. The results showed that the S100A12 level was significantly increased in OA SF and inflammatory chondrocytes. The OA synovium had more severe synovitis and higher levels of S100A12 than non-OA synovium. Exogenous S100A12 upregulated the levels of M1 markers and phosphorylated p65 and promoted p65 nuclear translocation, while pretreatment with BAY 11-7082 reversed these changes. It was also discovered that LINC00894 was upregulated in OA and significantly correlated with S100A12, potentially regulating S100A12 expression by acting as a miRNA sponge.

Conclusions: This study demonstrated that S100A12 promotes M1 macrophage polarization through the NF-κB pathway, and found that LINC00894 has the potential to regulate the expression of S100A12 as a therapeutic approach.

Osteoarthritis (OA) is a multifactorial joint disease characterized by articular cartilage degradation. Risk factors for OA include joint trauma, obesity, and inflammation, each of which can affect joint health independently, but their interaction and the associated consequences of such interaction were largely unexplored. Here, we studied compositional and structural alterations in knee joint cartilages of Sprague-Dawley rats exposed to two OA risk factors: joint injury and diet-induced obesity. Joint injury was imposed by surgical transection of anterior cruciate ligaments (ACLx), and obesity was induced by a high fat/high sucrose diet. Depth-dependent proteoglycan (PG) content and collagen structural network of cartilage were measured from histological sections collected previously in Collins et al.. (2015). We found that ACLx primarily affected the superficial cartilages. Compositionally, ACLx led to reduced PG content in lean animals, but increased PG content in obese rats. Structurally, ACLx caused disorganization of collagenous network in both lean and obese animals through increased collagen orientation in the superficial tissues and a change in the degree of fibrous alignment. However, the cartilage degradation attributed to joint injury and obesity was not necessarily additive when the two risk factors were present simultaneously, particularly for PG content and collagen orientation in the superficial tissues. Interestingly, sham surgeries caused a through-thickness disorganization of collagen network in lean and obese animals. We conclude that the interactions of multiple OA risk factors are complex and their combined effects cannot be understood by superposition principle. Further research is required to elucidate the interactive mechanism between OA subtypes.