Connective Tissue Research最新文献

Purpose: Dapagliflozin (DAP), an SGLT2 inhibitor commonly prescribed for type 2 diabetes, has been recognized for its anti-inflammatory and antioxidative effects in various disease contexts. However, its impact on hyperlipidemic tenocytes-particularly within the framework of obesity-induced tendinopathy-remains underexplored. This study investigated the protective role of DAP in palmitate-exposed tenocytes, which simulate lipid-induced tendon degeneration.

Methods: Protein expression was analyzed via Western blotting, while apoptosis was assessed through cell viability assays, caspase-3 activity, and TUNEL staining. Oxidative stress was evaluated through the quantification of H₂O₂, malondialdehyde (MDA), and reactive oxygen species (ROS). PPARα gene silencing was conducted via siRNA transfection.

Results: DAP treatment significantly attenuated apoptosis and oxidative stress, restored the extracellular matrix (ECM) balance, and enhanced tenocyte migration. These protective effects were associated with the upregulation of PPARα, PGC1α, and Nrf2, along with increased activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase. Notably, silencing PPARα negated the beneficial effects of DAP, underscoring its central role. Furthermore, irisin-a myokine upregulated by DAP in myocytes-was also found to reduce oxidative stress and apoptosis in palmitate-treated tenocytes.

Conclusions: This study provides novel insights into the mechanistic actions of DAP in musculoskeletal repair and highlights its potential in mitigating the cellular consequences of metabolic stress. By advancing therapeutic strategies rooted in metabolic regulation and cellular resilience, these findings support the development of safer, more effective interventions for chronic degenerative conditions associated with obesity.

Purpose: Cartilage-derived chondroprogenitors, with inherent chondrogenic capacity and low hypertrophic potential, represent a promising avenue for cartilage regeneration. For clinical translation, assessment of cellular genomic stability is a quality control mandate. Since culturing cells to higher passage numbers for achieving the cell requirement is indispensable, it is necessary to evaluate the possibility of culture-driven mutations before transplantation. Being a relatively newly discovered cell subset, the information on the genetic profile of these cartilage-resident cells is notably limited.

Methods: The study investigated the genomic stability of fibronectin adhesion assay-derived chondroprogenitors(FAA-CP), migratory chondroprogenitors(MCP) and chondrocytes (n = 3). Conventional karyotyping and microarray analysis were performed on early and late passage cells to assess their genomic integrity under standard culture conditions and any groups that showed variations were further evaluated for their tumorigenic potential using the soft-agar assay.

Results: Chondrocytes exhibited a higher propensity for culture-induced genetic aberrations, including chromosomal losses, gains, inversions, and translocations. In contrast, both the chondroprogenitor groups demonstrated greater genomic stability throughout culture, with an instance of Trisomy-7 observed in early passage and a loss of gonosome in the later passage MCP group. Microarray analysis of chondroprogenitors showed a normal genomic profile, and soft agar assays indicated a non-tumorigenic profile for all cell groups that showed abnormal cytogenetic profiles.

Conclusions: The study highlights the importance of distinguishing between inherent genetic abnormalities and those acquired during culture, particularly when considering cells for therapeutic applications. While the observed genetic variations did not confer tumorigenic potential, careful consideration is essential prior to therapy.

Low back pain (LBP), one of the most common health problems, is the leading cause of disability globally. Intervertebral disc degeneration (IDD) accounts for most LBP. However, the molecular mechanism underlying IDD remains unclear, and the existing treatment strategy for IDD is still limited. A growing body of evidences suggest that the Hedgehog (HH) pathway plays an essential role in the formation, maintenance, and degeneration of intervertebral discs (IVDs), with Sonic HH (SHH) being primarily involved in the development and maturation of the IVDs and a strong link between Indian HH(IHH) and disc calcification. This review provides an overview of the role of the HH signaling pathway in the developmental maturation and degeneration of IVDs and suggests potential therapeutic targets for IDD that may interfere with HH signaling.

Purpose/aim: Dysregulation of well-ordered chondrocyte proliferation and differentiation leads to distorted architecture of the growth plate, resulting in skeletal dysplasia with impaired longitudinal bone growth. Histone deacetylase 4 (HDAC4) is essential for chondrocyte hypertrophy and endochondral bone formation, but its role in postnatal bone development remains unexplored due to early lethality in Hdac4-ablated mice. Furthermore, a direct in vivo effect of Hdac4 on mesenchymal cell specification and bone development has not been investigated.

Materials and methods: We generated Prx1-Cre;Hdac4^fl/fl, Sp7-Cre;Hdac4^fl/fl, Acan-CreERT2;Hdac4^fl/fl, and Hdac4^fl/fl transgenic mice, respectively. The genotyping of transgenic mice was performed via conventional PCR. Whole-body radiographs and x-ray analyses of limbs were conducted. Trabecular and cortical bone microstructures of tibias from 21-day-old mice were evaluated using micro-computed tomography. EdU label-retention assay investigated cell proliferation, while histological analyses included H&E, TRAP, and Von Kossa staining. RT-qPCR and Immunohistochemistry to detect the pro-osteogenic function of HDAC4.

Results: Hdac4 inactivation in limb mesenchyme cells resulted in limb shortening, premature growth plate closure, abnormal bone morphologies, and loss of the rounded articular surface. HDAC4 was crucial for regulating chondrocyte proliferation and secondary ossification center formation. Micro-computed tomography showed increased trabecular and cortical bone Prx1-Cre;Hdac4^fl/fl mice at 3 weeks, with altered microarchitecture. .

Conclusions: Hdac4 in limb mesenchymal cells plays an indispensable role in chondrocyte proliferation, maintenance of the growth plate and formation of secondary ossification centers, its pro-osteogenic role was accomplished through premature differentiation of chondrocytes, along with accelerated cartilage-to-bone conversion.

Purpose/aim: Caspase-1 inhibition is a promising option for degenerative joint diseases such as osteoarthritis; however, there is still a long way to go toward clinical use. One of the open challenges is associated with the non-inflammatory role of this caspase in the inflammatory environment as well as under physiological conditions. This study therefore focuses on two already pre-clinically tested caspase-1 inhibitors, VX-765 and VX-740, to specify their effects on chondrogenic cells.

Materials and methods: The analysis was performed on mouse micromass cultures where chondrocyte differentiation, inflammatory cytokine release, and gene expression were examined.

Results: Our data indicate that the inhibitor VX-740 increases chondrogenesis, suggesting osteocalcin as a target molecule. In the inflammatory environment induced by IL-1β, there was an increase in chondrogenic nodules and partial compensation of differentiation for both investigated inhibitors. Morphological changes were not primarily due to changes in chondrogenic/osteogenic gene expression, but different levels of inflammatory molecules were found in the culture supernatant. While an increase in anti-inflammatory cytokine levels was observed with VX-765, a decrease in pro-inflammatory cytokines was recorded in the case of VX-740 treatment.

Conclusions: The results demonstrate the differential effects of the caspase-1 inhibitors VX-765 and VX-740 on chondrogenic cell cultures and point to molecules that may be potential targets for use in the local treatment of osteoarthritis.

Purpose/aim: Zic family member 2 (ZIC2) is closely associated with cancer development, however, its role in the progression of osteosarcoma (OS) remains unknown. This study aims to reveal the function of ZIC2 in OS cell tumor progression and the underlying mechanism.

Materials and methods: This work performed quantitative real-time polymerase chain reaction to detect mRNAlevels of ZIC2, retinoic acid receptor alpha (RARA) and methyltransferase 3,N6-adenosine-methyltransferase complex catalytic subunit (METTL3),whereas protein level was detected by western blotting assay and immunohistochemistry assay. CCK-8 together with 5-Ethynyl-2'-deoxyuridine (EdU) assay were performed to analyze cell growth. Cell apoptosis was assessed by flow cytometry. Transwell assay and wound-healing assay were used for measuring cell invasion and migration. A xenograft mouse model assay was conducted to reveal the effect of ZIC2 on tumor formation invivo. The association of ZIC2 and METTL3 was identified by m6A RNA immunoprecipitation assay, dual-luciferase reporter gene assay, Actinomycin D assay and co-immunoprecipitation assay.

Results: ZIC2and METTL3 mRNA expression were upregulated in OS tissues relative to paracancerous normal tissues. ZIC2 was a prognostic biomarker for OS,and its expression was significantly associated with TNM stage, lymph node metastasis, and tumor size of OS patients. Additionally, ZIC2depletion inhibited proliferation, invasion and migration and induced apoptosis of OS cells, but ZIC2 overexpression had the opposite effects. Moreover, ZIC2 knockdown delayed tumor formation invivo. METTL3stabilized ZIC2 mRNA through m6A methylation modification. Further,METTL3 deficiency repressed OS cell malignancy by reducing ZIC2expression.

Conclusions: METTL3-mediated m6Amodification of ZIC2 contributed to OS development, providing therapeutic targets for OS.

Purpose/aim: Bone regeneration and repair are critical research areas within regenerative medicine, aiming to address the challenges posed by critical-sized bone defects. Bioscaffolds and cell-based therapies have been explored to enhance osteogenesis and promote effective bone regeneration. This study aimed to assess the regenerative potential of rabbit amniotic membrane (rAM) derived bioscaffold and its comparative evaluation with another bioscaffold, the decellularized periosteum (DP) of the buffalo rib, which was recellularized with rAM derived mesenchymal stem cells (MSCs).

Materials and methods: Passage 3 (P3) rAM-MSCs were validated for positive and negative stemness marker expression by PCR, immunolocalization and trilineage differentiation. Fresh rAM was cryopreserved for three months. An autologous rabbit model was used to assess the osteogenic capacity of different bioscaffolds: fresh rAM (Group II), frozen-thawed rAM (Group III), DP (Group IV), and DP enriched with rAM-MSCs (Group V) and control (Group I) in critical-size defect of 10 mm in radius bone. Radiographic evaluation was performed on the 1^st, 60^th and 90^th days, and ultramicroscopic and histomorphological evaluations were performed.

Results: Groups II and V presented the highest levels of remodeling and osteogenesis, a reduction in the defect size and total radiological scores. Groups II and V had the highest levels of osteogenesis, bone marrow development, cortical bone production, and medullary bone formation and the highest total histology score.

Conclusions: These findings revealed that fresh rAM, a rich source of MSCs, and DP enhanced with rAM-MSCs are the preferred bioscaffolds for critical-sized bone defect repair.

Purpose/aim: Osteoarthritis is a common cause of disability worldwide. Exosomes are extracellular vesicles and can exert paracrine and endocrine actions. DPSCs exosomes offer a new avenue of research that may elucidate various functions related to cell proliferation, differentiation, and immunomodulation. We hypothesized that DPSC exosomes produced under hypoxia-induced culture conditions may have an anti-inflammatory effect on osteoarthritic chondrocytes and may re-regulate the inflammatory response that is increased in osteoarthritis. We also hypothesized that the decreased glycosaminoglycan production in osteoarthritis may be re-induced by DPSC exosomes produced under hypoxia.

Materials and methods: Exosomes were isolated from DPSCs under hypoxic (3% O₂) and normoxic conditions (21% O₂) separately and were applied to OA chondrocyte cells. Quantification, morphology and analysis of tetraspanin markers were performed to characterize the exosomes. After the OA chondrocytes were treated with exosomes for 48 hours, they were prepared for cell proliferation, apoptosis, viability, glycosaminoglycan tests, and inflammatory cytokine analysis.

Results: Our results show that the pro-inflammatory cytokines were significantly suppressed in osteoarthritic chondrocytes by DPSC exosomes produced under hypoxia (p < 0.05). Exosomes of DPSCs grown in a hypoxia environment dramatically increase the amount of GAG in OA chondrocytes, giving clues that they can be used in cartilage regeneration (p < 0.001).

Conclusions: Considering that OA is associated with inflammatory components, DPSC exosome produced under hypoxic conditions prevents the formation of proinflammatory cytokines in osteoarthritic chondrocytes and shows therapeutic effects on osteoarthritic chondrocytes. Our study provides the first evidence showing the efficacy of DPSC-derived exosomes produced under hypoxia on osteoarthritic chondrocytes.

Purpose/aim: Temsirolimus is a water-soluble mammalian target of rapamycin (mTOR) complex inhibitor, potentially suitable for intra-articular administration. The present study aims to evaluate the therapeutic effects of intra-articular administration of temsirolimus on human chondrocytes and osteoarthritis (OA) progression in mice.

Materials and methods: The beneficial effects of temsirolimus treatment were evaluated in human chondrocytes (Normal Human Articular Chondrocyte-Knee cells) with or without treatment with IL-1β in vitro by real-time polymerase chain reaction, TUNEL staining for apoptosis, and CYTO-ID(R) staining for autophagy. The therapeutic effect of intra-articular injection of temsirolimus was evaluated in OA models (destabilized medial meniscus in C57BL/6J and senescence accelerated mice prone 8 (SAMP8)) in vivo, by histological and immunohistochemical analyses.

Results: Temsirolimus treatment upregulated COL2A1 and aggrecan (a major proteoglycan in the articular cartilage) expression in human chondrocytes. In addition, temsirolimus treatment recovered IL-1β-induced down-reregulated COL2A1 and aggrecan expression, while it partially decreased upregulated MMP-1, MMP-13, ADAMTS-4, ADAMTS-5, IL-1β, and IL-6 expression and apoptosis in human chondrocytes. Further, temsirolimus treatment enhanced autophagic activity in human chondrocytes. The intra-articular injection of temsirolimus to 12-week and 1-year old wild-type surgically induced OA model mice and SAMP8 mice delayed OA progression as compared to that in the control mice.

Conclusions: Temsirolimus treatment protected human chondrocytes from IL-1β-induced OA gene expression changes and apoptosis. Intra-articular injection of temsirolimus delayed OA progression in the mouse OA model and in SAMP8 mice. Thus, the intra-articular administration of temsirolimus is a promising therapeutic approach to inhibit articular cartilage degradation.

Purpose/aim: Dupuytren's disease (DD) is a condition affecting the palmar fascia that may reduce the mobility of several fingers. Despite its clinical relevance, the genetic and structural mechanisms associated with this disease are still not well understood. In this work, we have carried out a genome-wide gene expression analysis to identify relevant genes associated with DD.

Materials and methods: A genome-wide gene expression analysis was carried out using next generation sequencing (NGS) followed by a histological, histochemical and immunohistochemical analysis of some major components of the palmar fascia in 26 DD patients and 17 control subjects without the disease (CTR).

Results: We found 237 genes or sequences differentially expressed between DD and CTR, with those genes corresponding to several gene pathways and functions related to contractility, development and morphogenesis, differentiation, extracellular matrix (ECM), migration and ossification. In turn, the histological analysis confirmed that DD tissues showed a disorganized ECM, with nonaligned fibers, and abundant cells were found scattered along the whole tissue. CTR showed significantly higher amounts of proteoglycans revealed by alcian blue, along with versican, keratan-sulfate, myoglobin, tropomyosin 3, filamin C and titin, whereas DD showed significantly enriched in collagen fibers, especially collagens type-I and V, MMP-14, S-100, tubulin-beta, SMA and tenascin C, with disorganization of the elastic fibers.

Conclusions: In general, these results confirm that a significant alteration of the tissue organization, extracellular matrix and structure is related to DD. These results could contribute to the future development of diagnostic and treatment strategies for this disease.