Connective Tissue Research最新文献

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.

Osteoarthritis (OA) is a progressive joint disorder that leads to pain and disability for millions of people worldwide. Post-traumatic OA (PTOA), a form of OA, arises secondary to joint injury and often impacts younger individuals. Among the most common joint injuries leading to disrupted joint homeostasis and PTOA is anterior cruciate ligament (ACL) rupture. Even with successful surgical stabilization, the risk of developing PTOA persists due to several factors, including altered biology that contributes to disease progression. Recent research into the biology of ACL injuries has advanced our understanding of the mechanisms by which PTOA develops, including the inflammatory pathways involved, the expression of biomarkers specific to ACL injuries, and their interaction with factors such as the chronicity of the injury. Evidence suggests that homeostatic balance of anabolic and catabolic processes in the knee is disturbed after ACL tears, triggering a catabolic and degenerative phenotype, ultimately leading to premature joint degeneration, pain, and disability. Several key knowledge gaps exist, such as the determinants of the transition from acute to chronic inflammation, inter-patient variability in inflammatory responses, and influence of systemic factors on disease development. PTOA research faces numerous challenges, including protracted nature of the disease, the complexity of joint biology, and difficulties in translating molecular discoveries into clinical practice. Future research should prioritize improving biomarker precision for early detection, developing targeted therapies, and leveraging emerging technologies like machine learning to personalize treatment. This approach will enhance our understanding of the biological basis of PTOA resulting from ACL injuries and identify opportunities to mitigate the long-term consequences of these injuries.

Knee osteoarthritis (OA) is a debilitating condition with limited treatment options beyond symptom management or total knee arthroplasty (TKA). For younger patients, TKA presents challenges, including higher failure rates and revision surgeries. Knee joint distraction (KJD) has emerged as a promising joint-preserving alternative for end-stage knee OA, demonstrating significant improvements in pain, function, and quality of life in clinical trials and clinical practice. Almost 20 years of research has highlighted KJD's capacity to delay or prevent TKA by promoting cartilage and subchondral bone repair through whole-joint remodeling. Recent studies, including a multicenter trial with a purpose-built distraction device, confirm the treatment's efficacy and durability, with benefits lasting up to 10 years. However, long-term outcomes remain limited, and variability in patient response underscores the need for refined predictive tools. Challenges include the high incidence of pin tract infections during treatment and integrating KJD into routine clinical practice, as highlighted by limited trial enrollment in the UK KARDS trial and variability in healthcare system compatibility. Future research should focus on minimizing complications, improving patient selection through advanced imaging and biomarker analyses, and further understanding the mechanisms underlying KJD-induced joint remodeling. Large-scale trials like the ongoing Dutch GODIVA study are poised to provide robust evidence for KJD's broader adoption, implementation, and reimbursement in healthcare systems. With continued advancements, KJD holds the potential to transform the management of knee OA, offering a viable alternative to TKA for younger patients and addressing a critical unmet need in OA care.