Connective Tissue Research最新文献

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.

Purpose/aim of the study: Tendon healing in Murphy Ross Large (MRL/MpJ) mice was examined using an Achilles tendon tenotomy model, a full transection model, to compare with previous studies in which regenerative tendon healing was shown in a patellar tendon focal injury model.

Materials and methods: Achilles tendons of MRL/MpJ and C57BL/6J mice were fully transected. Tensile testing and proteomics analysis were performed after four weeks of healing. MicroCT was performed after 10 weeks of healing.

Results: The Achilles tendons of MRL/MpJ mice healed via scar formation, regardless of sex, as did C57BL/6J mice. Tensile testing found that mechanical properties of injured tendons of both MRL/MpJ female and male mice were similar to those of C57BL/6J female and male mice, respectively, after four weeks of healing, which is during the remodeling phase. After 10 weeks of healing, injured tendons of MRL/MpJ mice possessed smaller heterotopic ossification volumes than those of C57BL/6J mice. Proteomics analysis revealed similar alterations to signaling pathways in injured tendons of MRL/MpJ and C57BL/6J male mice after four weeks of healing. However, among the altered pathways, actin cytoskeleton and integrin signaling pathways were two of the top pathways that were more prominently activated in C57BL/6J males than in MRL/MpJ males.

Conclusions: These findings indicate that MRL/MpJ mice possess limited capacity to regenerate injured tendons after complete rupture, and that tenotomized Achilles tendons of MRL/MpJ male mice have lesser induction of heterotopic ossification and lower activation of the signaling pathways also induced with injury in C57BL/6J male mice.

Purpose/aim: Post-traumatic joint contracture (PTJC) commonly occurs after elbow injury. Previous findings implied immune system activation in capsules of contracted joints; however, quantifying immune cell populations in rodent joint tissues is challenging due to small size and low cellularity. Here, we used flow cytometry to investigate the temporal immune response and enumerate cell populations in the rat elbow capsule after traumatic injury.

Materials and methods: After inducing PTJC, capsules were harvested from injured, sham, and control rat elbows at multiple time points, stained with surface markers for immune cells, and quantified via flow cytometry. Results were compared to previously published mechanics and histology data from the same model. Another injured group was treated with celecoxib to determine if changes due to anti-inflammatory treatment could be detected.

Results: Compared to control, injured animals displayed elevated leukocytes, T cells, and natural killer cells. CD45+ cells exhibited similar temporal changes as mechanics, which increased and then decreased after injury. CD3+, CD4+, and CD8a+ T cells followed a similar pattern as histology scores, which increased and remained elevated. Treating injured animals with celecoxib increased leukocytes but decreased several immune subpopulations.

Conclusions: A method for flow cytometry on rat elbow capsule was established and used to quantify immune cell populations, which changed in response to injury and anti-inflammatory treatment. Comparisons between flow cytometry and previously published mechanics and histology revealed additional insights about temporal patterns in cell-, tissue-, and joint-level changes. Future work will investigate whether changes in immune cells attenuate PTJC symptoms.

Objective: Lumican, a small leucine-rich proteoglycan, is implicated in diverse biological functions. This study investigates the role of lumican in osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) and its therapeutic efficacy against osteoporosis (OP), while preliminarily elucidating its mechanism.

Materials and methods: hBMSCs were cultured under osteogenic induction. Cell proliferation and migration were assessed using CCK-8 and Transwell assays. Osteogenic differentiation was evaluated by ALP/ARS staining, with osteogenic marker genes (Runx2, Osterix, OCN) measured via RT-qPCR. An ovariectomized (OVX) rat OP model was established. Bone microstructure was analyzed by HE staining and serum OCN levels by ELISA. Glycolysis was assessed through glucose uptake, lactate production, ATP levels, and key glycolytic enzyme expression. The glycolytic inhibitor 2-DG was used for rescue experiments. Histone lactylation (H3K18la) and its promoter enrichment were analyzed by Western blot and ChIP.

Results: Lumican expression increased during osteogenic induction and dose-dependently enhanced hBMSC proliferation, migration, and osteogenic differentiation. ALP/ARS staining showed. Enhanced osteogenic differentiation, while RT-qPCR further confirmed upregulated Runx2, Osterix, and OCN. In OVX rats, lumican improved trabecular microstructure and increased serum OCN and osteogenic gene expression in bone tissues. Mechanistically, lumican promoted glycolysis in hBMSCs, indicated by increased glucose uptake, lactate production, ATP levels, and glycolytic enzyme expression. The lumican-induced osteogenic effects were abolished by 2-DG. Furthermore, lumican enhanced histone lactylation, particularly increasing H3K18la enrichment at osteogenic gene promoters, which was suppressed by 2-DG.

Conclusions: Lumican promotes hBMSC osteogenic differentiation and ameliorates OP by enhancing glycolysis and histone lactylation, providing a potential therapeutic target.

Purpose/aim: Mitochondria are vital dynamic organelles released by cells into extracellular space, endocytosed in or transferred between cells in contact. Mitochondria from healthy bone marrow stem cells (MSCs) show rescue effects on chondrocytes, accordingly a concept of using healthy MSC mitochondria for cartilage regeneration is put forward. Therefore, whether mitochondria from healthy MSCs help to save chondrocytes in damaged cartilage microenvironment is intriguing. We answered this question by considering coexistent MSCs and chondrocytes, and their released mitochondria in damaged joint.

Materials and methods: Mitochondria were extracted from primarily cultured MSCs and chondrocytes of osteoarthritis (OA) human patients to represent mitochondria released endogenously by MSCs and chondrocytes in damaged joint. While mitochondria were extracted from healthy rats to represent mitochondria exogenously added during MSC mitochondrial repair for the inaccessibility of healthy human. The mitochondria were co-cultured with another type of cells. Endocytosing and afterward positioning of exogenous mitochondria, as well as induced alterations in mitochondria and cellular behaviors of recipient cells were assayed.

Result: Our results suggested that although mitochondria from healthy MSCs advantaged remedy for inflammatory chondrocytes, mitochondria from healthy and OA chondrocytes, as well as from OA MSCs disadvantaged chondrocytes remedy, no matter the mitochondria were from the same or different species. However, reactive oxygen species (ROS) modulation alleviated the disadvantage.

Conclusions: Our results provide a reminder for careful consideration of mitochondrial therapy, and explanation for unsuccessful repair of damaged cartilage by MSCs from aspect of mitochondria, as well as potential remedy through ROS modulation.

Purpose/aim: Parathyroid hormone -related protein (PTHrP) regulates skeletal development by controlling epiphyseal growth cartilage. In mice, PTHrP contains three functional domains: the N-terminus, nuclear localization sequence (NLS), and C-terminus. The PTHrP (67 -139) region contains both the NLS and the C-terminus. Our research group previously generated C57BL/6 mice lacking this region (Pthrp Δ/Δ), resulting in reduced postnatal growth and shorter stature. This study aimed to define the functional role of PTHrP (67 -139) in chondrocytes in vitro and ex vivo.

Materials and methods: Epiphyseal growth cartilage from 1 -2-day-old Pthrp Δ/Δ mice was evaluated using histology, immunofluorescence, and qPCR. Primary chondrocytes from Pthrp Δ/Δ mice and PTHrP-transfected chondrocytes were cultured to assess proliferation and gene expression.

Results: Pthrp Δ/Δ mice showed significantly reduced epiphyseal cartilage height, including decreased resting, proliferative, and hypertrophic zone lengths. This was accompanied by increased mRNA expression of hypertrophic markers (Ihh, Col10a1). Epiphyseal cartilage from Pthrp Δ/Δ mice also exhibited elevated Adamts5 and Mmp13 expression, indicating enhanced extracellular matrix degradation. Primary chondrocytes from Pthrp Δ/Δ mice and chondrocytes transiently transfected with the PTHrP deletion construct (ΔNLS+CTERM) showed reduced proliferation and matrix production. Chondrocytes lacking PTHrP (67 -139) had decreased expression of Col2a1 and Acan, along with reduced IGF-1/IGF-1R expression, suggesting impaired IGF-1 signaling.

Conclusions: Loss of the PTHrP (67 -139) domain causes impaired proliferation, reduced matrix production, and increased extracellular matrix degradation in epiphyseal chondrocytes. These findings demonstrate that PTHrP (67 -139) is required to maintain chondrocytes in an immature state and that its absence leads to premature differentiation of epiphyseal growth plate chondrocytes.

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.