Connective Tissue Research最新文献

Purpose: Rheumatoid arthritis is characterized by inflammatory erosions and increased prevalence in females. Androgen, the dominant sex hormone in males, is protective against bone loss. Here, we investigated the cellular targets of androgen and hypothesized that androgen negatively regulates TNFα-driven osteoclastogenesis.

Materials and methods: Bone marrow-derived cells from a C57BL/6J mouse femur and tibia were plated with 10^-9M of 5α-dihydrotestosterone (DHT) or vehicle then collected for flow cytometry and flow sorting. Osteoclast precursors (OCPs), myeloid-like CD11b+Gr-1- cells, were grown to osteoclasts then fixed and TRAP-stained. Osteoclasts were quantified by cell counting. Single-cell RNA-sequencing was performed on DHT or vehicle-treated TNFα-stimulated cells, and the differences in cell populations, gene expression, gene ontology, and cell trajectory were analyzed via bioinformatic approaches. RT-qPCR validated sequencing results.

Results: DHT-treated OCPs were decreased versus vehicle-treated OCPs (Vehicle = 12.43 ± 0.53%, DHT = 9.93 ± 0.85%, p < 0.05), and there was decreased differentiation from OCPs to osteoclasts (Vehicle = 403.3 ± 141.5, DHT = 10.67 ± 14.36, p < 0.01). Analysis identified osteoclastogenesis-related clusters that varied between samples and interferon-related processes upregulated with DHT treatment. Analysis also determined that DHT treatment may be associated with expression of the transcription factor Irf7. Isg15, the most differentially expressed gene in the DHT-treated clusters, may also be involved in osteoclastogenesis.

Conclusions: In summary, androgen targets OCPs and limits their differentiation into osteoclasts. In myeloid populations, DHT treatment results in differential expression of interferon-related processes and genes and alters the trajectory of cells, potentially regulating osteoclastogenesis.

Purpose: After anterior cruciate ligament reconstruction surgeries, tendon grafts lose their mechanical integrity in vivo. Both auto- and allografts contain dead cells, and remaining viable cells are restricted temporarily from abundant nutrient access. To evaluate a possible connection, nutrient deprivation was hypothesized to reduce cell viability, compromising tissue mechanics as cells adopt a catabolic state.

Materials and methods: Taking inspiration from the graft environment, collagenous tendon-mimics with tendon-derived cells were reconstituted in vitro. Subsequently, tendon-mimics were cultured at varying glucose and serum concentrations, ranging from complete medium (5 mM glucose, 2.5% serum), to mild deprivation (0.5 mM glucose, no serum), extreme deprivation (0 mM glucose, no serum), and a frozen control group (0 mM glucose, no serum).

Results: In the complete medium group, cell viability remained stable, improving tendon-mimic mechanics. Tendon cells were resilient to mild nutrient deprivation, maintaining viability. However, at extreme nutrient deprivation or tissue freezing, cell viability dropped significantly. Surprisingly, mechanical properties remained unaltered in all the groups, except the complete medium group. MMP8 secretion and activation confirmed a catabolic potential only in the complete medium group, while no further MMPs were secreted or activated in the nutrient-deprived groups. This suggests that viable cells that are exposed to a nutrient deprived environment likely lack the energy to exert contractile forces or produce/activate MMPs.

Conclusions: This study revealed that tendon-derived cells do not compromise collagenous tendon-mimic integrity under nutrient-deprived conditions in an in vitro model, highlighting the resilience of tenocytes in maintaining tissue structure.

Purpose: 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 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: Long noncoding RNA (lncRNA) small nucleolar RNA host gene 16 (Snhg16) has been confirmed to accelerate osteoarthritis (OA) progress. However, its regulatory mechanism has not been fully elucidated.

Materials and methods: Interleukin-1β (IL-1β)-induced ATDC5 chondrocytes were used to mimic OA cell models, and destabilization of medial meniscus (DMM) surgery was performed to construct OA mice models. The expression levels of Snhg16, glutathione peroxidase 4 (Gpx4), staphylococcal nuclease domain-containing 1 (Snd1), methyltransferase-like 3 (Mettl3) and extracellular matrix (ECM) degradation-related markers were detected by qRT-PCR or western blot. Besides, glutathione (GSH), malondialdehyde (MDA), Fe²⁺ and lipid reactive oxygen species (ROS) levels were detected to evaluate cell ferroptosis. The interaction between Snd1 and Snhg16 or Gpx4 was evaluated by RNA pull-down and RNA immunoprecipitation (RIP) assays. The stability of Gpx4 mRNA and Snhg16 was examined using actinomycin D assay. Methylated RNA immunoprecipitation (MeRIP) assay was used to measure the regulation of Mettl3 on Snhg16.

Results: Snhg16 downregulation repressed IL-1β-induced chondrocyte ferroptosis and ECM degradation. Also, silencing of Snhg16 alleviated cartilage tissue damage in OA mice models. In the terms of mechanism, Snhg16 inhibited the stability of Gpx4 mRNA via interacting with Snd1. Moreover, Mettl3 enhanced Snhg16 stability by m6A modification. Functional experiments showed that Mettl3 knockdown suppressed IL-1β-induced ferroptosis and ECM degradation through regulating the Snhg16/Snd1/Gpx4 axis.

Conclusions: Mettl3-mediated m6A modification of Snhg16 facilitated ferroptosis and ECM degradation to aggravate OA process via regulating Snd1/Gpx4 axis, providing a novel target for OA treatment.

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.

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.

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.