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1.
Mol Med ; 29(1): 17, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36721094

RESUMO

BACKGROUND: Osteoarthritis (OA) is a common chronic disease characterized by chronic inflammation and extracellular matrix degradation. Indole-3-propionic acid (IPA) is a tryptophan metabolite secreted by intestinal flora, which can exert anti-inflammatory effects in a variety of diseases. In this study, we further investigated the potential therapeutic role of IPA in OA and the underlying mechanism. METHODS: IL-1ß was utilized to induce chondrocyte inflammation. Then, the cytotoxicity of IPA on rat chondrocytes was assessed. Meanwhile, RT-qPCR, Griess reaction, ELISA, Western blot and immunofluorescence were performed to evaluate the expression of inflammatory factors and stromal proteins, and the NF-κB pathway in chondrocytes treated with IL-1ß alone, with IPA or with aryl hydrocarbon receptor (AhR) knockdown. An OA rat model was established by anterior cruciate ligament transection, and hematoxylin-eosin staining, Safranin-O/Fast Green staining and immunochemistry were applied to estimate OA severity. RESULTS: IPA did not affect cellular viability at concentrations up to 80 µM. IPA significantly inhibited the IL-1ß-induced expression of inflammatory factors (Nitric oxide, PGE2, TNF-α, IL-6, iNOS and COX-2) and matrix-degrading enzymes (MMP-3, MMP-13 and ADAMTS-5), upregulated the expression of anabolic markers (aggrecan and collagen-II) and inactivated the NF-κB pathway. However, AhR knockdown could abolish the above protection capabilities and the suppression of the NF-κB pathway induced by IPA. Furthermore, IPA significantly reduced serum inflammatory cytokines expression, cartilage destruction and synovitis in vivo, demonstrating its protective role in OA progression. CONCLUSION: IPA improved IL-1ß-induced chondrocyte inflammation and extracellular matrix degradation through the AhR/NF-κB axis, which provides an innovative therapeutic strategy for OA.

2.
J Orthop Res ; 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36722421

RESUMO

Many clinical studies have indicated an association between biomechanical factors and the incidence and pathological progression of Adolescent idiopathic scoliosis (AIS). However, at present, the research on AIS is mainly focused on the etiology, and there are few studies reporting the causes of progressive aggravation of AIS. In the present study we aim to investigate the role of Piezo1 in compressive stress induced mouse spinal vertebral growth plate chondrocytes apoptosis. Firstly, a scoliosis mouse model was established, and the expression of Piezo1 as well as the degree of apoptosis were investigated. We found that the expression of Piezo1 and the degree of apoptosis were significantly higher on the concave sides than that on the convex sides of the vertebral growth plate in mice with scoliosis. Spinal vertebral growth plate chondrocytes were further isolated and treated with Yoda 1 to mimic Piezo1 overload. Excess Piezo1 significantly promoted apoptosis of spinal vertebral growth plate chondrocytes. Moreover, static gas compressive stress was used to simulate the increased concave compressive stress in the process of scoliosis with or without GsMTx4, an Piezo inhibitor. It was observed that with the increase of static compressive stress, the expression of Piezo1 increased, and the chondrocytes of vertebral growth plate treated with Piezo1 inhibitor GsMTx4 weakened the above phenomena. In conclusion, our results indicated that compressive stress is strongly associated with the different degree of apoptosis on both sides on the convex and concave sides of the vertebral growth plate in scoliosis via inducing different expression of Piezo1. Reducing the expression of Piezo1 in the concave side of the vertebral growth plate and inhibiting the apoptosis of chondrocytes in the bilateral vertebral growth plate caused by asymmetric stress on both sides of the concave vertebral body may be a promising treatment strategy for AIS. This article is protected by copyright. All rights reserved.

3.
Elife ; 122023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36715686

RESUMO

Induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine. The iPSCs exhibit a preference for lineage differentiation to the donor cell type indicating the existence of memory of origin. Although the intrinsic effect of the donor cell type on differentiation of iPSCs is well recognized, whether disease-specific factors of donor cells influence the differentiation capacity of iPSC remains unknown. Using viral based reprogramming, we demonstrated the generation of iPSCs from chondrocytes isolated from healthy (AC-iPSCs) and osteoarthritis cartilage (OA-iPSCs). These reprogrammed cells acquired markers of pluripotency and differentiated into uncommitted mesenchymal-like progenitors. Interestingly, AC-iPSCs exhibited enhanced chondrogenic potential as compared OA-iPSCs and showed increased expression of chondrogenic genes. Pan-transcriptome analysis showed that chondrocytes derived from AC-iPSCs were enriched in molecular pathways related to energy metabolism and epigenetic regulation, together with distinct expression signature that distinguishes them from OA-iPSCs. Our molecular tracing data demonstrated that dysregulation of epigenetic and metabolic factors seen in OA chondrocytes relative to healthy chondrocytes persisted following iPSC reprogramming and differentiation toward mesenchymal progenitors. Our results suggest that the epigenetic and metabolic memory of disease may predispose OA-iPSCs for their reduced chondrogenic differentiation and thus regulation at epigenetic and metabolic level may be an effective strategy for controlling the chondrogenic potential of iPSCs.


Assuntos
Células-Tronco Pluripotentes Induzidas , Osteoartrite , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Transcriptoma , Epigênese Genética , Cartilagem , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Osteoartrite/genética , Osteoartrite/metabolismo
4.
Ann Rheum Dis ; 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36627169

RESUMO

OBJECTIVES: CHRFAM7A is a uniquely human fusion gene that functions as a dominant negative regulator of alpha 7 acetylcholine nicotinic receptor (α7nAChR) in vitro. This study determined the impact of CHRFAM7A on α7nAChR agonist responses, osteoarthritis (OA) severity and pain behaviours and investigated mechanisms. METHODS: Transgenic CHRFAM7A (TgCHRFAM7A) mice were used to determine the impact of CHRFAM7A on knee OA histology, pain severity in OA and other pain models, response to nAchR agonist and IL-1ß. Mouse and human cells were used for mechanistic studies. RESULTS: Transgenic (Tg) TgCHRFAM7A mice developed more severe structural damage and increased mechanical allodynia than wild type (WT) mice in the destabilisation of medial meniscus model of OA. This was associated with a decreased suppression of inflammation by α7nAchR agonist. TgCHRFAM7A mice displayed a higher basal sensitivity to pain stimuli and increased pain behaviour in the monoiodoacetate and formalin models. Dorsal root ganglia of TgCHRFAM7A mice showed increased macrophage infiltration and expression of the chemokine fractalkine and also had a compromised antinociceptive response to the α7nAchR agonist nicotine. Both native CHRNA7 and CHRFAM7A subunits were expressed in human joint tissues and the CHRFAM7A/CHRNA7 ratio was increased in OA cartilage. Human chondrocytes with two copies of CHRFAM7A had reduced anti-inflammatory responses to nicotine. CONCLUSION: CHRFAM7A is an aggravating factor for OA-associated inflammation and tissue damage and a novel genetic risk factor and therapeutic target for pain.

5.
Int Immunopharmacol ; 115: 109683, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36630751

RESUMO

Osteoarthritis (OA) is a joint disease that is characterized by articular cartilage degeneration and destruction. Stevioside (SVS) is a diterpenoid glycoside extracted from Stevia rebaudiana Bertoni with some specific effects against inflammatory and apoptotic, whereas it is still unclear what function SVS has in osteoarthritis. This study focuses on the anti-inflammatory and anti-apoptosis functions of SVS on chondrocytes induced by interleukin (IL)-1beta, and the role of SVS in an osteoarthritis model for mice. We can detect the production of inflammatory factors such as nitric oxide (NO) and prostaglandin E2 (PGE2) using real-time quantitative polymerase chain reaction (RT-qPCR), the Griess reaction, and enzyme linked immunosorbent assay (ELISA). On the basis of Western blot, we have observed the protein expressions of cartilage matrix metabolism, inflammatory factors, and apoptosis of chondrocytes. Simultaneously, the pharmacological effects of SVS in mice were evaluated by hematoxylin and eosin (HE), toluidine blue, Safranin O, and immunohistochemical staining. The results show that SVS slows extracellular matrix degradation and chondrocyte apoptosis. In addition, SVS mediates its cellular effect by inhibiting the activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways. Meanwhile, molecular docking studies revealed that SVS has excellent binding capabilities to p65, extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). The study suggests that SVS can be developed as a potential osteoarthritis treatment.

6.
Artigo em Inglês | MEDLINE | ID: mdl-36621590

RESUMO

OBJECTIVE: Osteoarthritis-related cartilage extracellular matrix remodeling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. METHODS: Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n = 5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analyzed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. RESULTS: We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9-14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33-64%]). This resulted in an altered ribosome translation modus (e.g., CrPV IRES, FC 3, 95% CI [2.2-4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3-2.0]). CONCLUSIONS: Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.

7.
JBMR Plus ; 7(1): e10701, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36699640

RESUMO

Relevance of mineralized nodules in two-dimensional (2D) osteoblast/osteocyte cultures to bone biology, pathology, and engineering is a decades old question, but a comprehensive answer appears to be still wanting. Bone-like cells, extracellular matrix (ECM), and mineral were all reported but so were non-bone-like ones. Many studies described seemingly bone-like cell-ECM structures based on similarity to few select bone features in vivo, yet no studies examined multiple bone features simultaneously and none systematically studied all types of structures coexisting in the same culture. Here, we report such comprehensive analysis of 2D cultures based on light and electron microscopies, Raman microspectroscopy, gene expression, and in situ messenger RNA (mRNA) hybridization. We demonstrate that 2D cultures of primary cells from mouse calvaria do form bona fide bone. Cells, ECM, and mineral within it exhibit morphology, structure, ultrastructure, composition, spatial-temporal gene expression pattern, and growth consistent with intramembranous ossification. However, this bone is just one of at least five different types of cell-ECM structures coexisting in the same 2D culture, which vary widely in their resemblance to bone and ability to mineralize. We show that the other two mineralizing structures may represent abnormal (disrupted) bone and cartilage-like structure with chondrocyte-to-osteoblast transdifferentiation. The two nonmineralizing cell-ECM structures may mimic periosteal cambium and pathological, nonmineralizing osteoid. Importantly, the most commonly used culture conditions (10mM ß-glycerophosphate) induce artificial mineralization of all cell-ECM structures, which then become barely distinguishable. We therefore discuss conditions and approaches promoting formation of bona fide bone and simple means for distinguishing it from the other cell-ECM structures. Our findings may improve osteoblast differentiation and function analyses based on 2D cultures and extend applications of these cultures to general bone biology and tissue engineering research. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

8.
Int J Mol Sci ; 24(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36675010

RESUMO

Loose bodies (LBs) from patients with osteochondritis dissecans (OCD) are usually removed and discarded during surgical treatment of the defect. In this study, we address the question of whether these LBs contain sufficient viable and functional chondrocytes that could serve as a source for autologous chondrocyte implantation (ACI) and how the required prolonged in vitro expansion affects their phenotype. Chondrocytes were isolated from LBs of 18 patients and compared with control chondrocyte from non-weight-bearing joint regions (n = 7) and bone marrow mesenchymal stromal cells (BMSCs, n = 6) obtained during primary arthroplasty. No significant differences in the initial cell yield per isolation and the expression of the chondrocyte progenitor cell markers CD44 + /CD146+ were found between chondrocyte populations from LBs (LB-CH) and control patients (Ctrl-CH). During long-term expansion, LB-CH exhibited comparable viability and proliferation rates to control cells and no ultimate cell cycle arrest was observed within 12 passages respectively 15.3 ± 1.1 mean cumulative populations doublings (CPD). The chondrogenic differentiation potential was comparable between LB-CH and Ctrl-CH, but both groups showed a significantly higher ability to form a hyaline cartilage matrix in vitro than BMSC. Our data suggest that LBs are a promising cell source for obtaining qualitatively and quantitatively suitable chondrocytes for therapeutic applications, thereby circumventing donor site morbidity as a consequence of the biopsies required for the current ACI procedure.


Assuntos
Cartilagem Articular , Condrócitos , Procedimentos Ortopédicos , Cartilagem , Cartilagem Articular/patologia , Diferenciação Celular , Condrócitos/metabolismo , Condrócitos/transplante , Células-Tronco Mesenquimais/metabolismo , Procedimentos Ortopédicos/métodos , Transplante Autólogo/métodos
9.
Mol Biol Rep ; 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36696024

RESUMO

BACKGROUND: Chondrocytes are the only cell components in the cartilage, which has the poor regeneration ability. Thus, repairing damaged cartilage remains a huge challenge. Sika deer antlers are mainly composed of cartilaginous tissues that have an astonishing capacity for repair and renewal. Our previous study has demonstrated the transforming growth factor ß (TGF-ß1) is considered to be a key molecule involved in rapid growth, with the strongest expression in the cartilage layer. However, it remains to be clarified whether deer TGF-ß1 has significantly different function from other species such as mouse, and what is the molecular mechanism of regulating cartilage growth. METHODS: Primary chondrocytes was collected from new born mouse rib cartilage. The effect of TGF-ß1 on primary chondrocytes viability was elucidated by RNA sequencing (RNA-seq) technology combined with validation methods such as quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence assay (IFA). Differential expression genes were identified using the DEGseq package. RESULTS: Our results demonstrated that the overexpression of deer TGF-ß1 possibly promoted chondrocyte proliferation and extracellular matrix (ECM) synthesis, while simultaneously suppressing chondrocyte differentiation through regulating transcription factors, growth factors, ECM related genes, proliferation and differentiation marker genes, such as Comp, Fgfr3, Atf4, Stat1 etc., and signaling pathways such as the MAPK signaling pathway, inflammatory mediator regulation of TRP channels etc. In addition, by comparing the amino acid sequence and structures between the deer TGF-ß1 and mouse TGF-ß1, we found that deer TGF-ß1 and mouse TGF-ß1 proteins are mainly structurally different in arm domains, which is the main functional domain. Phenotypic identification results showed that deer TGF-ß1 may has stronger function than mouse TGF-ß1. CONCLUSION: ​These results suggested that deer TGF-ß1 has the ability to promote chondrogenesis by regulating chondrocyte proliferation, differentiation and ECM synthesis. This study provides insights into the molecular mechanisms underlying the effects of deer TGF-ß1 on chondrocyte viability.

10.
Gene ; 859: 147188, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36632912

RESUMO

BACKGROUND: Mesenchymal stem cells (MSC) are a type of multipotent stem cell whose differentiation into cartilage cells has been considered in recent years. Platelet-rich plasma (PRP) may impair cartilage differentiation due to its richness in growth factors and hyperosmolarity due to its proximity to the required cartilage environment. OBJECTIVES: The main purpose of this study was to treat human adipose tissue-derived MSCs concurrently with PRP and hyperosmolarity to investigate the expression profile of micro-RNA (miRNA) involved in the cartilage process differentiation. We examined the effect of PRP and the increase in osmolarity on the expression of miR-27, miR-101, miR-140, miR-145, miR-146, and miR-199. METHODS: Mesenchymal stem cells were extracted from human adipose tissue and differentiated into chondrocytes and the effect of baseline cultures (diff), PRP (prp), hyperosmolarity (os), base plus hyperosmolarity (diff + os), PRP plus hyperosmolarity (prp + os) next to the control group were studied in cartilage differentiation using specific stains such as Alcian blue, hematoxylin and eosin, and collagen type 2 and 10 immunohistochemistry. In addition, the expression of miR-27, miR-140, miR-199, miR-146, miR-101, and miR-145 was evaluated using real-time PCR. CONCLUSION: Human adipose tissue-derived MSCs with the ability to differentiate into adipocytes and osteocytes showed the properties of chondrocytes in all differentiation groups. Alkaline phosphatase (ALP) enzyme activity and calcium deposition were lower in the diff + os group than in other groups. Therefore, the diff + os group may be a more suitable environment for cartilage differentiation. Furthermore, 5% PRP concentration and hyperosmolarity showed a positive effect on miR-140, miR-199, miR-27, and, miR-146 and a negative effect on miR-101 and miR-145 on cartilage differentiation.

11.
Bone Joint Res ; 12(2): 121-132, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36718653

RESUMO

AIMS: Pellino1 (Peli1) has been reported to regulate various inflammatory diseases. This study aims to explore the role of Peli1 in the occurrence and development of osteoarthritis (OA), so as to find new targets for the treatment of OA. METHODS: After inhibiting Peli1 expression in chondrocytes with small interfering RNA (siRNA), interleukin (IL)-1ß was used to simulate inflammation, and OA-related indicators such as synthesis, decomposition, inflammation, and apoptosis were detected. Toll-like receptor (TLR) and nuclear factor-kappa B (NF-κB) signalling pathway were detected. After inhibiting the expression of Peli1 in macrophages Raw 264.7 with siRNA and intervening with lipopolysaccharide (LPS), the polarization index of macrophages was detected, and the supernatant of macrophage medium was extracted as conditioned medium to act on chondrocytes and detect the apoptosis index. The OA model of mice was established by destabilized medial meniscus (DMM) surgery, and adenovirus was injected into the knee cavity to reduce the expression of Peli1. The degree of cartilage destruction and synovitis were evaluated by haematoxylin and eosin (H&E) staining, Safranin O/Fast Green staining, and immunohistochemistry. RESULTS: In chondrocytes, knockdown of Peli1 produced anti-inflammatory and anti-apoptotic effects by targeting the TLR and NF-κB signalling pathways. We found that in macrophages, knockdown of Peli1 can inhibit M1-type polarization of macrophages. In addition, the corresponding conditioned culture medium of macrophages applied to chondrocytes can also produce an anti-apoptotic effect. During in vivo experiments, the results have also shown that knockdown Peli1 reduces cartilage destruction and synovial inflammation. CONCLUSION: Knockdown of Peli1 has a therapeutic effect on OA, which therefore makes it a potential therapeutic target for OA.Cite this article: Bone Joint Res 2023;12(2):121-132.

12.
Bone Joint Res ; 12(1): 33-45, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36642417

RESUMO

AIMS: Circular RNA (circRNA) is involved in the regulation of articular cartilage degeneration induced by inflammatory factors or oxidative stress. In a previous study, we found that the expression of circStrn3 was significantly reduced in chondrocytes of osteoarthritis (OA) patients and OA mice. Therefore, the aim of this paper was to explore the role and mechanism of circStrn3 in osteoarthritis. METHODS: Minus RNA sequencing, fluorescence in situ hybridization, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of circStrn3 in human and mouse OA cartilage tissues and chondrocytes. Chondrocytes were then stimulated to secrete exosomal miR-9-5p by cyclic tensile strain. Intra-articular injection of exosomal miR-9-5p into the model induced by destabilized medial meniscus (DMM) surgery was conducted to alleviate OA progression. RESULTS: Tensile strain could decrease the expression of circStrn3 in chondrocytes. CircStrn3 expression was significantly decreased in human and mouse OA cartilage tissues and chondrocytes. CircStrn3 could inhibit matrix metabolism of chondrocytes through competitively 'sponging' miRNA-9-5p targeting Kruppel-like factor 5 (KLF5), indicating that the decrease in circStrn3 might be a protective factor in mechanical instability-induced OA. The tensile strain stimulated chondrocytes to secrete exosomal miR-9-5p. Exosomes with high miR-9-5p expression from chondrocytes could inhibit osteoblast differentiation by targeting KLF5. Intra-articular injection of exosomal miR-9-5p alleviated the progression of OA induced by destabilized medial meniscus surgery in mice. CONCLUSION: Taken together, these results demonstrate that reduction of circStrn3 causes an increase in miR-9-5p, which acts as a protective factor in mechanical instability-induced OA, and provides a novel mechanism of communication among joint components and a potential application for the treatment of OA.Cite this article: Bone Joint Res 2023;12(1):33-45.

13.
Heliyon ; 9(1): e13152, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36711315

RESUMO

Osteoarthritis (OA) has been proven as the second primary cause of pain and disability in the elderly population, impact patients both physically and mentally, as well as imposing a heavy burden on the global healthcare system. Current treatment methods, whether conservative or surgical, that aim at relieving symptoms can not delay or reverse the degenerative process in the structure. Scientists and clinicians are facing a revolution in OA treatment strategies. The emergence of exosomes brings hope for OA treatment based on pathology, which is attributed to its full potential in protecting chondrocytes from excessive death, alleviating inflammation, maintaining cartilage matrix metabolism, and regulating angiogenesis and subchondral bone remodeling. Therefore, we summarized the recent studies of exosomes in OA, aiming to comprehensively understand the functions and mechanisms of exosomes in OA treatment, which may provide direction and theoretical support for formulating therapeutic strategies in the future.

14.
World J Clin Cases ; 11(1): 17-29, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36687189

RESUMO

The degenerative disease of the intervertebral disc is nowadays an important health problem, which has still not been understood and solved adequately. The vertebral endplate is regarded as one of the vital elements in the structure of the intervertebral disc. Its constituent cells, the chondrocytes in the endplate, may also be involved in the process of the intervertebral disc degeneration and their role is central both under physiological and pathological conditions. They main functions include a role in homeostasis of the extracellular environment of the intervertebral disc, metabolic support and nutrition of the discal nucleus and annulus beneath and the preservation of the extracellular matrix. Therefore, it is understandable that the cells in the endplate have been in the centre of research from several viewpoints, such as development, degeneration and growth, reparation and remodelling, as well as treatment strategies. In this article, we briefly review the importance of vertebral endplate, which are often overlooked, in the intervertebral disc degeneration.

15.
Bone Joint Res ; 12(1): 46-57, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36647698

RESUMO

AIMS: After a few passages of in vitro culture, primary human articular chondrocytes undergo senescence and loss of their phenotype. Most of the available chondrocyte cell lines have been obtained from cartilage tissues different from diarthrodial joints, and their utility for osteoarthritis (OA) research is reduced. Thus, the goal of this research was the development of immortalized chondrocyte cell lines proceeded from the articular cartilage of patients with and without OA. METHODS: Using telomerase reverse transcriptase (hTERT) and SV40 large T antigen (SV40LT), we transduced primary OA articular chondrocytes. Proliferative capacity, degree of senescence, and chondrocyte surface antigen expression in transduced chondrocytes were evaluated. In addition, the capacity of transduced chondrocytes to synthesize a tissue similar to cartilage and to respond to interleukin (IL)-1ß was assessed. RESULTS: Coexpression of both transgenes (SV40 and hTERT) were observed in the nuclei of transduced chondrocytes. Generated chondrocyte cell lines showed a high proliferation capacity and less than 2% of senescent cells. These cell lines were able to form 3D aggregates analogous to those generated by primary articular chondrocytes, but were unsuccessful in synthesizing cartilage-like tissue when seeded on type I collagen sponges. However, generated chondrocyte cell lines maintained the potential to respond to IL-1ß stimulation. CONCLUSION: Through SV40LT and hTERT transduction, we successfully immortalized chondrocytes. These immortalized chondrocytes were able to overcome senescence in vitro, but were incapable of synthesizing cartilage-like tissue under the experimental conditions. Nonetheless, these chondrocyte cell lines could be advantageous for OA investigation since, similarly to primary articular chondrocytes, they showed capacity to upregulate inflammatory mediators in response to the IL-1ß cytokine.Cite this article: Bone Joint Res 2023;12(1):46-57.

16.
Adv Healthc Mater ; : e2202390, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36623538

RESUMO

The repair of damaged cartilage still remains a great challenge in clinic. It is demonstrated that bone marrow stromal cells (BMSCs)-chondrocytes communication is of great significance for cartilage repair. Moreover, BMSCs have been confirmed to enhance biological function of chondrocytes via exosome-mediated paracrine pathway. Lithium-containing scaffolds have been reported to effectively promote cartilage regeneration; however, whether lithium-containing biomaterial could facilitate cartilage regeneration through regulating BMSCs-derived exosomes has not been illustrated. In the study, the model lithium-substituted bioglass ceramic (Li-BGC) is selected and regulatory effects of BMSCs-derived exosomes after Li-BGC treatment (Li-BGC-Exo) are systemically evaluated. The data reveal that Li-BGC-Exo notably promotes chondrogenesis, which attributes to the upregulated exosomal miR-455-3p transfer, consequently leads to suppression of histone deacetylase 2 (HDAC2) and enhanced histone H3 acetylation in chondrocytes. Notably, BMSCs-derived exosomes after LiCl treatment (LiCl-Exo) exhibits the similar regulatory effect with Li-BGC-Exo, indicating that the pro-chondrogenesis capability of them is mainly owing to the lithium ions. Furthermore, the in vivo study proves that LiCl-Exo remarkably facilitates cartilage regeneration. The research may provide novel possibility for the intrinsic mechanism of chondrogenesis trigged by lithium-containing biomaterials, and suggests that application of lithium-containing scaffolds may be a promising strategy for cartilage regeneration.

17.
Cell Signal ; : 110604, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36669606

RESUMO

Articular osteochondral injury is a common and frequently occurring disease in orthopedics that is caused by aging, disease, and trauma. The cytokine interleukin-1ß (IL-1ß) is a crucial mediator of the inflammatory response, which exacerbates damage during chronic disease and acute tissue injury. Human Wharton's jelly mesenchymal stem cell (HWJMSC) extracellular vesicles (HWJMSC-EVs) have been shown to promote cartilage regeneration. The study aimed to investigate the influence and mechanisms of HWJMSC-EVs on the viability, apoptosis, and cell cycle of IL-1ß-induced chondrocytes. HWJMSC-EVs were isolated by Ribo™ Exosome Isolation Reagent kit. Nanoparticle tracking analysis was used to determine the size and concentration of HWJMSC-EVs. We characterized HWJMSC-EVs by western blot and transmission electron microscope. The differentiation, viability, and protein level of chondrocytes were measured by Alcian blue staining, Cell Counting Kit-8, and western blot, respectively. Flow cytometer was used to determine apoptosis and cell cycle of chondrocytes. The results showed that HWJMSCs relieved IL-1ß-induced chondrocyte injury by inhibiting apoptosis and elevating viability and cell cycle of chondrocyte, which was reversed with exosome inhibitor (GW4869). HWJMSC-EVs were successfully extracted and proven to be uptake by chondrocytes. HWJMSC-EVs ameliorate IL-1ß-induced chondrocyte injury by inhibiting cell apoptosis and elevating viability and cycle of cell, but these effects were effectively reversed by knockdown of transferrin receptor (TFRC). Notably, using bone morphogenetic protein 2 (BMP2) pathway agonist and inhibitor suggested that HWJMSC-EVs ameliorate IL-1ß-induced chondrocyte injury through activating the BMP2 pathway via up-regulation TFRC. Furthermore, over-expression of runt-related transcription factor 2 (RUNX2) reversed the effects of BMP2 pathway inhibitor promotion of IL-1ß-induced chondrocyte injury. These results suggested that HWJMSC-EVs ameliorate IL-1ß-induced chondrocyte injury by regulating the BMP2/RUNX2 axis via up-regulation TFRC. HWJMSC-EVs may play a new insight for early medical interventions in patients with articular osteochondral injury.

18.
Endocrinology ; 164(3)2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36592126

RESUMO

Peripheral neuropeptide Y (NPY) has been reported to regulate bone metabolism and homeostasis; however, its potential roles in growth plate chondrogenesis remain unclear. Here, we found that NPY expression decreased during chondrocyte differentiation in vitro and in vivo. NPY was required for chondrocyte proliferation; in contrast, knockdown of NPY facilitated chondrocyte hypertrophic differentiation. Administration of recombinant NPY in rat chondrocytes and metatarsal bones uncoupled normal proliferation and hypertrophic differentiation during chondrogenesis and thereby inhibited growth plate chondrogenesis and longitudinal bone growth. Remarkably, NPY activated the mTORC1 pathway in chondrocytes, whereas attenuation of mTORC1 activity by administration of rapamycin in vitro partially abrogated NPY-mediated effects on chondrocyte proliferation and hypertrophic differentiation. In addition, a combination of Y2R antagonist but not Y1R antagonist with NPY abolished NPY-mediated inhibition of metatarsal growth and growth plate chondrogenesis. Mechanistically, NPY activated Erk1/2 by NPY2R, then phosphorylated ERK1/2 activated mTORC1 to initiate PTHrP expression, which in turn promoted chondrocyte proliferation and inhibited chondrocyte hypertrophic differentiation. In conclusion, our data identified NPY as a crucial regulator of chondrogenesis and may provide a promising therapeutic strategy for skeletal diseases.


Assuntos
Condrócitos , Neuropeptídeo Y , Ratos , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Condrócitos/metabolismo , Neuropeptídeo Y/farmacologia , Neuropeptídeo Y/metabolismo , Hipertrofia/metabolismo , Proliferação de Células , Diferenciação Celular , Condrogênese/fisiologia
19.
J Orthop Surg Res ; 18(1): 34, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36635778

RESUMO

OBJECTIVE: This study aims to investigate the correlation of long non-coding RNA HOX transcript antisense RNA (lncRNA HOTAIR) with the PTEN/PI3K/AKT pathway and clinical-related indicators in osteoarthritis (OA) and determine the effect of baicalin intervention. METHODS: The levels of clinical lipid metabolism indexes and immune-inflammatory indexes in OA patients and normal controls was detected. OA chondrocytes (OA-CHs) were induced with peripheral blood mononuclear cells (PBMCs), followed by baicalin treatment (50 ug/mL). RT-qPCR was performed to measure lncRNA HOTAIR expression. The levels of inflammatory cytokines and adiponectin were detected using ELISA kits. CCK-8 assay was used to assess the viability of CHs. The related protein expression was measured using Western blot analysis. RESULTS: LncRNA HOTAIR might act as a biomarker of OA in vivo. LncRNA HOTAIR was positively correlated with TC, hs-CRP, IgA, TNF-α, and VAS score. Overexpression of lncRNA HOTAIR in vitro inhibited cell proliferation, reduced IL-10 and PTEN expression, but augmented TNF-α, p-PI3K, and p-AKT proteins in OA-CHs stimulated by OA-PBMCs. The changes of above indexes were also observed in OA-CHs stimulated by OA-PBMCs treated with si-lncRNA HOTAIR or baicalin, implying the synergistic effects of baicalin and lncRNA HOTAIR silencing on OA. CONCLUSIONS: Conclusively, lncRNA HOTAIR was highly expressed in OA-CHs, which facilitated OA inflammatory responses by orchestrating inflammatory cytokines and the PTEN/PI3K/AKT pathway. Baicalin exerted therapeutic effects by inhibiting the expression of lncRNA HOTAIR, decreasing the protein levels of p-PI3K and p-AKT, and increasing the protein levels of PTEN, APN, and ADIPOR1.


Assuntos
Osteoartrite , RNA Longo não Codificante , Humanos , Apoptose/genética , Condrócitos/metabolismo , Citocinas/metabolismo , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , Osteoartrite/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
Injury ; 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36621360

RESUMO

BACKGROUND: Drilling is a common technique used in orthopedic surgery procedures but causes increases in temperature that can lead to cell damage and death. The extent of this depends largely on the magnitude of the increase in temperature. The commonly accepted limit to prevent osteonecrosis is less than 47 °C for 60 s. There is controversy when it comes to the optimal drilling parameters that limit temperature increases and cell death. In addition to this, less research has been done on the drilling effects in the osteochondral area of joints. Osteochondral tissue damage can interfere with the daily lives of patients and if severe enough will need to be treated. We hypothesize that increasing tool speed and drill bit size will increase temperature that could be above the osteonecrosis limit. METHODS: Ex-vivo experiments were conducted on porcine shoulder joints that tested the thermal effects of different tool speeds and drill bit sizes. A thermal camera was used to record and measure real time temperature changes while drilling. Three drill bit sizes and five tool speeds were used. Statistical analyses includes Welch's ANOVA with Games-Howell Post Hoc analyses, multivariate linear regression, and surface response regression were used to explore the association of tool speeds and drill bit size on temperature. RESULTS AND CONCLUSIONS: All the tool speed and drill bit size combinations lead to an increase in temperature that were under the commonly accepted limit. The highest temperature reached was 44 °C with a tool speed of 1150 RPM and 3070 RPM and drill bit size 5.159 mm. It was found that increasing the tool speed increased the temperature change and increasing the drill bit size increased the temperature change.

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