Regenerative Potential of Platelet Concentrate Lysate in Mechanically Injured Cartilage and Matrix-Associated Chondrocyte Implantation In Vitro

Adjuvant therapy in autologous chondrocyte implantation (ACI) can control the post-traumatic environment and guide graft maturation to support cartilage repair. To investigate both aspects, we examined potential chondro-regenerative effects of lysed platelet concentrate (PC) and supplementary interleukin 10 (IL-10) on mechanically injured cartilage and on clinically used ACI scaffolds. ACI remnants and human cartilage explants, which were applied to an uniaxial unconfined compression as injury model, were treated with human IL-10 and/or PC from thrombocyte concentrates. We analyzed nuclear blebbing/TUNEL, sGAG content, immunohistochemistry, and the expression of COL1A1, COL2A1, COL10A1, SOX9, and ACAN. Post-injuriously, PC was associated with less cell death, increased COL2A1 expression, and decreased COL10A1 expression and, interestingly, the combination with Il-10 or Il-10 alone had no additional effects, except on COL10A1, which was most effectively decreased by the combination of PC and Il-10. The expression of COL2A1 or SOX9 was statistically not modulated by these substances. In contrast, in chondrocytes in ACI grafts the combination of PC and IL-10 had the most pronounced effects on all parameters except ACAN. Thus, using adjuvants such as PC and IL-10, preferably in combination, is a promising strategy for enhancing repair and graft maturation of autologous transplanted chondrocytes after cartilage injury.

Cyclic Mechanical Stretch Ameliorates the Degeneration of Nucleus Pulposus Cells through Promoting the ITGA2/PI3K/AKT Signaling Pathway

Background. Intervertebral disc degeneration (IVDD) is one of the major causes of low back pain and motor deficiency. Nucleus pulposus (NP) degeneration plays a key role in the process of IVDD. The mechanical and biological interactions involved in NP degeneration have not been elucidated. The present study is aimed at investigating the effect and mechanism of cyclic mechanical stretch in regulating the function and degeneration of NP cells. Methods. NP cells were subjected to cyclic tensile stress (10% deformation) of 0.1 Hz for 8640 cycles. Cell proliferation was conducted through the MTT assay. The cell cycle and apoptosis were detected by flow cytometry. A gene expression profile chip was used to analyze the differentially expressed genes between the tensile stress group and the control group. Enrichment analysis of Gene Ontology (GO) annotation and signaling pathways were analyzed. Western blot and RNA interference were carried out to investigate the role of the ITGA2/PI3K/AKT pathway in the effect of cyclic mechanical stretch on NP cells. Results. NP cells exhibited a greater ( P < 0.05 ) growth rate in the tensile stress group compared to the control group. Cyclic mechanical stress significantly promoted the cell cycle transition of NP cells from the S phase to the G2/M phase. A fewer proportion of apoptotic cells were found in the tensile stress group ( P < 0.05 ), indicating that cyclic mechanical stretch inhibits NP cell apoptosis. Microarray analysis revealed 689 significant differentially expressed genes between the two groups ( P < 0.05 ), of which 333 genes were upregulated and another 356 genes were downregulated. Cyclic mechanical stretch altered the expression of 31 genes involved in the ITGA2/PI3K/AKT pathway and remarkably promoted this pathway in NP cells. Downregulation of ITGA2 and AKT further demonstrated that the PI3K/AKT pathway was responsible for the proliferation and COL2A1 expression of NP cells upon cyclic mechanical stretch. Conclusions. Cyclic mechanical stretch promoted the proliferation and cell cycle and reversely inhibited the apoptosis of NP cells. Cyclic mechanical stretch promoted COL2A1 expression and ameliorated the degeneration of NP cells via regulation of the ITGA2/PI3K/AKT signaling pathway. Our results may provide a potential target and a possibility of IVDD disease treatment by ameliorating the degenerative changes.

SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation

Epigenetic modification is a key driver of differentiation and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC-chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, at different development stages. Activation of SIRT1 during 3D-pellet culture led to significant increases in expression of ECM genes for type-II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the ACAN –10 enhancer. Unexpectedly, while ACAN was enhanced, GAG retention in the matrix was reduced when SIRT1 was activated. Significantly, ARID5B and COL2A1 were positively correlated, with Co-IP indicating association of ARID5B with SIRT1 suggesting that COL2A1 expression is promoted by an ARID5B and SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, whilst altering ECM composition and suppressing GAG content during cartilage development. These results suggest that SIRT1 activity can be beneficial to cartilage development and matrix protein synthesis but tailored by addition of other positive GAG mediators.

Celecoxib Attenuates Cartilage Matrix Damage in Arthritis Rats by Inhibiting NF-κ B

Objective: Celecoxib selectively inhibits the activity of COX-2 and the production of prostaglandin (PG), and plays a therapeutic role in treating osteoarthritis (OA). NF-κB signaling and IL-1α and TNFα are involved in OA pathogenesis. This study explored whether Celecoxib might exert therapeutic effects on OA through regulating NF-κB signaling and IL-1β and TNF release in OA rat model. Method : The contents of MMP-13, Hyp, IL-1β and TNFα in synovial fluid were detected by ELISA. The protein expressions of NF-κ B p-p65, COL2A1 and the activity of caspase-3 were detected. OA model rats were separated into OA group and OA+ Celecoxib group followed by analysis of MMP-13, Hyp, IL-1β and TNF level in articular fluid by ELISA and p-p65 and COL2A1 level and caspase-3 activity by western blot. Rat cartilage tissue was cultured and divided into control group, LPS group and LPS+ Celecoxib group followed by analysis of expressions of p-p65 and COL2A1 in cartilage tissue, IL-1 and TNF content in culture supernatant, and chondrocyte apoptosis. Results: Compared with Sham group, p-p65 expression and caspase-3 activity in cartilage tissue of OA rats was increased and COL2A1 level was reduced. Meanwhile the expression of MMP-13, Hyp, IL-1β and TNF in articular fluid of OA rats was increased. Compared to OA group, p-p65 expression and caspase-3 activity was decreased and COL2A1 expression was increased in OA+ Celecoxib treatment group along with decreased MMP-13, Hyp, IL-1β and TNF level in articular fluid. p-p65 expression and caspase-3 activity in LPS group was increased and COL2A1 expression was decreased with increased IL-beta; and TNF content. Compared to LPS group, p-p65 expression and caspase-3 activity was decreased and COL2A1 expression was increased in LPS+ Celecoxib group with decreased content of IL-1β and TNFα. Conclusion: Celecoxib can protect cartilage in OA by inhibiting NF-κB activation and IL-1β and TNF release, and decreasing cell apoptosis in inflammatory environment.

miR-211 Reduces the Degradation of Articular Cartilage Matrix by Targeting Matrix Metalloproteninase 9

Matrix metalloproteninase 9 (MMP9) promotes osteoarthritis (OA) cartilage matrix degradation. Abnormal miR-211 expression is associated with OA. Bioinformatics analysis showed a targeted relationship between miR-211 and MMP9 mRNA 3′-UTR. Our study intends to evaluate miR-211’s role in the destruction of chondrocyte matrix in OA model rats. The OA model rats were divided into OA group, OA group+agomir-NC group, OA group+agomir-211 group, followed by analysis of the arthritis Mankin score, Hyp and IL-1β content by ELISA, MMP-9 and COL2A1 expression by western blot and miR-211 level by qRT-PCR. The cartilage tissues were divided into control group, IL-1β+ agomir-NC group, IL-1β+ agomir-211 group, and the expression of MMP-9 and COL2A1 in chondrocytes were detected. Compared with Sham group, IL-1β and Hyp levels in the joint cavity fluid of the OA group were significantly increased and miR-211 expression was significantly decreased with increased MMP9 expression and decreased COL2A1 expression. Injection of agomir-211 into the joint cavity of OA rats significantly reduced MMP9 expression in cartilage tissue, decreased Hyp content in the joint cavity fluid, increased COL2A1 expression, and decreased Mankin score and cartilage damage. IL-1β treatment significantly up-regulated MMP9 expression and decreased COL2A1 expression; miR-211 overexpression attenuated IL-1β-induced cartilage matrix degradation and cartilage destruction. miR-211 plays a role in regulating MMP9 expression and promoting OA. miR-211 overexpression inhibits MMP9 expression, reduces the degradation of cartilage matrix and increases collagen synthesis, thus ameliorating OA development.

miR-155 Regulates GSK-3β Expression and Affects Cartilage Matrix Degradation in the Pathogenesis of Osteoarthritis

Abnormal expression of GSK-3β is associated with the pathogenesis of osteoarthritis (OA). The miR-155 expression in cartilage tissue of OA patients was significantly elevated. A relationship between miR-155 and GSK-3β is predicted by the software. This study investigated whether miR155 regulates GSK-3β expression, affects Wnt/β-catenin pathway activity in OA. The cartilage tissues of OA patients and normal cartilage tissues after traumatic amputation were collected. The OA rat model was established and divided into OA + antagomir control group, OA + miR-155 antagomir group followed by analysis of expression of miR-155, GSK-3β, β-catenin and COL2A1 by qRT-PCR, Hyp content by ELISA, caspase-3 activity, and GSK-3β, β-catenin, COL2A1 expression by western blot. Compared to control group, the expression of miR-155 and -catenin in cartilage tissue of OA patients was significantly elevated and GSK-3β level was reduced. There was a targeted regulation relationship between miR-155 and GSK-3β. OA group had significantly higher miR-155 and β-catenin expression and lower GSK-3 and COL2A1 level in the cartilage than sham group. Compared with OA + antagomir control group, miR-155 and β-catenin expression and caspase-3 activity in OA + miR-155 antagomir group were significantly decreased, with increased expression of GSK3β and COL2A1 and reduced Hyp content. Increased miR-155 expression can decrease GSK-3β expression, enhance Wnt/β-catenin pathway activity, promote the degradation and destruction of cartilage matrix and inhibit miR-155 expression, thus ameliorating the development and progress of OA.

Does Nimodipine, a Selective Calcium Channel Blocker, Impair Chondrocyte Proliferation or Damage Extracellular Matrix Structures?

Background: The study aimed to investigate the effects of the active ingredient, nimodipine, on chondrocyte proliferation and extracellular matrix (ECM) structures in cartilage tissue cells. Methods: Chondrocyte cultures were prepared from tissues resected via surgical operations. Nimodipine was then applied to these cultures and molecular analysis was performed. The data obtained were statistically calculated. Results: Both, the results of the (3-(4,5 dimethylthiazol2-yl)-2,5-diphenyltetrazolium (MTT) assay and the fluorescence microscope analysis [a membrane permeability test carried out with acridine orange/ propidium iodide staining (AO/PI)] confirmed that the active ingredient, nimodipine, negatively affects the cell cultures. Conclusion: Nimodipine was reported to suppress cellular proliferation; chondroadherin (CHAD) and hypoxia-inducible factor-1 alpha (HIF-1α) expression thus decreased by 2.4 and 1.7 times, respectively, at 24 hrs when compared to the control group (p < 0.05). Furthermore, type II collagen (COL2A1) expression was not detected (p<0.05). The risk that a drug prescribed by a clinician in an innocuous manner to treat a patient by relieving the symptoms of a disease may affect the proliferation, differentiation, and viability of other cells and/or tissues at the molecular level, beyond its known side effects or adverse events, should not be forgotten.

miR-21-5p protects IL-1β-induced human chondrocytes from degradation

Objective Osteoarthritis (OA) is a prevalent degenerative disease caused by various factors. MicroRNAs are important regulators in OA. MiR-21-5p expression is decreased in OA cartilage, but the effects of modulating miR-21-5p on cartilage regeneration are unknown. Therefore, our aim was to investigate the effects of miR-21-5p on cartilage metabolism of OA chondrocytes. Design We used IL-1β (10 ng/ml) to mimic OA chondrocytes. OA chondrocytes were transfected with miR-21-5p, the gene expression of COL2A1, MMP13, and ADAMTS5 was detected by qPCR. At the same time, COL2A1, MMP13, and ADAMTS5 were analyzed at the protein level by Western blot. CCK8 measured the cell’s viability and SA-β-gal detected the cell’s senescence. Results Upregulation of miR-21-5p had increased COL2A1 expression and decreased MM P13 and ADAMTS5 expression, which were in accord with Western blot data. SA-β-gal activity significantly increased, the viability was decreased in OA chondrocytes, and upregulation of miR-21-5p can decrease the SA-β-gal activity and increase cell viability. Conclusion MiR-21-5p might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production. These results provided that novel insights into the important function in OA pathological development.