Objective To summarize the role of chondrocytes mitochondrial biogenesis in the pathogenesis of osteoarthritis (OA), and analyze the applications in the treatment of OA. Methods A review of recent literature was conducted to summarize the changes in mitochondrial biogenesis in the course of OA, the role of major signaling molecules in OA chondrocytes, and the prospects for OA therapeutic applications. Results Recent studies reveales that mitochondria are significant energy metabolic centers in chondrocytes and its dysfunction has been considered as an essential mechanism in the pathogenesis of OA. Mitochondrial biogenesis is one of the key processes maintaining the normal quantity and function of mitochondria, and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is the central regulator of this process. A regulatory network of mitochondrial biogenesis with PGC-1α as the center, adenosine monophosphate-activated protein kinase, sirtuin1/3, and cyclic adenosine monophosphate response element-binding protein as the main upstream regulatory molecules, and nuclear respiratory factor 1, estrogen-related receptor α, and nuclear respiratory factor 2 as the main downstream regulatory molecules has been reported. However, the role of mitochondrial biogenesis in OA chondrocytes still needs further validation and in-depth exploration. It has been demonstrated that substances such as puerarin and omentin-1 can retard the development of OA by activating the damaged mitochondrial biogenesis in OA chondrocytes, which proves the potential to be used in the treatment OA. ConclusionMitochondrial biogenesis in chondrocytes plays an important role in the pathogenesis of OA, and further exploring the related mechanisms is of great clinical significance.
Objective To investigate the effects of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) conjugated chitosan (CS) (NNSCS) mediated the transfection of microRNA-140 (miR-140) in rabbit articular chondrocytes in vitro. Methods Recombinant plasmid GV268-miR-140 and empty plasmid GV268 were combined with NNSCS to form NNSCS/pDNA complexes, respectively. Chondrocytes were isolated and cultured through trypsin and collagenase digestion from articular cartilage of newborn New Zealand white rabbits. The second generation chondrocytes were divided into 3 intervention groups: normal cell control group (group A), NNSCS/GV268 empty plasmid transfection group (group B), and NNSCS/GV268-miR-140 transfection group (group C). NNSCS/GV268 and NNSCS/GV268-miR- 140 complexes were transiently transfected into cells of groups B and C. After transfection, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expressions of exogenous miR-140; Annexin Ⅴ-FITC/PI double staining and MTT assay were used to detect the effect of exogenous miR-140 on apoptosis and proliferation of transfected chondrocytes; the expressions of Sox9, Aggrecan, and histone deacetylase 4 (Hdac4) were detected by RT-qPCR. Results RT-qPCR showed that the expression of miR-140 in group C was significantly higher than that in groups A and B (P<0.05). Compared with groups A and B, the apoptosis rate in group C was decreased and the proliferation activity was improved, Sox9 and Aggrecan gene expressions were significantly up-regulated, and Hdac4 gene expression was significantly down-regulated (P<0.05). There was no significant difference in above indexes between groups A and B (P>0.05). Conclusion Exogenous gene can be carried into the chondrocytes by NNSCS and expressed efficiently, the high expression of miR-140 can improve the biological activity of chondrocytes cultured in vitro, which provides important experimental basis for the treatment of cartilage damage diseases.
Objective To investigate the effect of epigallocatechin gallate (EGCG) on chondrocyte senescence and its mechanism. Methods The chondrocytes were isolated from the articular cartilage of 4-week-old Sprague Dawley rats, and cultured with type Ⅱcollagenase and passaged. The cells were identified by toluidine blue staining, alcian blue staining, and immunocytochemical staining for type Ⅱ collagen. The second passage (P2) cells were divided into blank control group, 10 ng/mL IL-1β group, and 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 μmol/L EGCG+10 ng/mL IL-1β group. The chondrocyte activity was measured with cell counting kit 8 after 24 hours of corresponding culture, and the optimal drug concentration of EGCG was selected for the subsequent experiment. The P2 chondrocytes were further divided into blank control group (group A), 10 ng/mL IL-1β group (group B), EGCG+10 ng/mL IL-1β group (group C), and EGCG+10 ng/mL IL-1β+5 mmol/L 3-methyladenine (3-MA) group (group D). After cultured, the degree of cell senescence was detected by β-galactosidase staining, the autophagy by monodansylcadaverine method, and the expression levels of chondrocyte-related genes [type Ⅱ collagen, matrix metalloproteinase 3 (MMP-3), MMP-13] by real-time fluorescent quantitative PCR, the expression levels of chondrocyte-related proteins (Beclin-1, LC3, MMP-3, MMP-13, type Ⅱ collagen, P16, mTOR, AKT) by Western blot. Results The cultured cells were identified as chondrocytes. Compared with the blank control group, the cell activity of 10 ng/mL IL-1β group significantly decreased (P<0.05). Compared with the 10 ng/mL IL-1β group, the cell activity of EGCG+10 ng/mL IL-1β groups increased, and the 50.0, 100.0, and 200.0 μmol/L EGCG significantly promoted the activity of chondrocytes (P<0.05). The 100.0 μmol/L EGCG was selected for subsequent experiments. Compared with group A, the cells in group B showed senescence changes. Compared with group B, the senescence rate of chondrocytes in group C decreased, autophagy increased, the relative expression of type Ⅱ collagen mRNA increased, and relative expressions of MMP-3 and MMP-13 mRNAs decreased; the relative expressions of Beclin-1, LC3, and type Ⅱ collagen proteins increased, but the relative expressions of P16, MMP-3, MMP-13, mTOR, and AKT proteins decreased; the above differences were significant (P<0.05). Compared with group C, when 3-MA was added in group D, the senescence rate of chondrocytes increased, autophagy decreased, and the relative expressions of the target proteins and mRNAs showed an opposite trend (P<0.05). ConclusionEGCG regulates the autophagy of chondrocytes through the PI3K/AKT/mTOR signaling pathway and exerts anti-senescence effects.
ObjectiveTo study the effect of chemical extraction of allogeneic tendon and allogeneic chondrocytes for reconstruction of anterior labrum of shoulder joint in rabbits.MethodsThe body weight of 45 adult New Zealand white rabbits ranged from 2.5 to 3.0 kg. The Achilles tendons of 15 rabbits were taken and the allogeneic tendons were prepared by chemical extraction with antigen inactivation. The extracted tendons were compared with untreated tendons by HE and Masson stainings. Chondrocytes were isolated and cultured by trypsin method and identified by immunohistochemical staining of collagen type Ⅱ. The remaining 30 rabbits were used to prepare the model of anterior labrum defect of shoulder joint. After the allogeneic tendon was transplanted to the damaged labrum, the rabbits was randomly divided into two groups (15 in each group). In group A, the allogeneic chondrocytes were injected into the joint immediately after transplantation, while in group B, no treatment was made. At 4, 6, and 8 weeks after operation, 5 transplanted tendons of each group were taken. After general observation, HE staining was used to observe the number of nuclei, Masson staining was used to observe the expression of collagen fibers in muscle fiber tissues, and AB staining was used to detect the glycosaminoglycan level after transplantation, to evaluate the cell growth in the tissues of the two groups of allogeneic tendon.ResultsBy HE and Masson stainings, the allogeneic tendon antigen prepared by chemical extraction method was inactivated and the fibrous tissue structure was intact; collagen type Ⅱ immunohisto-chemistry staining showed that the cultured cells were chondrocytes. After tendon transplantation, the content of glycosaminoglycan in group A was significantly higher than that in group B (P<0.05). At 6 weeks after operation, HE staining showed that the nuclear in tendon tissue of group A was significantly more than that of group B (t=20.043, P=0.000). Masson staining showed that the number of nuclei in tendon tissue of group A was significantly increased, the muscle fibers and collagen fibers were interlaced, the tissue structure was more compact, and the tendon tissue was mainly blue stained; while the number of nuclei in group B was less, mainly collagen fibers of the original graft.ConclusionThe allogeneic tendon inactivated by chemical extraction can be used to reconstruct the defect of anterior labrum of shoulder joint in rabbits, and the combination of allogeneic chondrocytes can promote the healing of tendon transplantation.
The aim of this article is to study how andrographolide-releasing collagen scaffolds influence rabbit articular chondrocytes in maintaining their specific phenotype under inflammatory environment. Physical blending combined with vacuum freeze-drying method was utilized to prepare the andrographolide-releasing collagen scaffold. The characteristics of scaffold including its surface morphology and porosity were detected with environmental scanning electron microscope (ESEM) and a density instrument. Then, the release of andrographolide from prepared scaffolds was measured by UV-visible spectroscopy. Rabbit chondrocytes were isolated and cultured in vitro and seeded on andrographolide-releasing collagen scaffolds. Following culture with normal medium for 3 d, seeded chondrocytes were cultured with medium containing interleukin-1 beta (IL-1β) to stimulate inflammation in vitro for 7 d. The proliferation, morphology and gene transcription of tested chondrocytes were detected with Alamar Blue assay, fluorescein diacetate (FDA) staining and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test respectively. The results showed that the collagen scaffolds prepared by vacuum freeze-dry possess a high porosity close to 96%, and well-interconnected chambers around (120.7±17.8) μm. The andrographolide-releasing collagen scaffold continuously released andrographolide to the PBS solution within 15 d, and collagen scaffolds containing 2.22% andrographolide significantly inhibit the proliferation of chondrocytes. Compared with collagen scaffolds, 0.44% andrographolide-containing collagen scaffolds facilitate chondrocytes to keep specific normal morphologies following 7 d IL-1β induction. The results obtained by RT-qPCR confirmed this effect by enhancing the transcription of tissue inhibitor of metalloproteinase-1 (TIMP-1), collagen II (COL II), aggrecan (Aggrecan) and the ratio of COL II/ collagen I(COL I), meanwhile, reversing the promoted transcription of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13). In conclusion, our research reveals that andrographolide-releasing (0.44%) collagen scaffolds enhance the ability of chondrocytes to maintain their specific morphologies by up-regulating the transcription of genes like COL II, Aggrecan and TIMP-1, while down-regulating the transcription of genes like MMP-1 and MMP-13 which are bad for phenotypic maintenance under IL-1β simulated inflammatory environment. These results implied the potential use of andrographolide-releasing collagen scaffold in osteoarthritic cartilage repair.
Objective Corticosteroids can destroy the cartilage. To investigate the effect of dexamethasone (Dexa) on the apoptosis and expression of Fas/FasL of human articular chondrocytes (HACs) in vitro so as to explore the mechanism ofpro-apoptotic role of Dexa on HACs. Methods Following full agreement of patients, the cartilage specimens were collectedfrom the patients with osteoarthritis undergoing knee replacement. The second passage HACs were incubated in cell culture media containing 0.125, 1.25, 12.5, 25, and 50 μg/mL Dexa for 48 hours respectively to determine the optimal concentration of Dexa by MTT. The apoptosis was assessed by TMRE/Hoechst/Annexin V-FITC/7-AAD quadruple staining after culture for 0, 24, and 48 hours. The mRNA expressions of Fas and FasL were determined by real-time quantitative PCR after culture for 48 hours. The protein expressions of Fas and FasL were determined by immunohistochemistry staining analysis after culture for 24 hours and 48 hours. Results The cell inhibitory rate of 25 μg/mL Dexa was significantly higher than that of 50 μg/mL Dexa (P lt; 0.05), and there were significant differences when compared with that at other concentrations of Dexa (P lt; 0.05), so 25 μg/mL Dexa was appropriately selected as an optimal concentration of Dexa. The apoptotic rates of HACs were 5.8% ± 0.3%, 27.0% ± 2.6%, and 36.0% ± 3.1% at 0, 24, and 48 hours, respectively, in a time dependent manner (P lt; 0.05). The expressions of Fas mRNA were (8.93 ± 1.12) × 10—3 in the experimental group and (3.31 ± 0.37) × 10—3 in the control group, showing significant difference (P lt; 0.05). The expressions of FasL mRNA were (5.92 ± 0.66) × 10—3 in the experimental group and (2.31 ± 0.35) × 10—3in the control group, showing significant difference (P lt; 0.05). The expressions of Fas and FasL proteins showed an increasing tendency with time in the experimental group and the expressions were significantly higher than those in the control group after culture for 24 hours and 48 hours (P lt; 0.05). Conclusion Dexa can induce the apoptosis and significantly upregulate the apoptotic gene expression of Fas/FasL, which can provide the experimental evidence to further investigate the role of Fas/FasL signaling pathway in Dexa-induced HACs apoptosis.
Objective To investigate the effects and underlying mechanisms of VX765 on osteoarthritis (OA) and chondrocytes inflammation in rats. MethodsChondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley (SD) rats. The third-generation cells were subjected to cell counting kit 8 (CCK-8) analysis to assess the impact of various concentrations (0, 1, 5, 10, 20, 50, 100 μmol/L) of VX765 on rat chondrocyte activity. An in vitro lipopolysaccharide (LPS) induced cell inflammation model was employed, dividing cells into control group, LPS group, VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity. Western blot, real-time fluorescence quantitative PCR, and ELISA were conducted to measure the expression levels of inflammatory factors—transforming growth factor β1 (TGF-β1), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Additionally, Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Thirty-two SD rats were randomly assigned to sham surgery group (group A), OA group (group B), OA+VX765 (50 mg/kg) group (group C), and OA+VX765 (100 mg/kg) group (group D), with 8 rats in each group. Group A underwent a sham operation with a medial incision, while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament, with removal of the medial meniscus. One week post-surgery, groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765, respectively, while groups A and B received an equivalent volume of saline. Histopathological examination using HE and safranin-fast green staining was performed, and Mankin scoring was utilized for evaluation. Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13 (MMP-13) and collagen type Ⅱ. ResultsThe CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 μmol/L (P<0.05), so 4 μmol/L and 8 μmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments. Following LPS induction, the expressions of TGF-β1, IL-6, and TNF-α in cells significantly increased when compared with the control group (P<0.05). However, intervention with 4 μmol/L and 8 μmol/L VX765 led to a significant decrease in expression compared to the LPS group (P<0.05). Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765 (P<0.05), indicating Nrf2 pathway activation. Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that, compared to group B, treatment with VX765 in groups C and D improved joint structural damage in rat OA, alleviated inflammatory reactions, downregulated MMP-13 expression, and increased collagen type Ⅱ expression.ConclusionVX765 can improve rat OA and reduce chondrocyte inflammation, possibly through the activation of the Nrf2 pathway.
Objective To investigate the effects of the misshapen auricular chondrocytes from microtia in inducing chondrogenesis of human adipose derived stem cells (ADSCs) in vitro. Methods Human ADSCs at passage 3 and misshapen auricular chondrocytes at passage 2 were harvested and mixed at a ratio of 7 ∶ 3 as experimental group (group A, 1.0 × 106 mixed cells). Misshapen auricular chondrocytes or ADSCs at the same cell number served as control groups (groups B and C, respectively). All samples were incubated in the centrifuge tubes. At 28 days after incubation, the morphological examination was done and the wet weight was measured; the content of glycosaminoglycan (GAG) was detected by Alcian blue colorimetry; the expressions of collagen type II and Aggrecan were determined with RT-PCR; and HE staining, toluidine blue staining, Safranin O staining of GAG, and collagen type II immunohistochemical staining were used for histological and immunohistochemical observations. Results At 28 days after incubation, all specimens formed disc tissue that was translucent and white with smooth surface and good elasticity in groups A and B; the specimens shrank into yellow spherical tissue without elasticity in group C. The wet weight and GAG content of specimens in groups A and B were significantly higher than those in group C (P lt; 0.05), but no significant difference was found between groups A and B in the wet weight (t=1.820 3, P=0.068 7) and in GAG content (t=1.861 4, P=0.062 7). In groups A and B, obvious expressions of collagen type II and Aggrecan mRNA could be detected by RT-PCR, but no obvious expressions were observed in group C; the expressions in groups A and B were significantly higher than those in group C (P lt; 0.05), but no significant difference was found between groups A and B in collagen type II mRNA expression (t=1.457 6, P=0.144 9) and Aggrecan mRNA expression (t=1.519 5, P=0.128 6). Mature cartilage lacunas and different degrees of dyeing for the extracellular matrix could be observed in groups A and B; no mature cartilage lacunas or collagen type II could be observed in group C. The expression of collagen type II around cartilage lacuna was observed in groups A and B, but no expression in group C; the gray values of groups A and B were significantly lower than that of group C (P lt; 0.01), but no significant difference was found between groups A and B (t=1.661 5, P=0.09 7 0). Conclusion Misshapen auricular chondrocytes from microtia can induce chondrogenic differentiation of human ADSCs in vitro.
Objective To investigate the effect of dynamic compression and rotation motion on chondrogenesis of the 3rd passage cell-loaded three-dimensional scaffold in a joint-specific bioreactor in vitro so as to provide theoretical basis of the autologous chondrocyte transplantation in clinical practice. Methods Primary chondrocytes were isolated and cultured from the knee cartilage of 3-4 months old calves. The 3rd passage cells were seeded onto fibrin-polyurethane scaffolds (8 mm × 4 mm). Experiment included 5 groups: unloaded culture for 2 weeks (group A), direct load for 2 weeks (group B), unloaded culture for 4 weeks (group C), direct load for 4 weeks (group D), and unload for 2 weeks followed by load for 2 weeks (group E). The cell-scaffold was incubated in incubator (unload) or in a joint-specific bioreactor (load culture). At different time points, the samples were collected for DNA and glycosaminoglycan (GAG) quantification detect; mRNA expressions of chondrogenic marker genes such as collagen type I, collagen type II, Aggrecan, cartilage oligomeric matrix protein (COMP), and superficial zone protein (SZP) were detected by real-time quantitative PCR; and histology observations were done by toluidine blue staining and immunohistochemistry staining. Results No significant difference was found in DNA content, GAG content, and the ratio of GAG to DNA among 5 groups (P gt; 0.05). After load, there was a large number of GAG in the medium, and the GAG significantly increased with time (P lt; 0.05). The mRNA expression of collagen type I showed no significant difference among 5 groups (P gt; 0.05). The mRNA expression of collagen type II in group B was significantly increased when compared with group A (P lt; 0.01), and groups D and E were significantly higher than group C (P lt; 0.01); the mRNA expression of Aggrecan in groups D and E were significantly increased when compared with group C (P lt; 0.01), and group E was significantly higher than group D (P lt; 0.01); the mRNA expression of COMP in group B was significantly increased when compared with group A (P lt; 0.01), and group E was significantly higher than group C (P lt; 0.01); and the mRNA expression of SZP in group E was significantly increased when compared with groups C and D (P lt; 0.05). The toluidine blue staining and immunohistochemistry staining displayed that synthesis and secretion of GAG could be enhanced after load; no intensity changes of collagen type I and collagen type II were observed, but intensity enhancement of Agrrecan was seen in groups D and E. Conclusion Different dynamic loads can promote chondrogenesis of the 3rd passage chondrocytes. Culture by load after unload may be the best culture for chondrogenesis, while the 3rd passage chondrocytes induced by mechanical load hold less capacity of chondrogenesis.
Objective To observe the effect of Melittin on collagen type II (Col-II) expression of rat endplate chondrocytes (EPCs) induced by interleukin 1β (IL-1β). Methods Primary EPCs from the lumbar vertebra of 4-week-old Sprague Dawley rats were culturedin vitro and identified by morphological observation, toluidine blue staining and Col-II immunofluorescence staining. Then, MTT assay was used to determine the optimal concentration of IL-1 and Melittin. Next, EPCs at passage 3 were randomly divided into 4 groups: no treatment was done in group A as control group; the optimal concentration of IL-1β, Melittin, and both IL-1β and Melittin were used in groups B, C, and D respectively. The expression of Col-II was detected by Western blot after 48 hours intervention. Results Under inverted microscope, the first generation EPCs were polygonal; cell proliferation decreased after fifth generation, and cell morphology changed into fusiform. The acidic mucosubstance in the cytoplasm (such as Aggrecan) was stained dark blue by toluidine blue. After marking Col-II by immunofluorescence, the positive expression of cytoskeleton (green fluorescence) could be observed. MTT assay showed that IL-1β and Melittin could inhibit the EPCs in a dose-dependent manner after intervention of 24 and 48 hours, and the optimal concentrations of IL-1β and Melittin intervention were 10 ng/mL and 1.0 μg/mL respectively. Compared with group A, the expression of Col-II was significantly reduced in group B, and was significantly increased in group C by Western blot assay, but there was no significant difference between group D and group A. The Col-II expression levels of groups A, B, C, and D were 0.991±0.024, 0.474±0.127, 1.913±0.350, and 1.159±0.297 respectively, showing significant difference between the other groups (P<0.05) except between group A and group D (P>0.05). Conclusion Melittin has a protective effect on endplate cartilage, and the research results provide experimental basis for the prevention and treatment of spinal degenerative disease.