As a kind of mechanical effector cells, chondrocytes can produce a variety of physical and chemical signals under the stimulation of multiaxial load in vivo, which affect their own growth, development and apoptosis. Therefore, simulating the mechanical environment in vivo has become a research hotspot in the culture of chondrocytes in vitro. Although a large number of reports have fully proved that different mechanical stimulation can regulate the metabolism of chondrocytes, the loading scheme has not been agreed. Starting from different mechanical forms, this review will explore the differences in the regulation of chondrocyte metabolism by different mechanical stimuli, so as to find an advantage scheme to promote the growth and proliferation of chondrocytes and to develop a more stable, effective and reliable experimental strategy.
Objective To explore the effects of low-intensity pulsed ultrasound (LIPUS) on anabolism, apoptosis and intraflagellar transport 88 (IFT88) expression in mouse chondrocytes after interleukin (IL)-1β intervention, and the correlation of cartilage repairment by LIPUS with primary cilia. Methods IL-1β intervention, LIPUS intervention and lentiviral carrying IFT88-specifific short hairpin RNA (sh-IFT88) transfection were performed on mouse chondrocytes, respectively. The groups included: normal chondrocyte group (N group), chondrocyte after IL-1β intervention group (OA group), chondrocyte after IL-1β intervention+LIPUS group (OA+U group), sh-IFT88+IL-1β intervention chondrocyte group (KO+OA group), and sh-IFT88+LIPUS+IL-1β treated chondrocyte group (KO+OA+U group). Real-time polymerase chain reaction and immunofluorescence were used to determine the expression of collagen Ⅱ, aggrecan, and primary cilia, and apoptosis was measured by flow cytometry. All experimental data were statistically analyzed using the GraphPad Prism 9.5 software. Results The expression of collagen Ⅱ and aggrecan increased, the apoptosis decreased, and the incidence of primary cilia in chondrocytes of mice increased in the OA+U group compared with those in the OA group (P<0.05). The collagen Ⅱ and aggrecan expression decreased and the apoptosis increased in the KO+OA+U group compared with those in the OA+U group (P<0.05). Conclusion LIPUS can reduce the apoptosis of chondrocytes in C57 mice after IL-1β intervention, and increase the expression of collagen Ⅱ and aggrecan in chondrocyte matrix, and the effect is related to primary cilia.
Objective To sum up the experimental and clinical history as wellas latest development of repair of growth plate injury Methods Recent articles about repair of growth plate injury were extensively reviewed and major reparative methods were introduced, especially including tissue engineering research on growth plate.Results Repair of growth plate injury was a great difficulty inexperimental study and clinical treatment of pediatric orthopedics. Transplantation of free growth plate and cartilage were unfavorably used because of lack ofblood supplement. Although circulation problem was solved by transplantation ofvascularized growth plate, autografts of epiphyseal cartilage were involved in limitation of donor, and allografts of epiphyseal cartilage induced immunological reaction. Noncartilaginous tissue and material could only prevent formation of bony bridge in small defect of growth plate and lacked ability of regenerative repair. Transplantationof tissue engineered cartilage and chondrocytes might be a choice for repair ofgrowth plate injury Conclusion Owing to lack of safe and effective methods ofrepairing growth plate injury, research on chondrocyte and tissue engineered cartilage should be further done.
ObjectiveTo observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage.MethodsChondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining).ResultsAfter chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining.ConclusionACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.
Objective To determine the short-term effectiveness of matrix-induced autologous chondrocyte implantation (MACI) for femoral trochlea cartilage injury. Methods A retrospective analysis was performed on the clinical data of 10 patients with femoral trochlea cartilage injury treated with MACI between June 2012 and October 2014. There were 6 males and 4 females, aged from 15 to 48 years (mean, 33 years). The left knee was involved in 3 cases and the right knee in 7 cases. Nine patients had a history of trauma, and 1 case suffered from osteochondritis dissecans. Combined injuries included meniscus injury in 1 case, anterior cruciate ligament injury in 3 cases, and lateral collateral ligament tear in 2 cases. The mean lesion depth was 2.80 mm (range, 2-7 mm), with the mean defect size of 84.85 mm2 (range, 28.26-153.86 mm2). The mean duration of definite diagnosis was 14 days (range, 5 days to 3 months). By using arthroscopic biopsy, 200-300 mg healthy articular cartilage at non weight-bearing area of the knee femoral trochlea was collected as a source of seed cells, which were isolated and cultured to prepare MACI membrane. The adhesion activity, growth rate, and mechanical properties of the chondrocytes on the Bio-gide collagen scaffold were evaluated. In addition, the stretch rate, tensile strength, and suture strength of scaffold were tested. MACI membrane was implanted after 2 weeks to 6 months. The visual analogou scale (VAS), Lysholm score, and Tegner movement level score at preoperation and last follow-up were used to assess the function. Results The MACI membrane was successfully prepared, and the human chondrocytes adhered and grew well on the Bio-gide collagen scaffold. Mechanical test showed that MACI membrane had the stretch rate of 65.27%, the tensile strength of 26.81 MPa, and the suture strength of 6.49 N, indicating good mechanical properties. MACI membrane was successfully implanted. The mean operation time was 58.5 minutes (range, 43-99 minutes), and the mean hospitalization time was 7 days (range, 6-15 days). All incisions healed well. Ten cases were followed up 9 to 16 months (mean, 12 months). Four cases underwent iliac bone graft surgery. The mean healing time was 14 weeks (range, 12-16 weeks). No complications of osteochondrolysis, knee pain, nerve and vascular injury, deep vein thrombosis, and knee adhesion occurred during follow-up. The VAS score, Lysholm score, and Tegner score at last follow-up were significantly improved when compared with preoperative scores (t=12.060,P=0.000;t=–9.200,P=0.000;t=–14.000,P=0.000). Conclusion MACI for femoral trochlea cartilage injury has good short-term effectiveness, with less injury and fast function recovery.
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.
ObjectiveTo review the research progress of different cell seeding densities and cell ratios in cartilage tissue engineering. MethodsThe literature about tissue engineered cartilage constructed with three-dimensional scaffold was extensively reviewed, and the seeding densities and ratios of most commonly used seed cells were summarized. ResultsArticular chondrocytes (ACHs) and bone marrow mesenchymal stem cells (BMSCs) are the most commonly used seed cells, and they can induce hyaline cartilage formation in vitro and in vivo. Cell seeding density and cell ratio both play important roles in cartilage formation. Tissue engineered cartilage with good quality can be produced when the cell seeding density of ACHs or BMSCs reaches or exceeds that in normal articular cartilage. Under the same culture conditions, the ability of pure BMSCs to build hyaline cartilage is weeker than that of pure ACHs or co-culture of both. ConclusionDue to the effect of scaffold materials, growth factors, and cell passages, optimal cell seeding density and cell ratio need further study.
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 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.
This study was to explore a better three-dimensional (3-D) culture method of chondrocyte. The interpenetrating network (IPN) gel beads were developed through a photo-cross linking reaction with mixed barium ions and calcium ions at the ratio of 5:5 with the methacrylic alginate (MA), which was a chemically conjugated alginate with methacrylic groups. The second generation of primary cartilage cells was encapsulated in the MA gel beads for three weeks. In the designated timing, HE stain, Alamar blue method and Scanning electron microscopic were used to determine the cartilage cells growth, proliferation and the cell distribution in the scaffolds, respectively. The expression of typeⅡcollagen was investigated by an immunohistochemistry assay and the glycosaminoglycan content was quantitatively evaluated with the spectrophotometry of 1, 9 dimethylene blue assay. Compared to the alginate control group, the deposition of glycosaminoglycan was significantly upregulated in IPN-MA gel beads with higher cell proliferation. The secretion of extracellular matrix and proliferation of chondrocyte in methacrylic alginate gel beads were higher than that in Alginate beads. Cells were able to attach, to grow well on the scaffolds under scanning electron microscopy. The result of immunohistochemistry staining of collagen typeⅡwas positive, confirming the maintenance of chondrocyte phenotype in methacrylic alginate gel beads. This study shows a great potential for three-dimensional culture of cartilage.