Objective To explore the effects of the basic fibroblast growth factor（bFGF） gene transfection on the meniscal fibrochondrocytes with the reconstructed lentivirus and to observe the response of the meniscal fibrochondrocytes to the bFGF gene transfection. Methods The cultured meniscal fibrochondrocytes were isolated from the same 3-monthold New Zealand rabbit. The cultured first-generation meniscal fibrochondrocytes were divided into 3 groups:Group A (experimental group), Group B (control group), and Group C (blank group). Each group comprised the cells in a 24hole flask in which each hole contained 2×104 cells. At the confluence of 60%, the fibrochondrocytes in Group A were cultured with the reconstructed lentivirus carrying the bFGF gene. The fibrochondrocytes in Group B were cultured with the lentivirus carrying no bFGF gene. The fibrochondrocytes in Group C were cultured without any intervention. After 48 h, the cell cycle, the collagen synthesis ability, the expression of bFGF, and the cell proliferation ability in each group were investigated. Results In Group A, the bFGF expression of 870±60 pg/ml was detected in the cells 48 h afterthe co-culture; however, in Group B and Group C, no expression of bFGF was found. After the co-culture for 6 days, the results of the MTT colorimetry revealed that the cells in Group A had an absorbtance of 0.427±0.037, which had a significant difference when compared with that in Group B and Group C (0.320±0.042,0.308±0.034,Plt;0.01). The cell cycle was significantly shorter in GroupA than in Group B and Group C (Plt;0.05); The durations of G1, S and G2M of the cells in Group A were 16.28, 12.60 and 11.04 h, but those in Group B and Group C were 23.61, 16.90, 21.33 h and 21.56, 19.80, 21.41 h, respectively. The disintegration per minute of the cells was significantly greater in Group A than in Group B and Group C (7281.69±805.50 vs 5916.40±698.11 and 5883.57±922.63，Plt;0.05). Conclusion The lentivirus vector can transfer the bFGF gene into the meniscal fibrochondrocytes, resulting in an increase of the cell proliferation and the collagen synthesis.
Objective To examine the biological characteristic changes in thededifferenciated human articular chondrocytes by the bioreactor culturing in vitvo.Methods The cartilage tissue was obtained from the joints of the adult human. The chondrocytes were isolated from the cartilage tissue with the type Ⅱ collagenase digestion（0.2%, 37℃, 3 h）and were cultured in DMEMF12 supplemented with 20% fetal bovine serum (FBS) with 1 ng/ml of TGF-β1and 5 ng/mlof FGF-2. After about 20 passages by the monolayer culture，the cells were then transferred to the bioreactor culturing of the rotational cell culture system (RCCS) for a 3-week sequence culture. The cell counting was performed with the platelet counter, and the doubling time for each passage of thecells was determined. The frozen section was stained with HE. The differentiated phenotype was evaluated by histochemistry or immunohistochemistry. Results When the monolayer culture was performed without any growth factors, the chondrocytes were rapidly proliferated within 3 passages (average doubling time, 59 h),but at the same time, dedifferentiation was also progressing rapidly. After the4th passage, most of the cells were dedifferenciated and the proliferation was decreased. With the growth factors (TGF-β1/FGF-2), the speed of the expansion was accelerated (average doubling time, 47 h), but the speed of the dedifferentiation was slowed down. After 20 passages were performed with the monolayer culture, the dedifferentiated chondrocytes could be redifferentiated when they were cultured for 3 weeks with RCCS. Then, the Safranine-O staining was bly positive for the cells, positive for aggrecan and collagen Ⅱ, but negative for collagen Ⅰ, with a wellregained phenotype. Conclusion The bioreactor culturing of the dedifferenciated human articular condrocytes can regain the differentiated phenotype and it is a useful method of obtaining the human articular chondrocytes in large amounts and in a differentiated phenotype in vitro.
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.
Objective To evaluate the immunological reaction and the outcome of allogeneic chondrocyte transplantation in repairing articular cartilage defects in porcins. Methods Full articular cartilage from the knee of two Shanghai white porcins about one-month-old was removed and cut mechanically, digested by 0.25% trypsin and 0.2% type Ⅱ collagenase and cultured in 10％ DMEM medium. Defects of 0.5 cm×0.5 cm involving the subchodral bone were created in both the left and right femur condyloid in 8 two-month-old Yunnai bama porcins. Allogeneic chondrocyte transplantation were implanted in defects at a density of (1.0-2.0)×106,0.2 ml. The lymphocytes from the receivers’ blood were collected before transplantation and after 3, 5, 7 and 12 weeks of transplantation, then mixed with allogeneic chondrocytes to determin the lymphocyte stimulation index(SI) in vitro. The histological observation in vivo was made after 5, 7 and 24 weeks of transplantation. Results Lymphocyte SI at 3, 5, 7 and 12 weeks(1.457±0.062,1.739±0.142,1.548±0.047，1.216±0.028) after transplantation was higher than that before transplantation(1.102±0.034，Plt;0.05). SI began to increase in the 3rd week and reached the peak value in the 5th week, then gradually declined at the 7th and 12th weeks, showing significant differences when compared with in the 5th week (Plt;0.05). Inflammation and lymphocytes infiltration could be seen in subchondral bone and the intergration area between repair tissue and normal cartilage in the 5th week, and then decreased and limited in subchondral bone in the 7th week. Defects were filled with cartilage tissue, which had good intergration with subchondral bone at 24 weeks after transplantation. Conclusion Immunological reactions can be found at early stage of allogeneic chondrocyte transplantation and then decreased with the time, the fullthickness articular cartilage defects could be repaired mainlywith hyaline cartilage by the allogeneic chondrocyte transplantation. This may provide a new method to repair articular cartilage defects clinically.
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 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 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 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.
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.