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.
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.
ObjectiveTo study the effect of down-regulated leptin receptor by small interfering RNA (siRNA) in inhibiting the messenger RNA (mRNA) expressions of interleukin (IL)-1β and nitric oxide (NO) of human osteoarthritis chondrocytes, in order to provide reference for basic clinical research. MethodsCartilage was harvested under sterile conditions from osteoarthritis knee joints in patients undergoing total knee arthroplasty. Human articular chondrocytes were isolated and the cells were cultured in vitro. The cells in the 3rd passage were transferred by siRNA Ob-Rb (experimental group) and blank Ob-Rb (control group), respectively. Then mRNA expressions of IL-1β and NO were tested by quantitative polymerase chain reaction at hour 24, 48 and 72 after successful transfection. ResultsThe mRNA expressions of IL-1β increased slightly and that of NO declined slightly at hour 24, 48 and 72 after transfection in the treatment group, but they all were significantly lower than those in the control group (P ＜ 0.05) , and the differences became much larger as time went on. ConclusionLeptin receptor under siRNA technology can significantly inhibit the mRNA expressions of IL-1β and NO in human osteoarthritis chondrocytes.
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 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.
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.
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 Toreview theresearch progress of nucleus pulposus cells phenot ypic markers. Methods The domestic and international l iterature about nucleus pulposus cells phenotypic markers was reviewed extensively and summarized. Results Due to different biomechanical properties，nucleus pulposus cells and articular chondrocytes have differences in morphology and extracellular components such as the ratio of aggrecan to collagen type II α1. Nucleus pulposus cells can be identified by surface marker (CD24), gene markers (hypoxia inducible factor 1α, glucosetransporter protein 1, matrix metalloproteinase 2, vascular endothel ial growth factor A, etc), and various markers (keratin 19 and glypican 3，paired box 1, forkhead box F1 and integrin-binding sialoprotein, etc). Conclusion Nucleus pulposus cells and articular chondrocytes have different phenotypic markers, but nucleus pulposus cells are still lack of specific markers.
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 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.