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 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 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.
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 explore the expression and significance of hypoxia-inducible factor 1α (HIF-1α) in endplate chondrocytes, and to study the relations between HIF-1α expression and endplate chondrocytes apoptosis. Methods Eight Sprague Dawley rats were selected to obtain the L1-5 intervertebral disc endplate; the endplate chondrocytes were isolated by enzyme digestion method, and the endplate chondrocytes at passage 3 were cultured under 20% O2 condition (group A), and under 0.5% O2 condition (group B). Cell morphology was observed by inverted phase contrast microscope and cell apoptosis was detected using flow cytometry after cultured for 24 hours; the mRNA expression of HIF-1α was detected by real-time fluorescent quantitative PCR, the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1α mRNA. HIF-1α specific RNAi sequence compound was constructed and transfected into cells. The transfected endplate chondrocytes at passage 3 were cultured under 0.5% O2 condition in group C and group D (HIF-1α gene was silenced). After cultured for 24 hours, cells were observed via immunofluorescence staining of HIF-1α, and cell apoptosis was detected using flow cytometry. Meanwhile, the mRNA expressions of HIF-1α, collagen type II (COL II), Aggrecan, and SOX9 were detected by real-time fluorescent quantitative PCR, and the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Results At 24 hours after culture, small amount of vacuoles necrotic cells could be observed in group A and group B; there was no significant difference in apoptosis rate between groups A and B (t=1.026,P=0.471), and HIF-1α mRNA and protein expressions in group B were significantly higher than those in group A (t=22.672,P=0.015;t=18.396,P=0.013), but, there was no significant difference in protein expressions of Bax and Bcl-2 between groups A and B (t=0.594,P=0.781;t=1.251,P=0.342). The number of vacuolar necrosis cells in group D was significantly higher than that in group C, and HIF-1α positive cells were observed in group D. The apoptosis rate of group D was significantly higher than that of group C (t=27.143,P=0.002). The mRNA expressions of HIF-1α, COL II, Aggrecan, and SOX9 in group D were significantly lower than those in group C (t=21.097,P=0.015;t=34.829,P=0.002;t=18.673,P=0.022;t=31.949,P=0.007). The protein expressions of HIF-1α and Bcl-2 in group D were significantly lower than those in group C (t=37.648,P=0.006;t=16.729,P=0.036), but the protein expression of Bax in group D was significantly higher than that in group C (t=25.583,P=0.011). Conclusion HIF-1α mRNA expression is up-regulated under hypoxia condition, which will increase the hypoxia tolerance of endplate chondrocytes. Cell apoptosis is suppressed by the activation of HIF-1α in endplate chondrocytes under hypoxia condition.
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 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 and mechanism of miR-4287, a chondrogenesis associated microRNA, regulated the expression of aggrecanase-1 (a disintegrin and metalloproteinase with thrombospondin motif 4, ADAMTS4) in human chondrocytes. Methods First, the voluntarily donated normal and osteoarthritic knee articular cartilages were used to detect the expressions of miR-4287 and ADAMTS4 mRNA by real-time fluorescence quantitative PCR. Then, chondrocytes were isolated from knee articular cartilages. The effect of IL-1β on the expression of miR-4287 and ADAMTS4 mRNA was validated by the first generation of osteoarthritic chondrocytes. To confirm the influence of IL-1β signal pathways on the expression of miR-4287 and ADAMTS4 mRNA, osteoarthritic chondrocytes were pretreated with MAPK signal pathway inhibitor SP600125, NF-κB pathway inhibitor SN50, and finally stimulated with IL-1β. Chondro cytes were transfected with miR-4287 mimics and mimics negative control, inhibitors and inhibitors negative control respectively to value the effect of miR-4287 on ADAMTS4 expression. Luciferase reporter assay was used to verify the direct interaction between miR-4287 and putative site in the 3-untranslated region (3’UTR) of ADAMTS4 mRNA. Results Compared with normal knee articular cartilages, the miR-4287 expression was markedly diminished and conversely ADAMTS4 mRNA expression was raised in osteoarthritis cartilages (P<0.05). Stimulation with IL-1β led to a reduction in miR-4287 expression and upregulation in ADAMTS4 mRNA expression, showing significant difference when compared with the untreated groups (P<0.05). Pretreatment with IL-1β signal pathway inhibitors induced miR-4287 expression and attenuated ADAMTS4 mRNA expression in human chondrocytes, which were significantly different from that of unstimulated cells (P<0.05). ADAMTS4 mRNA and protein were suppressed by transfection with miR-4287 mimics (P<0.05) and elevated by transfection with miR-4287 inhibitors (P<0.05). As luciferase reporter assay showed, overexpression miR-4287 failed to alter the luciferase activity of a reporter construct containing either wild or mutant 3’UTR of ADAMTS4 mRNA (P>0.05). Conclusion miR-4287, a chondrogenesis associated microRNA, may play an important role in cartilage degeneration. miRNA-4287 is able to regulate ADAMTS4 expression in human chondrocytes, but not by means of directly targeted the ADAMTS4 mRNA 3’UTR. The exact mechanisms need to be further addressed.
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.