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.
A solid-liquid two-phase finite element model of articular cartilage and a microscopic finite element model of chondrocytes were established using the finite element software COMSOL in this study. The purpose of the study is to investigate the mechanics environment and the liquid flow field of the host cartilage chondrocytes in each layer by multi-scale method, under physiological load, with the different elastic modulus of artificial cartilage to repair cartilage defect. The simulation results showed that the uniform elastic modulus of artificial cartilage had different influences on the microenvironment of different layer chondrocytes. With the increase of the elastic modulus of artificial cartilage, the stress of the shallow surface layer and the intermediate layer chondrocytes increased and the stress of deep layer chondrocytes decreased. The flow field direction of the middle layer and the bottom layer of cartilage can also be changed by artificial cartilage implantation, as well as the ways of nourishment supply of the middle layer and underlying chondrocytes change. A barrier to underlying chondrocytes nutrition supply may be caused by this, thus resulting in the uncertainty of the repair results. With cross-scale finite element model simulation analysis of chondrocytes, we can quantitatively evaluate the mechanical environment of chondrocytes in each layer of the host cartilage. It is helpful to assess the clinical effect of cartilage defect reparation more accurately.
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 explore the DNA repair effect of rat adipose-derived stem cells (ADSCs) on chond-rocytes exposed to ultraviolet (UV) radiation. Methods ADSCs were isolated and cultured from the inguinal adipose tissue of Sprague Dawley rat by digestion with collagenase type I. ADSCs cell phenotype was assayed with flow cytometry. Multiple differentiation capability of ADSCs at passage 3 was identified with osteogenic and adipogenic induction. The chondrocytes were obtained from rat articular cartilage by digestion with collagenase type II and were identified with toluidine blue staining. The chondrocytes at passage 3 were irradiated with 40 J/m2 UV and cultured with normal medium (irradiated group), and medium containing the ADSCs supernatant (ADSCs supernatant group) or ADSCs was used for co-culture (ADSCs group) for 24 hours; no irradiation chondrocytes served as control group. The cell proliferation was estimated by MTS method. The expression of phosphorylated histone family 2A variant (γH2AX) was detected by immunofluorescence and Western blot. Results ADSCs presented CD29(+), CD44(+), CD106(-), and CD34(-); and results of the alizarin red staining and oil red O staining were positive after osteogenic and adipogenic induction. Cell proliferation assay demonstrated the absorbance (A) values were 2.20±0.10 (control group), 1.34±0.04 (irradiated group), and 1.57±0.06 (ADSCs supernatant group), showing significant difference between groups (P<0.05). Immunofluorescence and Western blot showed that the γH2AX protein expression was significantly increased in irradiated group, ADSCs supernatant group, and ADSCs group when compared with control group (P<0.05), and the expression was significantly decreased in ADSCs supernatant group and ADSCs group when compared with irradiated group (P<0.05), but no significant difference was found between ADSCs supernatant group and ADSCs group (P>0.05). Conclusion ADSCs can increase the cell proliferation and down-regulate the γH2AX protein expression of irradiated cells, indicating ADSCs contribute to the repair of irradiated chondrocyte.
ObjectiveTo summarize the tissue engineering techniques for cartilage repair on the combination fields of the three elements of tissue engineering:cells, scaffolds and signals. MethodsThe literature on cell-scaffold-based cartilage repair techniques, cell-free scaffolds, and scaffold-free approaches was reviewed and summarized. ResultsThe cell-scaffold-based cartilage repair techniques such as matrix-induced autologous chondrocyte implantation (chondrocytes are seeded on the scaffold) are able to enhance the survival of the cells; cell-free scaffolds can promote cell recruitment with chemoatractants; and scaffold-free approaches have better hyaline-like properties and can avoid the toxic effect of scaffold degradation products. ConclusionCombination fields of the three elements of tissue engineering provide a more biomimetic environment for cartilage repair and have broad prospects.
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 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 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 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.