ObjectiveTo investigate the effect of cyclic stretch stress on the osteogenic differentiation of human cartilage endplate-derived stem cells (CESCs). MethodsCESCs were isolated from the endplate cartilage tissues by the method of agarose suspension culture system. The endplate cartilage tissue was harvested for immunohistochemical staining. Flexercell-4000TM Tension Plus system was used to apply cyclic stretch on CESCs at a frequency of 1 Hz and at a stretch rate of 10% for 1, 6, 12, or 24 hours (experimental group). No stretch stress was performed on CESCs in the same culture condition (control group). After mechanical loading, the protein expression of bone morphogenetic protein 2 (BMP-2) was measured by Western blot, and gene expressions of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and SOX9 were detected by real-time fluorescent quantitative PCR. ResultsImmunohistochemical staining showed BMP-2 protein expression in chondrocytes. The continuous cyclic stretch stress of 10% can increase the expression of BMP-2 protein in CESCs. Significant differences were observed in the expressions of BMP-2 protein (P<0.05) between 2 groups at the other time points except at 1 hour (P>0.05), in a time-dependent manner. The real-time fluorescent quantitative PCR indicated that the gene expressions of Runx2 and ALP showed an increasing tendency with time in the experimental group when compared with the control group, but there was down-regulated expression of SOX9. Significant difference was found in mRNA expressions of Runx2 and ALP at 12 and 24 hours and in mRNA expressions of SOX9 at 6, 12, and 24 hours between 2 groups (P<0.05), in a time-dependent manner. ConclusionCyclic stretch stress may induce osteogenic differentiation of CESCs by regulating the expressions of some genes related osteogenesis in CESCs.
ObjectiveTo study the effect of inhibitor of differentiation 1 (Id1) gene transfection on bone morphogenetic protein 2 (BMP-2) promoting the expressions of collagen type Ⅱ (COL Ⅱ) and aggrecan (ACAN) in intervertebral cartilage endplate cells (EPCs). MethodsEPCs were harvested from the New Zealand white rabbits, the 2nd generation EPCs were used for experiment. The transfection efficiency of green fluorescent protein blank lentivirus, high expression of Id1 lentivirus, RNA interference (RNAi) Id1 lentivirus transfection in the EPCs were observed by the fluorescence microscopy, real-time fluorescence quantitative PCR, and Western blot. Blank vector, single BMP-2 gene, BMP-2 and Id1 genes were transfected into EPCs, respectively. The cell morphology and the expressions of COL Ⅱ and ACAN in each group were observed. ResultsLentiviral transfection had no significant effect on the cell morphology. The EPCs were effectively transfected by the high expression Id1 lentivirus and RNAi Id1 lentivirus; the expression of Id1 mRNA was also significantly interfered. The expressions of COL Ⅱ and ACAN mRNA and synthesis of COL Ⅱ and ACAN protein were significantly higher in BMP-2 lentivirus and high expression Id1 lentivirus groups than control group (P<0.05). The expression of COL Ⅱ and ACAN protein were down regulated in the cartilage endplate cells when the expression of Id1 gene was decreased (P<0.05). ConclusionUp-regulation of Id1 gene expression can enhance the effects of BMP-2 on the synthesis of COL Ⅱ and ACAN in EPCs.
ObjectiveTo quantitatively determine the levels of type Ⅰ and type Ⅱ collagen mRNA in the intervertebral disc cartilage endplate of injured animal model, and to clarify the cytological function of intervertebral disc chondrocytes during fibrosis repair after intervertebral disc injury.MethodsForty healthy New Zealand rabbits were randomly divided into fibrosus puncture group, upper cartilage endplate single puncture group, upper and lower cartilage endplate multiple puncture group, and sham group. Two experimental animals were randomly selected from each group on the 2nd day, and the 2nd, 8th, 12th, and 24th week after the animal modeling operation to obtain intervertebral disc specimens. The levels of type Ⅰ collagen and type Ⅱ collagen in cartilage endplate cells of the intervertebral disc were determined by reverse transcriptase polymerase chain reaction (PCR). RNA was extracted from the endplate of the intervertebral disc, and the RNA concentration and the ratio of RNA concentration to protein concentration were determined by nucleic acid analyzer. Reverse transcription was performed by Revertaid M-Mulv reverse transcriptase, type Ⅰ and type Ⅱ collagen primers were designed to establish a PCR reaction system, 2% agarose gel electrophoresis (120 V, 40 min) was prepared by using 0.5×TBE electrophoresis buffer. The amplification results were observed under ultraviolet light, and the gray values of different electrophoresis bands were determined.ResultsThe level of type Ⅰ collagen mRNA in each operation group showed a progressive increase after 8 weeks, and the magnitude of the increase was related to the degree of injury. The level of type Ⅱ collagen mRNA showed a transient increase in the fibrosis puncture group and the upper endplate single-puncture group in the first two weeks after the endplate punctures were completed, and then began to decline progressively; in the multiple puncture group, it showed a downward trend from the beginning of the operation. ConclusionsThe synthesis of type Ⅰ collagen in chondrocytes of the injured nucleus pulposus tissue continues to increase with time, while the synthesis of type Ⅱ collagen begin to decrease progressively after a small increase. The formation and change of type Ⅰ and type Ⅱ collagen in injured intervertebral disc chondrocytes are different from natural degeneration.