| There are two manners, endochondral ossification and intromembrane ossification, involved in bone formation. Besides parietal bone, frontal bone, collarbone and part of the mandible, endochondral ossification forms all the other bones including truncal bones and limbs bones. Endochondral ossification is a complex process involving proliferation, differentiation and apoptosis of chondrocyte, mesenchymal stem cells, preosteoblasts and osteoclasts cells. During the endochondral ossification process, the proliferation and differentiation of cells is regulated by series of molecular regulation of mechanism, such as growth factors, hormones, as well as cells extracellular matrix. Parathyroid hormone (PTH) is the regulator of calcium and phosphorus, and important regulatory factor of bone growth and development and bone remodeling. Previous researches suggest that PTH can produce a dual effect in promoting or inhibiting bone formation depending on the different administration methods of PTH. In this study, through the following three parts, detection experiments of cell proliferation, ALP activity assay, alizarin red staining. Western blot and Real-time PCR were carried out to study the effects of intermittent and continuous PTH treatment on proliferation and differentiation of condylar chondrocytes and bone marrow stromal stem cells.Part One Effects of PTH on condylar chondrocyte proliferation and differentiation Objective:To study the effects of PTH on proliferation and hypertrophic differentiation of rat condylar chondrocyte in continuous and intermittent manners.Methods:After being separated from female rats born within24h, the condylar cartilage was digested by0.2%type Ⅱ collagenase enzyme for to obtain the primary rat condylar chondrocytes. The primary chondrocytes were cultured in DMEM containing10%FBS and100μg/mL penicillin/streptomycin. When the primary cultured condylar chondrocytes were approximately80%confluent, the culture medium was changed to the osteogenic culture medium, which is DMEM containing10%FBS,5mM β-glycerophosphate,50μg/mL ascorbic acid.100nM dexamethasone and100μg/mL penicillin/streptomycin. According to the PTH administration methods, condylar chondrocytes were divided into three groups:the continuous PTH administration group, the intermittent PTH administration group, and the control group. The continuous PTH administration group:the first6h and the rest42h of each48h. the osteogenic mediums both contained10nM PTH. The intermittent PTH administration group:lOnM PTH was added to the osteogenic medium at the beginning6h, while it was PTH free during the following42h of each48h. The control group:there was no PTH during all the48h. The effect of different PTH administration on proliferation of condylar chondrocyte was detected by MTT experiment every two days after PTH treatment started. Alizarin Red S staining was carried out to detect the mineral nodules formation of three groups after6and14days PTH treatment. After total mRNA or protein were extracted from three different PTH treatment groups at day2. day6or day14, ALP activity detection kit was used to detect the ALP activity:Western blot and Real-time PCR were carried to detect the expression levels of chondrocyte proliferation related, differentiation related and bone formation related markers, including RUNX2, BSP, BMP2, COL2a, COL10al, ALP, BMP2, OCN, COL1al and OSX.Results:The proliferation ability of condylar chondrocyte in continuous PTH treatment group is much higher than that in intermittent PTH treatment group and control group (P<0.05). and the proliferation ability in control group is higher than that in intermittent PTH treatment group (P<0.05). The result of Alizarin Red S staining shows that chondrocytes in intermittent PTH group form the most amount mineral nodules, while the least nodules is formed in the continuous PTH group. After6days PTH treatment, ALP activity in intermittent PTH group and control group is obviously higher than that in the continuous PTH group (P<0.05); ALP activity in intermittent PTH group is higher than control group, but there is no statistical difference between them. While after14days PTH treatment, ALP activity in intermittent PTH group is obviously higher than that in the continuous PTH group and control group (P<0.05); ALP activity in continuous PTH group is higher than control, but there is no statistical difference between them. Western bolt results show that protein expression levels of RUNX2, BSP and BMP2in intermittent PTH group are remarkable higher than that in continuous PTH group and control group (P<0.05); protein expression levels of RUNX2, and BSP in continuous PTH group are obviously lower than that in control group (P<0.05); protein expression level of BMP2in continuous PTH group is obviously higher than that in control group (P<0.05). COL2al mRNA expression in continuous PTH group is significantly up-regulated than that in intermittent PTH group (P<0.05), while COL10al mRNA expression in intermittent PTH group is obviously up-regulated than that in continuous PTH group (P<0.05). Expression levels of RUNX2and ALP mRNA in intermittent PTH group are significantly up-regulated than that in continuous PTH group (P<0.05). Expression levels of bone formation related genes, OCN. BMP2. COL1al and OSX, were significantly up-regulated in intermittent PTH group than continuous PTH group.Conclusion:Continuous PTH administration enhanced condylar chondrocyte proliferation and suppressed simultaneously condylar chondrocyte differentiation, whereas intermittent PTH administration promoted condylar chondrocyte differentiation and bone formation and inhibited its proliferation.Part Two Effects of PTH on bone marrow stromal cells proliferation and differentiationObjective:To study effects of PTH on proliferation and hypertrophic differentiation of rat bone marrow stromal cells (BMSCs) in continuous or intermittent manners.Methods:Femurs and tibias were dissected from4-6weeks old rat under sterile conditions, and the primary rat BMSCs were obtained with full bone marrow culture method. When cultured to the second passage, the BMSCs were divided into three groups:the continuous PTH administration group, the intermittent PTH administration group, and the control group (same as Part one). Effects of different PTH administration on proliferation activity of BMSCs were measured by MTT experiment. Alizarin Red S staining was carried out after14days PTH treatment to detect the mineral nodules formation. Total mRNA and protein were extracted from different groups after2.6.14days PTH treatment. ALP activity was measured with ALP activity detection kit. Real-time PCR was applied to study the osteogenic differentiation related and bone formation related genes expression, including RUNX2, ALP, BMP2, OCN, COLIal, OSX and bone formation inhibitor SOST.Results:The proliferation ability of BMSCs in continuous PTH treatment group is much higher than that in intermittent PTH treatment group and control group (P<0.05). The proliferation ability of BMSCs in intermittent PTH treatment group is much higher than that in continuous PTH treatment group, but there is no statistical difference between them. Mineral nodules formed in intermittent PTH treatment group are larger and more than that in continuous PTH treatment group and control group, while continuous PTH treatment group formed the least and smallest mineral nodules among the three groups. ALP activity of intermittent PTH treatment group BMSCs is obviously higher than continuous PTH treatment group and control group (P<0.O5). After6days and14days PTH treatment. ALP activity of continuous PTH treatment group is higher than control group (P<0.05). The expression levels of osteogenic differentiation markers of BMSCs, RUNX2and ALP. are significantly up-regulated than continuous PTH treatment group (P<0.05). Bone formation related genes. COL1al、BMP2、OCN、OSX. are significantly higher expressed in intermittent PTH treatment group than continuous PTH treatment group and control group after2days and/or6days PTH treatment (P<0.05). Bone formation antagonist SOST expression level is significantly up-regulated in continuous PTH treatment group than in intermittent PTH treatment group and control group (P<0.05).Conclusion:Continuous PTH administration enhanced rat BMSCs proliferation and suppressed its osteogenic differentiation. Intermittent PTH administration promoted rat BMSCs osteogenic differentiation and bone formation, but its proliferation hadn’t been inhibited obviously.Part Three Effects of PTH on the proliferation and osteogenic differentiation of co-cultured condylar chondrocyte and BMSCsObjective:To study the effects of continuous and intermittent PTH treatment on osteogenic differentiation and bone formation of co-cultured rat condylar chondrocyte and BMSCs.Methods:Co-culture the primary condylar chondrocyte and the second passage BMSCs with the ratio of1:1. When the co-cultured mixed cells were approximately80%confluent, the culture medium was changed to osteogenic culture medium. And according to the PTH administration manner, the co-cultured chondrocyte and BMSCs were divided into three groups:the continuous PTH administration group, the intermittent PTH administration group, and the control group (same as Part one). Alizarin Red S staining was carried out after6days and14days PTH treatment to detect the mineral nodules formation. Total mRNA and/or protein of different three groups were extracted after2days,6days and14days. ALP activity was measured with ALP detection kit. Western blot and Real-time PCR were applied to check out the expression of oteogenic differentiation related and bone formation related protein or genes, including COL2a1, COL10a1, RUNX2, BSP, MMP13, ALP, OCN, OSX, and the bone formation antagonist SOST mRNA expression.Results:Co-cultured condylar chondrocyte and BMSCs in intermittent PTH treatment group formed the most mineral nodules after6days and14days PTH treatment, and continuous PTH treatment group formed the least among the three different PTH treatment group. After6days and14days PTH treatment. ALP activity of intermittent PTH treatment group BMSCs was significantly higher than continuous PTH treatment group and control group (P<0.05), while ALP activity of intermittent PTH treatment group BMSCs was significantly lower than control group (P<0.05). Results of Western blot showed that the protein expression of RUNX2. BSP and MMP13in intermittent PTH treatment group were remarkably up-regulated than continuous PTH treatment group and control group (P<0.05). And these proteins were lower expressed in continuous PTH treatment group than in control group (P<0.05). Expression level of COL2al mRNA in continuous PTH treatment group was significantly up-regulated than intermittent PTH treatment group (P<0.05), while expression level of COL10al mRNA in intermittent PTH treatment group was obviously up-regulated than continuous PTH treatment group (P<0.05). Osteogenic differentiation related genes, RUNX2and ALP, and bone formation related genes. OCN and OSX, were obviously higher expressed in intermittent PTH treatment group than that in continuous PTH treatment group (P<0.05). Bone formation antagonist SOST expression level in continuous group was significantly up-regulated than intermittent PTH group and control group (P<0.05).Conclusion:Co-cultured condylar chondrocyte and BMSCs could imitate the process of endochondral ossification. Intermittent PTH administration promoted osteogenic differentiation and bone formation of co-cultured condylar chondrocyte and BMSCs. while continuous PTH administration inhibited this process. |
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