| Estrogen deficiency usually happens in post-menopause women and causes systemic osteoporosis. Osteoporosis is characterized by decreased bone mineral density (BMD) and microarchitectural deterioration of the bones due to more rapid bone resorption than formation. Because of the consequent bone fragility and an increased risk of fractures, osteoporosis has become one of the most prevalent and complex skeletal disorders for post-menopausal women, the aged and those associated with other medical conditions, or as a result of certain therapeutic interventions.Post-menopausal osteoporosis, as a matter of fact, is a metabolic disease due to imbalance between osteoclast-related bone resorption and osteoblast-related bone formation which could lead to bone loss, or even worse, fracture. And post-menopausal osteoporosis is also one of the major factors for the loss of the alveolar bone. The subsequent complications including alveolar bone absorption, gingival atrophy, root exposure, periodontal attachment loss, loosening of the teeth, and tooth loss could cause great difficulties during orthodontic treatment. However, many therapies have been failed to achieve the ideal efficacy at present because of severe side effects.A positive correlation has also been detected between post-menopausal osteoporosis and certain oral diseases. Since osteoporosis can reduce BMD of the mandible and condyle and promote the severity of alveolar residual ridge resorption, more patients with osteoporosis suffer from periodontal diseases than the general population. Chronic metabolic bone diseases including osteoporosis and osteogenesis imperfect can cause bone tissue destruction. And this kind of destruction will affect the health of teeth, periodontal tissue, and the alveolar bone. Some studies suggest that systemic bone metabolism disorder can destroy the microenvironment of the bone marrow mesenchymal stem cells (BMMSCs). Then BMMSCs’ characteristics and functions are impacted which leads to the weakening of its self-repair ability. BMMSCs are a population of multipotent stem cells. They can self-renew and differentiate into osteogenic lineage. In addition, BMMSCs have been applied to bone reconstruction for the bone resorption. However, if BMMSCs are used to repair the bone defects with metabolism disorder, that will not make it because the cellular activity and function of BMMSCs are decreased due to unhealthy microenvironment.Recently, Sphingosine-1-phosphate and its analog FTY-720 (fingolimod) have showed an great advance in promoting angiogenesis and improving microenvironment. Therefore, different concentration gradients (1nM、10nM、10nM) of FTY-720 were co-cultured with rat BMMSCs to attempt to further promote the stem cells’ function and activity in our study, which would help us to find a better method of enhancing BMMSCs’ osteogenic differentiation and investigating its osteogenetic mechanism so as to develop a promising strategy to cure bone defects with post-menopausal osteoporosis.Objectives1. To isolate and culture BMMSCs obtained from OVX and Sham rats, and identify the biological characters of them.2. To determine whether different concentration gradients (lnM、10nM、100nM) of FTY-720 can enhance osteogenic differentiation of BMMSCs derived from OVX and Sham rats.3. To explore the molecular mechanism of FTY-720 on enhancing osteogenesis of BMMSCs by studying the role of Smad4 on osteogenic differentiation of rat BMMSCs in FTY-720 co-culture medium system.4. To evaluate the effects of bone formation on cranial defect by lentivirus-mediated Smad4 shRNA transfection and lentivirus-mediated nonsense strands (Negative).Methods1. The improved whole bone marrow adherence for screening method to isolate and culture rat bone marrow mesenchymal stem cells. Flow cytometry analysis was used to analyze the phenotypes of rBMMSCs. MTT was used to determine the proliferation potential of rBMMSCs. Alizarin Red S and Oil red O staining was used to determine the multi-differentiation potential of rBMMSCs after osteogenic and adiopogenic induction.2. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of genes of the osteogenesis marker including Runx2, Sp7, OCN and ALP. And protein expression of Runx2, Sp7, OCN and ALP were detected by western blot analysis 3 weeks after induction by different concentration gradients (lnM、10nM、100nM) of FTY-720.3.3 small interfering RNAs (siRNA) and 1 scramble RNA-Negative as control were designed based on the sequence of the rat Smad4 cDNA HEK-293T cells were infected with the 3 shRNAs (short hairpin RNAs) using LipofectamineTM 2000. The relative Smad4 mRNA and protein levels were assessed by quantitative RT-PCR and Western blot. Among 3 shRNAs examined, shRNA2 was selected as the most efficient shRNA for use. Lentivirus was then packaged into HEK-293T cells. We construct lentivirus mediated Smad4 shRNA2 and infected rBMMSCs-OVX to overexpress shRNA2 in the cells. The expression levels of Smad4 mRNA and protein in rBMMSCs-OVX were detected by qRT-PCR and Western blot respectively. Then qRT-PCR and Western blot analysis were used to assess the osteogenic differentiation of rBMMSCs after induction by FTY-720.4. Established the rat cranial defect model and collected the BMMSCs that infected by lentivirus mediated Smad4 shRNA2 and lentivirus mediated negative strands, and then immediately implanted the combination of cells and gelatin sponge into the cranial defects. Micro-CT was used to observe and value the bone formation.Results1. Cultured rBMMSCs showed elongated spindle or rhomboid-shaped and displayed a rather homogeneous confluent population growth. MTT assay showed that rBMMSCs-OVX have higher self-renewal ability The cells were immunopositive for surface markers of mesenchymal, but were negative for the hematopoietic markers. Osteogenic and adipogenic induction results showed that the cells could differentiate into adipocytes and osteoblasts when cultured in induction media.2. The expression levels of osteogenesis-related genes including Runx2, Sp7, OCN and ALP (mRNA and protein) were increased during osteogenic differentation of rBMMSCs-OVX and rBMMSCs-SHAM after induction by different concentration gradients (lnM、10nM、100nM) of FTY-720.3. In order to further study the molecular mechanisms of FTY-720 in regulation of rBMMSCs osteogenic differentiation, shRNA2 was selected to construct lentiviral vector. After the lentivirus-mediated Smad4-shRNA2 transfecting into rBMMSCs-OVX, both qRT-PCR and Western blot analysis showed that the endogenous Smad4 gene and protein were significantly reduced. The results showed the effective Smad4-shRNA2 lentiviral vector was successfully constructed. The Runx2, Sp7, OCN, ALP gene and protein expression of lentivirus-mediated Smad4-shRNA2 group (Lv-S2-OVX) was significantly lower than that of the lentivirus-mediated Negative strands group (Lv-Negative).4. Micro-CT results showd that the lentivirus-mediated Negative strands group (Lv-Negative) markedly promoted the bone formation in vivo, on the contrary, lentivirus-mediated Smad4-shRNA2 group (Lv-S2-OVX) had no bone formation in the cranial defect.Conclusions1. First a rat model of post-menopausal osteoporosis was established successfully. BMMSCs isolated from OVX rats had higher self-renewal ability and several specific surface markers.2. All different gradients of FTY-720 (1nM,10nM、100nM) enhanced osteogenic differentiation of rBMMSCs from either OVX or Sham rats, and 10 nM FTY-720 could induce highest level of bone-related gene expression in the present study.3. Lentivirus-mediated Smad4 shRNA2 was constructed successfully, and could transfect rBMMSCs-OVX in vitro efficiently and suppress the expression of Smad4 both at gene and protein.4. Once the endogenous Smad4 gene in rBMMSCs-OVX was suppressed by Lentivirus-mediated Smad4 shRNA2, FTY-720 could not enhance osteogenic differentiation of rBMMSCs, that indicates the osteogenesis enhancement of FTY-720 is achieved by Smad signaling pathway.5. Once the endogenous Smad4 gene in rBMMSCs-OVX was suppressed, the new bone formation would not activated by FTY-720. |
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