| Objective:The osteogenic growth peptide (OGP) is a well-defined, 14-amino-acid peptide thatdistributes extensively in mammals as the same form. The OGP synthesized by CASproved to be significantly effective in osteogenesis both in vitro and in vivo. Bonemarrow mesenchymal stem cells (BMSCs) are adult stem cells with multilineagepotential, which is interacted closely with the surrounding micro-circumstance. Thestudy is composed by the following four parts with the purpose to clarify the effects ofsOGP in the differentiation of BMSCs and its therapeutic effects against osteoporosis.The first part focused on the osteogenesis and adipogenesis of BMSCs isolated fromdifferent clinical conditions in different micro-enviromonet. The second part studiedthe effects of sOGP on proliferation and differentiation of BMSCs from differentclinical conditions. According to previous studies, the hematopoietic differentiation ofbone marrow cells is via RhoA cascade, while RhoA/ROCK pathway plays animportant role in BMSCs' osteogenesis and adipogenesis differentiation. To elucidatethe possible mechanisms of sOGP's effects on BMSCs, the molecular changes ofRhoA/ROCK pathway was investigated in the BMSCs treated with sOGP in the thirdpart. In the forth part, sOGP's anti-osteoporosis effects were evaluated in theosteoprotegerin dificient mice, an osteoporosis model characterized by high turnoverrate, to provide in vivo data for the further clinical application.Methods:Part One: BMSCs were obtained by density gradients centrifuge and cell attachmentseparation method from normal young person, person with postmenopausalosteoporosis and old person with osteoporosis respectively. Morphologic changes ofthe BMSCs isolated from the three different clicnial conditions were observed underreverse phase microscope and electronic microscope in normal media, osteogenicmedia with dexamathasone and adipogenic media with rosiglitazone. Cell proliferation of the BMSCs was determined by MTT assay and the growth curve wascalculated. The expression of alkine phosphatase (ALP) by the BMSCs underosteogenic media with dexamathasone was measured by Real-time PCR, chemicalquantitation and histochemistry. Real-time PCR was used to measure PPAR-γ2mRNA expression in BMSCs induced by rosiglitazone, oil red O staining was used toexamine the lipid expression, the histochemistry and immunohistochemistry was usedto determine the expression of typeâ… collagen (Col-â… ) and osteocalcin (OC) in BMSCstreated by dexamathasone. All the data was compared between each group andanalyzed quantitively.Part Tow: This part of our experiment mainly focused on the effects of sOGP onproliferation and differentiation of the three kinds of BMSCs. The technique used wassame as the first part. Then we compared the results between or within each group andanalyzed quantitively.Part Three: Changes of RhoA/ROCK cytoskeleton pathway in BMSCs treated bysOGP were studied in the third part. ROCK and actin expression varieties and cellcytoskeleton changes were determined by immunofluorecence staining. Changes ofexpression levels of pFAK, RhoA, cofilin and their corresponding active forms, whichinvolved in RhoA/ROCK pathway, were determined by Western Blot. Theexpressions of the above proteins were determined again after the cells were treatedby the ROCK pathway specific inhibitor Y-27632, and the effect of Y-27632 onosteogenic effect of sOGP was also determined.Part Four: The effect of sOGP on osteogenesis in osteoprotegerin deficient mice(OPG-/-) model was investigated. First of all, the differences in bone mineral density(the body and the vertebral trabecula), blood biochemical index (ALP, ACP and OC),radio radiographs and scan electron microscope among 7-week, 13-week OPG-/- andOPG-WT mice were compared. Then, the OPG-/- mice were treated by sOPG (10-7mol/kg.day, 10-9 mol/kg.day and 10-11 mol/kg.day) or combined with salmoncalcitonin. The OPG-/- mice were also treated by phosphate saline or salmon calcitoninas negative and positive control respectively. Samples were collected from all thesemice, and the same indexes were detected to investigate whether sOGP can stimulateosteogenesis in the OPG-/- mice model.Results:Part one: BMSCs from old people with osteoporosis showed no significant differencefrom the other two kinds of BMSCs. However, the cell proliferation rate was lower and the number of rough endoplasmic reticulum and mitochondria were less than thatof the normal cell, which indicated that the metabolism of the cell was on a low level.After the osteogenic induction by dexamathasone, ALP's mRNA and proteinexpression levels of the BMSCs from normal young person and person withpostmenopausal osteoporosis were significantly higher than that of the BMSCs fromold people with osteoporosis, and the expression level of Col-â… and the calcium nodeformation showed the same patterns in the three kinds of BMSCs.After the adipogenic induction by rosiglitazone hydrochloride, lipid formation wasobserved in the BMSCs from the person with osteoporosis before it appeared in theBMSCs from the normal young person, and the PPAR-γ2 mRNA expression level andthe lipid formation degree were also higher than the BMSCs from the normal youngperson.Part Tow: Treated by the sOGP, BMSCs showed morphological changes, and thedense cell nodes appeared after several days' culture, in which the time course of thecell nodes appearing of the BMSCs from the old people with osteoporosis was longer.The proliferation promoting effect of sOGP on BMSCs was related to the BMSCscondition and concentration of the sOGP. sOGP with the concentration from 10-7-10-9mol/L stimulated the proliferation of the BMSCs from normal young person andperson with postmenopausal osteoporosis mildly, while sOGP showed no effect to theBMSCs from old people with osteoporosis except the cells were treated by the sOGPwith the highest concentration.Data from Real-time PCR, RT-PCR, cell staining and ALP quantitation revealed thatsOGP at the concentration of 10-9 mol/L had the strongest osteogenic effect to theBMSCs, and the effect was higher than that induced by dexamathasone.The expression level of PPAR-γ2 mRNA in the BMSCs treated by sOGP androsiglitazone was lower than that in the BMSCs treated only by rosiglitazone, and thesame result was observed in the oil red O assay.Part Three: ROCK expression level, which was detected by immuno-fluorescencestaining, was increased at the fifth days after the cells were treated by sOGE Theexpression level reached the summit at the 8-1lth day and then decreased. TheBMSCs showed spindle shape before they were treated by sOGP, while the cellsshowed polygonal shape and expressed thick actin protein in cell circumferences.Expression level of cofilin and RhoA never exchanged at each time point duringsOGP treatment, while pFAK expression increased at the first day of treatment and kept increasing till the level decreased after 8 days of treatment. The expression ofactived RhoA elevated at the beginning of day 2, and reached the highest level at the4-8th day. Phosphorylated cofilin level increased at the fifth day of sOGP treatment,and reached the highest level at the 8th day, and kept at a high level till the 14th daywith a little depression.When the BMSCs were treated by sOGP and Y-27632, a specific ROCK inhibitor, theexpression of ALP was inhibited, few calcium nodules formed, and the oil red ostaining results became significantly positive. Western blot results showed that p-FAKand the p-cofilin were expressed in a very low level in the cells.Part Four: Comparing to OPG-WT mice, the level of serum ALP, ACP in both OPG-/-mice groups were significantly increased (P＜0.01), BMD in OPG-/- groups weremarkedly decreased (P＜0.01). There was no significant difference of serum OCbetween control group and 7-week-old OPG-/- group. Bone mass loss, cortical bonethinning, trabecular bone porosity and collapse were all found in radiographs ofOPG-/- groups. Scan electron microscope (SEM) showed that vertebral trabecula ofOPG-/- groups were thin and discontinuous, with expanded trabecular space andpartial collapsing of trabeculae.After sOGP treatment, the data showed that the BMD in all the treatment groups weresignificantly higher than the saline treated control group (p＜0.01). In combinedtreatment groups, BMD of mice treated by medium and low dose of sOGP combinedwith salmon calcitonin were higher than BMD of the salmon calcitonin treated micein positive group. In all drug treated groups, concentrations of serum ALP and OCwere higher than that in control group (p＜0.05), while the serum ACP concentrationwas lower (p＜0.05). Compared to the control group, increased BMD and thickenedcortical bone were found by x-radiographs and thickened, dense and integratedvertebral trabecula were observed by SEM in all sOGP treatment groups.Conclusion:1,The difference of osteogenesis and adipogenesis differentiation abilitiesamong different BMSCs was significant.2,sOGP showed a significant concentration-dependent osteogenesispromotion and adipogenesis inhibition effect in BMSCs, with theconcentration of 10-9 mol/L presenting the lowest effect.3,sOGP stimulated the proliferation of BMSCs and the effect was related with the sOGP concentration and BMSCs condition. For the BMSCs fromnormal young person and person with postmenopausal osteoporosis, sOGPat all the concentrations had its growth promoting effect, while for theBMSCs from old person with osteoporosis, only sOGP at the 10-7 mol/Lhad the effect.4,RhoA/ROCK signaling pathway played an important role in the BMSCsosteogenesis differentiation induced by sOGP, and it was assumed to bethe initial step. Blocking the RhoA/ROCK signaling pathway led to theinhibition of the osteogenesis differentiation and stimulation of theadipogenesis differentiation.5,The OPG gene knock-out mice, an osteoporosis animal model with highturnover rate, provided an excellent platform for our research.6,sOGP treatment led to the BMD increase with inhibition of osteoclasticbone resorption in OPG-/- mice, and showed a synergistic effect whencombined with the salmon calcitonin. The data suggested a newtherapeutic method for the treatment of osteoporosis and the mechanism ofinhibition would be investigated in the future.
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