Effects Of Akermanite And Slit2 On Osteoblastic Differentiation

[Objectives]1. Previous studies showed akermanite bioactive ceramics possess improved mechanical properties such as fracture toughness, bending strength and Young's modulus as compared withβ–TCP ceramics. Furthermore, they possess bonelike apatite-formation ability and can release soluble ionic products to stimulate osteoblast proliferation, which indicates good bioactivity. However, bone marrow stromal cells (BMSC) play an important role in bone regeneration and the effects of calcium magnesium silicate bioactive ceramics on differentiation of MSC have not been reported. So, the first objective is to study the effects of akermaninte on proliferation and osteogenic differentiation of hBMSC.2. The differential effects between akermanite andβ-TCP on the proliferation and osteoblastic differentiation of hBMSC may be explained by differences in chemical composition, and the ionic products of the akermanite dissolution might play an important role in the stimulatory process. Then, the second objective is to study the effects of extracts from different biomaterias on proliferation and osteogenic differentiation of hBMSC. Furthermore, we focus on the effects of ionic products on osteogenic differentiation of mesenchymal stem cells.3. The third objective is to study the biocompatibility, osteointegration and ability of ingrowth of new bone tissue in vivo.4. We all know that the differentiation of osteoblastic cells are controlled and regulated by many factors in vivo. Expressions and roles in bone tissue of the neuron guidance molecules Slit/Robo, which play the main role in nerve system, haven't been reported then. So the fourth objective is to study the expression and the roles of Slit/Robo in osteoblastic cells.5. The last objective is to study the mechanism of Slit/Robo regulation of osteoblastic cells differentiation. [Methods]1. Marrow aspirates were harvested from healthy male donors using a bone marrow biopsy needle inserted through the cortical bone. hBMSC were selected on the basis of adhesion and proliferation on tissue culture plastic substrate. hBMSC were identified by osteogenic and adipogenic induction using the ALP, Alizen Red and Oil Red O staining respectively. The hBMSC'proliferation of the different passage has been compared by cell number count. Akermanite andβ-TCP disks were seeded with hBMSC and kept in growth medium or osteogenic medium for 10 days. Adhesion of hBMSC on the different surfaces was evaluated by SEM 24h after seeded. Proliferation was evaluated on day 1, 4, 7 and 10 using the alarmblue assay and lactic acid production assay. The ALP activity was identified by ALP staining and pNPP assay. The analysis of osteoblast-related genes, including ALP, osteopontin (OPN), bone sialoprotein(BSP) and osteocalcin(OCN), was conducted by RT-PCR, GAPDH as the house keeping gene.2. The extracts from akermanite andβ-TCP powder were harvested 24h after the biomaterials soaked in growth medium without serum. hBMSC were cultured in growth medium supplement with the extract of akermanite orβ-TCP. The proliferation of hBMSC in the different extracts was evaluated by MTT assay. The analysis of osteoblast-related genes were conducted by Real Time PCR assay, including ALP, osteopontin(OPN), steocalcin(OCN). Additional, the stimulatory effects on the C3H10T1/2 osteoblastic differentiation of Mg was conducted using a pNPP assay.3. By using a trephine drill, a bone defect of 5.5mm in diameter and 6mm in depth in all the animals, was performed in the lateral aspect of distal right femoral epiphysis. The implantations were evaluated by Van Gieson's picro-fuchsine staining at 4 and 12weeks after implantation.4. RT-PCR for the detection of Slit2/Robo mRNA expression was done at 0, 4, 8, 12 and 16d after Rat-MSC, calvarial osteoblasts derived from newborn rats (Rat-OB), C3H10T1/2 and MC3T3-E1 growth in osteogenic medium. Western blots for the detection of Slit2/Robo protein expression were done at 0, 8, and 12d after Rat-MSC (the left panel) or Rat-OB (the right panel) growth in osteogenic medium. Western blots were performed using antibodies for Robo1, Robo2 andβ-Actin.5. The recombinant slit2 was harvested using the slit2-myc/HEK 293cell line which can secrete the recombinant slit2 to medium. The osteogenic differentiation of Rat-OB and MC3T3-E1 was identified by pNPP assay and RT-PCR when the medium supplement with the recombinant slit2 protein. Further more, the inhibition of slit2 was in a dose dependent manner. In order to study the way of slit2's inhibition through, we expressed the recombinant HA-RoboN protein using the expression plasmid RoboN /pCS2+. We harvested the medium containing the recombinant HA-RoboN after the HEK 293cells transfected with RoboN /pCS2+. The inhibition of slit2 can be rescued by the medium supplement with the recombinant HA-RoboN, which was the extracellular domain of rat Robo1 protein. The role of RhoA/ROCK pathway in the effects of Slit2/Robo was evaluated by activated or inhibited by the agonist LPA or antagonist Y27632.[Results]1. hBMSC spread well on both biomaterials examined by SEM. The data from the alarmblue assay and lactic acid production assay showed that hBMSC proliferated more significantly on akermanite than onβ-TCP. The analysis of osteoblast-related genes, including ALP, OPN, BSP and OCN, indicated that akermanite ceramics enhanced the expression of osteoblast-related genes, but COLⅠshowed no noticeable difference among akermanite andβ-TCP ceramics.2. The data from the MTT assay showed that hBMSC proliferated more significantly in growth medium supplement with the low concentration extract of akermanite than ofβ-TCP. The analysis of osteoblast-related genes by Real Time PCR assay, including ALP, OPN and OCN, indicated that extract of akermanite enhanced the expression of osteoblast-related genes. Additional, the stimulatory effects on the C3H10T1/2 osteoblastic differentiation of Mg was conducted using a pNPP assay.3. The data from the implantation in vivo, showed good osteointegration, biocompatibility and effective ingrowth of new bone tissue. 4. The data indicated that the mRNA of Slit2, Robo1 and Robo2 were expressed all the time during Rat OB and MC3T3-E1's osteogenic differentiation. But the mRNA of Slit2, Robo1 and Robo2 weren't expressed until Rat-MSC and C3H10T1/2 after osteogenic differentiation. The Robo3 and Robo4 were not detected through the osteogenic differentiation of the all the cells. In the protein level, we didn't find the expression of Robo1 and Robo2 in the Rat-OB by Western blots.5. The data from Alp activity and RT-PCR, indicated the osteogenic differentiation of Rat-OB and MC3T3-E1 was inhibited when the medium supplement with the recombinant slit2 protein. Further more, the inhibition of slit2 was in a dose dependent manner. The inhibition of slit2 can be rescued by the medium supplement with the recombinant slit2 HA-RoboN, which was the extracellular domain of rat Robo1 protein. Our data also showed that slit2's inhibition of osteogenic differentiation was in a independent of RhoA/ROCK manner, for the activation or inhibition by the agonist LPA or antagonist Y27632 to RhoA/ROCK neither can rescue the inhibition of slit2.[Conclusions]1. This result suggests that akermanite can promote proliferation and osteoblastic differentiation of hBMSC in vitro even without osteogenic reagents.2. The differential effects between akermanite andβ-TCP on the proliferation and osteoblastic differentiation of hBMSC may be explained by differences in chemical composition, and the ionic products of the akermanite dissolution played an important role in the stimulatory process.3. These results indicate that akermanite has a good bioactivity both in vitro and in vivo and may be used as a hopeful material for bone regeneration and tissue engineering applications.4. For the first time, we found that the slit2, robo1 and robo2, were expressed during the osteogenic differentiation in the osteoblastic cells.5. The inhibition of slit2 through binding its receptor Robo was in a dose dependent manner, although independent of RhoA/ROCK pathway. These results suggest that slit2 play an important role during bone tissue development and may be the potential pharmacal target for treat bone disease.