OGP Regulation Mechanism And Promotion On MC3T3-E1Cell Proliferation And Adhesion

Objectives:Osteogenic growth peptide (OGP) is a14-mer polypeptide that widely exists in the sera of mammalian animals and has the functions in regulation of osteoblastic proliferation and promotion on the proliferation of marrow stromal cells, bone formation and hematopoiesis. OGP(10-14) is the C-terminal5-mer peptide of OGP and possesses the role of the full-length OGP. Chemically synthesized OGP(10-14)(shortly as sOGP(10-14) has the identical structure and function of the native OGP(10-14). The first part of this thesis investigates the effect of sOGP(10-14) on the proliferation of MC3T3-E1cells and explores the role of protein kinase A (PKA) in this process. The second part focuses on the investigation of the effect of sOGP(10-14) in combination with extracorporeal shock wave (ESW) on the proliferation of osteoblastic MC3T3E1cells and the exploration of the possibility in treating bone fracture and nonunion with sOGP(10-14) in combination with ESW. The third part of this thesis investigates the construction of biocompatible surfaces and explores the surface chemistry preference and OGP (10-14) promotion of osteoblast adhesion and spreading, monitored by quartz crystal microbalance. The research in this thesis is intended to provide theoretical basis for the clinic application of sOGP(10-14).MethodsPart I:Passage cells of MC3T3-E1were cultured in the OGP(10-14) solution with different concentrations. The celluler proliferation was examined by MTT assay. The expressions of PKA mRNA and p-CREB of substrate of PKA in different conditions acted on MC3T3-E1cells were analysised by RT-PCR and Western blotting, respectively.Part â…¡:Passaged cells were divided into four groups for different treatments, in which Control Group, Experimental group (intervened with OGP+ESW), ESW group (intervened with ESW), and OGP group (intervened with OGP). After the respective treatments, cells were cultured for24h,48h, and72h, respectively, for cell counting with MTT, observation with inverted fluorescence microscope at24h, and immunohistochemical examination of PKA activity, RT-PC determination of PKA mRNA expression at24h and48h.Part â…¢:The self assembly technique was used to construct the self assembled monolayers bearing surface functionalities hydroxyl, carboxyl, dextran, and carboxymethyl dextran, respectively; Contact angle measurement and ATR-FTIR were employed for the characterization of the wettability and structures of these self assembled monolayers; Quartz crystal microbalance (QCM) was used to monitor the adhesion of osteoblastic cells on these surfaces in real-time format; Fluorescence microscopic observation was performed and sOGP9(10-14) in promoting osteoblastic cell adhesion explored. ResultsPart I:The MTT result showed OGP(10-14)at the concentration of10-13mol*L-1exhibited an obvious role in promoting MC3T3-E1cells proliferation; the agonists FSK and OGP(10-14) enhanced the PKA mRNA expression with a significance (P<0.05) as assayed by RT-PCR, and OGP(10-14) was more significant than FSK (P<0.05).Whereas the OGP(10-14) set produced the p-CREB expression with significant increases both in4h and48h (P<0.05),and higher thanin the FSk set. The H-89set showed a low p-CREB expression and the OGP(10-14)+H-89set produced resulted in a less significant increase in the expression.Part â…¡:Cell counting revealed that cell proliferations in groups Experimental, ESW and OGP were significantly promoted as compared with that in Control group (P<0.05); while the level of cell proliferation in Experimental group was the highest (P<0.05). The immunohistochemical examination showed the positive staining intensities in group Experimental, ESW and OGP were obviously higher than that in Control group; while the positive staining intensity in Experimental group was the highest. RT-PC exhibited the expression levels of PKA activity in group Experimental, ESW and OGP were significantly higher than that in Control group and the level in Experimental group is the highest.Part â…¢:The surface with terminal carboxymethyl dextran possessed the highest biocompatibility and capacity for osteoblast adhesion, nearly2-fold of that of carboxyl surface (MUA) and5-fold of that of hydroxyl surface (MUO and dextran). The correlation of QCM response Af and the adherent cell number was established and instrumental sensitivity is ca.2.7mHz/cell. There existed a lag response for resonance resistance as compared with the resonance frequency response, meaning the fast cell attachment and retarded cell spreading process. sOGP(10-14) could promote the cell adhesion on the carboxymethyl dextran surface by a factor of30%.Conclusions1. sOGP(10-14) possesses the role in promoting the adhesion of osteoblastic MC3T3-E1cells, and the best suitable concentration is about10-13mol*L-12. The PKA pathway exhibits a regulation role in the OGP(10-14) promotion on the MC3T3-E1cells proliferation and the PKA signaling in this process was identified.3. sOGP(10-14) and ESW have a synergistic effect on the proliferation and maturation of osteoblastic cells.4. The surface with carboxymethyl dextran can significantly improve the osteoblastic adhesion capacity; while sOGP(10-14) can promote the cell adhesion on the surface even further with a factor of30%.5. Utilizing carboxymethyl dextran as the implant surface modifying material and employing sOGP(10-14) as the growth factor, it is expected to improve the integration of implants with the surrounding tissues.