Study On Function Of GSK-3? In Regulation Of Bone Homeostasis

Purpose of study:Maxillofacial injuries and secondary secection of head and neck tumor lead to large facial defects.To restore the patient's facial defects and emotional well being,maxillofacial prosthesis is applied by prosthodontic with a high level of expertise.The concept of restorative medicine provides new methods for maxillofacial restoration.As is known,facial part consists of complicated bone and cartilage system,which is the key to the restoration.During the process of osteogenesis,blood vessels are needed to provide nutrition and cytokines,etc.,meanwhile,draining the metabolic waste.Scientists found out a special subtype of blood vessel called 'H' type vessel directly coupled with osteogenesis.It indicates a spatial-temporal accordance between angiogenesis and osteogenesis,and they promote with each other.In this case,study on angiogenesis-osteogenesis coupling is to be of significance in the study of bone regeneration and modification.GSK-3? is a serine-threonine kinase that participate in the regulation of many signaling pathways.Preliminary experiments showed that gene-modified mice with increased in-vivo activity of GSK-3? would not suffer from osteoporosis after OVX compared with their wildtype littermates,with the expression of 'H' type vessel significantly higher.We hypothesize that GSK-3? can maintain bone via angiogenesis-osteogenesis coupling.We focus on the mechanism of how GSK-3? regulate 'H' type vessel by rich different methods in-vivo and in-vitro.We explore the possibility of applying GSK-3? as a target for bone modification and provide some new evidences for further research.Methods:1)We established and purified the mouse strain GSK-3?-/-with stable increased GSK-3? activity universally in-vivo.2)We applied LiCl as an inhibitor to inhibit activity of GSK-3? in-vitro.3)We utilized neutralizing antibody to neutralize PDGF-BB in-vivo and in-vitro.4)We adopted OVX model as a standard model of osteoporosis.5)We used micro-CT to analyze bone structure and bone mass.6)We cultured preosteoclasts BMSCs and ECs.7)We applied tube formation experiment to evaluate the power of angiogenesis for different conditional culture medium.8)We used PCR,Western Blot and Elisa assays.9)We used TRAP staining,immunohistological techniques and observed with laser confocal microscope.Results:1)We successfully bred and purified mouse strain GSK-3? +/-and proved a universally higher activity level of GSK-3? in the strain compared with its wildtype counterparts by GSK-3? activity kit and immunohistological technologies.This mouse strain is perfect for study on GSK-3? and its effect on angiogenesis-osteogenesis coupling.2)By establishing OVX models on GSK-3 ? +/-mice,we discovered that high activitylevel of GSK-3? could prevent the osteoporosis brought by OVX,meanwhile,a high expression of 'H' type vessel was detected compared with wildtype littermates,indicating the prevention was correlated with angiogenesis-osteogenesis coupling.3)With the help of protein chip and biostatistics,we screened two cytokines of significantly different in expression in serum,one of which is VEGF,an angiogenesis related factor;the other one is m-csf,a osteoclastogenesis related factor.We further did assays on osteoclastogenesis and osteogenesis and proved a higher level of osteoclastogensis in the GSK-3? +/-mice where activity of GSK-3?is increased.Thus,the mechanism of how GSK-3? regulate bone mass with angiogenesis-osteogenesis coupling may be correlated with osteoclastogenesis.4)PDGF-BB is the key factor to regulate 'H' type vessel,secreted mainly by preosteoclasts.We discovered that the activity of GSK-3? had a direct effect on level of PDGF-BB and further effect on angiogenesis.Tube formation showed that conditional media form preosteoclasts with high level of GSK-3? led to a high amount of tube formation,Vice Versa.The effect could be not only carried by PDGF-BB but also by a higher secretion of VEGF.Analysis on different conditional media revealed a significantly different level of PDGF-BB in them.By utilizing anti-PDGF-BB neutralizing antibody,we destroyed the effect of angiogenesis by high level of GSK-3? activity.The results indicated the key role of PDGF-BB in the mechanism of how GSK-3? regulates bone homeostasis.5)TRAP staining of tissue of cells indicated that high level of GSK-3? can lead to more preosteoclasts in-vivo and a faster induction to preosteoclasts in-vitro.This results indicated that GSK-3? may exert effect on angiogenesis-osteogenesis coupling by regulating the number of preosteoclasts thus further regulates PDGF-BB.Conclusions:In a summary,our study revealed a significant effect of GSK-3? on regulation of bone homeostasis.High level of GSK-3? resisted osteoporosis by angiogenesis-osteogenesis coupling.The effect may be related to promotion of number of preosteoclats and an increased secretion of PDGF-BB.PDGF-BB played a role in this chain while being neutralized compromised the positive effect of GSK-3? on maintenance of bone mass.The results shows a possibility of settingGSK-3? as a target to osteogenesis and bone formation and provides new scientific theory to further study on bone regeneration and angiogenesis-osteogenesis coupling.