The Mechanism Study Of Enhanced Osteogenesis By Brain Gray Matter Extracts (A Possible Role Of Adrenoceptors)

As we know, the nerve system composed of central and peripheral parts dominates multiple systems and organs of human body and regulates their complex function and responses. This is evident in bone and joint since nerve fibers of different kinds were found in bone by histological studies and close clinical relations between nerve function disorders and altered bone healing and remodeling were discovered. Attention was further attracted when the phenomenon was reported that those who suffered from traumatic brain injury (TBI) always achieved enhanced fracture healing, among whom some even got heterotopic ossificant (HO). What is the underlying mechamism? Many authors claimed their prosthesises while no one was acknoledged widely. The main idea which is one of the basis of current study that some inherent components of brain tissues may be released into blood circulation that enhance the fracture healing is somewhat supported by other authors. At least most of the growth factors seems to be the wanted one but the relevant evidents is yet specific enough. The recent findings that sympathetic nerve system was one of the most important elements transfering the leptin dependent central nerve system messages of bone formation and resorption was encouraging. The adrenoceptors at multiple cell lines in bone marrow and substantia ossea may transfer the downstream messages from pathways abovementioned which opened a new area for study.Our former studies have proved that both brain gray matter and white matter extracts did enhance rat's osteoblasts proliferation and differentiation amone which the gray matter was more significant. Also the gray matter extracts enhanced the Bone Marrow Stromal Cells (BMSCs) proliferation and differentiation to osteoblasts. Then some questions still need to be answered. Firstly, does higher concentration of brain tissue extracts enhance the osteoblasts'proliferation and differentiation more significantly? And does this enhancement work when the extracts and osteoblasts are from different species? Lastly what is the role of adrenoceptors on osteoblasts and BMSCs proliferation and differentiation and how it will influence the gray matter's stimulation of osteoblasts and BMSCs proliferation and differentiation?ObjectiveTo investigate whether the gray matter extracts enhance the proliferation and differentiation of osteoblasts in a dose-depenpdent pattern, and the effect of the extracts from porcine brain components on rat's osteoblasts proliferation. Furthermore the adrenoceptors'role in brain gray matter osteogenic stimulation is to be studied.MethodsThe research can be divided into 4 parts below:1. The brain gray matter extracts of SD rats were collected and diluted 1000,100 and 10 times with 10% MCS-MEM culture medium, then the rat's osteoblasts were cultured by these diluted extracts of gray matter respectively for 4 days. The proliferation of osteoblasts was compared by MTT assay and ALP of them by PNPP assay.2. The gray matter, white matter, pituitary gland and skeletal muscle of both swine and S-D rats were collected. The rat's osteoblasts were cultured by the extracts of these tissues respectively. The proliferation of osteoblasts was compared by MTT assay at day 1, 4,7 and 10.3. The extracts of rat's gray matter were made to culture the osteoblasts, and luM propranolol and luM phentolamine were added to the culture respectively. On day 1,4, and 7, the proliferation of the osteoblasts by MTT assay was assessed and simutaniously ALP assay was done. The propranolol and phentolamine's influence on the osteoblasts'proliferation and differentiation were compared with control group by ANOVA of SPSS15.0.4. The rats'BMSCs were cultured by the gray matter extracts of rats (X 100 diluted), propranolol(1μM), phentolamine(1μM) and epinephrine(0.1μM) respectively and combined. Quantitative fluorescence RT-PCR of RUNX2,ALP,osteocalcin,RANKL and osteoprotegerin mRNA was detected after 24 hours of cultivation.Results1. The enhancement of proliferation and differentiation by brain gray matter extracts was in a dose-dependent pattern.1) In terms of the osteoblasts proliferation, the gray matter extracts did enhance the process when diluted in 100 and 10 times comparing with the control group (P<0.05). But no significant proliferation stimulation by the extracts diluted in 1000 times was found comparing with the control group (P>0.05). The ANOVA Turkey analysis shew that differences among the extracts diluted in 1000,100 and 10 times was significant (P<0.05). According to the trend of the MTT increasement it indicated that the enhancement potential of brain gray matter extracts is dose-dependent.2) Considering the osteoblasts differentiation by ALP assay, the gray matter extracts did enhance the process when di luted in 1000,100 and 10 times comparing with the control group (P<0.05). The ANOVA Turkey analysis shew that differences among the extracts diluted in 1000,100 and 10 times were significant (P<0.05) too. According to the trend of the ALP volume increasement it indicated that all studied concentration of gray matter extracts had the potential of enhancing osteoblasts differentiation in a dose-dependent style.2. All porcine brain extracts including gray matter, white matter and pituitary stimulated significantly the proliferation of rats'osteoblasts (P<0.05) at day 1,4,7 and 10 among which the gray matter topped the proliferative stimulation capacity. While the extracts of porcine muscle did not change the proliferation characters (P>0.05). The strength of rat's osteoblasts proliferation enhancement of porcine brain components extracts is similar to their counterparts of rats' extracts. Concerning the temporal proliferation distribution the promotive effects of the brain extractives were more obvious at day 4 and day 7.3. The effect of a andβadrenoceptors'function on rat's osteoblasts proliferation and differentiation.1) Neither 1μM propranolol nor 1 u M phentolamine alone changed the proliferation of rat's osteobalsts significantly when the MTT assay was measured on day 1, 4 and 7 comparing with that of the control group (P>0.05).2) Neither 1μM propranolol nor 1 u M phentolamine alone changed the ALP volume of rat's osteobalsts significantly whose differentiation was indicated when the PNPP assay was measured on day 1,4 and 7 comparing with that of the control group (P>0.05)3) When 1μM propranolol or 1μM phentolamine was added to the culture system with the brain gray matter extracts respectively, both adrenergic receptors'blockers didn't interfere with the osteoblasts proliferation measured by MTT assay.4) When 1μM propranolol nor 1μM phentolamine were added to the culture system with the brain gray matter extracts respectively, the ALP volume were mildly suppressed by propranolol on 7 day's assay with no ALP's change in the day 1 and 4. Phentolamine presented no influence on the ALP assay. 4. The effect ofαandβadrenoceptors'function on rat's BMSCs mRNA transcription regulation relevant to the osteogenesis such as RUNX2, ALP, osteocalcin, RANKL and osteoprotegerin.1) The gray matter extracts increased the transcription of osteocalcin mRNA independently (P<0.05), but without effect on RUNX2,ALP mRNA or RANKL/OPG Ratio significantly(P>0.05)2) The epinephrine(0.1μM) cut down the RANKL/OPG ratio of BMSCs (P<0.05), while it didn't change the RUNX2,ALP or osteocalcin mRNA level significantly。3) Propranolol (1μM) didn't change all detected mRNA levels including RUNX2, ALP, osteocalcin, RANKL and osteoprotegerin(P>0.05).4) Phentolamine(1μM) increased the transcription of osteocalcin mRNA independently (P<0.05), but without effect on RUNX2,ALP mRNA or RANKL/OPG Ratio significantly(P>0.05)5) The brain gray matter extracts combined with epinephrine(0.1μM) greatly push the RUNX2,ALP and osteocalcin mRNA higher both than the control group and the groups cultured by them respectively (P<0.05). But the combined culture didn't change the RANKL/OPG mRNA Ratio significantly(P>0.05)6) The brain gray matter combined with propranolol(1μM) increased the osteocalcin mRNA (P<0.05), without effect on RUNX2 or ALP mRNA(P>0.05). Also they didn't change the RANKL/OPG mRNA ratioConclusion1. The enhancement of brain gray matter on osteoblasts proliferation and differentiation is evident and in a dose-dependent pattern.2. The porcine brain inherent components can stimulate the proliferation of different species'osteoblasts (rat osteoblasts), similar to that of the rats'counterparts, among which the gray matter topped the proliferative stimulation.3. a andβadrenergic receptors blockers don't directly change the proliferation and differentiation of rats'osteoblasts independently. Theβadrenergic receptor may indirectly participate the mechanism of enhanced proliferation and differentiation of osteoblasts caused by gray matter extractives.4. The a andβadrenoceptors are involved in the regulation of the BMSCs gene transcription caused by brain gray matter, which is related to bone formation and resorption. But the exact mechanism is still to be further investigated.