Effects Of Huo Xue Hua Yu(HXHY) Decoction To Bone Formation Function Of Cultured Osteoblast In Vitro

Objective: To observe the effects of Huo Xue Hua Yu(HXHY) decoction to bone formation function of cultured osteoblast which was from the skull of newborn SD rat in vitro, in order to explicate the mechanism by which HXHY decoction and HXHY method act on fracture healing.Methods: To obtain osteoblasts from neonatal SD rat's skull adopting the method of Collagenase-pancreatic enzyme digestion, conduct primary culture and sub-culturing, take the second generation cells as experimental model after identification, and then respectively culture these cells with different concentrations of the HXHY extracted fluid and drug-containing serum fluid together. Both of two experiments took the same method as function of cell proliferation was measured by MTT method(OD value at wave-length 490nm)and function of cell differentiation was measured by Golden's method(ALP activity). Directly incentive experiment used alizarin red staining method to measure function of cell mineralization (mineralized nodes/field of vision).Results: Relatively rarefied osteoblasts could be obtained by means of cell culture in vitro using the method of Collagenase-pancreatic enzyme digestion. Three days after communal cultivation, certain concentrations of HXHY extracted fluid and drug-containing serum could stimulate the proliferation of the OB2, and the former acted in dose-dependent manner in field of 100-5000ug/ml. Contrast with the control group, HXHY groups could promote the proliferation of the OB2 in the concentration of 100ug/ml and 500ug/ml whereas they had insignificant difference with control group(.P>0.05).1000ug/ml and 5000ug/ml groups had significant difference (P<0.05),especially the 5000ug/ml group(P<0.01), 10000ug/ml groupshowed descending tendency .Both of the 10% and 20% of drug-containing sera had significant difference with blank drug serum control group(P<0.05).Five days later, the activity of alkaline-phosphotase was more or less increased in experimental groups which were compared with control group respectively,5000ug/ml group had the striking result(P<0.01).Experiment of Drug-containing serum showed that 10% of drug-containing serum group had significant difference (P<0.05), and 20% of drug-containing serum group had even significant difference (P<0.01).Eighteen days later, there was insignificant difference between the experimental groups and control group according to the effects on mineralization (P>0.05).Conclusions: HXHY decoction can promote bone formation by accelerating the proliferation and the differentiation of osteoblast in vitro in early period. Promoting bone formation function of cultured osteoblast is one of the mechanism by which HXHY decoction and HXHY method effectively promote fracture healing. Results also suggest us that we should differentiate the symptoms and signs by stages while we treat the fracture clinically. HXHY decoction is fit for treating the fracture in early period.