Experimental Study Of BFGF And RhBMP-2 Polylactic Acid Sustained Release Nanospheres Accelerating Healing Process Of Mandubular Fracture

Mandubular fracture is a common disease and frequently occurring illness in oral maxilifacial region. With increases of the traffic accident rate progressively year by year, incidence of maxillofacial region fracture increases progressively. At present, research on diagnose and therapy of maxillofacial region's fracture is becoming one of hot spots. Compared to other site, maxillofacial region fracture has self characteristic: (1) It has a strict requirement in reposition and fixation. (2) After reposition and fixation, foodintake restriction will effect patients'nutrition,accordingly effect fracture healing. (3) Besides functional restoration,we must satisfy patients'requirement for contour. Accordingly we should try to ensure bone fracture's healing fastly.Research on molecular biology indicated that union of fracture is a complicated process by many growth factors participating in regulation. Among the total, basic fibroblast growth factor(bFGF) and bone morphogenetic protein-2(BMP-2) have a close relationship with tissue healing, especially union of fracture. bFGF have an evident stimulatory function in proliferation and differentiation of chonseoblast and osteoblast, consequently it play an important role in union of fracture. BMP-2 is as an initiator in union of fracture, it regulates related gene expression in cell nucleus, thereby induce cells'differentiation to promote generation of cartilage and bone.With the development of molecular traurmatology,it became a chance of using exogenous growth factors promoting fracture union.But at present there are some defect in using exogenous growth factor promoting fracture union.It is showed that growth factors have some characteristic: they have a strong biology activity,at the same time their half-life period is short in vivo, and have a high clearance rate. And their biological availability is low in non-injection administration. Some scholars attempted to use various kinds methods,including injecting bFGF and BMP-2 in region ,or preparing many bFGF and BMP-2 delivery system.But they degradated fast,and their effect was brief. At the same time the manipulation was complicated,so that they can't bring about stable biological effects lastingly.Nanometer technology is a synthetic technology system that materials'opreparation and industrialization in nanometer dimension. Nanospheres can extend pharmacodynamic action significantly and elevate availability after they were absorbed.we can carry out nanospheres'slow-releasing and target distribution by regulating their carrier.On account of above analysis,this subject first prepared pharmacy experiment.We used polylactic acid(PLA)as carrier to prepare bFGF PLA nanospheres(bFGF-PLA-Ns)and rhBMP-2 PLA nanospheres(rhBMP-2-PLA-Ns),to study bFGF-PLA-Ns and rhBMP-2-PLA-Ns accelerating mandubular fracture healing.bFGF-PLA-Ns and rhBMP-2-PLA-Ns were prepared into colloids and freeze drying powders;Then we studied the shape and particle diameter distribution, drug-loading,entrapment rate of bFGF-PLA-Ns and rhBMP-2-PLA-Ns.Rabbit osteoblast was primary cultured.We observed effects on cell proliferation by add different concentration of bFGF-PLA-Ns or rhBMP-2-PLA-Ns.And their effects on proliferating cell nuclear antigen(PCNA),alkaline phosphatase(ALP) activity and mineralization. rhBMP-2-PLA-Ns effecting on osteoblast vascular endothelial growth factor(VEGF)autocrine was observed.Rabbit mandubular fracture model were established.We used HE staining ,immunohistochemistry study, ambramycin fluorescent staining and in situ hybridization histochemistry in studying bFGF-PLA-Ns and rhBMP-2-PLA-Ns'effects on mandible fracture healing.methods and results:1. bFGF-PLA-Ns and rhBMP-2-PLA-Ns'preparation and their characteristic:PLA nanospheres were preparated,and their colloid mean diameter were detected by Malvern laser particle size distribution instrument.Results of three groups were 161nm,154nm,160nm. Degree of precision and stability in organics of bFGF and rhBMP-2 were detected. The results confirmed that bFGF and rhBMP-2 were reliable.bFGF-PLA-Ns and rhBMP-2-PLA-Ns were preparated by multiple emulsion volatilizing method. 1% galactosylglucose was selectde as supporting frame through optimizing.Then detected their mean diameter by electron microscope.The results showed that nanospheres'diameter were 30～50nm,bFGF-PLA-Ns and rhBMP-2-PLA-Ns were even and uniform. The drug loading amount and encapsulation efficiency of bFGF-PLA-Ns were (91.72±1.31)%and [(27.65±0.44)×10^-3]%.The drug loading amount and encapsulation efficiency of rhBMP-2-PLA-Ns were(90.54±1.32)% and[(124.73±0.41)×10^-3]%.bFGF-PLA-Ns and rhBMP-2-PLA-Ns can not only sustain release bFGF and rhBMP-2, but also their concentration can maintain homologous level in 14 days in simulated body environment. bFGF-PLA-Ns'average concentration were (72.47±6.26)ng/ml. rhBMP-2-PLA-Ns'average concentration were (73.44±5.38)ng/ml. In vitro release regularity of bFGF-PLA-Ns and rhBMP-2-PLA-Ns were figured by Higuichi equation: Q=27.7636 t^1/2-13.4247,r=0 9975 and Q=26.7687t^1/2-12.3283,r=0 9962.2. bFGF-PLA-Ns and rhBMP-2-PLA-Ns'biological effects on osteoblasts in vitro:Trypsin digestion method was select to primary culture rabbit osteoblasts.Osteoblasts were observed and identified from endothelial cells and fibroblasts. Microculture tetrazolium test(MTT) was used to observe bFGF-PLA-Ns and rhBMP-2-PLA-Ns'proliferation on osteoblasts. The result indicated that bFGF-PLA-Ns and rhBMP-2-PLA-Ns both can significantly increase osteoblasts'proliferation(p<0.05).We utilized alizarin monosulfonate staining method to detect mineralization nodus formation.The results indicated that bFGF-PLA-Ns and rhBMP-2-PLA-Ns have distinguished stimulant function(p<0.05).Our experiment about osteoblastic ALP activity showed that bFGF-PLA-Ns can notable promote osteoblast differentiation(p<0.05).We designed immunohistochemistry method to research bFGF-PLA-Ns effect on PCNA.The results showed bFGF-PLA-Ns can enhance PCNA activity (p<0.05).Immunofluorescence method result showed that rhBMP-2-PLA-Ns can enhance PCNA activity and expression. Western blot were used to research rhBMP-2-PLA-Ns'effect on VEGF expression.The results indicated that rhBMP-2-PLA-Ns can promote osteoblasts autocrining VEGF through semiquantitative analysis.3. Animal experiment of bFGF-PLA-Ns and rhBMP-2-PLA-Ns accelerating healing process of mandubular fracture:We set up rabbit mandible fracture model. experiment were divided into five groups: group control, group simple bFGF, group bFGF-PLA-Ns gel, group simple rhBMP-2, group rhBMP-2-PLA-Ns gel. Animal were executed in order after 1,2,4,8w. The bone fracture site's pathological section were stained by haematoxylin-eosin(HE), The results showed that experiment groups has newly formed bone trabecula compared to other two groups after 1w. Experiment groups had more woven bone formation after 2w, and had a high degree of maturity.Generous new vessels can be seen, bony callus ratio obviously exceeded other three groups after 4w.VEGF mRNA was detected by hybridization in situ.The results showed that group rhBMP-2-PLA-Ns was higher than group control and group simple rhBMP-2(P<0.05).PCNA was detected in sections of organ by immunohistochemistry.The results showed that amounts of PCNA positive cells in experiment groups were more than that of other groups(P<0.05).Immunohistochemistry study showed that bFGF-PLA-Ns can stimulateⅧfos-related antigen hypso-expression in vascular endothelial cells(P<0.05).Ambramycin fluorescent light staining method detected that bFGF-PLA-Ns and rhBMP-2-PLA-Ns could accelerate new bone formation,had a statistical significance (P<0.05).Conclusion:1.We select PLA as carrier to preparate bFGF-PLA-Ns and rhBMP-2-PLA-Ns by multiple emulsion volatilizing method.Drugs can completely satisfy requirement of nanospheres. components of nanospheres have no immunogenicity. And their biological safety can be warranted.2.Our experiments confirmed our nanospheres technique is mature.Our technique possess creativity,so that we have applied for a patent in China.3. Study showed that bFGF-PLA-Ns and rhBMP-2-PLA-Ns have a prominent biological effects on rabbit osteoblasts in vitro.4.Our study indicated that gels of bFGF-PLA-Ns and rhBMP-2-PLA-Ns can accelerate healing process of mandubular fracture. Above-mentioned study showed that bFGF-PLA-Ns and rhBMP-2-PLA-Ns have a better prospect in union of fracture.