Experimental Study On The Effect Of Chitosan/Alendronate Composite Materials On Proliferation And Differentiation Of Osteoblasts In Rats

【Objective】 To isolate and culture of neonate SD rats skull osteoblasts in vitro, and thenidentify their morphological and biological characteristic. Make three kinds ofchitosan/alendronate composite material, which the ratio of the amount of substance ofalendronate and chitosan was respective1:5,1:10and1:20. Observe their biocompatibility, andresearch the effect of these three composite materials on proliferation and differentiation ofosteoblast in rats.【Methods】The osteoblasts were isolated from neonate SD rats by being digested withcollagenase and trypsin. Culture them in vitro. Observe their morphologic characters by theinverted phase contrast microscope and HE coloration. Portray the growth curve of the thirdand fourth generation of osteoblasts by MTT assay. Determinate the contents of alkalinephosphatase in osteoblasts by alkaline phosphatase staining method. Alizarin red S staining andVon kossa staining methods were used to observe the mineral node of cultured osteoblasts.Make three kinds of chitosan/alendronate composite materials which the ratio of the amount ofsubstance of alendronate and chitosan was respective1:5,1:10and1:20, and make a kind ofchitosan support material without alendronate simultaneously. The third generation ofosteoblasts were co-cultured with the above four support materials for some time, and thenobserve the cell adhesion and growth situation on the surface of the four support materials byscanning electron microscope. Evaluate the effects of the four support materials on cellproliferation by MTT assay. Observe the chemotaxis of the four support materials on the cellsby transwell assay. Analyze the cell cycle by flow cytometry. Determinate the contents ofalkaline phosphatase in the cell culture supernatant by enzyme standard colorimetric method,and determinate the contents of bone gla protein in the cell culture supernatant by ELISAmethod. By these methods we can get started with the effect of these support materials onproliferation and differentiation of osteoblast in rats.【Results】In this experiment we successfully isolated the osteoblasts from neonate SD rats byenzyme digestion method. The result of MTT assay showed that the osteoblasts had Strongproliferation ability and increased rapidly. The osteoblasts, observed by alkaline phosphatasestaining method, contained large amounts of alkaline phosphatase particles, and it suggested that they had a good secretion function. After two weeks’ culture, we could observe that theosteoblasts had formed a mass of calcified nodules by alizarin red S staining and Von kossastaining methods. The results measured by scanning electron microscope and MTT assayindicated that the four support materials had good biocompatibility. The chitosan/alendronatecomposite material, which the ratio of the amount of substance of alendronate and chitosan was1:10, had the most significant effect of proliferation on osteoblasts of the four support materialsdetermined by MTT assay and flow cytometry, it also had the most significant effect ofdifferentiation on osteoblasts from the results of the contents of alkaline phosphatase and bonegla protein in the cell culture supernatant. The other two kinds of chitosan/alendronatecomposite materials, which the ratio of the amount of substance of alendronate and chitosanwas respective1:5and1:20, also played a role in proliferation and differentiation of osteoblasts,and the former had the strongest chemotaxis on osteoblasts by transwell assay. The chitosansupport material had no effect to proliferation and differentiation of osteoblasts.【Conclusions】 We succeeded in isolating the osteoblasts from neonate SD rats by enzymedigestion method, and the cells we had isolated have Strong proliferation ability and Increasedrapidly with higher purity. Their morphological and biological characteristics were stable.Chitosan/alendronate composite material is a new type of composite material with goodbiocompatibility. We had proved that the chitosan/alendronate composite material, which theratio of the amount of substance of alendronate and chitosan was1:10, had obvious significanteffect of proliferation and differentiation on osteoblasts. It also played a role in chemotaxis onosteoblasts. It may be useful to provide preliminary theoretical basis to greatly improve theefficiency of ossification in vivo and restore bone defect.