| Chronic periapical periodontitis and periodontitis are two common diseases of stamatology, Which can cause severe damage or deform of bone tissue. Osteoblasts are loc ated at the surface of bone tissue, They are functional cell, play an important role in bone ?forming or reparing stages. In many cases of bone tissue damage or deform, The loss of osteoblasts is not attribute to forming new bone tissue or matrix , But occurring apoptosis. That is the reason why present therapy methods may not obtain satisfying results, therefore, A new biological therapy method should be investigated to solve such kind of problem. How can we take adventage of the biological function of some biological active factor in order to increase the proliferation and differentiation of osteoblasts and decrease the rate of apoptosis at the same time,become a focus question in stomatology. With the development of bone tissue engineering, The application of biological therapy of bone damage or deform has clinical significance.Fibronectin ( FN) is a heterodimeric extracellular matrix (ECM) glycopro-tein which has been shown to regulate adhesion,migration, proliferation, differentiation and apoptosis of mesenchymal cells. Present study has been proved that FN is a survival factor for differentiated osteoblasts, and also plays a unique role in osteoblasts apoptosis. therefore, The clinical application of FN as a new biological agent is a promising and valuble therapy method.The purpose of this study is to establish an ideal and effective cultivation system including the methods of isolation, purification , cultivation and identification of rat calvaria osteoblasts in vitro and to investigate the effects of bovine plasma fibronectin on the proliferation, differentiation and apoptosis of rat osteoblasts in vitro in order to offer an experimental basis for the clinical application.Methods1. Isolation , purification , cultivation and identification of Osteoblasts Calvariae from 2d - old new bone rats were removed aseptically. The peri-osteal layers on both sides were carefully stripped off with tweezers under D -Hanks. Then, the bone specimens were treated by trypsinization for 20 min at 37 X,, and the supernatant was discarded. After that the specimens were digested by collagenase for 90 min, the supernatant was collected and centrifuged. The collagenase - release cells were plated in 199 medium supplemented with 15% fetal bovine serum (FBS). The medium was changed twice per week until the cells reached confluence. The differential adherence method was used to purify the osteoblasts. The immunohistochemical method of type I collagen was used to identify the osteoblasts.2. The morphologic observation of osteoblastsObserve shapes and structures of cultured osteoblasts with phase contrast microscope, Scan electron microscope ( SEM) and transmission electron microscope (TEM).3. Effects of bovine plasma FN on the proliferation of rat osteoblasts Osteoblasts were collected using 0. 25% trypsin and replated at a density of5 104cells/ml in 96 - well flat - bottomed plates. The medium was changed to serum - free medium after 24 h. The next day FN with different concentration (0 ug/ml, 10 ug/ml, 20 ug/ml, 30 ug/ml, 40 ug/ml, 50 ug/ml) was administrated. The proliferative response to FN was determined by the percents of reduced AlamarBlue4. Effects of bovine plasma FN on the differentiation of rat osteoblasts Osteoblasts were collected using 0. 25% trypsin and replated at a density of5 x 104cells/ml in 96 - well flat - bottomed plates. The medium was changed to serum -free medium after 24h. The next day FN with different concentration (0 ug/ml, 10 ug/ml, 20 ug/ml, 30 ug/ml, 40ug/ml, 50ug/ml) was administrated. The activity of alkaline phosphatase (ALP) was measured by Elisa method.5. Analysis the cell cycle and apoptosis effects by FNOsteoblasts were plated in flasks at a density of 1 x 105cells/ml, and allowed to attach for 3d. After that the medium was changed to serum - free medium. FN with different concen... |
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