摘要
目的:骨形成过程由骨形成蛋白诱导开始,并受到多种生长因子的综合调控。为建立良好的细胞培养体系和配制含有多种生长因子的活性材料,观察不同浓度的重组人骨形成蛋白2和碱性成纤维细胞生长因子对犬骨髓基质细胞增殖、成骨分化的影响。方法:实验于2007-05/07在上海交通大学医学院附属第九人民医院口腔生物工程实验室完成。①实验材料:1岁beagle犬3只。重组骨形成蛋白2,碱性成纤维细胞生长因子(Peprotech)。②实验方法:犬肌注麻醉后,穿刺抽取2mL骨髓,离心制备骨髓基质细胞,进行原代培养。待细胞80%汇合时,胰酶+乙二胺四乙酸消化传代。③实验评估:取传至第3代的细胞,接种后分别加入重组骨形成蛋白2和碱性成纤维细胞生长因子,各自分为0,25,50,100,200μg/L组。于第1,2,3,4,5,6天倒置显微镜下动态观察细胞形态;MTT比色法测定细胞增殖水平;碱性磷酸酶活性定量检测细胞成骨分化情况;于第6天行VonKossa及碱性磷酸酶染色。结果:①细胞形态观察:第3代骨髓基质细胞诱导培养3d后,多角形细胞增多,细胞逐渐向梭形或多边形转化,胞体膨大,存在大小形态不一的胞浆突起,胞核较大,位于细胞体一侧,偶见2个核仁;约6d细胞融合,仍以长梭形或线形为主,集落生长趋势明显。②细胞增殖测定:与空白对照组比较,100,200μg/L重组骨形成蛋白2能显著促进骨髓基质细胞生长,且浓度为100μg/L时随作用时间的延长细胞生长速度增加尤为明显(P<0.01);50μg/L碱性成纤维细胞生长因子能显著促进骨髓基质细胞生长(P<0.05)。③细胞成骨分化测定:与空白对照组比较,25μg/L重组骨形成蛋白2能显著促进犬骨髓基质细胞分化,且随作用时间的延长细胞分化尤为明显(P<0.01)。碱性成纤维细胞生长因子无显著促进犬骨髓基质细胞分化作用。④VonKossa染色结果:各组均有明显钙结节形成。⑤碱性磷酸酶染色结果:各组胞质内均可见紫色颗粒,呈阳性表达。结论:100μg/L重组人骨形成蛋白2与50μg/L碱性成纤维细胞生长因子均能有效地促进犬骨髓基质细胞增殖,25μg/L重组人骨形成蛋白2可明显促进犬骨髓基质细胞成骨分化,为构建活性骨替代材料提供良好的实验平台。
AIM： Bone morphogenetic protein-2 is reported to induce bone formation in the regulation of numerous cytokines. The effects of different concentration recombinant human bone morphogenetic protein-2 （rhBMP-2） and basic fibroblast growth factor （bFGF） on proliferation and differentiation of bone marrow stromal cells （BMSCs） in dogs were investigated to develop a culture-cell system and an activity biomaterial.
METHODS： The experiment was completed at Lab of Bioengineering, Ninth People＇s Hospital Affiliated to Medical College of Shanghai Jiao Tong University from May to July 2007. ①Materials included three beagle dogs aged 1 year, rhBMP-2 and bFGF （Peprotech）. ②Methods： Under general anesthesia, 2 mL bone marrow was aspirated from dog caput femoris. The cells were cultured in delbecco＇s modified eagle medium （DMEM） after centrifuged. When 80% cells were confluent, they were liberated by trypsin/EDTA. ③Measurement： The 3^th generated cells were seeded treated with rhBMP-2 and bFGF （0,25,50,100,200 μg/L） respectively. Cell appearance was observed under inverted microscope. The proliferation and differentiation of BMSCs were observed by MTT and alkaline phosphatase （ALP） at days 1, 2, 3, 4, 5 and 6. ALP and Von Kossa staining were performed on the 6th day.
RESULTS： ①Microscope observation： After induced for 3 days, the cells became polygonal or shaped like shuttle with kytoplasm pustute and big karyons at one side, and two nucleoli appeared. The cells were confluent at day 6, mainly shaped like shuttle or line, and colony growth appeared obviously. ②Measurement of proliferation： The concentration of 100 μg/L and 200 μg/L rhBMP-2 could significantly promote BMSCs proliferation, especially at 100 μg/L when the action time was prolonged （P 〈 0.01）. 50 μg/L bFGF also could significantly accelerate BMSCs proliferation （P 〈 0.05）. ③Measurement of differentiation： 25 μg/L rhBMP-2 could promote BMSC differentiation, especially when the action time was prolonged （P 〈 0.01） and bFGF had no effect on differentiation. ④Von Kossa staining proofed the positive expression of calcic node. ⑤ALP staining grooved the purple positive expression. CONCLUSION： 100 μg/L rhBMP-2 and 50 μg/L bFGF can promote BMSCs proliferation. 25 μg/L rhBMP-2 promote BMSCs differentiation for replacement bone.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第46期9276-9280,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
