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Title: [Content of bone morphogenetic protein 2 in demineralized bone matrix prepared from different long bones and study of the osteogenic properties in vitro].
Author: Zhao Y, Yin G, Du R, Wang L, Deng M, Guan G, Sun G, Liu Y.
Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2023 Aug 15; 37(8):945-951. PubMed ID: 37586793.
Abstract:
OBJECTIVE: To measure the concentration of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) prepared from different long bones and to evaluate the osteoinductivity of different DBM on MC3T3-E1 cells. METHODS: Different bones from the same cadaver donor were used as the initial materials for making DBM, which were divided into ulna group (uDBM), humerus group (hDBM), tibia group (tDBM), and femur group (fDBM) according to the origins, and boiled DBM (cDBM) was taken as the control group. The proteins of DBM were extracted by guanidine hydrochloride, and the concentrations of BMP-2 were determined by ELISA assay. Then the DBM were co-cultured with MC3T3-E1 cells, the proliferation of MC3T3-E1 cells was observed by cell counting kit 8 (CCK-8) assay. The osteogenic differentiation ability of MC3T3-E1 cells was qualitatively observed by alizarin red, alkaline phosphatase (ALP), and Van Gieson staining, and the osteogenic differentiation ability of MC3T3-E1 cells was quantitatively analyzed by ALP content. Linear regression was used to analyze the effect of BMP-2 concentration in DBM on ALP synthesis. RESULTS: There were significant differences in the concentration of BMP-2 among the DBM groups (P<0.05). The concentrations of BMP-2 in the lower limb long bone were higher than those in the upper limb long bone, and the concentration of BMP-2 in the fDBM group was about 35.5 times that in the uDBM group. CCK-8 assay showed that the cells in each group continued to proliferate within 5 days of co-culture, and the absorbance (A) values at different time points were in the order of cDBM group<uDBM group<hDBM group<tDBM group<fDBM group. After co-culture for 14 days, the expressions of ALP, calcified nodules, and collagen fibers in each group were consistent with the distribution of BMP-2 concentration in DBM. The order of ALP content from low to high was cDBM group<uDBM group<hDBM group<tDBM group<fDBM group, and the differences between the groups were significant (P<0.05). Linear regression analysis showed that y^=0.361+0.017x, the effect of BMP-2 concentration in DBM on cellular ALP content was significant (t=3.552, P=0.005); for every 1 ng/g increase in BMP-2 concentration, ALP content would increase by 0.017 [95%CI (0.006, 0.027)] U/100 mL. CONCLUSION: The concentration of natural BMP-2 in different long bones varies greatly, and the lower limb long bone is higher than the upper limb long bone. The harvested location of bone material was an important factor affecting the osteoinductivity of DBM. 目的: 测量不同长骨制备的脱矿骨基质（demineralized bone matrix，DBM）中BMP-2的浓度，评价不同DBM诱导MC3T3-E1细胞成骨分化的能力。. 方法: 取同一尸体标本不同长骨制备DBM，按取材部位分成尺骨组（uDBM组）、肱骨组（hDBM组）、胫骨组（tDBM组）和股骨组（fDBM组），以煮沸后的DBM为对照组（cDBM组）。将各组DBM经盐酸胍提取蛋白后采用ELISA法检测BMP-2含量；然后将其与MC3T3-E1细胞共培养，细胞计数试剂盒8（cell counting kit 8，CCK-8）法观察各组细胞增殖情况，行茜素红、ALP、Van Gieson染色定性观察和ALP含量定量分析各组细胞成骨分化能力；并采用线性回归分析DBM中BMP-2浓度对细胞合成ALP的影响。. 结果: 各DBM组间BMP-2浓度比较差异均有统计学意义（P<0.05），下肢长骨中BMP-2浓度高于上肢长骨，其中fDBM组BMP-2浓度约为uDBM组的35.5倍。CCK-8法检测示，共培养5 d内各组细胞持续增殖，5 d时各组吸光度（A）值从高到低依次为fDBM组>tDBM组>hDBM组>uDBM组>cDBM组。共培养14 d，各组细胞表达ALP、钙化结节和胶原纤维的程度与DBM中BMP-2浓度分布趋势一致。ALP含量从低到高依次为cDBM组<uDBM组<hDBM组<tDBM组<fDBM组，组间差异均有统计学意义（P<0.05）。线性回归分析示，y^=0.361+0.017x，DBM中BMP-2浓度对细胞ALP含量的影响有统计学意义（t=3.552，P=0.005）；BMP-2浓度每增加1 ng/g，ALP含量将增加0.017 ［95%CI（0.006，0.027）］ U/100 mL。. 结论: 不同长骨中天然BMP-2浓度差异较大，下肢长骨高于上肢长骨，骨骼取材部位是影响DBM骨诱导能力的重要因素。.

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