改良酶消化联合植块法培养兔骨膜成骨细胞

背景:骨膜成骨细胞具有很强的增殖能力和分化成骨潜能,而且取材方便,但以往常规培养方法时间较长,如伺能够缩短培养时间,是骨膜源性成骨细胞成为理想种子细胞的关键之一.目的:应用改良酶消化法培养兔骨膜原代成骨细胞,观察其在喷砂钛片上的黏附及增殖情况.方法:切取雄性日本大耳白兔胫骨近端前内侧面骨膜0.5～1.0 cm~2:①常规方法:以0.25%胰蛋白酶在37℃消化30 min后,用0.1%Ⅰ型胶原酶在37℃消化30 min,不断振荡,弃掉胰蛋白酶后植入培养瓶,干涸培养2 h,加入含体积分数15%胎牛血清的DMEM完全培养基培养.②改良方法:将Ⅰ型胶原酶消化时间由30 min改为1 h.碱性磷酸酶染色、钙结节染色鉴定成骨细胞.将原代培养成骨细胞与喷砂钛片联合培养,采用扫描电镜、MTT法检测不同时间点成骨细胞在喷砂处理钛片上的黏附及增殖情况.结果与结论:培养第5天,改良法培养的骨膜成骨细胞从组织块周围爬出,第25天细胞汇聚成单层,细胞呈三角形或多角形;传代培养1个月后,可见细胞成复层生长,并有黑色钙结节生成,具有典型的成骨细胞形态特征,碱性磷酸酶染色、钙结节染色呈阳性.常规法培养的细胞爬满单层时间推迟12 d左右.在喷砂处理的钛片表面上成骨细胞立体感强,有伪足伸出,伪足嵌入小的孔洞内,细胞表面有基质分泌.两种方法培养的成骨细胞在钛片表面的黏附及增殖差异无显著性意义.提示改良消化法可明显缩短成骨细胞培养时间,对成骨细胞的黏附及增殖能力无影响.

Culture of rabbit periosteal osteoblasts using modified enzymatic digestion combined with explant method

BACKGROUND:Periosteal osteoblasts possess strong reproductive activity,as well as osteoblastic differentiation potential,which is an ideal seed cell if can shorten the culture time.OBJECTIVE:Modified enzymatic digestion was used to culture rabbits'osteoblasts.and to study the adherence and proliferation of osteoblasts on the sudace of sandblasting titanium.MEITHODS:Periostea were harvested from the theanteromedial surface of the proximal tibia of male,Japanese white rabbits,and cultured as follow:①Routine method:Digested with 0.25%trypsinase at 37 ℃ for 30 minutes,followed by digestion with 0.1%type I collagenase at 37 ℃ for 30 minutes,vibration.removed trypsinase and dried.After 2 hours,DMEM containing 15% fetal bovine serums were added.②Modified method:30 minutes culture of type I collagenase was prolonged to 1 hour.The osteoblasts were identified by alkaline phosphatase staining and calcium node staining.The adherence and proliferation of osteoblasts cultured on sandblasting surface were measured by scanning electron microscopy and MTT.RESULTS AND CONCLUSION:Five days after culture.the periosteal steoblasts crawled out from tissues,gathered as monolayer with tdangle or polygon at after 25 days of modified culture.After 1 month of culture,superposition growth of calcium nodus appeared.The cultured cells possessed the morphological characteristic and biological behavior of osteoblasts.which were positive to alkaline phosphorase and calcium node staining.The time of cells cultured with routine method covered flask delayed 12 days than modified method.The osteoblasts were inseted into sandblasting titatium with pseudopodium.However,the adherence and proliferation of osteoblasts cultured on sandblasting surface had no obviously difference between two culture methods.The results suggested that modified enzymatic digestion can sho～en the culture time without effect on adherence and proliferation of osteoblasts.