犬上颌窦底黏膜骨原细胞的培养和成骨性能的研究

Osteogenic Property of Osteoprogenitor Cells from Canine Maxillary Sinus Floor Mucosa

【目的】研究犬上颌窦底黏膜骨原细胞的成骨性能。【方法】取Beagle犬上颌窦底黏膜,显微镜下观察其组织学形态。分离培养犬上颌窦底黏膜骨原细胞。流式细胞术检测一代细胞的表面抗原CD44、CD146和CD34。一代细胞经成骨诱导培养用于研究其体外成骨性能。诱导3 d、7 d和12 d时分别检测其碱性磷酸酶活性,对照组细胞用基础培养基培养。诱导16 d时,进行BMP-2免疫组化染色,对照组细胞经基础培养基培养。Western blot检测诱导0 d、7 d和16 d时BMP-2的表达。诱导28 d时,茜素红染色和Von Kossa染色观察钙结节形成情况。二代细胞成骨诱导培养7 d,与Bio-Oss复合后,植入裸鼠背部皮下,观察体内成骨情况,对照组植入Bio-Oss颗粒。【结果】犬上颌窦底黏膜由上皮层、固有层、黏膜下层和骨膜层组成,黏膜下层富含毛细血管。流式细胞术检测显示CD44和CD146阳性,CD34阴性。在3 d、7 d和12 d时,培养细胞碱性磷酸酶活性逐渐升高,成骨诱导培养细胞高于基础培养细胞。16 d时,与基础培养基中细胞相比,经成骨诱导的细胞BMP-2表达增强。Western blot检测发现诱导0 d、7 d和16 d时BMP-2的表达明显,且逐渐升高。诱导培养28 d时,可矿化形成明显的钙结节。细胞与Bio-Oss复合物植入裸鼠皮下可观察到成骨现象,对照组没有成骨表现。【结论】与人上颌窦底黏膜相同,犬上颌窦底黏膜含有骨原细胞,具有成骨能力。

[Objective]To investigate the osteogenesis property of osteoprogenitor cells in canine maxillary sinus floor mucosa.[Methods] Beagle maxillary sinus mucosa was obtained and the histological morphology was observed under microscope.Osteoprogenitor cells were harvested from canine maxillary sinus floor mucosa,then cultured.Cellular surface antigens of CD44,CD146,and CD34 at passage one were determined by flow cytometry.Cells at passage one were cultured in osteogenic inductive medium to investigate the osteogenesis property in vitro.Alkaline phosphatase activity was tested at day 3,7,and 12 after induction,respectively.Cells in the control group were cultured in basal medium.Immunohistochemistry stain of BMP-2 was performed at day 16 after induction,and cells in the control group were cultured in basal medium.The protein expression level of BMP-2 at 0 d,7 d,and 16 d after induction were tested by Western blot analysis.When induction for 28 d,alizarin red staining and Von Kossa staining were used to observe the formation of mineralized nodules.After being cultured in osteogenic inductive medium for 7 days,cells of passage two were composited with Bio-Oss,implanted into the back of nude mouse subcutaneously to observe the bone formation ability in vivo.And Bio-Oss particles were implanted in control group.[Results] The canine maxillary sinus floor mucosa was composed of epithelial layer,lamina propria,submucosa and periosteum.The submucosa was rich of capillaries.Flow cytometry detection showed CD44 and CD146 were positive,CD34 was negative.At day 3,7,and 12,alkaline phosphatase activity increased gradually,and that of cells cultured in osteogenic inductive medium was higher than in basal medium.Compared with in basal medium,BMP-2 protein expression enhanced when cells were cultured in osteogenic inductive medium.The protein expression level of BMP-2,which determined by Western blot,was evident and increased step by step at 0 d,7 d,and 16 d after induction.Obvious mineralized nodules formed when cultured in inductive medium for 28 d.Bone formation behavior was observed after implantion of cells and BioOss composite in nude mouse subcutaneously,no bone formation in control group.[Conclusion] Just the same as that of humans,canine maxillary sinus floor mucosa contains osteoprogenitor cells,which possess the ability of osteogenesis.