仿生磷酸钙缓释涂层改性三维支架的制备及性能研究

Study on preparation and performance of biomimetic calcium phosphate sustained-release coating modified 3D scaffold

目的运用仿生磷酸钙涂层技术将淫羊藿苷(icariin,ICA)负载到去蛋白牛骨无机材料(deproteinized bovine bone,DBB)表面,检测其成骨诱导性能。方法取2周龄C57雄鼠的腹股沟脂肪,采用酶消化培养法分离培养得到脂肪干细胞(adipose derived mesenchymal stem cells,ASCs)。显微镜下观察细胞形态,并对ASCs进行成骨和成脂诱导,通过茜素红染色和油红O染色检测其多向分化潜能。在DBB表面制备磷酸钙(calcium phosphate,Ca P)涂层,将ASCs接种在材料上,通过扫描电镜观察材料表面形貌和细胞的附着情况。制备负载ICA的Ca P涂层,将材料分为三组:DBB组(未经处理的DBB),Ca P组(Ca P涂层改性的DBB),ICA-CaP组(Ca P涂层内含有ICA的DBB)。通过碱性磷酸酶(alkaline phosphatase,ALP)活性实验检测三组支架材料的成骨诱导性能。结果显微镜下观察培养细胞呈纤维细胞样梭形,呈簇或散在分布。成骨分化茜素红染色及成脂分化油红O染色结果都呈强阳性。扫描电镜观察结果显示DBB表面为不平坦的沟壑状形貌,Ca P涂层表面为相互交错的网格状结晶体,Ca P涂层促进ASCs在DBB表面的附着。ICA-CaP组的ALP活性显著高于DBB组和Ca P组(P <0. 05)。结论 ICA-CaP涂层改性的DBB具有成骨诱导性能。

Objective To explore the osteoinductive property of deproteinized bovine bone(DBB)after biomimetic calcium phosphate coating technology was used to load icariin(ICA)onto the surface of DBB.Methods Inguinal fat pads were dissected from 2-week-old male C57 mice,and adipose derived mesenchymal stem cells(ASCs)were isolated by collagenase digestion.Cell morphology was observed under light microscope,and the multi-potency property of ASCs was identified by osteoblasts and adipocytes induction and tested by Alizarin red staining and oil red O staining respectively.Calcium phosphate(CaP)coating was developed on DBB,then ASCs were seeded on scaffolds,and the surface morphology of scaffolds and the attachment property of cells were observed by scanning electron microscope.ICA-loaded CaP coating was developed on DBB,then scaffolds were divided into three groups:DBB group(native DBB granules),CaP group(CaP coating modified DBB),ICA-CaP group(ICA-loaded CaP coating modified DBB).The alkaline phosphatase(ALP)activity experiment was employed to detect the osteoinductive property of scaffolds.Results ASCs appeared as spindle-shaped cells in both scattered individuals or small colonies.The differentiation of ASCs into osteoblast and adipocyte-like cells were confirmed by positive Alizarin red staining and oil red O staining.Under the scanning electron microscope,the surface of DBB was parallel arrays of ridges and troughs,the biomimetic CaP coating on DBB displayed an interlacing network of crystalline surface,and CaP coating enhanced initial cell attachment.ALP activity in ICA-CaP group was significantly higher than that in DBB group and CaP group(P<0.05).Conclusion ICA-CaP coating-modified DBB possess the osteoinductive property.