骨髓间充质干细胞膜片复合3D打印马鹿角粉/丝素蛋白/聚乙烯醇支架的体内成骨

Osteogenesis using bone marrow mesenchymal stem cell sheets combined with three-dimensional printed Cervus elaphus antler powder/silk fibroin/polyvinyl alcohol scaffold in vivo

背景:马鹿角粉是一种生物相容性及机械性能良好的组织工程骨材料。采用细胞膜片技术、3D打印技术构建组织工程骨,可实现极限骨缺损的个性化治疗。目的:探讨骨髓间充质干细胞膜片复合3D打印马鹿角粉/丝素蛋白/聚乙烯醇骨支架修复羊下颌骨极限缺损的能力及成骨效果。方法:全骨髓法培养羊骨髓间充质干细胞,采用细胞膜片技术及3D打印技术构建骨髓间充质干细胞膜片复合3D打印马鹿角粉/丝素蛋白/聚乙烯醇支架的组织工程骨。取12只新疆阿勒泰大尾羊,随机分为术后1,2,3个月组,每组4只,于双侧下颌骨制备20 mm×3 mm×5 mm骨缺损,每组2只羊一侧植入细胞膜片复合3D打印鹿角粉/丝素蛋白/聚乙烯醇支架,另一侧植入细胞膜片复合明胶海绵,每组另外2只羊,一侧植入细胞膜片复合纳米羟基磷灰石/丝素蛋白/聚乙烯醇支架,另一侧植入细胞膜片复合明胶海绵。植入1,2,3个月末处死实验动物,取下颌骨标本进行大体观察、锥形束CT、组织学观察及RT-PCR检测相关成骨指标。结果与结论:①锥形束CT观察:第1,2个月末,细胞膜片复合马鹿角粉/丝素蛋白/聚乙烯醇组骨缺损区呈稀薄云雾状,支架吸收较多,细胞膜片复合纳米羟基磷灰石/丝素蛋白/聚乙烯醇组吸收相对较少;第3个月末,细胞膜片复合马鹿角粉/丝素蛋白/聚乙烯醇组有大量的新骨形成,密度接近周围骨质,但细胞膜片复合纳米羟基磷灰石/丝素蛋白/聚乙烯醇组骨缺损区未长满,骨密度较低。细胞膜片复合明胶海绵组3个月内变化均不明显,均没有新骨生成;②组织学观察:第3个月末,与其他两组相比,细胞膜片复合马鹿角粉/丝素蛋白/聚乙烯醇组支架材料吸收较多,可见骨小梁排列规则及成熟板状骨,细胞膜片复合纳米羟基磷灰石/丝素蛋白/聚乙烯醇组也有少量新骨形成,细胞膜片复合明胶海绵组3个月内变化均不明显;③RT-PCR检测:细胞膜片复合马鹿角粉/丝素蛋白/聚乙烯醇组骨桥蛋白、骨钙蛋白、Ⅰ型胶原的mRNA表达水平均高于其他两组,在第3个月时成骨基因的表达量达到最高;④结果表明,骨髓间充质干细胞膜片复合3D打印马鹿角粉/丝素蛋白/聚乙烯醇支架的组织工程骨能够修复羊下颌骨极限骨缺损,可满足羊颌骨实验性缺损的修复重建。

BACKGROUND:Cervus elaphus antler powder is a bone tissue engineering material with good biocompatibility and mechanical properties.The use of cell patch technology and three-dimensional(3D)printing technology to build tissue engineered bone can accomplish individualized treatment of limit bone defects.OBJECTIVE:To explore the ability and osteogenesis effect of bone marrow mesenchymal stem cell sheet combined with 3D printed Cervus elaphus antler powder/silk fibroin/polyvinyl alcohol bone scaffold to repair the limit defect of sheep mandible.METHODS:The whole bone marrow method was used to cultivate sheep bone marrow mesenchymal stem cells,and the cell sheets technology and 3D printing technology were used to construct the bone marrow mesenchymal stem cell sheets combined with 3D printed Cervus elaphus antler powder/silk fibroin/polyvinyl alcohol scaffold tissue engineered bone.Totally 12 Xinjiang Altay big-tail sheep were randomly divided into 1-,2-,and 3-month groups(n=4 per group).An experimental bone defect area of 20 mm×3 mm×5 mm was prepared in the bilateral mandible edentulous area.Two sheep in each group were implanted with cell membrane combined with 3D printed Cervus elaphus antler powder/silk fibroin/polyvinyl alcohol scaffold.Cell sheets combined with gelatin sponge was implanted on the other side.The other two sheep was composited with nano-hydroxyapatite/silk fibroin/polyvinyl alcohol scaffold,and the other side was implanted with cell sheets combined with gelatin sponge.The experimental animals were sacrificed at the end of 1,2,and 3 months after implantation,and the mandible specimens were taken for gross observation,cone beam CT,histological observation,and RT-PCR to detect related osteogenic indicators.RESULTS AND CONCLUSION:(1)Cone beam CT:At the end of the 1^(st) and 2^(nd) months,the bone defect area in the cell sheets compounded with Cervus elaphus antler powder/silk fibroin/polyvinyl alcohol group was thin and cloudy;the scaffold absorbed more.The absorption of the cell sheets compounded with nanohydroxyapatite/silk fibroin/polyvinyl alcohol group was relatively low.At the end of the 3^(rd) month,a large amount of new bone was formed in the cell sheets composite Cervus elaphus antler powder/silk fibroin/polyvinyl alcohol group,and the density was close to the surrounding bone;however,the bone defect area in the composite nano-hydroxyapatite/silk fibroin/polyvinyl alcohol group was not overgrown and the bone density was lower.The cell sheets composite gelatin sponge group did not change significantly within 3 months,and there was no new bone formation.(2)Histological observation:At the end of the 3rd month,compared with the other two groups,the scaffold material of the cell sheets composite Cervus elaphus antler powder/silk fibroin/polyvinyl alcohol group was more absorbed,showing the regular arrangement of bone trabeculae and mature plate-like bone.There was also a small amount of new bone formation in the composite nano-hydroxyapatite/silk fibroin/polyvinyl alcohol group,and the cell sheets composite gelatin sponge group did not change significantly within 3 months.(3)RT-PCR:The mRNA expression levels of osteopontin,osteocalcin and type Ⅰ collagen were higher than those of the other two groups in the cell sheets composite Cervus elaphus antler powder/silk fibroin/polyvinyl alcohol group,and the expression of osteogenic genes reached the highest level at the third month.(4)The results show that the bone marrow mesenchymal stem cell sheets combined with 3D printed Cervus elaphus antler powder/silk fibroin/polyvinyl alcohol scaffold tissue engineered bone can repair the limit bone defect of the sheep mandible,and can meet the repair and reconstruction of the experimental sheep jaw defect.