可注射性分化微控单元和细胞单元体内构建大尺寸骨组织的研究

Fabrication of large-sized bone tissue in vivo by injectable differentiation-control modules and cell-culture modules

目的:探索一种操作简单、实用性强的体内构建大尺寸骨组织的方法。方法:采用大鼠双侧股骨及胫骨原代培养骨髓间充质干细胞(BMSCs),应用流式细胞仪鉴定BMSCs表面标志物、倒置显微镜观察细胞形态、Von Kossa矿化结节染色观察BMSCs成骨分化、油红O染色观察BMSCs的成脂分化情况。用CBD-BMP2(具有胶原结合区的BMP2)和微米级胶原丝结合构建分化微控单元,用大鼠骨髓间充质干细胞(BMSCs)和Cultispher-s胶原微球构建细胞单元。分别用注射器把分化微控单元-细胞单元混合组(实验组)、细胞单元组(对照组1)、分化微控单元组(对照组2)注入到11只SD雄性大鼠背部皮肤下,3组大鼠养至30 d后处死,取出皮下培养出的仿生组织。电镜及显微(Micro)CT扫描观察仿生组织表面和内部结构,对仿生组织进行HE染色、碱性磷酸酶(ALP)染色来观察组织内细胞分布及成骨成分的染色表达。采用5-溴-4-氯-3-吲哚-磷酸盐(BCIP)/四唑淡蓝(NBT)ALP显色试剂盒对仿生组织进行碱性磷酸酶活性检测;能谱分析测定仿生组织的钙离子含量;压缩模量检测仿生组织的生物力学强度。结果:P3代BMSCs表面阳性抗原CD44和CD90表达比例分别为95.11%、96.18%;造血表面标志CD11b和CD45的比例分别为1.18%、1.82%。BMSCs呈梭形、融合成片、旋涡状。BMSCs成骨诱导后可见大量的黑色矿化结节形成;BMSCs成脂诱导后可见大量的圆形红色的脂滴形成。实验组培养出大小约为1.3 cm×2.1 cm×0.6 cm、质地较硬的骨样组织,对照组1组培养出大小为1.0 cm×1.0 cm×0.4 cm、质地软的组织,对照组2未培养出成形组织。电镜及Micro-CT可见实验组仿生组织的外表为高信号的骨性结构,内部也充满大范围的高信号的骨性结构;对照组1仿生组织则未发现高信号结构。HE染色显示实验组形成大量骨组织,对照组1则未形成骨组织。实验组较对照组1产生更多的ALP,且实验组ALP活性高于对照组1[(0.023±0.005)nmol·mg ^(-1)·min ^(-1)比(0.005±0.002)nmol·mg ^(-1)·min ^(-1), P<0.05]。实验组钙离子含量为7.42%,对照组1为0.34%。实验组压缩模量高于对照组1[(2.62±1.41)MPa比(0.03±0.01)MPa, P<0.05]。 结论:利用细胞单元和分化调节单元在体内构建出大尺寸骨组织简单可行。细胞单元和分化微控单元具有可注射性,可以直接将其注入到骨折或者骨缺损处进行治疗,为组织工程走进临床试验提供了新思路。

Objective To explore a simple and practical method for fabricating large-sized bone tissue in vivo.Methods Bone marrow mesenchymal stem cells(BMSCs)were primarily cultured in bilateral femurs and tibias of rats.The surface markers of BMSCs were identified by flow cytometry.The Cell morphology was observed by inverted microscope.The osteogenic differentiation of BMSCs was observed by Von Kossa mineralized nodule staining,and the adipogenic differentiation of BMSCs was observed by oil red O staining.The differentiation-control modules were constructed by binding CBD-BMP2(bone morphogenetic protein-2 containing collagen-binding domains)to micron-scale collagen filaments,and the cell-culture modules were constructed using rat bone marrow mesenchymal stem cells(BMSCs)and Cultispher-s gelatin beads.Then,differentiation-control modules and cell-culture modules mixture(experimental group),cell-culture modules alone(control group 1),and differentiation-control modules alone(control group 2),contained in a syringe,were injected subcutaneously into the back of 11 male SD male rats,respectively.The rats in the three groups were sacrificed after being raised for 30 days.The biomimetic tissues cultured subcutaneously wereharvested.Electron microscope and micro-CT scanning were used to observe the surface and internal structure of the biomimetic tissues.HE staining and alkaline phosphatase staining of the biomimetic tissue were used to observe the distribution of cells and the stained expression of osteogenic componentsin the tissues.Alkaline phosphatase activity in the biomimetic tissues was examined using 5-bromo-4-chloro-3-indole-phosphate(BCIP)/Nitrobluetetrazolium(NBT)alkaline phosphatase chromogenic kit.Energy spectrum analysis was used to determine the level of calcium ions in the biomimetic tissues.Compressive modulus was used to measure the biomechanical strength of the biomimetic tissues.Results The expression rates of CD44 and CD90,the surface positive antigens of P3 generation BMSCs,were 95.11%and 96.18%,respectively.The proportions of hematopoietic surface markers CD11b and CD45 were 1.18%and 1.82%,respectively.BMSCs are spindle shaped,fusing into sheets and swirling.After osteogenesis induction of BMSCs,a large number of black mineralized nodules were formed.A large number of round red lipid droplets were formed after adipogenesis induction of BMSCs.The bone-like tissues cultured were approximately 1.3 cm×2.1 cm×0.6 cm in size with a fairly firm texture in the experimental group,and 1.0 cm×1.0 cm×0.4 cm in size with a soft texture in the control group 1.No formed tissues were cultured in the control group 2.Electron microscope and Micro-CT scanning showed that the biomimetic tissues in the experimental group presented a bony structure consisting of a surface with high signal intensity and an internalspace massively filled high signal intensity of bony components,but in the control group 1,no structure with high signal intensity was found in the biomimetic tissue.HE staining showed formation of bony tissues massively in the experimental group,but not the least in the control groups.The experimental group showed more production of alkaline phosphatase than did the control group 1,as well,the alkaline phosphatase activity was higher in the experimental group than that in the control group 1[(0.023±0.005)nmol·mg^(-1)·min^(-1) vs(0.005±0.002)nmol·mg^(-1)·min^(-1),P<0.05].The percentage content of calcium ion in the experimental group was 7.42%vs 0.34%in the control group 1.The compressive modulus was higher in the experimental group than that in the control group 1[(2.62±1.41)MPa vs(0.03±0.01)MPa,P<0.05].Conclusion Fabrication of large-sized bone tissue in vivo by differentiation-control modules and cell-culture modules can be simple and feasible.The injectability of cell-culture modules and differentiation-control modules allows infusion of these modules into bone fracture spaces or defects for therapeutic purpose,and therefore provides a new ideation for introducing tissue engineering into clinical trials.