Experimental Artificial Lamina Of Vertebral Arch Construction With Tissue Engineered Bone Combined With Altogeneic Rabbit Umbilical Cord Mesenchymal Stem Cells

Objective:①In order to provide seed cells for the construction of tissue engineered bone, we isolated, purified and cultivate rabbit umbilical cord mesenchymal stem cells (rUCMSCs) in vitro via collagenase digestion combined with adhere culture. ②Identificate cells we obtained as rUCMSCs by multiple methods.③Evaluate and compare the biocompatibilityof nano-hydroxyapatie -chitosan (nHA-CS) and chitosan (CS) scaffolds between rUCMSCs. Evaluate the osteogenic capability of BMSCs after osteo-induction in vitro. And quantitatively evaluate osteogenic potential of rUCMSCs after bone induction by PCR ④ Comparatively analyze the ectopic bone formation between hydroxyapatite-chitosan and chitosan scaffolds in vivo. ④Investigate the effectiveness of the rUCMSCs-nHA-CS scaffold complex and rUCMSCs-CS scaffold complex for artificial lamina of vertebral arch construction.Methods:①The umbilical cords are collected from 21 days pregnant rabbits. Cut the umbilical cords into about 1 mm3 tissue blocks. Single morphological adhere cells were isolated via collagenase digestion combined with adhere culture.②Identification of rUCMSCs by morphology, surface markers by flow cytometry and PCR methods, three differentiation staining and evaluation three differentiation by quantitative PCR. ③The experiment was divided into three groups:nHA-CS group, CS group, and simple rUCMSCs group. Planting the P4 rUCMSCs into nHA-CS, CS and control wells. After co-culture for 24h, the cell adhesion rate of three groups was measured and compared by counting the number of seeding cells and unattached cells, and calculating in the formula:cell adhesion rate=(cell seeding number-unattached cell number)/cell seeding number. The proliferation of rUCMSCs around the scaffolds was observed by microscope. The cell proliferation in nHA-CS group, CS group, and simple rUCMSCs group was examined by CCK-8 assay, and then the proliferation curves were drawn. And the cell seeding efficacy was observed by PI staining and scanning electron microscopy. And evaluate the levels of Runx2 expression at 1,2,3, 4 weeks by quantitative PCR methods.④150 healthy male White Zealand rabbits which weighed from 2.0kg to 2.3kg, at an average of(2.13±0.11)kg were randomly divided into five groups:rUCMSCs combined nHA-CS scaffolds group (group Ⅰ), rUCMSCs combined CS scaffolds groups (group Ⅱ), single nHA-CS scaffolds group (group Ⅲ), single CS scaffolds group (group IV) and the control group (group Ⅴ). All animals underwent vertebral defects in L5. The size of defect is about 10mm(length) ×8mm(width). All cells-scaffolds composites or single scaffolds were implanted into the defect area respectively. At 2,4,8,12,16 weeks after surgery, surgical lumbar were scanned by CT and MRI respectively. Then the surgical lumbar were harvested for Micro-CT scan, HE staining, BMP-2 staining.osteocalcin histological examination, to evaluate the new bone formation construct the vertebral arch.Results: ①By collagenase digestion and adherent culture get a lot of relatively uniform in shape, fusiform and fibroblast-shaped, directional arrangement of adherent cells. A lot of adherent cells, obtained via collagenase digestion combined with adhere culture, exhibited a typical morphology as the fusiform and elongated fibroblast-like shape and directional arrangement.②the purified adherent P4 cells expressed CD105, CD73 and CD90, but were negative (or dim positive) for CD45, CD34 By flow cytometry and PCR methods. After Tri-lineage differentiation(osteogenic, adipogenic and chondrogenic), the cells were revealed by Alizarin red staining and oil red O staining, and the levels of Runx2, PPARy and Sox9 gene expression increased at differentiated cells, which indicating that the cells could differentiate into bone, fat and cartilage cells. Thus we can identified the cells as rUCMScs.③nHA-CS group, CS group, and no scaffold control group at 2h,4h,6h,8h,12h after co-culture respectively were:The cell adhesion rates of nHA-CS group, CS group and simple cell group after 24h co-culture were respectively:(89.48+1.54)%、(88.58± 2.12)%、(89.52±1.01)%. Statistical analysis showed that there was no significant statistical difference among the cell adhesion rates at the same time of the three groups.After 24-hour co-culture of cells and scaffolds, cells, observed around the nHA-CS scaffolds, were fusiform, translucent, and in good condition by microscope. In the three proliferation curves, the proliferation was slow in the first and second days, and the third-fifth days proliferation increased significantly, then in sixth-eighth days the proliferation slowed down, arriving at platforms. The overall trend of three curves seems the same, like "S" shape, and there were no statistically significant differences. After 48h co-culture numerous cells adhered to the hole wall of nHA-CS scaffolds and CS scaffolds by PI staining and the electron microscope scan. After osteogenic differentiation the levels of Runx2 expression increased from7 th days to 28th days, indicating that rUCMSCs were differentiated into osteoblasts.④New bone formation were observed in nHA-CS scaffolds with cells group, CS scaffolds with cells group, and nHA-CS scaffold group in 2 weeks postoperatively, while so did it for CS scaffolds group in 6 weeks postoperatively. The amount of newly formed bone gradually increased along with time, and the amount in the same time point was more in nHA-CS scaffolds with cells group than nHA-CS scaffolds group and in CS scaffolds with cells group than CS scaffolds group.The shadow of bone tissue were observed in nHA-CS combined cells group and simple nHA-CS group in 2 weeks, while so did it for CS combined cells group and simple nHA-CS group in 4 and 8 weeks postoperatively. The density of the bone tissue shadow, the trabecular number and thickness gradually increased along with time, and the trabecular interval became narrow. In 16 weeks nHA-CS combined cells group and CS combined both formed complete and smooth vertebral plate, the curvature of which coincided with the back surface of duramaters. HE staining showed that the osteoid tissue deposition in scaffold intervals was observed at two weeks, and gradually increased along with the time. The void of nHA-CS combined cells group and CS combined cells group were filled with ossification, and gradually increased along with the time. Single nHA-CS group and single CS group osteogenesis slower than the previous two. In 16 weeks nHA-CS combined cells group and CS combined both formed vertebral plate, while Single nHA-CS group and single CS group were just filled with ossification. Immunohistochemistry has shown that nHA-CS combined cells group expressed BMP-2 and osteocalcin.Conclusion:(DCollagenase digestion combined with adhere culture was an ideal method for rUCMSCs separation.②Through the result of cells morphology, cell surface markers, the abilities of multi-differentiation, we define the cells as rUCMSCs. ③Both the nHA-CS scaffolds and the CS scaffolds exhibited a good biocompatibility with BMSCs. There was no significant difference between two scaffolds in the influence to cell proliferation and adhesion. After osteogenic differentiation the levels of Runx2, osteoblast-specific transcription factor, significantly increased, indicating that rUCMSCs have osteogenic potential, and could gradually become more mature osteoblasts.④nHA-CS and CS combined with osteogenic differentiated rUCMSCs could construct the vertebral arch. Osteogenic differentiated rUCMSCs and hydroxyapatite materials have osteogenic properties.