Comparative Analysis Of Ectopic Bone Formation Between Hydroxyapatite-chitosan And Chitosan Scaffolds

Objective:①In order to provide seed cells for the construction of tissue engineered bone, we isolated, purified and cultivate bone marrow mesenchymal stem cells (BMSCs) in vitro via Percoll density gradient centrifugation combined with adhere culture.②To comparatively evaluate the biocompatibility with BMSCs in vitro between nano-hydroxyapatie-chitosan (nHA-CS) and chitosan (CS) scaffolds.③To evaluate the osteogenic capability of BMSCs after osteo-induction in vitro.④To comparatively analyze the ectopic bone formation between hydroxyapatite-chitosan and chitosan scaffolds in vivo.Methods:①8-week-old SD rats were sacrificed. Bone marrow was obtained from rat aseptically separated femur by flushing marrow cavity. Single morphological BMSCs were isolated via percoll density gradient centrifugation combined with adhere culture.②The experiment was divided into three groups:nHA-CS group, CS group, and no scaffold control group. Cell adhesion at2h,4h,6h,8h,12h after co-culture, was determined by counting the number of seeding cells and unattached cells, and calculated in the formula:cell adhesion rate=(cell seeding number unattached cell number)/cell seeding number. The cell proliferation in the scaffolds was examined by CCK-8assay, and then the proliferation curves were drawn. BMSCs proliferation around the scaffolds was observed by microscope. And the cell seeding efficacy was observed by laser scanning confocal microscope.③After three days osteogenic induction, BMSCs were stained by calcium-cobalt staining to detect the intracellular ALP expression, and were stained by Von Kossa staining and Alizarin red staining to detect the extracellular calcium deposition after twenty-first-day induction.④The experiment applied twenty-five two-month-old SD healthy male rats of SPF level, which weighed from186.4g to217.5g, at an average of (202.9±7.2)g. The rats were randomly divided into five groups:I group:nHA-CS scaffolds with cells; II group:CS scaffolds with cells; III group:nHA-CS scaffold; IV group:CS scaffold; V group:control group. All scaffolds/cells composites or scaffolds were implanted intramuscularly. In order to investigate de novo bone formation, CT scanning of rat femurs, HE stain and Masson trichrome staining of species were implemented at2,4,6,8, and12weeks after implantation.Results:①BMSCs obtained via Percoll density gradient centrifugation combined with adhere culture exhibited a typical morphology as the fusiform and elongated fibroblast-like shape and directional arrangement.②The cell adhesion rates of nHA-CS group, CS group, and no scaffold control group at2h,4h,6h,8h,12h after co-culture respectively were:(80.23±4.73)%,(84.48±5.17),(86.77±3.10)%,(86.77±3.10)%,(88.02±2.07)%;(78.49±5.25)%,(83.12±5.45)%,(83.12±5.45)%,(87.04±3.95)%,(87.04±3.95)%;(84.42±4.27)%,(87.04±3.20)%,(89.38±3.02)%,(90.69±2.48)%,(90.69±2.48)%. Statistical analysis indicated that there was no significant difference among the cell adhesion rates at the same time of the three groups. During the first four days after cells were seeded onto scaffolds, BMSCs proliferated at the normal speed, while in the fifth day the rate began to decline, and from the sixth day on the rate significantly slowed down and the curves formed three proliferating platforms, which were of the same trend and "S" shaped. After24-hour co-culture of cells and scaffolds, cells, which were fusiform, translucent, and in good condition, could be observed around the scaffolds by microscope.both scaffolds could be seen that numerous labeled BMSCs adhere to the pore surfaces by confocal laser scanning microscopy scan.③After3-day osteogenic induction, calcium-cobalt staining showed a large number of black granular deposition in the cell cytoplasm, indicating the intracellular ALP expression, and after21-day induction, Von Kossa staining and alizarin red staining respectively showed the extracellular matrix brown and red calcium nodules.④Postoperative CT scan showed implants were located close to the femur in the thigh muscle tissue. New bone formation were observed in nHA-CS scaffolds with cells group, CS scaffolds with cells group, and nHA-CS scaffold group in2weeks postoperatively, while so did it for CS scaffolds group in6weeks 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 osteoid tissue deposition in scaffold pores was observed in HE staining at two weeks, and gradually increased along with the time, and subsequently cartilage-like tissue formation and ossification were observed either. Statistical analysis of the proportion of de novo bone area indicated that the proportions gradually increased along with time in each group (P<0.05), while in the same time point there were significant differences among four groups:the proportion in nHA-CS scaffolds with cells group was the largest, followed by CS scaffolds with cells group, nHA-CS scaffolds group and CS scaffolds group respectively (P<0.05). Statistical analysis of the proportion of remaining scaffold area indicated that the proportions gradually decreased along with time in each group (P<0.05), while in the same time point there were significant differences among four groups:the proportion in CS scaffolds group was the largest, followed by nHA-CS scaffolds group, CS scaffolds with cells group and nHA-CS scaffolds with cells group respectively (P<0.05). Masson staining showed the collagen first formed among osteoid depositions, and then gradually increased, interleaved together to form reticular and lamellar structures, and ultimately constituted the extracellular matrix of osteoblast. Statistical analysis of the proportion of collagen area indicated that the proportions gradually increased along with time in each group (P<0.05), while in the same time point there were significant differences among four groups:the proportion in nHA-CS scaffolds with cells group was the largest, followed by CS scaffolds with cells group, nHA-CS scaffolds group and CS scaffolds group respectively (P<0.05).Conclusion:①Percoll density gradient centrifugation combined with adhere culture was an ideal method for BMSCs separation.②Both 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.③Basic culture medium supplemented10mM β-glycerophosphate,10-8M dexamethasone and50mg/L L-ascorbic acid was a successful osteogenic inducing medium. BMSCs induced in osteogenic medium exhibited excellent osteogenic performance.④Both the nHA-CS scaffolds and the CS scaffolds exhibited excellent ability of ectopic bone formation in vivo, while the nHA-CS scaffolds obtained more de novo bone than CS scaffolds, which demonstrated that nHA-CS scaffolds were compromising materials for bone tissue engineering.