Studies Of Repairing Rabbit Skull Defects By Coral Hydroxyapatite Embedded With Mesenchymal Stem Cells

Objective Large skull defects are very common in neurosurgical practice, and the key point is the sources of repairing materials. Although there have been many kinds of materials, all of them have their own disadvantages. Bone tissue engineering may provide another innovative and ideal choice. We studied the biological features of bone mesenchymal stem cells (BMSCs) of rabbits, and observed the effect of repairing rabbit skull defects by 10% coral hydroxyapatite (CHA) embedded with BMSCs. 1. Study of biological features and osteogenic differentiation of rabbit-derivedBMSCsMethods BMSCs were isolated from rabbit bone marrow by density gradient centrifugation with lymphocyte separation media (1.073g/ml) and plastic-adherence screening, then cultured and expanded in L-DMEM culture without(A group) or with(B group) osteogenic inductors (10^-9mol/L DEX, 10mmol/L β-Sodium Glycerophosphate and 50mg/L L-ASC). Changes of growth and morphous of BMSCs were observed through inverted microscope everyday, and the proliferation of BMSCs was analyzed with MTT method. The symbols of osteogenic differentiation were detected, including: the expression of ALP after 1 , 2, 3, 4 weeks with quantificational method, collagen-I after 4 weeks with immuno-histochemistry method and mineralized nodes after 5 weeks with acheomycin sign. Results Fusiform-shaped BMSCs were obtained by density gradient centrifugation and plastic-adherence screening. BMSCs of both groups proliferated quickly after 3 days and reached the peak after 8 days, but the cell number of B group was more than that of A and there was significant difference between them after 3-8 days. Theamount of ALP expressions in B group was more than A and there was significant difference between them after 2-4 weeks, and the amounts in both groups increased obviously after 4 weeks. Cells excreting collagen-I, which was brown in cytoplasm after being manipulated with immuno-histochemistry methods could be seen in B group after 4 weeks, and not in B group. After 5 weeks there were mineralized nodes (stained golden by acheomycin) in B group, and not in A.Conclusions Bone marrow is one of good sources of BMSCs, which can be isolated with density gradient centrifugation and have great ability of proliferation and multipotency. BMSCs could express ALP, excrete collagen-I, and form mineralized nodes after being induced by 10⁹mol/L DEX - lOmmol/L P-Sodium Glycerophosphate and 50mg/L L-ASC. BMSCs could be identified through observation of morphous and detecting symbols of osteogenic differentiation. 2. Repairing rabbit skull defects by coral hydroxyapatite Embedded withMesenchymal Stem CellsMethods BMSCs (induced the way described above) were collected at fourth passage, and combined with 10%CHAin the density of 5><10"7/ml. After cultured 1 > 3^ or 7 days, we detected them by electron microscope scanning. 10%CHA+BMSCs Complexes (cultured for 7 days) were auto grafted into the skull defects in the diameter of 1.5cm (A group, n=8);CHA alone was implanted (B group, n=4) and nothing was implanted (C group, n=4) respectively as controls. Six or twelve months after operations, animals were sacrificed and new bone tissue in the defects was investigated by X-ray examination -. gross specimen inspection and histological observation.Results Electron microscope scanning showed that BMSCs could grow well on 10% CHAs after seeded for 3-7 days. Investigations showed that there was new boneregeneration in A group which had hard texture and close integration with auto-bone after 6 and 12 weeks;and materials with coarse surface^ particle-shape and encasement of connective tissue in B group after 12 weeks;and only connective tissue filled in C group after 12 weeks.Conclusions 10%CHAhas good biological compatibility^ cell-scaffold interface and proper rate of degradation;And BMSCs can adhere tightly and proliferate commendably on the surface of 10%CHA. 10%CHA+BMSCs Complexes can form new bone tissue in rabbit's skull defects without obvious immunological rejection. 10%CHA+BMSCs Complexes, as a method of bone tissue engineering, may become one ideal kind of materials of repairing the skull defects in clinical practice.