Study Of Repairing Skull Defects By Coral Hydroxyapatite Embeded With BMSCs

Objective Large skull defects are very common in neurosurgical practice, and the keypoint is the sources of repairing materials. Although there have been many kinds ofmaterials, all of them have their own disadvantages. Bone tissue engineering mayprovide another innovative and ideal choice. We studied the biological features ofbone mesenchymal stem cells (BMSCs) of rabbits, and observed the effect ofrepairing rabbit skull defects by 10% coral hydroxyapatite (CHA) embedded withBMSCs.1. Study of biological features and osteogenic differentiation of rat-derivedBMSCsMethods BMSCs were isolated from rat bone marrow by d plastic-adherencescreening, then cultured and expanded in L-DMEM culture without(A group) orwith(B group) osteogenic inductors (10^-9mol/L DEX, 10mmol/Lβ-SodiumGlycerophosphate and 50mg/L L-ASC). Changes of growth and morphous of BMSCswere observed through inverted microscope everyday, and the proliferation ofBMSCs was analyzed with MTT method. The symbols of osteogenic differentiationwere detected, including: the expression of ALP after 5,10,15,20 days withquantificational method, collagen-â… after 4 weeks with immuno-histochemistrymethod and mineralized nodes after 5 weeks with acheomycin sign.Results Fusiform-shaped BMSCs were obtained by density gradient centrifugationand plastic-adherence screening. BMSCs of both groups proliferated quickly after 3days and reached the peak after 8 days, but the cell number of B group was more thanthat 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 significantdifference between them after 15 days. Cells excreting collagen-â… , which was brownin 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 weremineralized 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 isolatedwith density gradient centrifugation and have great ability of proliferation andmultipotency. BMSCs could express ALP, excrete collagen-â… , and form mineralizednodes after being induced by 10^-9mol/L DEX,10mmol/Lβ-Sodium Glycerophosphateand 50mg/L L-ASC. BMSCs could be identified through observation of morphousand detecting symbols of osteogenic differentiation.2. Repairing rabbit skull defects by coral hydroxyapatite Embedded withMesenehymal Stem CellsMethods BMSCs (induced the way described above) were collected at fourth passage,and combined with 10%CHA in the density of 5×10⁷/ml. After cultured 1,3,or 7days, we detected them by electron microscope scanning. 10%CHA+BMSCsComplexes (cultured for 7 days) were auto grafted into the skull defects in thediameter of 2.5cm (A group, n=8); CHA alone was implanted (B group, n=4) andnothing was implanted (C group, n=4) respectively as controls. Six or twelve monthsafter operations, animals were sacrificed and new bone tissue in the defects wasinvestigated by X-ray examination, gross specimen inspection and histologicalobservation.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-boneafter 6 and 12 weeks; and materials with coarse surface, particle-shape andencasement of connective tissue in B group after 12 weeks; and only connectivetissue filled in C group after 12 weeks.Conclusions 10%CHA has good biological compatibility,cell-scaffold interface and proper rate of degradation; And BMSCs can adhere tightly and proliferatecommendably on the surface of 10%CHA. 10%CHA+BMSCs Complexes can formnew bone tissue in rabbit's skull defects without obvious immunological rejection.10%CHA+BMSCs Complexes, as a method of bone tissue engineering, may becomeone ideal kind of materials of repairing the skull defects in clinical practice.