Celluar Compatibilty And Capacity Of Repair Bone Defect Of The Nano-Hydroxyapatite/Collagen/Calcium Alginate In Rabbits

It is a problem in clinical to repair of bone defects caused by varieties factors. Traditionally,it is including autogenous bone graft and allograft bone graft,but both of them exist certain flawed.The source of autologous graft is limitied,and the shape of bone is not appropriate,what is more,it would increased the surgical trauma and risk of complications,so the use of it be restricted.Allograft bone graft has the faults of the inevitable rejection,and also have problems in individual differences, size,bone source limited,diseases spread,ect.With the development of the medical,especially the development of tissue engineering science.The tissue engineering bone play an important role in repairing bone defects.Traditional tissue engineering bone must prefabricated shape in vitro and implantated it in vivo by surgery,which often raise additional risks of complications and trauma.With the development of minimally invasive surgery, injectable plasticity of bone repair materials has got increasing attention.In this research,we composited the calcium alginate,nano-hydroxyapatite and collagen by a certain percentage to made a composite bone repair material,which is an injection,can be arbitrarily shaped and with good bone conduction as well as good bone-induced.Early experiments proved that it has good physical properties and can meet the requirements of the clinical.But it is not enough of bone repair material only able to meet required for clinical application of physical and chemical properties, which must has good biocompatibility and bone repair capacity.To ensure that its clinical safety and efficacy,in this study,we through the calcium alginate nano-hydroxyapatite collagen composite with rabbit bone marrow mesenchymal stem cells co-culture experiments and calcium alginate nano-hydroxyapatite collagen composite repair of radial bone defects of rabbits experiment,to know their biological compatibility and bone repair capacity.PartⅠ:The study of nano-hydroxyapatite collagen calcium alginate composite material with rabbit bone marrow stromal cells in vitro biocompatibilityObjecte:Test the compatibility of nano-hydroxyapatite collagen calcium alginate composite materials(nano-Hydroxyapatite/Collagen/Calcium Alginate,n-HACCA) and rabbit bone marrow stromal cells(BMSCs).Materials and methods:1.Materials 2-week-old healthy New Zealand white rabbits,DMEM / F12, D-Hank's,trypsin,fetal bovine serum,double-antibody,goat anti-mouse-Cy3,goat anti-mouse-FITC,CCK-8 Kit,CO2 incubator,fluorescence microscopy,flow cytometry,scanning electron microscopy,WellScan MK22-based microplate reader. Nano-hydroxyapatite collagen calcium alginate composite.2,Primary culture and subculture the bone marrow-derived mesenchymal cells which extracted from the bone marrow of rabbit,and then,identify them by general observation and flow cytometry.3,To observe how cells growth in the n-HACCA materials by the inverted microscope,scanning electron microscope and compare the growth of the group A (control group,pure cell culture) with the group B(experimental group,n-HACCA materials and cell co-culture) byCCK-8 kit.Results:1,The bone marrow mesenchymal stem cells(BMSCs) of rabbit Culture and identification.1).Most primary cultured BMSCs cells adherent in 48h.They morphology were more consistent short-fusiform,polygonal.In 5～7d,it can be seen clearly that them colony formation and proliferation fast,at about two weeks,cells fused each other,and looks like fibroblast-like,with positive spiral,fish-shaped.Adherent of subculture BMSCs are more soon(12h most cells had adherent),arranged fusiform, more uniform morphology,growth vigorous,about 90%can be fusion about 7d,when about 10d,they are growth gradually slow.2).BMSCs surface antigen In the vast majority of cells,bone marrow stromal cells characteristic signs of the CD29 and CD44 positive,well as a characteristic of hematopoietic stem cells marked by CD34 and CD45 negative. which suggested that the 5^th generation BMSCs isolated by the above passage ways have basically been purified.Concrete results:CD29-positive rate was 99.62%, CD44-positive rate was 94.28%;CD34-negative rate of 99.80%,CD45-negative rate of 99.57%.2.The results of rabbit bone marrow mesenchymal stem cells(BMSCs) which were cultured in nano-hydroxyapatite collagen calcium alginate composite (n-HACCAA)1).To observe the cells which co-culture with the n-HACCA/BMSCs composite material by the inverted microscope.Because of the material is opaque,so it is difficult to observe the cells which in the surface of the material under the inverted microscope.But it could be seen cells attached around n-HACCA material,and with the culture time extended,the cells attached to the material gradually extended and adherent.2).The observation of n-HACCA/BMSCs co-culture by SEM After compound cultivated 5d,it can be see cells attachment in both n-HACCA materials surface and porosity,them have extended into a fusiform or polygonal,extended pseudopodia and adherted on materials.Much microvilli was shows on cells surfaces, which suggesting that the cells have a good status.3).Detect the cells proliferation which on n-HACCA materials by CCK-8 assay From the growth curve drawn,which draw by OD value of the cell activity,it can be seen,after the cells inoculation,the cells activity gradual increase with time extent both in n-HACCA material and blank control group,the cells in n-HACCA material can still maintain normal proliferation rate,no significant difference(P＞0.05).can be find between the two groupsConclusion:Rabbit bone marrow stromal cells(BMSCs) can normally adhesion,proliferation, growth in the nano-hydroxyapatite calcium alginate collagen composite material,and their activity was not effected by nano-hydroxyapatite calcium alginat collagen composite materials.That is say,the nano-hydroxyapatite calcium alginat collagen composite materials have good biocompatibility. PartⅡ:The study of repair rabbit radial bone defects in rabbits by nano-hydroxyapatite collagen calcium alginate composite materialsObject:Discuss the osteogenic capacity of nano-hydroxyapatite collagen calcium alginate composite materials which implanted in animalsMaterials and methods:1,Materials 18 3-month-old healthy New Zealand white rabbits,which have prepared of middle radius 1.5 cm bone defect model.Nano-hydroxyapatite collagen calcium alginate composite materials.2,Methods Model rabbits were randomly divided into 2 groups with a random number table --experimental group and control group,there are 9 rabbits in each group.Nano-hydroxyapatite collagen calcium alginate composite materials was implanted in radial bone defects of experimental group rabbits while nothing was implanted in radial bone defects of the control group.Postoperative observation of animals in daily life situation.Three animals were sacrificed separately 4,8,12 weeks postoperatively in each group.Remove the specimens,detected them of defect sites into bone by the gross observation,X-ray observation and histological.Results:All the animals were entered the final analysis,non-shedding.1.After 1～2 d of surgery all the rabbits has bad spirit,eating less,less activity,no signs of fever chills,no rash.Surgical wound without swelling,exudate, etc.3 d after the surgery the spirit of all rabbits improvement,normal diet,activity increased.2.The general observation of bone defect site of specimens At different time after the surgery,the organizational level of operation area(around materials) is clear, no clear swelling,adhesion,and other signs of inflammation.4 weeks after operation, bone fragments was closely combinated with the material,no obvious cracks,Materials begins degradation,there is category osteoid tissue fill in materials, they has a certain intensity.After 8 weeks,boundaries between bone ends and materials were disappeared.Materials filled by bone-like tissue completely and have greater intensity.12 weeks after operation,materials have ossificated texture harded have no significant difference between normal bone.Blank control group,after 4 weeks of bone stump see periosteal reaction,8 weeks stump into fibrosis,no bone formation,12 weeks stump bone sclerosis.3.Observe bone surgical areas by X-ray.In the experimental group,at 4 weeks, bone formation can be seen clearly in radial bone defect area,they look like clouds, uniformly distributed in the bone defect area,its low density,substantial gaps exist;at 8 weeks,it showed that implants have been with the stump of bone defect healing, while cortical bone continuity was bad;12 weeks,the implants with the stump of bone defect healing,better continuity of cortical bone,medullary cavity potency. Blank control group at different time points have shown that bone defect area has not yet been repaired,leave large gaps,it was showed that bone sclerosis in the end in the 12 weeks.4.Histological observation of specimens We can see there have vary degrees of new bone formation in the rabbit radial defect at all stages of the experimental group.At 4 weeks,there have fibrous tissue regeneration in materials and part of them has been absorbed,there are newborn fibroblasts and vascular enveloped the material,but few of them are the same as the leukocyte inflammatory cells and the remaining materials continue to be absorbed by osteoclasts.After 8 weeks,there can be seen broad new active osteoblasts,woven bone structure,isolated as well as new bone neovascularization in the materials.At the surface of materials lamellar bone can be seen.which suggest that they have a very strong ability of bone remodeling. There can be seen much trabecular bone,bone matrix as well as the type of bone marrow cavity within materials.After 12 weeks,the woven bone matrix and neovascularization that in materials have disappeared,defect was filled with new bone,they have a lot of features like normal bone,such as trabecular bone,bone marrow cavity,there can be seen a lot of active osteoblasts at the surface of the endometrium of medullary cavityConclusion:The nano-hydroxyapatite collagen calcium alginate composite material has good osteogenic potential.Summary:1.It's able to get bone marrow-derived mesenchymal stem cells by the way of washing the rabbit bone marrow successfully.2.Nano-hydroxyapatite collagen Calcium alginate composite bone repair material has good biocompatibility and osteogenic capacity.