Vascular Pedicle Implantation Of Collagen-based Nanocrystalline Calcium Phosphate Bone Vascularization Animal Studies

Background&Objectives:The critical bone defect, which is caused by trauma, tumor, infection and tuberculosis, is difficult to repair and it is still a big challenge for the orthopedists. Bone grafting is necessary for the procedures. Autogenous bone grafts represents the gold standard and are frequently used clinically for osseus reconstruction. But bone harvesting is associated with considerable donor site morbidity, and the amount of autogenous bone for transplantation is limited. Allogenic and Xenogenic bone are alternatives which can overcome the limitation for amounts and volumes, but they are used with reservation because of the immune rejection and risk of disease transmission. The artificial bone materials have good biocompatibility, similar microscopic pore structure and pore ratio; they can be used to repair small bone defects with satisfactory effect. For the repair of massive bone defect, the key point is to obtain vascularization of grafting bones. Before artificial bone scaffolds are be grafted, they can be prevascularized with many kinds of methods in vitro or in vivo."Nano-calcium phosphates/collagen based bone substitute" is an artificial scaffold with nano structure identical to natural bone, which has been applied as bone grafting material in clinic practice and achieved satisfied outcomes for repair of small bone defects. In order to repair a large volume of bone defect with the scaffold in the future, we designed the research to study the property of axial vascularization through application of vascular pedicle implantation in animal models.Methods:1. Anatomical study was conducted in4New-Zealand white rabbits. The anatomy of the medial thigh and groin region was investigated with special regard to identification of an easily accessible vascular axis, which is suitable for surgical creation of a vascular pedicle. The femoral vessels were dissociated, then ligated and cut off distally. There was a vascular pedicle including femoral artery and vein with2-2.5cm length, and the pedicle could be used to axially vascularize the scaffolds.2.30New-Zealand white rabbits were randomly divided into3groups.10rabbits were included in blank material group (Group A):the vascular pedicle was made in left groin of animal, then the cylinder (diameter1.0cm, height1.0cm) of Nano-calcium phosphates/collagen based bone substitute with a predrilled hollow was embedded and the hollow was implanted with the pedicle; in the left groin, the same material was embedded in muscles without vascular pedicles as control.10rabbits were included in the bone marrow material group (Group B):in this group, before embedding and implantation with the pedicle, the material cylinder was loaded with osteogenic material from bone marrow.10rabbits were included in the VEGF material group (Group â…¤): before embedding and implantation, the material cylinder was composited with VEGF 165. At2week,6week and10week postoperatively,3animals of every groups were sacrificed, and the embedded material cylinders were taken out for histological observation. After decalcification, specimens were undergone HE stain and CD31immunohistochemistry luorescence labeling. Microvessel density (MVD) were assessed in order to evaluate angiogenesis in the materials. The MVDs of materials with and without the pedicle implantation in different time point in Group A were compared, and the MVDs of materials with the pedicle implantation in Group A, B, V were compared, too.Results:At2week postoperatively, the histological observation showed inflammatory response in HE stain and a small number of microvessel formation. At6week postoperatively, the inflammatory cells disappeared and formation of microvessel became obvious, especially in material with pedicle implantation.10weeks after surgery, the microvessel induced by vascular pedicle implantation became more remarkable. CD31immunohistochemistry luorescence labeling show gradually enhanced positive with time after surgery, which meant new formation of vascular endothelial cells.In Group A, the MVDs of material with pedicle implantation were significantly higher than those without pedicle implantation at2,6,8week after surgery (P<0.05), which indicated that the material was suitable to axial vaseularization with the vascular pedicles.The MVDs of materials with the pedicle implantation in Group A, B, V had no significant difference at2week postoperatively. At6week postoperatively, the MVDs of Group B were significant higher than tow other groups, and no difference between Group A and V. At10week postoperatively, there was no difference between Group B and V, but both of them higher significantly than Group A. It demonstrated that combination with bone marrow or VEGF improve the vaseularization of the material.Conclusions:The femoral vessels of New-Zealand white rabbits are suitable to fabricate vascular pedicles to induce axial vascularization of artificial bone scaffolds. The vascularization is obvious around artery pedicle. Nano-calcium phosphates/collagen based bone substitute is a kind of material with good potential to be vascularized, which makes it a candidate of artificial bone scaffold with large size. Based on pedicle implantation, combination with bone marrow material or VEGF can promote vascularization. In animal models, microvessel can observed6weeks after embedding, and in10weeks arteriole-like vessel can be found.