The Experimental Study Of Fabrication Of The Nano-ceramic Artificial Bone Combinated With VEGF

The Puprose of this research is,basing on clinical challenges,to fabricate a novel Artificial Compound Substiuttion Bone Biomaterial through the combination of the compound artificial bone transplantation theory and tissue engineering fabrication technique.The advnatages of the novel artificial bone are not only the cortical bone crude components of bioactive protein,organic and inorganic elements were deserved,but also the 3D structure and formation were controllable.The experimental data were provided by the study on physic-chemical,biological,histologic and immunohistologic technique,by the establishment of massive osseous defect animal model which was repaired by the biomaterial.Facing the clinical practice,the sutdy presented a foundation of overcoming the challenge of segmental bone defect treatment.【Objeetive】(1)To explore the biggest VEGF quantity can combinate with the nano-ceramic artificial bone,using the method of compositing the cell factor with the artificial bone material directly,and the rule of the VEGF releasing in vitro.(2)To find the capability of the nano-ceramic artificial bone composited with different density VEGF to promote the rabbit marrow matrix stem cells(MSCs)to adhere with the artificial bone material,proliferate and osteogeny,and the dependablity of the VEGF concentration.(3)To demonstrate the improved bone defect model in the upper portion of the rabbit radius is an ideal complete bone defect animal model by the animal expedment.(4)To study the effect of the VEGF /nano-ceramic composite artificial bone to treat the complete bone defect,and seek it's action mechanism and the characteristic.(5)To study the effects of nanometer ceramics artificial bone transplantation in the reconstruction of the bone defect and the effection of VEGF.【Methods】(1)Put the solution made of the incommensurability VEGF powder(400ng,350ng,300ng,250ng,200ng)to the nano-ceramic artificial bone strip by drops.then put the artificial bone in the thermostat for one night.Then soak it in the slow-release fluid of PBS for 3 times,in order to get rid of the VEGF not compound. The pickling fluid is collectted for every time,Then detect the quantity of VEGF with the ELISA device.Then calculate the VEGF quantity compounded in artificial bone. Immers the VEGF/nano-ceramic composite artificial bone strip in the PBS slow-release fluid with embryo cow blood serum,Then chang the fluid after 2 hours and every 24 hours later,and collect the liquid separately.Detect the VEGF quantity with the ELISA device.(2)Set them to 5 groups according to the quantity of VEGF.The passage bone marrow matrix stem cells were cocultured with them in vitro.Take the examination of the cell carriage rate,counting the cell quantity,alkalinity phosphatase activeness,the scanning electron microscope observation and the bone area computation in different time.(3)In the literature foundation,The complete bone defect model in the upper portion of the rabbit radius is choiced as the experimental animal model,and improves it's manufacture method.Take the autograft bone as the control group.Observate the wound heals,the body activity situation,the bone cicatrization situation,the ossein quantity,X-ray observation ossein density,quantity and so on;in different time.(4)Foundated at the forecited research,We choice the nano-ceramic artificial bone compound with VEGF300ng/cm³ for the experimental group(A group),taking the nano-ceramic artificial bone compound with BMP(B group)and the pure nano-ceramie artificial bone(C group)as the control groups,carries on the animal experiment in vivo.Examinate the VEGF and the alkalinity phosphatase content in the blood,the specimens come from different animals are taken the histology and immunohistologic examination of the VEGF acceptor and vessels with antibodies,for evaluating the quantity of the cells with the VEGF acceptor and the area of vessels per field of view.X-ray observation,the histology observation and the bone volume computation in the different time After operation.(5)From March of 2005 to November of 2007,43 cases of nanometer ceramics artificial bone transplantations were completed in patients with extremity bone defects which included 26 patients with traumatic bone defects andl7 patients with tumor induced bone defects,and be divided into two groups at randomly,and be treated with nano-ceramic artificial boneonly or nano-ceramic artificial bone and tablet of Jiegu Qili.Measuring B-ALP in the peripheral venous blood in the 1st and 2th weeks and 1st,3th,6th months after operation.【Results】(1)The quantity of VEGF bond to the artificial bone in the 400ng group and the 350ng group are close,but it increase along with the VEGF quantity added in below 350ng,and them are in present correlation.The complex can release VEGF slowly in the PBS solution approximately ten days,The VEGF quantity released from the artificial bone is big incipient,reduces rapidly afterwards,tends to steady gradually aider the 2nd day,The VEGF quantity released from the complex is dependent on the VEGF density compound to the artificial bone.(2)In vitro cell raise experiment,It was discovered that cell adherence rate of the four groups compound of VEGF obviously higher than group E's(P＜0.05),but it was not obvious difference between the four groups(P＞0.05),It was observated that the bone formation of the four groups compound of VEGF was earlier and more than the group E's with the scanning electron microscope at the 3rd,7th and 14th day.The bone quantity of group A＞group B＞group C＞group D at each time;Cell multiplicated count of group A＞group B＞group C＞group D＞group E(P＜0.05),group A,group B, group C and group D are higher obviously than group E(P＜0.01);As the same time, the quantity of alkalinity phosphatase of group A＞group B＞group C＞group D＞group E(P＜0.05),group A,group B,group C and groupD are obviously higher than group E(P＜0.01);The area of bone regeneration increased grudually in the 3rd,7th,14th day(P＜0.01),The area of bone regeneration of group A＞group B＞group C＞group D(P＜0.05),and the four groups' with VEGF was obviously more than group E without VEGF(P＜0.01).(3)The wound of animals all can heal smoothly, It discovered that only the fiber structure grown ino the defect of the blank experimental group in the 2nd week,the 4th week,8th week,12th week,The bone defect cannot heal,but the defect of the control allograft group was filled with regenerated bone in the 8th week,and the quality of new bone was similar.to the host bone in the 12th week;(4)The material volume of the group A and group B reduced obviously in the 4th week,but the group C's was not reduced obviously,The artificial material of the group A and group B degraded further in the 8^thweek,and it degrated nearly completely in the 12^thweek,The appearance of regenrated bone was similar with the periphery bone tissue,but the materials of group C still degrade partly;The quantity of alkalinity phosphatase in the blood serum of each group increased after the operation(P＜0.05).The comparison in 4th week:group A's＞group B's＞group C's; The comparison during from the 4^thto 8^thweek:group B's＞group A's＞group C's;The comparison in the 12^thweek:group C's＞group B's＞group A's;The density of VEGF in the blood serum increased obviously after operation(P＜0.05);The histology section discovered:The material degeneration and the osteogenesis of group A is quicker,more and earlier than group B and group C in the 8 weeks after operation; The X-ray inspection discovered:the material degrading of group A was slightly quicker than group B,obviously quicker than group C,The osteogenesis of group A was more than group B,and group B was more than group C,The difference between the group A and group B was not obvious during the 8^thweek to 12^thweek(P＞0.05), but was higher than group C;Lane-Sandhu histology grading elevated gradually (P＜0.05),in the different time,group A's＞group B's＞group C's(P＜0.05);,It was discovered that the matrial of group A has the blood vessel growthing into by the vessel immunohistologic dyeing,It is earlier obviously than group B and group C;It was discovered that the group A has massive blood vessel regenerated in the early time by the blood vessel counting,The vessel count of the group A elevated gradually in four weeks after the operation(P＜0.05),and descented gradually after the 8^th week(P＜0.05);The cell counting through the immunohistologic VEGF acceptor dyeing of the group A was more than group B and group C,and found massive dyeing cells in the pore of the artificial bone in the 2nd day,and many cytoblast,osteoblast and endothelial cell which dye in the periphery of the artificial bone,the dyeing cell counting of group A elevated in four weeks after operation(P＜0.05),then droped gradually(P＜0.05);group A's＞group B's＞group C's in 4 weeks(P＜0.01),group B's＞group A's＞group C's after the 8^thweek(P＜0.05).(5)Followed-up ranged from 9 to 24 months with an average of 15 months.The artificial bone has no immunogenieity,no rejection.But has the activity of osteoblasts.Porosis appeared at 1 to 3 months approved by radiograms.The average healing time of shaft was 18 months,and 9 months at osteoepiphyseal interface.The tablet of Jiegu Qili can decurtate the average healing time.【Conclusion】(1)Using the compound method introduced by this article,the maximum quantity of VEGF can bond to this nanometer ceramic artificial bone is 300ng/cm³,below this density,the VEGF quantity bond to the artificial bone increases along with the VEGF quantity put into,and them are positive correlation. The VEGF can release slowly from the compound nano-ceramic artificial bone in PBS slow-release solution for approximately ten days in vitro,the quantity is bigger at the begin,then reduces rapiddly,and tends to steady gradually after the 2nd day, The VEGF quantity released from the artificial bone is positive correlated to the VEGF quantity compound.(2)VEGF can promote the marrow matrix stem cell to adhere to this nano-ceramic artificial bone,can promote the marrow matrix stem cell to multiplicate obviously,and promote the bone formation through enhancing the ossify activity of the marrow matrix stem cell,this effect is positive correlation with the density of VEGF between 150ng/cm³ï½ž300ng/cm³;(3)This animal model manufacture method is simple,It can provide more stability of graft and more similarity with clinical bone defect.It can be considered reliably as a ideal complete osseous defect mode;(4)The implantation of this nano-eeramic artificial bone combinated with VEGF cannot provoke the VEGF density in the animal blood serum to upgrade obviously,VEGF can promote this nano-ceramic artificial bone early vaseularizing and bone formation obviously,Its bone repairing capacity is compared to the nano-ceramie artificial bone eombinated with BMP,and even slightly greater.It is an ideal bone repairing substitution for segmental bone defect.(5)It is affirmed that nanometer ceramics artificial bone is used to fill the smaller bone defect on clinical,the tablet of Jiegu Qili can elevate its effect of repairing for bone defect.