Angiogenesis Of Tissue Engineered-Bone In The Repair Of Rhesus Tibia Defects

OBJECTIVE: The study and the clinical application of tissue engineered bonegraft have got more and more recognition, but there are some key problems whichneed solving. Many experiments indicate that the angiogenesis plays a key role in theosteogenesis.An experimental pattern was set up designed to prepare a kind ofvascularized engineered-bone graft for repairing rhesus tibia defects and analyze thecharacters of the angiogenesis and osteogenesis in vivo by rontgenographic andmorphological approaches.We have ever repaired the 20mm tibia defect of big animals (goats) withvascularized tissue engineered bone, and achieved good results. In order to supplymore detailed and practical approaches for the clinical experiments, we chooserhesuses as experimental animal this time, and put forward the approach of furtherangiogenesis after the anatomical observation, that is, allowing the coexistence andco-growth of both blood vessels and fascia by specially designing the scaffoldconformation can not only accelerate the angiogensis of the tissue engineered bone,but also can greatly improve the quality of the osteogenesis and shorten the healingperiod.This study aims at: detecting the compatibility of BMSCs andβ-TCPscaffold, setting up an animal repairing rhesus tibia defects with engineered bone anddetecting the angiogensis of the tissue engineered bone and the quality of theosteogenesis. METHODS:The first part: The third passage of rBMSCs were cultured withβ-TCP withthe cells cultured without the materials as the control. The morphology andproliferation of cells were observed by inverted phase contrast microscope andscanning electron microscope(SEM). MTT assay was used to semiquantitativelyevaluate the cell proliferation.The second part: A 20 mm tibia defect was made in each of both sides of the 9rhesuses and fixed with a plate. The 18 tibia defects were randomly divided into twogroups equally. The gaps in experiment group were plugged with the engineeredbones constructed in vitro by bone marrow-derived stroma cells(BMSCs) andβ-TCPscaffolds in each one with a slit and a central tunnel. In addition, a correspondingportion of saphenous artery and veins was moved to run through along the centraltunnel in the scaffold with the "gate" help of the slit, and the engineered bone wastotally hugged by a sheet of pedicled deep fascia. The gaps in control group, however,were inserted engineered bones only. The vascularization for each treatment wasassessed by physical, histopathological and X-ray examinations at time intervals of 4,8, 12 weeks after operation.The third part-The 36 bilateral tibia 20mm defects were randomly divided into4 groups equally. The gaps in experimental groups were plugged with engineeredcomposites which were totally hugged by a sheet of pedicled deep fascia andadditionally a corresponding portion of saphenous artery and veins were moved to runthrough along the central tunnel in the scaffold with the "gate" help of the slit. Thegaps in the three other control groups, however, were inserted with fascia-coatedcompounds, compounds only and pure scaffold only respectively. In addition, tworhesuses were picked up as blanks. The angiogenesis and osteogenesis for eachtreatment was assessed by macroscopical, histological and roentgenographic analyses at time intervals of 4, 8, 12 weeks postoperative.RESULTSThe first part: Rhesus BMSCs showed a active ability of proliferation and amountthe highest level at 6th day at third passage. Osteogenic differentiation were carriedout at the third day after induction. The scaffold material presented finebiocompatibility with rhesus BMSCs while coculturing together but the disintegrationof material might have certain influence after cells seeding onto the scaffold.The second part: Through the special design to engineered bone features andapplying double vacularization methods of saphenous bundle insertion and pedicleddeep fascia coated, the experimental animal modal was set up. The samplesharvested from the subjects were analyzed by gross observation, roentgenographicand histologic examination. The modal and its procedure were recognized a feasibleway to get engineered bone revascularizaton but the results needed further researchin order to supply theoretic evidences for final clinical trials.The third part:General results4 weeks postoperatively: There is no significant differences among 4 groupswhich all have some connective or chondroid tissue formation on two contactsurfaces, the back of the material and the side of the plate. The samples presented noabsorption and its contact surface could be easily broken.8weeks postoperatively: There are differences among 4 groups. In group A,besides what has mentioned before, other surfaces and the contact surfaces with thebone are all wrapped up with bonelike tissues which also fills the slit and the centraltunnel. The implanted blood vessels can clearly be seen, and the bone are not easy tobe broken, 1/3 materials has been absorbed; in group B, no bonelike tissues form onthe medial surface and the front of the implanted material, and the same with the central tunnel. A few bonelike tissues appear on the other surfaces and the contactsurfaces with the bone; in group C, few bonelike tissues form on the contact surfacesbetween the implanted material, which are easy to be broken and the material has notbeen significantly absorbed, in group D, the condition is relatively the same as that ingroup C.12 weeks postoperatively: There are significant differences among 4 groups. Ingroup A, all the surfaces of the implanted material and the central part are whollywrapped up or replaced by bonelike tissues which are hard and cannot be broken. And2/3 materials have been absorbed; in group B and C, partial materials of the medialsurface and the front have not been replaced by bonelike tissues yet, which can bebroken with force, and 1/3 material has been absorbed; in group D, rare boneliketissues were seen at the contact interfaces, and no material has been absorbed.Blanks: At 12 week, the defect has been completely filled with granulationtissues, without osteogenesis.Microscopic observationGroup A: At 4w postoperatively, a few new bonelike tissues, woven bone,form on the edges, the sections and the center of the implanted material, near which,new bone and small alveoli-like ink-stained structures also appear in the holes of thematerial. The implanted blood vessels lumens are stained with ink, and the material isfairly complete, the holes have not dilated. At 8w, new bone and ink-stained bloodvessel structures have significantly increased, most of which are close to the externalmargin and the center. Broken materials, like fine particles, can be seen at thejunctions between the new bone and the material, and the implanted blood vesselshave not been ink-stained. At 12w, the implanted material has been completely coatedwith the bonelike tissues which separate the material into several small parts withbone trabecula structure formation. The density of the blood vessels structures greatly increases in the new bone and the left material. Those in the new bone are quite thick,straight and have less branches, while those in the left material are mostly alveolialike, rosary like and have more branches, with holes connected with each other.Quite a lot broken materials, like small particles, are seen at the junctions between thenew bone and the material, where the amount of ink-stained blood vessels structuresis maximal.Group B: At 4w postoperatively, a few newborn bone tissues, woven bonetissues form on the edge and sects of the implanted material, and there are also newbone and small alveoli like structures within the holes of the material. The lumen ofthe implanted blood vessel is ink-stained, the material is fairly complete, and theholes have not dilated. At 8w, more new bones form, and much more ink-stainedblood vessels appear. Many of them are near to the external edge, and brokenmaterials are visible at the junctions between the new bone and the material. Thelumens of the implanted blood vessel are not ink-stained. At 12w, most of the materialis wrapped up by the new bone and bone trabeculae have formed. Few blood vesselscan be seen in the center of the material.Group C: At 4w, no new bone can be seen on the edge and the center of theimplanted material, a few woven bones have formed only on the sections, and smallalveoli like ink-stained structures can be seen only on the external edge of thematerial back. The material is fairly complete and the holes have not dilated. At 8w, afew new bones appear on the edge and the sections of the implanted material, andink-stained blood vessels are seldom seen except that quite a lot appear at thejunctions between the new bone and the material. At 12w, the implanted material hasbeen slightly absorbed. The new bone and the blood vessel have increased comparedwith those at 8w, but still very few.Group D: No newbone formed at 4 weeks postoperatively, but a few of "ink-staining" structure showed up at interfaces. The "ink-staining" structureincreased quantitatively and a little of bonelike tissue could be seen at interfaces at 8and 12 weeks.Analyses of angiogenic and osteogenie areaTake the central tunnel and its vicinity within 2mm as the center portion, and theother places as the surrounding portion.There were statistical significance for bothangiogenic and osteogenic areas comparing the vaso-areas of both central andperipheral parts in group A to those of group B, C and D(P＜0.01). No significancecould be seen between group B and C for comparing the angiogenic areas at centralparts (P＞0.05), while it is significant comparing the angiogenic areas at peripheralparts(P＜0.05).X-rays ObservationAt 8w, group A's images show that the density of the implanted materialdecreases, and continual bony callus forms at the interfaces. At 12w, group A's imagespresent obviously decreased density which is lower than that of the normal bone inindividual areas, and the continual bony callus is manifest. At 8w and 12w, group Band C's images show no decreased density and the continual bony callus appear onthe sections. Group D shows no changes at every check intervals.Roentgenographic scoresFrom the blocking density values at the bone defects, it can be seen that nodifferences exist among the 4 groups at 4w, and no differences exist between group Band C at all observing time points; At 8w and 12w, group A is different from group Band C, and there are differences between all the time intervals of group A(P＜0.01).The density gradually decreases with the time, which is probably due tothe absorption of the implanted material. ConclusionEffects on the rhesus' activities seemed to be rare. This study shows us a feasibleand effective angiogenesis approach which can accelerate osteogenesis in vivo ofengineered bone.