Experimental Study On Repair Of Big Segmental Long Bone Defect In Goat Tibia With Tissue Engineered Bone Constructed By Improved Establishment Xenogenic Deproteinizated Bone

Background: The repairing of big segmental long bone defect has always been one of many difficult problems in orthopedics department, the general way is bone graft. Now the implantation materials include mainly autogenous bone, homogeneous bone, xenogenic bone and artificial bone. Autogenous bone graft has limited sources, complication, twice operation. Homogeneous bone has ethics, spreading infectious diseases, immunological rejection and limited sources also. The largest graft obstacle of xenogenic bone is immunological rejection except ethics and spreading infectious diseases. And artificial bone has only been as substitute materials in clinically, because it is hard to degradated or degradation velocity is inconsistency with bone grow, and to influence osteogenesis or function. Now the rise of tissue engineering technology brings new hope for repairing big segmental bone defect. It is not far of dream to rebuild function of bone to constructing organic tissue with compound culture of seeds cells amplification combined with degradable biomaterial in vitro to repair bone defects. Currently the study on bone tissue engineering of autologous cell sources in animal experiments have achieved impressive results, and preliminary clinical trial have achieved some success. But scaffold material is still the key to restrict extensive clinical application of tissue engineering bone. Currently the study on the scaffold about preparation rich source of low price, good performance is one of the hot researches in orthopedics. Recently although xenogenic bone has been reported application in bone tissue engineering as scaffold such as Keil bone, Bio-Oss bone. But the immunogenicity and biomechanics of xenogenic bone has not been solved completely. So this study was to prepare xenogenic deproteinization bone by pig bone, improve the technology of preparation, retain collagen protein as far as possible, remove noncollagen protein, peptid, lipoid and cells and lower antigenicity as well as retaining mechanics intensity. Its physico-chemical properties, Biological safety, cells compatibility and small animal osteogenesis were detected. The tissue engineringed bone composites were constructed by the combination of xenogenic deproteinizated bone as tissue engineering scaffold compound goat autologous mesenchymal stem cells plus osteoinductive factor rhBMP2 to repaired 20% segmental bone defect of goat tibia, which may provide clinical experimental basis for xenogenic deproteinizated bone as tissue engineering scaffolds to clinical application.Objectives: to evaluate the ability of xenogenic deproteinizated bone prepared by modified method to repair of big segmental long bone defect of big animal, and to investigate the change of immunology, biomechanics, histology and imageology during repairing procedure, and provide experimental basis for clinical application of xenogenic deproteinizated bone.Methods: 1. Improved establishment and physico-chemical properties study of scaffold material of xenogenic deproteinizated bone(pig). Refer to Improved method, to take them out of -80℃ultra cold freezer after 3 months, vacuum packaging, Co60 postirradiation and preservation in -20℃. Calculate every piece of xenogenic deproteinizated bone, average quality and size. Observe pore diamete and factor of porosity by scanning electron microscope. Detect amino acids category and content by amino acid analyzer. Detect scaffold material anti-compress, anti-bend mechnics index by almighty biodynamics instrument. And some piece of xenogenic deproteinizated bone were immersed 2 hours in rhBMP2 concentration 75ng/ml before it be grafted. 2. Culture, identification and bionomics of goat MSCs. To isolate, culture, and collect MSCs, observe bionomics. To direction location induce differentiation and identification. 3. Study on xenogenic deproteinizated bone scaffold material compatibility. To compound culture of xenogenic deproteinizated bone scaffold material and MSCs. Study on immunology and tracer in vivo of xenogenic deproteinizated bone. 4. Clinical observation on repair of big segmental long bone defect in goat tibia with tissue engineered bone constructed by xenogenic deproteinizated bone. 30 goats, to divide group blank, 6 goats and group test, 24 goats. Group test divide again 4 groups, named group A, B, C and D, 6 goats per group. to result in 20 percent left tibia middle and inferior diaphyseal defect of every goat and fixed with half-ring sulcated external fixator. According to group difference graft different materials, group blank, graft nothing. Group A, only simple xenogenic deproteinizated bone. Group B, xenogenic deproteinizated bone and autogenous MSCs. Group C, autogenous bone. Group D, xenogenic deproteinizated bone and autogenous MSCs and rhBMP2.To carry out imageology detect after surgery to 24 weeks every 4 weeks, and to detect vascularization, bone density and bone mine content, vitodynamics and histology.Results: 1. Improved establishment and nature study of scaffold material of xenogenic deproteinizated bone(pig). Gross appearance: Newly XDPB is white and yellow a little, shape in long strip, quality nature hard and crisp, and has a great quantity Honeycomb appearance ventage. Light microscope: bone block raritas porosity, ventage tegulation and few residue in it. Scanning electron microscope: this scaffold has primitive trabecula, rabecular spaces and bone lumens system, average pore (386.55±25.64)μm, factor of porosity (75.33±2.35) percent. Aanlysis of mino acid:category collagen AA content such as glycy, arginine, lysine ridge high and aromatic AA content such as tyrosine and cysteine ridge vanish. Vitodynamics detect anti-compress, anti-bend and anti-torsion load maximum load are nonsignificant except elastic modulus. 2. Culture, identification and bionomics of goat MSCs. Origin generation MSCs post inoculation adherence in 1～2 days, form colony in 5～6 days, and grow monolayer in 12～14 days. A bulk of cells is fusiform, morphous uniform, aniso-distribute. Passage cell fufil adherence in 2～4 hours, spread out full, and more morphous uniform, homogeneous distribution, array regular. Cell population increase obviously after 3 days, peak in 6 days and doubing time is 35.5 hours. 2. Identification of MSCs: bone induction cells show osteocyte feature, calcify nodus, ALP,collagenⅠ,osteocalcin dyeing masculine. Lipin induce culture show round lipid droplet, oil red dyeing masculine. 3. Study on xenogenic deproteinizated bone scaffold material compatibility. To compound culture of xenogenic deproteinizated bone scaffold material and MSCs. inverted microscope, scanning electron microscope manifest MSCs grow, differentiate and proliferation in scaffold material ventage. At the same time, cell proliferation peak value, cell population in group compound culture and group pure culture are nonsignificant. Study on immunology and tracer in vivo of xenogenic deproteinizated bone. Cell immunity and humoral immunity index in group autogenous bone and group xenogenic deproteinizated bone are nonsignificant. BrdU labeled autogeneic MSCs can be detected in xenogenic deproteinizated bone mesh after 4 weeks. 4. Clinical observation on repair of big segmental long bone defect in goat tibia with tissue engineered bone constructed by xenogenic deproteinizated bone. group blank has no bone formed in defect and extremities harden, cavitas medullaris closed. Test group: at the same time, X ray, vascularization, bone density and bone mine content, vitodynamics and histology, group C exceed group D exceed group B exceed group A. Group D compare with group C, group A and group B are nonsignificant, but group A, B and group C, D are significant.Conclusions: 1. This improved establishment xenogenic deproteinizated bone scaffold do not revoke obvious cell immunity and humoral immune reaction and possess favourable compatibility. 2. Through BrdU cell labeled technology show autogeneic MSCs grow, proliferation in xenogenic deproteinizated bone ventage. 3. This improved establishment xenogenic deproteinizated bone can as tissue engineered bone scaffold materials repair big segmental long bone big segmental defect. 4. This improved establishment xenogenic deproteinizated bone scaffold immunogenicity is lower, do not affect autogeneic MSCs ossify, and if it compound with rhBMP2, its ossify ability equivalency with autogenous bone. 5. Simple xenogenic deproteinizated bone is worst to repair bone defect, xenogenic deproteinizated bone and autogenous MSCs better a little. The ossify ability of compound of xenogenic deproteinizated bone and autogenous MSCs and rhBMP2 is equivalent with autogenous bone.