The Expression Of Bone Formation-Correlative Factors In Allograft Bones Implantation

Compared with autogenous bone grafts, allografts come from many routes andtake a irreplacemental position in bone transplantation, because of the characteristicsof native structure, shape and strenth and a few induction activities. But theimplantation will spread hepatitis and AIDS potentially without strict medicalinspections. To eminilate or weaken the antigenicity of allograft, all kinds of phisicsand chemical methods are always used, and the common and vertified effectivemethods cryoapplication, lyopyilization and decalcification, which are all appliedclinically mainly on bone cavity obturation, bone section implantation, joint fusionand prosthesis rebuilding. The decalcificated bones eliminating soluble alloantigensare treated with chemical sterilization, antigen extraction and autolysis digestionprocess, and get more bone formation after imbedded than fresh bones, freezed totalbones or irradiated freezed bones by Co60.Growth factors can promote cell proliferation, cell differentiation and synthesisof extracellular matrix, and affect the initiate, development, regulation and rebuildingof bone fracture repair extremely. Respecting the reason, it is natural to apply growthfactors to hasten the heal of bones. And animal experimental and clinical application resultsrevealed that growth factors result in good sequences to delayed conjunction of bonefracture or sejunctus bones. Though many factors take part in the modulation of bonehealing, while to nowadays, only few are known, such as bone morphogeneticprotein(BMP), transforming growth factorβ(TGF-β), VEGF, and so on, which areinvestigated more or less. These factors are synthesized by inflammatory cells,osteoblasts and cartilage cells during the whole time of healing after fracture, whichshow objectivity to the evaluation of the bone healing process and the effects of boneimplantations. After bone are implanted, bone formation in organism is a undirect course andthe judgement is hard to quantitied. Now, there are several methods used likeimmunohistochemistry, visualization quantitative assessment of irradiated bones,biomechanical analysis and quantitative assessment of RT-PCR, while the effects arenot the same.(1) Histological analysis: The histological research demonstrates thatnew bone formation can be a predictable process, if moulding and rebuilding developtotally as normal after endochondral bone formation, rhBMP-2 can be used tospondylodesis effectively like the expostside spondylodesis fusion modle of dog, inwhich tough bulk fusion appeared in 3 months after rhBMP-2 was imbedded, whilelittle fusion achived in 8 months after the autogenous bone graft. Nowadays, manyclinical researches on rhBMP-2' use in bone fracture are in progress, yet the resultsare preliminary. It is reported from four large trauma center in USA, in 12 cases ofimbedding of rhBMP-2 with quantities of 3.4 mg or 6.8 mg combined withcomplicated collagen vector into open fracture of tibia atⅱ,ⅲA orⅲB degree fixedby bone nails or external fixation device, nine cases(75％) are Cured one-off, the otherthree need an implantation again, and two cases got antibodies specific to BMP-2.(2)Visualization quantitative assessment of irradiated bones: the major inorganic salt inbones is hydroxyapatite crystal, whose structure is similar to ion exchange column,and the great surface area makes the metabolism of phosphate and other elementsrefreshed from blood by chemical adsorption and ion exchange. Bone tendencyradioactive drugs like 99mTc—phosphate is deposited inside bones after injected andabsorpted quickly by crystal surface, and disclose metabolism images of bonesspecially. When lesions of local bones take place, such as inflammation, tumor,fracture and the following change of regional blood flow and/or skeleto-mineral saltmetabolism, radioactivity ehibitted on corresponding position increase anormaly. Asthese abnormality appear obviously at the earlier stage of disease, early diagnosis andclear localization could be made to various kinds of bone diseases. Bone imagemonitoring of graft is uniquely valuable to judge whether the graft will survive. Afterthe implantation is done and the injury reactions of soft tissues depress, when theradioactivity of recipient region is no less than normal tissues in regional boneimaging, it is clear that blood transpotion is good and the graft survive.(3)Vitodynamics assessment: rhBMP-2-combining-collagen vector is sticked at the sitesof rabbit ulnar bones truncated as exteriorized graft, and compared with pure collagen vectoror the situation in which untreatment is confered after bone truncation, afterrhBMP-2-combining-collagen vector is implanted, bone healing speed up obviouslyrevealed by X ray and vitodynamic test and measurement. In 3 weeks, there is bridge grafting bone in rhBMP-2 group instead of the control group, and in 4 weeks, theanti-wrench strenth is as strong as the strenth of the control group in 6 weeks.Roughly same result is achieved when rhBMP-2-combining-collagen vector isimplanted into the segmental defect of rabbit femoral bone in 2.5 cm, that is to say,new bones form in 1 month, and internal fixation can be removed in 4 months, whenbone union will grow up in broken ends of fractured bone and bone mineral content isas the normal femoral bone. In a year, new os integumentale will form and camerapulpi recanalization will be finished.This research is divided into three parts.Ⅰ. This section concentrates on the preparation of animal model, for extraction ofbonycallus in next step.Ⅱ. This section focuses on rabbit radial bone graft, extracts semental bony callusand total RNA at 4, 6, 8, 10 and 12 week after allograft and autogenous boneimplantation, detects expressions and changing tendency of mRNA of BMP-2, TGF-βand VEGF by a meaning of RT-PCR, and compares allograft's multiple factors'expressions with that of autogenous bone implantation.Ⅱ. The above results were statistically treated and analyzed by method of SPSS13.0 software, to examine the time-effect relationshis between bone formation afterbone implantation and the expressions of BMP-2, TGF-βand VEGF, and compareautogenous bone implantation and allograft bone. Firstly, Dunnett method wasutilized to execute multiple comparision, to analyze the differences of mRNAexpressions of BMP-2(VEGF/TGF-β) and statistical significance was judged whenp＜0.05. It was discoveried that there were extreme significant deviation amongmRNA exopressions of BMP-2, TGF-βand VEGF at the time point of 4, 6, 8, 10and 12 week after autogenous bone implantation, and F values were respectively1418.186, 1127.803 and 347.603, and P values were all smaller than 0.05; and therewere extreme significant deviation among mRNA exopressions of BMP-2, TGF-βand VEGF at the time point of 4, 6, 8, 10 and 12 week after allograft, and F valueswere respectively 1429.903, 1288.781 and 384.228, and P values were all smallerthan 0.05; to compare the combinant effects, there were extreme significantdeviation among mRNA exopressions of BMP-2, TGF-βand VEGF at the timepoint of 4, 6, 8, 10 and 12 week after implantation, and F values were respectively2845.873, 2413.591 and 726.773, and P values were all smaller than 0.05. On thebasis, respecting the fact that average mRNA expressions of BMP-2, TGF-βandVEGF of autoplastic and allograft groups at the time point of 8 week were higherthan that at 4(6\10\12) week, it was hinted that this time point was a optimal postoperation juncture because of the highest values of bone formation relatedfactors in all time points were profit of bone formation. Secondly, factor analysismethod was applied to compare the bone formation related fators' expression at 4, 6,8,10 or 12 week after impalntation between autograft group and allograft group, andstatistical significance was judged when p＜0.05. It was indicated that theexpressions of BMP-2, TGF-βand VEGF were influenced by two factors ofgrouping and time: To VEGF, there was no reciprocation effect between groupingand time, and F=2.226, P=0.079＞0.05, and on the basis, there was no significantdifferences of VEGF expressions between autograft and allograft group, andF=0.002, P=0.965＞0.05; To BMP-2, there was no reciprocation effect betweengrouping and time, and F=2.341, P=0.068＞0.05, while on the basis, there wassignificant differences of BMP-2 expressions between autograft and allograft group,and F=10.340, P=0.002＜0.05; and to TGF-β, there was a reciprocation effectbetween grouping and time, and F=3.035, P=0.026＜0.05, so grouping differences ofevery time point instead of all the time course could be judged, and it was indicatedthat there ware no significant differences of TGF-βexpressions between autograftand allograft group at 4, 6, 8, 10 or 12 week, and F values were respectively 1.244,0.560, 4.492, 0.325 and 0.730, and P values were respectively 0.291, 0.471, 0.060,0.581and 0.413, which were all higher than 0.05.On the basis of above results, the conclusions are as follows:ⅰ. There are continual secreting expressions of BMP-2, TGF-βand VEGF in bonetissues after allograft and autogenous bone implantation, and the tendencies arepositive correlated with the bone formation activities of osteoblasts, and the peakamplitudes were obtained at the time point 8 week after operation, hinting that it to bea good opportunity for intervention.ⅱ. There were various time and grouping sensitivity of several factors'expressions between autograft and allograft group. To VEGF, there was noreciprocation effect between grouping and time, and F=2.226, P=0.079＞0.05, andon the basis, there was no significant differences of VEGF expressions betweenautograft and allograft group, and F=0.002, P=0.965＞0.05; To BMP-2, there wasno reciprocation effect between grouping and time, and F=2.341, P=0.068＞0.05,while on the basis, there was significant differences of BMP-2 expressions betweenautograft and allograft group, and F=10.340, P=0.002＜0.05; and to TGF-β, there wasa reciprocation effect between grouping and time, and F=3.035, P=0.026＜0.05, sogrouping differences of every time point instead of all the time course could be judged, and it was indicated that there ware no significant differences of TGF-βexpressions between autograft and allograft group at 4, 6, 8, 10 or 12 week, and Fvalues were respectively 1.244, 0.560, 4.492, 0.325 and 0.730, and P values wererespectively 0.291, 0.471, 0.060, 0.581and 0.413, which were all higher than 0.05.