| 一The backgroud and the significance of the researchThe fixation is the important link and basic method to treat the bone fracture, and it is the fundamental condition that the bone fracture can concrescent on time. At present, the main schools of the technique of the fracture fixation in clinic over the world include: AO(Arbeits gemeinschaft for Osbeosynthese fragen) school ; BO ( Biological Osteosynthesis) school, ilizarov external fixation school, that is DO ( distraction Osteosynthesis)school; samiento external fixation clamp stand school; CO (Chinese Osteosynthesis) school.Aomong the total, AO is the method of rigidity fixation, the others are elastic fixation, CO is the classic representative of them.The internal fixation technique of AO established by the Switzerland scholar Muller is founded on the vitodynamics, stuctural and functional anatomy. The AO technique integrate standard fixator design, specialized fixed technique and matched fixing equipment, which is fashionable about 40 years over the world. It can solve effectively the multiple and complicated fracture and fracture in privileged site, such as intraarticular fracture. But at the same time, accompanied by the spreading in clinic and building experience, AO school, which is in governed position in the department of orthopaedics in present ages, make substantial alteration in recent years on the theory and technology system of fracture fixation. The scholars of AO gradually realize the defect of the thought of rigidity fixation which they kept before, such as the negative effect caused by the "stressing block" , etc.So, in recent years, they gradually abandon the opinion that the fracture should "complete rest, absolute fixation" , improve positively the methods of internal and external fixation, consummate and develope technique of the AO internal fixation, thread a needle external fixation and local external fixation.The new opinion of BO is produced on the basis of the AO, whose fundamental content is minute trauma operate, its nucleus is that we do not enforce the the reestablish the anatomic position, but emphasis on the recovery of the line of force and the bone length, and the new idea think highly of the protection of the blood supply of the area of the fracture posion and of the earlier functional exercise after the operation. BO is the improvement of AO, but it is not new theory, and it donot reflect the rich achivement on the bioresearch of the union of the fracture in the 21 century , it cannot explain some important theory problems such as stressing block and the mechanism of delayed healing of the fracture as well.But the producing of the BO makes the research on the union of th fracture go back to the correct track of reconstruction of the effect of the bony callus, which is the regression on more higher level from AO to BO on the history.The raise of BO prognosticate the new trend of the healing of the fracture, The change of the AO school, which is from rigidity fixation to elastic fixation, fit on the more higher request of people to the health, whose medical thought and technique connotat ion is undoubtedly influenced by the principle of elastic fixation deposited in the method of small splint fixation. We should see that, in recent years, there is similar change in spinal column surgery. In international orthopedics area, scholars pay more attentions to the elastic fixation than the rigidity fixation at least in the domain of fixation of bone fracture and spinal column. But until today, BO just is a method of healing, not a theory system, the origin of BO is to make up the shortage of AO, whose opinion influenced by the principle of the "dynamic and static combination, lay equal stress on bones and muscles" . DO technique in the elastic fixation is the result enlightened by the opinion of CO. DO means that draw the bone by the stretch device in invariably velocity and frequency, stimulate the formation of the bony callus in the drawing distance, and then formate the bone. It emphasis on the grasping the dynamic and static relationship. CO technique is origined by the small splint technique from China, which is the result of improvement of the traditional small splint by the mordern vitodynamics, structure and functional anatomy, whose essence is elastic fixation.CO absorb the essence of the traditional Chinese medicine, and tightly combine the development of the mordern science and technology, whose guiding idea is "dynamic and static combination, lay equal stress on bones and muscles, exterior and interior treating, doctor and patient cooperate" , CO methods is primary on no-operate healing, assist with limited operation, which is similar to the idea of BO, that is to say, the idea of CO is advanced, it fit on the new trend of the healing of the bone fracture.So, after a lot of clinical and laboratory research, the international bone and wound department aera mainly recognize that elastic fixation is the most reasonable healing thought and technology , which is most profit for the union of fracture[9]. So called "elastic fixation" means that in the period of the union of fracture, the jog in the broken ends of the fracture is permitted, which is more benefit to the union of fracture, but too more activity between the broken ends is obviously no benefit to the union of fracture, and even make disunion and delayed union, which is the fact received by the commonwealth of learning and confirmed by the clinical practice. The "jog" is a healing method to promote the union of fracture which is admitted by the schollars all over the world, but some experiments are still in the verification and research of its mechanism of the promotion effect to the union to fracture by internal and external fixation clamp stand, and the influence on the union of fracture by the fixation of small flint is still vague.The jog and stress in the broken ends is the one of the most important motive power of the happening and developing of the fracture union. The local external fixation by the flint is not trying to fix the broken ends absolutely, but just keep the corresponding anatomy relationship of the broken ends. The activity of the limbs can make the broken ends accept some pressure and stress, which is a sort of physiological stimulate, it is benefit to the union of fracture.The procedure is sensible to the change of local mechanics environment, the stress change in the broken ends is decided by the loading on the fracture and the organizational characteristics,The fixation of small flint provides the relative mechanics environment of elastic fixation, the small flint, which has reasonable configuration and mechanics superiority, possess keeping bone on axial direction, put the principle of functional activity into effect, which can promote the formate of the bone callus, and can make the bone callus get favourable moulding in the situation of functional status, so the fixation of splint has visible merit in the bone functional adaptation.The splint fixation have no stress block effect, and it has no destruction to the blood supply of the os integumentale, it has no interference to the procedure of the natural union of fracture. But, if the splint is too loose, there is still shear force or shearing stress in the local position of the broken ends, which can interfere the formation of the bony callus, and the aversion can happened in the procedure of the fixation, so the expected result cannot be reached. On the other hand, if the strap is banded too tight, intumesce can be found in the period of the fixation, and then necrosis happens.The healing of long bone shaft fracture by fixation of small splint is the characteristic technique in the Chinese medicine, which has long history of thousands of years, and accumulate abundant of experience too. Small splint has the superiority such as simple operation, low price, easy to functional excise, easy to care for, little damage to the tissue arounding, and having no influence on the blood supply, etc. This superiority lies in the difference with the "rigidity fixation" , the small splint is a kind of elastic fixation of "dynamic and static combination" , which is its essece and soul. The small splint fixation can afford the relative immovable mechanics environment in the broken ends, which permits activity in the broken ends on the longitudinal axis. This activity is benefit to the union of fracture, which can promote the absorb of the blood tumor, periosteum reactive thickening and hyperplasy, bone cells differentiation accelerate, blood vessels regenerate plentifully, bony callus grow and calcify quickly, the accreting time is ahead of schedule. ShangTianyu' s research demonstrates that the local external fixation by the splint can control the activity that disadvantage to the union of fracture. It balances the the body gravity and the muscle stress which can cause the aversion again in the broken ends by the power of the restrainting of the strap, the lever power of the splint and the effective power of the pressure pad.The mechan-balance is implement by the two main mechanism of the soft tissue of the limb: one is the no compress ibility, the intermission filled with muscle form a invariably system when it is confined by the splint, and the broken ends wrapped by the muscle can get a relative stable situation as well.Besides, this fixation need not decoherence the periosteum, the damage to soft tissue is light, the destroying to the local blood supply is slight too, which is benefit to the union of the fracture; at the same time, the fixation by the small splint do not fix the joint, so it has no influence on the activity of the joint, which make the procedure of the union of the fracture and the functional recovery of the limb at equal pace. The aboving is special fit to the common medical idea of the international medical science community puisue to---take the medical "ending point target" as principle.So we can see that, the science connotation contained in the technique of healing fracture by small splint is very abundant, which is scientific, resonable, advanced, is a shining part of the Chinese medical science, and it is precious cultural and techincal heritage of our nation.However, what is regret that, for thousands years, it still stay in the state of the experience, whose mechanics essence and quantity relation is still not known, it lacks to be systematic and scientific unearthed, arranged, elevated, and be assigned new era connotation, and it has the trend of being drown without knowing. With a sense of responsibility of the history, using the mordern technique, we arrange, dig, improve this traditional technique, interpret the mechanism deeply, succeed and bring new ideas, make this technique have new period characterstic, and spread the clinical application, what is the background and the significance to carry out this research.My research use the traditional Chinese connect bone operation and small splint fixation in the experimental fracture for the first time over the world; we adopt advanced test index to evaluate the auality of the union of the fracture, and to monitor the procedure of the union of fracture and the mechanism how it can promote or be benefit to the union of fracture; we use the baroceptor in the external fixation device, so we can measure the jog extent in the broken ends fairly exactly, and then provide the consideration to design the new external ifxation device; the research result we got afford the experimental and theoretical foundation, which can change the traditional fixation technique of small splint from expirical to sciencetific, This research not only have fairly high practical value to guide the clinical work, but also can help us to dig, arrange, improve the essence of bone and wound technique of our traditional medical science, and it can mix the technique together with the mordern science, and to promote the traditional technique to bring benefit to people, so the research has important academic and theoretical signifcance.This research affords scientific base to the method of healing the bone fracture of four limbs by fixation of the smal1 splint, itestablishes new index to spread this technique, it offers theoretic data and experimental foundation to develop elastic fixed production; it hints the rule we shold followed to explore the new method of fixation of the fracture, so it has prospective guiding significance to prospect of the development of the healing the fracture by the means of combined treatment of traditional Chinese medicine and western medicine.二The purpose of the researchIn the research, we take the animal model of experimental fracture of rabbit tibia as our study object, fixed respectivily by small splint and steel plate. In the fixation of small splint, we let the broken ends accept different pressure by adapt the tightness of the strap. Observe the influence on the union of fracture in broken ends by the action of two different pressure, and compare with the steel fixed group, the main index that we observe are:To detect the express of VEGF and TGF-β1 by the means of immunity classify.To observe the fine structure of the bony callur tissue at different stage of the union of fracture by the technique of the electron microscopy; to observe the histology change at different stage of the union of fracture by the technique of light microscope; observe the vitodynamics influence on the bony callus in the procedure of the union of fracture by the different fixation, and then comprehend the quality of the union of fracture.Observe the influence on the bone density of the bony callus by the different fixation, observe the contents of the S-SAP in the different period after the rabbit experimental fracture, and the radioactivity observation to the local fracure. To evaluate the influence on the union of fracture by the elastic fixation of small splint from different point of view, monitor the procedure of the union of fracture, approach the mechanism how the fixation of small splint can promote the the union of the fracture, and then demonstrate the jog can stimulate the formation of the bony callus, to accelerate the union of fracture. Through this research , we can afford scientific base of healing bone fracture of four limbs by the means of fixation by small splint, so that we can spread this technique and offers theoretic data and experimental foundation to develop elastic fixed production.三Method for the study Experiment one Experiment material1.1Animal 45 rabbits, weigh 2-2. 5KG, afforded by the animal experiment center of hubei science research institute1.2 reagentAnti-VEGF and anti-TGF-β1 are all afforded by Zhongshan Reagent company1. 3 equipment light microscope immunity equipmentSplint: self-made splint is made by Shan tree barks which have good elastic, which is used in defferent place of bone fracture dividely , such as exterior, interior, front and behind. The upward part of the splint is wide and the downward part is narrow. The front splint is 11cm long, and its superior extremity is 2. 4cm wide; The back splint is 10cm long, and its superior extremity is 2. 4cm wide; the interior and exterior splint are both 10 cm long, their superior extremity is 1. 9cm wide. We sting a eyelet of 1.5 mm in the small splint which is used in front and behind part.Steel: It is provided by medical limited company of JiangSu Colden Deer GroupBaroceptor: is afforded by the WuHan ChaoYU detect and control technology company, its structure as follow:The baroceptor is composed by the pressure box, air duct, three way joint, infusing air hope, sensitive element and semiconductor straining piece. Whose principle is that: before measuring the pressure, infuse the air by infusing air hope, then the air duct is filled with air, that means the pressure can be kept balance at two sides of the sensitive element in the baroceptor, so the sensitive element will not be affected by the press. When the pressure affect on the pressure box, semiconductor straining piece pasted on the sensitive element changes the stressing produced by the change of pressure into the change of electric resistance, then establish the fixed quantity relation between the pressure and the electric resistance. We can measure the electric resistance by the measuring electron bridge in order to measure the change of the pressure. When using it, we put the baroceptor into the splint, then we know how tight the bandage is by the number of baroceptor.2. empirical method2.1We select 45 healthy rabbits, get a transverse fracture pattern of 3mm in the meta-infer 1/3 of left shin bone, divide into group A, B and C, In group A and B, we fix the fracture by cypsum, 5 days later, we change the fixation into external fixation by small splint. In group C, the broken bone is fixed by steel plate.Cross a Kirschner wire whose diameter is 1. 5 mm into the rabbit tibial tubercle, and then penetrate into the hole of circa side splint so that the splint cannot slip, the baroceptor is placed in the inner surface of the ventri-splint in the broken ends, and tie up the four splint by four bandage, the tightness of each group is not same, the wag of bandage in group A, B is 3mm and 7mm, and the number of baroceptor is 18kPa in group A, and which in group B is 12kPa.The collection and mamagement of the samplePut the rabbits to death 14 days, 24 days and 34 days dividedly after the operation, saw the sample of shin bone in the place of 1 cm above and below the fracture, hold it by 10% formaldehyde solution, decalcificate by 5% nitric acid, flush by lotic water, dewater by the grad alchol, embed by par, cut sheet in the longth wise, every piece is 4 um thick.The slice is deparaffinaged by the dimethyl benzene, incubated by 3% H2O2 Deionized water For 5 minutes to deactivate the endogenous peroxydase. Washed by the distilled water, soaked in PBS for 5 minutes, repaired by the micro-wave.Dropwise goat setum to block, rabbit anti- by 3% H2O2 Deionized water iAntibody and mice anti-VEGF antibody diluted by the proportion of 1: 100, stay overnight at 4℃. Wash by PBS for 3 minutes, and then dropwise biotinylation anti-2 and incubate for 15 minutes in the room temperature.Wash by PBS for 3 minutes, three times, drop horseradish enzyme labelling strepto-antibiotin, incubate for 15 minutes in the room temperature. Wash by PBS for 3 minutes, three times, colorate by the DAB, wash by the tap water, after stain by hematoxylin. dehydtate by Grad alcoholic, mounting after transpatent.2.3 Observe index: the express of VEGF, TGF-β1 Experiment two1 subject1. 1 Experimental animals: the same as experiment one1.2 equipmentlight microscopeTransmission electron microscope: H-500 (which is produced by Japanese Hitachi company). It is provided by WuHan military area army Gen Hospital.Light microscope (which is produced by Japanese Olympus Company). Transmission electron microscope: H-500 (which is produced by Japanese Hitachi company). It is provided by WuHan military area army Gen Hospital. Ultramicrotome: LKB-2, which is produced by Sweden LKB company, is provided by WuHan University medical college.2. Empirical method2.1 The animal model building and grouping is the same as experiment 12.2 Observe index and method2.2.1 macroscopic observation: We observe external,internal bony callus and bridge bony callus after the death of rabbits.2. 2. 2 histomorphology observation: Put the rabbits to death by means ofaeroembolism in the ear vein, kill the first batch 14 days after theoperation, kill the second batch after 24 days, kill the third batch after34 days, about 5 rabbits every time in each group. saw the sample of shinbone in the place of 1 cm above and below the fracture, hold it by 10%formaldehyde solution, decalcificate by 5% nitric acid, flush by loticwater, dewater by the grad alchol, embed by par, cut sheet in the longthwise, every piece is 5 cm thick, color it by campeachy-eosin in routinemethod, then observe it in light microscope.2. 2. 3 Fine structure observation: We get a sample in the size of 1 mm3in each period, fix it by the 2.5% GA solution, decalcificate it by 1%EATA solution, wash it three times by balanced solution (pH 7. 2-7. 4), thenfix it by 1% osmic acid 2 hours, dehyd it by alchol, Embed it by epoxyresin 812, get extra thin section, color by lead uranium, observe by TEM.experiment 31 Meterial and method1.1 Select 18 healthy rabbit, divided into group A, B and C on random,The animal model building and grouping is the same as experiment 11. 2 Detect item1. 2. 1 S-ALP: get the heart blood 2ml/kg by using number 9 pinhead beforeand 2, 4, 6 weeks after the operation. Detect the S-ALP. (detect by theHUBei Chinese medical hospital)1.2.2 Vitodynamic test tensile strength testThe three group animals are all put to death by the means of aeroemblism from ear vein, dissect left shin bone, get rid of soft tissue, wrapped up by the gauze, soak by the isotornic Na chloride.Put the experimental bone in the temperature of 20±5℃for at least 3h, detect within 12 hours. Then fix the rabbit shin bone with a side of the steel-wire, another side is hanged on the tongs of the LJ-500 tensile force equipment. Carry out the elongation test at the speed of 25mm/min, till pull and break the shin bone, so we can get the number of power of pull and break, the pull break cross-section should around the place of theunion of fracture, otherwise, abandon the data.Every way of fixation can get 6 data of effective pull break power, compute the bone' s section area by the outside areaof the bone, use pull breal power division the section area, we can get the value of tensible strength, sort the total six data ,2 Make statistic treatment to the experimental result Experiment four1 Experimental meterial1. 1 Animal: 18 rabbits, weigh 2-2. 5KG, afforded by the animal experiment center of hubei science research institute1.2 Equipment: QDR 2000plus type DEXA2 Experiment method2.1 Bulit the animal mode and divide the groupSelect 18 healthy rabbits, devide into group A, B, and C on random, make mode and group the animals just as the experiment one, put the animals to death after the end of 6 weeks, dismantle the exterior and interior fixation, disengage the knee joint of the two posterior limb, reject the whole soft tissue, get the two shin bones, and remain the periosteum.2.2 Objective index2.2.1 Observation from the x-rayTake photographs of x-ray before putting every animal to death, make sure that no pseudpartoculation formates, and observe the condition of the growth of the bony calllus and of the union of fracture.2.2.2 bone densitometryAdopting the QDR 2000plus type DEXA, using the absorption method of the two power x-ray, detect the BMD in the middle tibia of fracture, choose the region of interest R1 in the same condition of the fracture, take the place of fracture as center, scan a distance of 10 cm from near place to the far place. Calculate the BMD of the Regions of Interest ( ROIs) about 1 cm arround the place of fracure using the data measured by the analysis software. Scan the corresponding position of the other side being not operated, the data we get are took as the contrasting data. Every sample are measured three times, we reset the sample every time after the measuring. The microcomputer can automatic analysis and print the result by the DEXA, calculate the ratio of the bone density of every couple of the samples.3 Data processing and statistics analysis: the formula to calculate the ratio of the parameter is that: ratio of bone density=value of corresponding side of being not operated/value of the side of being operated×100%. The value of bone density of every group is expressed as mean±standard deviation (x±s)四The result of the research1. The result of immunity classify: in the every phases of the union of fracture, the cell expression of TGF-β1 increases, and the cell expression of VEGF increases too, and there is invariably rapport between them. In the early phase and the middle phase of the union of fracture, the amount of the blood vessel formation and of the bony callus formation in group A are all more than which in group B and group C, and the time of the blood vessel formation in group A is earlier than which in group B and group C too. So the masculine express in group A is earlier than which in group B and group C, the masculine level in group A surpass obviously than which in group B and group C.2. Macroscopic observation of bony callus in the period of the union of fracture: In group A, the portion of fracture conjugate primarily 14 days after operation, we can see a 1 ittle fusion shape external callus, 24 days later, the callus are in fushion shap, callus bridge the two broken ends, 24 days later, the callus conjugate the two ends firmly, there is no range of motion in the broken ends.Group B: we put 5 rabbits to death at the time of 14 days, dislocation can be seen in one rabbits at the place of fracture; in the time of 24 days, there is partly conjugation in the broken ends, but the range of motion is large, the external callus is less than which in group A, the bony callus is moderate, taking Fusiform shape 34 days later. The broken ends are not connected completely by the bony callus, the fracture line still can be seen clearly.In group C: the broken ends have no dislocation in every period, we can see fibrous joint after 14 days, and we can see sporadic external callus in medium dose in 24 days, it align regularly, joint of Fushion form shape can not be see in the broken end, 34 days later, part of the external callus form Fushion shape, but not very firmly.3. Electron microscope shows that: in group A, in the procedure of union of fracture, osteoblast appears earlier, the quantity of it is more than which in the SF group, besides, its function is active. 34 days later, generous bone cell of active function can be seen in the broken ends. In group B, 24 days later, chondroblast can be seen, and 34 days later, hypertrophic cartilage cells still can be seen.4. The lighe microscope shows that: In group A , periosteumcallus, bridge callus can formate earlier rapidly, then become uniting callus gradually, 34 days later, the callus can connect the broken ends, where the woven bone transformate lamellar bone. Croup B: 14 days later, there is still blood tumor and granulation tissue in the broken ends, the amount of the bony callus is small. 24 days later, there are partly cartilage connection, woven bone is badly to see. 34 days later, the broken ends are not connected completely, and the hypertrophic cartilage cell still can be seen. In group C, 14 days later, a little fibrous callus can be seen in the broken ends, and granulation tissue still can be seen there, 24 days later, slight cartilage connects, 34 days later, the callus step over the broken ends, but not connect completely.5. The vitodynamic measuring in the broken ends6 weeks after the fracture, the tensile atrength in group A is more better than which in group B and the steel fixed group.6. The result of the S-ALP:different extent of jog influences on the the content of S-ALP in the experimental union of the rabbit fracture, the extent of the changing of AKP is different in the different period and in the different group, which is more higher in group A than in group B, C in 14 days.7. The result of the imageology: group A: the broken ends are filled with compact bony callus, the fracture line is vague, external callus is compact, partily pulp cavity is recanalization.Group B: there is many bony callus in the broken ends, pathy shadow can be seen in the broken ends, but the fracture line is still there.Group C: the distance between the broken ends can be seen clearly, there is small raritas bony callus through the broken ends, small external callus formate.8. The measure result of bony densityThere is highly significant deviation between group A and B after 42 days, the value of BMD in group A surpass the value in group B obviously, and there is no significant deviation between group B and C.五The result of the research of this topicThe jog can promote the union of fracture obviously; the time of the union of fracture is shortest in group A which is fixed by the small splint, whose velocity and qualitatively of the union of fracture has conspicuous superiority. Proper fixation by the small splint can improve the blood cycle of the local fracture, increase the amount of the cell, promote the cell differentiation, maturity, facilitate the depositing of the mineral matter in the broken ends of fracture, elavate the quality of union of fracture, shorten the time of union of fracture, and raise the strength of the broken ends.
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