| Bone tissue repaired is an important problem about medical health, in manyyears ago, Bone tissue repaired technology is autoallergic or variant bone graft,but quite a few patience give up curing because of quantity and price limited. Ourcountry with a lot of people, need large amount of bone tissue repaired materials,with improving people's living level and strengthening health protection witting.Autoallergic bone graft is so limited and will induce to 10-30percent complicationin region field;variant bone graft will exist mmunological rejection, AIDS virusinfection, slow bone substitute, small volume of new bone and so on. So it hasbeen spot of orthopaedic surgery research that people try to get ideal bonerepaired materials by natural or synthesis route. In tens of years, people haveprepared so many bone tissue repaired materials by all kinds of methods and route,and have got some success, especially bone tissue engineering have good future.Biomimetic synthesis of nano-Hydroxyapatite/Collagen/PLA is a large futureroute that may prepare excellent materials substitude for bone, which have beenused in clinic and accepted by people. But extracting collagen protein will cost alot of money and materials, and be easy to spread AIDS and mad cow disease.Silk-protein, a protein with good biocompatibility, has been showed solicitude. Under biomimetic synthesis and CPC, imitating procedure of preparingbiology mineralizing matetials that professor Cuifuzhai of tinghua universitybiological matetials dep have done, and procedure of bone materials synthsis inDoctor Kongxiangdong's paper (Fibroin Regulated Biomineralization of CalciumPhosphate), this study have prepared a nanocomposite for repairing bone defect.Meanwhile, we used cheap silk protein substitute for collagen protein that maycut down material cost, avoiding costing and wasting. This paper will payattention to study its biomineralization and biodegradability in body and vitro,observe its bone defect prepare. This will provide new-type and high efficientmaterials for clinical curing bone defect, and investigate reason and clinicalapplication prospect of this materials repairing bone defect.Chapter 1 Synthesis and function evaluation of nano-Hydroxyapatite/Fibroin (nHAF)1. objectiveThis research synthesized nHAF/PLA and evaluated function.2. synthesis of nano-Hydroxyapatite/Fibroin (nHAF) and TG analysisWe got fibroin by Bombyxmori silkworm cocoon taking off sericin, took itinto different concentration solution, dipped them into water solution with calcium,continue to mix it, then dipped phosphate radical with proportion of Ca: P=1.67;dipped NaOH solution maintaining pH (7.4~8), we found the solution appearedsediment when pH is 5~6, white suspension when pH is 7, then continued to mixit more than 24 hours, after static 24 hours, get rid of supernate, centrifugation itby 5000rpm, washed it with deionized water, freezed it and dried it withlyophilizer. At last we got dry powder by milling-in. This research showed by TG:We synthesized several kinds of nHAF with different content ratio by adding thedifferent concentration of fibroin in the initial solution. One kind of nHAF that itsTG curve have similarity with bone contained water 4.6%, fibroin 27.2%,inorganic 68.2%. this showed we may prepare bone materials that have similarratio of organic and inorganic composition, with natural bone by controllinginitial concentration of fibroin.3. synthesis and function evaluation of nano-Hydroxyapatite/Fibroin/polylactide (nHAF/PLA)10% Chloroform as dissolvent dissolved PLA completely, dipped NaCl asbuilding hole doses, dipped nHAF powder, then dispersed it for 30min withsupersonic, we took above solution into tooting made of Politef, freezing under-20℃ , at last, made Chloroform volatilized, NaCl dissolved, then dried it. Wecould get nHAF/PLA. There is nonvisulization on X-ray, nHAF/PLA was easy tobe sterilized, there was no change about material characteristics before and afterthat, its interval porosity was 83%, its density was 0.116g/ml, its average holediameter was 176.3μm, hole was polygon, no regularly, its wall of hole was equaland even thick, connected with each other, its density was even in systhesis andmolding of nHAF/PLA, its intensity was 1.5±0.3Mpa, like natural bone, this resultwas corresponding with interval porosity.4. conclusionWe synthesized several kinds of nHAF with different content ratio of fibroinby controlling the initial concentration of fibroin in the solution. That means wecould synthesized bone materials that have similar concentration ratio with naturalbone in organic and inorganic composition, and looked it as research object. Thisresult showed nHAF/PLA have similar with natural bone in microarchitecture andcomponent, have suitable physical and mechanical function, is easy to sterilize,there is no change about material characteristics after it.Chapter 2 Research of cytotoxicity test for nano-Hydroxyapatite/Fibroin/ polylactide (nHAF/PLA)1. objectiveResearch of biocompatibility of nHAF/PLA by cytotoxicity test.2. method and resultWe colcultivated NIH3T3 cell and nHAF/PLA, PLA and blank as contrast,observed cytotoxicity of nHAF/PLA, the result that observed by microscope show:NIH3T3 cell in 24 hole board adherence, generation and immigration nomally,and adherence in 2 days, full 4 days, better reflect light, appeared shuttle andpolygon cell, every group cells appeared dense and regular, there is no suspendingdeath cell. MTT method showed: NIH3T3 cell grew well on nHAF/PLA, and cellquantity continued to increase, RGR of nHAF/PLA ≥100%, cytotoxicity 0, ODincreased accompanying with cell growing, that showed nHAF/PLA hadsuperordinary biological function, and excellent cellular affinity.3. conclusionnHAF/PLA have excellent cellular affinity with histiocyte, no cell-cytotoxicreaction, superordinary biocompatibility.Chapter 3 Research of vitro degradation of nano-Hydroxyapatite/Fibroin/ polylactide (nHAF/PLA)1. objectiveObservation of biodegradation of nHAF/PLA by vitro co-cultivation ofnHAF/PLA and degradation solution.2. method and resultTaking materials sterilized into degradation solution, then set it to 37℃sterile cupboard, we got sample in differet time and differet observation location.We weight nHAF/PLA and analyze change proporation of component, in order toobserve its degradation, measure its speed of degradation. The result showed: itshad biodegradation, in first week, speed of materials was so quick that weight ofmaterials decreased 10%, in fourth week, decreased 20%. There was differentspeed of degradation in three component of nHAF/PLA, silk-fibroin is best fast,HA and PLA are secondary. But SEM showed: nHAF/PLA maintained ownframework, but wall of hole got thin, surface of wall of hole got roughen.3. conclusionnHAF/PLA have some biodegradability and its speed of degradation matchwith union of frature.Chapter 4 Observing biocompatibility and biodegradability of nano-Hydroxyapatite/Fibroin/polylactide (nHAF/PLA)1. objectiveTo embed nHAF/PLA that have been sterilized into abdominal cavity andmuscle of back of rats, to observe biocompatibility and biodegradability.2. method20 adult male SD rats, weight 230-300g, were divided into 4 group random.After abdominal cavity anaesthesia, (1) embedding abdominal cavity: rats wereplaced in rats plate with dorsal decubitus, four limbs were abducted, abdominalskin were sterilized by complexation, with median abdominal incision, 0.5-0.9cmlong, we cut off skin, hypoderma, deep fascia, Linea alba abdominis, andabdominal membrane, used automatic micro-retractor to pull away abdominalmembrane, put bone materials into abdominal cavity, then closed it. (2)embedding muscle: rats were placed in rats plate with ventral decubitus, fourlimbs were abducted, back skin were sterilized by complexation, with medianback incision, 0.5-0.9cm long, we cut off skin, hypoderma, deep fascia, backmuscle: latissimus dorsi muscle, split fascia musculares, put bone materials intoback muscle, then closed it one by one. After operation, in 2, 4, 6, 8 weeks, onegroup were random sudden death, bone materials were taken out of .in this study,we observe conglutination degree, and environmental tissue alteration by directobservation and HE dyeing observation.3. resultThere was no rats death, no ankylenteron and no bowel obstruction in periodof observation, Operative wound is all primary healing. (1) direct observation: in2 weeks, basic framework of bone materials still existed, were wraped up byintraabdominal colic omentum. Inflammatory reaction appeared in the backmuscle, then lessen gradually, texture of bone materials turned to soft. in 8 weeks,phenomenon of Intraabdominal colic omentum package disappeared andInflammatory reaction disappeared in the back muscle. (2) HE dyeing: In 2 weeks,basic framework of bone materials still existed, Inflammatory reaction appearedobviously, bone materials were wrapped up by a great quantity cells, some fibrousconnective tissue grow into interal. Gradually, materials turn to collapse,Inflammatory reaction disappeared, cells at edge of materials decreased, butpenetrated deeply into interal materials. After that, considerable fibrousconnective tissue grow into interal materials from different direction,cell nucleusshape happen change from spindle to macro-spindle. at same time, cellsphagocytized materials. in 8 weeks, matetials has been collapsed completely, denovo connective tissue distribute extremely , fill in materials internal, cellsbegin to approach materials particle, tight pasted surface of materials. thickness ofhole wall turned to thinningz, there is a large amount of epimatrix in holes.4. conclusionnHAF/PLA are safe, its degradation products have no toxic reaction, havesuperordinary biocompatibility and eligible biodegradability, which is idealsubstitute for bone defect.Chapter 5 Observation of nano-Hydroxyapatite/Fibroin/polylactide(nHAF/PLA) repairing segmental bone defect of rabbit radial1. objectiveTo embed mineralized silk-fibroin bone materials (nHAF/PLA) that havebeen sterilized into segmental bone defect of rabbit radial, to observe its ability ofrepairing bone defect, furtherly to observe its biocompatibility andbiodegradability.2. method15 adult male New-Zealand rabbits, weight 3000-4000g, were divided into 3group random. After ear iv injection anaesthesia, rabbits were placed in animalexperiment bench with ventral decubitus, four limbs were abducted, right forelimbwere sterilized by complexation, with median radial incision, 2 cm long, we cutoff skin, hypoderma, deep fascia, and muscle, used scalpel to pull awayperiosteum, then come off it, fixed on radius with electrodrill, put away 1.5cmradius with scroll saw, after that, moulded bone materials, put bone materials intoradial bone defect, then closed it. The same operation repeated on the left forelimbwithout embedding bone materials as contrast. After operation, in 4, 8, 12 weeks,one group were random sudden death, bone materials were taken out of. In thisstudy, we observe conglutination degree, environmental tissue alteration and bonedefect repaired by direct observation, X-ray and HE dyeing observation.3. resultThere was no rabbits death, no bone fracture in period of observation,Operative wound is all primary healing. In 4 weeks, there is some excrescence,then turned to small and disappeared, (1) X-ray: The bone defect of contrastinggroup still were not restored, in areas of bone defect, only periosteal reactionappeared. In 4 weeks, there were osteotylus on the bone defect of experimentalgroup, basic framework of bone materials still existed, new bone tissue begin tomolding. In 8 weeks, bone density gradually increased in bone defect. In 12 weeks,new bone tissue substituted bone materials, cavum medullare appeared, bonedefect have been cured. (2) HE dyeing: In 4 weeks, at edge of bone materials,there were synostosis, a great deal of fibrous connective tissue and new bonetissue filled materials hole. Gradually, bone materials turned to collapse. In 12weeks, a great amount of neoplastic bone matrix and trabecula of bone appeared,new bone tissue substituted bone materials, and bone materials disappear, bonedefect have been cured. (3) Through image analytical technique, We calculatedinternal degradation speed, in 4 weeks, bone materials degradation 27percent, in 8weeks, 39 percent, in 12 weeks, 44 percent. (4) SEM observation: When 4 weeksthe massive staggered fibrous bundles wrap around the material, the material stillmaintained its porous portal frame construction, but the material opening wallchanged thinly, the material surface also changed relatively quite is smooth. Afterimplants 8 weeks to approach the material inner layer region, may see has themassive bone cells existence, the cell is taller and slenderer as well as extendspseudopodium.4. conclusionnHAF/PLA have superordinary biocompatibility and eligible biodegrad-ability, favourable osteoconduction and osteoinduction, could repair bone defectin normal bone healing time, which is an ideal substitute for bone defect. |
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