| Objectivetissue engineering technique provid a new method for theconstruction for organ or tissue. we used tissue engineering technique andmicrosurgical technique to construct vascularized and nervalized tissue-engineered bone in animal vivo,and repair large section bonedefect.many methods were used to observation and assessment to discussthe effect of vascularization and neurotization in the construction oftissue- engineered bone. The experiment purpose:1.To investigate the possibility about the the new porousβ—TCP asscaffold for bone tissue engineering.2. To study the bone defect reparation rule that tissue- engineeredbone wraped up by blood supplied fascia was used to repair large bonedefect.对比3. To contrast study the ossification effect on tissue- engineeredbone by transplanting nerval bundles of different types and discuss theconstruction methods about neurotization of tissue- engineered boneMethods The first part A study of the new porousβ—tricalcium phosphate asscaffold for bone tissue engineeringLight microscope and scanning electron microscope were used toobservation, bone marrow stromal cells(BMSCs) were cultivate withβ—TCP, andMTT ways were use to judge the biocompatibility;the cytotoxicity were analysisby the method that use the different density leaching liquor gained fromβ—TCPto raise BMSCs; BMSCs were induced to be osteoblast,and were compoundwithβ—TCP, which were used to repair large radius bone defect in rabbit.The second part Early observations of tissue- engineered bone wrapedup by blood supplied fascia to repair rabbit large bone defectThe Newzeland rabbit bone marrow stromal cells(BMSCs) were induce toosteoblast and were cultivated withβ—TCP to repair large bone defect (1.5cm) inrabbit, s two radiuses;the tissue engineering bone were wraped up by bloodsupplied fascia in rabbit, s light radius,as the control,the treatment wasn, t done toright radius. At 2, 4, 6, 8and 12 weeks after operation, specimen observation,histology, radiology, bone density and radionuclide bone scan were use to judgethe osteogenesis and vascularize.The third part Preliminary experimental contrast study on constructionmethods about neurotization of tissue- engineered boneThe Newzeland rabbit BMSCs (bone marrow stromal cells, BMSCs) wereinduce to osteoblast and were subsequently compounded withβ—TCP (β—tricalcium phosphate,β—TCP)to make tissue- engineered bone,and different kinds ofnerval bundles were transplanted into these tissue- engineered bone. All Newzelandrabbits were divided into 5 groups according to the different types of nerval bundles:Group A, tissue- engineered bones without nerval bundles transplantation; Group B,tissue- engineered bones transplanted with sensory nerval bundles; Group C, tissue- engineered bones transplanted with motor nerval bundles; Group D, tissue-engineered bones transplanted with vascular bundles (autonomic nerves contained);Group E, tissue- engineered bones transplanted with both sensory and motor nervalbundles.Then, 1.5cm bone defect in femur of Newzeland rabbit were repairedseparately by these methods.At 4,8 and 12 week after operation, radiologicalexamination was used to show the bone reconstitution.At 12 week, gross specimenobservation, histology observation and bone density detection was separately used tocompare the result of bone repairation in different group.ResultA study of the new porousβ—tricalcium phosphate as scaffold for bonetissue engineering1. observation by microscope, theβ—TCP has uniformity aperture and all hole iscommunicate; observation by scanning electron microscope, superficies intema issmooth and glossy, spheroidal aperture is 500±150 micrometer, conjunction path is150±50 micrometer.2. cell adhesion is good by phase contrast microscope.MTT is use to determinatecell proliferation, The D570 increase gradually follow time,12d is up to the zenith.experimental groups and control group have no significant difference(t=0. 599,t=2.074,t=1.401,t=1.579, P＞0.563)3. cell proliferation rate is super 100%cultivated by different density leachingliquor lixiviated fromβ-TCP. refer to corresponding relationship of cytotoxicity andcell proliferation rate, cytotoxicity ofβ-TCP is zero grade.4.the osteoblast were cultivated withβ—TCP to repair large bone defect (1.5cm)in rabbit's radiuse, histology, ECT and X-ray were used to determination,at the 12w, the bone defect is rebuild.Early observations of tissue- engineered bone wraped up by blood supplied fascia to repair rabbit large bone defectFrom 2 to 4 weeks after operation, there is a crest-time in vascularize. after 4weeks later, every time observation point, experimental group is better than controlgroup not only in vascularize degree, new bone formation and materials degradationspeed but also in bone repair time and bone rebuild quality(all statistics analysisp＜0.05)Preliminary experimental contrast study on construction methods aboutneurotization of tissue- engineered bone1. specimen observation: The 5 groups of specimen observation at 12 week afteroperation: Massive fibrous connective tissue was found surrounding bone defect andbone defect was not all repair in group Aand group C; large of os integumentale wasformation and the bone defct was completely reconditioning in group B,group D andgroup E2. X-ray observation: The 5 groups of X-ray observation at 4 week afteroperation: in group A,group B,group Cand group E,area surrounding materialbecame blur and dotted or flocced radioresistant images was found,but the materialand the crevice in bone defect were still clear; group D,the material was packaged byabundant bony callus, the crevice in bone defect were blur;The 5 groups of X-rayobservation at 8 week after operation: In group A and group C, patchingradioresistant images were foung in the area surrounding material and bonedefect,more bony callus made the crevice in bone defec blur;in group B,groupDand group E, larger patching radioresistant images were foung in the areasurrounding material and bone defect,the material turned anomalism because ofdegradation and absorption, massive bony callus made the crevice in bone defec blur;The 5 groups of X-ray observation at 12 week after operation: In group A and groupC, large patching radioresistant images were found in the area between material and bone defect, bony callus turned maturity,the material was continueddegradation;in group B,group Dand group E, larger high density patchingradioresistant images were foung in the area surrounding material and bone defect,crevice between material and bone was disappear, the material was not distinguishbecause of degradation and absorption. Refer to Yang score, values of callus densityand roentgenographic scores was statistics,there is difference in them(values of callusdensity: F=218.272, P＜0.001; roentgenographic scores F=4.426, P=0.003). allresult were: group D were higher than other 4 group(P＜0.05)and there were nodistinc difference between the other 4 group at 4 week. (P＞0.05).At 8 and 12 week,the relative values in group B,D,E were each higher than each group A,C(P＜0.05),and there were no distinc difference between group B,D,E (P＞0.05) andbetween group A,C (P＞0.05).3.histology observation(Masson): calcification and degradation ofβ—TCP wasfound in all groups at 12 week after operation, the 5 groups of specimen histologyobservation at 12 week after operation: In group Aand group C, massive material wasnot complete degradation,the degree of ossification around the material was lower;ingroup B,group Dand group E, material was divide into min particle by bonetissue, the degree of ossification around the material was higher. Draw the callusfrom specimen, In group A and group C, the degree of ossification was lower, bonelamella was not found instead of disorder osteocyte; in group B,group Dand groupE, bone lamella was clearly found which showed the higher degree of ossification..4. bone density observation: The 5 groups of bone density observation at 12week after operation: Group A(a) and group C(c), bone repair was not complete;group B(b),group D(d)and group E(e), bone repair was already complete. The bonedensity in group B,D,E were each higher than each group A,C(P＜0.01, and therewere no distinc difference between group B,D,E (P＞0.05) and between group A, C(P＞0.05)Conclusions1. The new porousβ—TCP is good in cell adhesion and biocompatibility, it hasno cytotoxicity.Because of its adqulis and communicated pore, it can repair largeradius bone defect in rabbit, Because of its distinct 3D stereochemical structure andsuperordinary physico-chemical property, the new porousβ—TCP is a good scaffoldfor bone tissue engineering.2.Blood-supplied fascial flap improved vascularization of tissue- engineeredbone greatly.3. The criticality period that blood-supplied fascial flap improvedvascularization of tissue- engineered bone is earlier period.4. volant vascularize in earlier period is the important influential factor toossific of tissue- engineered bone.5. nerve factor is the important facilitative factor to ossific process of tissue-engineered bone6. nerval bundles embedded in tissue- engineered bone can be a constructionmethod about neurotization of tissue- engineered bone7. Sensory nerval can effectively accelerate the ossification of tissue-engineered bone8. vascular bundles and Sensory nerval bundles have the same effect onaccelerating the ossification of tissue- engineered bone... |
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