| 1. BACKGROUNDBone defects caused by trauma, deformation, tumors and other diseases pose seriouschallenges to orthopedic surgeons. The conventional means of bone grafting includeautogenic and allogenic bone graft.Although considered as the "golden standard" for bonerepair policy, the clinical application of autogenic bone grafting is limited due to its limitedsources as well as postoperative complications. Allogenic bone graft, although withadequate sources, has low transplant success rate, which is attributed to the severe rejectiontriggered by the exogenous bones. Therefore, the development of bone graft material tomake up for above shortages has become a hot and difficult spot in current research. Inrecent years, the rapidly developed tissue engineering technology is expected to shoulderthis task.2. OBJECTIVEWith inorganic hydroxyapatite and organic polycaprolactone as raw materials, artificialbone scaffold is built by Selective Laser Sintering technology, a rapid molding technology.The scaffold is then used to prepare for the tissue engineered nano-hydroxyapatite/polycaprolactone bone scaffold by being combined with seed cells continuously secretinghuman bone morphogenetic protein-7. The clinical application of tissue engineered nano-hydroxyapatite/polycaprolactone bone bracket is approved after various examination andanalysis, including characterization, in-vitro biocompatibility, bioactivity as well as repairability for bone defects, laying the experimental foundation for its future application in thetreatment of bone defects.3. MATERIALS&METHODSPart one. Preparation of Nano-hydroxyapatite/polycaprolactone bone scaffolds:Nano-scale hydroxyapatite and micron-scale polycaprolactone were raw materials in thisstudy. According to the reaction formula：10Ca(NO3)2+6(NH4)2HPO4+8NH3H2Oâ†'Ca10(PO4)6(OH)2+20NH4NO3+6H2O,nano-scale hydroxyapatite powder was prepared. And micron-scale polycaprolactonepowder was obtained by cryogenic pulverization technique. As characterized byScanning electron microscope (SEM) and X-ray diffraction, these two prepared powder materials proved to be the needed substances and comply with the demand of the presentstudy in respect of particle size.These two powder were mixed in V-type blender for5min,15min,30min,45min,1h and2h respectively before being amounted to SEM to observethe evenness of mixing and determine the optimum mixing time. The mixed powder ofnano-scale hydroxyapatite and polycaprolactone was prepared in different weight ratioaccording to the optimum mixing time. The optimal parameters for the preparation of fnano–scale hydroxyapatite/polycaprolactone bone scaffolds were determined byexploring and optimizing the parameters of selective Laser Sintering Machine. Theresulted scaffoldss were tested with macroscopic observation, scanning electronmicroscope, porosity, compressive strength, MTT, alkaline phosphatase staining andalizarin red staining to examine the morphology, mechanical strength, biocompatibility andbiological activity. The nano-scale hydroxyapatite/polycaprolactone scaffoldss with bestcomprehensive performance were selected after comparison and applied as samples in thefollowed in-vivo experiments.Part two. Preparation of genetically modified seed cells:In this part of experiment, bone marrow was isolated from New Zealand rabbit femur toextract mesenchymal stem cells which were cultivated and passaged. The3rd generation ofthese stem cells could be used for experimental purposes. According to the gene sequences(genetic serial number: NM001719) of human bone morphogenetic protein-7(hBMP-7) inGenBank, a recombinant adenoviral vector Ad-CMV-hBMP7-ires-eGFP was preparedwith genetic engineering techniques and verified with enzyme digestion and sequencing.The correctly verified reconstructed adenoviral vector was then subjected to packaging,amplification and purification. The titer of recombinant adenovirus was determined by293cell infectious dosage method.hBMP-7was transfected into the previously obtainedmesenchymal stem cells of rabbit bone marrow through in-vitro gene transfectionmethod to produce transgenic seed cells needed in this study. With RT-PCR, Western blotand calcium nodules staining, the expression of targeted gene hBMP-7cells of transgenicseed cells in-vitro environment and their effect on osteogenic differentiation ofmesenchymal stem cell from bone marrow were observed.Part three. Composition of nano-hydroxyapatite/polycaprolactone bone scaffoldsand genetically modified seed cellsIn this part of study, the model of New Zealand rabbit femur bone defect and largesegmental radial bone defect were established to implant mere polycaprolactone stents, nano-scale hydroxyapatite/polycaprolactone bracket, and nano-scale hydroxyapatite/polycaprolactone ester+genetically modified seeds cell stents into bone defect sites. Therepair of New Zealand rabbit bone defects was observed and assessed by imaging andhistological examination at different time to determine if the nano-scale hydroxyapatite/polycaprolactone bone scaffold combined with seed cells carrying the targeted genehBMP-7had the ability of synergistically promoting the differentiation of mesenchymalstem cells from bone marrow into the osteoblast.4. RESULTSPart one.As shown by Scanning Electron Microscope and X-ray diffraction, the scale of preparedhydroxyapatite and polycaprolactone powder had reached the nano level and micron levelrespectively with99%sample in compliance with standard, indicating that these twomaterials powder reached the requirements for raw material in this study. The SEMscanning results of powder mixed with V-type blender for5min,15min,30min,45min,1hand2h respectively had shown that the evenness of resulted powder mixed for2h wassimilar to that mixed for1h and higher than those mixed for5min,15min,30min and45min, indicating that the1h was the optimum mixing time. The two raw material powderwere mixed in accordance with the ratio of "Nano-scale hydroxyapatite: polycaprolactone=0:100,5:95,10:90,15:85,20:80," to sinter Nano-scale hydroxyapatite/polycaprolactonebone scaffold with the optimized selective laser sintering machine parameters. Theprepared artificial bone scaffold was then examined with Scanning Electron microscope,porosity, compressive strength, MTT, alkaline phosphatase staining and alizarin redstaining. It was shown in these tests that the scaffold produced with selective laser sinteringmachine had exhibited three-dimensional interconnected pore structure. The porosity of thebone scaffold decreased slightly in line with the increase of proportion of Nano-scalehydroxyapatite, while mechanical strength had significantly increased, reaching up to7Mp. All prepared artificial bone scaffold demonstrated good biocompatibility; At sametime, the research of biological activity showed that the bone group containing nano-scalehydroxyapatite took on much better biological activity than pure polycaprolactone group,and the biological activity increased in line with content of nano-scale hydroxyapatite.Part two.Mesenchymal stem cells separated and extracted from the New Zealand rabbit bonemarrow were cultured and passaged. The3rd generation was then used for experimental purposes. The recombinant adenoviral vector Ad-CMV-hBMP7-ires-eGFP wassuccessfully constructed with genetic engineering techniques and then tested with enzymedigested sequencing and verified. It was shown by the results that sequence of the nucleicacid was100%identical to the (NM-001719) coding sequence of hBMP7sequencepublished by PUBMED, which could prove the fragment has been inserted in rightdirection. The293cells infected with recombinant adenovirus vectorAd-CMV-hBMP7-ires-eGFP were gradiently diluted and then subjected to virus infectioustiter calculation with fluorescent counting method. The calculated titer for recombinantadenoviral vector Ad-CMV-hBMP7-ires-eGFP was approximately1.1×1011v.p./ml.1-3dfluorescent counting was recorded for the3rd generation of mesenchymal stem cells fromrabbit bone marrow transfected with the recombinant adenoviral vectorAd-CMV-hBMP7-ires-eGFP. Using relatively high cell transfection efficiency, relativelyslight cell toxicity and more economic virus application as criteria, the optimummultiplicity of infection (MOI) of mesenchymal stem cells by recombinant adenovirusvector Ad-CMV-hBMP7-ires-eGFP was50:1. Mesenchymal stem cells from rabbit bonemarrow was then infected with optimum multiplicity of infection (MOI=50:1).Theexperimental group (mesenchymal stem cells from rabbit bone marrow infected withrecombinant adenoviral vector Ad-CMV-hBMP7-ires-eGFP), the control group(mesenchymal stem cells from rabbit bone marrow infected with the Ad-CMV-ires-eGFP)and the blank control group (unprocessed mesenchymal stem cells from rabbit bonemarrow) were continuously cultivated for three weeks under induction. The real-time PCRquantitative determination and Western blot results of hBMP7sampled on day4,7,14,21,28had shown that mesenchymal stem cells from rabbit bone marrow were capable ofeffectively intervening the expression of the foreign target gene after being transfectedwith recombinant adenoviral vector Ad-CMV-hBMP7-ires-eGFP. The expression oftransfected hBMP7reached a peak on day14and then reduced after day21. It wasobserved under inverted microscope during culture period, the cells of the experimentalgroup gradually developed osteoinductive-like morphology change with prolongedcultivation time. The cells transformed from spindle into cubes and polygons, along withemergence of calcified plaque.At the same time, the results of ALP staining, collagentype-I PCR quantitative real-time determination and alizarin red staining of samples takenon day4,7,14,21,28under induction culture condition had shown that: the positivestrength of ALP staining for samples from experimental group was the highest, along with the most strong expression of type-I collagen. It also had the largest number of calciumnodules with obvious formation, which indicated that transfected mesenchymal stem cellsfrom rabbit bone marrow in experimental group had been successfully induced and begunthe differentiation into osteoblasts.Part three.The model of New Zealand rabbit femur bone defect at epicondyle,6mm in diameterand10mm in depth and large segmental radial bone defect at middle part,20mm in lengthwere established respectively. The defect sites were then transplanted with correspondingmaterial and sutured with incision. Conventional injection of intramuscular antibiotics wasconducted for7days after operation. It has shown that no infection phenomenon isobserved during postoperative1-14days. The wound animals for experimental purposehad been healed well, with all incisions taking on grade I healing. And these resultsshowed that the implanted material had no significant adverse effects on experimentalanimals. The Micro-CT results for the samples taken in postoperative3w,6w,9w and12whad shown that the group implanted with nano-scale hydroxyapatite/polycaprolactone+genetically modified seed cell stent produced better bone defect repair than other3groups.The repair of local bone defects including cortical bone had almost completed at9w. Fulland continuous new bone structure exhibited at12w. The channel of marrow cavity and thedefects had almost be repaired. And no significant adverse inflammatory or immuneresponse were observed as shown by biopsy implanted in the same region. The groupimplanted with nano-scale hydroxyapatite/polycaprolactone+genetically modified seedcells stent had generated much more new bones than the rest group. The implanted materialhad been observed with relatively obvious degradation, which was then replaced by fibroustissue or new bones.5. CONCLUSIONSAs one of rapid formation technology, Selective Laser Sintering technology has hugepotential in applications of tissue engineering and even orthopedic clinical treatment,thanks to its advantage of rapid production and batch processing with three-dimensionalcomputer model.It has been proved through experiment that the artificial bone scaffold—prepared withnano–scale hydroxyapatite and polycaprolactone powder as raw material and selectivelaser sintering technology—has highly connected three-dimensional pore structure,relatively high porosity, the more outstanding mechanical strength as well as excellent biocompatibility and biological activity.After being transfected with human bone morphogenetic protein-7gene throughadenoviral vector, the mesenchymal stem cells from the rabbit bone marrow cansignificantly promote the induced differentiation of mesenchymal stem cells from rabbitbone marrow into osteoblasts.Compared with relatively simple artificial bone scaffold, the tissue-engineered bonescaffold constructed with nano-scale hydroxyapatite/polycaprolactone+geneticallymodified seed cells has more significant bone defect repair capacity, displaying its greatapplication potential in the treatment of clinical bone defects. |
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