| At present,autograft,allograft,heterogenous bone and artificial bone are mainly used to repair bone defects.The problems of immune rejection and histocompatibility exist in allograft bone graft.The problem of immune rejection inevitably exists in heterogenous bone,which may even lead to infection of animal diseases.The use of artificial bone substitute materials,such as various metal alloys and macromolecular polymers,is not only limited to histocompatibility,biological or mechanical properties,but also may cause other diseases and pain due to foreign body reaction.Bone tissue engineering provides a new direction for the repair of alveolar bone defects.Bone tissue engineering is a combination of medical principle and engineering technology.Under the induction of various factors,the cells inoculated on the scaffold materials are continuously proliferated to gradually achieve the goal of repairing bone defect.The key elements are cells,scaffolds and induction factors.Studies have shown that bone marrow mesenchymal cells can differentiate into osteoblasts,chondrocytes and muscle cells under appropriate conditions,which are ideal seed cells for tissue engineering,but how to regulate directional differentiation is still a problem.Bioglass promoting tissue repair has outstanding physical and chemical properties,the fiber deposition equipment to print sol-gel type micro/nano three-dimensional porous scaffolds,bioactive glass can be on the premise of guarantee the mechanical properties,naturally formed by micro glass ball stack nanoscale pore,promote cell adhesion,migration and differentiation,endowed with bioactive glass scaffolds more excellent performance;At the same time,the fiber deposition technology avoids the conventional hot melt,which is beneficial to retain the biological activity of the material or the biological factors that may be added.3D printing can prepare 3D supports with specific geometry,size and diversified internal pore structure to meet different clinical needs.Chitosan CS is a high quality carrier with biological characteristics.Nel-like type I molecular-1(NELL1)is a new and effective osteogenic factor,and its related researches are being explored and perfected.In this experiment,CS nanoparticles loaded with nell-1 DNA(pDNA-NELL1)were constructed,and the nanoparticles were combined with the 3d-printed sol-gel micro-nano bioactive glasses(BG).In rhesus monkeys between bone marrow mesenchymal stem cells(ipads marrow mesenchymal stem cells,BMSCs)as seed cells,pDNA-NELLI as induced factor,BG for support,composite material of bone tissue engineering,into the preparation of rhesus monkeys have alveolar bone defect model,to evaluate the in situ osteogenesis ability of tissue engineering bone graft material,under the observation of BMSCs mediated gene modified micro/nano biological glass composites in rhesus monkeys alveolar bone defect and regenerative effect.In order to provide a more effective and feasible tissue-engineered bone material for clinical repair of alveolar bone with different geometric shapes and sizes,as well as bone defects,tissue engineering bone repair has taken a great step towards personalized development.This experiment is divided into the following two parts:Part OneThe preparation of NEll1-DNA tissue-engineered bone materials using 3D printed micronanometer bioactive glass scaffold mediated by BMSC.Object:Construct a novel tissue engineered bone composed of 3D-printed bioactive glass and chitosan nanoparticles(BG/CSn)composites loaded with Nel-like type I molecular-1 DNA(pDNA-NELL1)and/or bone marrow mesenchymal stem cells(BMSCs).Methods:1.Relevant studies have shown that bioactive glass has good bioactivity and degradation,which can provide three-dimensional environment of connected porous bone tissue repair,while micropore size in the range of 150-500 pore m and porosity not less than 40%can promote the proliferation of bone cells.Used in the experiment of 3D printing micro/nano bioactive glass scaffolds by south China university of technology national engineering research center for human tissue function reconstruction provides,in view of the particularity and the requirements of this experiment,integrated with sol-gel technique and organic template self-assembly technique was used to prepare mesoporous glass microspheres,with a diameter of about 350 um of bioactive glass microspheres as printed material,polyvinyl alcohol as binder,printing bioactive glass fiber deposition equipment of three-dimensional porous scaffolds,the successful preparation of 10 mm × 10 mm × 5mm rectangle bioglass bracket,the aperture width is 250microns.High concentration of PBS buffer and sodium alginate phosphate complex were applied on the surface of the scaffold to form K3CaH(PO4)2 deposition surface to maximize the compressive strength of bioactive glass scaffold materials,and have good in vitro apatite formation activity.Its related performance,the research center has done a complete experiment and research and published results.2.Extracting and culturing BMSCs:Six-year-old healthy adult female rhesus monkeys weighing 3-5kg were selected for iliac bone marrow puncture.8-10mL marrow was extracted and transferred to a flask,and then cultured in DMEM/F12 medium with 10%fetal bovine serum and 1%penicillin-streptomycin double antibiotic in a cell incubator.The third and fourth generations of the cells were collected for preparing the tissue engineered bone.3.Preparation of growth factor pDNA-NELL1 nanoparticles carried by chitosan:10 g chitosan was dissolved in 360 mL 1-methy l-2-pyrrolidone(NMP)and added with 35 mL NaOH(15%,aq)and 57.5 mL CH3I while stirring.Starch test paper was used to detect iodine residue,and the sediment was dried by lyophilization,labelled as TMC(trimenthyl chitosan).1 mg TMC was dissolved in 1 mL bone salivary protein(BSP)to prepare TMC solution with a concentration of 1 mg/mL.Then 0.4 mL of pDNA-NELL1 was dissolved in 1.2 mL above-mentioned TMC-BSP solution to prepare DNA solution with a concentration of 0.5 mg/mL.The mixed solution was shaken vigorously immediately after mixing,and then placed in a 37?water bath for 30 min,then the nanoparticle solution loaded with pDNA-NELL1 with an N/P value of 10 was prepared.4.BMSCs mediated the preparation and experimental grouping of 3D printed sol-gel bioactive glass 3D scaffold composite Nelll tissue engineering bone materials.The national research center of human tissue function reconstruction engineering and technology of south China university of technology provides the experimental cuboid bioglass scaffold with a diameter of 250 mm.The experiment was divided into 4 groups,control group 3 and experimental group.Construction of tissue engineered graft materials:Four types of graft material were constructed:BG,BG+BMSCs,BG\CSn + BMSCs,and BG\CSn(pDNA-NELL1)+ BMSCs.For each type,BG scaffold was pretreated by sterilization using UV radiation and washed by PBS solution for 5 times,3 min at a time;then the BG scaffold was immersed into low glucose DMEM within a 50mL tube for pre-wetting overnight.Before the implantation,lmg/mL chitosan solution and BMSCs suspension with 5×106 cells/mL were prepared,then the graft materials were prepared as follows.?.BG:220?L saline was added to 3D printed BG scaffold and infiltrated into the scaffold for 4-6h,then 200 ?L saline was added to the scaffold.?.BG+BMSCs:220 ?L saline was added to 3D printed BG scaffold and infiltrated into the scaffold for 4-6h,then 200 ?L BMSCs suspension was added to the scaffold.?.BG\CSn + BMSCs:200 ?L chitosan solution and 20 ?L saline were mixed with vibration for 30s,then the mixture was added to the 3D printed BG scaffold and infiltrated into the scaffold for 4-6h,followed by adding 200 ?L BMSCs suspension to the scaffold.?.BG\CSn(pDNA-NELL1)+ BMSCs:200 pL chitosan solution and 20 ?L above mentioned pDNA-NELL1 solution were mixed with vibration for 30s,then the mixture was added to the 3D printed BG scaffold and infiltrated into the scaffold for 4-6h,followed by adding 200 ?L BMSCs suspension to the scaffold.For above graft materials 2-4,1×106 cells were seeded on each scaffold.The implantation was performed immediately after the preparation of each scaffold.Results:1.The microstructure of the scaffold was observed by scanning electronic microscopy(SEM)and transmission electron microscopy(TEM).Homogeneous BG microspheres were combined together to form porous structure with connected pores.2.BMSCs of rhesus monkeys were observed under the inverted microscope,displaying uniform cell morphologies,which were spindle cell-like with compactar rangement and manifested as convergence-like or vortex-shaped colony arrangement growth.3.Construction of tissue engineered graft materials:Four types of graft material were constructed:BG,BG+BMSCs,BG\CSn + BMSCs,and BG\CSn(pDNA-NELL1)+ BMSCs.Conclusion:Construct a novel tissue engineered bone composed of BG\CSn(pDNA-NELL1)+ BMSCs.Part TwoAnimal experiments on the restoration of alveolar bone defects in rhesus monkeys by tissue engineeringExperiment 1.Animal experiment:Establishment of the alveolar bone defect model in rhesus monkeys and Bone tissue engineering material implantationObject:Establish of the alveolar bone defect model and implant biological material implantation,to study their osteogenic activities by repairing bone defects in rhesus monkeys.Methods:1.Establishment of the alveolar bone defect modelFour adult female rhesus monkeys were selected and randomly coded as A,B,C and D,and their(upper,lower,left,and right)jaws were labelled as 1,2,3,4,respectively according to the international oral area.Thus,there was a total of 16 jaws from the group of four monkeys.The animals fasted from water and food for 24h prior to the operation,and were administered general anesthesia with 3%pentobarbital sodium via a slow intravenous bonus injection.Following disinfection,the first and second premolar teeth from each jaw were removed,where the gum was also separated using a gum detacher.Buccal mucoperiosteal flap of the surgical area was turned over;and a dental high-speed hand piece was used to remove the peripheral socket bone and buccal bone cortex,as well as intact lingual bone cortex was retained to prevent the loss of material.A bone defect area of approximately 10mm×10mm×5mm was established for each site,and the cancellous bone was exposed.2.Biological material implantation and group:The tissues at the defect areas were rinsed with saline and the bone debris were removed before the implantation.The four types of graft materials(BG,BG+BMSCs,BG\CSn + BMSCs,and BG\CSn(pDNA-NELL1)+ BMSCs)were implanted to ensure that there were four materials in each jaw region of each rhesus,so as to eliminate the differences between the different monkeys and jaw regions.After the implantation,the graft materials were completely covered by buccal mucoperiosteum and fixed using gingival restoration and apposition suture.The animal experiments set three control groups(1,2,and 3)and one experimental group.Control group 1:BG;control group 2:BG+BMSCs;and control group 3:BG\CSn + BMSCs.The experimental group used BG\CSn(pDNA-NELL1)+BMSCs.3.X-ray examinations were performed immediately post-operation to verify correct implantation of the tissue engineered bone into the bone defect area,4.postoperative care and observation:Following gingival suture and local disinfection,the animals were fed with soft food for two weeks after the operation.To prevent systemic infection,1 g ceftriaxone sodium and 4 mg dexamethasone were administered via an intramuscular injection for seven consecutive days,and 50 mL of 2%chlorhexidine was used twice daily for two weeks for oral rinsing and surgical site disinfection.The wound was closely observed for signs of inflammations(e.g.,redness,swelling,or exudation)and complications(e.g.,suppuration and necrosis),and the suture was removed two weeks later if the abovementioned conditions were absent.Results:1.After establishment of the alveolar bone defect model,the four types of graft materials(BG,BG+BMSCs,BG\CSn+BMSCs,and BG\CSn(pDNA-NELL1)+BMSCs)were implanted to ensure that there were four materials in each jaw region of each rhesus,so as to eliminate the differences between the different monkeys and jaw regions.2.X-ray examinations post-operation displayed correct implantation of the tissue engineered bone into the bone defect area.3.The reactivity of animals in each group was well after operation,and all wounds showed healing by first intention.No local redness and swelling,fester and necrosis were found.Conclusion:The alveolar bone defect models of rhesus monkeys were established,and the four types of graft materials(BG,BG+BMSCs,BG\CSn + BMSCs,and BG\CSn(pDNA-NELL1)+ BMSCs)were implanted.to ensure that there were four materials in each jaw region of each rhesus,so as to eliminate the differences between the different monkeys and jaw regions.The reactivity of animals in each group was well after operation,and all wounds showed healing by first intention.No local redness and swelling,fester and necrosis were found.Experiment 2.The evaluation of the bone tissue engineering material implantation for the alveolar bone defect model of rhesus monkeysObject:Observations and detections were carried out 12 weeks after the implantation,study the repair effect of BG\CSn(pDNA-NELL1)+BMSCs on the regeneration of large alveolar bone defect in rhesus monkeys.Methods:1.X-ray film of bone graft area was photographed at 12w after materials being implanted,and the growth of bone was observed.2.All the jaw samples containing the repair regions were obtaned from the rhesus.Before taking the specimen,the animals were treated similar to the procedures prior to the implantation.For each animal,the canine teeth of each jaw were removed following disinfection.A trapezoid incision was made using a scalpel at the sampling location.A gum detacher was used to separate the buccal and lingual mucoperiosteal flap in the surgical region.A high-speed dental hand piece was used to expand and take the specimen,which was a bone block of 15mm × 12mm × 7mm.Each specimen was labelled immediately after the sampling.3.The up and down as well as left and right four alveolar bones at bone graft area were cut out from each animal after 12 weeks,which were examined by micro-computed tomography(Micro-CT).4.Histopathological observations:The specimen was fixed in a 10%formaldehyde solution for seven days and hematoxylin-eosin(HE)staining.Results:1.An x-ray of the experimental group(BG\CSn(pDNA-NELL1)+BMSCs)suggested an unclear boundary between the bone graft materials and the surrounding bone.The bone graft materials were almost completely absorbed and reconstructed.A large amount of regenerated trabecular bone was observed,which was clearly arranged in a porous network structure uniformly.The alveolar crest was higher than those of the control groups;however,it was similar to that of the peripheral host bone.A representative x-ray observation from control group 1(BG)is shown a clear boundary between the grafting materials and the surrounding bone.The more similar the materials used for absorption and reconstruction were to the surrounding bone,the better the observed regenerative effect.There scattered loose bone trabecula and grafting materials were clearly observed near the alveolar crest.A representative x-ray observation from control group 2(BG + BMSCs)is shown an unclear boundary between the grafting materials and the surrounding bone.The bone graft materials were almost completely absorbed and reconstructed.The trabecula was clear in structure,less in quantity,and exhibited a scattered arrangement.A representative x-ray observation from control group 3(BG\CSn + BMSCs)is shown an unclear boundary between the grafting materials and surrounding bones.The bone graft materials were almost completely absorbed and reconstructed.The trabecula was clear in structure,fewer but thicker than the BG + BMSCs group,and exhibited a scattered arrangement.The radiodensity shadow was darker in the grafting region than that of the BG + BMSCs group,indicating better osteogenesis effect of the BG\CSn + BMSCs group than the BG + BMSCs group.2.New bone formation was observed by naked eye on the specimens.The size of the new bone was similar to that of the implanted materials,and the boundary between the new bone and the surrounding normal bone tissue was not obvious.The new bone exhibited a smooth surface,without obvious depression or protruding,and was covered by periosteum.3.Micro-CT 3D reconstructionThe models revealed that new bone formed in both the experimental and the control groups.Compared with the control groups,the trabecula of the experimental group was thicker,denser and more closely arranged.In the BG group,the trabecula was disorganized and sparsely formed with cavities.The trabecula was clear in structure with large quantities in both BG + BMSC and BG\CSn + BMSC group without cavities.The new bone was integrated with the surrounding bone without obvious boundary,and the bone defect healed well.4.Histopathological observationsHematoxylin-eosin(HE)stained tissue sections at 12 weeks post-operation revealed that the new bone was completely fused with the host bone in the experimental group(BG\CSn(pDNA-NELL1)+ BMSCs),while the scaffold was completely resorbed.Dense new bone with regular osseous lamella has formed,and there was a large number of mature osteocytes and new vessels.New bone in the control group 1(BG)exhibited several cavities and connective tissue with unabsorbed scaffolds,and few osteocytes formed,which were sparsely distributed with inflammatory cell infiltration.The density and quantity of the new bone were lower in the BG + BMSCs(control group 2)and BG\CSn + BMSCs(control group 3)groups than those of the experimental group,but higher than those of the control group 1(BG),and had fewer number of cavities and connective tissue than that of the control group 1(BG).Unabsorbed scaffolds with macrophages on the edge were also observed in control groups 2 and 3.Conclusion:Tissue engineered bone BG\CSn(pDNA-NELL 1)+BMSCs of this study exhibited good osteoconductivity for promoting the formation of new alveolar bone tissue,and NELL1 gene played a promotional role in bone regeneration. |
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