| Objective Preparation of HA/ZrO2 porous nanomaterials using three-dimensional printing technology based on micro-CT scan data of cervical spine and lumbar spine in macaques;isolation and culture of bone marrow mesenchymal stem cells（BMSCs）from macaque monkeys;promotion of third generation monkeys as seed cells Bone;2.Preparation of gelatin/chitosan gel sustained-release system as a sustained-release carrier of bone morphogenetic protein（BMP-2）to maintain stable release of BMP-2;3.Construction and loading of third-generation macaque BMSCs BMP-2 Gelatin/Chitosan Gel Sustained Release System HA/ZrO2 novel tissue engineered bone,and studied the ability to repair large defects in macaques.Methods 1、The macaque lumbar vertebral body defect model was placed in animal dedicated Micro CT for volume scanning.The collected images are transmitted to the graphic workstation via the digital interface and the STL files are output in DICOM data format.A new type of HA / ZrO2 gradient composite material is designed and prepared according to the scanned data;Intramuscular injection of rhesus macaques monkey experimental monkey collected bone marrow,and isolated and cultured rhesus monkey bone marrow mesenchymal stem cells,and its subculture,the cell growth curve;2、The gelatin / chitosan hydrogel sustained-release system and compound BMP-2 were prepared by double emulsion cross-linking method.The drug loading,entrapment efficiency and sustained-release properties of BMP-2 were detected by enzyme-linked immunosorbent assay.3、BMP-2 gelatin / chitosan hydrogel sustained-release system wrapped third-generation rhesus monkey BMSCs planted in HA / ZrO2 gradient composite materials to build a new tissue engineering bone;4,The new HA / ZrO2 tissue engineered bone implanted in the cervical spine intervertebral space and lumbar vertebral body defect model: 24 healthy adult male rhesus monkeys were randomly divided into cervical spine group and lumbar spine group,12,cervical vertebra group A: BMP 5 gelatin / chitosan hydrogel sustained-release system and the third generation of BMSCs three-dimensional printing preparation of new gradient composite HA / ZrO2 tissue engineering bone 5（n = 5）;cervical B group: blank gradient composite HA / ZrO2 nano Five scaffolds（n = 5）;cervical C group: two from autologous iliac group（n = 2）;A group of lumbar vertebrae: 5（n = 5）new gradient composite HA / ZrO2 tissue engineering bone loaded with BMP-2 gelatin / chitosan hydrogel sustained-release system and the third generation of BMSCs were prepared,lumbar group B: Five blank HA / ZrO2 scaffolds with blank gradient and C group of lumbar vertebrae（n = 5）: 2 in autologous iliac group（n = 2）;The X-ray images of the experimental rhesus monkeys were observed at the immediately postoperative,8th,and 16 th week respectively.The animals were sacrificed 16 weeks after the operation.The specimens of group A and group B were taken out and examined by Micro-CT scanning.The osteogenic proteins of group A and group B were detected by RT-PCR,Western Blot and immunofluorescence at 16 weeks after operation.Type I collagen,osteoprotegerin（OPG）,bone morphogenetic protein 4（BMP-4）,bone morphogenetic protein 7（bmp-7）,osteocalcin（OCN）,osteopontin（OPN）basic fibroblast growth factor（b FGF）（ALP）and vascular endothelial growth factor（VEGF）.Biomechanical tests were performed on the experimental specimens of group A,group B and group C at 16 weeks after operation to evaluate the ability of repairing bone defects of new type of tissue engineering bone.Result Based on the STL file obtained by Micro-CT scanning,the porosity was designed and the HA / ZrO2 gradient composite scaffold material was prepared by photocuring molding technology（SLA）.Macroscopically,the macroscopical gradient composite scaffolds of the monkeys were observed with oval shape on the surface,and the pores of the materials were uniform.The macaque lumbar gradient composite scaffolds were semi-cylindrical with uniform pore size and uniform cross-section;The morphological observation of BMSCs isolated from whole bone marrow adherent separation showed that primary cultured BMSCs were adherent to adherent cells 48 h after culture,and the adherent cells were basically plated on the 7th to 10 th day.The mature BMSCs were mostly Shuttle type,mostly whirlpool-like distribution,and showed the trend of accumulation of growth.The 1: 3 ratio of passage cells were basically the same growth pattern,the proliferation rate was significantly higher than the primary cells cultured 3 4d can basically covered the bottom of the Petri dish.After passage 6,BMSCs in multiple passage experiments showed that: the growth rate of cells was significantly slowed down,and the morphology of cells appeared triangular,polygonal and irregular shape.If subculture continued,vacuoles appeared inside the cells,Aging,apoptosis begins.Morphological observation of adherent growth of cells showed that it was consistent with the characteristics of BMSCs;the growth curve of BMSCs from the 1st,3rd,6th,and 9th generations was drawn by MTT method;The drug loading,entrapment efficiency and sustained-release properties of BMP-2 were detected by enzyme-linked immunosorbent assay.The drug loading rate and entrapment efficiency of BMP-2 in the first two days were 63.47 ± 0.03% and 95.21 ± 0.05% respectively,The drug loading rate and entrapment efficiency decreased to 34.29 ± 0.1% and 51.43 ± 0.15% respectively on the 9th day,and decreased slowly from the 9th day to the 12 th day.The drug loading rate and entrapment efficiency decreased To 31.18 ± 0.12)% and 46.78 ± 0.18)%,respectively,and the first 15 d BMP-2 drug loading rate and entrapment efficiency changed to 31.06 ± 0.14% and 45.86 ± 0.14%,indicating that it entered into a relatively stable phase from the 12 th day to the 15 th day.3,RT-PCR and Western Blot of type I collagen,OPG,BMP-4,BMP-7,OCN,OPN,b FGF and ALPVEGF after operation showed that the expression of group A gene in cervical and lumbar group（P <0.05）.The result of 16 CT micro-CT scan and immunofluorescence showed that the bone mass in group A was significantly higher than that in group B（P <0.05）The results of pressure test showed that the cervical spine A group was 18.72 ± 2.33 MPa;the B group was 12.81 ± 2.13 MPa;the C group was 20.31 ± 3.61 MPa.Lumbar A group 38.72 ± 1.33 MPa;B group 20.24 ± 1.64 MPa;C group 40.41 ± 2.73 MPa.The ultimate compressive strength of group A and group B had no significant difference（P> 0.05）,group A was greater than group C（P <0.05）;Conclusion Bone marrow mesenchymal stem cells（BMP-2）coated with BMP-2 gelatin / chitosan gel system have the biocompatibility and biomechanical strength to implant HA / ZrO2 porous nanomaterials prepared by three-dimensional printing technology.Therapeutic principles,can be a good repair of vertebral bone defects in rhesus monkeys,is an ideal tissue engineering bone material. |
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