| Objective:1.The midface of beagle dogs are in traction with a self designed rigid external fixed traction device to simulate suture traction in the treatment of middle facial dysplasia.It is objective that the animal model can provide the basis for the follow-up study of trans-suture traction osteogenesis.2.In the study,we use adenovirus as the carrier and gene therapy is used to transfect Ad-BMP2 into bone marrow mesenchymal stem cells and study the promoting effect of bone formation.The purpose is to provide theoretical basis for the treatment of craniofacial bone deformity and shorten the period of treatment.Methods:1.Four to five months beagle dogs were randomly divided into two groups: two in experiment group and one in control group.Implant nails were implanted as the bone marker in the bilateral zygomatic temporal suture,zygomandibular maxillary suture and palatine transverse suture in experimental group.The traction of the maxilla was carried out by the external cranial traction frame with canine fossa as bearing point,800 g force each side,elastic traction for 15 days.The control group only implanted the implant nail as the bone marker on both sides of the bone suture.The distance between two implant nails was measured by vernier calipers and X-ray examination,compared with preoperative and postoperative changes.X-ray and cephalometric measurements were used to measure change in the basilar-maxillary angle.HE staining was used to observethe width of the bone seams,the morphology and structure of the cells and the tissue of the new bone under the phase contrast microscope.2.Bone marrow mesenchymal stem cells were Isolated and cultured by whole bone marrow adherence method and identified of osteogenic and adipogenic induction.The growth curve of P3 cells was determined by CCK-8.The optimal number of Ad-BMP2 infection was measured and the expression of the target gene was detected by qRT-PCR and Western-blot in the cells of third days after the best infection complex number.Fourteen beagle dogs were divided into 3 groups and 4 beagles in each group.After the completion of the traction,the dogs were divided into 3 groups.Group A was injected with the same amount of normal saline,group B was injected with BMSCs without transfection of BMP-2,group C was injected with BMSCs transfected with Ad-BMP2,and maintained for 2 weeks and 4 weeks.The animals were killed and harvested according to the preset time nodes(2 in traction immediately,2 in each group at 2 and 4weeks).HE staining was used to measure the width of bone suture and thickness of trabecular bone.Masson staining is used to observe the degree of bone maturity.Immunohistochemical staining of type I collagen was used to measure IOD.The results were analyzed statistically.Results:1.The test group had a good retention of the beagle traction frame.In the experimental group,the maxillaries of dogs were protrudent in the process of traction gradually and the occlusal relationship changed to type II malocclusion.When the traction is 15 days,the coverage distance is about 8-9mm.Before and after the traction,the distance between landmark points indicated that the spacing between the transverse palatine suture was the largest,and zygomaticotemporal suture was the second,and zygomaticomaxillary suture was less.The skull base angle was no change before and after operation.Histology showed that the collagenous fibers in the suture of the control group were denser and the osteoblasts were visible on the edge of the suture,showing osteogenic activity.The experimental group significantly widened suture,the number offibroblasts increased significantly with loose collagen fiber.The direction of cell and fiber arrangement were parallel to the traction force.There were more vessels and bone marrow cavities and the active osteogenesis was shown.Osteogenic action was active.2.Bone marrow mesenchymal stem cells were isolated and cultured,and alizarin red staining was positive for 21 days after induction of osteogenesis.Lipid droplets of different sizes were found in the cells after 20 days of lipid induction,and the oil red O staining was positive.The growth curve of P3 generation BMSCs was “S” shape,and had strong proliferation ability.Compared with BMSCs group,qRT-PCR results showed that BMP-2 in Ad-BMP2 transfected BMSCs group increased significantly(P<0.05).Western-blot results showed that Ad-BMP2 protein expression in BMSCs group increased significantly.HE staining showed that the width of bone suture in three groups of A,B and C increased in turn,and the thickness of bone trabecula increased in turn.There was significant difference between the width of bone suture and the thickness of trabecular bone in any two groups(P<0.05).The width of bone suture gradually decreased in 0 weeks,2 weeks and 4 weeks,and the thickness of bone trabecula increased gradually.There was significant difference between the bone suture width and the thickness of trabecular bone between any two time points(P<0.05).When Masson staining was fixed for 4 weeks,the red staining area around B suture was more than that in group A,and C group was the most.The IOD of type I collagen immunohistochemical staining showed that the IOD increased in turn in group A,B and C,and the difference of IOD values was statistically significant in any two groups(P<0.05).At 0 weeks,2 weeks and 4 weeks,the IOD value increased first and then decreased,and the IOD value at any two time points was statistically significant(P<0.05).Conclusion:1.Young beagles can be used to establish an animal model of suture traction for experimental study.The traction of the cranial traction frame designed by ourselves in this experiment can be applied to the follow-up experiment.In the experiment,Kirschners are penetrated the base plane of the pyriform hole of the maxilla(the posteriorpart of the canine fossa),and the traction force and the center of resistance are similar.The traction direction is basically the same as the growth direction,and the maxilla is basically parallel to the front.2.Bone marrow mesenchymal stem cells cultured with all bone marrow adherent method have the ability of osteogenic differentiation.Ad-BMP2 can be successfully transfected into bone marrow mesenchymal stem cells and express BMP-2.Bone marrow mesenchymal stem cells can be used as seed cells for gene therapy.Bone marrow mesenchymal stem cells can promote the formation and reconstruction of new bone in bone suture traction area.The effect of Ad-BMP2 transfected on BMSC is more significant.Clinical application may shorten the treatment process of seture traction osteogenesis. |
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