| Dental implants as mostly used and ideal treatment for patients without teeth.Titanium and its alloys are the most widely used implant materials due to their excellent mechanical strength,chemical inertness,and high biocompatibility.With the increasing morbidity of type 2 diabetes mellitus,the demand for implant surgery in type 2 diabetes mellitus patients without teeth is also increased.However,the poor osteogenic ability of patients with type 2 diabetes mellitus leading to poor implant osseointegration has become a prominent issure.Recently,many studies have focused on improving implant stability by managing implant surface characteristics to improve the success rate of the implantation surgery and the survival rate of the implants in type 2 diabetes mellitus patients.Selective laser melting technology has attracted extensive attention in recent years,which is conducive to the rapid production of implants with complex shapes with high material utilization rate,uniform density distribution,and minimum post-treatment requirements.However,whether the surface of titanium implant treated by SLM can improve the level of osseointegration in T2 DM state,thereby increasing the success rate of implantation treatment in T2 DM patients,remains to be confirmed.In this study,Ti6Al4 V alloy powder was used manufactured titanium samples by selective laser melting.In order to evaluate whether selective laser melting titanium implants lead to a better effect of early bone integration in type 2 diabetic rats.A series of in vitro and in vivo experiments were carried out.The surface properties and the mechanical property of the whole selective laser melting implant were researched.And its effect on the adhesion and proliferation of human gingival fibroblasts,and osteogenic differentiation of osteoblasts was investigated in vitro.Part 1: Design,manufacture and analysis of selective laser melting implants Objective: To design and 3D printed manufacture selective laser melting titanium implants and analyze their surface.Methods: Selective laser melting implants and titanium plates were designed using the 3D design software Pro Engineer 5.0.Pure titanium implants and disks were manufactured using the control milling machine.The pure titanium implants and disks with the same sizes as the products were divided into three groups based on the surface treatment: basic polishing treatment,sandblasting and large-grits etching treatment and selective laser melting treatment using Ti6Al4 V powders.The surface morphology of implant was observed by SEM.The surface elements of implant were analyzed by X-ray photo electron spectroscopy.The wetting ability of implant surface was measured by the contact Angle measurement system.The surface morphologies of the three samples were observed by atomic force microscope and the roughness of the implant surface was measured.Yield strength of the implants was measured by uniaxial compression tests using an electronic universal testing machine.Results: The sample surface of the selective laser cladding group is in the shape of peaks and valleys;the surface of the sandblasting acid etching group has a porous structure Shallow,relatively flat,flat structure is seen on the surface of samples without treatment group.Selective laser cladding implants with high titanium content In the control group.The wettability experiment showed that the surface wettability of the selective laser cladding group was better than that of the control group.Rough The measurement results show that the surface roughness of the selective laser cladding group is higher than that of the control group.Mechanical test results Now,the elastic modulus of the implants in the selective laser cladding group is lower than that of the other two groups.Conclusion: Selective laser melting implants have better surface morphology and mechanical property than the other groups.Part 2: The effect of selective laser melting implants on the adhesion and proliferation of human gingival fibroblasts,and osteogenic differentiation of osteoblasts and biosafety assessment Objective: To observe the effect of selective laser melting implants on the adhesion and proliferation of human gingival fibroblasts,and osteogenic differentiation of osteoblasts in vitro.Methods: MG63,HGF-C339 and L929 cells were resuscitated,cell passage was carried out and cells were in high sugar medium cultured into 12-well plates with different titanium plates according to experimental groups.HGF-C339 cells were stained with acridine orange/ethidium bromide reagent after 3-and 72-hours’ culture.Cell morphology and spreading on the surface of samples were observed by an inverted fluorescence microscope.When the MG63 cell growth reached 80% density,changed the medium with osteogenic induction medium.4 and 8 days later,osteogenic related genes Col I,Runx2,ALP,OC,OSX,OPN were detected,and alizarin red staining was performed.Results: Compared with pure titanium group and sandblasting and large-grits etching group,HGF-C339 cells of selective laser melting showed more proliferation and displayed membrane protrusions.After 72 hours,cell number was increased,adjacent HGF-C339 cells were connected via cellular bridges,filamentous pseudopods were also observed.In contrast,while HGF-C3339 cells grown on pure titanium group and sandblasting and large-grits etching group had no obvious proliferation.Osteogenesis gene expression of MG63 cells The trend of q PCR and Western-Blot detection results is consistent: Compared with the pure titanium group,SLA and SLM have a certain promotion effect on the expression of osteogenic genes.SLM has a more obvious up-regulation effect on MG63 osteogenic gene expression.Cultured cell on the surface of titanium sheet for 4 days.After8 days,the expression of COL I,Runx2,OPN,COL I,OPN and OC in the SLA group and SLM group were significantly up-regulated compared to the 8-day culture.Alizarin red staining showed that after 21 days of osteogenesis induction,larger and denser mineralized nodules were induced on the surface of titanium plates in the selective laser melting group compared with sandblasting and large-grits etching group and pure titanium group.The biological safety evaluation of the titanium sheet material showed that the four groups of differently treated titanium sheet L929 cells grew well.Conclusion: Selective laser melting samples biological safety test is non-toxic,can promote adhesion and proliferation of HGF-C339 cells and the early osteogenic differentiation of MG63 cells,and also may promote the formation of collagen in the process of osteogenesis.Part 3: The peri-implant osseointegration effect of selective laser melting implants in type 2 diabetes mellitus rats Objective: To observe the effect of selective laser melting on peri-implant osseointegration in type 2 diabetes mellitus rats.Methods: The animal model of type 2 diabetes mellitus rats was constructed.36 type 2diabetes mellitus rats were randomly selected and divided into 3 groups(n=12)for implant surgery.Micro-CT scanner was used to evaluate the osteogenesis status after implant surgery at 4 and 8 weeks.Bone volume/total volume,bone surface/bone volume,trabecular thickness,trabecular number and trabecular spacing were calculated.The bone tissue around the implant was collected,and the protein content of Western-blot was analyzed.The implant-bone samples were stained by Van Gieson to observe new bone formation,and the bone-implant contact rate was calculated.Then,each implant was fixed with a removal tool and the maximum removal torque was measured.Removal tool connecting with digital torque meter was used to remove the implants.Results: Two weeks after the injection of streptozocin,blood glucose of type 2 diabetes mellitus group rats raised quickly to 28 mmol/L,and was stable within 27 ～ 28.5 mmol/L over the next four weeks,which mean the type 2 diabetes mellites rat models were successfully established.The micro-CT images showed that selective laser melting group,compared with other group,contains more bone deposition around the implant,more regular and continuous bone permutation and morethe bone volume on the surface of the implant.Statistical analysis of trabecular parameters showed that,compared with the other two groups,trabecular thickness and bone surface/ bone volume in selective laser melting group were significantly increased at 4 weeks after implant surgery(P < 0.05),while trabecular spacing was significantly decreased(P < 0.05).At 8 weeks after implant operation,the trabecular number,bone surface/ bone volume and bone volume/total volume of selective laser melting group was significantly increased(P < 0.05)while trabecular spacing was significantly decreased than the other groups(P < 0.05),which suggested a tighter bone formation around the selective laser melting implant.Western-blot showed that the protein expression of each group was similar.Histological images of the three groups showed obvious newly formed bone tissues stained by van gieson in the selective laser melting group and sandblasting and large-grits etching group implants.The new bone in the selective laser melting group was significantly more than that in the sandblasting and large-grits etching group,and the selective laser melting group had the highest bone-implant contact ratio,while there were almost no bone connections around the pure titanium implants at this stage.After 8 weeks of recovery,bone connections were observed on selective laser melting and sandblasting and large-grits etching samples’ surfaces,while bone implant connections were still weak in the pure titanium group.The degree of osseointegration was further measured by the torque removal test.At 4 and 8 weeks,the torque removal of selective laser melting group was significantly higher than that of sandblasting and large-grits etching group and pure titanium group.Conclusion: These results directly confirm that selective laser melting implant showed great effect on osseointegration in type 2 diabetes mellitus rats at the early stage of osseous healing.Selective laser melting implant may be a new type of implant for type 2diabetes mellitus patients.The main research conclusions of this experimental study are as follows:(1)SLM titanium implants have higher surface roughness,higher purity,better hydrophilicity and porous structure,and suitable mechanical strength.(2)The biosafety detection of SLM titanium implants is non-toxic,which is beneficial to promote the adhesion and growth of gingival fibroblasts,and induce the proliferation and early osteogenic differentiation of MG63 osteoblasts.(3)The early osseointegration of implants by SLM technology in type 2 diabetic rats shows the advantages of this technology in promoting implant osseointegration at both the cellular and molecular levels. |
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