| BackgroundAutologous bone graft is the "gold standard" reconstruction method of large mandibular defects, due to the trauma, inflammation and tumor. However, the disadvantages such as limited supply, pain, infection, donor site morbidty and other complications restrict the clinical application of autologous bone graft. The allogenic mandible has affinis contour and internal structure to the host mandible, and avoids the secondary injury caused by the harvest of autologous bone, which is considered as an alternative method of autologous bone graft. The immunogenicity of the allogenic bone is low and the biomechanical strength is closed to that of the host bone, confirmed by many studies. Consequently,allogenic bone is a more suitable option for the reconstruction of large bone defects[1-4].Study showed that clinical healing and satisfied outcome were obtaineded using the frozen/freeze-dried allogenic mandibles to reconstruct the temporomandibular joint and mandibular defects in beaglesf3l. The feasibility of freeze-dried allogenic mandibles in the application of large mandibular defects reconstruction was comfirmed by studies[4].However, the bone union of allogeneic bone graft was slow and often failed due to the infection, bone resorption or immunological rejection[5-7]. In order to improve the survival rate, autologous cancellous particles, autologous bone marrow or growth factors were combined with the allogenic bone[8-10]. A study reported that the frozen allogenic mandibles loaded with autologous iliac cancellous bone particles successfully restored 4 cases of large mandibular defects[10]. Nevertheless, the source of autologous bone marrow is limited and the osteogenic capacity of that is related to the age of donor, which restrict its clinical application.Bone tissue engineering raises an alternative method for mandibular reconstruction. The scaffolds for large mandibular defects reconstruction require the similar outline and mechanical strength to the defects. However, the mechanical strength of scaffolds in bone tissue engineering cannot achieve this requirement. Consequently, we need to find more suitable scaffold materials. The allogenic mandible can satisfy this requirement and would be a suitable scaffold of bone tissue engineering. Titanium alloy is commonly used as clinical artificial material to repair bone defects. With the development of computer-aided design software and 3D printing equipments, individual and meshy titanium alloy scaffold fabricated by 3D printing technology has identical structure and adjustable mechanical strength to the defect area. This scaffold may be an appropriate option for bone tissue engineering.ObjectiveTo investigate and compare the feasibility of individual titanium alloy fabricated by 3D printing technique and freeze-dried allogenic mandible on the mandibular reconstruction and provide reference for their clinical application.Method1 Mesenchymal stem cells were isolated from bone marrow and identified through cell surface labeling and multi-directional differentiation ability detection.2 Allogenic mandibles were collected from sacrificed beagle dogs of previous study and treated through frozen and dried. Segmental defects were created on the left mandibles of 27 beagles and were randomly repaired using allogenic freeze-dried mandible alone,freeze-dried bone combined with autologous bone marrow mesenchymal stem cells or allogenic bone combined with allogenic mesenchymal stem cells. The reconstruction effect was evaluated by clinical observation, CT scanning,micro-CT scanning and histological examination.3 To evaluate and compare the biocompatibility of the titanium alloys fabricated by 3D printing technique through cytotoxicity test, haemolytic test, dermal irritation test and skin sensitivity test.4 Three dimensional meshy titanium alloy scaffolds were fabricated by electron beam melting. Allogenic bone marrow mesenchymal stem cells were encapsulated in the thermosensitive hydrogel prepared using chitosan, Bio-Oss and P-sodium glycerophosphate. The cell viability and proliferation was investigated by CCK-8 test and live/dead staining. Critical defects were created on the right mandibles of beagles and randomly repaired using meshy titanium alloy scaffolds, titanium alloy scaffolds combined with chitosan/ Bio-Oss hydrogel or titanium alloy scaffolds combined with chitosan/Bio-Oss/cells. The reconstruction effect was evaluated by clinical observation, CT scanning,micro-CT scanning and histological examination.5 Hemi-mandibular defects including temporomandibular joint were created and randomly reconstructed by freeze-dried allogenic mandibles, individual solid and meshy titanium alloy fabricated by electron beam melting. Preliminary comparison of reconstruction effect among the three methods was conducted through the operative procedure, appearance contour and influence on the non-surgical temporomandibular joint, liver and kidney.Results1 Bone marrow mesenchymal stem cells were successfully separated by density gradient centrifugation and have high reproductive activity and multi-directional differentiation ability.2 Completely healing achieved in the three groups. The outline of the mandibular defects in the three groups were well restored. CT examination showed that bone resorption of allogenic grafts was observed in the two groups which combined with mesenchymal stem cells, while no resorption was observed in the sole allogenic graft. The size of the implants in the two composite allogenic grafts was smaller than that of the original ones. There was no statistical difference of micro-architectural parameters of allogenic grafts between the two composite groups (P> 0.05). Compared to the sole allogenic grafts group, more new bone was formed in the two composite allogenic grafts, which was showed by histology examination and micro-CT examination.3 The results of cytotoxicity test demonstrated that the titanium alloys fabricated by electron beam melting and selective laser melting had no toxic effect on the proliferation and osteogenic ability of mesenchymal stem cells. There was no significant difference between the two samples (p>0.05). Haemolytic ratio of the two titanium alloys was2.24% and 2.46% respectively, which was lower than 5%?Haemolytic test demonstrated both of the two titanium alloys had good hemocompatibility. Dermal irritation test showed the primary irritation index of both titanium alloy samples was 0, which demonstrated no skin irritation. Delayed-type hypersensitivity test showed the skin hypersensitivity grade of the two titanium alloys was 0, which illustrated no skin sensitization.4 Chitosan/Bio-Oss was liquid at room temperature and transformed into solid hydrogel at 37? within 10-12min. The cells viability and proliferation capacity of bone marrow mesenchymal stem cells encapsulated in the Chitosan/Bio-Oss hydrogel was not affected.All animals achieved the clinical healing and no infection, graft fracture or exposure happened to the titanium alloy implants in the three groups. CT showed meshy titanium scaffolds were firmly fixed with host bone and no bone resorption was observed. More new bone formations in the implants of meshy titanium alloy combined with Chitosan/Bio-Oss/1BMSCs group were observed, compared to the other two groups,showed by Micro-CT (p<0.05) . Six month postoperative, histological analysis showed new bone formed significantly in the meshy scaffolds of the three groups six month postoperative. New bone formation was almost filled in the pores of meshy scaffolds in the groups of meshy titanium alloy combined with Chitosan/Bio-Oss/BMSCs.5 Freeze-dried allogenic mandible, 3D printing individualized solid and meshy titanium alloys were all easily to be operated. Allogenic mandibles were needed to be shaped to match to the defects, while the two titanium alloy implants can be directly fitting. Esthetic consequence and good occlusal relationship were achieved in the three groups. No significant difference in the location of bilateral temporomandibular joint among the three implants, observed by postoperative CT examination. Degeneration occurred in the temporomandibular joint discs of non-surgical side in the three groups, which showed by scanning electron microscopy and histological examination.Conclusion1 Both of the titanium alloys fabricated by electron beam melting and selective laser melting titanium had good biocompatibility. There was no statistical difference between the two alloys.2 Freeze-dried allogenic mandibles and meshy titanium alloys fabricated by electron beam selective melting can be used as scaffolds of bone tissue engineering in mandibular reconstruction. Allogenic mandibles combined with bone marrow mesenchymal stem cells had superior osteogenic ability. The composition of mesencymal stem cells and osteogenic materials could promote the formation of new bone, however, the bone regeneration rate was slow and the new bone formation was insufficient. The bone growth factors and vascular growth factors were needed to be combined to improve bone regeneration of implants.3 Allogenic bone marrow mesenchymal stem cells had identical ability to the autologous bone marrow mesenchymal stem cells, which could promote the speed and improve the quality of bone regeneration. No statistically significant difference between the two cells.Allogenic mesenchymal stem cells were an alternative choice to autologous stem cell in the application of bone tissue engineering.4 The advantages of freeze-dried allogenic mandibles and 3D printing titanium alloys were convenient operation and individualized reconstruction. Satisfied outcome of the temporomandibular joint and hemi-mandibular defects was achieved in all of the three grafts, which demonstrated suitable methods for mandibular reconstruction. |
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