| BackgroundNowadays,limb salvage surgery has become a standard treatment for malignant bone tumors of the extremities.In limb salvage surgery,the step of soft tissue reconstruction after tumor removal and bone defect reconstruction is very important,which usually includes excellent soft tissue coverage,tendon and ligament attachment point reconstruction,and joint capsule repair,etc.These factors are closely related to the postoperative functional recovery of the affected limb and the occurrence of postoperative complications,and even to the success of limb salvage surgery.However,there are some limitations in the current clinical approach for soft tissue reconstruction.In soft tissue coverage reconstruction,the skin or subcutaneous tissue or transferred muscle flaps were usually covered directly over the surface of the smooth prosthesis,which was easily to form a liquid dead space at the prosthesis-soft tissue interface after post-operative bleeding and exudation,thus delaying wound healing and increasing the risk of post-operative prosthesis infection,exposure,or even prosthesis failure and removal.On the other hand,the important tendons and ligaments preserved during tumor resection were usually suspended and fixed directly by sutures on the holes structure reserved for the prosthesis,which were prone to sliding with the smooth prosthetic surface and failed to form an effective biological connection,thus easily lead to complications such as postoperative joint instability and poor functional recovery of the affected limb.With the rapid development of 3D printing technology,3D printed porous titanium prosthesis has become one of the standard treatment for bone defect reconstruction in limb salvage surgery.The porous structure not only reduced the elastic modulus of the prosthesis,thus reducing stress shielding,but also effectively promoted the growth of bone and soft tissue into the porous interface to form bio-integration.However,most of the commonly used 3D printed porous titanium prostheses only focused on the integration of prosthesis and bone tissue interface,but ignored the effective integration of the prosthesis and soft tissue,and were not specifically designed with a porous structure suitable for soft tissue ingrowth,which still did not effectively solve the above-mentioned deficiencies in the soft tissue reconstruction of limb salvage surgery.Moreover,the parameters in the pore size and porosity of the prosthesis were often designed and prepared according to the bone tissue growth-in characteristics,which may not be suitable for the growth of soft tissue,and the optimal pore size parameters suitable for soft tissue ingrowth were not yet clear.Therefore,our group firstly propose to build in vivo and in vitro experiments to explore the optimal pore size for soft tissue ingrowth.Secondly,to address the shortcomings of the above-mentioned soft tissue reconstruction methods,we would apply the optimal pore size for soft tissue growth into the optimization and preparation of 3D printed prosthesis,and add porous structures to the soft tissue coverage of the prosthesis and important tendons and ligaments attachment points,so as to promote good integration and effective biological healing at the interface between prosthesis and soft tissue.We will then evaluate the safety and efficacy of the optimized 3D printed porous titanium prosthesis in clinical limb-salvage surgical applications,especially the clinical outcome of prosthesis integration with soft tissue,and eventually provide a more stable and biocompatible 3D printed porous titanium prosthesis for limb salvage surgery,providing insights and solutions for soft tissue reconstruction in limb salvage surgery.Objective1.We fabricated 3D printed porous titanium scaffolds with different pore sizes to investigate the most suitable pore size for soft tissue growth from cytological and histological perspectives,providing a theoretical basis for further clinical applications.2.The 3D printed porous titanium alloy prosthesis was designed to optimize pore size suitable for soft tissue ingrowth on the surface of the prosthesis and at the attachment points of important tendons and ligaments,to observe the clinical treatment effect of the 3D printed prosthesis in limb salvage surgery,especially in the area of soft tissue reconstruction,and to evaluate the safety and efficacy of the prosthesis,and finally provide a more personalized,stable and biocompatible 3D printed prosthesis for bone tumor surgical treatment,and provide new ideas and solutions for soft tissue reconstruction in limb salvage surgery.Methods1.The porous titanium alloy scaffolds were designed by computer-aided design(CAD)software.Porous titanium alloy discs(14 mm in diameter and 2 mm in height)and porous titanium alloy cylinders(10 mm in diameter and 10 mm in height)with pore sizes of 600μm,800 μm,1000 μm and 1200 μm and filament diameters of 400 μm were designed and fabricated,respectively.The actual pore size,porosity and other parameters of the scaffolds were measured by Micro-CT;the hydrophilicity of scaffolds surface were measured by wetting angle tester;the mechanical properties of the scaffolds with different pore sizes were measured by a microcomputer-controlled mechanical testing machine.2.In vitro experiments,fibroblasts(L929)were cultivated on porous titanium alloy discs with different pore sizes to investigate the effect of pore size on fibroblast proliferation.The number and morphology of fibroblasts on scaffolds with different pore sizes were observed by scanning electron microscopy;the proliferation activity of fibroblasts on scaffolds with different pore sizes was detected by CCK-8;the effect of pore size on the number of fibroblasts alive and dead was observed by live-dead cell staining.3.In vivo experiments,the rat subcutaneous implantation model and the rabbit erector spinae muscle implantation model were established to investigate the optimal porous pore size for soft tissue ingrowth.Firstly,after 7,14 and 28 days of implantation,the hard tissue sections were stained with HE to observe the subcutaneous soft tissue growth in the different pore size porous titanium discs and to measure the ratio of growth-in area to pore area(per unit area).Secondly,after 7,14 and 28 days,the hard tissue sections were stained with HE staining to observe the muscle growth in the porous titanium alloy cylinders and to measure the ratio of growth-in area to pore area(per unit area).4.The pore size suitable for soft tissue ingrowth was applied to the design and preparation of 3D printed porous titanium alloy prosthesis,and the porous structure suitable for soft tissue ingrowth was added at the soft tissue coverage on the surface of the prosthesis and at the attachment points of important tendons and ligaments.The 3D printed porous titanium alloy prosthesis was used in the limb surgery that met the application indications to observe the clinical application effect of the prosthesis in limb preservation surgery,especially in the area of soft tissue reconstruction.The 3D printed porous titanium alloy prostheses were used in limb salvage surgery of tibial malignant tumor which met the application indications,to observe the clinical effect of the prostheses in limb salvage surgery,especially in the area of soft tissue reconstruction.Outcome1.Four different pore sizes of porous titanium alloy scaffolds were manufactured by SLM techniques,and the pore sizes of the same pore size scaffolds were uniform and structurally homogeneous.The actual pore diameters of the 600 μm,800 μm,1000 μm and1200 μm scaffolds were 591.18 ± 11.2,796.12 ± 13.21,999.53 ± 5.06 and 1276.19 ± 5.66μm,respectively,measured by Micro-CT.The elastic modulus of porous scaffolds with pore sizes of 600 μm,800 μm,1000 μm,and 1200 μm were measured as 6253.03 ± 203.35,4628.73 ± 185.88,3012.91 ± 275.36,and 2172.98 ± 196.08 MPa,respectively;the compressive strengths were measured as 228.94 ± 15.56,146.48 ± 9.37,93.15 ± 11.25,and63.39 ± 10.88 MPa,respectively,using a microcomputer-controlled mechanical testing machine.2.In vitro experiments,fibroblasts were cultured on porous titanium scaffolds with four different pore sizes(600,800,1000,1200 μm).Scanning electron microscopic observations of fibroblasts cultured on the scaffolds at 3 and 7 days showed that as the pore size of the scaffolds increased,the number of fibroblasts per unit area was higher and the cell spreading area was larger,while more extracellular matrix was secreted on the cell surface and more filamentous pseudopods were seen to extend.The results of CCK-8 test suggested that the overall trend of cell proliferation activity on scaffolds of different pore sizes was proportional to the size of the scaffolds and the larger of the pore size,the stronger the cell proliferation activity in the scaffold.The results of live-dead cell staining of fibroblasts on scaffolds with different pore sizes suggested that the number of live cells per unit area in the pore plate improved as the pore size of porous scaffolds increased,and the ratio of the number of live-dead cells also increased.3.In the rat subcutaneous implantation experiments,porous titanium alloy discs of different pore sizes were implanted subcutaneously in rats for 7,14 and 28 days,respectively.The results of HE staining of the hard tissue sections of the specimens indicated that at 7days,the 1000 μm group had the best soft tissue ingrowth compared with the other three groups(p < 0.05),followed by 800 μm(p < 0.05),and the 1200 μm group had the smallest soft tissue ingrowth area ratio(p < 0.05).At 14 and 28 days,the subcutaneous soft tissues fully grew into the scaffolds of different pore sizes,and no statistically significant differences were found in the ratio of the growth area to the void area between the groups(p > 0.05).In the rabbit erector spinae muscle implantation experiment,porous titanium alloy cylinders of different pore sizes were implanted in the rabbit erector spinae muscle bag for 7,14,and 28 days,respectively.the results of HE staining of hard tissue sections suggested that at 7 days of implantation,the 1000 μm group had the best soft tissue growth,followed by the 800 μm group(p < 0.05),and the 1200 μm group had the least soft tissue growth and the most voids in the scaffold(p < 0.05).At 14 days,there was no significant difference in soft tissue ingrowth between the 800 μm,1000 μm,and 1200 μm groups(p >0.05),and the 600 μm group had the smallest soft tissue ingrowth area ratio compared with the other three groups(p < 0.05).At 28 days,there was no significant difference in soft tissue ingrowth between the groups(p > 0.05).Based on the in vitro and in vivo results,we concluded that a porous pore size of 1000 μm is more suitable for early soft tissue growthin.4.The 3D-printed titanium tibial segmental prostheses were made with porous structures(pore size: 1000 μm)suitable for soft tissue ingrowth at the surface of prostheses and at important tendon and ligament attachment points,and the prostheses were applied in two cases of primary tibial malignant tumor limb salvage surgery.At 7 days postoperatively,no signs of infection such as redness,swelling,or fever were seen in the wounds of the 2patients,and no obvious fluid dead space around the subcutaneous prostheses could be palpated.The results of the 6-month postoperative follow-up indicated that the two patients had good functional recovery of the affected limbs,with MSTS-93 limb scores of 27 and28,respectively,and no adverse events such as prosthetic infection or exposure occurred in either case.The appearance of hard tissue sections and HE staining after removal of the prosthesis in case 2 indicated that the soft tissue was well integrated with the porous titanium prosthesis at 12-month follow-up,and the interior of the porous prosthesis was completely filled with soft tissue.Result1.Based on the results of in vivo and in vitro studies,we concluded that the porous titanium alloy pore size of 1000 μm is more suitable for early soft tissue ingrowth.2.The results of the 2 case reports initially validated the safety and effectiveness of3 D printed porous titanium alloy tibial prosthesis.Soft tissues can be effectively integrated with the porous structure of the prostheses,thus enhancing early functional recovery of the affected limb,reducing postoperative complications,and effectively improving the biological stability of the prosthesis.The application of porous structures in 3D printed titanium prostheses is promising to provide a new option for soft tissue reconstruction in limb salvage surgery. |
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