| Osseointegration(OI)has been defined as the direct connection between strictly defined implant and ordered living bone in structure and function,thereby the symbiosis between the implant and the continuously remodeled bone tissue has been rebuilt.Since this concept was proposed by Professor Br?nemark in the 1950s,with the deepening of research and understanding,it has been widely accepted by osteologists,stomatologists and medical biomaterials scientists.Moreover,based on this concept,new disciplines or emerging research fields have been developed such as biomaterials,dental implants,clinical bone integration,etc.In recent years,titanium implants have been implanted into the bone marrow cavity of amputee stump and the technology of percutaneous osseointegrated prosthetic(POP)has been evolving.The external connectors which are integrated with the mature bone tissue at the broken end through the skin,have three advantages compared with traditional socket prosthetic devices.The first advantage is that the POP provides solid support and is not easy for shedding.The second advantage is the immobilization method makes the movement of the limb more controllable and the direction of movement more flexible.The last but not the least advantage is that the symbiosis between the implant and bone is more likely to produce stronger feedback perception of the limb,which brings hope for the improvement of the quality of life for patients with bone integration prosthesis.At present,it has been noted that the endosteum tissue structure and the internal environment of the bone marrow cavity are damaged by such intramedullary implantation.Moreover,bone resorption often occurs in the bone at the edge of the amputation due to the force shielding,followed by the retraction of the soft tissue around the implant and bacterial infection,leading to the fall of the implant and the failure of the treatment.Therefore,in this study,based on the principle of preserving bone tissue(osteocytes and endosteum)and reducing lateral stress shielding,a new type of implant was designed by optimizing and improving the geometry of the implant.Osseointegrated implants are integrated with reconstructed mature bone tissues,and local histological changes are the gold standards for evaluating the effect of osseointegration.In this study,osseointegration has been achieved by changing the implantation method,improvement of the shape design of the implant,maintainment of the tissues in the bone marrow cavity and the reduction of the bone resorption at the end of the residual limb caused by stress shielding.However,in recent years,the data from in vivo experiments of osseointegration lacks the histological changes of tissue repair at different stages after integration,including cell chemotaxis,differentiation and the fate of these cells.In this process,bone mesenchymal stem cells(BMSCs),as an important stem cell reserve,are the key to the dynamic osseointegration and bone remodeling.However,the molecular mechanisms of how stem cells perceive the physical signals of implants and regulate the osteogenic differentiation is still needed to be further elucidated.It is speculated that in this process,the integrin molecules act as initiation molecules to receive mechanical signals,and then cooperate with BMPR to trigger the cascade reaction of downstream molecules,regulating the transcription of the osteogenic gene RUNX2.The purpose of this study is to investigate whether the new implant can achieve successful osseointegration and reduce the bone resorption by changing the design of the implant and implantation method,as well as the role of integrin in the process of osteointegration and bone regeneration in vivo;how the physical factor stiffness,as a design parameter of the implant,affects the osteogenic differentiation of MSCs.This study intends to optimize the parameter design of transplant,regulated the differentiation of MSCs,and provide theoretical and experimental basis for the clinical application and mechanism research of osseointegration in bone defect repair.Methods:1.Modification and stress analysis of osseointegration implants:(1)Modification and processing of osseointegration implants:A new type of percutaneous osseointegration implant,that is,external cap-shaped titanium implant fixed outside of the medullary cavity,was designed by UG NX 10.0 software.X-ray imaging was used to measure the inner/outer diameters of the cortical bone of the rabbit tibia,and the outer diameter of the internal cylindrical implant and the internal diameter of the external cap-shaped implant were determined according to the average value.Auto cad software was used to draw the machining diagram of the implant which was made of grade 4 pure titanium.The surfaces of two kinds of implants were treated by sandblasting and acid etching,respectively.The threaded cylindrical titanium implant implanted in the medullary cavity is used as a control.(2)Stress analysis of osseointegration implants:Then,the implants and bone tissues with full weight bearing were analyzed by three-dimensional finite element method which was carried out by using ANSYS workbench 17.0 software.The finite element method of meshing was used to simulate the load-bearing function of the two implants under the same load after implantation in the tibia,as well as the load-bearing stress distribution of the bone-implant interface.2.Improvement,osseointegration and assessment of the implants in rabbit tibia:(1)Improved implantation in rabbit tibia:The tibial amputation model was established in New Zealand white rabbits.The external cap-shaped titanium implant and the traditional threaded cylindrical titanium implant(intramedullary implantation)were implanted into the rabbit tibia respectively.After the rabbit tibial amputation model was constructed,the control group was screwed the built-in cylindrical implant into the medullary canal used the traditional intramedullary implantation method.In the experimental group,an improved transplantation method was used to prepare threads on the surface of the extramedullary cortical bone of the tibia,and 3-4 holes with a diameter of 1 mm were arranged on the cortical bone to ensure blood supply.The cap implant was implanted and fixed on the cortical bone outside the medullary cavity.After the implantation was complete,the skin was sutured and closed.(2)Osseointegration of the implants in rabbit tibia:(a)The X-ray examination was done after the implantation.Micro-CT scan was used to observe the osseointegration of the implant-bone interface.(b)Scanning electron microscopy(SEM):At the same time,the two implants were removed from the tibial implant site after sampling,and the surface morphology of osteoid tissue after the osseointegration was observed by SEM.(3)Histopathological assessment of the osseointegration induced by implants:Hard tissue sectioning and paraffin sectioning were performed from the bone samples.The changes in the tissue structure of the implantos seointegration interface,the bone around the implant and the distribution of new bone matrix were observed by HE staining and toluidine blue staining.Immunohistochemistry was performed to detect the expression and distribution of the markers of osteogenic differentiation of bone MSCs,including osteoprotegerin,osteocalcin,periosteal protein,as well as integrin ?1.Histologically,it is studied whether integrin ?1 plays an important role in osseointegration and bone regeneration around implants,and whether bone marrow MSCs in vivo differentiate towards osteogenic direction.3.Effect and mechanism of stiff titanium promoting osteogenic differentiation of hBMSCs:(1)Promotion of osteogenic differentiation of hBMSCs by stiff titanium:Polyacrylamide hydrogels with stiffness of 1-10kPa were prepared as control,and the titanium plates were laid in the cell culture dishes.The stiffness of 1-10kPa and titanium plates(?110GPa)were coated with the same concentration of fibronectin,in order to avoid the influence of different materials on cell differentiation,the surface morphology including porosity and roughness was observed by SEM.(2)Mechanism of stiff titanium promoting osteogenic differentiation of hBMSCs:The mRNA expression levels of integrin ?5,?1,mechanically sensitive cation channel protein 1(Piezo 1),BMPRIA,BMPRIB were detected by RT-qPCR.After treatment with BMPRI inhibitor(LDN193189)on these two stiffness groups for 7 days,Western Blot(WB)was used to detect the protein expression of BMPRIA and p-SMAD1/5;RTqPCR was performed to detect the expression of osteoblast differentiation related gene Runx2,integrin ?1 and Piezo 1;WB was performed to detect the protein expression of p-SMAD1/5,FAK,p-AKT and p-ERK.Results:1.Modification and stress analysis of osseointegration implants:(1)Modification and processing of osseointegration implants:The mechanical flow diagrams were drawn according to the experimental purpose and design concept.And the new geometric shape external capshaped titanium implant and the traditional shape built-in cylindrical titanium implant were made by the mechanical processing method.The results suggest that the positions of osseointegration are different:the former was located outside the bone marrow cavity and directly integrated with the cortex,forming extramedullary osseointegration,saving more bone marrow tissue in the marrow cavity;the latter was located inside the marrow cavity,forming intramedullary osseointegration.(2)Stress analysis of osseointegration implants:Three-dimensional finite element analysis of the weight-bearing implants showed that when the same force was given to the two implants,the maximum stress in the traditional intramedullary columnar implant was concentrated on the connecting rod and the upper end away from the end of the residual limb bone.The cortical bone at the end of the residual limb was shieled by force,which led to bone resorption,and the connecting rod deformation of the implant was easy to occur.The maximum stress of the external cap implant was concentrated on the implant base and the end of the residual limb bone.This was the maximum circumference of the implant and was not easy to deform.The force here could avoid the bone resorption which led to bone regeneration.2.Improvement,osseointegration and assessment of the implants in rabbit tibia:(1)Improved implantation in rabbit tibia:A successful and stable rabbit tibia amputation model was established,and the graft was also stably implanted into the designed site in vivo.(2)Osseointegration of implant in rabbit tibia:(a)Micro-CT scans showed new bone formation and osseointegration at the BIC interface of both implants.However,external cap implants had substantial bone regeneration in the cortical bone space and the medullary cavity.Moreover,this new type of external titanium implants didn't cause bone resorption at the end of the stump,but induce periosteal ossification.(b)SEM scanning of the implant surface showed that osteocytes spread on the surface of both the external cap implant and the internal columnar implant.The extended protrusions were anchored to the surface of titanium and connected with other protrusions.It was suggested that the external cap implant and the internal column implant had the same effect on osseointegration.(3)Histopathological assessment of the osseointegration induced by implants:The distribution of osteoblasts and new bone matrix shown by HE staining and toluidine blue staining were consistent with the CT results.All these results showed that the interface between the new external titanium implant and bone tissue had undergone osseointegration.And the cortical bone at the end of the residual limb became denser instead of bone resorption.IHC showed that during bone regeneration in the contact area of external titanium implant,osteoprotegerin,osteocalcin,periosteal protein,and integrin?1 were highly expressed,indicating that integrin?1 played an important role in the process of inducing differentiation of bone marrow MSCs in vivo.3.Effect and mechanism of titanium hardness promoting osteogenic differentiation of hBMSCs:(1)Promotion of osteogenic differentiation of hBMSCs by stiff titanium:hBMSCs were cultured on soft substrate(1-10kPa)and hard substrate titanium(Ti).RT-qPCR showed that the expression levels of osteogenic markers RUNX2,COL-1 and ALP were highly expressed on the 1st and 7th day on titanium surface.Moreover,the expression of RUNX2 showed an upward trend,suggesting that the differentiation ability of hBMSCs in osteoblasts was gradually enhanced.(2)Mechanism of stiff titanium promoting osteogenic differentiation of hBMSCs:RT-qPCR showed that the expression levels of integrin ?5,?1 and BMPR1 were significantly higher on titanium surface on the 1st day,and decreased on the 7th day,but Piezo 1 was significantly higher on the rough titanium surface that day.After inhibition of BMPR(LDN-193189),WB showed that the protein expression of BMPRIA and p-SMAD1/5 decreased significantly,and RT-qPCR showed that RUNX2 expression was down-regulated,suggesting that BMPRI promoted osteogenic differentiation of BMSCs.The expression of integrin ?1 was also inhibited after inhibition of BMPRI,while the expression of Piezol was increased.The protein expressions of FAK,p-AKT and p-ERK were significantly down-regulated.In summary,these results suggested that the hardness factor of titanium promoted the osteogenic differentiation of hBMSCs,integrin ?1 played an important role,possibly by influencing BMPR1A/B and Piezol might be also involved in the different stages of osteogenic differentiation.Conclusions:1.A novel external cap implant was designed and fabricated for the first time.The maximum stress of the external cap implant is concentrated at the bottom of the implant and the end of the bone stump,which is the largest diameter of the implant.It is not easy to be deformed,providing the mechanical principle of reducing bone absorption at the end of the stump.2.The new external cap implant can cause effective osseointegration after transplantation,and its local histopathological changes,including stem cell chemotaxis,osteogenic differentiation,bone matrix secretion and ossification,are basically the same as those of the intramedullary implant.However,the bone resorption at the transplant site is significantly reduced,and a large amount of new osteoid tissue is observed in the medullary cavity,which is important for clinical application.3.Titanium could induce osteogenic differentiation of bone marrow MSCs both in vitro and in vivo,and integrin ?1 plays an important effect.The hardness of titanium could promote the osteogenic differentiation of hBMSCs,and integrin ?1 and BMPRI have synergistic effect during this process. |
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