Experimental Study On Design And Osseointegration Of Custom-made Talar Component For Total Ankle Replacement Prostheses

BackgroundTotal ankle Replacement（TAR）is an effective treatment for end-stage ankle arthritis.TAR has the outstanding advantages of retaining ankle joint function,relieving pain,reducing infection rate and avoiding secondary adjacent joint degeneration.At present,the shortcomings of previous research on TAR mainly are as following:First,the high failure rates are likely due to the designs of current TAR prosthesis,especially the designs of the talar component,which are not patient-specific and anatomy-based,disadapting to the complex biomechanical and kinematic requirements of ankle joints.Further,due to the inconsistent interface materials and structural materials of current TAR prosthesis,the osseointegration of the prosthesis-bone interface is difficult,and the complications such as infection,loosening and sinking of the prosthesis are still common.Third,it is difficult in adaptation design of new TAR prosthesis with microporous processing interface.How to rapidly manufacture custom-made talar component for TAR prostheses need to be further studied.Based on the above problems,the aims of this study are therefore to establish a three-dimensional model of weight-bearing foot and ankle by using three-dimensional digital reconstruction,to analyze the osseointegration mechanism of new antibacterial Cu-bearing Co-Cr alloys porous scaffolds by simplifying the prosthesis-bone interface structure,to rapidly manufacture custom-made talar component samples for TAR prostheses by using biomedical 3D-printing after standardizing the matching design of prosthetic model and prosthesis-bone interface.It is likely to provide theoretical and practical basises for the advent and clinical application of new TAR prosthesis.Materials and Methods1 Digital reconstruction of three-dimensional model of weight-bearing foot and ankle1.1 Collected imaging datas in DICOM format of weight-bearing foot and ankle obtained by a healthy volunteer,whom performed cone-beam CT（CBCT）scan by PedCAT scaner（Curvebeam,USA）.At the same time,the scan time and imaging time were measured.1.2 After segmenting threshold,the three-dimensional reconstruction and the partition mask were edited.The 3D model of weight-bearing foot and ankle was established in Mimics software,and the talar model was extracted separately in the software.1.3 In Geomagic software,mesh checking,error correction and smoothing on the model werw performed to form a curved CAD 3D model and generate package files for output in IGES format.2 Design and finite element analysis of custom-made talar component model for TAR prostheses2.1 Designed the dome and geometric fixture of talar component for TAR prosthesis with reference to current TAR prosthesis parameters and design concept.2.2 Designed different osteotomy methods in talar component for TAR prosthesis,according to current TAR prosthesis,which were acuate or corner osteotomy in the sagittal plane,and with or without the lateral side osteotomy.All prosthetic models were designed to be fully covered with the osteotomy surface,and the amount and area of osteotomy of each model were calculated.2.3 Performed three-dimensional finite element analysis（FEA）on various models.In FEA,selected the contact force as the input load when standing on both feet,and calculated the stress distribution of every prosthesis modle,and analyzed the static stability of the prosthesis.3 Effect of new antibacterial Cu-bearing Co-Cr alloys porous scaffolds on biological behavior of cells3.1 Designed the three-dimensional scaffold model by computer aid,and fabricated porous cylindrical by selective laser melting（SLM）with new antibacterial Cu-bearing Co-Cr alloys powder.The diameter of the scaffolds was 10mm,and the total height of which was3.5mm.The bottom layer of the scaffolds was solid,and the height of which was 0.5 mm.The porous layer of the scaffolds was a diamond positive octahedron structure,and the height of which was 3 mm.3.2 Conducted experimental grouping based on the porosity of the scaffolds with 40%,60%and 80%porosity,and the corresponding pore sizes were 201μm,373μm and 454μm.3.3 Carried out compression experiments to test the mechanical properties of the three groups of scaffolds,and observed the porous structure of the three groups of scaffolds by scanning electron microscopy,and tested the toxicity of the materials by leaching solution.3.4 After the porous scaffolds were washed and sterilized,L929 cells were planted on the scaffolds and cultured in high glucose DMEM medium,and incubated for 2,4 and 8 days respectively,and performed the related tests at time points of incubation.3.5 Used whole bone marrow method to isolate and culture BMSCs,and implanted BMSCs on the scaffolds and cultured in osteogenic induction medium,and incubated for 2,4and 8 days respectively,and performed the related tests at time points of incubation.4 Mechanism of new Cu-bearing antibacterial Co-Cr alloy porous scaffold to promote osseointegration4.1 Designed and fabricated Co-Cr alloy porous cylindrical scaffold with a porosity of373μm and a pore diameter of 373μm according to the method in the first part of this chapter.The diameter of the scaffolds was 5mm,and the total height of which was 5.1mm.The bottom layer of hollow structure,and the height of which was 0.1 mm.The porous layer of the scaffolds was a diamond positive octahedron structure,and the height of which was 5 mm.4.2 The experimental group was based on Co-Cr alloy composition,including copper-free and Cu-bearing groups.4.3 The mechanical properties of the two groups of scaffolds were tested by compression experiments,and the two groups of porous structures were observed under scanning electron microscopy.4.4 After the porous scaffold was washed and sterilized,the BMSCs were planted on the two sets of scaffolds,and the osteogenic induction medium was cultured for 2 days,4 days and 8 days.The detailed methods,steps and contents were the same as those in the first part of this chapter.4.5 After the porous scaffold was cleaned and sterilized,9 goats were operated.The porous scaffolds of the two different components described above were implanted into the distal femur of the goat.（1）Before the operation and on the first day,the 30th day,the 45th day,and the 60th day after the operation,the internal jugular vein blood of the goat was collected,and the flame atomic absorption method was used to determine the copper ion concentration of the goat serum.（2）On the 90th day after the operation,the goat was sacrificed and the right knee joint of the goat was subjected to anterior and posterior radiographs.（3）The goat femur specimens implanted in the porous scaffold were taken out,and Micro-ct examination was performed.The data was imported into the DataViewer software for analysis,and the gray value of the bone tissue volume around the scaffold was used to estimate the bone mineral density（BMD）difference between the two groups.（4）The goat femur specimens implanted in the scaffold were embedded,trimmed,dehydrated,and photopolymerized.The specimens were then patched,sectioned and ground in an EXAKT hard tissue microtome.Finally,two consecutive hard tissue sections were made.The sections were numbered corresponding to the specimen and the position for the adjacent implantation position of the same specimen when performing the tissue section analysis.（5）One of the two hard tissue sections obtained from each of the above brackets was was subjected to spectral identification under a Raman spectrometer,scanning step scanning was selected,and combined with environmental scanning electron microscopy.The differences in tissue composition of the bone-interface of the two groups of porous scaffolds were analyzed.（6）Another of the 2 hard tissue sections obtained from each of the above brackets was stained with methylene blue-acid fuchsin.Tissue sections that have been scanned by Raman spectroscopy and electron microscopy.The tissue section that have been scanned by Raman spectroscopy and electron microscopy was stained with Goldner trichrome.All of the tissue sections were magnified 20 times in an Olympus digital slicing workstation and stored digitally.（7）The digital section was opened in the software,and the osteogenesis area inside and around the two groups of porous structures was calculated,and the differences between the two groups were compared and analyzed.5 Manufacture of custom-made talar component prosthetic samples for total ankle replacement prostheses5.1 The experimental results of the first two chapters were used to fit the porous bone interface of talar component of TAR prosthesis with COA+LO,and the print model was exported in STL format.5.2 The print model was introduced into the SLM printer with a 45°tilt support,and a new Cu-bearing anti-microbial Co-Cr alloy talar component was fabricated.5.3 After the support of the prosthesis was removed,the prosthesis was polished and cleaned.The prosthesis was generally observed and the shape of the prosthesis and the joint surface and porous structure of the trochlear were observed under a metallographic microscope.6 Statistical methodsResults were expressed as mean and standard deviation.One-way ANOVA or T-test analysis was performed using SPSS 20.0 statistical software.The results were statistically significant when P<0.05 was defined.Results1 CBCT scan was performed,and the data of bilateral weight-bearing foot and ankle was extracted.The PedCAT scanning time was 20 seconds,and the imaging time was 3 minutes.The three-dimensional model of weight-bearing foot and ankle comprehensively reproduced the spatial position and structure of the bone of full foot and ankle when the feet are standing.Meanwhile,the three-dimensional model of the talus was successfully established.2 Completed the design of the dome and geometric fixture of talar component for TAR prosthesis,and the dome was a custom-made and anatomical design,and the geometric fixture was consists of a hollow cylindrical fixed stem and 2 pins.Four different osteotomy models of talar component for TAR prosthesis were designed,and the amount and area of osteotomy of each model were calculated were calculated.The specific content is as follows.（1）Arcuate osteotomy of the apical talus（AOA）:3153.8 mm³,877.9 mm².（2）Arcuate osteotomy of the apical talus and lateral osteotomy of talus（AOA+LO）:3920.3 mm³,1030.4mm².（3）Corner osteotomy of the apical talus（COA）;3892.4 mm³,947.6 mm².（4）Corner osteotomy of the apical talus and lateral osteotomy of talus（COA+LO）;4432.9 mm³,1114.3mm².After established the FEA models of the four prostheses,the stress distributions of the prosthesises and talus after osteotomy were calculated.The results showed that the stress distribution of two groups with lateral osteotomy（the AOA+LO group and the COA+LO group）was better than that of other two groups wihtout lateral osteotomy（AOA group and COA group）,and the difference in stress distribution between arcuate osteotomy and corner osteotomy was small.3 The results of compression experiments suggest that the compressive strengths of the three porous scaffolds with different porosity meet the clinical requirements.It was observed under electron microscope that the porous structure of the 40%porosity group was blocked by the residual molten powder particles,while the phenomena have not been observed in the pore size and shape of the porous structure of the 60%and 80%porosity groups.The cell culture and scratch test related to leaching solution showed that the new antibacterial Cu-bearing Co-Cr alloys material was not cytotoxic.Incubated results of L929 cells on the scaffold:（1）After the cells were double stained with FM and PI,it was observed that cell proliferation was superior in the 40%porosity group at 2 days of incubation,but the 60%porosity group showed significant cell proliferation and less apoptosis relative to the 40%or 80%porosity groups at 4 and 8 days of incubation.（2）The cells in the viable state were scanned by electron microscopy.It was observed that the number of cells changed with the increase of incubation time,which showed a gradual increase trend.When cultured for 8 days,the number of cells in the 60%porosity group was significantly higher than that in other groups,and there was statistical difference.Osteoinductive culture results of BMSCs on the scaffold:（1）The results of CCK-8showed no statistical difference between the three groups at each time points of incubation,but the total number of cells on 60%porosity group was higher than the other groups.（2）Laser scanning microscope scans showed that the 60%porosity group had the most obvious cell proliferation and less apoptosis at each time points of incubation.（3）At 2 and 4 days of incubation,the OD value representing ALP activity was not significantly different among the three groups.At 8 days of incubation,the 60%porosity group was significantly larger than the 40%and 80%porosity groups.At each incubation time point,the OCN content of the 60%porosity group was greater than that of the other groups.4 The three-dimensional space of the two sets of porous scaffolds was observed by electron microscopy,and no blockage of powder particles was observed.The results of compression experiments suggest that the compressive strength of the two groups of porous scaffolds with different compositions meet the clinical requirements.The osteogenic induction incubation results of BMSCs on the two groups of scaffolds are as follows.（1）The CCK-8 results showed no statistical difference between the Cu-bearing group and the copper-free group at each incubation time point.（2）There was no significant difference in cell proliferation and apoptosis between the Cu-bearing group and the copper-free group under laser confocal microscopy at each incubation time point.（3）The ALP and OCN results representing the osteogenic differentiation trend of BMSCs at each incubation time point were not significantly different between the Cu-bearing group and the copper-free group.The experimental results of the porous scaffold after being implanted into the goat are as follows.（1）One of the goats died at 31 days after surgery,and the datas were not included in the statistical analysis.（2）The complete data of each time node of 8 goats was obtained,and the results of statistical analysis were obtained.After implantation of the prosthesis,the serum copper ion value of the goat gradually increased,reaching a peak on the 15th day,being16.28±3.43μmol/L.（3）In the knee joint anteroposterior radiographs of 8 specimens after surgery,no prosthesis dislodgement and displacement were observed.（4）The volume gray value that the Micro-ct data was converted into in the software.One area was randomly selected for each scaffold,and 16 results were obtained for each group.The statistical analysis showed that the mean value of the gray value of the copper-containing group was 142±12,and that of the copper-free group was 121±10.The result showed that the Cu-bearing group was higher than the copper-free group.（5）16 hard tissue sections in each group were completed the Raman spectroscopy and scanning electron microscopy analysis,and the phosphoric acid vibration peak at 965 cm^-1 was defined as the characteristic peak of bone tissue.The phosphate content and bone content of the Cu-bearing scaffolds-bone interface were significantly higher than those without Cu-bearing group.（6）16 hard tissue sections of each group were stained with methylene blue-acid fuchsin,and another 16 hard tissue sections of each group were stained with Goldner trichrome.The digitized sections were compared and analyzed under software,and it was found that the Cu-bearing group had an advantage in bone integration.Finally,the area of bone formation inside and around the porous structure of32 sections was calculated.The result of statistical analysis of the obtained datas was that the Cu-bearing group was more than the copper-free group.5 The talar component of TAR prosthesis with COA+LO was manufactured using SLM technology,and the prosthesis was consistent with the design model.The dome of the prosthesis was smooth and reflective,and the porous structure of the bone-interface side of the prosthesis was integrated with the main body of the prosthesis.The surface of the prosthesis was observed by the microscope to be smooth and no voids.When the prosthesis was corroded,the visible microstructure was consistent with the laser scanning path.The porous structure was observed to have the same pore size and shape,and the three-dimensional space between the pores was connected to each other without powder particle clogging.Conclutions1 The three-dimensional digital model of the weight-bearing foot and ankle truly restores the three-dimensional structure of the ankle-to-foot bone when the human body is standing,providing a basis for the design and analysis of the talus component.2 In the individualized talar component model for TAR prosthesis with different osteotomy methods,the prosthetic model with Corner osteotomy of the apical talus and lateral osteotomy of talus（COA+LO）was selected for its advantages over others.3 Porous Cu--bearing Co-Cr alloy scaffold with 60%porosity and pore size of 373μm is beneficial to cell proliferation and osteogenic differentiation of BMSCs.4 There was no significant difference in the biological behavior of BMSCs between Cu-bearing and copper-free porous Co-Cr alloy scaffolds with the same porosity and pore size.In vivo experiments in animals suggest that Cu-bearing porous Co-Cr alloy scaffolds are beneficial for osseointegration.5 A method for interactive design of individualized prosthetic porous bone interface was proposed,and a new talar component for TAR prosthesis was fabricated with Cu-bearing antibacterial Co-Cr alloy.