| PrefaceArtificial joint replacement is a common way in orthopedic repair.Due to the loss of natural insertions of tendons or ligaments,fixation of tendon and ligaments on metal joint prostheses is necessary for joint replacement in patients with bone tumors,bone infections or revision prosthetic arthroplasty.At present,tendon-prosthesis integration mainly relies on mechanical fixation,wihch may become loose and lead to fixation failure over time.How to promote the effective integration of the tendon-prosthesis interface is of great significance to the success of operation and the recovery of patients’ motor function.Titanium alloy is the commonly used material of metal prostheses.Due to its biological inertia,the tendon cannot attach effectively on the metal surface.The stress concentration at the interface of tendon-prosthesis insertion due to the sudden change of physical properties of tissue is also a major reason for the failure of tendon-prosthesis insertion.Studies have reported that porous titanium alloy combined with BMP-2 can induce osteogenesis at the tendon-metal interface,and the combined application of porous structure and osteogenesis can enhance the fixation.However,the current structural design of porous titanium alloy materials focuses on the effect of bone integration,and the structural design suitable for tendon,ligament has not been reported.Compared with BMP-2 and other cytokines,europium has better stability,and has the potential to be used as osteogenic induction material at the tendon-metal interface.In the study,we design porous structure,and manufacture with titanium alloy material by3 D printing,than use chitosan hydrogel containing europium for its biological activity,to obtain the porous titanium alloy-europium containing hydrogel composite material,explore its role in the tendon-metal integration.Providing experimental basis for the promotion of integration of soft tissue-metal prosthesis.1 Preparation of 3D printed porous titanium alloy material and its effect on tendon ingrowth.1.1 Method:1.1.1 triply periodic minimal surface models with different pore sizes were designed by implicit function method.Ti6Al4 V alloy was used to manufacture the corresponding porous titanium alloy samples by selective laser melting.According to the pore size,the samples were named Ti300,Ti500 and Ti700.1.1.2 The ratio of apparent volume to actual volume was used to calculate the actual porosity.The samples were observed with scanning electronic microscope and micro-CT.1.1.3 The leach liquor of porous titanium alloy samples with different pore sizes were extracted for calculation of cytotoxicity to tendon-derived stem cells.Use relative proliferation rate to evaluate cytotoxicity.1.1.4 The fixation model of rabbit patellar tendon to porous titanium alloy was established.According to the pore diameter of the implanted sample,it was divided into group solid,ti30,ti500 and Ti700.1.1.5 Hard tissue sections were stained with hematoxylin-eosin and Sirius red to evaluate the growth of tendon tissue in different groups of porous samples.1.1.6 Biomechanics was used to detect the maximum pulling force of tendon in different groups of samples,and to determine the influence of samples with different pore sizes on the fixation strength of tendon.1.2 Result:1.2.1 The surface of different groups of titanium alloy samples was observed by scanning electron microscope,which all rough on surface,presenting small curved angle and smooth between pillars consistent with the design.Micro-CT observed that the metal pillars and pores in the material were evenly arranged,and the pores were interconnected.The porosity of different groups was 78.12%±0.45% for Ti300,82.76%±0.54% for Ti500 and 83.01%±0.58% for Ti700.1.2.2 The relative proliferation rate of tendon-derived stem cells cultured with leach Liquor of titanium alloy with different pore sizes was not significantly different from that of the control group cultured with DMED low sugar medium.1.2.3 HE staining of hard tissue sections showed that the pores of Ti500 group and Ti700 group were more filled than those of Ti300 group at all time points postoperative,and the fibers in the pores of Ti500 group were more orderly at the 12 th week.Sirius red staining showed that more type I collagen was produced in the pores of ti500 group at the 12 th week.1.2.4 At the 12 th week,the maximum pulling force of Ti300,Ti500 and Ti700 were 44.46±11.26 N,101.62± 13.69 N,and 54.66±11.17 N respectively.Ti500 group was significantly higher than the other two groups.1.3 Summary1.3.1 The porous Ti6Al4 V alloy samples fabricated by selective laser melting is in line with the pre-design,with regular pores and interconnected pore structure and fair biocompatibility.1.3.2 The 500 μm aperture triply periodic minimal surface porous titanium alloy sample has better tendon ingrowth,better collagen remodeling and higher mechanical strength.2 Preparation of europium-containing chitosan hydrogel and its effect on ossification in tendon2.1 Method2.1.1 Tendon-derived stem cells were extracted from rat Achilles tendon by enzyme digestion,and the stem cell characteristics and multidirectional differentiation ability were identified.2.1.2 The tendon-derived stem cells were cultured in induction medium with different concentrations of Europium.The m RNA levels of osteogenic differentiation marker in different groups were detected by real-time fluorescence quantitative PCR,and the effect of the 7th day was detected by Western blot.2.1.3 Chitosan hydrogels containing 1 μM,10 μM,100 μM Europium and no Europium were prepared.The physical and chemical properties of chitosan hydrogels were detected by scanning electron microscopy,optical profiler and X-ray photoelectron spectroscopy.2.1.4 The cells were co-cultured with leach liquor of hydrogels with different concentrations of Europium for calculation of their cytotoxicity.The proliferation and mortality of tendon derived stem cells on hydrogels of different groups were detected by living and dead cell staining.2.1.5 Chitosan hydrogels containing 10 μM Europium was used as the experimental group(Eu10),and chitosan hydrogels without Europium was used as the control group(Eu0).The in vivo osteoinduction effect of Europium was verified by injection of hydrogels into rat Achilles tendon.Osteogenesis effect was assessed by Micro-CT and histological observations at week 4,8,and 12 after injection.2.2 Result2.2.1 The P1 generation cells obtained from rat Achilles tendon showed clonal colony growth.Immunofluorescence staining for CD31 and CD34 was negative,CD44 and CD90 were positive.After culture in the corresponding induction medium,they could differentiate into osteogenic,chondrogenic and adipogenic directions respectively.2.2.2 On day 7 of bone induction culture,10 μM Europium significantly promoted the transcription of m RNA ALP,BMP-2 and Runx2.Increased the expression levels of ALP,BMP-2,Runx2 and OPN protein.2.2.3 Chitosan hydrogels showed lamellar-porous mixed structure under electron microscope.The hydrogels containing Europium have more complex 3D structure pores under electron microscope,and the pore wall surface is rougher.It also shows higher surface roughness on optical profiler.X-ray energy dispersive spectroscopy and X-ray photoelectron spectroscopy confirmed the successful doping of Europium element in chitosan hydrogels.2.2.4 The hydrogel leach liquor without Europium showed mild toxicity,while the hydrogel Leach Liquor contain Europium in all groups showed no biological toxicity.Tendon-derived stem cells cultured in different groups of hydrogels showed that the Europium-containing hydrogels had more cell growth on day 5 than the Europium-free hydrogels.2.2.5 At week 8 and 12 after injection,the volume of Achilles tendon mineralization in Eu10 group was 1.462±0.283 mm~3 and 2.978±0.769 mm~3,which was significantly higher than Eu0 group at the same time point.Immunohistochemical staining results showed that the positive rates of osteogenic markers ALP and BMP-2,chondrogenic markers Aggrecan and endochondral ossification marker Col X in the tissues around hydrogel in Eu10 group were significantly higher than those in Eu0 group at week 8 and 12 after injection.In Eu10 group,red stained cartilaginous areas were observed by His red O-solid green staining at week 12.2.3 Summary2.3.1 Stem cells from rat tendon were extracted successfully.2.3.2 The optimum concentration of Europium in vitro osteogenesis was 10 μM.2.3.3 Chitosan hydrogels with Europium was successfully prepared.2.3.4 Chitosan hydrogels with 10 μM Europium can significantly promote the osteogenesis in rat Achilles tendon compared with hydrogels non-Europium.3 Porous titanium alloy-Europium containing chitosan hydrogel composite can promote ossification in tendon reinforce tendon fixation3.1 Method3.1.1 Porous titanic-hydrogel composites were prepared by freeze-drying method.The samples without or with Europium in the hydrogel were designated Ti-G group and Ti-E group.The structure of the composite was observed by scanning electron microscope.3.1.2 The cell seeding efficiency and cell proliferation of the samples were tested to determine their cytocompatibility.3.1.3 The model of rabbit patellar tendon fixed on titanium alloy-hydrogel composite was established.3.1.4 At week 4,8 and 12 after the establishment of rabbit model,the osteogenesis effect was evaluated by micro-CT and histological approach.Micro-CT and methylene blue-acidic fuchsine staining of hard tissue sections were used to detect the osteogenesis effect in the tissues.3.1.5 At the 12 th week after the establishment of rabbit model,biomechanics was used to detect the maximum pulling force of different groups to determine the effect of porous titanium alloy-EUROPIUM-containing hydrogel composite samples on the fixation strength of tendon.3.2 Result3.2.1 Scanning electron microscope(SEM)observed that the hydrogel was completely filled in the original pores of the metal pillars,showing lamellar and porous.The hydrogel pores of Ti-E group were more complex and uniform.X-ray energy dispersive spectrum confirm that the ratio of Europium atoms to oxygen atoms is about 0.33% in Ti-E group,which conforms to the designed Europium concentration.3.2.2 The cell seeding efficiency of TI-G group and Ti-E group was 73.36%±5.48% and 76.26%±3.53%,which was higher than simple metal material.The cells could proliferate well on the material surface after seeding.3.2.3 At the 4th week after surgery,hard tissue section staining can identify the mineralized area on the surface of the sample in Ti-E group.At the 8th and 12 th weeks after surgery,hard tissue section and Micro-CT could identify the mineralized area on the surface of the sample in Ti-E group,and the osteogenic volume was 1.293±0.332 mm~3 and 1.316±0.702 mm~3 through micro-CT calculation.There is no mineralization observed in Ti-G group.3.3 Summary3.3.1 The metal-hydrogel composites were successfully prepared.Both groups had good biocompatibility.3.3.2 Metal-Europium-containing chitosan hydrogel composite showed good osteoinduction effect in rabbit patellar tendon.3.3.3 The fixation strength of tendon on the surface was enhanced on metalEuropium-containing chitosan hydrogel composite.Conclusion:In this study,we optimized the porous structure of the fixation point on porous titanium and determined the suitable concentration of Europium in the chitosan hydrogel for osteoinduction in tendon tissue,then prepared composite material with Europium hydrogel in porous titanium alloy.Finally,it was verified that composite material induces bone formation in tendon tissue and reinforce tendon fixation on the metal implant. |
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