Sustained Release Of Infliximab From ADSCs-Loaded Hydrogel For Promoting 3D Printed Metal Microporous Interface Osseointegration In RheumatoidArthritis

Background:Rheumatoid arthritis(RA)is a global multiple disease,and the proportion of joint replacement surgery has increased year by year.Although the RA conventional prosthesis can achieve early temporary fixation,it can not form a good interface osseointegration,which affects the long-term stability of the prosthesis.Therefore,we need to develop artificial joint prosthesis with bioactive interface for the pathogenesis and pathological basis of RA,which has important clinical significance for solving the problem of high incidence of RA prosthesis loosening.Studies have shown that prosthesis loosening after RA is mainly related to the design of traditional prosthesis and the level of peripheral inflammation.The elastic modulus between the plants in the traditional titanium alloy and the host bone does not match,and the stress shielding effect is easily generated.At present,the 3D printed plants prepared by electron beam melting(EBM)technology have suitable elastic modulus,which can reduce stress shielding and bone absorption.In addition,the metal microporous structure can provide bone ingrowth space,thereby improving the osseointegration ability and long-term stability of the inner plant.However,the biological inertness of metal scaffolds and the inflammatory microenvironment of RA are not conducive to osseointegration,so we need to construct a new "biologically active interface" on the surface of metal micropores.As a carrier of biologically active substances,chitosan hydrogel can improve the osseointegration efficiency of the metal microporous interface.By carrying exogenous mesenchymal stem cells(MSCs),hydrogels not only improve cell adhesion at the microporous interface,but also help to exert immunomodulatory effects of MSCs and reduce local inflammatory levels,thereby facilitating the development of osteogenic differentiation potential.Among various MSCs,adipose-derived stem cells(ADSCs)have strong immunomodulatory ability.However,the immunomodulatory ability of MSCs alone is low,and the local combination of infliximab is expected to exert synergistic anti-inflammatory effects.Therefore,this study attempts to construct a bioactive interface of "3D printed metal microporous/hydrogel/inflixima/ADSCs",which has a synergistic immunomodulatory effect on the sustained release of infliximab and ADSCs,and is constructed to facilitate osteogenesis.The differentiated microenvironment promotes the integration of the metal micropore-bone interface.This study can provide experimental support for artificial prosthesis interface design in patients with articular replacement such as RA.Part ?: In vitro isolation and culture of adipose-derived stem cells(ADSCs)and their multi-directional differentiation potential and cell surface markersThe collection and separation of ADSCs from Adipose tissue: Adipose tissue was obtained from the subcutaneous fat pad of rabbit inguinal region,and ADSCs were isolated with enzymatic digestion.The ADSCs morphology was similar in 5th passage,which mainly showed spindle and polygon morphology.Flow cytometry showed that cells were positively labeled by CD44 and Integrin-?,and low positively labeled by WT,CD34,CD31,c-kit and VCAM1.After culture for 21 days under adipogenic condition,the mineralized nodules were stained by alizarin red;after cultured for 14 days,lipid-filled vesicles were stained by oil red.Part ?: Construction of bioactive interface for 3D printed metal microporous/hydrogel3D printed titanium scaffolds using EBM technique have high porosity(67±4%)and proper pore size(242±15 ?m)with fully interconnected pores.The compression strength and Young's modulus of scaffolds were 104±3 MPa and 4.9±0.2 GPa,respectively.The SEM measurement displayed that the surface roughness was high.The mixed sol can pass through the No.26 syringe with no clogging phenomenon;at room temperature,the sol can be converted into a hydrogel by mixing for about20 s.After 1 hour of incubation,the hydrogel exhibited a balanced swelling state with a maximum swelling ratio of 50%.The hydrogel remained in its original form and no significant weight loss was observed.In vitro degradation experiments: The hydrogel weight was reduced by about 50% at 14 days and completely degraded at30 days.In vivo degradation experiments: hydrogel weight decreased by about 50%at 14 days and complete degradation at 30 days.Inflixoxib/ADSCs/A sol mixture was injected into the space of the stent from top to bottom with a 1 mL syringe syringe,and then the B sol was also injected into the space of the stent,and allowed to stand for 20 s for liquid-solid transformation to complete the 3D printed metal microporous/ Construction of a hydrogel bioactive interface.The SEM showed that a hydrogel having a uniform porous structure was observed inside the pores of the rough titanium alloy.Part ?: Loading of ADSCs hydrogel sustained-release infliximab to promote osseointegration of 3D printed metal microporous interface in RAThe hydrogel system can achieve a slow release of infliximab,and the cumulative release rate of infliximab in about 28 days is 71.06%.The results of living dead cell staining showed that the composite system had good cytocompatibility;the results of phalloidin staining showed that the cells were spherical in the hydrogel system.The RA inflammation microenvironment was simulated and the cell proliferation levels of each group were evaluated.The CCK-8experiment showed that the cell proliferation ability of the hydrogel/infliximab/3D titanium alloy stent group(MSHI group)was superior to the other groups after 4,7and 14 days of culture,and the difference was statistically significant.The RA microenvironment was simulated and the osteogenic differentiation of each group was evaluated.The ALP activity test showed that the ALP level in the MSHI group was higher than that in the other groups after 4 and 7 days of culture,and the difference was statistically significant.The alizarin red staining and semi-quantitative results showed that the mineralization nodule formation ability of MSHI group was better than that of other groups at 14 and 21 days of culture,and the difference was statistically significant.The results of osteogenic related gene expression showed that the expression levels of Runx2,ALP,Osterix and Col-I in MSHI group were better than those in other groups,and the difference was statistically significant.Each composite system was implanted into the femoral condyle of the RA model animal,and the level of joint inflammation and the osseointegration ability of the stent were evaluated 12 weeks after surgery.Joint inflammation level: MSHI group and(infliximab/ADSCs/hydrogel/3D printed titanium alloy stent)The jointdiameter of MSHIA group was significantly lower than other groups,the difference was statistically significant;MSHIA group TNF-?,IL The levels of IL-6 and ovalbumin were lower than those of the other groups.The difference was statistically significant.The levels of synovial inflammation in MSHIA group and MSHI group were lower than those in other groups.The difference was statistically significant.Micro-CT results showed that the bone length,bone density and trabecular structure of the MSHIA group were superior to those of other groups.The results of H&E staining of the scaffold tissue sections showed that there were more new bones around the scaffold in the MSHIA group,and the bones were ingrowthed inside the scaffold,which was superior to other groups.The stent introduction experiment showed that the MSHIA group had better scaffolding power than the other groups,and the difference was statistically significant.