In Vitro Study Of 3D Printed Porous β-tricalcium Phosphate Doped Lanthanum Scaffold For Bone Tissue Repair

【Objective】Bone defects caused by tumors,inflammation,trauma,and surgery could seriously affect patients’ quality of life.To this day,autograft remained the gold standard in the treatment of bone defects.However,the use of bone repair materials had been limited because of the donor source and immune rejection,so the field of bone graft therapy had been a hot spot.I’m looking forward to using 3D printing technology,combined with biological effects of beta tricalcium phosphate and lanthanum elements,build a bionic structure of artificial bone repair scaffold,expect to give it bone inductivity,regulate the performance of the inflammatory microenvironmentand inhibiting osteoclast progenitor cell differentiation,thus has important potential,research value,and it’s hopeful to become an ideal artificial bone repair scaffold.【Method】1.La-β-TCP scaffold preparation,physical characterization,in vitro simu Lated degradation experiment and cytotoxicity experimentThe x La-β-TCP scaffolds were prepared using solid phase sintering and 3D printing techniques,(x=0,1,3,5,x is the percentage of substance La is doped into,referred to as La-β-TCP).The material phase composition and crystal structure,chemical groups and surface morphology of the composites were observed using X-ray diffraction(XRD),Raman spectroscopy and scanning electron microscopy(SEM).The material was immersed in a degradation solution to simulate in vitro degradation.The scaffold’s mineralisation capacity was measured by measuring the p H in the degradation solution and the surface structural observed by SEM.Finally the scaffold was tested for cytotoxicity by MTT assay.2.Effect of La-β-TCP scaffold on osteogenic differentiation,macrophage polarization and osteoclast differentiation of bone marrow mesenchymal stem cellsRat bone marrow mesenchymal stem cells(BMSCs)were cultured using La-β-TCP scaffold extracts and its effect on the proliferative activity of BMSCs was measured using CCK-8,live/dead staining on days 1 and 3;The expression of osteogenic marker genes(Runx-2,Col I,OCN)was examined after 7 and 14 days of treatment of BMSCs with stent extracts;BMSCs were treated with scaffold extracts,stained with ALP on days 7 and 10,the expression of alkaline phosphatase,the osteogenic marker protein,was measured by the ALP assay;and the effect of scaffold extracts on the mineralization capacity of BMSCs was measured by alizarin red staining on day 14 of the extract treatment;The RAW264.7 cell line was treated with La-β-TCP scaffold extract and the effect of the scaffold on the proliferative activity of RAW264.7 cells was measured on the third day using the CCK-8 kit;The expression of inflammation-related genes(IL1,CD206,TNF-α,ARG-1)was measured after 3 days of treatment with stent extracts;the release of pro-inflammatory factors(TNF-α)and anti-inflammatory factors(IL-4)was measured using ELISA kits;and their surface antigens(CD86,CD206)were detected using flow cytometry;The stent infusion was intervened on RAW264.7 cells and the expression of the osteoblast phenotype gene(TRAP)was assayed;anti-tartrate acid phosphatase(TRAP),the osteoblast-specific protein,was assayed using an anti-tartrate acid phosphatase assay kit on day 5.【Result】1.La was successfully incorporated into the structure of β-TCP by solid-phase sintering and causing corresponding lattice expansion;La-β-TCP scaffolds(x La-β-TCP,x=0,1,3,5)with essentially uniform pore size and morphological homogeneity were successfully prepared by 3D printing technology;and the in vitro simulations of the scaffolds showed good mineralization and no hidden toxicity of La-β-TCP scaffolds.2.BMSCs isolated by the whole bone marrow apposition method were microscopically observed to be long shuttle-shaped or triangular in shape and clustered in fish groups when they were passed to the third generation;flow analysis indicated high cell purity and osteogenic and lipogenic induction assays suggested that they had the capacity for directed differentiation.3.SEM results show that BMSCs adhere well to the surface of the scaffold.4.After intervention with BMSCs and RAW264.7 using scaffold extracts,CKK-8assay and cell viability staining suggested good biocompatibility of the scaffold;ALP assay,ALP staining,alizarin red staining and RT-PCR assay indicated that the La-β-TCP scaffold had good osteogenic properties.5.After intervention with scaffold extracts on RAW264.7 cells,RT-PCR,ELISA and flow cytometry assays suggested that with La incorporation,the La-β-TCP scaffold possessed the biological properties to inhibit the polarization of RAW264.7cells towards the M1 phenotype and promote them towards the M2 phenotype while inhibiting their release of the inflammatory factor TNF-α.6.After intervention with scaffold extracts on RAW264.7 cells,RT-PCR and TRAP assays suggested that the La-doped β-TCP scaffold had the ability to regulate the differentiation of macrophages into osteoblasts.【Conclusion】The La-β-TCP scaffold(x La-β-TCP,x=0,1,3,5)was successfully prepared using 3D printing technology combined with solid-phase sintering.The scaffold is biocompatible and has not only a high ability to induce osteogenic differentiation in rat BMSCs,but also certain effects on inhibiting inflammation and osteoclastogenesis.