Continuously Released Zn²⁺ In 3D-Printed PLGA/β-TCP/Zn Scaffolds For Bone Defects Repair By Improving The Osteoinductivity And Anti-inflammatory Ability

Background:Bone defects can usually be caused by a variety of orthopedic diseases,and the most common treatment for bone defects is bone grafting.In recent years,as the shortcomings of autologous and allogeneic bone grafts have been gradually confirmed,traditional bone grafts have been unable to meet the huge demand for bone grafting.It is urgent to develop a more ideal new type of bone repair bioscaffold to solve this problem.In the studies of other researchers,the poly(lactic-co-glycolic acid)/β-tricalcium phosphate(PLGA/β-TCP,PT)scaffolds,due to their good osteoconductivity and adjustable degradation rate,have been used in the field of bone repair and have been shown to be effective.However,obvious problems such as insufficient mechanical properties and lack of osteoinductivity limit its further application and development.Metal elements such as magnesium and zinc(Zn)have been studied in improving mechanical properties and osteoinductivity.Compared with magnesium,Zn has a more moderate degradation rate and has an anti-inflammatory ability that is beneficial to bone repair,and is more suitable for solving the problems of PT scaffolds.However,the excessive concentration of zinc ion(Zn2+)generated by Zn degradation will cause cytotoxicity,so it is particularly important to control the content of Zn and to release Zn2+stably.Nanoparticles have good stability and are easy to add and prepare.Studies have confirmed that zinc nanoparticles(Zn-NPs)can be added to materials to control the content of Zn and stably release Zn2+.Therefore,adding Zn-NPs to PT scaffolds is expected to make up for the lack of mechanical properties and osteoinductivity of PT scaffolds by controlling Zn addition content and stably releasing Zn2+,thereby developing a more ideal new type of bone repair bioscaffolds.Objective:This study intends to uniformly add Zn-NPs into PT scaffolds by lowtemperature 3D printing technology.The mechanical properties,osteoinductivity and antiinflammatory abilities of the scaffolds are expected to be enhanced and release a safe dose of Zn2+.At the same time,the scaffold was structurally biomimetic to develop a new PLGA/β-TCP/Zn(PTZ)scaffold to repair bone defects,and to explore its osteogenesisrelated molecular mechanism.Methods:PLGA(P),PT,and PTZ scaffolds were prepared by low-temperature 3D printing technology,and the composition ratio of scaffolds was selected by evaluating the proliferation of rat bone marrow derived mesenchymal stem cells(BMSCs).The surface morphology,internal structure and mechanical properties of the scaffolds were characterized.Meanwhile,the release of Calcium ion(Ca2+)and Zn2+ in vitro of PTZ scaffolds was tested.The effects of three scaffolds on the viability,adhesion and osteogenic differentiation of BMSCs were observed in vitro.Western blot(WB)and proteomics were used to analyze the molecular mechanism of PTZ scaffold promoting osteogenic differentiation of BMSCs.Finally,in vivo experiments,the effect of implanted scaffolds on the repair of rat femoral condyle bone defects was observed.Results:The composition ratio of the PTZ scaffold was chosen as the ratio of PLGA and βTCP was 2:1,and the Zn content was 1 wt%.The PTZ scaffolds continuously released Zn2+within 16 weeks without cytotoxicity and had good cell proliferation ability.The internal pores of PTZ scaffolds were connected,and its large pore size(-500 μm)was conducive to the growth of BMSCs.The small-sized micropores(<1 μm)on the surface of PTZ scaffolds and the stable release of Zn2+ from PTZ scaffolds promoted the osteogenic differentiation of BMSCs.Compared with PT scaffolds,the addition of Zn-NPs to PTZ scaffolds reduced the agglomeration of β-TCP particles on the surface of the scaffolds,making the PTZ scaffolds more dense and improving the compressive strength of the scaffolds.In vitro,none of the three groups of scaffolds had adverse effects on cell viability.Meanwhile,the PTZ scaffolds promoted the adhesion and osteogenic differentiation of BMSCs due to the sustained release of Ca2+and Zn2+,respectively.Preliminary analysis of in vitro WB and proteomic results indicated that compared with PT scaffolds,PTZ scaffolds might upregulate Runx2 by activating the Wnt/β-catenin pathway,thereby promoting the osteogenic differentiation process.At the same time,the activation of P38 MAPK pathway and the inhibition of NFkB pathway also generated an immune microenvironment favorable for osteogenesis.In vivo experiments also found that PTZ scaffolds compared with PT scaffolds accelerated new bone formation and produced less hyperplastic fibrous tissue after 4 weeks,8 weeks,and 12 weeks of implantation into rat femoral condyle defects with a diameter and depth of 3 mm.At the same time,tissues implanted with PTZ scaffolds expressed higher osteogenesis-related factors BMP2,OPN and anti-inflammatory factor IL10,and lower pro-inflammatory factor TNF-α.Conclusion:We developed a novel PTZ scaffold in this study.The internal pore connectivity of the PTZ scaffold was good,and the multi-level pore structure of the scaffold with both macropores and micropores was similar to that of natural cancellous bone.The addition of Zn-NPs in the PTZ scaffolds reduced the agglomeration of(3-TCP particles,which was beneficial to the formation of a denser structure,and the CS of the PTZ scaffolds was improved.The E and improved CS of PTZ scaffolds were close to those of trabecular bone.PTZ scaffolds had no effect on the vability of BMSCs.At the same time,the PTZ scaffold was beneficial to the proliferation and adhesion of BMSCs,and the sustained release of Zn2+promoted the osteogenic differentiation of BMSCs and maintained longterm non-toxicity.Therefore,PTZ scaffolds could create a bone physiological microenvironment that was favorable for the ingrowth and osteogenic differentiation of BMSCs.In addition,the PTZ scaffolds enhanced the osteoinductivity and antiinflammatory ability in vivo through the sustained release of Zn2+,thereby promoting the repair of bone defects.This study preliminarily explored the molecular mechanism of PTZ scaffold as a novel scaffold for bone defect repair and future orthopaedic therapy.