Osteoinduction and long-term osseointegration promoted by combined effects of nitrogen and manganese elements in high nitrogen nickel-free stainless steel

High nitrogen nickel-free stainless steel (HNS) has attracted particular attention in recent years, because of its potential application in orthopedic implants. However, the effects of HNS on osteoblast differentiation of bone marrow mesenchymal stem cells and long-term osseointegration are still unclear. In this study, a newly developed HNS with high manganese (Mn) content (Fe-21Cr-16Mn-2Mo-0.93N) was successfully manufactured. The HNS exhibited the improved bio-corrosion resistance and surface hydrophilicity compared to 316L stainless steel (316L SS) due to its high nitrogen (N) content. The hydrophilic surface of HNS could promote initial adhesion and spreading of rat bone marrow mesenchymal stem cells (rBMSCs) via the upregulation of the gene expression of integrin alpha 1 and integrin beta 1. The high Mn and nickel-free content of HNS facilitated rBMSC proliferation. More importantly, it was revealed that ionic manganese from HNS could enhance the expression levels of osteoblast-related genes (RUNX-2, ALP, OCN and OPN) of rBMSCs, as well as promote ALP activity and OCN protein expression. Moreover, the activation of extracellular signal-related kinases (ERK) 1/2 pathway, via which HNS might induce osteogenic differentiation, was observed in rBMSCs cultured on HNS. Finally, based on HNS implantation in rabbit tibiae for 4, 12 and 26 weeks with 316L SS as a control, Micro-CT evaluation, sequential fluorescent labeling and histological analysis consistently demonstrated that HNS evidently promoted new bone formation and bone-implant contact around implants. Biomechanical tests also confirmed the increased bonding strength at the bone-HNS implant interface in both early and late stages. These results suggest that HNS can induce osteogenic differentiation of rBMSCs and promote rapid and long-term osseointegration of implants, which may be attributed to the combined effects of N and Mn elements.