Evaluation Of The Cytocompatibility And Osteoinduction Of N-acetyl Cysteine Loaded Silk Fibroin/α-tricalcium Phosphate (α-TCP/SF-NAC) Cement

Objective:To prepare N-acetyl cysteine loaded silk fibroin/α-tricalcium phosphate cement(α-TCP/SF-NAC) and evaluate the release kinetics, in vitro cytotoxicity and osteogenic capacity.Methods:A range of concentrations(0 mM, 10 m M and 25 mM) of NAC-loaded SF aqueous solutions was prepared by adding different amounts of NAC stock solution to the SF aqueous solution, and the final concentration of SF was 6%(w/v).The NAC-loaded SF/α-TCP cement(α-TCP/SF-NAC) sample was prepared by mixing the NAC-loaded SF solution(L) andα-TCP powder(P) with an L/P ratio of 0.4 ml/g.UV spectrophotometer was used to determine the concentration of released NAC and total release percentage was calculated. rBMSCs, isolated from the femurs of Sprague–Dawley(SD)-rats, were cultured in cement extracts and cell proliferation was determined using MTS assay andlactate dehydrogenase(LDH) assay was performed to evaluate cell damage. Cell morphology was observed after immunofluorescence staining. Cells were seeded on the surface of the cements and then were subjected to osteoinduction medium. The alkaline phosphatase(ALP) activity in culture medium was determined using ALP activity kit and the relative expression of ALP, Col-Ⅰ and Runx2 genes were measured using RT-PCR.Results:NAC loaded-silk fibroin/α-tricalcium phosphate cement(α-TCP/SF-NAC) was prepared. NAC release kinetics from the composite cement was concentration dependent. 45.25±2.55%(α-TCP/SF-NAC10mM) and 59.15±5.73%(α-TCP/SF-NAC25mM) of total drug was released after 3 weeks. The cytotoxicity of α-TCP/SF-NAC was grade 0 or 1. The cells remained elongated shape in cement extract. The LDH levels of α-TCP/SF-NAC were lower than the other two groups. A significantly higher level of ALP activity of α-TCP/SF-NAC25 mM than the other three groups was measured after 3 days of incubation(p<0.01), the ALP activity level of α-TCP/SF-NAC10 mM was higher than α-TCP/SF and the control group(p<0.05). Both α-TCP/SF-NAC10 mM group and α-TCP/SF-NAC25 mM group had higher relative expression of osteogenic related genes than the other two groups.Conclusion: NAC could be sustained released form the composite cements. NAC loaded-silk fibroin/α-tricalcium phosphate cement(α-TCP/SF-NAC) presented no obvious cytotoxicity. α-TCP/SF-NAC could induce osteogenic differentiation of rBMSCs...