Heparin-LL37 complexes are less cytotoxic for human dental pulp cells and have undiminished antimicrobial and LPS-neutralizing abilities.

ABSTRACT

AIM: To investigate whether glycosaminoglycans (GAGs) binding to high-dose LL37 eliminates its cytotoxicity to dental pulp cells (hDPCs) whilst retaining undiminished antimicrobial and LPS-neutralizing abilities. METHODOLOGY: hDPCs were stimulated with varying concentrations of LL37, and their cell viability was analysed by MTT. Then, high-dose LL37 (10 µmol L-1 ) was bound to varying concentrations of three GAGs, heparin, chondroitin sulphate and hyaluronic acid, and their cytotoxic effects on hDPCs and antimicrobial effects were evaluated and compared. Furthermore, the LPS-neutralizing ability of heparin (5 µg mL-1 )-LL37 (10 µmol L-1 ) complexes, which were found to be less cytotoxic for hDPCs with undiminished antimicrobial ability, was investigated. Statistical analysis was performed using one-way analysis of variance (anova), followed by Dunnett's test. P values below 0.05 were considered significant. RESULTS: LL37 significantly reduced the cell viability of hDPCs in a dose-dependent manner (P < 0.01). LL37 (10 µmol L-1 ) binding to heparin within a limited concentration range (2~6 µg mL-1 ) eliminated the cytotoxicity for hDPCs (P < 0.01) whilst exerting potent antimicrobial effects against Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Aggegatibacter actinomycetemcomitans and Escherichia coli. LL37 (10 µmol L-1 ) binding to chondroitin sulphate exhibited similar functions (P < 0.01); however, the effective chondroitin sulphate concentration was highly restricted (3 µg mL-1 ). LL37 (10 µmol L-1 ) binding to hyaluronic acid was unable to abrogate the cytotoxicity of LL37 even at higher concentrations (10 and 100 µg mL-1 ). Moreover, exogenous addition of LPS dose-dependently reduced the amount of LL37 precipitated with the heparin-LL37 agarose beads (P < 0.01), and the released LL37 simultaneously neutralized the pro-inflammatory ability of LPS in macrophages (P < 0.01). CONCLUSIONS: Heparin-LL37 complexes generated at suitable concentration ratios are easy to make, are less cytotoxic and are broad-range antimicrobial materials that can neutralize LPS by providing LL37 in accordance with the amount of free LPS. They may be a potential treatment to save dental pulp tissue from the acute inflammation exacerbated by invading bacteria and the LPS they release.