Heparin–LL37 complexes are less cytotoxic for human dental pulp cells and have undiminished antimicrobial and LPS‐neutralizing abilities. (11th May 2019)

• Record Type: Journal Article
• Authors: Yoshida, K.; Suzuki, S.; Kawada‐Matsuo, M.; Nakanishi, J.; Hirata‐Tsuchiya, S.; Komatsuzawa, H.; Yamada, S.; Shiba, H.
• Is Part Of: International endontic journal. Volume 52:Number 9(2019)
• Page Start: 1327
• Page End: 1343
• Publication Date: 2019-05-11
• Subjects: antimicrobial peptides -- cytotoxicity -- human dental pulp cells -- inflammatory cytokine release -- LL37 -- LPS neutralization; Endodontics -- Periodicals; 617.6342
• DOI: 10.1111/iej.13130
• Languages: English
• ISSNs: 0143-2885

Abstract

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.