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Heparin– LL 37 complexes are less cytotoxic for human dental pulp cells and have undiminished antimicrobial and LPS ‐neutralizing abilities
Author(s) -
Yoshida K.,
Suzuki S.,
KawadaMatsuo M.,
Nakanishi J.,
HirataTsuchiya S.,
Komatsuzawa H.,
Yamada S.,
Shiba H.
Publication year - 2019
Publication title -
international endodontic journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.988
H-Index - 119
eISSN - 1365-2591
pISSN - 0143-2885
DOI - 10.1111/iej.13130
Subject(s) - cytotoxicity , chondroitin sulfate , antimicrobial , chemistry , cytotoxic t cell , glycosaminoglycan , streptococcus mutans , heparin , microbiology and biotechnology , biochemistry , in vitro , bacteria , biology , genetics
Aim To investigate whether glycosaminoglycans ( GAG s) binding to high‐dose LL 37 eliminates its cytotoxicity to dental pulp cells ( hDPC s) whilst retaining undiminished antimicrobial and LPS ‐neutralizing abilities. Methodology hDPC s were stimulated with varying concentrations of LL 37, and their cell viability was analysed by MTT . Then, high‐dose LL 37 (10 μmol L −1 ) was bound to varying concentrations of three GAG s, heparin, chondroitin sulphate and hyaluronic acid, and their cytotoxic effects on hDPC s and antimicrobial effects were evaluated and compared. Furthermore, the LPS ‐neutralizing ability of heparin (5 μg mL −1 )– LL 37 (10 μmol L −1 ) complexes, which were found to be less cytotoxic for hDPC s 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 LL 37 significantly reduced the cell viability of hDPC s in a dose‐dependent manner ( P < 0.01). LL 37 (10 μmol L −1 ) binding to heparin within a limited concentration range (2~6 μg mL −1 ) eliminated the cytotoxicity for hDPC s ( P < 0.01) whilst exerting potent antimicrobial effects against Streptococcus mutans , Streptococcus sobrinus , Streptococcus salivarius , Aggegatibacter actinomycetemcomitans and Escherichia coli . LL 37 (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 ). LL 37 (10 μmol L −1 ) binding to hyaluronic acid was unable to abrogate the cytotoxicity of LL 37 even at higher concentrations (10 and 100 μg mL −1 ). Moreover, exogenous addition of LPS dose‐dependently reduced the amount of LL 37 precipitated with the heparin– LL 37 agarose beads ( P < 0.01), and the released LL 37 simultaneously neutralized the pro‐inflammatory ability of LPS in macrophages ( P < 0.01). Conclusions Heparin– LL 37 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 LL 37 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.