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

K. Yoshida, S. Suzuki, M. Kawada-Matsuo, J. Nakanishi, S. Hirata-Tsuchiya, H. Komatsuzawa, S. Yamada, H. Shiba

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1327-1343
Number of pages17
JournalInternational Endodontic Journal
Volume52
Issue number9
DOIs
Publication statusPublished - 2019 Jan 1

Keywords

  • LL37
  • LPS neutralization
  • antimicrobial peptides
  • cytotoxicity
  • human dental pulp cells
  • inflammatory cytokine release

ASJC Scopus subject areas

  • Dentistry(all)

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