HaCaT Keratinocytes Response on Antimicrobial Atelocollagen Substrates: Extent of Cytotoxicity, Cell Viability and Proliferation

Abstract

The effective and widely tested biocides: Benzalkonium chloride, bronopol, chitosan, chlorhexidine and irgasan were added in different concentrations to atelocollagen matrices. In order to assess how these antibacterial agents influence keratinocytes cell growth, cell viability and proliferation were determined by using MTT assay. Acquired data indicated a low toxicity by employing any of these chemical substances. Furthermore, cell viability and proliferation were comparatively similar to the samples where there were no biocides. It means that regardless of the agent, collagen-cell-attachment properties are not drastically affected by the incorporation of those biocides into the substrate. Therefore, these findings suggest that these atelocollagen substrates enhanced by the addition of one or more of these agents may render effectiveness against bacterial stains and biofilm formation, being the samples referred to herein as "antimicrobial substrates" a promising view in the design of novel antimicrobial biomaterials potentially suitable for tissue engineering applications.

Figure 1

Chemical structure of employed antibacterial agents.

Figure 2

Chitosan chemical structure.

Figure 3

Effect of various concentrations of target compounds on HaCaT cell viability. Cell line seeded on atelocollagen matrix with concentration of agent (A) 2.0% (w/w); (B) 1.0% (w/w); (C) 0.5% (w/w); (D) 0.2% (w/w); (E) 0.1% (w/w); (F) 0.02% (w/w) determined by MTT assay (the error bars depict standard deviations and dashed lines define cytotoxicity ranges: non-cytotoxicity > 80%; weak > 60%; moderate > 40%; strong < 40%). *p < 0.05 and **p < 0.01, compared with pristine atelocollagen film.

Figure 4

Concentration-viability curve of studied agents on HaCaT keratinocyte cell line.

Figure 5

Comparison of HaCaT cell growth upon atelocollagen films with and without bactericides determined by measuring absorbance of Formazan product by MTT assay at 570 nm (the error bars signify standard deviation). * p < 0.05 and ** p < 0.01, compared with free of biocide film.

Figure 6

Photomicrographs of Human skin HaCaT keratinocytes in culture upon two atelocollagen films compared with control: (A) Atelocollagen without biocides; (B) An endowed film with cell viability above 80% (non-cytotoxic).