Challenging applicability of ISO 10993-5 for calcium phosphate biomaterials evaluation: Towards more accurate in vitro cytotoxicity assessment

Biomater Adv. 2024 Jun:160:213866. doi: 10.1016/j.bioadv.2024.213866. Epub 2024 Apr 18.

Abstract

Research on biomaterials typically starts with cytocompatibility evaluation, using the ISO 10993-5 standard as a reference that relies on extract tests to determine whether the material is safe (cell metabolic activity should exceed 70 %). However, the generalized approach within the standard may not accurately reflect the material's behavior in direct contact with cells, raising concerns about its effectiveness. Calcium phosphates (CaPs) are a group of materials that, despite being highly biocompatible and promoting bone formation, still exhibit inconsistencies in basic cytotoxicity evaluations. Hence, in order to test the cytocompatibility dependence on different experimental setups and material-cell interactions, we used amorphous calcium phosphate, α-tricalcium phosphate, hydroxyapatite, and octacalcium phosphate (0.1 mg/mL to 5 mg/mL) with core cell lines of bone microenvironment: mesenchymal stem cells, osteoblast-like and endothelial cells. All materials have been characterized for their physicochemical properties before and after cellular contact and once in vitro assays were finalized, groups identified as 'cytotoxic' were further analyzed using a modified Annexin V apoptosis assay to accurately determine cell death. The obtained results showed that indirect contact following ISO standards had no sensitivity of tested cells to the materials, but direct contact tests at physiological concentrations revealed decreased metabolic activity and viability. In summary, our findings offer valuable guidelines for handling biomaterials, especially in powder form, to better evaluate their biological properties and avoid false negatives commonly associated with the traditional standard approach.

Keywords: Apoptosis; Calcium phosphates; Cytocompatibility guideline; Cytotoxic; Direct contact; Indirect contact; Material characterization; Necrosis; Standards.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Biocompatible Materials* / pharmacology
  • Biocompatible Materials* / toxicity
  • Calcium Phosphates* / chemistry
  • Cell Line
  • Cell Survival / drug effects
  • Durapatite*
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism
  • Humans
  • Materials Testing* / methods
  • Materials Testing* / standards
  • Mesenchymal Stem Cells* / drug effects
  • Osteoblasts* / drug effects
  • Osteoblasts* / metabolism

Substances

  • Calcium Phosphates
  • Biocompatible Materials
  • Durapatite
  • octacalcium phosphate
  • alpha-tricalcium phosphate
  • calcium phosphate