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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

Abstract

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Grünwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

Introduction

Biocompatibility can be defined as the capacity of a material to integrate tissue and induce a favorable therapeutic response, free of local and systemic damages1,2,3. Biocompatibility evaluation is crucial for the development of any material intended for medical use. Therefore, this protocol provides a systematic and comprehensive approach for every researcher aiming to develop new biomaterials or studying new applications for existing biomaterials.

In vitro cytotoxicity tests are widely used as the first phase for biocompatibility evaluation, usi....

Protocol

1. Pellets preparation

  1. Prepare the polyvinyl chloride (PVC) molds by performing circular-shaped holes of known dimensions in PVC plates.
    NOTE: PVC moldings can be made of different sizes. Calculate the contact surface of PVC molds, using the formula A= h(2πr)+2πr2 (r: radius of the cylinder; h: height of the cylinder).
  2. Prepare the biomaterial to be tested according to the manufacturer's instructions and as close as possible to the beginning of the experiment.
    NOTE: For the preparation of paste/paste formulation biomaterials, an adequate amount of base paste and catalyst are mixed manually with a mixing ....

Results

The representative results here refer to the study of dental biomaterials. The extract methodology allows to obtain a cytotoxicity profile and cell function after exposition to the dental materials, regarding effects on metabolic activity (Figure 2), cell viability, cell death profile and cell morphology (Figure 3), and specific proteins expression (Figure 4).

The MTT assay is used to obtain a quick overv.......

Discussion

This protocol was designed taking into consideration the ISO 10993-5, which refers to the evaluation of in vitro cytotoxicity of biomaterials that contact with the tissues, to evaluate the biocompatibility and to contribute to studies reproducibility21. This is a growing concern in science, and many authors are already following these recommendations in the experimental design of their in vitro studies15,22,

Disclosures

The authors have no competing financial interests or other conflicts of interest.

Acknowledgements

We thank the following for support: GAI 2013 (Faculdade de Medicina da Universidade de Coimbra); CIBB is funded by National Funds via FCT (Foundation for Science and Technology) through the Strategic Project UIDB/04539/2020 and UIDP/04539/2020 (CIBB). We thank to Jacques Nör, University of Michigan Dental School, for providing the cell line MDPC-23.

....

Materials

NameCompanyCatalog NumberComments
Absolute ethanolMerck Millipore100983
AccutaseGibcoA1110501StemPro Accutas Cell Dissociation Reagent
ALDH antibodySanta Cruz BiotechnologySC166362
Annexin V FITCBD Biosciences556547
Antibiotic antimycotic solutionSigmaA5955
BCA assayThermo Scientific23225Pierce BCA Protein Assay Kit
Bovine serum albuminSigmaA9418
CaCl2Sigma10035-04-8
CD133 antibodyMiteny Biotec293C3-APCAllophycocyanin (APC)
CD24 antibodyBD Biosciences658331Allophycocyanin-H7 (APC-H7)
CD44 antibodyBiolegend103020Pacific Blue (PB)
Cell strainerBD Falcon35234040 µM
Collagenase, type IVGibco17104-019
cOmplete MiniRoche118 361 700 0
DAB + ChromogenDakoK3468
DithiothreitolSigma43815
DMEM-F12SigmaD8900
DNAse IRoche11284932001
DSP (M-20) Antibody, 1: 100Santa Cruz BiotechnologyLS-C20939
ECC-1ATCCCRL-2923Human endometrium adenocarcinoma cell line
Epidermal growth factorSigmaE9644
Hepes 0.01 MSigmaMFCD00006158
Fibroblast growth factor basicSigmaF0291
Giemsa Stain, modified GS-500SigmaMFCD00081642
GlycerolDakoC0563
HaemocytometerVWRHERE1080339
HCC1806ATCCCRL-2335Human mammary squamous cell carcinoma cell line
Insulin, transferrin, selenium SolutionGibco41400045
May-Grünwald Stain MG500SigmaMFCD00131580
MCF7ATCCHTB-22Human mammary adenocarcinoma cell line
MethylcelluloseAlfaAesar45490
NaClJMGS37040005002212
Polyclonal Rabbit Anti-goat immunoglobulins / HRP, 1: 100DakoG-21234
Poly(2-hydroxyethyl-methacrylateSigmaP3932
PutrescineSigmaP7505
RL95-2ATCCCRL-1671Human endometrium carcinoma cell line
Sodium deoxycholic acidJMSEINECS 206-132-7
Sodium dodecyl sulfateSigma436143
Substrate BufferDako926605
TrisJMGS20360000BP152112
Triton-X 100Merck108603
Trypan blueSigmaT8154
Trypsin-EDTASigmaT4049
β-actin antibodySigmaA5316

References

  1. Williams, D. F. On the mechanisms of biocompatibility. Biomaterials. 29 (20), 2941-2953 (2008).
  2. Bruinink, A., Luginbuehl, R. Evaluation of biocompatibility using in vitro methods: interpretation and limitations.

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