An in-vitro medical device material biocompatibility study using primary cell cultures of rat osteoblasts

Authors

  • Fiayaz Shaama Department of Medical Sciences, University of The West Indies, Trinidad and Tobago, West Indies
  • Ann Wilson Department of Chemistry, University of The West Indies, Trinidad and Tobago, West Indies
  • Valerie Stoute V.A.S. Consulting, Port-of-Spain, Trinidad and Tobago

DOI:

https://doi.org/10.18203/issn.2454-2156.IntJSciRep20205020

Keywords:

Osteoblast, Bioactive wood, Biomarkers, Medical device testing, Alkaline phosphatase

Abstract

Background: We questioned if simple chemical methods could be applied as a possible biocompatibility test for biomaterials.

Methods: In a qualitative experiment, osteoblasts cells harvested from newly born (3-5) day old sprague-dawley rats were cultivated in growth medium in a controlled environment in the presence of titanium (Ti) (American Elements®), cobalt-chromium (Co-Cr) (Nobilium®), bio-activated rattan wood, orthopaedic bone cement (CEMEX®), implant fixtures (Astra TDC), and resorbable suture material (Poly-Gly-Lac) (VICRYL®). Sample aliquots were withdrawn periodically over 16 days. One and two-dimensional polyacrylamide gel electrophoresis (PAGE) as well as isoelectric focusing (IEF) produced spots that were subjected to enzyme digestion and molecular weight determination. In a follow-up quantitative experiment, the same samples, except for those containing CEMEX and VICRYL, were prepared. The alkaline phosphatase (ALP) activity was monitored continuously using the colorimetric Stanbio® kits. The ALP activities at days 2, 4, 8, 12, and 16, designated as a longitudinal variable, TIME, were analysed, using SPSS V.22, as a mixed ANOVA model with TIME as a repeated measure and the material type as an independent factor.

Results: The IEF and second dimension PAGE produced an additional spot for Ti at pH(I) of 5-6. This was identified as IQUB_RAT IQ, a ubiquitin-like domain, molecular weight 2.6 kDa. This method was able to find finite differences in osteoblast activity after initial exposure to a foreign body. Both the ALP activity changes from one day to the next for all materials and the TIME-material type interaction effects were significant (p=0.000).

Conclusions: This technique is suitable for use with human cell lines or clones. Experiments like these reduce the need for animal testing.  

Metrics

Metrics Loading ...

References

Zange R, Li Y and Kissel T. Biocompatibility testing of ABA triblock copolymers consisting of poly (L-lactic-co-glycolic Acid) A Blocks attached to a central poly (ethylene Oxide) B Block under in vitro conditions using different L929 mouse fibroblasts cell culture models. J Cont Relea. 1998; 56:249-58.

Thonemann B, Schmalz G, Hiller KA, Schweikl H. Responses of L929 mouse fibroblasts, primary and immortalized bovine dental papilla-derived cell lines to dental resin components. Dent Mater. 2002; 18:318-23.

Anderson JM, Rodriguez A, Chang DT. Foreign body reaction to Biomaterials. Semin Immunol. 2008;20:86-100.

Yamaguchi T, Chattopadhyay N, Kifor O, Butters RR Jr, Sugimoto T, Brown EM. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells. J Bone Miner Res. 1998;13:1530-8.

Pautke C, Schieker M, Tischer T, Kolk A, Neth P, Mutschler W, Milz S. Characterization of osteosarcoma cell lines MG-63, SaOs-2 and U-2 OS in comparison to human osteoblasts. Anticanc Res. 2004;24:3743-8.

Thonemann B, Schmalz G, Hiller KA and Schweikl H. Responses of L929 mouse fibroblasts, primary and immortalized bovine dental papilla-derived cell lines to dental resin components. Dent Mater. 2002:318-23.

Kaur G, Dufour J. Cell lines Valuable tools or useless artefacts. Spermatogenesis 2012;2:1-5.

ISO. Medical devises. Available at: https://www.iso.org/iso-13485-medical-devices.html. accessed 14 on March 2019.

FDA. Medical devices. Available at: https://www.fda.gov/downloads/medicaldevices/.../ucm348890.pdf. Accessed on 13 February 2019.

Flecknell P. Replacement, reduction and refinement. ALTEX 2002;19:73-8.

Russell WMS, Burch RL. John Hopkins Bloomberg school of Public Health. The Principles of Humane Experimental Technique. London: Methuen and Co. Ltd.; 1959.

FDA/CDRH Webinar [Online]. Training guidance. https://www.fda.gov/downloads/Training/CDRHLearn/UCM51240-9.pdf. Accessed on 21 February 2020.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-5.

Gronow M, Griffith G. Rapid isolation and separation of non-histone proteins of rat liver nuclei. FEBS Lett. 1971;15:340-4.

Wilson C. Staining of proteins on gels: comparisons of dyes and procedures. Meth Enzymol. 1983;91: 236-47.

O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975;250 10:4007-21.

Thierry R, Cécile L. Two-dimensional gel electrophoresis in proteomics: a tutorial. J Proteomics. 2011;74:1829-41.

Rekola J, Aho AJ, Gunn J, Mathinlinna J, Hirvonen J, Vitaniemi P et al. The effect of heat treatment of wood on osteoconductivity. Acta Biomat. 2009;5: 1596-604.

1Murdoch AH, Mathias KJ, Shepherd DE. Investigation into the material properties of beech wood and cortical bone. Biomed Mater Eng. 2004; 14:1-4.

Gross KA, Ezerietis E. Juniper wood as a possible implant material. J Biomed Mater Res A. 2003;64: 672-83.

Touey G. United States Patent Preparation of Cellulose Phosphates. US 2759924 A. Publication date. 1956.

Orriss IR, Taylor SE and Arnett TR. Rat osteoblast cultures. Methods Mol Biol. 2012;816:31-41.

Pickart CM, Eddins MJ. Ubiquitin: structures, functions, mechanisms. Biochim Biophys Acta. 2004;1695:55-72.

Xu G, Jaffrey SR. Proteomic Identification of Protein Ubiquitination Events. Biotechnol Genet Eng Rev. 2013;29:73-109.

Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998;67:425-79.

Hoeller D, Dikic I. Targeting the ubiquitin system in cancer therapy. Nature. 2009;458:438-44.

Cole GM, Timiras PS. Ubiquitin-protein conjugates in Alzheimer’s lesions. Neurosci Lett. 1987;79:207-12.

Ahmed N, Zeng M, Sinha I. The E3 ligase Itch and deubiquitinase Cyld act together to regulate Tak1 and inflammation. Nat Immunol. 2011;12:1176-83.

Xu G, Paige JS, Jaffrey SR. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol. 2010;28: 868-73.

Downloads

Published

2020-11-23

Issue

Section

Original Research Articles