DOI: http://dx.doi.org/10.18203/issn.2454-2156.IntJSciRep20205020

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

Fiayaz Shaama, Ann Wilson, Valerie Stoute

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 Sample aliquots were withdrawn periodically over 16 days.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.  


Keywords


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

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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.