Evaluation of the HGF-1 Cells on Titanium Abutments Fixed by Various Procedures

Öz

The aim of this study was to compare the effects of fixation protocols in terms of attachment and proliferation of human gingival fibroblasts (HGF-1) on titanium abutment surfaces. HGF-1 cells were seeded on Titanium alloy (Ti6Al4V) plates with dimensions of 10x10x1 cm3. Cells were evaluated at 48 hours. 8 groups were determined (n=4). The specimens were treated with glutaraldehyde (GA) for 30,45 and 60 minutes (Group GA30, GA45, GA60), with formaldehyde (FA) for 6, 12, and 24 hours (Group FA6,FA12,FA24) and with paraformaldehyde (PFA) for 2 hours and 20 minutes (Group PFA2, PFA20) for fixation. Specimens were evaluated under Scanning Electron Microscope (SEM) (n=2) and under Immonoflorescence (n=2). The contact areas of cells in all groups were measured and differences between hours was evaluated statistically. According to SEM images, HGF-1 cell were similar for FA groups as spindled and homogeneous. Cell contact area was 95.83% for FA12 which exhibited the best cell spread among all groups. In GA and PFA groups the cells were elongated, clustered in certain areas and layered with distinct nucleated. According to immunofluorescence images, the densities of actin filaments were observed at similar levels in cells in all three groups, and it was determined that the cell bodies on the titanium surface in the PFA groups were more prominent, and better spread. The fixation process is one of the most critical step of imaging in cell studies. Researchers are needed to consider the most appropriate fixation method in order to obtain successful image results.

Anahtar Kelimeler

• Ti6Al4V
• HGF-1
• Fixation
• Immunoflorescence

Titanyum Abutment Üzerinde Çeşitli Prosedürlerle Fikse Edilmiş HGF-1 Hücrelerinin Değerlendirilmesi

Öz

Bu çalışmanın amacı, titanyum abutment üzerinde, insan gingival fibroblast hücre hattının (HGF-1), tutunma ve proliferasyon açısından fiksasyon solüsyon ve sürelerinin etkilerinin SEM ve İmmunofloresan görüntüleme ile değerlendirilmesi ve kıyaslanmasıdır. Hazır temin edilen insan gingival hücre hattı (HGF-1) 10x10x1cm3 boyutunda 32 adet titanyum alaşım (Ti6Al4V) plaka üzerine ekildi. 8 grup belirlendi (n=4). 48 saat sonucunda hücreler değerlendirildi. Örnekler Gluteraldehit ile 30, 45, 60 dakika (Grup GA30, GA45, GA60), Formaldehit ile 6, 12, 24 saat (Grup FA6, FA12, FA24) ve Paraformeldehit ile 2 saat ve 20 dakika (Grup PFA2, PFA20) süre ile fikse edildi. Fiksasyon sonrası her gruptan 2 örnek Taramalı elektron mikroskobunda (SEM) ve 2 örnek İmmunofloresan mikroskobunda görüntülenmek için hazırlandı. Tüm gruplardaki hücrelerin temas alanları ölçüldü, saatler arasındaki farklılık istatistiksel olarak değerlendirildi. SEM değerlendirmesinde, en uygun HGF-1 hücre morfolojisi görüntülerinin formaldehit gruplarının iğsi şekilli homojen yayılım gösterdiği tespit edildi. FA12 grubunda hücre temas alanı %95,83 bulunmuş olup, tüm deney grupları içerisinde en iyi hücre yayılımını göstermiştir. Gluteraldehit ve paraformaldehit gruplarında birbirleriyle benzer şekilde hücrelerin uzamış, belirli alanlarda öbekleşmiş ve üst üste katmanlanmış belirgin çekirdekli hücre görüntüleri tespit edildi. İmmunofloresan görüntülerinde her üç (gluteraldehit, formaldehit, paraformaldehit) gruptaki hücrelerde de aktin filamentlerinin yoğunlukları benzer seviyelerde görülmesinin yanı sıra paraformaldehit gruplarında titanyum yüzeydeki hücre gövdelerinin diğer fiksatif gruplarına göre daha belirgin, iyi yayılmış ve daha büyük yüzey alanlarına sahip olduğu gözlendi. Fiksasyon hücre çalışmalarında görüntülemenin en kritik basamaklarından biridir. Araştırmacıların başarılı görüntü sonucu elde edebilmek için en uygun fiksasyon yöntemini göz önünde bulundurmaları gerekmektedir.

Anahtar Kelimeler

• Ti6Al4V
• HGF-1
• Fiksasyon
• İmmunofloresan

Kaynakça

1. 1. Garza-Ramos MA, Estup􀵴ñan-Lopez FH, Gaona-Tiburcio C, et al. Electrochemical behavior of Ti6Al4V alloy used in dental implants immersed in Streptococcus gordonii and Fusobacterium nucleatum solutions. Materials 2020; 13(18): 4185.
2. 2. Kalyoncuoğlu UT, Yılmaz B, Güngör S, et al. Evaluation of the chitosan-coatıng effectiveness on a dental titanium alloy in terms of microbial and fibroblastic attachment and the effect of aging. Mater Technol 2015; 49(6):925–931.
3. 3. Atsuta I, Ayukawa Y, Kondo R, et al. Soft tissue sealing around dental implants based on histological interpretation. J Pros Res 2016; 60(1):3–11.
4. 4. Pandoleon P, Bakopoulou A, Papadopoulou L, Koidis P. Evaluation of the biological behaviour of various dental implant abutment materials on attachment and viability of human gingival fibroblasts. Dent Mater 2019; 35(7):1053–1063.
5. 5. Zhang C, Zhou L, Quian S, J et al. Improved response of human gingival fibroblasts to titanium coated with micro-/nano-structures tantalum. Int J Impl Dent 2021; 36(7):1-12.
6. 6. Grenade C, Pauw-Gillet MC, Gailly P, et al. Biocompatibility of polymer-infiltrated-ceramic-network (PICN) materials with Human Gingival Fibroblasts (HGFs). Dent Mat 2016; 32(9):1152-1164.
7. 7. Wisse E, Braet F, Duimel H, et al. Fixation methods for electron microscopy of human and other liver. World J Gastroenterol 2010; 16(23):2851-2866.
8. 8.Pansani TN, Basso FG, Souza IR, Hebling J, Costa CAS.Characterization of titanium surface coated with wpidermal growth factor and its effect on human gingival fibroblasts. Arch Oral Bio 2019; 102:48-54.

9. 9.Chao Y, Zhang T. Optimization of fixation methods for observation of bacterial cell morpology and surface ultrastructures by atomic force microscopy. Appl Microbiol Biotechnol 2011; 92(2):381-392.
10. 10.Kashi AM, Tahermanesh K, Chaichian S, Joghataei MT, Moradi F. How to prepare biological samples and live tissuesfor Scanning Electron Microscopy (SEM). Galen Med J 2014; 3(2):63-80.
11. 11.Thavarajah R, Mudimbaimannar VK, Elizabeth J, Rao UK,Ranganathan K. Chemical and physical basics of routine formaldehyde fixation. J of Oral Maxillofac Pathol 2012; 16(3):400-405.
12. 12.Srinivasan M. Sedmak D, Jewell S. Effect of fixatives andtissue processing on the content and integrity of nucleic acids. Am J Path 2002; 161(6):1961-1971.
13. 13.Kiernan JA. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: What they are and what they do. Micros Today2000; 8(1):8‐12.
14. 14.Goding J. Monoclonal Antibodies Principles and practices. Third edition. Melbourne: Elsevier Ltd. eBook ISBN: 9780080536958
15. 15.Guida L, Oliva A, Basile MA, Giordano M, Nastri L. Human gingival fibroblast functions are stimulated by oxidized nano-structured titanium surfaces. J Dent 2013(10); 41:900-9007.
16. 16.Rausch MA, Shokoohi-Tabrisi H, Wehner C, et al. Impact of implant surface material and microscale roughness on the initial attachment and proliferation of primary human gingival fibroblasts. Biology 2021(5); 10:1-14.
17. 17.Martinez MAF, Balderrama IF, Karam PSBH, et al. Surface roughness of titanium disks influences the adhesion, proliferation and differentiation of osteogenetic properties derived from human. Int J Impl Dent 2020(6);46.
18. 18.Eltoum I, Fredenburgh J, Grizzle WE. Advanced Concepts in Fixation: 1. Effects of Fixation on Immunohistochemistry,Reversibility of Fixation and Recovery of Proteins, Nucleic Acids, and other Molecules from Fixed and Processed Tissues. 2.Developmental methods of fixation. J Histotech 2013; 24:201-210.
19. 19.Al Shehadat S, Gorduysus MO, Hamid SSA, et al.Optimization of scanning electron microscope technique for amniotic membrane investigation: A preliminary study. Eur JDent 2018; 12(4):574-578.
20. 20.Lee SW, Kim SY, Rhyu IC, et al. Influence of microgroovedimension on cell behavior of human gingival fibroblasts cultured on titanium substrata. Clin Oral Impl Res 2009(1); 20:56–66.
21. 21.Hobro A. Smith NI. An evaluation of fixation methods: Spatialand compositional cellular changes observed by Raman imaging. Vibr Spect 2017; 91:31-45.