Sains Malaysiana 48(9)(2019): 1927–1935

http://dx.doi.org/10.17576/jsm-2019-4809-14

Evaluation of the Human Amniotic Membrane as a Scaffold for Periodontal Ligament Fibroblast Attachment and Proliferation

(Penilaian Membran Amnion Manusia sebagai Perancah untuk Pelekatan dan Pengembangbiakan Fibroblas Ligamen Periodontal)

ASRAR ELAHI¹, HASLINA TAIB²*, ZURAIRAH BERAHIM², AHMAD AZLINA³ & SUZINA SHEIKH AB HAMID⁴

¹Department of Periodontics, College of Dentistry, Lahore Medical and Dental College, 53400, Lahore, Pakistan

²Periodontics Unit, School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

³Molecular Biology, School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

⁴Tissue Bank, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

Diserahkan: 20 November 2018/Diterima: 13 Jun 2019

ABSTRACT

This study was aimed at evaluating the ability of the human amniotic membrane (HAM) to act as a scaffold for the growth of the main cells in periodontal regeneration, human periodontal ligament fibroblasts (HPDLFs). The HAM has many biological properties that are suitable for periodontal tissue regeneration such as low immunogenicity, anti-fibrosis, anti-inflammation, and a rich extracellular matrix component. Commercially available HPDLFs were seeded onto the HAM, and the attachment and proliferation of the cells were observed through scanning electron microscopy (SEM) and histological analysis. Cell viability was assessed using the alamarBlue® proliferation assay at days 1, 3, 7, 14 and 21. Histologically, the HPDLFs showed a monolayer to multilayer attachment onto the HAM from day 1 to day 7. The SEM analysis demonstrated that the HPDLFs had attached appropriately onto the HAM surface at day 1 to day 3, and began overlapping at day 7, while maintaining their flat shape. However, by days 14 and 21, there was an alteration in the morphology of the cells, where they later became rounded. The proliferation assay showed that the viability of the HPDLFs on the HAM had increased significantly from day 1 to day 7 (p=0.012), but later showed significant reduction at day 14 (p=0.002) and day 21 (p=0.005). In conclusion, this study showed that the HAM was able to function well as a scaffold for HPDLFs within 7 days, and thus, it can be a promising scaffold for periodontal regeneration. However, the behaviour of the cells in relation to the membrane over longer culture duration warrants further investigation.

Keywords: Amniotic membrane; periodontal fibroblasts; periodontal regeneration; scaffold

ABSTRAK

Kajian ini bertujuan untuk menilai keupayaan membran amniotik manusia (HAM) untuk bertindak sebagai perancah bagi pertumbuhan sel-sel utama dalam regenerasi periodontal iaitu sel fibroblas ligamen periodontal manusia (HPDLFs). HAM mempunyai banyak ciri biologi yang sesuai untuk pertumbuhan semula tisu periodontal seperti keimunogenan yang rendah, anti-fibrosis, anti-keradangan dan kaya dengan komponen matriks luar sel. HPDLFs yang tersedia secara komersial telah dibiakkan ke atas HAM, dan pelekatan serta pengembangbiakan HPDLFs diperhatikan melalui pemeriksaan mikroskop imbasan elektron (SEM) dan analisis histologi. Daya tahan sel telah dinilai menggunakan ujian percambahan alamarBlue® pada hari 1, 3, 7, 14 dan 21. Secara histologi, HPDLFs menunjukkan pelekatan ekalapisan kepada beberapa lapisan ke atas HAM daripada hari pertama hingga ke-7. Analisis SEM menunjukkan bahawa HPDLFs telah melekat dengan baik pada permukaan HAM pada hari pertama hingga hari ke-3 dan mula bertindih pada hari ke-7, sambil mengekalkan bentuknya yang rata. Bagaimanapun, pada hari ke-14 dan ke-21, terdapat perubahan pada morfologi sel, dengan mereka kemudiannya menjadi bulat. Ujian percambahan menunjukkan bahawa daya tahan sel HPDLFs pada HAM meningkat dengan ketara daripada hari pertama hingga hari ke-7 (p=0.012) namun kemudiannya menunjukkan penurunan yang ketara pada hari ke-14 (p=0.002) dan hari ke-21 (p=0.005). Kesimpulannya, kajian ini menunjukkan bahawa HAM dapat berfungsi dengan baik sebagai perancah untuk HPDLFs dalam tempoh 7 hari, dengan itu mampu menjadi perancah untuk regenerasi periodontal. Walau bagaimanapun, tingkah laku sel berhubung dengan membran ini dalam tempoh yang lebih lama masih memerlukan kajian lanjut.

Kata kunci: Fibroblas periodontal; membran amnion; perancah; pertumbuhan semula periodontal

RUJUKAN

Adachi, K., Amemiya, T., Nakamura, T., Honjyo, K., Kumamoto, S., Yamamoto, T., Bentley, A.J., Fullwood, N.J., Kinoshita, S. & Kanamura, N. 2014. Human periodontal ligament cell sheets cultured on amniotic membrane substrate. Oral Diseases 20: 582-590.

Amemiya, T., Honjo, K.I., Adachi, K., Nishigaki, M., Oseko, F., Yamamoto, T. & Kanamura, N. 2014. Immunohistochemical study of periosteal-derived cell sheet cultured on amniotic membrane aiming at periodontal tissue regeneration. Journal of Oral and Maxillofacial Surgery 72: e176.

Barabino, S., Rolando, M., Bentivoglio, G., Mingari, C., Zanardi, S., Bellomo, R. & Calabria, G. 2003. Role of amniotic membrane transplantation for conjunctival reconstruction in ocular-cicatricial pemphigoid. Ophthalmology 110(3): 474-480.

Causa, F., Netti, P.A. & Ambrosio, L. 2007. A multi-functional scaffold for tissue regeneration: The need to engineer a tissue analogue. Biomaterials 28: 5093-5099.

Chau, D.Y., Brown, S.V., Mather, M.L., Hutter, V., Tint, N.L., Dua, H.S., Rose, F.R. & Ghaemmaghami, A.M. 2012. Tissue transglutaminase (TG-2) modified amniotic membrane: A novel scaffold for biomedical applications. Biomedical Materials 7(4): 045011.

Chen, Y.J., Chung, M.C., Jane Yao, C.C., Huang, C.H., Chang, H.H., Jeng, J.H. & Young, T.H. 2012. The effects of acellular amniotic membrane matrix on osteogenic differentiation and ERK1/2 signaling in human dental apical papilla cells. Biomaterials 33: 455-463.

Chopra, A. & Thomas, B.S. 2013. Amniotic Membrane: A novel material for regeneration and repair. Journal of Biomimetics Biomaterials and Tissue Engineering 18: 1-8.

Cooper, L.J., Kinoshita, S., German, M., Koizumi, N., Nakamura, T. & Fullwood, N.J. 2005. An investigation into the composition of amniotic membrane used for ocular surface reconstruction. Cornea 24: 722-729.

Fujisato, T., Tomihata, K., Tabata, Y., Iwamoto, Y., Burczak, K. & Ikada, Y. 1999. Cross-linking of amniotic membranes. Journal of Biomaterials Science. Polymer Edition 10(11): 1171-1181.

Garg, T., Singh, O., Arora, S. & Murthy, R. 2012. Scaffold: A novel carrier for cell and drug delivery. Critical Review in Therapeutic Drug Carrier Systems 29(1): 1-63.

Gholipourmalekabadi, M., Sameni, M., Radenkovic, D., Mozafari, M., Mossahebi-Mohammadi, M. & Seifalian, A.M. 2016. Decellularized human amniotic membrane: How viable is it as a delivery system for human adipose tissue-derived stromal cells? Cell Proliferation 49(1): 115-121.

Graham, H.K., Horn, M. & Trafford, A.W. 2008. Extracellular matrix profiles in the progression to heart failure. European Young Physiologists Symposium Keynote Lecture-Bratislava 2007. Acta Physiologica (Oxford, England) 194(1): 3-21.

Honjo, K.I., Yamamoto, T., Oseko, F., Amemiya, T., Kita, M., Mazda, O. & Kanamura, N. 2014. Examination of bone differentiation for human dental pulp-derived cells cultured on amniotic membrane. Journal of Oral and Maxillofacial Surgery 72: 178-179.

Iwasaki, K., Komaki, M., Yokoyama, N., Tanaka, Y., Taki, A., Honda, I., Kimura, Y., Takeda, M., Akazawa, K., Oda, S., Izumi, Y. & Morita, I. 2014. Periodontal regeneration using periodontal ligament stem cell-transferred amnion. Tissue Engineering Part A 20: 693-704.

Kiernan, J.A. 2000. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: What they are and what they do. Microscopy Today 8: 8-12.

Kim, Y.S., Shin, S.I., Kang, K.L., Chung, J.H., Herr, Y., Bae, W.J. & Kim, E.C. 2012. Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E2 by hypoxia-inducible factor-1alpha up-regulation in human periodontal ligament cells. Journal of Periodontal Research 47(6): 719-728.

Kruse, F.E., Joussen, A.M., Rohrschneider, K., You, L., Sinn, B., Baumann, J. & Völcker, H.E. 2000. Cryopreserved human amniotic membrane for ocular surface reconstruction. Graefe’s Archive for Clinical and Experimental Ophthalmology 238(1): 68-75.

Kukacka, J., Prusa, R., Kotaska, K. & Pelouch, V. 2005. Matrix metalloproteinases and their function in myocardium. Biomedical Papers 149(2): 225-236.

Lai, J.Y. 2014. Photo-cross-linking of amniotic membranes for limbal epithelial cell cultivation. Material Science and Engineering. C, Materials for Biological Applications 45: 313-319.

Lai, J.Y. & Ma, D.H. 2013. Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics. International Journal of Nanomedicine 8: 4157-4168.

Lai, J.Y., Wang, P.R., Luo, L.J. & Chen, S.T. 2014. Stabilization of collagen nanofibers with L-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells. International Journal of Nanomedicine 9: 5117-5130.

Lai, J.Y., Lue, S.J., Cheng, H.Y. & Ma, D.H.K. 2013. Effect of matrix nanostructure on the functionality of carbodiimide cross-linked amniotic membranes as limbal epithelial cell scaffolds. Journal of Biomedical Nanotechnology 9(12): 2048-2062.

Lee, S.B., Li, D.Q., Tan, D.T., Meller, D.C. & Tseng, S.C. 2000. Suppression of TGF-beta signaling in both normal conjunctival fibroblasts and pterygial body fibroblasts by amniotic membrane. Current Eye Research 20(4): 325-334.

Lindner, D., Zietsch, C., Becher, P.M., Schulze, K., Schultheiss, H.P., Tschope, C. & Westermann, D. 2012. Differential expression of matrix metalloproteases in human fibroblasts with different origins. Biochemistry Research International 2012: 875742.

Lisboa, R.A., Lisboa, F.A., de Castro Santos, G., Andrade, M.V. & Cunha-Melo, J.R. 2009. Matrix metalloproteinase 2 activity decreases in human periodontal ligament fibroblast cultures submitted to simulated orthodontic force. In vitro Cellular and Development Biology. Animal 45(10): 614-621.

Litwiniuk, M. & Grzela, T. 2014. Amniotic membrane: New concepts for an old dressing. Wound Repair and Regeneration 22: 451-456.

Ma, D.H., Lai, J.Y., Cheng, H.Y., Tsai, C.C. & Yeh, L.K. 2010. Carbodiimide cross-linked amniotic membranes for cultivation of limbal epithelial cells. Biomaterials 31(25): 6647-6658.

Malhotra, C. & Jain, A.K. 2014. Human amniotic membrane transplantation: Different modalities of its use in opthalmology. World Journal of Transplantation 4: 111-121.

Mamede, A.C., Carvalho, M.J., Abrantes, A.M., Laranjo, M., Maia, C.J. & Botelho, M.F. 2012. Amniotic membrane: From structure and functions to clinical applications. Cell and Tissue Research 349: 447-458.

Nanci, A. & Ten Cate, A.R. 2013. Ten Cate's Oral Histology: Development, Structure, and Function. 8th edition. St. Louis: Elsevier.

Nanci, A. & Bosshardt, D.D. 2006. Structure of periodontal tissues in health and disease. Periodontology 2000 40: 11-28.

Niknejad, H. & Yazdanpanah, G. 2014. Anticancer effects of human amniotic membrane and its epithelial cells. Medical Hypotheses 82: 488-489.

Oren, R. & Kohn, A. 1969. Density dependent inhibition of cell growth in cultures of primary and established lines of cells. Journal of Cellular Physiology 74(3): 307-314.

Paolin, A., Cogliati, E., Trojan, D., Griffoni, C., Grassetto, A., Elbadawy, H.M. & Ponzin, D. 2016. Amniotic membranes in ophthalmology: Long term data on transplantation outcomes. Cell and Tissue Banking 17(1): 51-58.

Rai, B., Kaur, J., Jain, R. & Anand, S.C. 2010. Levels of gingival crevicular metalloproteinases-8 and -9 in periodontitis. Saudi Dental Journal 22(3): 129-131.

Shimauchi, H., Nemoto, E., Ishihata, H. & Shimomura, M. 2013. Possible functional scaffolds for periodontal regeneration. Japanese Dental Science Review 49(4): 118-130.

Sisson, K., Zhang, C., Farach-Carson, M.C., Chase, D.B. & Rabolt, J.F. 2010. Fiber diameters control osteoblastic cell migration and differentiation in electrospun gelatin. Journal of Biomedical Materials Research. Part A 94(4): 1312-1320.

Spinale, F.G. 2007. Myocardial matrix remodeling and the matrix metalloproteinases: Influence on cardiac form and function. Physiological Reviews 87(4): 1285-1342.

Spira, M., Liu, B., Xu, Z., Harrell, R. & Chahadeh, H. 1994. Human amnion collagen for soft tissue augmentation-biochemical characterizations and animal observations. Journal of Biomedical Materials Research 28(1): 91-96.

Spoerl, E., Wollensak, G., Reber, F. & Pillunat, L. 2004. Cross-linking of human amniotic membrane by glutaraldehyde. Ophthalmic Research 36(2): 71-77.

Taghiabadi, E., Nasri, S., Shafieyan, S., Jalili Firoozinezhad, S. & Aghdami, N. 2015. Fabrication and characterization of spongy denuded amniotic membrane based scaffold for tissue engineering. Cell Journal 16(4): 476-487.

Tseng, S.C., Li, D.Q. & Ma, X. 1999. Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. Journal of Cellular Physiology 179(3): 325-335.

*Pengarang untuk surat-menyurat; email: haslinakk@usm.my