Sains Malaysiana 52(1)(2023): 223-231

http://doi.org/10.17576/jsm-2023-5201-18

Effect of Dermal Fibroblast Conditioned Medium on Keratinocytes Irrespective of Age Group

(Kesan Medium Terkondisi Fibroblas Kulit pada Keratinosit Tanpa mengira Kumpulan Umur)

NURUL ‘IZZAH ABDUL GHANI¹, MANIRA MAAROF^1,*, SHIPLU ROY CHOWDHURY¹, AMINUDDIN BIN SAIM², RUSZYMAH BINTI HAJI IDRUS^1,3 & FAUZI MH BUSRA¹

¹Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

²KPJ Ampang Puteri Specialist Hospital, 1 Jalan Mamanda 9, 68000 Ampang, Kuala Lumpur, Federal Territory, Malaysia

³Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

Received: 16 March 2022/Accepted: 5 October 2022

Abstract

Skin aging causes delayed re-epithelialisation and impaired wound healing. Thus, supplementation of wound healing mediators and extracellular matrix (ECM) components may be a potential treatment strategy for age-related impaired wound healing. Fibroblasts secrete wound-healing factors and can be collected from used medium, i.e., dermal fibroblast conditioned medium (DFCM). In this study, we elucidated the effect of DFCM on the in vitro wound healing of keratinocytes isolated from different age groups (≥18–35, 36–54, ≥55 years) via cell attachment, growth rate, and wound healing rate assays. The DFCM was prepared by culturing confluent fibroblasts with serum-free keratinocyte-specific (DFCM-KM) and fibroblast-specific (DFCM-FM) medium. The cell attachment efficiency decreased with the increase of age. However, keratinocyte attachment was enhanced in the DFCM-KM group, where it was 1.24, 1.27, and 1.32 times higher of cells concentration for the ≥18–35-, 36–54-, and ≥55-year age groups, respectively, as compared to the control group. The keratinocytes from each age group demonstrated a similar growth profile for all culture conditions, where the DFCM-KM group exhibited a comparable growth rate with the control group whilst the DFCM-FM group exhibited a significantly lower growth rate compared to the other groups. In contrast, the DFCM-FM group demonstrated a significantly higher healing rate in all age groups as compared to the DFCM-KM and control groups. However, there was no significant difference between the healing rates of the DFCM-KM and control groups. In conclusion, DFCM-KM enhanced keratinocyte attachment while DFCM-FM enhanced the keratinocyte healing rate irrespective of donor age, which indicated the potential application of DFCM in wound healing in aged skin.

Keywords: Aging; conditioned medium; re-epithelialisation; wound

Abstrak

Penuaan kulit menyebabkan kelewatan dalam pengepiteliuman semula dan penyembuhan luka. Oleh itu, penambahan faktor penyembuhan luka dan komponen matriks ekstrasel (ECM) merupakan strategi rawatan yang berpotensi bagi penyembuhan luka yang terjejas akibat faktor usia. Fibroblas merupakan sel yang merembeskan faktor pertumbuhan yang terlibat dalam proses penyembuhan luka dan boleh dikumpul daripada medium terpakai, iaitu medium terkondisi daripada fibroblas kulit (DFCM). Kajian ini bertujuan untuk mengkaji kesan DFCM pada penyembuhan luka secara in vitro bagi sel keratinosit daripada kumpulan umur yang berbeza (≥18-35, 36-54 dan ≥55 tahun) melalui pelekatan sel, kadar pertumbuhan dan penyembuhan luka. DFCM disediakan daripada pengkulturan fibroblas di dalam media tanpa serum khusus keratinosit (DFCM-KM) dan khusus fibroblas (DFCM-FM). Tahap pelekatan sel berkurangan dengan peningkatan umur. Walau bagaimanapun, pelekatan keratinosit menunjukkan peningkatan dengan tambahan DFCM-KM berbanding kumpulan kawalan yang masing-masing 1.24, 1.27 dan 1.32 kali lebih tinggi kepekatan sel bagi umur ≥18-35, 36-54 dan ≥55 tahun. Keratinosit daripada kumpulan umur yang berbeza menunjukkan profil pertumbuhan yang sama untuk semua keadaan kultur, dengan DFCM-KM menunjukkan kadar yang setanding dengan kumpulan kawalan manakala DFCM-FM adalah jauh lebih rendah berbanding kumpulan lain. Sebaliknya, kumpulan DFCM-FM menunjukkan kadar penyembuhan yang jauh lebih tinggi dalam semua kumpulan umur berbanding DFCM-KM dan kumpulan kawalan. Walau bagaimanapun, tiada perbezaan yang ketara dalam kadar penyembuhan luka untuk keratinosit dengan tambahan DFCM-KM dan kumpulan kawalan. Kesimpulannya, tanpa mengira umur penderma, DFCM-KM meningkatkan pelekatan keratinosit manakala DFCM-FM meningkatkan kadar penyembuhan keratinosit, menunjukkan potensi penggunaan DFCM dalam penyembuhan luka pada kulit pesakit yang sudah tua.

Kata kunci: Luka; medium terkondisi; pengepiteliuman semula; penuaan

REFERENCES

Alam, W., Hasson, J. & Reed, M. 2021. Clinical approach to chronic wound management in older adults. Journal of the American Geriatrics Society 69(8): 2327-2334.

Chowdhury, S.R., Aminuddin, B.S. & Ruszymah, B.H. 2012. Effect of supplementation of dermal fibroblasts conditioned medium on expansion of keratinocytes through enhancing attachment. Indian J. Exp. Biol. 50(5): 332-339.

Chowdhury, S.R., Lim, S.J., Zolkafli, M.N.H.B., Mohd Abu Zarin, N.A.B., Wan Abdullah, W. A.B., Md Mothar, N.A.B., Maarof, M. & Abdullah, N.A.H. 2019. Exploring the potential of dermal fibroblast conditioned medium on skin wound healing and anti-ageing. Sains Malaysiana 48(3): 637-644.

Desjardins-Park, H.E., Foster, D.S. & Longaker, M.T. 2018. Fibroblasts and wound healing: An update. Future Medicine 13: 491-495.

Fivenson, D.P., Faria, D.T., Nickoloff, B.J., Poverini, P.J., Kunkel, S., Burdick, M. & Strieter, R.M. 1997. Chemokine and inflammatory cytokine changes during chronic wound healing. Wound Repair Regen 5(4): 310-322.

Gosain, A. & Dipietro, L.A. 2004. Aging and wound healing. World J. Surg. 28(3): 321-326.

He, S. & Sharpless, N.E. 2017. Senescence in health and disease. Cell 169(6): 1000-1011.

Ishak, M.F., Manira, M., Ng, M.H., Khairul, B., Gargy, L., Aminuddin, B.S. & Ruszymah, B.H.I. 2019. Long term effect of cryopreservation on primary human skin cells. Sains Malaysiana 48(1): 137-144.

Jayaraman, P., Nathan, P., Vasanthan, P., Musa, S. & Govindasamy, V. 2013. Stem cells conditioned medium: A new approach to skin wound healing management. Cell Biol. Int. 37(10): 1122-1128.

Lee, D.Y., Yang, J.M. & Park, K.H. 2007. A dermal equivalent developed from fibroblast culture alone: Effect of EGF and insulin. Wound Repair and Regeneration 15(6): 936-939.

Maarof, M., Chowdhury, S.R., Saim, A., Bt Hj Idrus, R. & Lokanathan, Y. 2020. Concentration dependent effect of human dermal fibroblast conditioned medium (DFCM) from three various origins on keratinocytes wound healing. Int. J. Mol. Sci. 21(8): 2929.

Maarof, M., Mh Busra, M.F., Lokanathan, Y., Bt Hj Idrus, R., Rajab, N.F. & Chowdhury, S.R. 2019. Safety and efficacy of dermal fibroblast conditioned medium (DFCM) fortified collagen hydrogel as acellular 3D skin patch. Drug Delivery and Translational Research 9(1): 144-161.

Maarof, M., Lokanathan, Y., Ruszymah, H.I., Saim, A. & Chowdhury, S.R. 2018. Proteomic analysis of human dermal fibroblast conditioned medium (DFCM). Protein J. 37(6): 589-607.

Maarof, M., Law, J.X., Chowdhury, S.R., Khairoji, K.A., Saim, A.B. & Idrus, R.B. 2016. Secretion of wound healing mediators by single and bi-layer skin substitutes. Cytotechnology 68(5): 1873-1884.

Makrantonaki, E. & Zouboulis, C.C. 2007. Characteristics and pathomechanisms of endogenously aged skin. Dermatology 214(4): 352-360.

Metcalfe, A.D. & Ferguson, M.W. 2007. Tissue engineering of replacement skin: The crossroads of biomaterials, wound healing, embryonic development, stem cells and regeneration. J. R. Soc. Interface 4(14): 413-437.

Naylor, E.C., Watson, R.E. & Sherratt, M.J. 2011. Molecular aspects of skin ageing. Maturitas 69(3): 249-256.

Quan, T., Shao, Y., He, T., Voorhees, J.J. & Fisher, G.J. 2010. Reduced expression of connective tissue growth factor (CTGF/CCN2) mediates collagen loss in chronologically aged human skin. J. Invest. Dermatol. 130(2): 415-424.

Riha, S.M., Maarof, M. & Fauzi, M.B. 2021. Synergistic effect of biomaterial and stem cell for skin tissue engineering in cutaneous wound healing: A concise review. Polymers 13(10): 1546.

Roig-Rosello, E. & Rousselle, P. 2020. The human epidermal basement membrane: A shaped and cell instructive platform that aging slowly alters. Biomolecules 10(12): 1607.

Shim, G., Devenport, D. & Cohen, D.J. 2021. Overriding native cell coordination enhances external programming of collective cell migration. Proc. Natl. Acad. Sci. USA 118(29): e2101352118

Smola, H., Thiekötter, G. & Fusenig, N.E. 1993. Mutual induction of growth factor gene expression by epidermal-dermal cell interaction. J. Cell Biol. 122(2): 417-429.

Spiekstra, S.W., Breetveld, M., Rustemeyer, T., Scheper, R.J. & Gibbs, S. 2007. Wound-healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes. Wound Repair Regen 15(5): 708-717.

Tsao, M.C., Walthall, B.J. & Ham, R.G. 1982. Clonal growth of normal human epidermal keratinocytes in a defined medium. Journal of Cellular Physiology 110(2): 219-229.

Wang, A.S. & Dreesen, O. 2018. Biomarkers of cellular senescence and skin aging. Frontiers in Genetics 9: 247.

Werner, S. & Grose, R. 2003. Regulation of wound healing by growth factors and cytokines. Physiological Reviews 83(3): 835-870.

Xie, J., Bian, H., Qi, S., Xu, Y., Tang, J., Li, T. & Liu, X. 2008. Effects of basic fibroblast growth factor on the expression of extracellular matrix and matrix metalloproteinase‐1 in wound healing. Clinical and Experimental Dermatology: Continuing Professional Development (CPD) 33(2): 176-182.

Zhang, S. & Duan, E. 2018. Fighting against skin aging: The way from bench to bedside. Cell Transplantation 27(5): 729-738.

^*Corresponding author; email: manira@ukm.edu.my