Ascorbic Acid Enhances the Metabolic Activity, Growth and Collagen Production of Human Dermal Fibroblasts Growing in Three-dimensional (3D) Culture

Tissue engineering (TE) enables the development of functional synthetic substitutes to be replaced with damaged tissues and organs instead of the use of auto or allografts. A wide range of biomaterials is currently in use as TE scaffolds. Among these materials, naturally sourced ones are favorable due to being highly biocompatible and supporting cell growth and function, whereas synthetic ones are advantageous because of the high tunability on mechanical and physical properties as well as being easy to process. Alongside the advantages of synthetic polymers, they mostly show hydrophobic behavior that limits biomaterial-cell interaction and, consequently, the functioning of the developed TE constructs. In this study, we assessed the impact of L-Ascorbic acid 2-phosphate (AA2P) on improving the culture conditions of human dermal fibroblasts (HDFs) growing on a three-dimensional (3D) scaffold made of polycaprolactone (PCL) using emulsion templating. Our results demonstrated that AA2P enhances the metabolic activity and growth of HDFs as well as collagen deposition by them when supplemented in their growth medium at 50 µg/mL concentration. It showed a great potential to be used as a growth medium supplement to circumvent the disadvantages of culturing human cells on a synthetic biomaterial that is not favored in default. AA2P's potential to improve cell growth and collagen deposition may prove an effective way to culture human cells on 3D PCL PolyHIPE scaffolds for various TE applications.

Keywords:

L-ascorbic acid, Human dermal fibroblasts, Emulsion templating, 3D culture Polycaprolactone,

PDF

___

[1] Langer, R., Vacanti, J.P., "Tissue engineering", Science, 260(5110): 920–926, (1993).
[2] Abruzzo, A., Fiorica, C., Palumbo, V.D., Altomare, R., Damiano, G., Gioviale, M.C., Tomasello, G., Licciardi, M., Palumbo, F.S., Giammona, G., Lo Monte, A.I., "Using polymeric scaffolds for vascular tissue engineering", International Journal of Polymer Science, 2014: 1–9, (2014).
[3] Cheng, Z., Teoh, S.H., "Surface modification of ultra thin poly (ε-caprolactone) films using acrylic acid and collagen", Biomaterials, 25(11): 1991–2001, (2004).
[4] Miroshnichenko, S., Timofeeva, V., Permyakova, E., Ershov, S., Kiryukhantsev-Korneev, P., Dvořaková, E., Shtansky, D., Zajíčková, L., Solovieva, A., Manakhov, A., "Plasma-Coated Polycaprolactone Nanofibers with Covalently Bonded Platelet-Rich Plasma Enhance Adhesion and Growth of Human Fibroblasts", Nanomaterials, 9(4): 637, (2019).
[5] Aldemir Dikici, B., Sherborne, C., Reilly, G.C., Claeyssens, F., "Emulsion templated scaffolds manufactured from photocurable polycaprolactone", Polymer, 175: 243–254, (2019).
[6] Woodruff, M.A., Hutmacher, D.W., "The return of a forgotten polymer - Polycaprolactone in the 21st century", Progress in Polymer Science (Oxford), 35(10): 1217–1256, (2010).
[7] Dikici, S., Claeyssens, F., MacNeil, S., "Pre-Seeding of Simple Electrospun Scaffolds with a Combination of Endothelial Cells and Fibroblasts Strongly Promotes Angiogenesis", Tissue Engineering and Regenerative Medicine, 17(4): 445–458, (2020).
[8] Kim, C.G., Han, K.S., Lee, S., Kim, M.C., Kim, S.Y., Nah, J., "Fabrication of biocompatible polycaprolactone–hydroxyapatite composite filaments for the FDM 3D printing of bone scaffolds", Applied Sciences (Switzerland), 11(14): 6351, (2021).
[9] Huang, A., Jiang, Y., Napiwocki, B., Mi, H., Peng, X., Turng, L.S., "Fabrication of poly(ϵ-caprolactone) tissue engineering scaffolds with fibrillated and interconnected pores utilizing microcellular injection molding and polymer leaching", RSC Advances, 7(69): 43432–43444, (2017).
[10] Thadavirul, N., Pavasant, P., Supaphol, P., "Development of polycaprolactone porous scaffolds by combining solvent casting, particulate leaching, and polymer leaching techniques for bone tissue engineering", Journal of Biomedical Materials Research - Part A, 102(10): 3379–3392, (2014).
[11] Dikici, S., Aldemir Dikici, B., Bhaloo, S.I., Balcells, M., Edelman, E.R., MacNeil, S., Reilly, G.C., Sherborne, C., Claeyssens, F., "Assessment of the angiogenic potential of 2-deoxy-D-ribose using a novel in vitro 3D dynamic model in comparison with established in vitro assays", Frontiers in Bioengineering and Biotechnology, 7: 451, (2019).
[12] Aldemir Dikici, B., Dikici, S., Reilly, G.C., MacNeil, S., Claeyssens, F., "A Novel Bilayer Polycaprolactone Membrane for Guided Bone Regeneration: Combining Electrospinning and Emulsion Templating", Materials, 12(16): 2643, (2019).
[13] Dikici, S., Aldemir Dikici, B., Macneil, S., Claeyssens, F., "Decellularised extracellular matrix decorated PCL PolyHIPE scaffolds for enhanced cellular activity, integration and angiogenesis", Biomaterials Science, 9(21): 7297–7310, (2021).
[14] Aldemir Dikici, B., Claeyssens, F., "Basic Principles of Emulsion Templating and Its Use as an Emerging Manufacturing Method of Tissue Engineering Scaffolds", Frontiers in Bioengineering and Biotechnology, 8: 875, (2020).
[15] Aldemir Dikici, B., Reilly, G.C., Claeyssens, F., "Boosting the Osteogenic and Angiogenic Performance of Multiscale Porous Polycaprolactone Scaffolds by in Vitro Generated Extracellular Matrix Decoration", ACS Applied Materials and Interfaces, 12(11): 12510–12524, (2020).
[16] Aldemir Dikici, B., Malayeri, A., Sherborne, C., Dikici, S., Paterson, T., Dew, L., Hatton, P., Ortega Asencio, I., MacNeil, S., Langford, C., Cameron, N.R., Claeyssens, F., "Thiolene- and Polycaprolactone Methacrylate-Based Polymerized High Internal Phase Emulsion (PolyHIPE) Scaffolds for Tissue Engineering", Biomacromolecules,: in press, (2021).
[17] Dikici, B.A., Dikici, S., Reilly, G.C., MacNeil, S., Claeyssens, F., "A novel bilayer polycaprolactone membrane for guided bone regeneration: Combining electrospinning and emulsion templating", Materials, 12(16): 2643, (2019).
[18] Gunes, S., Tamburaci, S., Tihminlioglu, F., "A novel bilayer zein/MMT nanocomposite incorporated with H. perforatum oil for wound healing", Journal of Materials Science: Materials in Medicine, 31(1): 2643, (2020).
[19] Dikici, S., Claeyssens, F., MacNeil, S., "Decellularised baby spinach leaves and their potential use in tissue engineering applications: Studying and promoting neovascularisation", Journal of Biomaterials Applications, 34(4): 546–559, (2019).
[20] Mangir, N., Bullock, A.J., Roman, S., Osman, N., Chapple, C., MacNeil, S., "Production of ascorbic acid releasing biomaterials for pelvic floor repair", Acta Biomaterialia, 29: 188–197, (2016).
[21] Prockop, D.J., Kivirikko, K.I., "Collagens: Molecular biology, diseases, and potentials for therapy", Annual Review of Biochemistry, 64: 403–434, (1995).
[22] Houglum, K.P., Brenner, D.A., Chojkier, M., "Ascorbic acid stimulation of collagen biosynthesis independent of hydroxylation", American Journal of Clinical Nutrition, 54(6 SUPPL.): 1141S–1143S., (1991).
[23] Kurata, S.I., Senoo, H., Hata, R.I., "Transcriptional activation of type I collagen genes by ascorbic acid 2-phosphate in human skin fibroblasts and Its failure in cells from a patient with α2(I)-chain-defective Ehlers-Danlos syndrome", Experimental Cell Research, 206(1): 63–71, (1993).
[24] Owen, R., Sherborne, C., Paterson, T., Green, N.H., Reilly, G.C., Claeyssens, F., "Emulsion templated scaffolds with tunable mechanical properties for bone tissue engineering", Journal of the Mechanical Behavior of Biomedical Materials, 54: 159–172, (2016).
[25] Barbetta, A., Cameron, N.R., "Morphology and surface area of emulsion-derived (PolyHIPE) solid foams prepared with oil-phase soluble porogenic solvents: Span 80 as surfactant", Macromolecules, 37(9): 3188–3201, (2004).
[26] Dikici, S., Bullock, A.J., Yar, M., Claeyssens, F., MacNeil, S., "2-deoxy-D-ribose (2dDR) upregulates vascular endothelial growth factor (VEGF) and stimulates angiogenesis", Microvascular Research, 131: 104035, (2020).
[27] Dikici, S., Claeyssens, F., MacNeil, S., "Bioengineering Vascular Networks to Study Angiogenesis and Vascularization of Physiologically Relevant Tissue Models in Vitro", ACS Biomaterials Science and Engineering, 6(6): 3513–3528, (2020).
[28] Dikici, S., "A “sweet” way to increase the metabolic activity and migratory response of cells associated with wound healing: deoxy-sugar incorporated polymer fibres as a bioactivwound patch", Turkish Journal of Biology, 46(1): 41–56, (2022).
[29] Dikici, S., Mangir, N., Claeyssens, F., Yar, M., MacNeil, S., "Exploration of 2-deoxy-D-ribose and 17β-Estradiol as alternatives to exogenous VEGF to promote angiogenesis in tissue-engineered constructs", Regenerative Medicine, 14(3): 179–197, (2019).
[30] Fischer, A.H., Jacobson, K.A., Rose, J., Zeller, R., "Hematoxylin and eosin staining of tissueand cell sections", Cold Spring Harbor Protocols, 3(5): 1–2, (2008).
[31] Junqueira, L.C.U., Bignolas, G., Brentani, R.R., "Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections", The Histochemical Journal, 11(4): 447–455, (1979).
[32] Fujisawa, K., Hara, K., Takami, T., Okada, S., Matsumoto, T., Yamamoto, N., Sakaida, I., "Evaluation of the effects of ascorbic acid on metabolism of human mesenchymal stem cells", Stem Cell Research and Therapy, 9(1): 93, (2018).
[33] Zhang, P., Li, J., Qi, Y., Zou, Y., Liu, L., Tang, X., Duan, J., Liu, H., Zeng, G., "Vitamin C promotes the proliferation of human adipose-derived stem cells via p53-p21 pathway", Organogenesis, 12(3): 143–151, (2016).
[34] Kouakanou, L., Xu, Y., Peters, C., He, J., Wu, Y., Yin, Z., Kabelitz, D., "Vitamin C promotes the proliferation and effector functions of human γδ T cells", Cellular and Molecular Immunology, 17(5): 462–473, (2020).
[35] Hata, R. ‐I, Senoo, H., "L‐ascorbic acid 2‐phosphate stimulates collagen accumulation, cell proliferation, and formation of a three‐dimensional tissuelike substance by skin fibroblasts", Journal of Cellular Physiology, 138(1): 8–16, (1989).
[36] Phillips, C.L., Combs, S.B., Pinnell, S.R., "Effects of ascorbic acid on proliferation and collagen synthesis in relation to the donor age of human dermal fibroblasts", Journal of Investigative Dermatology, 103(2): 228–232, (1994).
[37] Denk, P.O., Knorr, M., "In vitro effect of ascorbic acid on the proliferation of bovine scleral and tenon’s capsule fibroblasts", European Journal of Ophthalmology, 8(1): 37–41, (1998).
[38] Schafer, I.A., Silverman, L., Sullivan, J.C., Robertson, W. V., "Ascorbic acid deficiency in cultured human fibroblasts.", The Journal of cell biology, 34(1): 83–95, (1967).
[39] Hata, R.-I., Senoo, H., "Extracellular Matrıx System Regulates Cell Growth, Tıssue Formatıon, and Cellular Functıons", Tıssue Culture Research Communıcatıons, 11(3): 337–343, (1992).
[40] Kouki, M., Norio, M., Kyoko, F., Itaru, Y., "Comparison of ascorbic acid and ascorbic acid 2-O-α-glucoside on the cytotoxicity and bioavailability to low density cultures of fibroblasts", Biochemical Pharmacology, 44(11): 2191–2197, (1992).
[41] Comings, D.E., Okada, T.A., "Electron microscopy of human fibroblasts in tissue culture during logarithmic and confluent stages of growth", Experimental Cell Research, 61(2): 295–301, (1970).
[42] Chan, D., Lamande, S.R., Cole, W.G., Bateman, J.F., "Regulation of procollagen synthesis and processing during ascorbate-induced extracellular matrix accumulation in vitro", Biochemical Journal, 269(1): 175–181, (1990).
[43] Murad, S., Tajima, S., Johnson, G.R., Sivarajah, S., Pinnell, S.R., "Collagen synthesis in cultured human skin fibroblasts: Effect of ascorbic acid and its analogs", Journal of Investigative Dermatology, 81(2): 158–162, (1983).
[44] Prockop, D.J., Berg, R.A., Kivirikko, K.I., Uitto, J., "Intracellular steps in the biosynthesis of collagen.",. In: Biochemistry of Collagen. pp. 163–273. (1976)
[45] Tajima, S., Pinnell, S.R., "Regulation of collagen synthesis by ascorbic acid. Ascorbic acid increases type I procollagen mRNA", Biochemical and Biophysical Research Communications, 106(2): 632–637, (1982).
[46] Lyons, B.L., Schwarz, R.I., "Ascorbate stimulation of PAT cells causes an increase in transcription rates and a decrease in degradation rates of procollagen mRNA", Nucleic Acids Research, 12(5): 2569–2579, (1984).
[47] Levene, C.I., Bates, C.J., "Growth and macromolecular synthesis in the 3T6 mouse fibroblast. I. General description and the role of ascorbic acid.", Journal of Cell Science, 7(3): 671–682, (1970).
[48] Hata, R., Sunada, H., Arai, K., Nagai, Y., "Regulation of extracellular matrix metabolism by growth factors in human skin fibroblasts.", Progress in clinical and biological research, 217 B: 381–384, (1986).
[49] Schwartz, E., Bienkowski, R.S., Coltoff-Schiller, B., Goldfischer, S., Blumenfeld, O.O., "Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding", Journal of Cell Biology, 92(2): 462–470, (1982).
[50] HATA, R. ‐i, SUNADA, H., ARAI, K., SATO, T., NINOMIYA, Y., NAGAI, Y., SENOO, H., "Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts", European Journal of Biochemistry, 173(2): 261–267, (1988).
[51] Peterkofsky, B., "The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts", Archives of Biochemistry and Biophysics, 152(1): 318–328, (1972).
[52] Du, J., Cullen, J.J., Buettner, G.R., "Ascorbic acid: Chemistry, biology and the treatment of cancer", Biochimica et Biophysica Acta - Reviews on Cancer, 1826(2): 443–457, (2012).