Advertisement

Drug Delivery and Translational Research

, Volume 9, Issue 1, pp 144–161 | Cite as

Safety and efficacy of dermal fibroblast conditioned medium (DFCM) fortified collagen hydrogel as acellular 3D skin patch

  • Manira Maarof
  • Mh Busra Mohd Fauzi 
  • Yogeswaran Lokanathan
  • Bt Hj Idrus Ruszymah 
  • Rajab Nor Fadilah 
  • Shiplu Roy ChowdhuryEmail author
Original Article
  • 56 Downloads

Abstract

Skin substitutes are one of the main treatments for skin loss, and a skin substitute that is readily available would be the best treatment option. However, most cell-based skin substitutes require long production times, and therefore, patients endure long waiting times. The proteins secreted from the cells and tissues play vital roles in promoting wound healing. Thus, we aimed to develop an acellular three-dimensional (3D) skin patch with dermal fibroblast conditioned medium (DFCM) and collagen hydrogel for immediate treatment of skin loss. Fibroblasts from human skin samples were cultured using serum-free keratinocyte-specific media (KM1 or KM2) and serum-free fibroblast-specific medium (FM) to obtain DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively. The acellular 3D skin patch was soft, semi-solid, and translucent. Collagen mixed with DFCM-KM1 and DFCM-KM2 showed higher protein release compared to collagen plus DFCM-FM. In vitro and in vivo testing revealed that DFCM and collagen hydrogel did not induce an immune response. The implantation of the 3D skin patch with or without DFCM on the dorsum of BALB/c mice demonstrated a significantly faster healing rate compared to the no-treatment group 7 days after implantation, and all groups had complete re-epithelialization at day 17. Histological analysis confirmed the structure and integrity of the regenerated skin, with positive expression of cytokeratin 14 and type I collagen in the epidermal and dermal layer, respectively. These findings highlight the possibility of using fibroblast secretory factors together with collagen hydrogel in an acellular 3D skin patch that can be used allogeneically for immediate treatment of full-thickness skin loss.

Keywords

Fibroblasts Dermal fibroblast conditioned medium Acellular 3D skin patch Tissue engineering 

Notes

Acknowledgements

We are thankful to our colleague Dr. Shinsmon Jose, who provided expertise that greatly assisted part of the research. Some parts of this work were performed at the UKM Bioserasi Laboratory.

Compliance with ethical standards

Research Grants

This study was funded by the Science Fund (02–01-02-SF0964), UKM fundamental fund (FF-2015-204) and the Tissue Engineering Centre, UKM Medical Centre.

Ethics approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the ethical standards of the responsible committee on human experimentation (UKMREC), with approval code UKM FPR.4/244/FF-2015-204, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all patients included in the study. All institutional and national guidelines for the care and use of laboratory animals were followed with approval code PP/TEC/2015/SHIPLU/20-MAY/675-MAY-2015-DEC-2016.

Conflict of interest

The authors declare that they have no conflicts of interest.

References

  1. 1.
    Halim AS, Khoo TL, Mohd Yussof SJ. Biologic and synthetic skin substitutes: an overview. Indian J Plast Surg. 2010;43(Suppl):S23–8.  https://doi.org/10.4103/0970-0358.70712. CrossRefGoogle Scholar
  2. 2.
    Harrison CA, MacNeil S. The mechanism of skin graft contraction: an update on current research and potential future therapies. Burns. 2008;34(2):153–63.  https://doi.org/10.1016/j.burns.2007.08.011.CrossRefGoogle Scholar
  3. 3.
    Schneider JC, Holavanahalli R, Helm P, Goldstein R, Kowalske K. Contractures in burn injury: defining the problem. J Burn Care Res. 2006;27(4):508–14.  https://doi.org/10.1097/01.bcr.0000225994.75744.9d. CrossRefGoogle Scholar
  4. 4.
    Ojeh N, Akgül B, Tomic-Canic M, Philpott M, Navsaria H. In vitro skin models to study epithelial regeneration from the hair follicle. PLoS One. 2017;12(3):e0174389.  https://doi.org/10.1371/journal.pone.0174389.CrossRefGoogle Scholar
  5. 5.
    Coulomb B, Friteau L, Baruch J, Guilbaud J, Chretien-Marquet B, Glicenstein J, et al. Advantage of the presence of living dermal fibroblasts within in vitro reconstructed skin for grafting in humans. Plast Reconstr Surg. 1998;101(7):1891–903.CrossRefGoogle Scholar
  6. 6.
    Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975;6(3):331–43.CrossRefGoogle Scholar
  7. 7.
    Snyder DL, Sullivan N, Schoelles KM. AHRQ Technology Assessments. Skin substitutes for treating chronic wounds. Rockville (MD): Agency for Healthcare Research and Quality; 2012.Google Scholar
  8. 8.
    Berthiaume F, Maguire TJ, Yarmush ML. Tissue engineering and regenerative medicine: history, progress, and challenges. Annu Rev Chem Biomol Eng. 2011;2:403–30.CrossRefGoogle Scholar
  9. 9.
    Jones I, Currie L, Martin R. A guide to biological skin substitutes. Br J Plast Surg. 2002;55(3):185–93.  https://doi.org/10.1054/bjps.2002.3800.CrossRefGoogle Scholar
  10. 10.
    Varkey M, Ding J, Tredget EE. Advances in skin substitutes—potential of tissue engineered skin for facilitating anti-fibrotic healing. J Funct Biomater. 2015;6(3):547–63.  https://doi.org/10.3390/jfb6030547.CrossRefGoogle Scholar
  11. 11.
    Auger FA, Lacroix D, Germain L. Skin substitutes and wound healing. Skin Pharmacol Physiol. 2009;22(2):94–102.  https://doi.org/10.1159/000178868. CrossRefGoogle Scholar
  12. 12.
    Usui ML, Mansbridge JN, Carter WG, Fujita M, Olerud JE. Keratinocyte migration, proliferation, and differentiation in chronic ulcers from patients with diabetes and normal wounds. J Histochem Cytochem. 2008;56(7):687–96.  https://doi.org/10.1369/jhc.2008.951194.CrossRefGoogle Scholar
  13. 13.
    Spiekstra SW, Breetveld M, Rustemeyer T, Scheper RJ, Gibbs S. Wound-healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes. Wound Repair Regen. 2007;15(5):708–17.CrossRefGoogle Scholar
  14. 14.
    Hur W, Lee HY, Min HS, Wufuer M, Lee C, Hur JA, et al. Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium. Stem Cell Res Ther. 2017;8:92.  https://doi.org/10.1186/s13287-017-0520-7.CrossRefGoogle Scholar
  15. 15.
    Debels H, Hamdi M, Abberton K, Morrison W. Dermal matrices and bioengineered skin substitutes: a critical review of current options. Plast Reconstr Surg Glob Open. 2015;3(1):e284.  https://doi.org/10.1097/GOX.0000000000000219.CrossRefGoogle Scholar
  16. 16.
    Collawn SS, Mobley JA, Banerjee NS, Chow LT. Conditioned media from adipose-derived stromal cells accelerates Healing in 3-dimensional skin cultures. Ann Plast Surg. 2016;76(4):446–52.  https://doi.org/10.1097/sap.0000000000000754.CrossRefGoogle Scholar
  17. 17.
    Seet WT, Maarof M, Anuar KK, Chua K-H, Irfan AWA, Ng MH, et al. Shelf-life evaluation of bilayered human skin equivalent, MyDerm™. PLoS One. 2012;7(8):e40978.CrossRefGoogle Scholar
  18. 18.
    Mazlyzam AL, Aminuddin BS, Fuzina NH, Norhayati MM, Fauziah O, Isa MR, et al. Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold. Burns. 2007;33(3):355–63.  https://doi.org/10.1016/j.burns.2006.08.022.CrossRefGoogle Scholar
  19. 19.
    Idrus RBH Rameli MAbP, Low KC, Law JX, Chua KH, Latiff MbA, Saim Ab. Full-thickness skin wound healing using autologous keratinocytes and dermal fibroblasts with fibrin: bilayered versus single-layered substitute. Adv Skin Wound Care. 2013.Google Scholar
  20. 20.
    Maarof M, Law JX, Chowdhury SR, Khairoji KA, Saim AB, Idrus RB. Secretion of wound healing mediators by single and bi-layer skin substitutes. Cytotechnology. 2016;68:1873–84.  https://doi.org/10.1007/s10616-015-9940-3.CrossRefGoogle Scholar
  21. 21.
    Han G, Ceilley R. Chronic wound healing: a review of current management and treatments. Adv Ther. 2017;34(3):599–610.  https://doi.org/10.1007/s12325-017-0478-y.CrossRefGoogle Scholar
  22. 22.
    Braund R, Hook S, Medlicott NJ. The role of topical growth factors in chronic wounds. Curr Drug Deliv. 2007;4(3):195–204.CrossRefGoogle Scholar
  23. 23.
    Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound Repair Regen. 2008;16(5):585–601.  https://doi.org/10.1111/j.1524-475X.2008.00410.x. CrossRefGoogle Scholar
  24. 24.
    Wu L, Xia YP, Roth SI, Gruskin E, Mustoe TA. Transforming growth factor-beta1 fails to stimulate wound healing and impairs its signal transduction in an aged ischemic ulcer model: importance of oxygen and age. Am J Pathol. 1999;154(1):301–9.CrossRefGoogle Scholar
  25. 25.
    Emmerson E, Campbell L, Davies FC, Ross NL, Ashcroft GS, Krust A, et al. Insulin-like growth factor-1 promotes wound healing in estrogen-deprived mice: new insights into cutaneous IGF-1R/ERalpha cross talk. J Invest Dermatol. 2012;132(12):2838–48.  https://doi.org/10.1038/jid.2012.228. CrossRefGoogle Scholar
  26. 26.
    Manira MCS, Rosliza A, Yi Ling A, Abidah A, Vittarino J, Nurul ‘Izzah AG, et al. Concentration dependent effect of dermal fibroblast conditioned medium on in vitro wound healing properties of keratinocytes. Regenerative Research. 2014;3(2):3.Google Scholar
  27. 27.
    Chowdhury SR, Aminuddin BS, Ruszymah BH. Effect of supplementation of dermal fibroblasts conditioned medium on expansion of keratinocytes through enhancing attachment. Indian J Exp Biol. 2012;50(5):332–9.Google Scholar
  28. 28.
    Fauzi MB, Lokanathan Y, Aminuddin BS, Ruszymah BH, Chowdhury SR. Ovine tendon collagen: extraction, characterisation and fabrication of thin films for tissue engineering applications. Mater Sci Eng C Mater Biol Appl. 2016;68:163–71.  https://doi.org/10.1016/j.msec.2016.05.109.CrossRefGoogle Scholar
  29. 29.
    Sakamoto M, Morimoto N, Ogino S, Jinno C, Taira T, Suzuki S. Efficacy of gelatin gel sheets in sustaining the release of basic fibroblast growth factor for murine skin defects. J Surg Res. 2016;201(2):378–87.  https://doi.org/10.1016/j.jss.2015.11.045.CrossRefGoogle Scholar
  30. 30.
    Magnusson B, Kligman AM. The identification of contact allergens by animal assay. The guinea pig maximization test. J Invest Dermatol. 1969;52(3):268–76.CrossRefGoogle Scholar
  31. 31.
    Thejaswi K, Amarnath M, Srinivas G, Jerald MK, Raj TA, Singh S. Immune modulatory responses of mesenchymal stem cells from different sources in cultures and in vivo. Cell & Tissue Transplantation & Therapy. 2012;4(3374-CTTT-Immune-Modulatory-Responses-of-Mesenchymal-Stem-Cells-from-Different-S.pdf):1–13.  https://doi.org/10.4137/CTTT.S9812.
  32. 32.
    Wu Z, Fan L, Xu B, Lin Y, Zhang P, Wei X. Use of decellularized scaffolds combined with hyaluronic acid and basic fibroblast growth factor for skin tissue engineering. Tissue Eng A. 2015;21(1–2):390–402.  https://doi.org/10.1089/ten.TEA.2013.0260.CrossRefGoogle Scholar
  33. 33.
    Shakespeare PG. The role of skin substitutes in the treatment of burn injuries. Clin Dermatol. 2005;23(4):413–8.CrossRefGoogle Scholar
  34. 34.
    Manira M, Anuar KK, Seet WT, Irfan AWA, Ng MH, Chua KH, et al. Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression. Cell Tissue Bank. 2013:1–9.Google Scholar
  35. 35.
    Maarof M, Lokanathan Y, Ruszymah HI, Saim A, Chowdhury SR. Proteomic analysis of human dermal fibroblast conditioned medium (DFCM). Protein J. 2018;37(6):589–607.  https://doi.org/10.1007/s10930-018-9800-z.CrossRefGoogle Scholar
  36. 36.
    Zhu J, Marchant RE. Design properties of hydrogel tissue-engineering scaffolds. Expert Rev Med Devices. 2011;8(5):607–26.  https://doi.org/10.1586/erd.11.27.CrossRefGoogle Scholar
  37. 37.
    McBane JE, Vulesevic B, Padavan DT, McEwan KA, Korbutt GS, Suuronen EJ. Evaluation of a collagen-chitosan hydrogel for potential use as a pro-angiogenic site for islet transplantation. PLoS One. 2013;8(10):e77538.  https://doi.org/10.1371/journal.pone.0077538.CrossRefGoogle Scholar
  38. 38.
    Vulpe R, Popa M, Picton L, Balan V, Dulong V, Butnaru M, et al. Crosslinked hydrogels based on biological macromolecules with potential use in skin tissue engineering. Int J Biol Macromol. 2016;84:174–81.  https://doi.org/10.1016/j.ijbiomac.2015.12.019.CrossRefGoogle Scholar
  39. 39.
    Choi J, Park H, Kim T, Jeong Y, Oh MH, Hyeon T, et al. Engineered collagen hydrogels for the sustained release of biomolecules and imaging agents: promoting the growth of human gingival cells. Int J Nanomedicine. 2014;9:5189–201.  https://doi.org/10.2147/ijn.s71304.CrossRefGoogle Scholar
  40. 40.
    Aoki S, Takezawa T, Uchihashi K, Sugihara H, Toda S. Non-skin mesenchymal cell types support epidermal regeneration in a mesenchymal stem cell or myofibroblast phenotype-independent manner. Pathol Int. 2009;59(6):368–75.  https://doi.org/10.1111/j.1440-1827.2009.02379.x.CrossRefGoogle Scholar
  41. 41.
    Parenteau-Bareil R, Gauvin R, Cliche S, Gariepy C, Germain L, Berthod F. Comparative study of bovine, porcine and avian collagens for the production of a tissue engineered dermis. Acta Biomater. 2011;7(10):3757–65.  https://doi.org/10.1016/j.actbio.2011.06.020.CrossRefGoogle Scholar
  42. 42.
    Zhao J, Hu L, Gong N, Tang Q, Du L, Chen L. The effects of macrophage-stimulating protein on the migration, proliferation, and collagen synthesis of skin fibroblasts in vitro and in vivo. Tissue Eng A. 2015;21(5–6):982–91.CrossRefGoogle Scholar
  43. 43.
    Liu Y, Ma L, Gao C. Facile fabrication of the glutaraldehyde cross-linked collagen/chitosan porous scaffold for skin tissue engineering. Mater Sci Eng C. 2012;32(8):2361–6.  https://doi.org/10.1016/j.msec.2012.07.008.CrossRefGoogle Scholar
  44. 44.
    Shevchenko RV, James SL, James SE. A review of tissue-engineered skin bioconstructs available for skin reconstruction. J R Soc Interface. 2010;7(43):229–58.  https://doi.org/10.1098/rsif.2009.0403.CrossRefGoogle Scholar
  45. 45.
    Pourahmad J, Salimi A. Isolated human peripheral blood mononuclear cell (PBMC), a cost effective tool for predicting immunosuppressive effects of drugs and xenobiotics. Iran J Pharm Res. 2015;14(4):979.Google Scholar
  46. 46.
    Busra FM, Lokanathan Y, Nadzir MM, Saim A, Idrus RBH, Chowdhury SR. Attachment, proliferation, and morphological properties of human dermal fibroblasts on ovine tendon collagen scaffolds: a comparative study. Malays J Med Sci. 2017;24(2):33–43.  https://doi.org/10.21315/mjms2017.24.2.5. Google Scholar
  47. 47.
    Busra FM, Chowdhury SR, Saim AB, Idrus RB. Genotoxicity and cytotoxicity of ovine collagen on human dermal fibroblasts. Saudi Med J. 2011;32(12):1311–2.Google Scholar
  48. 48.
    Yamamoto A, Mishima S, Maruyama N, Sumita M. Quantitative evaluation of cell attachment to glass, polystyrene, and fibronectin- or collagen-coated polystyrene by measurement of cell adhesive shear force and cell detachment energy. J Biomed Mater Res. 2000;50(2):114–24.CrossRefGoogle Scholar
  49. 49.
    Vyas KS, Vasconez HC. Wound healing: biologics, skin substitutes, Biomembranes and Scaffolds. Healthcare (Basel). 2014;2(3):356–400.  https://doi.org/10.3390/healthcare2030356. CrossRefGoogle Scholar
  50. 50.
    Boyce ST, Lalley AL. Tissue engineering of skin and regenerative medicine for wound care. Burns Trauma. 2018;6(1):4.  https://doi.org/10.1186/s41038-017-0103-y. CrossRefGoogle Scholar
  51. 51.
    Alrubaiy L, Al-Rubaiy KK. Skin substitutes: a brief review of types and clinical applications. Oman Med J. 2009;24(1):4–6.  https://doi.org/10.5001/omj.2009.2.Google Scholar
  52. 52.
    Park JY, Lee TG, Kim JY, Lee MC, Chung YK, Lee WJ. Acellular dermal matrix to treat full thickness skin defects: follow-up subjective and objective skin quality assessments. Arch Craniofac Surg. 2014;15(1):14–21.  https://doi.org/10.7181/acfs.2014.15.1.14.CrossRefGoogle Scholar
  53. 53.
    Moore MA, Samsell B, Wallis G, Triplett S, Chen S, Jones AL, et al. Decellularization of human dermis using non-denaturing anionic detergent and endonuclease: a review. Cell Tissue Bank. 2015;16(2):249–59.  https://doi.org/10.1007/s10561-014-9467-4.CrossRefGoogle Scholar
  54. 54.
    Payushina OV, Butorina NN, Sheveleva ON, Domaratskaya EI. Effect of mesenchymal stromal cells and conditioned media on mealing of skin wound. Bull Exp Biol Med. 2018;165(4):572–5.  https://doi.org/10.1007/s10517-018-4215-6
  55. 55.
    Sun J, Zhang Y, Song X, Zhu J, Zhu Q. The healing effects of conditioned medium derived from mesenchymal stem cells on radiation-induced skin wounds in rats. Cell Transplant. 2018;963689718807410.  https://doi.org/10.1177/0963689718807410

Copyright information

© Controlled Release Society 2018

Authors and Affiliations

  1. 1.Tissue Engineering Centre, Faculty of MedicineUniversiti Kebangsaan MalaysiaKuala LumpurMalaysia
  2. 2.Department of Physiology, Faculty of MedicineUniversiti Kebangsaan MalaysiaKuala LumpurMalaysia
  3. 3.Bioserasi LaboratoryUniversiti Kebangsaan MalaysiaBangiMalaysia

Personalised recommendations