DEVELOPMENT OF CHITOSAN BASED SKIN SUBSTITUTE USING CO-CULTURE OF HUMAN FIBROBLASTS AND EPITHELIAL STEM CELLS DERIVED FROM HUMAN HAIR FOLLICLE AND ITS EFFECTIVENESS ON IMPAIRED WOUND HEALING IN RAT MODEL

Abu Bakar, Mohd Hilmi (2014) DEVELOPMENT OF CHITOSAN BASED SKIN SUBSTITUTE USING CO-CULTURE OF HUMAN FIBROBLASTS AND EPITHELIAL STEM CELLS DERIVED FROM HUMAN HAIR FOLLICLE AND ITS EFFECTIVENESS ON IMPAIRED WOUND HEALING IN RAT MODEL. PhD thesis, Universiti Sains Malaysia.

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Abstract

Tissue engineering is an interdisciplinary field in regenerative medicine that aims to replace damaged or diseased skin. It involves a multi-skilled in the area of stem cell, biomaterial, cellular and molecular biology, and animal modeling before the final application in clinical setting. The discoveries of epithelial stem cells derived from human hair follicle have encouraged researchers to utilize the hair follicle as a therapeutic source of stem cells for skin tissue engineering. Therefore, this current study aims to fabricate the skin substitute using epithelial stem cells. The chitosan has been used as scaffold for co-culture between the hair follicle stem cells (HFSCs) and human dermal fibroblasts (HDFs). Samples of human scalp were used for isolation of HFSCs and HDFs. It involved 35 samples of patient. The characterization of HFSCs was performed by using the stem cell marker of CD200 and K15. Additionally, HFSCs were induced to differentiate into an epidermal layer. The characterization of the epidermal layer was confirmed by the presence involucrin- and K6-positive cells. Meanwhile the characterization of HDFs was performed by using the surface marker of vimentin and versican. The novel skin substitute was fabricated via co-culture between HFSCs and HDFs on the chitosan. To evaluate healing process, the novel skin substitutes were used in full-thickness wound with impaired and non-impaired healing on rats. This in vivo study involved 30 male rats. The rats have been divided into two groups of irradiated and non-irradiated groups. Two months post-radiation, 1 cm by 1 cm full-thickness wound was excised at dorsum of rats. Each of wound was treated randomly using three different biomaterials. There are chitosan dermal substitute which refer to the chitosan without cells, chitosan skin substitute which refer to the chitosan with cells construct and duoderm CGF as positive control. On days 7, 14 and 21 post-wounds, the rats were euthanized for wounds excision. Irradiated wounds treated with the chitosan skin substitute showed the most re-epithelialization (33.2 ± 2.8%), longest epithelial tongue (1.62 ± 0.13 mm) and shortest migratory tongue distance (7.11 ± 0.25 mm). The scar size of wounds treated with the chitosan dermal substitute (0.13 ± 0.02 cm) and chitosan skin substitute (0.16 ± 0.05 cm) were significantly decreased (P<0.05) compared with duoderm CGF (0.45 ± 0.11 cm). In irradiated wounds, the expression of cytokeratin 10 (K10) along the migratory tongue was dysregulated on days 7 and 14. Conversely, K10 was highly expressed on day 21 proving that, it is useful as marker of neo-epidermis in an animal model of impaired wound healing. Human leukocyte antigen (HLA) expression on days 7, 14 and 21 revealed the presence of human hair follicle stem cells and fibroblasts that were incorporated into and surviving in the irradiated wound. This study has proven that a chitosan dermal substitute and chitosan skin substitute were suitable for wound healing in full-thickness wound that was impaired due to radiation.

Item Type: Thesis (PhD)
Subjects: Q Science > Q Science (General)
Faculty / Institute: Faculty Of Health Sciences
Depositing User: DR MOHD HILMI ABU BAKAR
Date Deposited: 16 Nov 2014 04:18
Last Modified: 27 Apr 2015 03:07
URI: http://erep.unisza.edu.my/id/eprint/1764

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