Skin is the largest organ in human system and plays a vital role as a barrier against environment and pathogens. Skin regeneration is important in tissue engineering especially in cases of chronic wounds. With the tissue engineering technology, these skins equivalent have been use clinically to repair burns and wounds. Consented redundant skin samples were obtained from patients aged 9 to 65 years old. Skin samples were digested with dispase, thus separating the epidermis and the dermis layer. The epidermis layer was trypsinized and cultured in DKSFM in 6-well plate at 37 degrees C and 5% CO2. Once confluent, the culture were trypsinized and the cells were pooled. Cells were counted using haemacytometer. Doubling time and viability were calculated and analysed. From the result, we conclude that doubling time and viability of in vitro keratinocytes cultured in DKSFM media is not age dependant.
Age Factors ; Burns/physiopathology ; Burns/*therapy ; Cell Aging/*physiology ; Cell Division/physiology ; Cell Survival/physiology ; Chronic Disease ; Keratinocytes/*cytology ; *Skin Transplantation ; Statistics ; Tissue Engineering/*methods ; Wound Healing/physiology ; Wounds and Injuries/physiopathology ; Wounds and Injuries/*therapy

The angiogenic potential of native skin (NS), keratinocytes single skin equivalent (SSE-K), fibroblasts single skin equivalent (SSE-F) and bilayered skin equivalent secreting angiogenic growth factors such as transforming growth factor beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) in the in vitro systems at 24, 48, 72 hours and 7 days was compared using Enzyme-Linked Immunosorbent Assay (ELISA). Bilayered skin equivalent exhibit highest release of growth factors within 24 hours to 7 days of culture compared to NS, SSE-K and SSE-F. This proved the potential of bilayered skin equivalent in producing and sustaining growth factors release to enhance angiogenesis, fibroblasts proliferation, matrix deposition, migration and growth of keratinocytes.

Our objective is to determine the quality of tissue engineered human skin via immunostaining, RT-PCR and electron microscopy (SEM and TEM). Culture-expanded human keratinocytes and fibroblasts were used to construct bilayer tissue-engineered skin. The in vitro skin construct was cultured for 5 days and implanted on the dorsum of athymic mice for 30 days. Immunostaining of the in vivo skin construct appeared positive for monoclonal mouse anti-human cytokeratin, anti-human involucrin and anti-human collagen type I. RT-PCR analysis revealed loss of the expression for keratin type 1, 10 and 5 and re-expression of keratin type 14, the marker for basal keratinocytes cells in normal skin. SEM showed fibroblasts proliferating in the 5 days in vitro skin. TEM of the in vivo skin construct showed an active fibrocyte cell secreting dense collagen fibrils. We have successfully constructed bilayer tissue engineered human skin that has similar features to normal human skin.
Fibroblasts/*cytology ; Keratinocytes/*cytology ; Mice, Nude ; Microscopy, Electron ; Microscopy, Electron, Scanning ; Quality Control ; Regeneration/physiology ; Skin/pathology ; Skin Transplantation/pathology ; Skin Transplantation/*standards ; Tissue Engineering/*standards

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) evaluation were carried out in the in vivo skin construct using fibrin as biomaterial. To investigate its progressive remodeling, nude mice were grafted and the Extracellular Matrix (ECM) components were studied at four and eight weeks post-grafting. It was discovered that by 4 weeks of remodeling the skin construct acquired its native structure.
Collagen/*physiology ; Extracellular Matrix/pathology ; Fibroblasts/pathology ; Mice, Nude ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Regeneration/*physiology ; Skin/*pathology ; *Skin Transplantation/pathology ; *Tissue Engineering

Chitosan has similar structure to glycosaminoglycans in the tissue, thus may be a good candidates as tissue engineering scaffold. However, to improve their cell attachment ability, we try to incorporate this natural polymer with collagen by combining it via cross-linking process. In this preliminary study we evaluate the cell attachment ability of chitosan-collagen scaffold versus chitosan scaffold alone. Chitosan and collagen were dissolved in 1% acetic acid and then were frozen for 24 hours before the lyophilizing process. Human skin fibroblasts were seeded into both scaffold and were cultured in F12: DMEM (1:1). Metabolic activity assay were used to evaluate cell attachment ability of scaffold for a period of 1, 3, 7 and 14 days. Scanning electron micrographs shows good cell morphology on chitosan-collagen hybrid scaffold. In conclusion, the incorporation of collagen to chitosan will enhance its cell attachment ability and will be a potential scaffold in tissue engineering.
Cell Adhesion/*physiology ; *Chitosan ; *Collagen ; Energy Metabolism/physiology ; Fibroblasts/cytology ; Microscopy, Electron, Scanning ; Organ Culture Techniques/*methods ; *Polymers ; Tissue Engineering/*methods

A major factor limiting survival following extensive thermal injury is insufficient availability of donor sites to provide enough skin for the required grafting procedures. Limitation of autologous grafting promotes the usage of allograft skin substitutes to promote wound healing. Here, we investigated the wound healing potential of allograft single layered tissue engineered skin which comprises of either keratinocytes (SLTES-K) or fibroblast (SLTES-F) with fibrin as the delivery system. Results from gross and microscopic evaluation showed our single layered tissue engineered skin constructed with keratinocytes or fibroblast after gamma radiation with the dosage of 2Gy could serve as allograft for the treatment of skin loss.