The Development of a Wound Healing Model Using Nerve Growth Factor(NGF) Expression in Raft Culture

Kim, Kyu Han; Lee, Dong Youn; Park, Kyoung Chan

Abstract

BACKGROUND
The wound healing process is impaired or delayed in aged patients. The development of a new wound healing model is needed. Nerve growth factor (NGF) plays a special role in wound healing because NGF is expressed only in proliferating tissues such as wounds.
OBJECTIVE
The aim of our study was to develop a wound healing model using a 3-dimensional culture system, raft culture, by comparing the level of NGF expression according to the wound stage after an artificial wound was made to the raft samples. We tried to specifically localize the site of NGF expression both in mRNA and protein level.
METHODS
Raft culture using normal human keratinocytes was done and a 2 mm slit wound was made in the center of the raft samples. Raft samples of no wound, 4 d, 7 d, and 9 d after wounding were prepared. In situ RT-PCR and immunohistochemistry were performed to detect and localize NGF expression after making wounds and the addition of substance P (SP).
RESULTS
We failed to localize NGF mRNA expression in raft samples by in situ RT-PCR. Immunohistochemistry showed NGF staining throughout the epidermis although a little more dense staining was found in the basal layer. NGF(+) cells tended to increase until 7 d after wounding, but there were no significant differences according to the wounding days. There was `a tendency that the SP(+) group showed more NGF(+) cells than the SP(-) group, but there were no statistical differences.
CONCLUSION
We think that our in vitro raft wound model using NGF expression could be used, at least in part, as an objective indicator for wound healing. In our raft model lacking nerve, NGF may not be suitable for representing wound healing process because this model can not reflect the interaction between the skin and the nervous system. Expression of growth factors or cytokines other than NGF need to be applied to our raft culture system.