Abstract
M.Tech.
The skin accounts for 14 percent of the total body weight and is the largest organ in the body (Edward & MacKie, 2001). Our skin serves as a protective barrier against the outside world, thus any break in it must be rapidly and efficiently mended. A wound may be defined as any disruption of the tissues of the body caused by injury (Vardaxis, 1995). Commonly recognised examples include bruises, grazes, incisions, ulcers and burns. While some wounds heal easily others become fatally infected. According to statistics in the United States, for the year 2000, approximately five million Americans suffered from chronic open sores that could become seriously infected (UMMC, 2000). Unfortunately, neither Statistics South Africa (STATS SA) nor the South African Medical Research Council have statistics on open sores.
The aim of this study was to determine the effectiveness of Low Level Laser Therapy (LLLT) and Calendula officinalis 3cH respectively as treatment protocols on wound healing in injured human skin fibroblasts (HSF) in-vitro. Furthermore the study aimed to investigate the effectiveness of a combination of both treatment modalities on wound healing.
Commercially available human skin fibroblast (HSF) cell lines (CRL1502 WS1) were obtained from the American Type Culture Collection (ATCC). Each week these fibroblasts were subcultured from 75cm² flasks to six, 3.3cm diameter culture plates. To simulate mechanical disruption of the cells a central scratch was performed across the confluent monolayer of fibroblasts according to the method described by Rigau et al., (1995), using a sterile Pasteur pipette of 2mm in diameter. Each scratch was irregular and the “wounds” ranged from 1-2mm in diameter. Five of the plates received the aforementioned scratch. Thereafter only four of the plates received a specific treatment modality. The remaining two plates served as controls.
To assess the effectiveness of each treatment modality, wound healing was measured using the following techniques: cell morphology using an inverted microscope (Olympus CKX41) to monitor cell migration and wound closure. Cell viability was measured using the Trypan blue and ATP cell viability assay. Cytotoxicity was measured using the Lactate dehydrogenase (LDH) membrane assay. Apoptosis of cells was detected with a Caspase assay. Alkaline phosphatase (ALP) assay was used as a marker for wound healing. Interleukin-6 (IL-6) is a cytokine released early in wound healing and was used to measure cell proliferation.
The experimental procedure was designed to determine if there was a difference in the wound healing outcome if cells were exposed to a single treatment application or a double treatment application with a 24 hour interval. The single treatment protocol was repeated four times (n=4) while the double treatment protocol was repeated six times (n=6). The data for both experimental procedures was analysed using Sigma Plot 8.0 computer software. The student t-test was utilised to examine the effect that the treatments (independent variables) had on the various aspects of wound healing (dependent variables). In each case a statistical difference was identified as P< 0.05.
Morphological changes indicate that a double treatment application of Calendula officinalis 3cH increases wound closure on wounded HSF cells in-vitro. Laser irradiation too showed morphological signs of increased wound closure for HSF cells receiving a double exposure. However, a synergistic relationship, based on morphology, was not established when combining the two treatments.
Based on statistical analysis the results showed that neither Calendula officinalis 3cH nor laser irradiation, as singular or combination treatment modalities, improved wound healing in wounded HSF cells in-vitro. However, findings were encouraging and minor changes evident on a cellular level may be more significant at a systemic level.