The Application of a Fibroblast Gel Contraction Model to Assess the Cytotoxicity of Topical Antimicrobial Agents

Disclosure: Financial support for this work was provided by ConvaTec Ltd., United Kingdom. Introduction Since the introduction of a fibroblast-populated collagen lattice in the late 1970s, this type of in-vitro model has been extensively used to study fibroblast function.[1,2] Contraction of fibroblast/collagen gels has been used as in-vitro models for investigating the biological mechanisms of wound contraction[3,4] and also the effects of various compounds aimed at stimulating (enhancing wound healing) or reducing (preventing scar formation) the rate of contraction.[5] The benefit of this model is that the fibroblasts are grown in a three-dimensional collagen gel culture, a matrix component native to the wound environment. Fibroblasts cultured in these conditions have distinct characteristics compared to those cultured on dishes. The fibroblasts cultured in collagen gels acquire a bipolar spindle form, while fibroblasts grown on culture dishes manifest a ruffled membrane that possesses one or more broad-banded pseudopodia.[6,7] In the gels, collagen production is decreased, proteinases and fibronectin production is increased,[8] and fibroblast proliferation is slower.[9] Generally, it appears that the culture of fibroblasts in these gels more closely resembles growth in an in-vivo situation, and this is important when considering cellular interactions and potential cytotoxic compounds. Fibroblast culture systems have been routinely used to investigate wound contraction in a wide range of experimental conditions, including irradiation effects,[10] inhibition of chronic inflammatory cell mediators,[11] and the biocompatability of wound management products.[12] In particular, these in-vitro cell systems routinely have been used to assess the cytotoxicity of topical antimicrobial agents[13] and dental materials.[14] In the current studies, the L929 immortalized mouse cell line, a recognized model for wound dressing compatibility studies,[12] was used along with equine fibroblasts, which were harvested and cultured from slow healing wounds or from granulating wounds with exuberant granulation tissue removed during normal surgical debridement.[15] There were two reasons for choosing equine fibroblasts. First, the chronic wound in the horse is considered to have a similar pathology to the human chronic wound.[2,16] These nonhealing wounds are only present on the lower limbs (i.e., below the knee) of horses and are highly inflamed with high levels of protease activity in wound tissue and wound fluid.[16] The second reason is that equine nonhealing wounds hyper-granulate, and excision of this tissue provides an excellent source of granulation tissue-derived fibroblasts. Equine granulation tissue fibroblasts (EGTF) have been shown to have similar growth rates to those of normal equine fibroblasts yet have a spread-out cuboidal appearance compared to the typical spindle-shaped morphology found in normal fibroblasts.[7] It was proposed in the first part of these studies to compare the rates of contraction of normal equine fibroblasts with those of EGTF against the well-characterized mouse fibroblast cell line (L929 cells) that is used routinely in cytotoxicity studies.[13–15] In the second part of the studies, collagen gels containing EGTF and L929 cells were compared by assessing their response to the application of potentially cytotoxic, iodine-containing topical antiseptic agents (Table 1). There is still much debate regarding the use of antiseptics in wound care,[17,18] although there is a growing consensus that the slower releasing formulations have a role to play.[18] Materials and Methods Cell culture. L929 cells were obtained from the European Collection of Cell Cultures, Centre for Applied Microbiology and Research, Porton Down, United Kingdom. Normal equine fibroblasts were obtained post mortem from horses that had been killed for non-related clinical reasons. Chronic wound (granulation) tissue fibroblasts were cultured from tissue taken from slow-healing wounds or from granulating wounds with exuberant granulation tissue during normal surgical debridement prior to skin grafting. The in-vitro gel contraction model used throughout these studies to evaluate tissue toxicity was in compliance with the ISO Standard for the Biological Evaluation of Medical Devices.[19] Briefly, appropriate samples were taken for fibroblast culture and immediately transferred to a dish, washed in Hank’s balanced salt solution (HBSS) (all cell culture materials were supplied by Gibco, United Kingdom, unless otherwise stated), cut into 5mm2 pieces, and placed into 25cm2 tissue culture flasks containing media. The media was made by the addition of Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10-percent fetal calf serum (FCS) (Sigma, United Kingdom), 20mM Hepes buffer, 100µg/mL gentamicin, and 0.5µg/mL amphotericin B. Cell cultures were incubated at 37 degrees C in a five-percent CO2/95-percent air environment. Readiness for subculturing was determined by the extent of fibroblast cell outgrowth (5–10 days). Cells were farmed successively in a 1 to 4 split ratio and passages 3 to 8 were used in these experiments. Fibroblasts were harvested from stock dishes and plated out in 35mm, six-well plates at 1x106 cells/mL in type I collagen (Fred Baker Scientific, United Kingdom) at 2mg/mL. Six wells were set up for each individual formulation and cell type (n=6). Untreated dishes containing fibroblasts in collagen as above (n=6) were established to use as controls. The dishes were incubated in a five-percent CO2/95-percent air environment at 37 degrees C. After a period of one hour when the collagen fibroblast gels had set sufficiently, the gel surface was washed with 1mL of HBSS, and 1mL of media was added. In the first series of studies investigating normal contraction rates, the cells were allowed to contract for a maximum of 144 hours. In the second series of experiments, either 0.5mL or 0.5g of a range of topical antiseptic formulations (Table 1) was separately applied to the surface of the gels. Gel contraction was measured using a set of calipers (mm) at 24-hour intervals and thereafter extending to a maximum period of 96 hours. There was no need to remove the dressings to measure the gels because gel contraction did not exceed surface area of the dressing. In these studies, povidone-iodine solution was used as a positive control at a clinically relevant concentration (i.e., 10% w/v). Gauze was soaked in 0.5mL povidone-iodine solution (10%) prior to application to the surface of the collagen gel. The negative control had no formulation applied to the gel surface. Statistical analysis. A multivariate analysis of variance (Duncan’s Multiple Comparison Test) was used to analyze the data. This test accommodates the experimental design used in these studies. Notably, they were two-factor experiments (i.e., different dressings and time points) with repeated measurements on one factor (i.e., contraction rate). These multifactor experiments have been described in detail by Winer.[20] Results The initial study showed that EGTF have greater contractile capacity than normal equine fibroblasts, and these results are in agreement with the results of Germain, et al., who studied human granulation fibroblasts (Figure 1).[4] She suggests that there may be a higher proportion of myofibroblasts present in wound fibroblast populations and that these may enhance wound contraction.[4] In the second series of experiments, EGTF were compared with L929 cells to evaluate a range of iodine-containing topical antiseptic agents (Table 1). The results are presented in Figures 2 and 3. Similar results were observed for both sets of fibroblasts. Only inadine appeared to offer a reduced level of cytotoxicity beyond 24 hours (i.e., greater level of contraction), but even this was seen to be significantly different to the control group (p