In-Vitro Wound Contraction in the Horse: Differences Between Body and Limb Wounds

Introduction Horses commonly have complications in the healing of wounds on the lower limb regions.[1–4] Upper body wounds, often extensive and deep, often heal well with few complications.[5] The speed of healing in different regions in the body and the limb may be attributed to wound contraction. The work of Wilmink, et al.,[6,7] showed that ponies have a higher rate of contraction than horses. There have been similar variations seen in some human wounds, especially human leg ulcers (Figure 1), which fail to heal despite advanced therapeutic intervention. Equine chronic wounds that are exuberant or indolent have similarities to human leg ulcers. In the horse and man, the problems manifest themselves in the lower limb and are often age related.[5] Wound contraction is a major contributor to the healing process, and the rate of contraction of wounds has been calculated from experimental studies involving experimental wounds of a standard size and shape.[8,9] It has been shown that wound contraction not only speeds up the healing process but enhances the tensile strength and cosmetic appearance of the healed wound. In chronic limb wounds, however, the opposite occurs, and the newly formed epithelium is fragile and lacks hair follicles.[2,10,11] Myofibroblasts differentiate from granulation tissue and develop ultrastructural and biochemical features of smooth muscle cells, including the presence of microfilament bundles and the expression of a-smooth muscle actin.[12] Myofibroblasts play a role in wound contraction and are the main cell type implicated in the synthesis of extracellular components, including type I collagen and fibronectin.[13] The contraction capacity of the cells and the local extracellular environment will determine the degree of contraction of the wound. Contraction of fibroblast/collagen gels has been used as an in-vitro model for investigating the biological mechanisms of wound contraction[14,15] and also the effects of various compounds aimed at stimulating (enhancing wound healing) or reducing (preventing scar formation) the rate of contraction.[16] The advantage of using this model is that the fibroblasts are grown in a three-dimensional collagen gel culture where collagen is a component native to the wound environment. The culture of fibroblasts in these gels more closely resembles growth in an in-vivo situation, and this is important when considering cellular interactions. The aim of this study was to compare the contractile abilities of differentiated fibroblasts harvested from five different sites of the body and the limbs of horses. A similar comparison was made between tissue harvested from chronic granulating wounds, normal skin, and L929 cells. The contractile capacity of the cells was determined by a decrease in gel area. 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. Equine dermal tissue (normal fibroblasts [NF]) was obtained post mortem. Six different anatomical sites were sampled; these were the eyelid, axilla, groin, medial thigh, ventral midline, and limb (fore, mid-dorsal cannon). Similar tissue was taken from chronic, slow-healing, granulating wounds (GT) located on horses’ hind limbs through normal surgical debridement prior to skin grafting.[13,17,18] Briefly, appropriate samples were taken for fibroblast culture and immediately transferred to a sterile dish, washed in Hanks balanced salt solution (HBSS) (all cell culture materials were supplied by Gibco, United Kingdom, unless otherwise stated), cut into 3- to 5mm2 pieces, and placed into 25cm2 tissue culture flasks containing media. The media consisted of Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10-percent fetal calf serum (FCS) (Sigma, UK), 20mM Hepes buffer, 100µg/mL gentamicin, and 0.5µg/mL amphotericin B. Cell cultures were incubated at 37 degrees C in a five-percent carbon dioxide air environment. Readiness for subculturing was determined by the extent of fibroblast cell outgrowth (5 to 10 days). Cells were farmed successively in a 1:4 split ratio to passage 3 to 8 times for experimental use. For seeding in the collagen matrix, NF from the six sites and GT from granulating limb wounds were harvested from tissue culture flasks with trypsin-EDTA washed and resuspended with DMEM. The cells were counted and resuspended at 1 x 106 cells/mL in DMEM medium. Preparation and testing of collagen gels. The cells were then resuspended into type I collagen (Fred Baker Scientific, United Kingdom) at 2mg/mL and plated into six well tissue culture plates. Six gels of 1mL were made for each cell type (n = 18). Gelation occurred within five minutes. The surface of the gels were washed with 2mL HBSS, and 2mL DMEM was added followed by incubation in a five-percent CO2 in air environment at 37?C. Analysis of contraction. Contraction of collagen lattices was determined by averaging the longest and shortest diameter of each lattice measured by calipers. Calculation of the diameters represents the mean and standard deviation of 18 lattices (n = 18). Cell viability. The viability of the fibroblasts was monitored using Trypan blue staining during the successive culture steps and the preparation of collagen gels. At the end of the experiment (96 hours), the cells were released from the collagen lattice by digestion of the collagen gel using 0.2-percent collagenase in cell culture medium for 10 minutes, resuspended in DMEM, and tested for viability. Viability was expressed as the percentage of viable cells remaining in the cell suspension. Photomicroscopy. Photomicrographs were taken at 48, 72, and 96 hours after incubation in the collagen lattice using an Olympus CK2 inverted microscope with a photomicrograph attachment PM10-AK-2 and an Olympus C35 DA/2 camera. Kodak Ektachrome color slide film was used. Statistical analysis. All of the results were expressed as the mean ± standard error (SEM). The statistical significance of the results was assessed using the Student’s t-test, and a P value of Contraction morphology of NF cultured from different sites of the body. The cells were evenly dispersed throughout the collagen lattice, spherical in shape, and showed signs of spreading out after 24 hours. By 48 hours, the cells were beginning to elongate and acquire the typical morphology of fibroblasts. In general, at 72 hours the cells showed a marked change in morphology and assumed their typical bipolar spindle form with several long processes (Figure 2). It was evident that the cells cultured from the limb and abdomen were able to arrange themselves in a more organized pattern. The contracted gels at 96 hours revealed a regular arrangement of fibroblasts with more and longer processes (Figure 3). In-situ morphological characteristics of GT and L929 cells in collagen lattices. Fibroblasts cultured from GT were spherical when seeded into the collagen lattice but within a few hours had rapidly elongated and become spindle shaped with long processes. In contrast, the L929 cells remained rounded for much longer (Figure 4) and did not show signs of elongating until 72 hours. Collagen contraction mediated by normal fibroblasts. The fibroblasts from the limb, axilla, and abdomen started to contract sooner than the other cells, and at 24 hours, there is a significant difference between the limb and the other cell types (Figure 5). At 48 hours, however, cells from the other sites were contracting faster, and there was only a significant difference between the limb and the eyelid fibroblasts. Fibroblast cells cultured from the axilla and thigh showed significant differences to the limb cells at 72 hours and by 96 hours, there was a significant difference between the limb and all of the other cell lines. Collagen contraction mediated by NF, GT, and L929 cells. All three cell lines contracted in a linear fashion over time (Figure 6). The fibroblasts derived from granulation tissue from nonhealing wounds contracted the gels much more rapidly than the normal cells. In contrast, the L929 cells were very slow to contract the collagen lattice. At all four time points, there was a significant difference between the GT, NF, and L929 cells (p Cell viability. The viability of fibroblasts prior to seeding into the collagen matrix exceeded 95 percent for all of the cell types used in this study. At 96 hours, the collagen gels were digested with collagenase, and viability was reassessed (Figure 7). There was no significance difference in viability between NF, GT, and L929 fibroblasts. Furthermore, there was no significant difference in viability between cells cultured from the limb or the other five sites of the horse. Discussion Wound healing myofibroblasts play a crucial role in tissue repair as well as in contraction. Previous studies have demonstrated the importance of these cells in the success of tissue remodeling and normal wound closure.[19,20] Fibroblasts are the main cellular type involved in extracellular deposition during tissue repair. To replace damaged tissue, fibroblasts actively participate in the synthesis of extracellular matrix components, such as tenascin, fibronectin, and collagen I and III. There are numerous factors that affect fibroblast contraction in collagen gels including collagen concentration[21] and type,[22] serum concentration,[23] and the presence of growth factors.[24] The rate of contraction of the collagen lattice will be determined by the cell density, viability, and proliferation rate. In the current research, the contractile capacity of equine NF taken from six different sites of the body was assessed. The contractile characteristics of cells cultured from tissue taken from the limb contracted the gels faster than cells from the other sites. There was a significant difference seen between the limb and various other sites at all the time points. However, at 96 hours, a significant difference in the rate of contraction was seen between the limb fibroblasts and cells cultured from the other sites. In the clinical situation, the opposite is seen; the difference may be related to the culture conditions used. Although the authors hoped to mimic the in-vivo situation, the model is limited in that there is no blood supply, and in turn there would be a lack of environmental factors, such as growth factors. The fibroblasts from the limb displayed a more differentiated phenotype in the gels, were better organized, and had longer cell processes. In contrast, fibroblasts from the other sites were less well organized in the gels and were smaller with shorter processes. A similar comparison was made between fibroblasts harvested from chronic granulating wounds, normal skin, and L929 cells. The GT fibroblasts contracted the gels at a significantly higher rate than the NF and L929 cells at all experimental time points. Fibroblasts in the granulation tissue of limb wounds in horses maintain their proliferative immature character for a long time, even after the wound is completely closed. This usually leads to the formation of exuberant granulation tissue even though these cells express a-smooth muscle actin (SMA).[6,7] Cell viability remained high throughout the experiment, and there was no significant difference between the three cell types at 96 hours. Although there was no significant difference in the viability between the cell types, a reduction was observed in the NF and GT cells. This was probably due to contact inhibition as the gels contracted, causing some of the cells to die. At 96 hours, the contracted gels containing NF and GT cells were very densely populated. There are anatomical and physiological differences seen in the horse when wound healing occurs.[5] The problems with the distal limb may be attributed to poor blood supply, which in turn could lead to a deficit in nutrients and other cell mediators, which are needed for normal wound healing. Conclusion It is concluded from these studies that during second-intention wound healing in the horse, the differences in wound contraction between wounds on the limbs and the body are caused by differences in the contractile capacity of fibroblasts. The extracellular environment plays a role in the behavior of fibroblast cells. However, local environmental factors, such as the response to inflammation, may play a role in determining the rate of contraction during wound healing. The value of this work has been to show that the contractile variation of fibroblasts from different tissues is highest in the limb. The results do not match the observed wound contraction of in-vivo limb injuries, and, therefore, further research should be considered to determine those factors within the in-vivo limb environment that would allow the limb fibroblasts to contract to their observed full in-vitro potential.