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