William J. Lindblad

Collagen expression by novel cell populations in the dermal wound environment

Numerous collagenous structures must be reconstituted following injury to the skin in order to return function to this tissue. The basement membrane zone, vascular basement membranes, and the dense connective tissue of the dermis are examples of structures that contain a number of different collagen types and that may need replacement following injury. In addition, a scar is deposited at the site of damage in order to substitute for elements lost in the trauma and for elements that cannot be successfully replaced. Clearly, cells resident within the different compartments of the skin are able to synthesize and deposit collagen to reform these multiple structures. However, accumulating experimental evidence suggests that in addition to these resident cells, blood‐borne cells may be responsible for the deposition of a portion of the newly synthesized collagen. Studies from this laboratory point to the activated monocyte as a potential source of collagen in the wound environment. Given the dynamics of the process, the hypothesis is proposed that during normal wound healing, the activated monocyte is a source of collagen essential for the rapid formation of a provisional matrix conducive for the subsequent formation of granulation tissue. Collagen synthesis also occurs by expanded populations of resident cells, under the influence of inflammatory cell‐derived mediators, which results in the major accumulation of collagen during normal wound repair. However, if a chronic inflammatory state is initiated, the activated monocytes may remain in sufficient numbers to deposit collagen leading to a pathological lesion.

Analysis of the effects of chitosan on inflammation, angiogenesis, fibroplasia, and collagen deposition in polyvinyl alcohol sponge implants in rat wounds.

Chitosan is a large molecular weight, positively charged polysaccharide extracted and purified from the chitin of crab shells. This compound has been shown to have hemostatic activity and has been suggested for use as a topical agent in tissue repair. The objective of this study was to analyze the effect of chitosan on the wound healing response to a standardized injury in the rat. The polyvinyl alcohol sponge implant model was used as a means to deliver either chitosan or its vehicle to a standardized subcutaneous wound on the backs of Sprague‐Dawley rats. On days 8 and 14, the chitosan‐treated implants contained primarily polymorphonuclear leukocytes compared with the vehicle controls which contained mainly macrophages, fibroblasts, collagen, and new blood vessels. High‐performance liquid chromatography analysis for hydroxy‐l‐proline deposited in the sponge implants showed significantly lower amounts on both days 8 and 14 in the chitosan treatment group. These histologic and biochemical studies suggest that chitosan modulates wound healing by first reducing the influx of activated tissue macrophages, which in turn reduces the subsequent events of angiogenesis, fibroplasia, and connective tissue deposition.

Altered collagen metabolism and delayed healing in a novel model of ischemic wounds

Cellular mechanisms occurring in the healing wound have been well described in various animal models. However, the events associated with wound healing seen in ischemic skin have not been as thoroughly defined. In this series of experiments, we created a novel model of excisional skin wounds under gradient ischemia to study the cellular and extracellular events leading to delayed healing. We hypothesized that altered collagen metabolism accounts for delayed wound healing in ischemic skin. Three pairs of 4 mm punch wounds were made 4 days after bipedicle skin flaps were created on the dorsum of rats. Sham‐operated control animals had the same punch wounds without flap creation. The kinetics of excisional wound healing were measured by means of computerized planimetry. In addition, wounds were excised with a 6 mm trephine, radiolabelled with (3H)‐proline and in vitro collagen synthesis determined as collagenase digestible protein along with quantitation of DNA content. Total collagen deposition was determined as 4‐hydroxy‐L‐proline by high‐performance liquid chromatography, and wounds were histologically evaluated. Data was analyzed by means of two‐way analysis of variance. Although control wounds healed by day 10, flap wounds consistently had greater surface area on days 2, 4, 6, 8, and 12 (p < 0.001). Relative collagen synthesis (% collagen/noncollagen protein), as measured by an in vitro synthesis method, showed no statistically significant differences between flap and controls wounds. However, the total collagen content (deposition) as measured by 4‐hydroxy‐l‐proline was significantly lower in flap wounds compared with controls on days 7 (p < 0.05) and 9 (p < 0.001). In addition, a significant increase occurred in DNA content in the flap wounds on days 7 (p < 0.05) and 9 (p < 0.001) versus control wounds. These data indicate that, in ischemic wounds, significantly less collagen is deposited despite the inherent ability of the tissue to synthesize appropriate levels of collagen. Because the in vitro collagen synthesis technique only assesses the ability of the tissue to synthesize collagen in a well oxygenated environment, one cannot be assured that the tissue expresses this potential in vivo. However, these data are consistent with the hypothesis that the delay in wound closure is due to an alteration in collagen metabolism which results in a net decrease in collagen accumulation. Because of the observed increase in DNA within the ischemic wounds, we suggest that there is prolonged inflammation in these wounds which may enhance collagen degradation through the release of proteases. In addition, there may be an inability of the tissue to maintain appropriate levels of collagen in this inflammatory wound environment.

Use of a cell‐based interactive wound dressing to enhance healing of excisional wounds in nude mice

The need to have viable, metabolically active cells to heal wounds is well recognized, because there is clear evidence that cellular dysfunction delays healing. This suggests that addition of metabolically active cells to a delayed healing tissue could enhance the healing of the tissue. Therefore, we examined the ability of an interactive wound dressing composed of human keratinocytes or fibroblasts grown on microporous bio‐reactor beads and placed into a polyethylene bag to facilitate the delayed healing of wounds in nude mice. A 1 × 1 cm wound was made on the backs of nude mice, and the dressing with or without viable cells was placed on the wound for 8 to 24 days, with dressing changes every other day. Wound area and time to heal measurements were compared after various interventions including freeze‐thawing. The data shows that the interactive wound dressing was more effective than the control dressings (p<0.05) and that keratinocytes were more effective than fibroblasts in wound healing (p<0.05). Freezing‐thawing of the interactive wound dressings destroyed the activity of the dressing. Studies examining cells using a live/dead viability assay showed that both keratinocytes and fibroblasts were alive after 2 days on the mice. Surprisingly, human fibroblasts appeared to exhibit bridging behavior that is indicative of fibroblast proliferation. We conclude that a simple interactive wound dressing using either keratinocytes or fibroblasts can enhance the healing of wounds in nude mice.