Liancun Wu

Acute and chronic animal models for excessive dermal scarring : Quantitative studies

&NA; Excessive scarring in the form of keloids and hypertrophic scars continues to be a clinical problem for some patients. The lack of an animal model for such scarring has been an obstacle to studying the cellular and molecular biology of these entities. Previous observations made by the authors that some surgical scars in the rabbit ear remain raised for months after wounding prompted us to investigate whether the rabbit ear might provide a model by which to study excessive dermal scarring. After establishing the model in preliminary study, 40 excisional wounds, 6 mm in diameter, were created over the ventral surface of rabbit ears. Elevated scars were treated with either intralesional triamcinolone acetonide or saline at day 16 postwounding. On day 22, 25 scar wounds were used for thorough histomorphometric analysis, 15 wounds were eliminated prior to analysis because of invagination of epithelial tissue, which made analysis difficult. Total area of scar and Hypertrophic Index, a ratio comparing scar prominence with the thickness of adjacent unwounded tissue, were measured for 25 (62 percent) of the resulting scars. Both total area of scar and Hypertrophic Index were found to be significantly decreased in the steroid‐treated group (p < 0.02 and < 0.03, respectively). In a chronic form of this model, in which larger excisions were taken, an excessive accumulation of both new collagen and cartilage over 9 months was observed. An animal model for excessive dermal scarring that allows quantitation of scar formation and, at an early stage, can be modulated in a predictable way with intralesional corticosteroid treatment is presented. This model may parallel hypertrophic scarring in humans and thus might provide a tool by which to study its pathophysiology and objectively evaluate therapeutic modalities. (Plast. Reconstr. Surg. 100: 674, 1997.)

Transforming Growth Factor-β1 Fails to Stimulate Wound Healing and Impairs Its Signal Transduction in an Aged Ischemic Ulcer Model : Importance of Oxygen and Age

Clinical trials of exogenous growth factors in treating chronic wounds have been less successful than expected. One possible explanation is that most studies used animal models of acute wounds in young animals, whereas most chronic wounds occur in elderly patients with tissue ischemia. We described an animal model of age- and ischemia-impaired wound healing and analyzed the wound-healing response as well as the transforming growth factor (TGF)-beta1 effect in this model. Rabbits of increasing ages were made ischemic in the ear where dermal ulcers were created. Histological analysis showed that epithelium ingrowth and granulation tissue deposition were significantly impaired with increased age under ischemia. TGF-beta1 stimulated wound repair under both ischemic and non-ischemic conditions in young animals, although it showed no statistical difference in aged animals. Procollagen mRNA expression decreased under ischemic conditions and with aging. Neither TGF-beta1 nor procollagen alpha1(I) mRNA expression increased in response to TGF-beta1 treatment under ischemia in aged animals. Therefore, the wound-healing process is impaired additively by aging and ischemia. The lack of a wound-healing response to TGF-beta1 in aged ischemic wounds may play a role in the chronic wounds.

Differential effects of oxygen on human dermal fibroblasts: acute versus chronic hypoxia†

The observation that many chronic wounds are ischemic has spurred a series of studies evaluating the response of cells exposed to hypoxia. To date, these studies have shown largely beneficial effects from hypoxia, such as increased cellular replication and procollagen synthesis. These findings are counter‐intuitive from a clinical standpoint because cellular growth and synthetic function are known to be retarded in chronic ischemic wounds. We have established an in vitro system in which human dermal fibroblasts grown chronically at 5 ± 3 mm Hg will proliferate at a rate three times slower than those fibroblasts grown under standard culture conditions (namely an oxygen partial pressure of 150 mm Hg). No phenotypic changes are noted in chronically hypoxic cells, and the growth‐retarding effects are reversible when the cells are returned to standard oxygen conditions. Competitive reverse transcription‐polymerase chain reaction showed that acute exposure to hypoxia (up to 1 week) results in a 6.3‐fold increase in the relative expression of transforming growth factor‐β1 messenger RNA, whereas chronic exposure to hypoxia leads to a 3.1‐fold decrease in this message. Collagen production measured at both the mRNA and protein level is also decreased in the setting of chronic hypoxia. We propose that this system may be the most appropriate setting for studying the role of oxygen on dermal fibroblasts in ischemic, nonhealing wounds.

Effect of ischemia on growth factor enhancement of incisional wound healing

BACKGROUND Several growth factors have been shown to enhance incisional wound healing in different models, but the models have been difficult to compare, and the effects under ischemic conditions are unknown. METHODS An ischemic model was developed by selective interruption of the rabbit ear circulation and placement of dorsal incisions. The model was defined during a 28-day period by use of serial blood gas analysis, breaking strength measurement, and histologic analysis. In a separate experiment, incisions were treated with topical growth factors with the contralateral ear serving as paired controls, and the wounds were evaluated similarly. RESULTS The ischemic ears were more hypoxic than controls through day 14 after wounding (48.5 versus 41 mm Hg, p < 0.03), and healing was impaired through day 28 (3.21 versus 1.90 newtons, p < or = 0.001). Transforming growth factor-beta (1 microgram) and Kaposis fibroblast growth factor (20 micrograms) increased breaking strengths under both normal (3.03 versus 2.41 and 2.83 versus 2.47 newtons, respectively; p < 0.05) and ischemic conditions (1.40 versus 1.11 and 1.56 versus 1.23 newtons, respectively; p < 0.05). Platelet-derived growth factor-BB (10 micrograms) was effective only under normal conditions (2.67 versus 2.15 newtons, p < 0.02), whereas basic fibroblast growth factor (20 micrograms) was ineffective under both conditions. CONCLUSIONS The rabbit ear incisional model is a reproducible ischemic incisional model allowing comparison of growth factor effects under ischemic and nonischemic conditions. Transforming growth factor-beta and Kaposis fibroblast growth factor are both effective under ischemic conditions, whereas basic fibroblast growth factor and platelet-derived growth factor-BB are not, suggesting that ischemia is an important parameter affecting growth factor actions.

Effects of Oxygen on Wound Responses to Growth Factors: Kaposi's FGF, but not Basic FGF Stimulates Repair in Ischemic Wounds

Kaposis fibroblast growth factor (K-FGF, FGF-4) is a newer member of FGF family with uncharacterized wound healing properties. Basic fibroblast growth factor (bFGF, FGF-2) has been well studied and accelerates repair in normal and impaired wound healing models. K-FGF and bFGF are known to have similar biological effects in tissue culture, and both stimulate fibroblast and endothelial cell proliferation. The rabbit dermal ulcer model was used to examine the effects of bFGF and K-FGF under ischemic and nonischemic conditions. We found bFGF was ineffective in stimulating healing under ischemic conditions even at high doses (30 micrograms/wound). However, when the ischemic wounds were treated with bFGF (5 micrograms/wound) plus hyperbaric oxygen therapy, it was highly effective again as previously found under nonischemic conditions (P < 0.05). In contrast K-FGF stimulated repair in both nonischemic and ischemic wounds (P < 0.05). These results suggest that wound oxygen content differentially regulates responsiveness to bFGF and that K-FGF is biologically active in hypoxic wounds.

differential Effects of Platelet-derived Growth Factor Bb in Accelerating Wound Healing in Aged versus Young Animals: The Impact of Tissue Hypoxia

&NA; Platelet‐derived growth factor BB (PDGF‐BB) plays a central role in wound healing. Platelet‐derived growth factor has been shown to accelerate healing in preclinical and clinical studies, but its wound‐healing effects in older animals have not been examined. An ischemic incisional model in young female (5 months) and aged male (60 months) rabbits was used to determine the influence of platelet‐derived growth factor on day 14 postwounding with reduced blood supply. Wounds in aged ischemic animals were severely impaired in breaking strength compared with their nonischemic control wounds and wounds in young animals (p < 0.001). Topical platelet‐derived growth factor (10 &mgr;g per wound) partially reversed the reduction in breaking strength in aged ischemic animals but was ineffective in young animals. Histologic studies showed that new granulation tissue deposition and mononuclear cell infiltration was dramatically lower in ischemic wounds of aged animals compared with their control wounds as well as wounds from young animals under both nonischemic and ischemic conditions. Platelet‐derived growth factor partially reversed this deficit in old ischemic wounds but not in young ischemic wounds. Epithelial growth was reduced in wounds from aged animals compared with wounds from young animals after 14 days of healing. Platelet‐derived growth factor treatment increased ischemic wound epithelial growth in aged ischemic animals about threefold compared with paired controls. In conclusion, wound healing in aged rabbits was severely impaired by ischemia, and a single topical platelet‐derived growth factor treatment partially reversed this deficit. (Plast. Reconstr. Surg. 99: 815, 1997.)

Platelet-derived growth factor-BB accelerates wound closure in a new mesentery culture model without macrophages.

Platelet-derived growth factor-BB has multiple effects in vitro and has been demonstrated to accelerate wound healing in several animal models, but its precise mechanisms of action in vivo have not been fully explained. A rat mesentery culture model was developed, and platelet-derived growth factor-BB (100 ng/ml) was added to the medium containing 2% fetal calf serum for examining its closure effects. The mesentery specimens were stained by routine hematoxylin and eosin and immunohistochemical staining for macrophages. A significant improvement in closure rate and complete closure rate was seen when plateletderived growth factor-BB was added to the medium compared with the controls (p < 0.05). There are significantly fewer macrophages in the in vitro specimen than in the in vivo specimen. In conclusion, platelet-derived growth factor-BB can contribute to wound closure of a mesenteric defect even without macrophages, suggesting a second mechanism in which it can act directly on mesenchymal cells during wound healing. This tissue-culture model is useful for gaining further insights into growth factor interactions and wound healing.