During wound healing, angiogenic capillary sprouts invade the fibrin/fibronectin-rich wound clot and within a few days organize into a microvascular network throughout the granulation tissue. As collagen accumulates in the granulation tissue to produce scar, the density of blood vessels diminishes. A dynamic interaction occurs among endothelial cells, angiogenic cytokines, such as FGF, VEGF, TGF-beta, angiopoietin, and mast cell tryptase, and the extracellular matrix (ECM) environment. Specific endothelial cell ECM receptors are critical for these morphogenetic changes in blood vessels during wound repair. In particular, alpha(v)beta3, the integrin receptor for fibrin and fibronectin, appears to be required for wound angiogenesis: alpha(v)beta3 is expressed on the tips of angiogenic capillary sprouts invading the wound clot, and functional inhibitors of alpha(v)beta3 transiently inhibit granulation tissue formation. Recent investigations have shown that the wound ECM can regulate angiogenesis in part by modulating integrin receptor expression. mRNA levels of alpha(v)beta3 in human dermal microvascular endothelial cells either plated on fibronectin or overlaid by fibrin gel were higher than in cells plated on collagen or overlaid by collagen gel. Wound angiogenesis also appears to be regulated by endothelial cell interaction with the specific three-dimensional ECM environment in the wound space. In an in vitro model of human sprout angiogenesis, three-dimensional fibrin gel, simulating early wound clot, but not collagen gel, simulating late granulation tissue, supported capillary sprout formation. Understanding the molecular mechanisms that regulate wound angiogenesis, particularly how ECM modulates ECM receptor and angiogenic factor requirements, may provide new approaches for treating chronic wounds.

Skin, the largest organ in the body, protects against toxins and microorganisms in the environment and serves to prevent dehydration of all non-aquatic animals. Immune surveillance, sensory detection, and self-healing are other critical functions of the skin. Loss of skin integrity because of injury or illness may result acutely in substantial physiologic imbalance and ultimately in significant disability or even death. It is estimated that, in 1992, there were 35.2 million cases of significant skin loss (US data) that required major therapeutic intervention. Of these, approximately 7 million wounds become chronic. Regardless of the specific advanced wound care product, the ideal goal would be to regenerate tissues such that both the structural and functional properties of the wounded tissue are restored to the levels before injury. The advent of tissue-engineered skin replacements revolutionized the therapeutic potential for recalcitrant wounds and for wounds that are not amenable to primary closure. This article will introduce the reader to the field of tissue engineering, briefly review tissue-engineered skin replacement from a historical perspective and then review current state-of-the-art concepts from our vantage point.

The presence of mast cells near capillary sprouting sites suggests an association between mast cells and angiogenesis. However, the role of mast cells in blood vessel development remains to be defined. In an attempt to elucidate this relationship, we investigated the effect of human mast cells

The neutrophil has been implicated as an important mediator of vascular injury, especially after endotoxemia. This study examines neutrophil-mediated injury to human microvascular endothelial cells in vitro. We found that neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP), the complement fragment C5a, or lipopolysaccharide (LPS) (1-1,000 ng/ ml) alone produced minimal endothelial injury over a 4-h assay. In contrast, neutrophils incubated with endothelial cells in the presence of low concentrations of LPS (1-10 ng/ml) could then be stimulated by FMLP or C5a to produce marked endothelial injury. Injury was maximal at concentrations of 100 ng/ml LPS and 1o-7 M FMLP. Pretreatment of neutrophils with LPS resulted in a similar degree of injury, suggesting that LPS effects were largely on the neutrophil. Endothelial cell injury produced by LPS-exposed, FMLP-stimulated neutrophils had a time course similar to that induced by the addition of purified human neutrophil elastase, and different from that induced by hydrogen peroxide (H202). Further, neutrophil-mediated injury was not inhibited by scavengers of a variety of oxygen radical species, and occurred with neutrophils from a patient with chronic granulomatous disease, which produced no H202. In contrast, the specific serine elastase inhibitor methoxy-succinyl-alanyl-alanylprolyl-valyl-chloromethyl ketone inhibited 63% of the neutrophilmediated injury and 64% of the neutrophil elastase-induced injury. However, neutrophil-mediated injury was not inhibited significantly by 50% serum, 50% plasma, or purified a, proteinase inhibitor. These results suggest that, in this system, chemotactic factor-stimulated human neutrophil injury of microvascular endothelial cells is enhanced by small amounts of LPS and may be mediated in large part by the action of neutrophil elastase.