The long-term objective of this research is to develop therapies to encourage skin strengthening in individuals at risk of skin breakdown from prolonged mechanical stress (e.g. prosthesis users). The specific aims are to develop, evaluate, and use an in vitro skin model system to investigate collagen structural and bioprocess changes that occur in skin adapting to repetitive mechanical stresses, and to apply that insight to an in vivo model to test a new therapy to encourage skin strengthening. For the in vitro model, a surgically-excised pig skin sample is put in culture at an air-media interface within a custom-designed flow chamber. Clinically relevant stresses, those experienced at the residual limb-prosthetic socket interface by lower-limb amputees, are applied to the explant surface for at least a 2-week period using a custom-designed, closed-loop, force-controlled load applicator. Mechanisms of collagen adaptation are investigated to pinpoint specific biomolecules responsible for promoting the adaptive processes resulting in a mechanically stronger structure. A key question to answer is if collagen fibril diameters are increased by adding to existing fibrils or by degrading old collagen and forming new fibrils. It is hypothesized that after a metalloproteinase concentration peak to degrade small existing collagen fibrils, proteoglycans decorin, biglycan, fibromodulin, lumican, and thrombospondin-2 as well as collagen-related integrin expression are upregulated; chondroitin sulfate and collagen V are downregulated; collagen I and III production and cross-linking increase; and new larger collagen fibrils are formed. As a potential therapy to facilitate skin adaptation, a graded-increase stress-application treatment is tested using an in vivo skin model. If started before the metalloproteinase peak has subsided, the treatment is expected to create an architecturally inferior and weak tissue. However, if started after the metalloproteinase peak during remodeling, the treatment should enhance collagen fibril diameter and skin strength. Similar should be the case if the treatment is initiated during the stabilization phase of adaptation, though fibril enlargement and strength should be even further enhanced since the treatment will likely re-initiate the entire adaptation process. If shown to enhance architecture and strength, the treatment regime would have direct clinical applicability. The health relatedness of this proposal is new knowledge that has potential application to the development of novel treatments for persons at risk of skin breakdown. By understanding the process of skin adaptation (natural skin strengthening) at both a cellular and molecular level, therapies to encourage skin adaptation before breakdown occurs can be intelligently pursued.