Dermal fibroblasts are a dynamic and diverse population of cells whose functions in skin in many respects remain unknown. Normal adult human skin contains at least three distinct subpopulations of fibroblasts, which occupy unique niches in the dermis. Fibroblasts from each of these niches exhibit distinctive differences when cultured separately. Specific differences in fibroblast physiology are evident in papillary dermal fibroblasts, which reside in the superficial dermis, and reticular fibroblasts, which reside in the deep dermis. Both of these subpopulations of fibroblasts differ from the fibroblasts that are associated with hair follicles. Fibroblasts engage in fibroblast-epidermal interactions during hair development and in interfollicular regions of skin. They also play an important role in cutaneous wound repair and an ever-increasing role in bioengineering of skin. Bioengineered skin currently performs important roles in providing (1) a basic understanding of skin biology, (2) a vehicle for testing topically applied products and (3) a resource for skin replacement.

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The effective delivery of bioactive molecules to wound sites hasten repair. Cellular therapies provide a means for the targeted delivery of a complex, multiple arrays of bioactive factors to wound sites. Thus, the identification of ideal therapeutic populations is an essential aspect of this approach. In vitro assays can provide an important first step toward this goal by selecting populations that are likely suitable for more expensive and time-consuming in vivo assays. In this study, bone marrow-derived mesenchymal stem cells (BM-MSCs) were integrated into a three-dimensional coculture system that supports the development and stabilization of vascular tube-like structures. The presence of a limited number of BM-MSCs resulted in their coalignment with vascular structures, and it further resulted in increased tubule numbers and complexity. Thus, these studies suggest that BM-MSCs functionally interacted with and were attracted to in vitro formed vascular structures. Further, these cells also provided sufficient bioactive factors and matrix molecules to support the formation of tubular arrays and the stabilization of these arrays. This in vitro system provides a means for assessing the function of BM-MSCs in aspects of the angiogenic component of wound repair.

The interfollicular dermis of adult human skin is partitioned into histologically and physiologically distinct papillary and reticular zones. Each of these zones contains a unique population of fibroblasts that differ in respect to their proliferation kinetics, rates at which they contract type I collagen gels, and in their relative production of decorin and versican. Here, site-matched papillary and reticular dermal fibroblasts couples were compared to determine whether each population interacted with keratinocytes in an equivalent or different manner. Papillary and reticular fibroblasts grown in monolayer culture differed significantly from each other in their release of keratinocyte growth factor (KGF) and granulocyte-macrophage colony stimulating factor (GM-CSF) into culture medium. Some matched fibroblast couples also differed in their constitutive release of interleukin-6 (IL-6). Papillary fibroblasts produced a higher ratio of GM-CSF to KGF than did corresponding reticular fibroblasts. Interactions between site-matched papillary and reticular couples were also assayed in a three-dimensional culture system where fibroblasts and keratinocytes were randomly mixed, incorporated into type I collagen gels, and allowed to sort. Keratinocytes formed distinctive cellular masses in which the keratinocytes were organized such that the exterior most layer of cells exhibited characteristics of basal keratinocytes and the interior most cells exhibited characteristics of terminally differentiated keratinocytes. In the presence of papillary dermal fibroblasts, keratinocyte masses were highly symmetrical and cells expressed all levels of differentiation markers. In contrast, keratinocyte masses that formed in the presence of reticular fibroblasts tended to have irregular shapes, and terminal differentiation was suppressed. Furthermore, basement membrane formation was retarded in the presence of reticular cells. These studies indicate that site-matched papillary and reticular dermal fibroblasts qualitatively differ in their support of epidermal cells, with papillary cells interacting more effectively than corresponding reticular cells.

Mesenchymal stem cells are a heterogeneous population of fibroblast-like cells found in most adult organs. However, most of our current knowledge is based on cells of bone marrow or interstitial adipose tissues. These cells are capable of differentiation along various mesenchymal lineages. In addition, they have demonstrated therapeutic characteristics in wounds and ischemic situations. The therapeutic characteristics of these cells are activated upon their entering wounds or other damaged tissues. A current problem is the development of strategies that ensure that these cells reach wound beds in a timely fashion and in sufficient numbers to maximize their therapeutic benefits. Currently, there are two basic delivery methods: systemic infusion of cells into the vascular circulation and direct application of therapeutic cells to wound sites. Skin wounds are optimal candidates for the topical delivery approach. However, the methods by which therapeutic cells are delivered to such wounds vary. This review outlines the basic methods used to deliver therapeutic cells to skin and other wounds. Upon entering wounds, therapeutic cells interact with other wound cells through paracrine mechanisms that are not yet well understood. Nonetheless, interactions with vascular endothelial cells and immunomodulation appear to play significant roles in accelerating wound healing and in reducing scar formation upon the completion of the healing process. Although the phenomenological body of evidence indicating the efficacy of therapeutic cells is substantial, considerable work is still required to better determine the molecular and cellular functions of these cells and to assess their fate and the long-term consequences of their application.

Dramatic changes occur in skin as a function of age, including changes in morphology, physiology, and mechanical properties. Changes in extracellular matrix molecules also occur, and these changes likely contribute to the overall age-related changes in the physical properties of skin. The major proteoglycans detected in extracts of human skin are decorin and versican. In addition, adult human skin contains a truncated form of decorin, whereas fetal skin contains virtually undetectable levels of this truncated decorin. Analysis of this molecule, herein referred to as decorunt, indicates that it is a catabolic fragment of decorin rather than a splice variant. With antibody probes to the core protein, decorunt is found to lack the carboxyl-terminal portion of decorin. Further analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry shows that the carboxyl terminus of decorunt is at Phe 170 of decorin. This result indicates that decorunt represents the amino-terminal 43% of the mature decorin molecule. Such a structure is inconsistent with alternative splicing of decorin and suggests that decorunt is a catabolic fragment of decorin. A neoepitope antiserum, anti-VRKVTF, was generated against the carboxyl terminus of decorunt. This antiserum does not recognize intact decorin in any skin proteoglycan sample tested on immunoblots but recognizes every sample of decorunt tested. The results with anti-VRKVTF confirm the identification of the carboxyl terminus of decorunt. Analysis of collagen binding by surface plasmon resonance indicates that the affinity of decorunt for type I collagen is 100-fold less than that of decorin. This observation correlates with the structural analysis of decorunt, in that it lacks regions of decorin previously shown to be important for interaction with type I collagen. The detection of a catabolic fragment of decorin suggests the existence of a specific catabolic pathway for this proteoglycan. Because of the capacity of decorin to influence collagen fibrillogenesis, catabolism of decorin may have important functional implications with respect to the dermal collagen network.The mechanical properties of the dermis are determined primarily by the extracellular matrix. These mechanical properties change dramatically as a function of age (1, 2), perhaps as a direct result of the known age-related changes in the molecules of the dermal extracellular matrix. Age-related differences have been shown for fibrillar collagens (3-7), which are the major extracellular matrix components of the dermis (8). In addition to collagen, the dermal extracellular matrix also contains proteoglycans, which show age-related differences (9 -16). Perhaps related to these changes in proteoglycans are age-related increases in the water content of the dermis (11) and in the content of mobile water (17).Although dermal proteoglycans are present in much lower abundance than collagen, evidence indicates that these molecules are important in the physiology of skin. For example, the small proteogl...

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The construction of vascularized connective tissues is an important goal in tissue engineering in that the presence of a patent bio-engineered vasculature should facilitate vascularization of an implant. Fibroblasts play an essential role in the angiogenic process through their production of extracellular matrix molecules and through their release of essential growth factors. Therefore, the aim of this study is to develop a thin 3-dimensional model in which fibroblasts support endothelial cells in the formation of tube-like structures. Macro- and microvascular endothelial cells were seeded onto confluent lawns of human fibroblasts and were cultured in the presence of high levels of ascorbate 2-phosphate to create a tissue-like structure in which endothelial cell organized into tube-like structures. The process was visualized in the culture dish through labeling of cells with a long-lasting fluorescent vital dye. Intact sheet-like structures were created in which endothelial cell tube-like structures were encased by fibroblasts and were surrounded by a basement membrane. These structures appeared to contain a lumen and remained stable for up to 5 weeks in culture. This culture system provides an in vitro method to study fibroblast-endothelial cell interactions and to study the effects of pro- and anti-angiogenic factors on endothelial cell differentiation. This system also provides an experimental basis for developing vascularized tissue-engineered connective tissue.