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Regular soft tissue healing relies on the well-organized interaction of different stromal cell types with endothelial cells. However, spatiotemporal conditions might provoke high densities of one special stromal cell type, potentially leading to impaired healing. Detailed knowledge of the functions of rivaling stromal cell types aiming for tissue contraction and stabilization as well as vascular support is mandatory. By the application of an in vitro approach comprising the evaluation of cell proliferation, cell morphology, myofibroblastoid differentiation, and cytokine release, we verified a density-dependent modulation of these functions among juvenile and adult fibroblasts, pericytes, and adipose-derived stem cells during their interaction with microvascular endothelial cells in cocultures. Results indicate that juvenile fibroblasts rather support angiogenesis via paracrine regulation at the early stage of healing, a role potentially compromised in adult fibroblasts. In contrast, pericytes showed a more versatile character aiming at angiogenesis, vessel stabilization, and tissue contraction. Such a universal character was even more pronounced among adipose-derived stem cells. The explicit knowledge of the characteristic functions of stromal cell types is a prerequisite for the development of new analytical and therapeutic approaches for impaired soft tissue healing. The present study delivers new considerations concerning the roles of rivaling stromal cell types within a granulation tissue, pointing to extraordinary properties of pericytes and adipose-derived stem cells., (© 2022 The Authors. Cell Biochemistry and Function published by John Wiley & Sons Ltd.)

There is growing evidence suggesting that healing of chronic soft tissue wounds profits from the presence of adipose-derived stem cells (ADSC). Among the large spectrum of mechanisms by which ADSC might act, especially the interaction with the microvascular endothelial cell, a main player during angiogenesis, is of special interest. In the present 2D model on the basis of endothelial cell ADSC co-cultures, we focused on the identification of characteristics of both cell types in response to a typical condition in acute and chronic wounds: hypoxia. Parameters like proliferation capacity, migration, myofibroblastoid differentiation of ADSC and the quantification of important paracrine factors related to angiogenesis and inflammation were used to correlate our experimental model with the in vivo situation of soft tissue healing. ADSC were not negatively affected by hypoxia in terms of proliferation, referring to their excellent hypoxia tolerance. Myofibroblastoid differentiation among ADSC was enhanced by hypoxia in mono- but not in co-culture. Furthermore, co-cultures were able to migrate under hypoxia. These effects might be caused to some extent by the distinct milieu created by interacting ADSC and endothelial cells, which was characterized by modulated levels of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor. The identification of these cell characteristics in the present 2D in vitro model provide new insights into the process of human soft tissue healing, and underpin a beneficial role of ADSC by regulating inflammation and angiogenesis.

The clinical phenomenon of inadequate soft tissue healing still remains an important issue. The occurrence of chronic wounds is correlated to the life span, which is still increasing in western countries. Tissue engineering products containing adipose-derived stem cells are discussed as a promising therapeutic approach. Several studies confirmed the value of these cells for soft tissue healing improvement, suggesting a paracrine as well as a direct effect on vessel repair and angiogenesis. In an attempt to figure out specific effects of adipose-derived stem cells on dermal microvascular endothelial cells with respect to the different phases of soft tissue healing, we designed a 3D in vitro model on the basis of spheroids. Basic parameters like spheroid volume, cell numbers, and rate of apoptotic cells were determined in dependence on culture time, on different oxygen conditions and using mono- as well as co-cultures of both cell types. Furthermore we focused on gene expression and protein levels of interleukin-6, interleukin-8, monocyte chemoattractant protein-1, and vascular endothelial growth factor, which are discussed against the background of therapies for chronic wounds. The visualization of α-smooth muscle actin allowed the estimation of the function of adipose-derived stem cells as stabilizer for dermal microvascular endothelial cells. The results of the present 3D model underscore a paracrine effect of adipose-derived stem cells on microvessel repair during early hypoxic conditions, whereas a stabilizing effect occurs during a later phase of soft tissue healing, simultaneously to reoxygenation.

The demographic change in western countries towards an older population is being shadowed by an increased appearance of chronic diseases influencing soft tissue healing in a negative manner. Although various promising therapeutic approaches are available for treating chronic wounds, no in vitro model exists that successfully allows the analysis of interacting cells and of the effect of therapeutic drugs within a wound. Granulation tissue assures wound stability, neo-angiogenesis and revascularization finally leading to functional soft tissue repair. As one of the first steps in developing a model for human granulation tissue, we examined microvascular endothelial cells and pericytes in conventional 2D and in 3D spheroid co-cultures. We determined which parameters could be used in a standardized manner and whether the cultures were responsive to hypoxia and to erythropoietin supplementation. The read-out parameters of cell migration, cell density, rate of apoptotic cells, spatial cell distribution in the spheroid and spheroid volume were shown to be excellent analytic measures. In addition, quantification of hypoxia-related genes identified a total of 13 genes that were up-regulated in spheroids after hypoxia. As these parameters delivered reliable results in the present approach and as the general morphological distribution of pericytes and endothelial cells within the spheroid occurred in a typical manner, we believe that this basic in vitro model will serve for the future study of diverse aspects of soft tissue healing.