A new in vitro wound model based on the co‐culture of human dermal microvascular endothelial cells and human dermal fibroblasts

Abstract

Background information. Different in vitro models, based on co-culturing techniques, can be used to investigate the behaviour of cell types, which are relevant for human wound and soft-tissue healing. Currently, no model exists to describe the behaviour of fibroblasts and microvascular endothelial cells under wound-specific conditions. In order to develop a suitable in vitro model, we characterized co-cultures comprising NHDFs (normal human dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells). The CCSWMA (co-culture scratch wound migration assay) developed was supported by direct visualization techniques in order to investigate a broad spectrum of cellular parameters, such as migration and proliferation activity, the differentiation of NHDFs into MFs (myofibroblasts) and the expression of endothelin-1 and ED-A-fibronectin (extra domain A fibronectin). The cellular response to hypoxia treatment, as one of the crucial conditions in wound healing, was monitored.Results. The comparison of the HDMEC-NHDF co-culture with the respective mono-cultures revealed that HDMECs showed a lower proliferation activity when co-cultured, but their number was stable throughout a period of 48 h. NHDFs in co-culture were slightly slower at proliferating than in the mono-culture. The MF population was stable for 48 h in the co-culture, as well as in NHDF mono-culture. Co-cultures and HDMEC mono-cultures were characterized by a slower migration rate than NHDF mono-cultures. Hypoxia decreased both cell proliferation and migration in the mono-cultures, as well as in the co-cultures, indicating the general suitability of the assay. Exclusively, in cocultures well-defined cell clusters comprising HDMECs and MFs formed at the edges of the in vitro wounds.Conclusions. On the basis of these results, the CCSWMA developed using co-cultures, including HDMECs, NHDFs and MFs, proved to be an effective tool to directly visualize cellular interaction. Therefore, it will serve in the future to evaluate the influence of wound-healing-related factors in vitro, as shown for hypoxia in the present study.

Modelling the interaction of keratinocytes and fibroblasts during normal and abnormal wound healing processes

“…Figure 6 shows the wound takes much longer to heal than in the normal case, which is in agreement with the conclusions of Oberringer et al [31]. Additionally, as seen in figure 5, our model predicts that collagen is produced at a slower rate than in the normal case.…”

“…In §4, we show that our model captures essential features of the normal healing process, and can be used to describe abnormal wound healing pathologies. It is widely accepted that persistent, chronic hypoxia inhibits healing [30,31] and is one of the most common causes of chronic wounds [32] in which healing fails to produce anatomical and functional integrity [33]. In addition to chronic hypoxia, we consider the case of an excessively long inflammatory response, following Murphy et al [24].…”

Cell-Based Vascularization Strategies for Skin Tissue Engineering

“…Microvascular EC can be isolated from skin samples 24 and from neonatal foreskin. 25 Human dermal microvascular EC (HDMEC) are also commercially available as a standardized EC line that is suitable for preclinical testing, such as in in vitro wound assays, 26 as well as in in vivo blood vessel formation assays. 27 Further refinement of the culture protocol has led to the development of serum-free culture techniques, making these cells safer for potential clinical use in STE.…”

On-chip assay of the effect of topographical microenvironment on cell growth and cell-cell interactions during wound healing

“…4 However, the complicated mechanism of interactions among these cells and each role in cutaneous wound healing have not yet been investigated thoroughly. Recently, on the basis of conventional in vivo (e.g., minipig 5 ) and in vitro (e.g., scratch assay 6 and transwell assay 7 ) models, a great deal of studies has greatly enhanced the understanding of cell interactions during wound healing process. For example, dermal fibroblasts would be activated and differentiated into myofibroblasts when co-cultured with dermal microvascular endothelial cells and then migrated to heal the wound.…”

“…For example, dermal fibroblasts would be activated and differentiated into myofibroblasts when co-cultured with dermal microvascular endothelial cells and then migrated to heal the wound. 6 Contacting with fibroblasts stimulated the migration and proliferation of keratinocytes during wound healing. 7 In return, the expression and synthesis of type I collagen (the predominant form of collagen in fibroblasts in human skin) was regulated by keratinocyte-releasable factors.…”