A new in vitro wound model based on the co-culture of human dermal microvascular endothelial cells and human dermal fibroblasts.
BACKGROUND
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 co-cultures 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.
Oberringer
Martin
M
Clinic of Trauma, Hand and Reconstructive Surgery, Saarland University, Kirrberger Strasse, Building 57, 66421 Homburg, Germany. oberringer@uniklinkum-saarland.de
Meins
Claudia
C
Bubel
Monika
M
Pohlemann
Tim
T
D000199:Actins / Q000145:classification / Q000378:metabolism; D002452:Cell Count; D002454:Cell Differentiation; D015687:Cell Hypoxia; D002465:Cell Movement; D049109:Cell Proliferation; D018920:Coculture Techniques; D020405:Dermis / Q000098:blood supply*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D019332:Endothelin-1 / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D005353:Fibronectins / Q000145:classification / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008954:Models, Biological; D010100:Oxygen / Q000494:pharmacology; D018800:Thy-1 Antigens / Q000378:metabolism; D013997:Time Factors; D014945:Wound Healing / Q000502:physiology*
D000199:Actins; D019332:Endothelin-1; D005353:Fibronectins; D018800:Thy-1 Antigens; D010100:Oxygen