EUROPEAN TISSUE REPAIR SOCIETY

TISSUE ENGINEERING I

LIVING SKIN EQUIVALENT; FIBROBLASTS

LIVING skin substitutes are becoming more and more important in the treatment of burns and chronic wounds. Fibroblasts play a central role in the regeneration of new skin tissue. Generally, it is assumed that wound healing is improved by the presence of more fibroblasts.^1-3 However, little is known about optimal concentrations, phenotype and whether or not allogeneic cells can be used rather than autologous.

We investigated in experimental wounds whether seeded fibroblasts could actually survive in an artificial dermal matrix made out of freeze-dried collagen/elastin extracellular matrix.⁴ We also examined what their contribution was in the process of wound healing in terms of effects on wound contraction, extracellular matrix formation and remodelling. In addition, we seeded increasing cell numbers in these substitutes and studied the effects on wound healing parameters.⁵ Allogeneic as well as autologous cell sources were used,⁶ and fibroblasts were harvested from different tissues in order to identify readily available cell sources for tissue engineering.⁷

Methods

Experimental full thickness wounds were created on the backs of Yorkshire pigs. The wounds were treated with dermal substitutes seeded with different concentrations of fibroblasts, with cell numbers varying from 1 to 5 x 10 cells cm^-2. The dermal substitutes were either transplanted immediately after seeding or cultured for ten days in vitro prior to transplantation. Allogeneic fibroblasts were harvested from donor pigs from either the same or from a different breeding strain and were seeded at 5 x 10 cells cm^-2. In some cases, fibroblasts were labelled with a fluorescent marker to be able to identify seeded cells in wound biopsies taken at a later time point.

Cultured cells harvested from different tissues were characterised by FACS (Fluorescence-Activated Cell Sorting). Fibroblasts were characterised further by determining the myofibroblast phenotype, contractile properties and morphological differences.

Results

Fluorescent labelling showed that fibroblasts survived the seeding and operating procedures, and actually started to proliferate (3 times higher numbers of labelled cells were detected after 5 days). The presence of seeded fibroblasts (1 x 10 cells cm^-2) significantly reduced the degradation rate of the dermal substitute. At later time points a reduction in granulation tissue and in the number of myofibroblasts, and an increase in mature collagen bundles were noted in the wounds treated with cell-seeded dermal substitutes. The effects were greater with higher numbers of fibroblasts seeded in the dermal matrix (Figure 1).

The use of allogeneic fibroblasts diminished the quality of wound healing: more wound contraction with more myofibroblasts, more inflammatory cells and more immature collagen were detected (Figure 2). Finally, we characterised cells from dermal, adipose and burn eschar tissue. The cells from dermal origin had the best characteristics for skin regeneration: the lowest number of myofibroblasts, the lowest contractile properties and the highest cell proliferation (Figure 3).

Discussion

The incorporation of living fibroblasts into products that aim at improving wound healing (in rate and/or quality) seems to be a logical and inevitable choice. Several beneficial effects have been shown by us as well as by others.^8,9 A more practical source of fibroblasts than autologous dermal tissue would facilitate the production process and feasibility of using a living dermal substitute. However, allogeneic cell sources seem to have unwanted side effects, because inflammation and scarring are induced by allogeneic fibroblasts.

The use of tissues other than dermis as a cell source might provide similar advantages. However, our data show that differences in phenotype exist between fibroblasts from different tissues.^7,10 Dermal fibroblasts showed the best growth characteristics in vitro, with lowest alpha-smooth muscle actin expression, and also possessed a different morphology from fibroblasts derived from subcutaneous fat or other tissue.

The myofibroblast phenotype particularly, which expresses alpha-smooth muscle actin, is associated with scarring and wound contraction. Therefore, we hypothesise that the phenotypic differences between dermal and adipose fibroblasts, and especially the higher prevalence of myofibroblasts in the cell population of the latter, are important in determining the outcome of burn wound healing with respect to scarring. Deep dermal defects, which give rise to enhanced scar formation, are thought to heal through the involvement of fibroblasts that have migrated out of the subcutaneous fat layer. The phenotypic characteristics of these fibroblasts are more likely to result in scar formation. Further research is required to learn how to influence these phenotypes in vitro in order to direct the healing process towards optimal healing.

Figure 1. Effect of seeding dermal substitutes with fibroblasts

1A: Polarized light micrographs showing birefringence of mature collagen. Acellular substitute, six weeks post-wounding; an area with immature collagen was present in the middle of the regenerated dermis. 1B: Polarised light microscopy, substitute seeded with 5 x 10⁵ cells cm^-2 and pre-cultured for 10 days; six weeks post-wounding; the whole dermal area contained mature collagen. 1C: Average percentages of dermal area containing mature collagen, bars indicate SD.

Figure 2. Comparison of dermal substitutes prepared with autologous or allogeneic fibroblasts

2A: Polarised light microscopy showing birefringence of mature collagen. Wounds treated with dermal substitutes seeded with autologous fibroblasts at 5 x 105 cells cm-2, six weeks post wounding. 2B: Polarised light microscopy, dermal substitutes were seeded with allogeneic fibroblasts at 5 x 105 cells cm-2, six weeks post wounding.
2C: Macroscopic appearance of A.
2D: Macroscopic appearance of B.

Figure 3, Phenotypic differences between dermal and subcutaneous fat fibroblasts

A and B. Morphology of cells on tissue culture plastic: A) dermal fibroblasts; B) subcutaneous fat fibroblasts (bar = 200 µm). C and D. Detection of various cell types on day 0 (C) or day 14 (D) in the different cell populations, determined by FACS analysis. (MC = mesenchymal cells, FB = fibroblasts, MF = myofibroblasts, KC = keratinocytes, MM = monocytes/macrophages, GC = granulocytes)

References

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8. Rennekampff HO, Hansbrough JF, Kiessig V, Abiezzi S, Woods V.Jr. Wound closure with human keratinocytes cultured on a polyurethane dressing overlaid on a cultured human dermal replacement. Surgery 1996; 120: 16–22.
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10. Dugina V, Alexandrova A, Chaponnier C, Vasiliev J, Gabbiani G. Rat fibroblasts cultured from various organs exhibit differences in alpha-smooth muscle actin expression, cytoskeletal pattern, and adhesive structure organization. Exp Cell Res 1998; 238: 481–490.

Corresponding author:
Esther Middelkoop
Research Department,
Dutch Burns Foundation,
PO Box 1015,
1940 EA Beverwijk,
the Netherlands