Production of a Novel Fibroblast-Populated Platelet Matrix Cocultured with Keratinocytes

Abstract

We have developed a new method for the production of a dermal matrix equivalent. Human platelets were used to dilute human fibroblasts. The platelet mix was placed in a cell culture well. Addition of 200 microL of a thrombin solution caused gel formation. Gels were overlaid with standard Iscove's growth medium supplemented with 10% fetal bovine serum, insulin, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer. Medium was exchanged regularly. Keratinocytes were plated on top of selected gels and elevated to the air-liquid interface. The gels were harvested weekly, fixed, cut, and stained with hematoxylin and eosin stains and immunostains for collagens I, III, and IV and cytokeratins. Digital image analysis was used to quantitate collagen production. Growth factors, including transforming growth factor-beta (TGF-beta), platelet-derived growth factor, and vitamin C were added. Staining identified fibroblasts within the gels with a surrounding fibrous matrix. Immunostaining for cytokeratin identified keratinocytes on the gel surface. Immunostaining revealed the fibrous matrix to be composed of collagen I and III and some collagen IV. Digital image analysis demonstrated that greater TGF-beta concentration resulted in greater collagen production. These differences were statistically significant. With development of this construct, a viable dermal/epidermal replacement may be possible. TGF-beta enhances collagen production by fibroblasts in this matrix.