Development of a cultured dermal substitute composed of a spongy matrix of hyaluronic acid and atelo-collagen combined with fibroblasts: fundamental evaluation

We have developed an allogeneic cultured dermal substitute (CDS) by cultivating fibroblasts on a 2-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Cot). The HA sponge was designed to have a honeycomb structure with many holes (0.5 mm diameter) separated by a distance of 4 mm. Part of the Cot sponge was able to penetrate into these holes, and the resulting anchoring structure allows binding of a HA spongy layer with a Cot spongy layer. The preparation of the CDS consists of two steps: (i) attachment of cells to the Cot surface of the hydrated 2-layered spongy matrix and (ii) proliferation of cells on this sponge immersed in culture medium. The aim of the present study was to assess properties of fresh and cryopreserved CDS. Fibroblasts seeded on the Cot surface of the 2-layered spongy matrix attached, proliferated and released vascular endothelial growth factor (VEGF) and fibronectin. The amount of VEGF released from cryopreserved CDS after thawing slowly in an incubator at 37degreesC and re-cultivation for 1 week was about 300 pg/ml. After thawing quickly in a water bath at 37degreesC and re-cultivation for I week, the amount of VEGF released was about 600 pg/ml. These findings indicate that the cryopreserved CDS maintained its ability to release a significant amount of VEGF. Retention of the therapeutic properties of CDS after cryopreservation is important for clinical use.