Biomaterials 28 (2007) 650–660 A Nd:YAG laser-microperforated poly(3-hydroxybutyrate-co-3- hydroxyvalerate)-basal membrane matrix composite film as substrate for keratinocytes Fernando Serrano a,Ã , Laura Lo´pez-G a , Maria Jadraque b , Marie¨lle Koper a , Gary Ellis c , Pilar Cano c , Margarita Martı´n b,1 , Leoncio Garrido c,1 a Fundacio´n Hospital de Alcorco´n, Avda Villaviciosa 1, Alcorco´n E-28922, Spain b Instituto de Quı´mica-Fı´sica Rocasolano, CSIC, Serrano 119, E-28006 Madrid, Spain c Instituto de Ciencia y Tecnologı´a de Polı´meros, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain Received 7 June 2006; accepted 14 September 2006 Available online 6 October 2006 Abstract Epithelia cultured for the treatment of ulcers, burns and for gene therapy applications require a flexible biomaterial for growth and transplantation that is adaptable to body contours. We tested several materials and found that a poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBHV) polyester provided support for keratinocytes, although adhesion to this material proved to be suboptimal. Since epithelia adhere to the mesoderm through basal membranes, we engineered a basal membrane surrogate by preparing composites of PHBHV with basal membrane matrix (BMM). To allow cell migration into injuried areas the polyester film was micromachined to insert high-density micropores through a Nd:YAG laser ablation process. These flexible composites provided firm attachment for keratinocytes from the outer root sheath of human hair allowing keratinocyte migration through micropores. Films of microperforated PHBHV-BMM may be of use for the replacement of diseased or injured skin epithelia. r 2006 Elsevier Ltd. All rights reserved. Keywords: PHBHV; Basal membrane matrix; Epithelium; Microperforated biomaterial 1. Introduction There is an increased demand for epithelium substitutes for the treatment of severe burns, congenital and acquired epidermal disorders (epidermolysis bullosa, pemphigoid), and as vehicles for gene therapy applications [1,2]. The integrity of an artificial epithelium relies on the existence of stem cells to maintain its ability to generate differentiated progeny [3,4]. Stem cells reside in a microenvironment termed the stem cell niche, where they receive intrinsic and extrinsic signals from neighboring cells, the extracellular matrix, basal membrane (BM) and growth factors. Whereas epidermal cells can be cultured and expanded to some degree [5], transplant of pure epidermal sheets is sometimes unsatisfactory [1,6]. Engraftment is poor when dermal integrity has been compromised (second-degree burns). This led to engineering of epidermal/dermal equivalents on which the cultured epidermis is supported by an underlying fibroblast layer that simulates the dermis, which is then able to graft [1,2,7–9]. In vivo, epithelial stem cells do not interact directly with the underlying connective tissue, but rather through a BM [10]. The principal BM components are collagen IV and laminins arranged to form a matrix (BMM) [11,12]. Laminins appear to maintain the undifferentiated status of the basal epithelial layer through interaction with integrins a3b1 and a6b4 [4]. The major epidermal laminin is laminin 5, which is also associated with other epithelial tissue types such as cornea, mammary gland, oral mucosa and pancreatic b cells [10,13–15]. When cultured keratino- cytes are placed in suspension, they withdraw from the cell ARTICLE IN PRESS www.elsevier.com/locate/biomaterials 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.09.018 Abbreviations: H-E, Hematoxilin-eosin; Ph/C, Phase contrast. Ã Corresponding author. Tel.: +34 91 5619845; fax: +34 91 5619845. E-mail address: fserrano@fhalcorcon.es (F. Serrano). 1 Contributed equally to this work.