Amino- and Carboxyfunctionalized Nano- and Microstructured Surfaces for Evaluating Adhesion, Proliferation and Differentiation of Primary Keratinocytes

Background: Tissue Engineering is an interdisciplinary research field with the goal to manufacture in vitro tissues and organs. A crucial factor is the functional cultivation of primary cells. Therefore, scaffolds have to be created, featuring structurally and functionally tailored substrates. To gain basic insight into the impact of non-biological features on cells' behaviour, keratinocytes were cultured on functionalized planar, nano- and microstructured surfaces. Adhesion, proliferation and differentiation of primary keratinocytes have been evaluated. Methods: Glass-slides were coated with nano- or microparticles and sintered at 560 degree C for 60min or 620 degree C for 90min. After sintering, glass-slides were functionalized with Aminopropyl-triethoxysilan or Carboxysilantriol. The surface was analyzed with ellipsometry, SEM, XPS and AFM. Cells were isolated from human foreskin and cultured at 37 degree C and 5%CO sub(2). Adhesion, proliferation and differentiation of primary keratinocytes on various surfaces were evaluated with cell proliferation tests and immuno-histochemical staining of cytoskeleton and differentiation markers. Results: Stable nano- and microstructured surfaces were prepared and amino- and carboxy-functionalized. Primary keratinocytes showed better adhesion and proliferation on amino- than on carboxy-functionalized surfaces. Increasing proliferation from microstructured to planar surfaces with amino-functionalization could be detected by WST-1 tests. Cytokeratin 14 as an early differentiation marker was detected, especially on modified surfaces, but no later markers for differentiation like cytokeratin 10 or filaggrin were found. Outlook: Insight into the cells' preferences concerning structure and chemical functionality of the substrate helps to built up improved cell culture systems, optimized biomaterials and tailored surfaces for implants without the use of cost-intensive biological components.