Engineered Lysozyme Amyloid Fibril Networks Support Cellular Growth and Spreading Nicholas P. Reynolds,* ,† Mirren Charnley, ‡ Raffaele Mezzenga, § and Patrick G. Hartley † † CSIRO, Materials Science and Engineering, Private Bag 10, Bayview Avenue, Clayton, Victoria 3169, Australia ‡ Centre for Micro-Photonics and Industrial Research Institute Swinburne, Swinburne University of Technology, Victoria 3122, Australia § ETH, Food & Soft Materials, Department of Health Science and Technology, Schmelzbergstrasse 9, 8092, Zurich, Switzerland * S Supporting Information ABSTRACT: Fibrous networks assembled from synthetic peptides are promising candidates for biomimetic cell culture platforms and implantable biomaterials. The ability of the materials to reproduce physiological cell-matrix interactions is essential. However, the synthetic complexity of such systems limits their applications, thus alternative materials are desirable. Here, we design lysozyme derived amyloid fibril networks with controllable topographies, and perform a comprehensive study of the response of cultured fibroblast and epithelial cells. At high surface coverage a favorable increase in spreading and the generation of focal adhesions was observed, due to a combination of biomimetic chemistry and morphology. Their ease of synthesis, makes the nanoscale fibrils presented here ideal materials for future clinical applications whereby large volumes of biomimetic biomaterials are required. Furthermore, the surface chemistry of the fibrils is sufficient for the promotion of focal adhesions with cultured cells, eliminating the need for complex protocols for fibril decoration with bioactive moieties. ■ INTRODUCTION A major challenge in the field of tissue engineering and regenerative medicine is the development of an artificial extracellular matrix (ECM) mimicking material. If such a material is to have clinical applications (e.g., soft tissue regeneration, or expansion of stem cells), it would need to be able to accurately mimic cell:matrix interactions and be available in large volumes at a reasonable cost. 1 These biomaterials should possess a nanoscale topographic morphol- ogy reminiscent of the ECM. Moreover the materials should have good cell adhesive properties. Self-assembling peptidic systems offer great potential in the design of nanoscale fibrous biomaterials with both in vitro 2-4 and in vivo 5,6 applications. These systems are attractive as they can self-assemble into nanoscale fibrillar morphologies that mimic the morphology of the fibrous proteins that make up the ECM. 3,7 Self-assembled systems are often preferred over animal derived matrices or coatings such as Matrigel 8 or collagen 9 due to the highly defined morphology, chemical composition, and purity. There are many examples of self-assembling synthetic peptides 2,6,10-14 and one common motif shared by all these systems is the presence of sequences which form β-sheet containing supra- molecular structures encouraging the formation of high aspect ratio nanoscale fibrils. Self-assembling peptides are attractive materials as they can be made in high purity and can be designed to display specific functional moieties on the fibril surface. 7 However, the design and synthesis of self-assembling short peptide sequences is nontrivial and requires considerable synthetic expertise. 15 In nature, nanoscale self-assembled fibrillar structures containing rigid β-sheets motifs (that drive self-assembly) are commonly found in the form of amyloid fibrils. 16 However, their connection to neurodegenerative diseases including Parkinson’s 17,18 and Alzheimer’s 19 has meant they were previously considered unsuitable as ECM mimics. More recent research has provided convincing evidence that the mature fibril is often a nontoxic byproduct of disease. 18,20-22 There have also been examples whereby amyloids have been found to possess beneficial functions. 23,24 Thus, there has been some inves- tigations into the suitability of using amyloids as self-assembling biomaterials 3,4 and biomimetic hybrids. 25 Amyloids can be assembled from short synthetic peptides 4,26 or from full proteins. 3,27 Full protein systems have some advantages over synthetic peptides; first the starting materials are readily available and inexpensive; 27 second as many of the proteins used are contained in foodstuffs(β-lactoglobin 28 from milk and lysozyme 27 from hen eggs), they are generally considered to be nontoxic. Full protein systems, however, offer no control over Received: November 7, 2013 Revised: January 15, 2014 Article pubs.acs.org/Biomac © XXXX American Chemical Society A dx.doi.org/10.1021/bm401646x | Biomacromolecules XXXX, XXX, XXX-XXX