PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/91452 Please be advised that this information was generated on 2021-10-10 and may be subject to change. Novel approaches to regenerative medicine of the skin Construction and comprehensive molecular analysis of collagenous biomaterials in vitro and in vivo Gerwen Lammers Research presented in this thesis was performed at the Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, and funded by the Dutch Program for Tissue Engineering. Cover image: www.istockphoto.com, scaffolding by Joe Gough Lay-out: G. Lammers Printed by: CPI Wöhrmann, Zutphen, The Netherlands ISBN: 978-90-8570-765-3 © 2011 by G. Lammers All rights reserved. No parts of this publication may be reproduced, stored in a retrieval sys- tem, or transported in any form or means, without prior written permission of the holder of the copyright. Novel approaches to regenerative medicine of the skin Construction and comprehensive molecular analysis of collagenous biomaterials in vitro and in vivo Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het college van decanen in het openbaar te verdedigen op woensdag 18 januari 2012 om 13.30 uur precies door Gerwen Lammers geboren op 25 oktober 1980 te Doetinchem Promotor: Prof. dr. R.E. Brock Copromotoren: Dr. T.H. van Kuppevelt Dr. W.F. Daamen Manuscriptcommissie: Prof. dr. J.A. Jansen (voorzitter) Prof. dr. E. Middelkoop, Vrije Universiteit Amsterdam Dr. P.L.J.M. Zeeuwen Table of contents Page Chapter 1 7 General introduction, aims and outline of this thesis Chapter 2 13 An overview of methods for the in vivo evaluation of tissue-engineered skin constructs Tissue Engineering part B – Reviews 2011 Feb;17(1):33-55. Chapter 3 47 A comparison of seven methods to analyse heparin in biomaterials: quantification, location and anticoagulant activity Tissue Engineering Part C - Methods 2011: 17(6):669-676 Chapter 4 63 A molecularly defined array based on native fibrillar collagen for the assessment of skin tissue engineering biomaterials Biomaterials 2009 Oct;30(31):6213-6220. Chapter 5 79 Cloning, large-scale production, and purification of active dimeric rat vascular endothelial growth factor (rrVEGF-164) Protein Expression and Purification 2010 Jan;69(1):76-82. Chapter 6 93 Design and in vivo evaluation of a molecularly-defined acellular skin construct: reduction of early contraction and increase in early blood vessel formation Acta Biomaterialia 2011 Mar;7(3):1063-71. Chapter 7 111 High density gene expression microarrays and gene ontology analysis for identifying processes in implanted tissue engineering constructs Biomaterials 2010 Nov;31(32):8299-312. Chapter 8 137 Construction of a microstructured collagen membrane mimicking the papillary dermis architecture and guiding keratinocyte morphology and gene expression Macromolecular Bioscience Provisionally accepted. Chapter 9 161 Summary and future directions Nederlandse samenvatting en toekomstvisie Chapter 10 169 Curriculum vitae, list of publications & dankwoord Chapter 1 General introduction Aims and outline of this thesis Chapter 1 General introduction The skin is the largest organ of the body and it plays an important role in the regulation of body temperature, the defence against pathogens, and the prevention from dehydration. Morphologically, the skin can be divided in a dermal and an epidermal layer, positioned on top of a subcutaneous fat layer (Fig. 1). Figure 1. Schematic representation of the human skin. Three different layers are visible: The epidermis (top), dermis (middle) and subcutaneous fat layer (bottom part). Blood and lymph vessels are present throughout the dermis and appendages like a hair follicle, sebaceous and sweat gland are indicated. Reprinted from: http://commons.wikimedia.org/wiki/File:HumanSkinDiagram.jpg. The dermis consists of soft connective tissue containing fibroblasts as the major cell type. It contains functional structures like hair follicles, sweat glands, sebaceous glands, blood and lymph vessels. The dermal extracellular matrix consists mainly of type I collagen providing the skin its structural strength, elastic fibres providing elasticity, and long, un- branched polysaccharides called glycosaminoglycans, which provide water-binding capacity and play an important role in the binding and modulation of growth factors and other effector molecules [1]. The top of the dermis has a wave-like pattern consisting of dermal papillae. 8 General introduction, aims and outline of this thesis The epidermis consists of a cell-dense layer of mainly keratinocytes, and these cells produce a basement membrane containing type IV collagen. The basal cells continuously divide and undergo differentiation when moving upwards, thereby outlining the different layers of the epidermis. The top layer, the stratum corneum, consists of dead keratinocytes filled with a highly crosslinked network of structural proteins like keratins that form a physical barrier. When the skin is damaged, its functions and architecture have to be restored as quickly as possible. Evolutionary, the fastest way to close large full-thickness wounds and burns is by contraction, but this often results in scarring [2]. Scar tissue may differ from normal skin by an aberrant colour, increased thickness, irregular surface area and poor functional quality and this can have functional, cosmetic, and psychological consequences [3]. A novel ap- proach in the reduction of contraction and its negative consequences may be found in the use of tissue engineering/regenerative medicine. Tissue engineering has been defined as an interdisciplinary field that applies the prin- ciples of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function [4]. Different approaches can be used, mostly requiring a natural or synthetic scaffold, often in combination with cells. Our approach is to construct molecularly-defined acellular scaffolds based on natural type I collagen [5,6]. By using natural components, the original extracellular matrix can be mimicked as closely as possible. Biological effector molecules can be incorporated to attract cells from the sur- rounding tissue to infiltrate the scaffold and to stimulate regeneration of the original tissue. Highly-purified constituents are used to facilitate the investigation of the effect of each indi- vidual component on the wound healing process. From a commercial point of view, acellular constructs are preferred, since cellular constructs require additional handling and storage conditions. In addition, the use of cells from the patient will require time, knowledge, and equipment for cell isolation, expansion and/or scaffold seeding. Growth factors are potent biological signalling molecules that regulate cellular processes like growth, proliferation, and differentiation. Binding of growth factor to specific receptors on the cell membrane of target cells leads to activation of a signalling pathway and finally re- sults in regulation of gene expression. Growth factors play an important role during devel- opment, tissue homeostasis and repair. Therefore, specific growth factors can be incorpo- rated in tissue-engineered constructs to guide the cellular regeneration process. Members of the fibroblast growth factor (FGF) family are involved in angiogenesis, wound healing and embryonic development [7], and members of the vascular endothelial growth factor (VEGF) family are involved more specifically in the process of angiogenesis. Like most FGFs, FGF2 (also known as basic FGF) has an effect on a large variety of different cell types and can bind to isoforms of all four FGF tyrosine kinase receptor types (FGFR1-4) [8]. FGF7 (also known as keratinocyte growth factor, KGF) is a special member of the FGF family and acts only on epithelial cells and specifically binds FGFR2b. Members of the VEGF family (VEGF A-VEGFE, and placental growth factor (PIGF)) also signal through tyrosine kinase receptors 9 Chapter 1 [9]. VEGFA, the best studied VEGF, exists in five isoforms which all bind to VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) homo- and heterodimers, and the isoform that we used (VEGF- A165) also binds receptors neurophilin 1 and 2. Receptor signalling of most FGFs and most VEGFA isoforms is dependent on simultaneous binding to glycosaminoglycans like heparin and heparan sulfate. We have previously demonstrated that incorporation of heparin, FGF2 and VEGF stimulates blood vessel formation and maturation in an acellular skin construct in vivo [10], and since FGF7 can stimulate keratinocytes this can be incorporated in the epi- dermal part of a construct to guide re-epithelialisation. A wide range of tissue-engineered skin constructs has been reviewed by others [11-17]. However, despite the multitude of constructs that have already been developed, perfect skin regeneration without complications is for large wounds still science fiction. Aims and outline of this thesis This thesis focuses on the development and evaluation of tissue-engineered collagen- based scaffolds for skin regeneration. Chapter 2 starts with an overview of methods