The interplay between biomaterial degradation and tissue properties : relevance for in situ cardiovascular tissue engineering Citation for published version (APA): Brugmans, M. C. P. (2015). The interplay between biomaterial degradation and tissue properties : relevance for in situ cardiovascular tissue engineering. Technische Universiteit Eindhoven. Document status and date: Published: 01/01/2015 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. 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Printed by Ipskamp Drukkers B.V., Enschede, the Netherlands. The research and printing of this thesis was supported by: Financial support by the Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged. This work was supported by a grant from the Dutch government to the Netherlands Institute for Regenerative Medicine (NIRM, grant No. FES0908). The interplay between biomaterial degradation and tissue properties Relevance for in situ cardiovascular tissue engineering PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Technische Universiteit Eindhoven, op gezag van de rector magnificus, prof.dr.ir. F.P.T. Baaijens, voor een commissie aangewezen door het College voor Promoties in het openbaar te verdedigen op woensdag 10 juni 2015 om 16.00 uur door Maria Cornelia Philomena Brugmans geboren te Veghel Dit proefschrift is goedgekeurd door de promotoren en de samenstelling van de promotiecommissie is als volgt: Voorzitter: prof. dr. P.A.J. Hilbers 1e promotor: prof.dr.ir. F.P.T. Baaijens 2e promotor: prof.dr. C.V.C. Bouten copromotor: dr. A. Driessen-Mol leden: dr. J. Kluin (UvA) dr. P. Habibovic (UM) dr.rer.nat. C. Ottmann adviseur: dr. P.Y.W. Dankers Contents Summary III Chapter 1: General introduction 1 1.1. Human cardiovascular tissues 2 1.1.1 Heart valves 2 1.1.2 The heart valve leaflets 3 1.1.3 Blood vessels 4 1.2 Cardiovascular diseases and current treatments 5 1.3 Cardiovascular tissue engineering approaches and challenges 7 1.4 Biomaterials 10 1.4.1 Natural biomaterials 11 1.4.2 Synthetic biomaterials 11 1.5 In vivo resorption of biomaterials 12 1.5.1 Resorption pathways 12 1.5.2 Variation in resorption of biomaterials 13 1.6 The host response to biomaterials 14 1.6.1 The phases of the natural healing response 14 1.6.2 Macrophage phenotypes 15 1.7 Rationale and outline 16 Chapter 2: Polycaprolactone scaffold and reduced in vitro cell culture: Beneficial effect on compaction and improved valvular tissue formation 19 2.1 Abstract 20 2.2 Introduction 21 2.3 Materials and Methods 23 2.4 Results 26 2.5 Discussion 33 2.6 Conclusion 38 Chapter 3: Superior tissue evolution in slow-degrading scaffolds for valvular tissue engineering 39 3.1 Abstract 40 3.2 Introduction 41 3.3 Materials and Methods 42 3.4 Results 44 3.5 Discussion 50 3.6 Conclusion 53 I Chapter 4: Hydrolytic and oxidative degradation of electrospun supramolecular biomaterials: In vitro degradation pathways 55 4.1 Abstract 56 4.2 Introduction 57 4.3 Materials and Methods 59 4.4 Results 62 4.5 Discussion 68 4.6 Conclusion 72 Chapter 5: Advanced electrospun scaffold degradation by inflammatory macrophages in comparison with healing macrophages 73 5.1 Abstract 74 5.2 Introduction 75 5.3 Materials and Methods 76 5.4 Results 81 5.5 Discussion 87 5.6 Conclusion 88 Chapter 6: General discussion 91 6.1 Main findings of the thesis 92 6.2 Towards the most promising tissue engineering approach and scaffold material 96 6.3 Study limitations and the future of in-situ cardiovascular tissue engineering 101 6.4 Conclusion 104 References 107 Nederlandse samenvatting 127 Dankwoord 129 Curriculum vitae 131 List of publications 133 II Summary The interplay between biomaterial degradation and tissue properties: Relevance for in situ cardiovascular tissue engineering Various tissue engineering (TE) approaches are currently under investigation to create cardiovascular tissue replacements. The most promising strategy is the in-situ TE approach, in which off-the-shelf available synthetic electrospun scaffolds are used to replace diseased vessels or heart valves. After implantation, a host inflammatory response is activated, leading to the infiltration of macrophages, which play a key role in both scaffold degradation and tissue formation. As a result, a living tissue that is able to remodel and adapt to the environmental changes is obtained in-situ. It is crucial to select the optimal scaffold material to ensure mechanical integrity immediately after implantation, which starts degrading as soon as sufficient tissue is formed to take over the native function. The aim of the research described in this thesis was to examine the interplay between scaffold degradation rates and the amount and composition of the formed tissue within the scaffold. Furthermore, degradation characteristics of scaffolds manufactured from different supramolecular biomaterials, were investigated. By imbalance between scaffold degradation and tissue formation, the mechanical integrity cannot be ensured and compaction and retraction of in-vitro TE heart valves occurs, causing regurgitation in-vivo. We studied whether compaction could be reduced by the use of slow-degrading polycaprolactone (PCL) instead of fast-degrading poly-4- hydroxybutyrate coated polyglycolic acid (PGA-P4HB) electrospun scaffolds and/or the use of a lower cell passage number to enhance tissue formation. The use of slow- degrading materials improved resistance to retraction of TE valvular leaflets and reduced compaction of TE rectangular scaffold strips. In addition, tissue formation, stiffness, and strength increased with decreasing cell passage number, but did not affect compaction of the engineered tissues. Thereafter, the effect of scaffold degradation rate on the amount and composition of tissue, the mechanical integrity, and the tissue to scaffold ratio were investigated. Slow- and fast-degrading scaffolds were seeded with vascular cells or kept unseeded. We hypothesized that the cells in fast-degrading scaffolds would compensate for the rapid loss of mechanical integrity by increased tissue production. Increasing amounts of tissue with time were shown in both scaffold groups, which was indeed more pronounced for PGA-P4HB-based tissues during the first two weeks of culture. Ultimately, PCL-based tissues resulted in the highest amount of tissue after 6 weeks. In addition, we described a method to correct for the amount of remaining scaffold weight, in order to allow a fair comparison between in-vitro engineered tissues grown on scaffolds with a different III Summary degradation rate and in-vitro engineered tissues and native tissues. By implementation of this correction, extracellular matrix values similar to values of native pulmonary heart valves were found. The amounts of collagen crosslinks were still below native values in all engineered tissues, but did display a continuing increase during culture. In-vivo, degradation of scaffold materials can be accomplished by the (enzymatic accelerated) hydrolytic and/or the oxidative pathway. To investigate both pathways, separately and in an accelerated fashion, in-vitro degradation assays were designed. For in-situ TE of cardiovascular tissues, the supramolecular materials PCL-2-ureido-[1H]- pyrimidin-4-one (PCL-UPy) and PCL-bisurea (PCL-BU) are used, due to their combination of strength and elastic properties. Degradation characteristics and susceptibility to the hydrolytic or the oxidative degradation pathway of these materials were investigated and compared with those of conventional PCL. Depending on the morphological and chemical composition of the materials, conventional and supramolecular PCL-based scaffolds responded differently to both degradation pathways. Conventional PCL is more prone to hydrolytic enzymatic degradation as compared to the supramolecular materials, while the opposite was shown when degraded by the oxidative pathway. We demonstrated the ability of tuning degradation characteristics by mix-and-match PCL backbones with supramolecular moieties.