Materials Science and Technology of Polymers at the University of Twente

Gradient Polymers in Tissue Engineering

Michel klein Gunnewiek thesis cover: Gradient Polymers in Tissue Engineering

The aim of this PhD Thesis was the development of polymer brush-based multi-dimensional gradient platforms for tissue engineering. This objective was achieved through the fabrication and application of different coatings “grafted-from” biodegradable polyester-based supports featuring both 2D and 3D scaffolds. A specific focus was devoted to the preparation of brush coatings with controllable properties (e.g. cell adhesion, flexibility and bioactivity) synthesized by surfaceinitiated atom transfer radical polymerization (SI-ATRP). Thermoresponsive poly(Nisopropyl acrylamide) (PNIPAM) layers were applied to thermally control cell adhesion, while poly(oligo(ethylene glycol) methacrylate) (POEGMA)-coated supports were used to vary the bioactivity of the coating via fibronectin (FN) and growth factors (GFs) conjugation.

Chapter 1 provided a short introduction to the topics discussed in this Thesis and the motivation for the reported research. The scope of this Thesis is also presented in this first section.

In Chapter 2 a literature overview on the application of polymer brush platforms for controlling adhesion and differentiation of different cell types was reported. The fabrication of thermoresponsive and bio-conjugated brush coatings were highlighted as supports for tissue engineering. In the last section, the application of these polymer brush coatings for the fabrication of supports for tissue engineering was presented. As an additional introductory Chapter, a literature overview on 2D and 3D supports presenting gradients in protein concentration was discussed in Chapter 3. The fabrication methods to produce protein gradients on flat substrates and on hydrogel surfaces were reviewed followed by the formation of protein gradients within 3D hydrogel supports and other porous polymeric scaffolds. Additionally, the employment of protein gradients for cell adhesion, migration and differentiation applications was discussed.

In Chapter 4 surface-initiated polymerization of NIPAM from poly(ε-caprolactone) (PCL) flat films was presented. The surface of PCL was activated by aminolysis and subsequent initiator coupling which allowed surface-initiated atom transfer radical polymerization (SI-ATRP) of NIPAM. Cells showed to attach, spread and grow on PNIPAM modified surface at 37°C. Cooling the media to 25°C released the cells in the form of sheets and showed to be viable after being harvested when cultured on tissue culture plates. Additionally, free-standing PCL films with a PNIPAM coating on one side proved the thermoresponsive activity by the bending and stretching behavior.

Chapter 5 focused on SI-ATRP of POEGMA from thin PCL films presenting different semicrystalline structures. By varying PCL thermal processing, spherulitic size and density was controlled between sub-micron (up to 1 μm) to several hundreds of microns. The different micro-/nano-topologies obtained were shown to alter the behavior of hMSCs upon attachment and spreading. Sub-100 nm, FNfunctionalized POEGMA brush coatings grafted from the different PCL films were finally demonstrated to efficiently decouple substrate topology and cell adhesion. Namely, cells showed to respond irrespective to the underlying PCL topology when different spherulite size/density were uniformly coated with sub-100 nm thick POEGMA-FN brush coatings.

Chapter 6 focused on the behavior of stem cells adhering on a POEMGA brush layer with various grafting densities and chain lengths (molecular weight) grafted from PCL. These substrates were obtained by spin-coating PCL and varying the initiator coverage or the polymerization time. Cell shape analysis showed that cells attached to these POEGMA coatings adopted different morphologies responding to variations in grafting density and brush thickness. Brush layers with low thicknesses (sub-20 nm) or low densities (0.035 chains/nm2)were shown to allow hMSCs adhesion, while thicker (60 nm) or dense (0.341 chains/nm2) layers showed cell-repulsive properties due to the efficient resistance by dense and long brushes against protein adsorption. Linear gradients of POEGMA brush thickness were conjugated with fibronectin (FN) to obtain cell adhesive films. Cells adhered uniformly on the gradient and the cell shape showed to be independent of the POEGMA thickness. On the contrary, differences in formation and morphology of focal adhesion (FA) complexes were related to the different brush-ligand flexibilities across the gradient samples.

In Chapter 7, a novel method to introduce multi-directional variations of (bio)chemical environments inside 3D porous structures is presented. The hydrophilic nature and the high functionality of POEGMA was coupled to a structured scaffold to trigger diffusion and subsequent covalent immobilization of proteins from solutions. This allowed the fabrication of 3D axial and radial protein gradients with tailored morphologies. Gradients of brush-supported FN controlled the immobilization of human mesenchymal stem cells (hMSCs) in spatially determined cultures. Furthermore, the application of growth factors coupled to POEGMA-coated PCL scaffolds proved as an effective strategy to induce hMSCs differentiation within these 3D environments. The application of multidirectional gradients of brush-supported GFs on these 3D porous scaffolds will have the potential to regenerate complex interfaces between different tissue types, such as bone and cartilage.