Characterising demixed polymer surfaces to stimulate mesenchymal stromal cell activity and influence tissue development during osteochrondral regeneration

Abstract

Osteoarthritis (OA) is defined clinically as the world’s leading cause of joint disease. Better understanding of the disease aetiology, contributing factors and improved imaging technologies has changed how we can treat this disease. Early intervention and long-term implant integration has inspired a new generation of biomaterials, able to influence local biological events in situ. The response of cells to their physical environment is known to regulate their proliferation, phenotype, gene expression and differentiation. To rebuild tissue with the appropriate structural and cellular organisation, temporal and spatial cues are required, possibly in combination with biomolecules and/or biomolecular motifs, to control musculoskeletal and adult mesenchymal stromal cell (MSC) phenotype and subsequent differentiation. Work presented here identifies a simple, adjustable and efficient technique for the investigation of how controlled chemical and distinct topographical surfaces can be used to influence MSC differentiation. Thin films of polymers were spread onto glass substrate by spin coating. Using a combination of immiscible polymers, Polystyrene (PS) and Poly (methyl methacrylate) (PMMA), phase separation generates a landscape of opposing structures. This is further accentuated by the addition of small quantities of water to produce pores. Low concentration polymer solutions (3%w/v) were demixed at various ratios (v/v) and spin coated at speeds > 8,000rpm under humid conditions. Polymer surfaces were evaluated for topography, pattern and chemistry using; atomic force microscopy (AFM), scanning electron microscopy (SEM) and X-ray photon spectroscopy (XPS). Biological response was evaluated using immunofluorescence, histological staining, and qPCR. Primary adult human mesenchymal stem cells (huMSC) were isolated the marrow of bone fragments. Multipotent cell populations were characterized by flow cytometry and trilineage differentiation. Immobilized proteins and other biomolecules functionalised the surfaces using zero length crosslinking, restrictively bound to discreet areas of the biphasic surfaces. Lineage specific growth factors (BMP-2, TGF-β and KRTGN) were immobilized on the surfaces and evaluated for their ability to influence MSC phenotype. A range of demixed polymer ratios, concentrations and solvents were evaluated and these combinations produced a variety of distinct surfaces. A selection of surfaces (PS:PMMA [(i)40:60,(ii)50:50,(iii)60:40] at 3%w/v in toluene) were chosen for further characterization based upon the reproducibility of their fabrication. Different ratios created opposing raised PMMA and low lying PS islands [(i) 8 μm2,(ii) 8-12 μm2,(iii)12-15 μm2]. Saturated humid conditions induced breath figure patterns producing average crater-like features, caldera, of (i) 0.5 μm, (ii) 0.7 μm,(iii)1 μm in height. Variations in these crater-like structures and their distribution were observed to III IVV created opposing raised PMMA and low lying PS islands [(i) 8 μm2,(ii) 8-12 μm2,(iii)12-15 μm2]. Saturated humid conditions induced breath figure patterns producing average crater-like features, caldera, of (i) 0.5 μm, (ii) 0.7 μm,(iii)1 μm in height. Variations in these crater-like structures and their distribution were observed to differ between demixed polymer ratios and this concurrently altered cell adherence profiles. Short-term (24h and 72h) effects displayed concentrated cell focal adhesion plaque interaction with the raised caldera, altered polymer demixed ratios consequently influence cell morphology. Changes in surface topography between surface i to ii altered cell morphology from polygonal to multipolar and hyperbolae shapes, in turn changing cell F-actin cytoskeleton. Long-term effects found higher commitment to a more chondrogenic lineage, with RNA expression levels elevated for SOX9 and ACAN. Effects were not isolated to individual cell behaviour but displayed mass cell condensation and aggregation. Histological analysis displayed altered ECM deposition of cell clusters. Immobilized large biomolecules to the polymer surface, using a fluorescein isothiocyanate labelled albumin (AL-FITC) identified restricted immobilization to opposing polymer chemistry. Functionalized surfaces with immobilized phosphatase enzyme illustrated the retention of bioactivity of a protein for up to 12 days. Growth factor immobilization showed slight phenotypic change to direct cell lineage commitment but this was not significant (IBMP2 and I-TGF-β). Surface fabrication methods described in this thesis identify a simple reproducible method for evaluation of cell response to their physical environment. Polymer demixing generates chemically distinct surfaces with additional adaptability for downstream processing of these surfaces with covalent crosslinking. Simple modification of the fabrication conditions by introducing humidity greatly enhanced the topographical features adding an altered height dynamic to these textured surfaces. Micro- and nano- topographical features influenced cell adhesion and cell morphology. This in turn resulted in altered gene expression and indicated potential for phenotype change. Surfaces of high caldera elevation and dispersion were associated with enhanced chondrogenic lineage expression by primary MSCs. Additionally, cell-cell interactions were influenced on these surfaces, promoting cell aggregation, condensation and localised matrix deposition. Demixed polymer blends provided tuneable topographies for the stimulation of multipotent cells through identified contact with distinct Caldera positioning. Utilizing the individual polymer chemistries of these topographies, biomolecules were able to be restrictively bound and retain bioactivity, but tailored surfaces immobilized with tissue specific growth factor only displayed slight lineage effects in the work presented here but is an area worthy of further study.