Cell adhesion on 3D printed submicron patterns: a quantitative study

Abstract

Objectives: With an increasing life expectancy in the western societies and more people practising extreme sports, the demand for orthopaedic implants is set to increase. Orthopaedic implant loosening is one of the main causes of revision surgeries, leading to increases in the costs of patient care and patient dissatisfaction. Better osseointegration could lead to higher success rates of primary interventions. Mesenchymal stem cells (MSC) can undergo osteogenic differentiation on the implant surfaces, promoting the implant osseointegration. Alternatively, they can commit to other differentiation paths (e.g., fibroblasts) and thus hinder the osseointegration and functionality of the implant. It is therefore fundamental to promote the osteogenic differentiation of MSC at the surface of the implant to ensure enhanced osseointegration and thus its long term success. Among the available strategies that promote osteogenic differentiation, nano and micro-scale physical patterns have proved effective but the underlying mechanisms that induce these cellular changes are not yet understood. Cellular adhesion to the surface is believed to be the key process regulating mechanotransduction (i.e., extrinsic cell signalling) and subsequently, the mesenchymal stem cell osteogenic differentiation. Nevertheless, no quantitative systematic study has been performed in order to establish the relationship between the cell adhesion and the differentiation behaviour. Furthermore, the available cellular adhesion studies are only qualitative or semi-quantitative in nature. In this work, novel fluid force microscopy (FFM) technique has been employed to characterize the adhesion properties induced by a set of osteogenic and a set of non-osteogenic submicron patterns. Methods: Two sets of substrates consisting of arrays of submicron pillars with known osteogenic potential were manufactured by means of two photon polymerization. Preosteoblast mouse cells were cultured on the patterns and on a flat control surface. The adhesion properties (i.e., the force and work of adhesion) were measured by FFM after 4 and 24 hours of cell culture. The cellular behaviour picture was completed by assessing the Young’s modulus of the living cells attached on the patterns and on the control surface. Likewise, the topographical and mechanical mapping was performed by using atomic force microscopy system. Results: In this study, the adhesion of preosteoblast cells was successfully quantified on two types of submicron pattern with known osteogenic potential, and compared to cellular adhesion data on a flat control. Therefore, the fluid force microscopy (FFM) was applied for the first time. The trend indicated that cells seeded on the osteogenic substrate adhered stronger (mean force of adhesion = 159 ± 74 nN) than cells on the non-osteogenic substrate (mean force of adhesion = 108 ± 104 nN) after 24h of incubation. Moreover, the cells adopted different morphologies and spatial distribution of the Young’s modulus depending on the culture substrate.