Elsevier

Biomaterials

Volume 26, Issue 34, December 2005, Pages 7057-7067
Biomaterials

Controlling the phenotype and function of mesenchymal stem cells in vitro by adhesion to silane-modified clean glass surfaces

https://doi.org/10.1016/j.biomaterials.2005.05.008Get rights and content

Abstract

The behaviour of human mesenchymal stem cells (hMSC) when cultured in contact with a range of silane-modified surfaces was examined to determine if changing the surface chemistry affected the early differentiation potential of mesenchymal stem cells in vitro over a 7-day period. Cells were cultured for 1 and 7 days in direct contact with glass which had been functionalized by surface treatment to provide a range of different surfaces: -CH3, -NH2, -SH, -OH, and -COOH modified surfaces and a clean glass reference (TAAB). Viable cell adhesion was quantified by Lactate Dehydrogenase assay, and morphology and viability was qualitatively evaluated using calcein AM, ethidium homodimer, cytoskeletal (F Actin), extra-cellular matrix (fibronectin and vitronectin) and Hoechst staining (nucleus). The expression of selected differentiation markers, Collagen II (chondrocytes), CBFA1 (bone transcription factor), Collagen I (MSC marker) and TGF-β3 (extra-cellular matrix production) was determined using real time polymerase chain reaction. The expression of ornithine decarboxylase was evaluated as a marker of proliferation. Surfaces of the -NH2 group demonstrated the greatest level of cell adhesion by the 7-day period, and mRNA expression profiles indicated osteogenic differentiation, increased CBFA1 and decreased Collagen II expression. Cells cultured in contact with the -COOH surfaces displayed different cell morphologies, fibronectin and vitronectin spatial distributions compared with the cells in contact with the -NH2 surfaces, in addition to an increase in Collagen II expression, indicative of chondrogenic differentiation. The modifications to the surface chemistry of glass did affect cell behaviour, both in terms of viable cell adhesion, morphology and profiles of mRNA expression, providing the means to alter the differentiation potential of the MSCs.

Introduction

Mesenchymal stem cells (MSC) are a pivotal element within the multi-disciplinary field of tissue engineering. This is largely due to their potential as an unlimited source of autologous multi-potent cells that can be expanded and differentiated with a view to produce tissue-engineered constructs in vitro for use in vivo. [1], [2], [3], [4], [5].

Manipulating the differentiation pathway of MSC, in vitro, using cocktails of growth factors and cytokines is now well documented. Indeed such techniques are often employed as a method of determining the plasticity of a population of isolated stem cells [6], [7], [8], [9], [10], [11], [12], [13], [14], due to the fact that practical definitive markers of MSC phenotype at the time of isolation are limited. Little generic knowledge has been gained to determine to what extent substrate material surface factors can control not only the adhesion, but also the differentiation and function of MSCs.

Research has focused on changing surface properties [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25] e.g. surface charge, nanotopography and microstructure, and evaluating how these changes affect a specific type of cell behaviour or a pre-selected differentiation pathway [26]. For example, we are slowly gathering information on how the changes in the surface properties of materials can influence the osteogenic capabilities of pre-osteoblast cells [20], or the adhesion of inflammatory cells [16], [27], but as yet no information has been gathered on the effect of changing surface chemistry on the generic multi-potency of MSCs.

Research utilising bone marrow-derived MSC has concentrated on how specific substrates and surface chemistries can be used to enhance specific differentiation pathways i.e. osteogenic [28], [29] and chondrogenic [30]. Whilst this research is important for bone bonding and other specific applications it fails to explore the basic question as to can surface chemistry be used to control initial cell adhesion and the subsequent related differentiation, or is there an optimum surface chemistry for the maintenance of the MSC phenotype?

The relationship between controlling the level of MSC adhesion, resultant morphology, and consequent differentiation has still to be determined. Earlier research has demonstrated that changes in surface chemistry results in differences in surface energy and subsequent protein and cell adhesion [31], [32], [33], [34], but as yet we have little knowledge as to how the changes in protein conformation and consequential cell adhesion controls, or influences the proliferative or differential potential of MSCs. The understanding that controlling the level of cell adhesion will influence the differentiation of the cell is derived from the fact that chondrocytes and osteoblast obtained from bone marrow MSCs demonstrate very different morphologies that contribute to the function of the cell [35], [36], [37], [38]. For example chondrocyte performance i.e. Collagen II expression is optimised when chondrocytes are rounded and the number of focal adhesions is reduced compared to well spread chondrocytes grown on standard tissue culture polystyrene (TCPS) [39], [40]. Therefore controlling the level of cell adhesion on a surface could provide a way to control or influence the differentiation process when MSCs are cultured in contact with surfaces.

To address this question, we have prepared a range of chemically defined modified surfaces of clean glass (TAAB) to provide the following functional groups on the surface of the glass: methyl (-CH3), hydroxyl (-OH), carboxyl (-COOH), amino (-NH2) and silane (-SH). All of these functional groups can be found naturally within biological systems. Human MSCs were cultured in direct contact with the surfaces for 1 and 7 days and the levels of cell adhesion were quantified and early morphology was visualised using a combination of cytoskeletal and viability stains with confocal microscopy. In addition the expression of a number of osteogenic and chondrogenic and proliferative markers was quantified using real time polymerase chain reaction.

Section snippets

Sample preparation

Glass coverslips (13 mm diameter, Borosilicate Glass Co. UK) were dipped into 5% NaOH solution for 1 h followed by concentrated HNO3 for 1 h. All the coverslips were then rinsed with ultra-pure water and then with 100% ethanol four times, dried at 120°C for 10 min, and stored in a vacuum desiccator prior to introduction of different functional groups on the surface (i.e. -CH3, -NH2, -SH, -OH, -COOH) by silanation.

To introduce the -CH3 functional group the coverslips were dipped into

Contact angle

The results of dynamic contact angle measurements using water as the solvent for glass and silane-modified surface are shown in Fig. 1. It can be seen that the clean glass surface (TAAB) was the most hydrophilic with a contact angle value of 62.16±2.33. The -OH and -COOH surfaces had slightly higher contact angle values (63.06±2.01, 68.89±4), as the -OH and -COOH are highly polar groups. The contact angles of the -NH2 and -SH surface were 78.71±5.69 and 84.96±4.11, respectively, these were

Discussion

This study indicated that mRNA expression, cell morphology and levels of fibronectin and vitronectin, and subsequent MSC behaviour were affected by changes in surface chemistry. This could provide a path of investigation that may well enable the development of materials for tissue engineering purposes which could control stem cell differentiation, either directly or in combination with growth factors.

Quantitative cell adhesion demonstrated that MSC cells cultured in contact with the -NH2

Conclusion

Utilisation of silane-modified surfaces provided us with a controlled method to investigate the effect of specific surface chemistries on MSC adherence, viability, proliferation and differentiation. Positively charged -NH2 surfaces not only sustained the greatest amount of viable cell adhesion, but also indicated an increase in osteogenic pathway differentiation. Cells cultured in contact with the-COOH surfaces maintained a more rounded morphology and a potential for chondrocytic

Acknowledgements

The authors would like to thank the EPSRC, MRC and BBSRC who fund the UK Centre for Tissue Engineering (UKCTE) for their continued support and funding.

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