Elsevier

Biomaterials

Volume 30, Issue 28, October 2009, Pages 5094-5103
Biomaterials

The use of nanoscale topography to modulate the dynamics of adhesion formation in primary osteoblasts and ERK/MAPK signalling in STRO-1+ enriched skeletal stem cells

https://doi.org/10.1016/j.biomaterials.2009.05.049Get rights and content

Abstract

The physiochemical characteristics of a material with in vivo applications are critical for the clinical success of the implant and regulate both cellular adhesion and differentiated cellular function. Topographical modification of an orthopaedic implant may be a viable method to guide tissue integration and has been shown in vitro to dramatically influence osteogenesis, inhibit bone resorption and regulate integrin mediated cell adhesion. Integrins function as force dependant mechanotransducers, acting via the actin cytoskeleton to translate tension applied at the tissue level to changes in cellular function via intricate signalling pathways. In particular the ERK/MAPK signalling cascade is a known regulator of osteospecific differentiation and function. Here we investigate the effects of nanoscale pits and grooves on focal adhesion formation in human osteoblasts (HOBs) and the ERK/MAPK signalling pathway in mesenchymal populations. Nanopit arrays disrupted adhesion formation and cellular spreading in HOBs and impaired osteospecific differentiation in skeletal stem cells. HOBs cultured on 10 μm wide groove/ridge arrays formed significantly less focal adhesions than cells cultured on planar substrates and displayed negligible differentiation along the osteospecific lineage, undergoing up-regulations in the expression of adipospecific genes. Conversely, osteospecific function was correlated to increased integrin mediated adhesion formation and cellular spreading as noted in HOBS cultured on 100 μm wide groove arrays. Here osteospecific differentiation and function was linked to focal adhesion growth and FAK mediated activation of the ERK/MAPK signalling pathway in mesenchymal populations.

Introduction

The triggering event in osteogenesis is the transition of skeletal or mesenchymal stem cells into bone-forming, differentiated osteoblast cells a process exquisitely controlled by sequential activation of diverse transcription factors that regulate the expression of osteospecific genes. It is becoming increasingly clear that epigenetic modulation of cellular behaviour and subsequent cellular differentiation is highly regulated by mechanotransductive events and that topographical modification may be a viable strategy to regulate this process.

The rationale for developing nanostructured materials for regenerative medicine stems from the complex structure of extracellular tissue in vivo, in essence a collection of composite micro and nanostructures composed of seemingly random fibers, pits and protrusions. The fact that many cell types are naturally adapted to function within a complex nanotopographical environment has facilitated a series of studies to investigate the cellular response toward the phenomenon of nanotopographically mediated mechanotransduction with studies indicating that most cells do react significantly to nanotopographical cues in vitro [1].

Cell–substrate interactions can be regarded as the defining factors of a biomaterial performance, ultimately determining the long-term performance of a device in situ. The processes that mediate an altered cellular response to nanoscale surface structure however are not well understood and may be direct [2], [3] (a result of the influence of the surface topography) or indirect [4], [5] (where the surface structure has affected the composition, orientation, or conformation of the adsorbed ECM components) [6].

Cells establish dynamic contacts with the underlying substrate termed focal adhesions, the regulation of which is highly complex and involves initial integrin binding to ECM components and the reinforcement of the adhesion plaque by further protein recruitment. In addition to their adhesive functions, integrins mediate bi-directional signalling between the ECM and the cell, activating signalling pathways that regulate transcription factor activity, and direct cell growth and differentiation.

Although integrin molecules provide a platform for intracellular signalling, they do not exhibit intrinsic enzymatic activity in their cytoplasmic domains [7]. Therefore, downstream signalling following integrin binding is mediated by non-receptor tyrosine kinases [8]. One such pathway that may regulate the proliferation and differentiation of skeletal stem cells is the extracellular signal-regulated kinase (ERK) signalling cascade, a member of the mitogen activated protein kinase (MAPK) pathway. ERK-MAPK signalling conveys physical information from the extracellular environment to the nucleus [9] and acts to regulate the cell cycle. In addition to playing a role in both proliferation and apoptosis, the ERK-MAPK pathway has important functions in cellular differentiation [10], [11], [12] and has been implicated in the differential response of bone cells to a variety of signals, including ECM-integrin binding [13], [14], and mechanical loading [15]. Furthermore, it has been shown that integrin mediated activation of the ERK/MAP kinase pathway results in phosphorylation and stimulation of RUNX2 activity, a transcription factor critical in early osteospecific differentiation.

In order to elucidate the effects of topographical cues on primary human osteoblast populations (HOBs), biologically relevant nanoscale substrates were fabricated by lithographic methods to examine the modulation of both cell–substratum adhesion in osteoblasts and ERK/MAPK signalling in STRO-1+ enriched skeletal stem cell populations. The dynamics of focal adhesion formation and growth on nanotopographic features were observed in HOBs over 36 h and sites of focal adhesion were quantified in S-phase populations. To investigate the topographical induction of skeletal stem cell differentiation, functional modification of the ERK/MAPK signalling pathway by experimental topographies was examined by microarray analysis.

Section snippets

Substrate fabrication

Experimental and control substrate fabrication was exclusively in poly(methyl methacrylate) (PMMA), a non-biodegradable polymer which has FDA approval for implants. All experimental and control substrates were first subjected to a sterilisation process prior to cell seeding, PMMA substrates were rinsed in 70% ethanol/water solution 3 times for 3 s. All PMMA replicates were next rinsed twice in HBSS pH 7.4 for 5 s to remove any ethanol residue. Planar PMMA (Cambridgeshire, Goodfellow, UK) replicas

Substrate characterisation

The planar PMMA control substrates possessed no significant topographical features under SEM evaluation and were verified to have an Ra of 1.2 nm over 10 × 10 μm2 by AFM (Fig. 1A and B). The embossing process resulted in the fabrication of nanopit, and microgrooved topographies in PMMA as verified by AFM (not shown) and SEM. Substrates originally fabricated by EBL and transferred to PMMA replicas via a Ni intermediary were arrays of ordered pits which were 100 nm deep, 120 nm in diameter and

Discussion

While cellular adhesion and the onset of differential function are known to be intimately associated, the regulatory mechanism for mechanical stress on cells of the osteoblastic differentiation cascade remains to be fully elucidated. This is due to the limited number of suitable in vitro model systems that can be used to examine osteoblastic differentiation from non-osteogenic cells. Previous studies have implicated integrin mediated cell adhesion as a critical regulator of osteogenic

Conclusion

It can be inferred from this study that topographical modification can modulate cellular differentiation in multipotent stem cell populations, a process which is regulated by the ERK/MAPK signalling pathway through integrin mediated cellular adhesion. We have shown that nanopit arrays in both square and hexagonal conformations disrupt adhesion formation and cellular spreading in HOB cells and prevent osteospecific differentiation in skeletal stem cell populations. Here up-regulation in protein

Acknowledgements

This work was funded by the AO Research Fund, Switzerland; Grant Number: 04-D81. MJD is supported by the BBSRC, ROCO is supported both by the BBSRC and EPSRC. The authors acknowledge the work of the Sir Henry Welcome Functional Genomics Facility and thank the following people: Prof Adam Curtis and Dr. Mathis Riehle for their interesting discussions and support, John Pedersen (SDC Dandisc A/S, Denmark) for the organising and preparation of nickel shims, Bill Monaghan, Mary Robertson and Sara

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