Mesenchymal stem cell differentiation on microstructured poly (methyl methacrylate) substrates

doi:10.1016/j.aanat.2008.07.013

Annals of Anatomy - Anatomischer Anzeiger

Volume 191, Issue 1, January 2009, Pages 136-144

https://doi.org/10.1016/j.aanat.2008.07.013 Get rights and content

Summary

Recent studies on 2D substrates have revealed the importance of surface properties in affecting cell behaviour. In particular, surface topography appears to influence and direct cell migration. The development of new technologies of hot embossing and micro-imprinting has made it possible to study cell interactions with controlled micro features and to determine how these features can affect cell behaviour. Several studies have been carried out on the effect of microstructures on cell adhesion, cell guidance and cell proliferation. However, there is still a lack of knowledge on how these features affect mesenchymal stem cell differentiation. This study was designed to evaluate whether highly controlled microstructures on PMMA could induce rMSC differentiation into an osteogenic lineage. Structured PMMA was seeded with rMSC and cell number; cell morphology and cell differentiation were evaluated. Results confirm that microstructures not only affect cell proliferation and alignment but also have a synergistic effect with osteogenic medium on rMSC differentiation into mature osteoblasts.

Introduction

Research on stem cells is advancing the body of knowledge on how an organism develops from a single cell and how healthy cells replace damaged cells in adult organisms. This promising area of science is also leading researchers to examine the possibility of cell-based therapies for treatment of disease, which is often referred to as regenerative or reparative medicine.

Research using embryonic stem cells is growing in interest due to their potential medical applications. Despite this potential, the ethical issues surrounding these cells have created intense controversy. This concern has promoted in-depth research into adult stem cells. Adult stem cells are undifferentiated cells found in differentiated tissue that can renew themselves and differentiate (within certain limitations) to give rise to all the specialized cell types of the tissue from which they originate. Such adult stem cells have been discovered and characterized in a multitude of tissues, which suggests that autologous clinical implantation or genetically engineered stem cells for protein or drug delivery could be used without risk of immunorejection (The National Institute of Health resource for stem cell research, 2007).

In adult stem cell research, one of the main fields is the differentiation of stem cells into an osteogenic lineage for subsequent use in bone tissue engineering and reconstructive medicine. Interest in this field is related to the increased incidence of osteodegenerative diseases (i.e. osteoporosis and osteoarthritis) in the rapidly aging populations of developed countries. Bone is a mineralized tissue that confers multiple mechanical and metabolic functions to the skeleton. Bone contains two distinct cell types: osteoblasts and osteoclasts. Osteoblasts are bone-forming cells of mesenchymal origin. Mesenchymal stem cells (MSCs) have the potential to differentiate into osteoblasts, chondrocytes, adipocytes, fibroblasts, marrow stroma and other tissues of mesenchymal origin. These MSCs can be obtained from many sources (blood, adipose tissue, periosteum, muscle, dermis, etc.), but bone marrow aspirate is the most common supply. Due to the variety of cell types and the recently discovered plasticity of adult stem cells (stem cells from one tissue may give rise to cell types of a completely different tissue), there is a need for well-defined and efficient protocols for directing the differentiation of stem cells into the osteogenic lineage.

Cell behaviour depends on interactions with the environment. Consequently, the contact between cells and implantable materials will determine the success or failure of an implantable material or a medical device. It is well known that the cell response is affected by the physicochemical parameters of the biomaterial surface, such as surface energy, surface charges or chemical composition. Topography is one of the most crucial physical cues for cells. Microtopography influences cell adhesion, proliferation and differentiation (Zinger et al., 2005; Boyan et al., 2002; Aparicio et al., 2002). More recently, it has become clear that nanotopography also guides cell behaviour (Dalby et al., 2007). Chemical cues in the form of various biomolecules, such as adhesive proteins (i.e. fibronectin and laminin), also influence cell behaviour in a crucial manner (Lan et al., 2005).

Here we evaluate the effect of various surface modifications with the aim of directing rat MSCs to differentiate into osteoblast lineage.

Section snippets

Polymer fabrication

Poly (methyl methacrylate) (PMMA, 125 μm thick) was purchased from Goodfellow (UK) and used as received. A nanoimprint lithography apparatus (Obducat, AB, Sweden) was used to fabricate free-standing PMMA samples with surface features that had lateral dimensions ranging from 50 to 2 μm, following a previously reported hot embossing procedure (Mills et al., 2007). Briefly, this technique uses a microstructured silicon mould, covered by a grown silicon oxide layer, which is pressed into the bulk

Cell isolation and culture

rMSC from bone marrow were isolated and passaged twice to evaluate their potential to become osteoprogenitors and mature osteoblasts. rMSC proliferated for 11 days. The cell population increased 5-fold. After 14 days of culture, 75% of the total cell population was ALP positive (Figure 1A). After 21 days of culture, mineralization nodules were seen (Figure 1B). Very few cells with adipocyte morphology were seen at the beginning of the cultures, and hardly any after 21 days.

Cell morphology, proliferation and differentiation

Cell morphology

rMSC

Discussion

MSCs are usually grown on PS treated with polylysine, to enhance cell adhesion and cell growth. The stability of these cultures on other 2D synthetic substrates has not been studied in depth. The most common polymers used for MSC cultures are natural polymers, such as collagen (mostly Type I), fibrin, alginates, hyaluronic acid, etc. However, synthetic polymers allow better control of physicochemical properties than natural polymers. In addition, the use of synthetic polymers reduces the risk

Acknowledgements

This study was carried out in the context of the CellPROM project, funded by the European Community as contract no. NMP4-CT-2004-500039. The information contained in this paper reflects the authors’ views only. The authors are grateful to the Spanish Ministry of Science and Education (MEC) for support provided through project TEC2004-06514-C03, and for the provision of grants by the Ramon y Cajal programme (EM). Drs. C. Moormann and T. Wahlbrink from AMO Gmb (Germany) are gratefully

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Mesenchymal stem cell differentiation on microstructured poly (methyl methacrylate) substrates

Summary

Introduction

Section snippets

Polymer fabrication

Cell isolation and culture

Cell morphology, proliferation and differentiation

Discussion

Acknowledgements

Matrix Biol.

Biomaterials

Dev. Cell

Mater. Sci. Eng. C – Biomimetic Supramol. Syst.

Biomaterials

Human osteoblasts proliferation and differentiation on grit-blasted and bioactive titanium for dental applications

J. Mater. Sci.: Mater. Med.

Titanium surface roughness alters responsiveness of MG63 osteoblast-like cells to 1α, 25-(OH)2D3

J. Biomed. Mater. Res.

Titanium surface roughness alters responsiveness of MG63 osteoblast-like cells to 1α, 25-(OH)₂D₃