Elsevier

Colloids and Surfaces B: Biointerfaces

Volume 97, 1 September 2012, Pages 211-220
Colloids and Surfaces B: Biointerfaces

Regulation of the behaviors of mesenchymal stem cells by surface nanostructured titanium

https://doi.org/10.1016/j.colsurfb.2012.04.029Get rights and content

Abstract

The study describes the influence of surface nanostructured titanium substrates on the growth behaviors of mesenchymal stem cells. Surface nanostructures of titanium were produced with surface mechanical attrition treatment (SMAT) technique. The morphologies of native titanium and surface nanostructured titanium substrates were characterized by field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and contact-angle measurements, respectively. A thin nanostructured layer was formed onto the surfaces of titanium substrates after SMAT treatment. The effects of the surface nanostructured titanium substrates on the adhesion, spreading, proliferation and differentiation of mesenchymal stem cells (MSCs) was examined at cellular and molecular levels in vitro. The results suggest that the surface nanostructured substrates were beneficial for the growth of MSCs, including adhesion, filament orientation, proliferation and gene expression. This approach for the fabrication of surface nanostructured titanium may be exploited in the development of high performance titanium-based implants.

Highlights

► Surface nanostructured titanium was fabricated by surface mechanical attrition treatment (SMAT) technique. ► Surface nanostructured titanium promoted the adhesion, proliferation and differentiation of MSCs. ► Specific bone related genes expressions were revealed at molecular level. ► The potential mechanism was proposed.

Introduction

Titanium and its alloys have been widely used to fabricate dental and orthopedic implants in clinical applications, mainly due to their bioinert and good mechanical properties [1]. However, improved osseo-integration of an implant to its surrounding natural bone tissue still remains an important issue [2]. Another objective is to maintain long-term stability of the titanium based implants, which is related to their wear resistant properties. The wear debris in the form of titanium particles derived from the implants would enter into the surrounding tissues to induce inflammation and bone resorption in the case of long term implantation, ultimately resulting in loosening and failure of the implant [3]. Therefore, it is essential to improve the biocompatibility and wear resistance of a titanium implant for its successful long-term survival.

The interactions between cells and biomaterials are considered as one of the most important factors leading to the successful clinical applications of biomaterials. Since cells initially contact with the surface of a biomaterial, cellular responses are mainly dominated by the surface properties of the biomaterial, such as roughness, stiffness, wettability, surface charges, chemical compositions and topography [4], [5], [6], [7], [8]. Recently, more and more studies were focused on the influence of nanotopographies and/or nanotextures of substrates on cell behaviors. For instance, Park et al. [9], [10] found that TiO2 nanotube arrays improved the growth behaviors of endothelial cells and mesenchymal stem cells. Tabrizian et al. [11] demonstrated that the attachments and growth rates of pre-osteoblasts were improved by ultrafine-grained titanium substrates produced by the high-pressure torsion (HPT) process. While Misra et al. [12] showed that the nanograined/ultrafine-grained structures of austenitic stainless steel produced by ingenious phase reversion technique were favored for the growth of pre-osteoblasts. However, understanding the underlying fundamental mechanisms in the field still needs further investigation.

In this study, we employed surface mechanical attrition treatment (SMAT) technique to fabricate surface nanostructured titanium as model substrates to investigate its effects on cell behaviors. The SMAT is a surface self-nanocrystallization technique that results in severe plastic deformation (SPD) on the top surface layer of a bulk material by means of repeated multidirectional impacts of flying balls [13]. SMAT treated materials with surface nanocrystalline structures have demonstrated superior physical properties for applications in the aerospace, automotive, and biomedical fields [13], [14], [15]. In particular, many groups successfully employed SMAT technique to improve the friction and wear resistance of different materials, such as low carbon steel [16], copper [17] and titanium [18].

MSCs were selected as object cells in this study, with clinical implication that titanium based implants would interact with bone marrow stem cells in a hip joint-replacement application. Mesenchymal stem cells isolated from bone marrow stroma have multi-lineage differentiation potentials to chondrocytes, osteoblasts, adipocytes and endothelial cells under desirable cellular microenvironments [19], [20], [21], [22]. Moreover, MSCs demonstrated themselves bright future in bone regeneration and cell therapy for bone diseases [23], [24].

The objective of this study was to investigate the effects of nanostructured substrates on the proliferation and differentiation of mesenchymal stem cells at cellular and molecular levels. The potential underlying mechanism was proposed as well.

Section snippets

Materials

Commercial pure Ti sheets were provided by the Northwest Institute for Non-ferrous Metal Research, China. Alkaline phosphatase (ALP), bicinchoninic acid (BCA) assay kit, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum album (BSA), alizarin red-S (ARS) and Hoechst 33258 were purchased from Sigma Chemical Co. (MO, USA). Anti-vinculin antibody and fibronectin (FN) were purchased from Santa Cruz Biotechnology Co. (USA). Mouse anti-goat fluorescein isothiocyanate

Sample characterization

In this study, the surface nanostructured titanium substrates were fabricated by the SMAT technique. The treated samples were characterized by SEM, AFM, TEM, XRD and contact angle measurement, respectively.

The morphologies of native titanium and surface nanostructured titanium substrates were visualized with optical microscope, SEM and AFM (Fig. 1), respectively. Slight difference in morphology was observed from optical micrographs of the native titanium and surface nanostructured titanium

Discussion

Titanium has widely been used as bone implant in clinical application. It is well known that titanium has much higher elastic modulus than that of natural bone, resulting in mismatch of mechanical property. Although various techniques such as high-pressure torsion (HPT) process [11] and equal channel angular pressing (ECAP) [32] have been developed to produce nanostructured titanium and improve cell growth behavior, the nanostructuring of bulk titanium highly enhanced its elastic modulus, which

Conclusion

In this study, surface nanostructured titanium substrates was fabricated with surface mechanical attrition treatment technique, which was verified by combined techniques of SEM, AFM, TEM, XRD and water contact angle measurements. In vitro tests confirmed that the surface nanostructured titanium promoted the focal adhesion, proliferation and differentiation of MSCs. The underlying mechanism was proposed based on mRNA expressions of osteogenesis related proteins and gene (OC, OPN, collagen I and

Acknowledgements

This work was financially supported by China Ministry of Science and Technology (973 Project No. 2009CB930000), Natural Science Foundation of China (11032012 and 51173216), Fok Ying Tung Education Foundation (121035) and Natural Science Foundation of Chongqing Municipal Government (CSTC, 2011JJJQ10004, 2010AB5116, 2011GGB10011 and 2011GGC10002).

References (49)

  • M. Long et al.

    Biomaterials

    (1998)
  • S.M. Sporer et al.

    Orthop. Clin. North Am.

    (2005)
  • T.P. Kunzler et al.

    Biomaterials

    (2007)
  • S. Bodhak et al.

    Acta Biomater.

    (2009)
  • K.Y. Zhu et al.

    Acta Mater.

    (2004)
  • Z.B. Wang et al.

    Mater. Sci. Eng. A

    (2003)
  • Y.S. Zhang et al.

    Wear

    (2006)
  • K.H. Choi et al.

    Biomaterials

    (2010)
  • H.N. Yang et al.

    Biomaterials

    (2011)
  • L. Huang et al.

    Surf. Coat. Technol.

    (2006)
  • M. Wen et al.

    Surf. Coat. Technol.

    (2008)
  • X. Wu et al.

    Acta Mater.

    (2005)
  • L.T. Allen et al.

    Biomaterials

    (2006)
  • D.C. Roy et al.

    Biomaterials

    (2011)
  • W.C. Clem et al.

    Biomaterials

    (2008)
  • C.M. Gundberg et al.

    Bone

    (2002)
  • K.Y. Cai et al.

    Acta Biomater.

    (2010)
  • J. Huang et al.

    Joint Bone Spine

    (2009)
  • J.C. Reichert et al.

    Biomaterials

    (2010)
  • C.A. St Pierre et al.

    J. Orthop. Res.

    (2010)
  • D.E. Discher et al.

    Science

    (2005)
  • A. Joy et al.

    Langmuir

    (2011)
  • E. Martinez et al.

    Nanomedicine

    (2009)
  • J. Park et al.

    Nano Lett.

    (2009)
  • Cited by (0)

    View full text