Skeletal stem cell and bone implant interactions are enhanced by LASER titanium modification

https://doi.org/10.1016/j.bbrc.2015.10.013Get rights and content

Highlights

  • Bone stem cells on LASER Ti surface display enhanced cell growth and viability.

  • Bone stem cells on LASER Ti surface exhibit marked biocompatibility.

  • Human bone stem cells on LASER Ti surface exhibit altered morphology.

  • LASER Ti enhance osteogenic differentiation of human bone skeletal stem cells.

  • LASER Ti provides a unique approach to enhance osseointegration with the material.

Abstract

Purpose

To evaluate the osteo-regenerative potential of Titanium (Ti) modified by Light Amplification by Stimulated Emission of Radiation (LASER) beam (Yb-YAG) upon culture with human Skeletal Stem Cells (hSSCs1).

Methods

Human skeletal cell populations were isolated from the bone marrow of haematologically normal patients undergoing primary total hip replacement following appropriate consent. STRO-1+ hSSC1 function was examined for 10 days across four groups using Ti discs: i) machined Ti surface group in basal media (Mb2), ii) machined Ti surface group in osteogenic media (Mo3), iii) LASER-modified Ti group in basal media (Lb4) and, iv) LASER-modified Ti group in osteogenic media (Lo5). Molecular analysis and qRT-PCR as well as functional analysis including biochemistry (DNA, Alkaline Phosphatase (ALP6) specific activity), live/dead immunostaining (Cell Tracker Green (CTG7)/Ethidium Homodimer-1 (EH-18)), and fluorescence staining (for vinculin and phalloidin) were undertaken. Inverted, confocal and Scanning Electron Microscopy (SEM) approaches were used to characterise cell adherence, proliferation, and phenotype.

Results

Enhanced cell spreading and morphological rearrangement, including focal adhesions were observed following culture of hSSCs1 on LASER surfaces in both basal and osteogenic conditions. Biochemical analysis demonstrated enhanced ALP6 specific activity on the hSSCs1-seeded on LASER-modified surface in basal culture media. Molecular analysis demonstrated enhanced ALP6 and osteopontin expression on titanium LASER treated surfaces in basal conditions. SEM, inverted microscopy and confocal laser scanning microscopy confirmed extensive proliferation and migration of human bone marrow stromal cells on all surfaces evaluated.

Conclusions

LASER-modified Ti surfaces modify the behaviour of hSSCs.1 In particular, SSC1 adhesion, osteogenic gene expression, cell morphology and cytoskeleton structure were affected. The current studies show Ti LASER modification can enhance the osseointegration between Ti and skeletal cells, with important implications for orthopaedic application.

Introduction

Research in the field of biomaterials has advanced significantly in recent years driven in part by the desire to develop biomaterials that will provide extended longevity and enhanced performance for an increasing ageing population [1]. Bone tissue engineering seeks to address the unmet need for new tissues lost as a consequence of disease, trauma or ageing, using a raft of interdisciplinary approaches including developmental biology, materials science, stem cells and bioengineering. Typically, the approach is to harness the therapeutic potential of stem cells together with an appropriate biomaterial [2], [3]. Ti has long been the gold standard for orthopaedic given the excellent biocompatibility, low corrosion, wear resistance and to promote osseointegration at the bone-implant interface [4]. For the development of osseointegration the recruitment of cells with osteogenic potential is essential. Subsequent colonisation by the cells is believed to occur through the release of growth factors and cytokines into the clot surrounding the site of implant placement, and it is widely accepted that SSCs1 are the first cells recruited to such sites in vivo [5]. While Ti implants have found clinical utility for many decades, the process of osseointegration remains, to date, unclear. The process is time dependent and is dependent upon the close relationship between the bone quality and the Ti surface, although the bone structure is naturally difficult to change, the Ti surface can be relatively easily modified [6]. There are two accepted approaches to enhance the material bone response – the first is the development of a rough topography optimised for bone response [7], and the second is the establishment of a high surface energy (wettability) rendering the surface super-hydrophilic, thereby facilitating initial cell contact and adherence [8], [9].

A number of approaches have been advocated to modify and improve the Ti surface, LASER treatment is an innovative approach that results in surfaces with increased surface area, enhanced wettability and, in preclinical (lapine) bone models, displays negligible corrosion and high removal torques of established implants [6]. As recently detailed in a number of studies, LASER treatment appears to provide a promising method for Ti implant generation, resulting in enhance and rapid onset of osseointegration [6], [10], [11], [12], [13].

Understanding how to control, manipulate, and enhance the intrinsic healing events modulated through osteogenic differentiation of SSCs1 through the application of modified surfaces offers significant potential for the orthopaedic field. It is clear that an exquisite interplay exists between the cells and the microtexture of a material. In vivo, cells encounter a number of topographical features ranging from protein folding to collagen banding [14]. Due to the ease of manufacture, the development of materials with a range of surface roughness has been widely used to further examine the bone material interface. Such a strategy provides useful information regarding the bone cell response to structured materials [15]; either as a consequence of surface modification that generates enhanced implant stability and/or indeed accelerated healing following implantation [16].

Based on the hypothesis that modified surfaces can modulate the initial osteo-inductive responses of cells, this study set out to examine the osteo-regenerative potential of Ti-modified by LASER beam (Yb-YAG) on hSSC1 compatibility and subsequent cell function.

Section snippets

Cell culture

Skeletal cell populations STRO-1+ hSSCs1 were isolated and cultured following previously described protocols [17] with the approval of the Local Research Ethics Committee (LREC 194/99).

2500 STRO-1+ hSSCs1 derived from the same patient were cultured on titanium discs in non-tissue culture plastic multiwell dishes for 10 days across four groups: Mb,2 Mo,3 Lb4 and Lo.5

hSSCs1 cultured on LASER-modified Ti surface display enhanced cell growth and viability

No significant differences were observed after 10 days of culture of hSSCs1 seeded on any of the Ti surfaces (Mb2 versus Lb4 (100% × 87.51%), Mo3 versus Lo5 (103% × 160.22%)) indicating cell survival and growth (Fig. 1). Cell viability and an absence of cell necrosis were confirmed by live/dead staining with CTG7/EH-18 after 10 days culture (Fig. 1A–D).

hSSCs1 cultured on LASER-modified Ti surface exhibit excellent biocompatibility, altered morphology, modified-cytoskeletal structures and focal adhesions

To analyse the effects of the Ti surfaces on the hSSC1 cytoskeleton, fluorescence staining was performed with vinculin monoclonal antibody and

Discussion

Ti surface modification to enhance implant function can be achieved using a variety of methods. In the current study we demonstrate the efficacy of LASER irradiation of titanium to generate a surface to improve skeletal stem cell function. LASER irradiation has been shown to be a promising method for Ti surface treatment, increasing the Ti surface area, wettability and, critically, offering a high degree of surface purity at relatively low cost [11], [12], [19]. Furthermore, studies with

Acknowledgements

The authors thank Franco Conforti, Atsushi Takahashi and Anton Page for technical advice; and Lindsey Goulston for critical advice on this paper. Funding for this work is gratefully acknowledged from the following: Science without border Scholarship code (10331-12-3) for K.E.S. from CAPES/Brazil. Work in the Oreffo laboratory is funded through the BBSRC (BB/GO105791), EU (MarieCurie IRSES) and MRC (MR/K026682/1). We gratefully acknowledge the provision of bone samples by the orthopaedic

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