lable at ScienceDirect Biochemical and Biophysical Research Communications 473 (2016) 719e725 Contents lists avai Biochemical and Biophysical Research Communications journal homepage: www.elsevier .com/locate/ybbrc Skeletal stem cell and bone implant interactions are enhanced by LASER titanium modification Karin E. Sisti a, b, c, *, María C. de Andr�es a, David Johnston a, Edson Almeida-Filho b, Antonio C. Guastaldi b, Richard O.C. Oreffo a a Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton SO16 6YD, UK b Biomaterials Group, Institute of Chemistry, S~ao Paulo State University (UNESP), Box 355, Araraquara, Brazil c Federal University of Mato Grosso do Sul (UFMS), Campo Grande, Brazil a r t i c l e i n f o Article history: Received 23 September 2015 Accepted 2 October 2015 Available online 9 October 2015 Keywords: Titanium surface Skeletal stem cell Tissue regeneration Bone formation LASER * Corresponding author. Bone and Joint Research Development, Stem Cells and Regeneration, Institute University of Southampton, Southampton SO16 6YD, E-mail address: karinellensisti@gmail.com (K.E. Si http://dx.doi.org/10.1016/j.bbrc.2015.10.013 0006-291X/© 2015 Elsevier Inc. All rights reserved. a b s t r a c t 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-1þ 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, prolif- eration, 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 osteo- pontin 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. © 2015 Elsevier Inc. All rights reserved. 1. Introduction Research in the field of biomaterials has advanced significantly Group, Centre for Human of Developmental Sciences, UK. sti). 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 conse- quence 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 Delta:1_given name Delta:1_surname Delta:1_given name Delta:1_surname Delta:1_given name Delta:1_surname mailto:karinellensisti@gmail.com http://crossmark.crossref.org/dialog/?doi=10.1016/j.bbrc.2015.10.013&domain=pdf www.sciencedirect.com/science/journal/0006291X www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2015.10.013 http://dx.doi.org/10.1016/j.bbrc.2015.10.013 http://dx.doi.org/10.1016/j.bbrc.2015.10.013 K.E. Sisti et al. / Biochemical and Biophysical Research Communications 473 (2016) 719e725720 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 sur- rounding 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 e 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 im- plants [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 osseointegra- tion [6,10e13]. Understanding how to control, manipulate, and enhance the intrinsic healing events modulated through osteogenic differenti- ation 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 fea- tures 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 informa- tion 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. 2. Methods 2.1. Cell culture Skeletal cell populations STRO-1þ hSSCs1 were isolated and cultured following previously described protocols [17] with the approval of the Local Research Ethics Committee (LREC 194/99). 2500 STRO-1þ 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 Basal 1 hSSCs(human Skeletal Stem Cells). 2 Mb(Machined Ti, basal media). 3 Mo(Machined Ti, osteogenic media). 4 Lb(LASER-modified Ti, basal media). 5 Lo(LASER-modified Ti, osteogenic media). media was DMEM with 10% FCS and osteogenic media included 10 nM dexamethasone. 2.2. Ti discs Ti discs were prepared at UNESP(Araraquara/Brazil). 180 Ti rods were cut into 8 mm diameter by 2 mm long cylinders, and the surfaces of 90 discs were modified by LASER beam as described previously [6,11,18]. All samples were sterilized by ethylene oxide. 2.3. Analysis of hSSC1 proliferation and viability Cell number was determined using a standard DNA PicoGreen assay [18]. Cell lysate was measured for DNA content using Pico- Green (Molecular Probes, Paisley, UK) analysed using a BioTek FLx- 800 microplate fluorescent reader. 2.4. Live/dead immunostaining CTG7 was used to label viable cells and EH-18 for necrotic cell nuclei. Cell images were assessed for cell viability using Zeiss Axi- ovision software Ver 3.0 via an AxioCam HR digital camera on an Axiovert 200 inverted microscope (Carl Zeiss, Hertfordshire, UK) under fluorescent light. 2.5. Analysis of the osteogenic differentiation of hSSCs1 ALP6 activity within the cell lysate was measured using p- nitrophenyl phosphate as the substrate in 2-amino-2-methyl-1- propanol alkaline buffer solution (Sigma, Poole, UK), analysed us- ing a BioTek ELx-800 microplate reader to provide specific enzyme activity (ALP6/DNA/hr) across samples. 2.6. Analysis of cell adherence and morphology, cytoskeleton structure and focal adhesion e confocal laser scanning microscopy The FAK100 j Actin Cytoskeleton/Focal Adhesion Staining Kit (Millipore®) was used to analyse cytoskeleton modifications following culture of the hSSCs1 on the different Ti samples. In brief, cells were fixed in 4% formaldehyde in PBS, permeabilised (0.1% Triton X-100 in PBS) and blocked (1% BSA in 1% PBS). Cells were incubated with a primary anti-vinculin mouse monoclonal anti- body (1:100) and then with FITC-conjugated goat anti-mouse sec- ondary antibody (1:100) and cultures were then stained simultaneously with TRITC-conjugated phalloidin (1:1000) (to enable labelling of actin filaments) and DAPI (1:1000 dilution of a 1 mg/ml sotck). The secondary antibody alone was used as a negative control. Images were taken using a confocal laser scanning microscope (Leica TCS SP5, Leica Biosystems, Wetzlar, Germany). 2.7. SEM Samples were fixed whole in 3% gluteraldehyde and 4% form- aldehyde in 0.1 M PIPES buffer at pH 7.2. A post-fixative of 1% osmium tetroxide was applied prior to dehydration through a se- ries of graded alcohols followed by critical point drying. The surface was putter-coated with gold-palladium and visualized with an FEI Quanta 200 SEM (FEI, Oregon, USA) to observe the morphology and attachment of cells on the Ti surfaces. 6 ALP(Alkaline Phosphatase). 7 CTG(Cell Tracker Green). 8 EH-1(Ethidium Homodmer-1). K.E. Sisti et al. / Biochemical and Biophysical Research Communications 473 (2016) 719e725 721 2.8. Molecular analysis Following incubation of hSSCs1 on the different surfaces, sam- ples were washed, incubated with collagenase IV, trypsinised and total RNA then extracted using RNeasy Plus Mini Kit (Invitrogen) to enable gene expression analysis. Extracted RNA was reverse tran- scribed using VILO cDNA synthesis kit (Invitrogen®) for RT-PCR. qRT-PCR was performed using a 96-well optical reaction plate and a 7500 Real Time PCR system (Applied Biosystems, Carlsbad, USA). Each sample was subjected to qRT-PCR against a panel of osteogenic gene primers (Table 1). Values were calculated using the comparative threshold cycle (Ct) method, normalized to b-actin expression and expressed as the mean ± SD. 2.9. Statistics All experiments were run three times using four independent samples. Data was expressed as the mean ± SD. Statistical analysis was performed using SPSS v 17.0 (SPSS Inc, Chicago, IL/USA). The Wilcoxon's signed rank test was used to compare between groups. P values less than 0.05 were deemed significant. 3. Results 3.1. 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. 1AeD). 3.2. 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 cytoskel- eton, fluorescence staining was performed with vinculin mono- clonal antibody and TRITC-conjugated phalloidin. Enhanced cell spreading and cytoskeletal (actin) structure rearrangement was observed in cells cultured on the LASER-modified surface as ana- lysed using confocal microscopy (Fig. 2B, D, F). Actin filaments (red) in hSSCs1 grown on machined surface were observed to be orga- nized parallel to the underlying surface topography (Fig. 2A, C and E), while cells cultured on LASER-modified surfaces exhibited actin Table 1 Human osteogenic gene primer sequences used for RT-PCR. Protein Gene Primer sequences B-Actin (housekeeping gene) B-Actin F:50- GGCATCCTCACCCTGAAGTA R:50- AGGTGTGGTGCCAGATTTC Alkaline phosphatase ALP F:50-GGAACTCCTGACCCTTGACC R:50-TCCTGTTCAGCTCGTACTGC Collagen type 1A1 COL1A1 F:50-GAGTGCTGTCCCGTCTGC R:50-TTTCTTGTTCGGTGGGTG Runt-related transcription factor 2 RUNX2 F:50-GTAGATGGACCTCGGGAACC R:50-GAGCTGGTCAGAACAAAC Sex-determining region Y, box 9 SOX9 F:50-CCCCAACAGATCGCCTACAG R:50-GAGTTCTGGTCGGTGTAGTC Osteopontin OPN F:50-GTTTCTCAGACCTGACATCC R:50-CATTCAACTCCTCGCTTTCC Osteocalcin OCN F:50-GGCAGCGAGGTAGTGAAGAG R:50-CTCACACACCTCCCTCCTG filaments arranged randomly (Fig. 2B, D, F). hSSC1 focal adhesion formation was evidenced by vinculin staining (green) and pre- sented at the cell periphery on LASERmodified surface (Fig. 2F, J). In contrast, the actin filaments of hSSCs1 cultured on machined sur- facewere oriented in a predominantly parallel manner (Fig. 2A, C, E, G, I, K). 3.3. SEM Used for cell adherence and morphology following growth of hSSCs.1 hSSCs1 exhibited discrete differences in cell morphology as a function of profile surface and media. All groups displayed healthy adherent cells on Ti surfaces (Fig. 3). On machined surface, cells presented as flattened structures, with a distinct spread morphology. On Mb2 surfaces, hSSCs1 displayed few protoplasmic processes attached to the surface (Fig. 3I). Cells were observed to be distributed over the LASER surface and to form cytoplasmic bridges of variable thickness, suspended above the peaks and depressions of the LASER-modified surface (Fig. 3B and D). On the LASER- modified surfaces, the hSSCs1 presented numerous protoplasmic processes (Fig. 3F, H, J and L). Filopodia were shown as a conse- quence of culture in osteogenic culture media (Fig. 3 K and L). 3.4. LASER-modified Ti surface enhance osteogenic differentiation of hSCCs1 To investigate the effect of hSSCs1 differentiation on LASER- modified surfaces after 10 days in culture, the expression of oste- ogenic markers was analysed using biochemical and molecular approaches. ALP6 specific activity was increased in hSSCs1 cultured on LASER-modified surfaces compared to hSSCs1 cultured on con- trol (machined) surfaces, in basal culture media (154.28% vs 100% Mb2 control) (Fig. 1). Molecular analysis showed that hSSCs1 seeded on Lb4 displayed enhanced osteogenic marker gene expression in comparison to hSSCs1 cultured on Mb.2 Specifically, ALP6 and OPN mRNA levels in hSSCs1 cultured on Lb4 were respectively 2-fold and 3.6-fold higher than those cultured in Mb2 (Fig. 4). 3.5. Osteogenic conditions do not modulate hSSCs1 cultured on LASER-modified Ti surface The induction of proliferation and osteogenic differentiation of hSSCs1 were assessed using standard osteogenic culture medium with samples from the same patients cultured on machined and LASER surfaces. Results revealed that the hSSCs1 did not show any statistically significant increases in cell proliferation (mean ± SD 103 ± 20.94 versus 160 ± 8.27) (Fig. 1), ALP6 specific activity (mean SD 124.71 ± 33.78 versus 134.78 ± 23.11) and osteogenic marker gene expression ALP6 (mean ± SD 4.82 ± 1.190 versus 8.814 ± 4.98) and OPN (mean ± SD 1.2 ± 0.84 versus 0.76 ± 0.09) (Fig. 4) or any cytoskeleton modification (Fig. 2). 4. 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 LASER-modified materials implanted in rabbit tibias and subsequently presenting high Fig. 1. Biochemical analysis (DNA, ALP6 and Specific Activity ALP6/DNA) of hSSCs1 on Ti discs (10 days). Error bars denote Standard Deviation. *p < 0.05. AeD Immunofluorescence (cell tracker green e inverted microscope 20X magnification, scale bar ¼ 100 mm) of hSSCs1 (10 days) on Mb,2 Lb,4 Mo3 and Lo.5 Fig. 2. Confocal images: cytoskeletal structure hSSCs1 (10 days) on Mb2 (A,E,I), Lb4 (B,F,J), Mo3 (C,G,K) and Lo5 (D,H,L). Red: actin. Blue: nucleus. Green: vinculin. Cell focal adhesion (arrows). Scale bar ¼ 50 mm. K.E. Sisti et al. / Biochemical and Biophysical Research Communications 473 (2016) 719e725722 Fig. 3. SEM: micrographs of hSSCs1 adherence and morphology (10 days) on Mb2 (A,E and I), Lb4 (B,F and J), Mo3 (C,G and K) and Lo5 (D,H and L). AeD scale bar ¼ 50 mm, EeH scale bar ¼ 20 mm and IeL scale bar ¼ 5 mm. Fig. 4. hSSC1 gene expression of osteogenic markers (ALP,6 RUNX2, COL1A1, OCN, OPN) and chondrogenic marker (SOX9) following culture for 10 days in basal and osteogenic conditions, on machined surface and LASER Ti surfaces (b-actin ¼ internal control). Values are mean ± SD of 4 independent samples, *p < 0.05). K.E. Sisti et al. / Biochemical and Biophysical Research Communications 473 (2016) 719e725 723 K.E. Sisti et al. / Biochemical and Biophysical Research Communications 473 (2016) 719e725724 removal torques [11,12,19] and properties that favour cell adhesion and proliferation make this an attractive approach [10,20]. We have previously shown that the topography of a Ti LASER-modified surface displays distinct topographies including a surface rough- ness with an appearance comparable to a “cauliflower” morphology that provide enhancedwettability and surface area [6]. The biocompatibility of biomaterials is closely related to cell viability and proliferation with attachment, adhesion, and spreading in the early phase of the cell/material interaction being critical in modulating the capacity of a cell to proliferate and differentiate [21]. The LASER treated surfaces in the current study, provide a topography that supported hSSC1 viability and prolifer- ation. A wealth of studies indicate rough Ti surfaces can enhance osseointegration in the clinic in comparison to smooth surfaces [6,7,12,22], although the cellular and molecular mechanisms that drive this process remain far from clear. ALP6 activity is typically used as a marker to follow the differentiation of osteoblasts from non-calcium-depositing to calcium-depositing cells [23], and as a marker of the early stages of osteogenic differentiation [24]. Studies suggest the ALP6 activity of a cell is surface-dependent. Thus, if the Ti surface is modified elevated ALP6 activity is a likely consequence [25]. In the current study, hSSCs1 on LASER-modified Ti surfaces, displayed enhanced differentiation as assessed by ALP6 activity. It is assumed this is a consequence of enhanced material surface reac- tivity [26] and enhanced physicalechemical properties [6]. In contrast, Takeuchi et al. showed that a modified surface can reduce cell proliferation whilst initially driving the expression of specific cell markers except for ALP6 [27]. The cellebiomaterial interface functions not only to define the boundary between tissue and implant, but also to act as a mediator of first stage protein in- teractions as well as later stage cell adhesion and orientation [28]. When blood cells arrive at the implant Ti surface, the blood cells express a variety of integrins, resulting in cytoskeletal changes. Changes in cytoskeleton tension have a direct effect on cell morphology as evidenced by actin staining. Alteration in cell morphology as a consequence of cytoskeletal tension has an indi- rect effect onmechano-transduction pathways, as demonstrated by expression changes in stem cell responses [14,29]. In the current studies cytoskeletal rearrangement were observed, including altered expression patterns of vinculin, a key structural component of focal adhesions [30]. On the LASER topography, vinculin dis- played arrangement around the cell cytoplasm periphery. Inter- estingly, expression of the vinculin marker was spread distinctly revealing an extensive non focal distribution in the cytoplasm of hSSCs1 on the machined surface. Furthermore, hSSCs1 cultured on machine surfaces displayed a flat morphology, primarily orientated along the discrete grooves, with relatively few protoplasmic pro- cesses attached to the Ti substrate. In contrast, cells cultured on LASER Ti discs displayed enhanced adherence to the modified surface, indicating the potential for modified cellular activity or tissue responses leading to greater osteogenesis [3]. We have previously reported that changes in cytoskeletal ten- sion in response to topography may modify interphase nucleus organisation and hence directly influence cell gene expression profiles [14,31]. The pattern of five specific osteogenic markers, RUNX2, ALP,6 COL1A1, OPN and OCN and, the chondrogenic marker SOX9, in primary hSSCs1 cells cultured on LASER Ti surfaces were compared with machined Ti surface substrates. RUNX2 is essential for osteoblast maturation and osteogenesis [32] and is a key regu- lator of OCN, COL1 and ALP6 genes [33]. ALP6 and COL1A1 are matrix-mineralizing proteins, and their expression has been shown to be important for bone matrix assembly [34]. In the present study ALP6 gene expression showed a statistically significant increase from Mb2 to Lb,4 in agreement with other studies [35]. The mech- anisms of bone remodelling underline the potential role of two non-collagenous matrix proteins, osteopontin and osteocalcin [36]. Osteopontin is a multifunctional phosphorylated glycoprotein secreted by osteoblasts, and has been suggested to be present at an early stage of bone development and to promote the attachment of osteoblasts to the extracellular matrix [37]. Osteopontin is involved in bone remodelling [38] whilst osteocalcin is a marker of primary bone formation and is produced by osteoblasts [39]. While the precise role of osteocalcin is still under examination, roles as an endocrine regulator of metabolism in the skeleton and as a regu- lator of mineralization have been proposed. Serum concentrations of osteocalcin have been shown to correlate with histo- morphometric indices of newly formed bone [40]. The present study show that hSSCs1 cultured on LASER-modified Ti surfaces display enhancee osteopontin expression, indicating the possible osteogenic potential of LASER-modified Ti surfaces. Interestingly, no significant changes in osteocalcin expression were observed in the current study, in agreement with previous studies [41]. This may potentially be as a consequence of osteocalcin being a late marker of bone cell differentiation and osteopontin an early marker [42]. Although, COL1A1, RUNX2 and OCN expression did not show statistical significant differences across the substrates, a trend of enhanced expression on LASER-modified surfaces was observed. As expected, SOX9 expression, a chondrocytic marker was unaffected. In conclusion, this study demonstrated the influence of the microtexture of LASER-modified Ti surfaces on the behaviour of hSSCs.1 Cell proliferation, adhesion, osteogenic gene expression, cell morphology and cytoskeleton structure were all affected by the modified topography of Ti surfaces that resulted from LASER irradi- ation. These studies show the potential of Ti LASER modification to enhance the osseointegration at the material-bone cell interface with important implications for orthopaedic and dental application. 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 lab- oratory is funded through the BBSRC (BB/GO105791), EU (Mar- ieCurie IRSES) and MRC (MR/K026682/1). We gratefully acknowledge the provision of bone samples by the orthopaedic surgeons at Southampton General Hospital. Transparency document Transparency document related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2015.10.013. References [1] M. Geetha, A.K. Singh, R. Asokamani, A.K. 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http://refhub.elsevier.com/S0006-291X(15)30714-2/sref42 http://refhub.elsevier.com/S0006-291X(15)30714-2/sref42 Skeletal stem cell and bone implant interactions are enhanced by LASER titanium modification 1. Introduction 2. Methods 2.1. Cell culture 2.2. Ti discs 2.3. Analysis of hSSC1 proliferation and viability 2.4. Live/dead immunostaining 2.5. Analysis of the osteogenic differentiation of hSSCs1 2.6. Analysis of cell adherence and morphology, cytoskeleton structure and focal adhesion – confocal laser scanning microscopy 2.7. SEM 2.8. Molecular analysis 2.9. Statistics 3. Results 3.1. hSSCs1 cultured on LASER-modified Ti surface display enhanced cell growth and viability 3.2. hSSCs1 cultured on LASER-modified Ti surface exhibit excellent biocompatibility, altered morphology, modified-cytoskeletal ... 3.3. SEM 3.4. LASER-modified Ti surface enhance osteogenic differentiation of hSCCs1 3.5. Osteogenic conditions do not modulate hSSCs1 cultured on LASER-modified Ti surface 4. Discussion Acknowledgements Transparency document References