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. 2014 Feb;10(2):651-60.
doi: 10.1016/j.actbio.2013.11.008. Epub 2013 Nov 16.

Osteogenic lineage restriction by osteoprogenitors cultured on nanometric grooved surfaces: the role of focal adhesion maturation

John W Cassidy  1 , Jemma N Roberts  2 , Carol-Anne Smith  2 , Mary Robertson  3 , Kate White  4 , Manus J Biggs  5 , Richard O C Oreffo  4 , Matthew J Dalby  2
Affiliations

Osteogenic lineage restriction by osteoprogenitors cultured on nanometric grooved surfaces: the role of focal adhesion maturation

John W Cassidy et al. Acta Biomater. 2014 Feb.

Abstract

The differentiation of progenitor cells is dependent on more than biochemical signalling. Topographical cues in natural bone extracellular matrix guide cellular differentiation through the formation of focal adhesions, contact guidance, cytoskeletal rearrangement and ultimately gene expression. Osteoarthritis and a number of bone disorders present as growing challenges for our society. Hence, there is a need for next generation implantable devices to substitute for, or guide, bone repair in vivo. Cellular responses to nanometric topographical cues need to be better understood in vitro in order to ensure the effective and efficient integration and performance of these orthopedic devices. In this study, the FDA-approved plastic polycaprolactone was embossed with nanometric grooves and the response of primary and immortalized osteoprogenitor cells observed. Nanometric groove dimensions were 240 nm or 540 nm deep and 12.5 μm wide. Cells cultured on test surfaces followed contact guidance along the length of groove edges, elongated along their major axis and showed nuclear distortion; they formed more focal complexes and lower proportions of mature adhesions relative to planar controls. Down-regulation of the osteoblast marker genes RUNX2 and BMPR2 in primary and immortalized cells was observed on grooved substrates. Down-regulation appeared to directly correlate with focal adhesion maturation, indicating the involvement of ERK 1/2 negative feedback pathways following integrin-mediated FAK activation.

Keywords: Bone formation; Contact guidance; Focal adhesion; Tissue engineering; Topography.

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Figures

Fig. 1
Fig. 1
Focal adhesion analysis (OPG cells). Greyscale images of immunolabelled adhesions were exported to Adobe Photoshop (panel A) and each identified adhesion traced with a 1-pixel straight line, creating an adhesion mask superimposed over the background image (panel B). Care was taken to avoid labeling bleed from the actin channel. The background image was removed (panel C) and the adhesion mask exported to ImageJ for analysis. Panel D shows a complete RGB image: red is actin, green vinculin and blue DAPI (nuclear). Bar is 25 μm.
Fig. 3
Fig. 3
Surface optimization. Water contact angle analysis (panel A) and AFM surface imaging (panel B) of untreated 540 nm grooved controls and the same surface after 30 s (panels C and D) and 2 min plasma treatment (panels E and F). Panel F shows groove integrity compromised, thought to be due to prolonged plasma treatment.
Fig. 4
Fig. 4
Cellular growth assays. Water contact angle analysis of surfaces after various plasma treatment times (trendline; right axis) with corresponding MG63 cell counts at 24 h (bar chart; left axis). There is a marked and statistically significant (***p < 0.001) fall in surface energy after 30 s treatment, as seen by WCA, and a corresponding increase in 24 h MG63 cell count over 10 random fields of view (***p < 0.001).
Fig. 5
Fig. 5
Focal adhesion analysis (OPGs). Adhesion analysis of OPG cells cultured on test and control substrates, statistical significance (t-test) relative to planar controls is noted (*p < 0.05, **p < 0.01 and ***p < 0.001). Panel A shows absolute adhesion subtype counts, panel B shows the percentage of each subtype and panel C shows total adhesion counts. In addition to differences relative to controls, the absolute numbers of SMAs between cells cultured on 540 nm and 240 nm grooves was found to be statistically significant (**p < 0.01). Adhesion classification rationale can be found in the methods (FX<1 μm
Fig. 6
Fig. 6
Mature adhesion analysis (OPGs). Graphs show mature adhesion breakdown of primary osteoprogenitors on test and control surfaces. In addition to super mature adhesions (SMAs > 5 μm), these data distinguish large SMAs (SMAs > 8 μm) and very large SMAs (>10 μm). Panel A shows absolute counts whereas panel B shows the proportion of each subtype. Statistical significance (t-test) relative to planar controls is noted (*p < 0.05, **p < 0.01 and ***p < 0.001).
Fig. 7
Fig. 7
MG63 focal adhesion analysis. Focal adhesion analysis of MG63 immortalized osteoblast-like cells cultured on test and control substrates. Panel A shows absolute adhesion subtype counts, panel B shows the percentage of each subtype and panel C shows total adhesion counts. Adhesion classification rationale can be found in the methods (FX < 1 μm < FA < 5 μm < SMA). Statistical significance (t-test) relative to planar controls is noted (*p < 0.05, **p < 0.01 and ***p < 0.001).
Fig. 2
Fig. 2
Nuclear distortion analysis (OPG cells). Greyscale images captured in the DAPI channel (panel A) were exported and nuclei traced along their long and short axes (panels C and D). Background images where removed (panels E and F) and axis lengths calculated in ImageJ. The corresponding ratio gives a measure of nuclear elongation along the major axis. Panel B shows a complete RGB image: red is actin, green vinculin and blue DAPI (nuclear). Bar is 50 μm.
Fig. 8
Fig. 8
Relative focal adhesion alignment and nuclear distortion (OPGs). Focal adhesion alignment quantified via FA and SMA angle standard deviation (panel B). Nuclear distortion is presented as a ratio of longest over shortest axis, higher values indicate elongated nuclei (panel A). Statistical significance (t-test) relative to planar is noted (*p < 0.05, **p < 0.01 and ***p < 0.001). Cells cultured on grooved surfaces showed significantly more elongated nuclei and less variant adhesion angles relative to those on planar controls. 540 nm-grooved surfaces tended to correspond to more angle variation and less distorted nuclei than 240 nm-grooved surfaces, though this trend was not significant.
Fig. 9
Fig. 9
Relative expression of target genes BMPR2 and RUNX2 (OPGs). Values presented are RQ (expression relative to planar control) and have been normalized to housekeeping gene GAPDH by the delta delta CT method. Each experiment consisted of three replicates per condition and each replicate was the result of four pooled samples. Both BMPR2 and RUNX2 expression was significantly (***p < 0.001 and **p < 0.01 respectively) down-regulated in both test conditions relative to planar controls. Expression of target genes was down-regulated to a greater extent on 240 nm compared to 540 nm-grooved substrates in each experiment, though this trend was not significant. MG63 down-regulation was significant (*p < 0.05) on 240 nm substrates but not on 540 nm (RUNX2 p = 0.07; BMPR2 p = 0.07) (panel B).
Supplementary Figure 1
Supplementary Figure 1
Validation of focal adhesion analysis (OPG cells). Greyscale images of immunolabelled adhesions labeled for paxillin were exported to Adobe Photoshop, and adhesions characterized as described in Fig. 1. Top panel shows RGB composite images of cells cultured on either planar or 540 nm-grooved PCL substrates. Red is actin, green paxillin and blue is nuclear; bar is 10 μm. Bottom panel shows focal adhesion subtypes and percentage subtype normalized to cell numbers (40 distinct cells). Relative proportions were similar to vinculin-based analysis, validating the use of vinculin as an adhesion marker. Statistical significance (t-test) relative to planar is noted (*p < 0.05, **p < 0.01 and ***p < 0.001).
Supplementary Figure 2
Supplementary Figure 2
Protein data from Oligo GEarrays (Stro-1 MSC cells). Fig. shows bone-related gene expression of MSC cells cultured on 10 μm wide and 330 nm deep grooved surfaces, embossed into polymethylmethacrylate (PMMA) with the arrays performed as per the manufacturer’s instructions. Panel A shows control and panel B shows 330 nm grooves. Panel C shows fold decrease in gene expression. 8 is osteocalcin (non-collagenous protein found in bone); 13 is BMP4 (bone morphogenic protein 4, involved in bone formation); 41 is collagen, type IX, alpha 2; 58 is VEGF (vascular endothelial growth factor, angiogenesis during bone formation); 63 is insulin-like growth factor 1; 66 is Integrin, alpha 1; 84 is RUNX2; 99 is Stratherin; 100 is Tuftelin interacting protein 11 (involved in mineralization process). 123 is GAPDH control.
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