Osteoblast and stem cell response to nanoscale topographies: a review

Figures & data

Figure 1. Scanning electron microscope (SEM) images of hFOB cells on (a) 11 nm, (b) 38 nm, and (c) 85 nm islands after cultured for 24 h. Additional SEM images of hFOB cells cultured on 85 nm islands after (d, e) 3 h and (f) 24 h. Arrowhead and arrow indicate the interaction with the top and other portions of island respectively. Images reproduced with permission from [42].

Figure 2. Atomic force microscopy (AFM) images of poly-lactic acid (PLLA)/PS demixed nanopit-textured films spin-cast at 0.5% solution concentration (forming 14 nm deep pits),1% solution concentration (forming 29 nm deep pits), and 1.5% solution concentration (forming 45 nm deep pits) and flat PLLA films. Images reproduced with permission from [46].

Figure 3. Paxillin (green) and vinculin (green) immunofluorescence staining double-labeled with actin (red) for hFOB cultured for 24 h on PLLA/PS demixed nanopit-textured films and flat PLLA films. Images reproduced with permission from [46].

Figure 4. SEM image of (A) human bone marrow stem cells (HMSCs) with normal morphology on planar control materials; (B, C) filopodia interaction with the 3:1000 substrates (arrowheads) and inset on (C) shows filopodia curving around an island; (D, E) filopodia interaction with the 3:3000 substrates (arrowheads) and inset on (E) shows filopodia curving around an island; (F) filopodia interaction with the hemi substrates (arrowheads); and (G) filopodia curving around a hemisphere. Images reproduced with permission from [47].

Figure 5. Osteocalcin (OCN) and osteopontin (OPN) fluorescence images of HMSCs cultured on control and test materials. Cells on planar control formed confluent layers but very little OCN or OPN stained was observed on day 21. Bone nodule formation can be seen on 3:1000. (Note: red = actin, green = OCN/OPN). Images reproduced with permission from [47].

Figure 6. The topmost row shows the images of nanotopographies fabricated by electron beam lithography (EBL). All the pits are 120 nm in diameter, 100 nm deep and have average 300 nm center–center spacing with square, displaced square 20 (±20 nm from true center), displaced square 50 (±50 nm from true center) and random arrangements. (a, f) MSC with fibroblastic appearance and absence of OPN or OCN positive cells on the control, (b, g) no OPN or OCN positive cells on SQ, (c, h) OPN positive cells but no OCN positive cells on DSQ 20, (d, i) OPN and OCN positive cells and nodule formation (arrows) on DSQ 50, (e, j) MSC with osteoblastic morphology and absence of OPN or OCN positive cells on RAND, (k) MSCs with fibroblastic morphology on control after 28 days of culture and (l) mature bone nodules on DSQ 50 after 28 days of culture. Images reproduced with permission from [49].

Figure 7. Expression of progenitor and osteoblast markers by MSCs cultured on SQ, NSQ50 and controls (i.e. flat and osteogenic media (OGM)) after four and eight weeks of culture and the insets show SEM images of the SQ and NSQ50 surfaces. (a) On the flat surface the cells had fibroblast-like morphology and the heterogeneous cell population, retained stromal precursor antigen-1 (STRO-1) and activated leukocyte cell adhesion molecule (ALCAM) expressions (i.e. the MSC markers) and expressed OCN and OPN markers. On the OGM control, the expression of the less specific progenitor marker, ALCAM, was retained while OCN and OPN expressions were noted. The cells had grown confluence on SQ and no expression of OCN or OPN was noted. STRO-1 and ALCAM markers were highly expressed on SQ but only low levels of STRO-1 were noted on NSQ50. (b) STRO-1 is a more stringent marker for MSC than ALCAM which is expressed by both stem cells and progenitor cells. Expression of ALCAM on NSQ50 at eight weeks suggests that there are still osteoprogenitor cells present although the actual MSC numbers have dwindled. In all images, green = phenotypic marker, red = actin (cell morphology) and blue = nucleus. Images reproduced with permission from [50].

Figure 8. Scanning electron micrographs of filopodia of osteoprogenitor cells cultured on (A) planar control; (B) square (SQ) nanopit arrays and (C) hexagonal (HEX) nanopit arrays. Images reproduced with permission from [52].

Figure 9. SEM images of (A, B) hBMCs with normal morphologies on planar control materials; (C) hBMC conforming to a groove edge of smaller and shallower pit (arrow); (D) hBMC with filopodia entering a small pit and inset (e) shows evidence of endogenous matrix formation; (E) filopodial guidance in larger and deeper pit (arrow); (F) filopodial guidance (arrow) and inset (e) shows evidence of endogenous matrix formation; (G, H) contact guidance of hBMC and their filopodia on narrow grooves; (I) hBMC aligning along the wide grooves (arrow) and spanning across grooves (double headed arrows); (J) filopodial guidance on the wide grooves (arrows). Images reproduced with permission from [54].

Figure 10. Fluorescence images of actin, tubulin and vimentin cytoskeletons and vinculin (focal adhesions) of HBMSCs cultured on control and test materials. Increased cytoskeleton organizations and numbers of focal adhesions were seen on the topographies compared to cells on planar control. (N) tubulin is seen condensing along grooves while in (P), adhesions are seen aligning to the grooves. Images reproduced with permission from [54].

Table 1. Summary of cell response to nanotopographies.

Technique	Materials used	Pattern and dimension	Cell type	Cell responses	Ref.
Polymer demixing	PS/PBrS	Nanoislands with an average height of 11, 38 and 85 nm respectively	hFOB	Higher cell adhesion and larger cell size on 11 nm nanoislands.	[42]
				Similar ALP activity on 85 nm nanoislands and TCPS
Polymer demixing	PMMA	3:1000 imprint (height:45 nm, diameter: 2.2 μm, center-to-center distance: 4.3 μm)	HMSCs	Increased cell spreading, enhanced expression of stress fibers, tubulin and vimentin networks and higher OCN and OPN expression on nanotopographies compared to control	[47]
		3:3000 imprint (height: 33 nm, diameter: 1.7 μm, center-to-center distance: 2.9 μm)		Cells extending filopodia on hemispheres and curling around imprints
		Hemispheres (height:10 ± 1 nm, diameter: 144 ± 11 nm, spacing:184 ± 24 nm)
Polymer demixing	PLLA/PS	Pit-shaped topography of 14 nm, 29 nm, 45 nm deep respectively	hFOB	Higher cell coverage, αv integrin and paxillin expression on 14 nm and 29 nm	[46]
				Greatest cell attachment on 14 nm followed by 29 nm and 45 nm pits
				Invariant vinculin expression on pits
Photolithography	PMMA	Pit diameter to depth ratio: 30:310 nm (denoted as 30:300P) 40:362 nm (denoted as 40:400P)	hBMCs	hBMCs conformed, formed filopodial contact and exhibited increased cell spreading, increased tubulin and vimentin networks as well as OCN and OPN expressions on the pit patterns	[54]
				Larger hBMCs cell area, more defined stress fibers and mature nodule formation (after 21 days) on 40:400P
		Groove width to depth ratio: 5:510 nm (denoted as 5:500G) 50:327 nm (denoted as 50:300G)		Better contact guidance, significant reduction of cell area, highly aligned stress fibers along the groove direction and tubulin condensing along the ridges on 5:500G
				Aligned vimentin and increased areas of OCN and OPN production on groove patterns
EBL	PMMA	100 nm deep (D: 120 nm) PMMA imprints arranged:	MSC	Elongated, and aligned morphology with fibroblastic appearance on SQ and planar control	[49]
		SQ: in square array with center-to-center spacing of 300 nm		Decreased osteoprogenitor density on HEX
		HEX: hexagonal array		Increased level of OPN, OCN and mineralization on DSQ50 after 28 days
		DSQ50: randomly up to 50 nm on both axes from their position in a true square		Osteoblastic morphology and expressed foci of OPN on DSQ20
		DSQ20: randomly up to 20 nm on both axes from their position in a true square		Denser cell growth on RAND compared with planar PMMA
		RAND: randomly over a 150 μm by 150 μm field, repeated to fill a 1 cm^2 area		Polygonal, osteoblastic morphology on RAND after 21 days
EBL	PC	PC imprints comprised of 120 nm diameter pits with 300 nm center-to-center spacing in SQ and HEX arrangements	Osteoprogenitor cell	Stellate cell structure, higher number of filopodia per μm of membrane and presence of cortical actin on PC imprints	[52]
				Higher cell spread on hexagonal arrangement and planar control Stress fibers on planar control
EBL	Polycaprolactone (PCL)	SQ: PCL imprints comprised of 120 nm deep pits in square arrangement with center-center spacing of 300 nm	MSC	SQ induced a switch from osteogenic stimulation to a surface conducive to MSC growth and permitted prolonged retention to MSC markers and multipotency	[50]
		NSQ50: PCL imprints comprised of 120 nm deep pits with ±50 nm offset in both x- and y- axes		MSC differentiated into osteogenic cells on NSQ50 and OGM controls after four and eight weeks
				STRO-1, ALCAM, OPN and OCN markers expressed by cells on NSQ50 and OGM
				Increased OPN expression on NSQ50 and OGM after a few weeks
				Cells exhibited raised/similar metabolomic profiles on NSQ50 and OGM compared to SQ

Abbreviation: OGM, osteogenic media.

Figure 11. SEM micrograph of (a) nanoislands around a gadolinia-doped ceria (GDC) particle after annealing at 1100 °C for 5 h with 10 °C min^–1 heating and 1 °C min^–1 cooling rates; and (b) smaller powder particles with relatively broader nanoisland coverage after heat treatment. Images reproduced with permission from [71].

Figure 12. SEM of morphology of representative SK-N-SH neuroblastoma cells on the (a) smooth control; (b) islands; (c) connected islands, and (d) pits. Images reproduced with permission from [74].

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