Kenneth E. Gonsalves, Craig R. Halberstadt, Cato T. Laurencin, Lakshmi S. Nair. Biomedical Nanostructures

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alignment and orientation of primary osteoblasts in the direction of grooves

[80]. The exposed patterned surface topography promoted the cell elongation,

cytoskeleton actin, and actinin molecules’ orientation in the direction of the

grooves. However, vinculin, a protein associated with focal adhesions,

appeared to concentrate at the opposite end of aligned cells presumably due

to the balancing tension generated from the surface anisotropy [80]. The

observed anisotropic behavior of osteoblasts on the nanogrooves might

enhance cell settlement on the nano topographic surface.

10.2.5 Cell Behavior Toward Nanotopographic Surfaces Created

by Self-Assembly

Self-assembly is a powerful technique to create nanostructures from various

polymers and biomolecules via molecular self-assembly. Molecules self-

assemble by virtue of weak noncovalent bonds. Such interaction forces may

be hydrogen bonds, electrostatic interactions, hydrophobic interactions, and

van der waals forces. All the biomacromolecules interact and self-assemble to

create complex functional defect-free and self-healing nanostructures. The

molecular self-assembly is simple, facile, and scalable. However, no direct

control over the fabrication process is possible, and also it is not possible, to

design speciﬁc geometric features. Using the bottom-up approach, many

nanostructures created were studied as scaffold materials for various

biomedical applications [81–95].

Self-assembled peptide nanoﬁber scaffolds (SAPNS) of branched and linear

peptide-amphiphile molecules as coating on poly(glycolic acid) scaffolds

resulted in preferential attachment of primary human smooth muscle cells on

branched than on linear peptide amphiphile and control [88]. The SAPNS as

sciatic nerve grafts could partially restore the optic tract and functional vision

in brachium transected experimental adult animals in vivo [94]. Rat

mesenchymal stem cells on SAPNS with RGD sequence resulted in increased

attachment, alkaline phosphatase (ALP) activity, and osteocalcin content

than the SAPNS without RGD sequence and tissue cultur e pol ystyrene [95].

Hybrid SAPNS scaffolds also supported MSC and resulted in homogeneous

bone formation in vivo in a rat subcutaneous model. In perfusion, in vitro

culture registered higher alkaline phosphatase activity and osteocalcin

content indicated enhanced osteogenic differentiation of MSC than control

and static culture [84]. The pentapeptide epitope isolucinelysinevaline

alaninevaline (IKVAV) containing SAPNS supported the neural progenitor

cells and caused selective rapid differentiation into neurons with lesser number

of astrocytes [87]. The IKVAV-incorporated SAPNS combine bioactive

epitope and nanotopography elicits the observed advantages [87]. MC3T3-E1

cells not only survive and proliferate on SAPNS but also possibly utilize

peptide molecules in their metabolic pathways [89]. Functionalized and

biomimetic SAPNS can elicit cellular events such as wound healing and tissue

regeneration by creating ECM recognition domains with nanofeatures [86].

276 BIOMEDICAL NANOSTRUCTURES

10.2.6 Cell Behavior Toward Nanotopographic Surfaces Created

by Phase Separation

The phase separation technique used for the fabrication of highly porous

scaffolds with controllable porosity can create a wide range of geometries and

dimensions including pits, ﬁbers, and irregular pore structures [81, 96– 99].

Phase separat ion either solidliquid or liquidliquid can be induced by

lowering the solution temperature. In brief, to a polymer solution in a low

melting point solvent, addition of a small quantity of water generates polymer-

rich and polymer-poor phases. Such a system cooled below solvent melting

point followed by vacuum drying to sublime the solvent produces the porous

scaffolds of both micro- and nanostructures. The technique can create highly

porous scaffolds with controllable porosity. The process is simple, facile, and

scalable and does not require any costly equipment. The inability to fabricate

well-organized patterns and speciﬁc geometric features is the limiting factor of

this technique.

Nanostructures formed by phase separation technique favored the adhesion,

proliferation, and differentiation of different cell lines studied. MC3T3-E1

showed better response on PLL A nanoﬁber scaffolds than the solid walled

controls created using reverse solid freeform fabrication and thermal phase

separation technique [96]. Signiﬁc antly higher cell number, and osteocalcin and

bone sialoprotein expressions were observed on nanoﬁber scaffolds. However,

observed lower expression of type I collagen on nanoﬁbers was presumably due

to the quicker differentiation of preosteoblasts [96]. PLLA nanoﬁber scaffolds

with an average diameter of 200 nm fabricated by phase separation technique

supported the nerve stem cell differentiation and neurite outgrowth [99].

Figure 10.5 present s the neonatal mouse cerebellum C17-2 stem cells on PLLA

nanoﬁber scaffolds presenting a differentiated cell with neurite penetration into

the scaffolds [99]. Thus, the nanoﬁber structures could mimic the ECM

FIGURE 10.5 SEM micrographs of PLLA nanoﬁber scaffolds fabricated by the phase

separation technique presenting neonatal mouse cerebellum stem cells seeded on these

materials after 1 day in culture (a) at a magniﬁcation of 500 and (b) differentiated cells

with neurite penetration into the nanoﬁber scaffolds identiﬁed by arrows at 2000.

(Reprinted with permission from Reference [99].)

CELL BEHAVIOR TOWARD NANO STRUCTURED SURFACES 277

structure and support the adhesion, proliferation, and phenotype expression of

the cells.

10.2.7 Cell Behavior Toward Nanotopographic Surfaces Created

by Colloidal Lithography

Colloidal lithography provides a means to pattern surfaces containing columns

and pits with controllable dimens ions. This technique utilizes nanocolloids as

an etch mask that is dispersed and self-assembled electrostatically into a

monolayer on the substrate surfaces. The area surrounding the nanocolloids is

etched away along with the surface itself leaving a patterned substrate using a

suitable etching technique. For example, surface nanotopographies containing

columns can be created using chemically assisted ion beam etching or directed

ion beam bombardment and pits with ﬁlm evaporation technique. Patterned

surface properties can be varied changing colloid size, colloid ionic strength,

and the monolayer coverage on the substrate surface. Colloidal lithography is

relatively cheap and less time consuming, and can pattern large surface area

with less effort; however, it is difﬁcult to create speciﬁc feature geometries.

Cells on these columns and grooves in nanoscale showed interesting structural,

morphological, an d phenotypic behaviors on these nanotopographies [37, 43,

44, 100–105].

Columns with an average height of 160 nm, diameter of 100 nm and with a

230 nm spacing between the features produced by colloidal lithography

resulted in less spreading of human ﬁbroblasts (h-tert BJ1) compared to

control plane surfaces [100]. Microarray analysis showed a shift in gene level

expression when compared to microtopographic surfaces. Upregulation of

several genes responsible for cellcell signaling, wound healing, proliferation,

and cell differentiation, and downre gulation of several genes such as collagen,

and cytoskeleton were observed. Such observed changes in gene level

expression were presumably due to the decreased cell spreadi ng on colloidal

nanotopography [100]. The ﬁbroblasts assumed more stellate and less well-

spread morphology on these nanocolumns. Fibroblasts produced increased

ﬁlopodia and were observed to interact with nanocolumns regularly with

ﬁlopodia [103]. An attempt to endocytose the nanocolumns by ﬁbroblasts

failed; however, some cells appeared to try and go further producing high levels

of Rac at sites of pseudopodia formation [37]. Human ﬁbroblasts cultured for a

short duration of time, 180 min, showed decreased cell adhesion and spreading

on these nanocolumns than the planar control [101]. Fibroblasts showed less

well-organized actin and vimentin cytoskeletons on these nanocolumns;

however, tubulin cytoskeleton was well organized with least number of

microtubules [101]. Silicon wafer surfaces modiﬁed with 50-nm colloidal gold

particles resulted in the direct interaction with the primary rat epitenon cells at

the peripheral cell membrane [102]. Nanocolumns with diameters of 58, 91,

111, and 166 nm resulted in increased spreading of rat pancreatic epithelial cells

when compared to ﬂat surfaces and spreading increased with increase in

278 BIOMEDICAL NANOSTRUCTURES

column diameter [44]. Epithelial cells (human bladder carcinoma, HTB4) on

nanotopographies containing continuous edged grooves with 184 nm depth

were seen to become align ed [43]. Cells on nanotopographies containing

hemispheres with 100 nm height and 167 nm diameter assumed less spread, less

round, and more stellate morphology than the control. Cells registered no

change in the production of cytokine on both the nanofeatures and a decreased

production of IL-6 and IL-8 on the nanofeatures than the control [43]. Mouse

mammary epithelial cells (HC11) on nanotopographies containing either

continuous or discontinuous edges of various depths ranging from 40 to

400 nm were seen to align on the grooves than the control [104]. Grooved

surfaces with continuous edges favored the orientation of cells with more

elongated cells than grooves with discontinuous edges [104]. Nanotopographies

containing heminanosp heres features with a height of 110 nm and various

packing densities of these features were coated with Ti oxide thin ﬁlms to

obtain a single chemistry [105]. These nanotopographies induced the release of

chemotactic macrophage activation agents and caused stress ﬁber and

ﬁbronectin formation from the primary human macrophages. Primary human

osteoblasts were seen to migrate away from these nanofeatures [105]. The

nanotopographies present physical cues for the cells that can elicit responses to

align the cells, increase or reduce cell adhesion and proliferation, alter

differentiation, and increase motility.

10.2.8 Cell Behavior Toward Nanotopographic Surfaces Com posed

of Random Nanoﬁbers Created by Electrospinning

Cells identify the exposed surface topo graphy and random nanoﬁber porous

matrices inﬂuence the adh esion, spreading, proliferation, and gene expression

of various cell types seeded on them. Mouse ﬁbroblasts adhered, migrated

through the pores, and integrated well with PLAGA nanoﬁbers, and the

development of the cell growth was guided by nanoﬁber architecture [106].

Fibroblasts changed morphology from a spread and ﬂat to a long and spindle

shape on nanoﬁbers when adhered to the nanoﬁbers detaching the ﬂat surfaces

[107]. Cross-linked gelatin nanoﬁbers with improved mechanical properties and

thermal stability supported human dermal ﬁbroblast proliferation, and a linear

increase in cell number was observed with time [108]. Scaffolds of polystyrene

nanoﬁber resulted in a signiﬁcant increase in smooth muscl e cell attachment

than control, and the ECN produced by oriented nanoﬁbers was similar to

native bladder tissue [109]. MC3T3-E1 cells showed signiﬁcantly increased cell

number and osteocalcin, alkaline phosphate production with increa sing culture

time on silk ﬁbroin nanoﬁbers [110, 111]. The silk ﬁbroin nanoﬁber scaff olds in

vivo showed good biocompatibility and enhanced bone regeneration without

any inﬂammatory reaction [110]. Human coronary artery SMCs showed

normal phenotypic shape on polycaprolactone and collagen nanoﬁbers, and

nanoﬁbers coated with collagen resulted in the migration of SM Cs inside the

nanoﬁber scaffolds and formation of smooth muscle tissue [112–114]. NIH 3T3

CELL BEHAVIOR TOWARD NANO STRUCTURED SURFACES 279

ﬁbroblasts and normal rat kidney cells showed the morphology and

characteristics of their counterparts in vivo on polyamide nanoﬁbers [115].

Bone marrow stromal cells’ (BMSCs) attachment and proliferation was

favored on gelatin/PCL nanoﬁbers, and cells were able to migrate inside the

scaffold [116]. Increased proliferation of H9c2 cardiac rat myoblasts was

observed on polyaniline gelatin blend nanoﬁbers with different cell morphol-

ogies at the initial time point; however, similar cell density and morphology

were attained after 1 week on all the nanoﬁber scaffolds as the cultures reached

conﬂuence [117, 118]. Cellnanoﬁber interaction and orientation is more

pronounced in the initial culture time, and once the conﬂuence is reached it is

difﬁcult to identify morphological changes. Hepatocytes cultured on the

galactosylated poly(e-caprolactone-co-ethyl ethylene phosphate) (PCLEEP)

nanoﬁbers exhibited similar functional proﬁles as on ﬂat control surfaces;

however, morphologicalchangeswere observed. Hepatocytesformed 50300-mm

spheroids on ﬂat surfaces whereas smaller aggregates of 20100 mm were

formed on nanoﬁber surfaces [119]. Random and fused surfa ce topographies

having ﬁber diameters in the range of 140 nm to 2.1 mm showed increased

MC3T3-E1 osteoprogenitor cell density in the presence of osteogenic factors

on the ﬁbers than the smooth surfaces. The cell density increased with

increasing ﬁber diameter while ALP expression was independent of surface

topography [120]. Osteoblast-like cells on starch/polycapolactone micronano-

ﬁbers showed interesting morphological and phenotype expression behavior

[121]. With the introduction of nanoﬁber structures on microﬁber scaffolds,

osteoblasts organized to bridge between microﬁbers and possessed much more

spread and stretched morphology in contrast to continuous monolayer on

microﬁbers. Additionally, the presence of nanostructure resulted in increased

ALP activity than the microﬁbers. Nanomicroscaffolds were fabricated by

electrospinning nanoﬁbers on PLAGA knitted scaffolds, and porcine bone

marrow stromal cells showed improved cellular behavior on nanoﬁber

combined scaff old than the control [122]. Increased cell attachment, faster

cell proliferation, and higher expression of collagen I, decorin, and biglycan

genes were observed on nanoﬁber-included scaffolds. Increased adhesion and

proliferation of human um bilical vein endothelial cells were observed on

poly(

L-lactide-co-e-caprolactone) nanoﬁber matrices (0.31.2 mm) whereas a

decline in cell adhesion and restricted spreading was observed on larger ﬁber

diameter (7.0 mm) [123].

10.2.9 Cell Behavior Toward Nanotopographic Surfaces Created

by Chemical Etching

Chemical etching i s a surface modiﬁcation process to create surf ace roughness

in nanometer scale length. In a typical procedure, material surfac es are soaked

in a variety of etchants such as sodium hydroxide (NaOH), nitric acid

(HNO

), and hydroﬂuoric acid (HF) to name a few and the selection of the

etchants depends on the material properties [124–127]. The material surface

280 BIOMEDICAL NANOSTRUCTURES

disintegrated as they were exposed to etchants, and the surface became rough

with pits and projections in nanometer scale length. It is possible to vary the

surface roughness b y varying exposure time, na ture, and concentration of

etchants to get various surface roughness dimensions in nanoscale . This

process is fa st, sim ple, and inexpensive; however, it is not possible to get

required geometry features since it is a surface treatment phenomenon. Thus,

created nanometer-scale roughness can potentially affect cellular b ehavior

[124–127].

Compacts of selenium (Se) metal particles in micron- and nanometer-size

range were tested as an anticarcinogenic orthop edic material [124]. These

compacts were chemically etched with different con centrations of sodium

hydroxide to create surfa ce roughness in nanometer scale. Osteoblast densities

increased on both nano- and microparticles with nanoscale roughness when

compared to reference wrought titanium and Se microparticles after 24 h of cell

culture under standard in vitro conditions [124]. The pillar patterned silicon

wafers were subjected to wet ch emical etch to produce nanometer-scale surface

roughness, and astrocytes showed preferential attachment to these surfaces

[125]. Transformed astrocytes preferred the wet etched surface to the

unmodiﬁed silicon substrate as seen in Fig. 10.6 [125]. The degree of selective

adhesion and spreading increased with duration of wet etch presumably due to

the increased surface area. Primary cortical astrocytes from neonatal rats

showed a preference for silicon glass over the wet etched surface. Such

observed cellular behaviors are presum ably due to the different cell types in the

study [125]. Nanostructures created on PLAGA and PU surfaces by chemical

etching showed improved adhesion and proliferation of bladder smooth muscle

cells [126]. Surface chemistry and increased nanometer surface roughness

created after prolonged chemical etching brought changes in the observed

cellular behavior. Nanostructures produced after longer duration of etching on

FIGURE 10.6 Transformed astrocytes cell line (LRM55) on (a) unmodiﬁed silicon

glass and wet etched glass surface, and (b) the same at higher magniﬁcation. Cells

appear ﬂat and well attached showing very few projections on wet etched surfaces,

whereas cells appear not closely adhered to the surface that contains the complex

projections and rufﬂes. (Reprinted with permission from Reference [125].)

CELL BEHAVIOR TOWARD NANO STRUCTURED SURFACES 281

PLAGA surfaces registered signiﬁcantly higher cell number compared to

submicron surfaces, ﬂat untreated and control glass surfaces. While

nanostructured surfaces of PU did not show increasing proliferation trend

with increasing nanometer surface roughness, chemically treated surfaces and

submicron dimension surface roughness showed higher cell number than the

control and conventional ﬂat surfaces [126]. Further nanometer surface

roughness on PLAGA resulted in absorption of signiﬁcantly more vitronectin

and ﬁbronectin from serum compared to untreated ﬂat PLAGA surfaces [127].

Signiﬁcantly higher amoun ts of proteins, na mely ﬁbronectin and vitronectin

adsorptions, enhanced the vascular smooth muscle cell and endothelial cell

density presumably due to the cellular recognition sites present on the proteins.

It is also observed that blocking of cell-binding epitopes of ﬁbronectin and

vitronectin on nanometer surface roughness resulted in signiﬁcantly decreased

vascular cell adhesion on nanostructured surfaces [127].

10.2.10 Cell Behavior Toward Nanotopographic Surfaces Created

by Incorporating Carbon Nanotubes/Nanoﬁbers

Carbon nanostructures such as carbon nanotubes and nanoﬁbers possess

outstanding physical and chemical properties and have a diversiﬁed application

range. Carbon nanotubes can be eithe r multiwalled nanotubes (MWNTs) or

single-walled nanotubes (SWNTs). Carbon nanoﬁbers are essentially of

ﬁlamentous structure and can assume various shapes such as straight, spiral,

or ﬁshbone depending on the metal catalyst used. These carbon nanostructures

are fabricated in industrial scale by three main methods, namely electric arc

discharge, laser ablation, and catalytic chemical vapor deposition (CVD).

Usually these processes simultaneously produce SWNTs, MWNTs, fullerenes,

and a considerable amount of soot and carbon nanoparticles and need further

puriﬁcation to isolate each component. However, the yield of individual

nanostructures varies depending on the method and fabrication condition

used. Thus, produced carbon nanostructures are 5 nm to several 100 nm in

diameter and several microns in length and have excellent mechanical,

electrical, and surface properties and a potential utility in various biomedical

applications including tissue engineering scaffolds [128–138].

Osteoblasts registered size-dependent behavior on multiwalled carbon

nanoﬁbers with diameters ranging from 60 to 200 nm in an in vitro culture of

21 days. Increased osteoblast proliferation, ALP synthesis, and calcium

depositions were observed on carbon nanoﬁbers with lesser diameter than the

control (larger borosilicate glass) [129]. Ost eoblasts, ﬁbroblasts, chondrocytes,

and smooth muscle cells showed dimension-dependent behavior on carbon

nanoﬁbers with the diameter range of 60200 nm [130]. Osteoblasts adhesion

increased with decreasing ﬁber diameter as previously observed [129], whereas

other cells were not inﬂuenced by ﬁber dimension. Adhesi on of ﬁbroblasts,

chondrocytes, and smooth muscle cells decreased with a decrease in nanoﬁber

diameter and were dependent on carbon nanoﬁber chemistry. Further,

282 BIOMEDICAL NANOSTRUCTURES

PLAGA carbon nanoﬁber composites enhanced the osteoblast adhesion that

increased with lower diameter nanoﬁber dispersions [130]. SWNTs showed

biocompatibility with cardiomyocytes in culture; however, slight modiﬁcation

in cell shape was observed microscopically due to the binding of carbon

nanotubes to cell membranes that affected the cell count and viability only

after 3 days of culture [128]. Polycarbonate urethane composites with different

weight percentages of carbon nanoﬁbers were used to study the adhesion

behavior of astrocytes and osteoblasts on these composites and register ed a

size-dependent adhesion behavior [131, 132]. Astrocytes preferentially adhered

and proliferated on carbon nanoﬁbers with higher ﬁber diameter. Osteoblasts

preferentially adhered and proliferated well on greater weight percentages of

carbon nanoﬁbers with least ﬁber diameter composites, whereas astrocytes

showed reduced adhesion and proliferation on these composites [131, 132].

Neurons also showed improved adhesion and proliferation on these

composites [131]. Such observed phenomenon is presumably due to the high

degree of surface roughness in the nanometer scale. Osteosarcoma ROS 17/2.8

cells cultured on chemically modiﬁed SWNTs and MWNTs supported

osteoblast proliferation. Nanotubes carrying neutral electric charge resulted

in increased cell growth and produced plate-shaped crystals. Osteoblasts

presented a dramatic change in cell morphology on MWNTs, which was

correlated with changes in plasma membrane functions [133]. Osteoblasts and

ﬁbroblasts exhibited better viability for high purity MWNTs, and nanotubes

induced an increase in collagen expression by these cells [135]. High purity

SWNTs and fullerenes offered a very low toxicity to human macrophage cells

and did not simulate nitric oxide release from murine macrophage cells in vitro

[134]. MWNTs induced the release of the proinﬂammatory cytokine interleukin

8 from human epidermal keratinocytes [136]. Composites of collagen-SWNT

gels showed biocompatibility and maintained smooth muscle cell viability more

than 85% in vitro for 7 days [137]. MWNTs’ and nano-onions’ exposure

resulted in increased apoptosis/necrosis of skin ﬁbroblasts that indicated a

strong immune and inﬂammatory response, which was further evident from the

changes in the expression of genes involved in cellular transport, metabolism,

cell cycle regulation, and stress response [138].

10.2.11 Cell Behavior Toward Nanotopographic Surfaces Created

by Polymer Demixing

Polymer demixing is a widely studied nanofabricating techni que to pattern

large surface area with relatively low cost and high efﬁciency [139, 140]. In

brief, in a typical experiment, polystyrene (PS) and poly(4-bromostyrene)

blends undergo spontaneous phase separation during spin casting on silicon

wafers. Various topographic features including pits, islands, or ribbons can be

created with a wide range of height and depth by varying the blend

concentration and composition. Different geometric shapes were fabricated

by changing the blend ratio while the blend concentration changed the sizes of

CELL BEHAVIOR TOWARD NANO STRUCTURED SURFACES 283

these shapes. Thus, created nanotopographic features are unordered and

covered randomly on the surfaces. This technique has precise control on

patterning in the vertical direction as opposed to the horizontal, and thus

allowed creating nanostructures with deﬁned heights or depths. These

unordered randomly placed nanotopographic features such as islands, pits,

or ribbons of different heights or depths were utilized to evaluate cellular

behavior [38, 41, 53, 139–149].

Dalby et al. have done extensive studies on ﬁbroblast interaction and their

behavior by changing heights of the islands [141–145, 149]. A detailed study

was performed to know the effect of cell nanotopography interactions on

different gene expressions. Human ﬁbroblasts on 13-nm-high polymer demixed

islands were studied for various gene expressions using a 1718-gene microarray

analysis. Many changes in genes involved in signaling, cytoskeleton, ECM gene

transcription, and protein translation were observed. A total of 584 genes

showed an upregulation in their expression when compared to ﬂat surfaces as

control. The upregulation of these genes suggests that the ﬁbroblasts were well

differentiated on the studied nanotopography than the control. Fibroblasts on

similar surface topography resulted in more spreading with the aid of many

ﬁlopodia projecting from the cell membranes as seen in Fig. 10.7 [53]. Increased

cell attachment, spreading, and well-developed cytoskeleton were also

observed. Fibroblast interaction with nanoislands of different height s 13, 35,

and 95 nm resulted in interesting cellular behavior [38]. Fibroblasts interaction

seemed to increase with increasing island size, which is evident by the observed

large pseudopodial projections on 95 nm. The 13-nm islands identiﬁed in the

FIGURE 10.7 Scanning electron micrographs of human ﬁbroblasts on 13-nm

nanoislands created by polymer demixing of polystyrenepoly(4-bromostyrene) blends.

Strong interaction between ﬁlopodia and nanoislands was seen on nanotopographies:

(a) low magniﬁcation; (b) high magniﬁcation. (Reprinted with permission from

Reference [53].)

284

BIOMEDICAL NANOSTRUCTURES

most proliferative cell population had well-developed actin, tubuli n, and

vimentin cytoskeletons, whereas the 95-nm islands were characterized by a low

level proliferative population and poorly developed cytoskeleton [38]. The

nylon tubes of internal diameters 0.5 and 1.5 mm were used to create

nanotopographic features with heights of 90 and 40 nm by polymer demixing

using the blends of polystyrene poly(n-butylmethacrylate) (PS/PnBMA) [142].

Fibroblasts on control tubes showed better spreading, well-arranged b-tubulin,

whereas nanotubular topog raphies assumed high stellate morphology with

rounded cell struc ture and were poorly organized. The cell membranes on

nanotubular topographies appeared rufﬂed with exceptionally long ﬁlopodia

interacting with nanoislands. However, no signiﬁcant differences in cellular

responses were observed for both the diameters with varying island heights.

Primary human ﬁbroblasts’ interaction with the nanoislands of 10 nm height

and controls resulted in increased cell adhesion, spreading, well-deﬁned

cytoskeleton formation, the ﬁbroblas tic morphologie s were maintained, and

produced lamellipodia with visi ble stress ﬁbers at any given time point studied

[143], whereas nanoislands with a height of 50 nm resulted in poor cell

adhesion, low percentage of spreading, poorly deﬁned cytoskeleton with very

few lamellae, and developed no stress ﬁbers at all the time points studied [143].

These topo graphies studied might help in designing surfaces with reduced cell

adhesion and could be utilized in biomaterial design for stents and heart valves.

Fibroblasts cultured for 4 days on the 27-nm nanoislands resulted in

signiﬁcantly greater areas than ﬂat controls used in the study [144]. Well-

organized cytoskeleton characterized by organized acti n and tubulin on both

the surfaces and stress ﬁbers were more often observed in cells on the 27-nm

islands. By day 30, vimentin was well organiz ed on the control whereas poorly

organized on the 27-nm islands. Fibroblasts on nanoislands of 95 nm height

registered temporal changes in both morphology and cytoskeleton [145].

Fibroblasts on these islands started producing lamellae and ﬁlopodia after

5 min of seeding and with progressing time ﬁlopodiaisland interactions

increased. Fibroblasts assumed different morphologies with increasing time

points and attained more stellate morphology with the large pseudopodial

processes. Cultures even after 3 weeks had few patches of grouped cells and a

large number of isolated cells. Isolated cells appeared to have a strong

interaction with these nanoislands. On the contrary, ﬂat control surfaces

maintained normal ﬁbroblast morphology and by 1 week cells were conﬂuent.

Many stress ﬁbers around the cell periphery were observed, and these ﬁbers

were seen stretched across the cytoplasm on the cells on the na noislands at the

initial time points. At the later time points, controls had a matured

cytoskeleton with distinct actin stress ﬁbers and organized tubulin whereas

cells on the nanoislands had less organized cytoskeleton [145]. Fibroblast

interaction with the nanoislands can be summarized as presented in Fig. 10.8

that indicates that controlling the island height has a direct effect on different

cellular activity.

CELL BEHAVIOR TOWARD NANO STRUCTURED SURFACES 285