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

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Technique

Topographic

features Merits Shortcomings Cell type used

Observed

change in

cellular

behaviors References

3 Electrospinning

(aligned

nanoﬁbers)

Aligned ﬁbers

in the nanometer

dimension and

can be extended

to several micron

thick ﬁbers

Simple, facile, wide

range of materials

available for

spinning ability to

incorporate

bioactive

molecules

Can create only

nonwoven

ﬁber structures

Endothelial,

neural stem

cells, ligament

ﬁbroblast, smooth

muscle cells,

chondrocyte

(HTB-94)

osteoblast

(MG-63)

Spreading,

proliferation,

phenotype

expression,

orientation,

alignment

[70–77]

4 Nanoimprinting Wide range of

structures such as

pillars, grooves,

ridges possible in

nanodimensions

Multilayer three-

dimensional

structures at

comparatively

lower cost than

electron beam

lithography

Expensive

equipment

and time

consuming

Osteoblasts,

smooth

muscle cells

Strong

alignment,

spreading,

migration,

phenotype

expression

[78–80]

Unordered nanotopographic surfaces

5 Self-assembly Can create ﬁbers of

any dimensions

based on the

start-up material

Process is simple,

facile, low tech,

can create complex

functional structures.

Structures are

defect-free and

self-healing

No direct control

over fabrication,

compared to

lithography

complex structure

fabrication is

difﬁcult.

MC3T3-E1,

neural,

bladder, smooth

muscle, aortic

endothelia and

neural progenitor

cells. In vivo

studies in Syrian

hamsters

Adhesion,

increased

proliferation,

differentiation,

and phenotype

expression.

[81–95]

TABLE 10.1 (Continued )

266

(continued )

Technique

Topographic

features Merits Shortcomings Cell type used

Observed

change in

cellular

behaviors References

6 Phase separation Wide range of

geometry

and dimensions

include pits, islands,

ﬁbers, and irregular

pore structures

Simple, facile, no

equipment needed,

highly porous

scaffolds with

control on porosity,

and scalable

No organized

patterns

OCT-1 osteoblast-

like cells, nerve

stem cells,

MC3T3-E1

preosteoblasts

Adhesion,

proliferation,

differentiation

[81, 96–99]

7 Colloidal

lithography

Columns and islands

can be created in

nanometer ranges

Relatively cheap and

less time consuming,

can pattern large

surface area with

less effort

Speciﬁc feature

geometries

hard to design

Epitenon cells,

pancreatic

epithelial cells

(AR4-2J),

mammary

epithelial

cells (HC11),

human bladder

carcinoma,

HTB-4, primary

human

osteoblasts,

macrophages/

monocytes

human

ﬁbroblast

Interaction,

spreading,

alignment,

migration,

phenotype

expression,

and

proliferation

(37, 43, 44,

100–105)

8 Electrospinning

(random

nanoﬁbers)

Fibers in the

nanometer

dimension

and can be

extended

to several micron

thick ﬁbers

Simple, facile, wide

range of materials

available for spinning

ability to incorporate

bioactive molecules

Can create

only ﬁbrous

structures

Fibroblasts,

MC3T3-E1

osteoblast-like,

SMCs, NIH3T3,

bone marrow

stromal cells

Spreading,

proliferation,

phenotype

expression,

orientation,

[106–123]

267

(continued )

Technique

Topographic

features Merits Shortcomings Cell type used

Observed

change in

cellular

behaviors References

9 Chemical etching Depends on the

nature and time of

etching agent used

Fast, simple, and

inexpensive

Difﬁcult to get

required geometry

and features

Human

osteoblasts

CRL-11372,

aortic smooth

muscle cells,

primary rat

smooth muscle

cells from

thoracic aorta,

bladder and

cortical cells,

LRM55

CNS tumor

Adhesion,

migration,

proliferation

and phenotype,

expression

[124–127]

10 Carbon

nanoﬁbers/

nanotubes

Fibers in

nanodimensions

5 nm to several

100 nm in diameter

and several microns

in length. Various

shapes straight,

spiral, ﬁshbone,

and so on

Excellent mechanical,

electrical, and surface

properties. Relatively

easy to fabricate and

no costly equipment

needed

Can create only

ﬁbers and tubes

and speciﬁc

geometries not

possible

Human

osteoblasts,

epidermal

keratinocytes,

macrophages

and skin

ﬁbroblast rat

astrocytes,

aortic smooth

muscle cells,

cardiac muscle

cells and cortical

cells, mouse skin

ﬁbroblasts, bovine

bladder smooth

muscle cells

Compatibility,

adhesion,

proliferation,

differentiation,

morphology

[128–138]

TABLE 10.1 (Continued )

268

nano

ﬁb

ers

nanotubes

nano

mens

ons

5 nm to several

100 nm in diameter

and several microns

in length. Various

shapes straight,

spiral, ﬁshbone,

ectr

,an

sur

ace

properties. Relatively

easy to fabricate and

no costly equipment

needed

ﬁb

ers an

and speciﬁc

geometries not

possible

osteo

asts,

epidermal

keratinocytes,

macrophages

and skin

ﬁbroblast rat

astrocytes,

aortic smooth

muscle cells,

cardiac muscle

cells and cortical

cells, mouse skin

ﬁbroblasts, bovine

bladder smooth

muscle cells

on,

proliferation,

differentiation,

morphology

11 Polymer demixing Nanotopographic

features such as

pits, islands, or

ribbons of wide

range of height or

depth possible

Can pattern large

surface area, low

cost, high

efﬁciency,

less effort

Can only create pits,

islands, or ribbons.

No other speciﬁc

geometries possible

Human ﬁbroblasts,

osteoblasts,

endothelial cells

Adhesion,

morphology,

proliferation,

differentiation,

gene expression

[139–149]

269

nanofabrication techniques in brief while discussing the importance of

nanotopographic features on cell behavior.

10.2.1 Cell Behavior Toward Nanotopographic Surfaces Created

by Electron Beam Lithography

Electron beam lithography is an attractive computer-controlled techn ique to

create precise geometries and patterns on nanoscale without the use of any

mask. In brief, either positive or negative resists are used to create programmed

nanotopographic features. Positive resists break down into lower molecular

weight fragments when irradiated by a high energy electron beam, whereas

negative resists form insoluble cross-linked networks. These irradiated resists

are developed in a suitable developer. The preprogrammed nanotopographic

features were created by leaching out of the resists in a developer. However, the

use of negative controls limits the resolution of these features due to swelling

while developing. Further, this technique requires expensive equipment and is

time consuming. Many nanotopographic features including square grooves,

ridges, and nanopillars, created using the current technique, were used to study

in vitro cellular behavior to elicit the importance of nanoscale features for a

variety of biomedical ap plications [52, 55 –62].

Micro- and nanotopographic features containing grooves and ridges with

dimensions varying from 400 to 4000 nm directed the alignment and migration

of SV40-transformed human corneal epithelial cells along the grooves and

ridges of all the dimensions [55]. It was observed that cell colonies migrated out

along grooves and ridges in circular zones and migrated perpendicular to ridges

in some cases. On ﬂat controls equal migration was observed in all directions.

Stress ﬁbers and focal adhesions were also aligned along the ridges and groves

[55]. Keratocytes showed a stronger alignment than epithelial cells to these

nanotopographic surfaces [20]. Keratocytes presented fewer stress ﬁbers and

focal adhesions on nanodimensions when compared to micro and ﬂat control

surfaces [56]. Nanotopographic features containing varying widths of grooves

and ridges ranging from nano- to micron dimensions provided stimulus for

human corneal epithelial cells [57]. Cells were found to align along the grooves

and the ridges of nanodimensions with elongated structures while rounded cells

were observed on smooth surfaces as seen in Fig. 10.2. On the nanopatterned

surfaces, cells periodically extend ﬁlopodia along the direction of the

nanofeature as shown in Fig. 10.2c [57]. When the culture medium was

changed from DMEM/F12 to Epilife medium, human corneal epithe lial cells

aligned perpendicular to the nanotopographic features as evident from Fig.

10.2d whereas parallel alignment was observed on microscale topographies

[58]. Such an observed change in alignment is presumably due to a change in

focal adhesion structure. Human gingival ﬁbroblasts and rat neurons on

patterned surfaces with grooves showed focal adhesion contacts and cells

aligned to the grooves [59, 60]. Xenopus neuritis aligned parallel to all groove

sizes whereas hippocampal neuritis aligned perpendicular to narrow, shallow

270 BIOMEDICAL NANOSTRUCTURES

grooves [60, 61]. Nanotopographic surfaces containing ordered features of

pillars, pits, cliffs, and random gold colloid particles showed topography

dependence in their adhesion. Ordered cliffs showed enhanced adhesion at the

cliff, edges. Reduction in cell adhesion was observed on pits and pillars when

compared to ﬂat and colloid particle surfaces [52]. Anisotropic features in the

size range of 2002000 nm on polyurethane surfaces showed decreasing

proliferation of corneal epithelial cells with decreasing dimensions when

compared to planar surfaces [62].

10.2.2 Cell Behavior Toward Nanotopographic Surfaces Created

by Photolithography

Photolithography is one of the popular techniques to create nanotopographic

surfaces containing many geometric features including grooves, ridges, and

round nodes from nanometers to several microns in size. Photolithography

involves a series of processes in brief; cleaned surfaces of silicon wafers are

coated with photoresist and soft baked to remove the solvents from photoresist

FIGURE 10.2 SEM micrographs of human corneal epithelial cells cultured in

DMEM/F12 on (a) patterned nanotopography elongated and aligned (b) patterned

surfaces ﬁlopodia extend in the direction of features. (c) Flat substrates with rounded

morphology. (d) Patterned surfaces cultured in Epilife medium showing perpendicular

alignment to nanotopographic features. (Reprinted with permission from (a, b and c)

Reference [57] and (d) Reference [58].)

CELL BEHAVIOR TOWARD NANO STRUCTURED SURFACES 271

that converts the resist photosensitive. The photomask containing previously

deﬁned geometric features aligned with the silicon surface and exposed to light

source followed by etching and developing leaves the predetermined patterned

surfaces. The hard baking process improves the adhesion of photoresist to

silicon wafer and hardens the photoresist. Photolithography can create precise

geometries and patterns but require expensive complicated instrumentation,

and mostly microsized topographies can be created easil y. Thus created

nanotopographic features elicit changes in cellular functions including

orientation, interaction, morphology, and differentiation based on the

topography and dimensions they are exp osed to [63–69].

Implant surfaces containing nodes of 2 and 5 mm diameter and having

different heights in nanometer length showed fewer mononuclear cells and

thinner ﬁbrous capsules when implanted in a rabbit model than the control

planar and 8-mm-diameter grooved surfaces [68]. Also, cells appeared to be

elongated and with more number of ﬁlopodia on nanotextured surfaces, whereas

cells assumed a rounded shape with less number of ﬁlopodia indicating less

interaction with the implant surfaces [68]. Nanotopographic features containing

rough surfaces were created by reactive ion etching, and smooth wet etched

surfaces were created on silicon wafers showing surface-dependent adhesion

behavior to rat astrocytes [69]. Transformed astrocytes showed preferential

attachment and a spread morphology on wet etched surfaces. Columnar

nanotopographic features created by reaction ion beam etching resulted in round

morphology, loose attachment, and exhibition of complex surface projections of

transformed cells [69]. Rat ﬁbroblasts on square grooves with submicron

dimension oriented and elongated along grooves [63]. P388D1 macrophages, rat

peritoneal macrophages, and chick embryo cerebral neurons showed increasing

orientation and spreading with increasing groove depth [64, 65]. Well-

characterized cytoskeleton and F-actin and vinculin accumulation was observed

along the edges of the grooves [64, 66]. Uromyces appendiculatus fungus cells

showed a high degree of orientation to the polystyrene nanoridge spacing of

0.56.7 mm, whereas ridge height of 500 nm showed maximum cell differentia-

tion compared to ridges of height greater than 1 mm or less than 0.25 mm [67].

10.2.3 Cell Behavior Toward Nanotopographic Surfaces Composed

of Aligned Nanoﬁbers by Electrospinning

Polymeric nanoﬁbers are created using a variety of techniques such as template

synthesis, phase separation, drawing, self-assembly, and electrospinning.

Among these techniques, electrospinning is extensively studied since the

process is simple, elegant, and facile, and can create polymeric ﬁbers in the

range of a few nanometers to several micron thicknesses using the same

experimental setsup [70–77]. During the process of electrospinning, ﬁbers

randomly deposited on the grounded collector create unordered surface

topographies. Aligned nanoﬁbers that could present ordered nanotopographies

can be created by using a high speed rotating collector, applying an auxiliary

272 BIOMEDICAL NANOSTRUCTURES

electric ﬁeld, and using a sharp edged thin wheel collector or a frame collector

in place of the stationary grounded collector [71–76]. Thus created

nanostructured surfaces both aligned and random were used as tissue

engineering constructs, and cells respond differently to the surfaces they were

exposed to [71–76].

Human coronary artery endothelial cells on aligned gelatin-modiﬁed

poly(caprolactone) (PCL) nanoﬁbers in the diameter range of 2001000 nm

provided improved adhesion, spreading, and proliferation than the control

PCL and random nanoﬁbers [71]. These aligned nanoﬁbers strongly cause the

orientation of endothelial cells parallel to the nanoﬁbers with spindle-like

structures; also well-deﬁned cytoskeleton and enhanced phenotypic expression

were observed. Shear stress caused by the blood ﬂow orients the endothelial

cells in the ﬂow direction in vivo, and surface-modiﬁed aligned nanoﬁbers

simulate the actual in vivo condition in a static culture that provides an

alternative to complex dynamic culture [71]. Neonatal mouse cerebellum C17.2

stem cells (NSCs) on PLLA-aligned and random nanoﬁbers attached well and

changed their original round shape to elongated spindle-like sh ape that

provided morphological evidence for NSC differentiation. The direction of

NSC elongation and its neurite outgrowth was exactly parallel to the direction

of aligned nanoﬁbers with a higher rate of NSC differentiation than the

control. Increased interaction between the ﬁlament-like structures produced by

NSCs was observed on aligned ﬁbers that were absent on random nanoﬁbers as

seen from Fig. 10.3 [72]. Such aligned nanoﬁ bers could be used as a potential

FIGURE 10.3 NSCs on PLL-aligned ﬁbers had an apparent bipolar elongated

morphology with neuritis, and ﬁlament-like structures marked with arrows (a) attach to

nanoﬁbers and (b) microﬁbers, and (c) absence of ﬁlament-like structures on random

nanoﬁbers. (Reprinted with permission from Reference [72].)

CELL BEHAVIOR TOWARD NANO STRUCTURED SURFACES 273

cell carrier for neural tissue engineering. Human ligament ﬁbroblast (HLF) on

aligned polyurethane nanoﬁbers also oriented in the direction of aligned

nanoﬁbers and had a spindle shape [73]. Fibroblasts synthesized signiﬁcantly

more collagen on aligned nanoﬁbers, and furt her HLFs were more sensitive to

the stress applied in longitudinal direction resulting in an increased production

of collagen in response to applied stra in [73]. Human coronary artery smooth

muscle cells (SMCs) on poly(

L-lactid-co-e-caprolactone)-aligned nanoﬁbers

attached and migrated along the direction of the nanoﬁber orientation and

maintained a spindle-like structure [74, 76]. SMCs resulted in increased

adhesion and proliferation on aligned nanoﬁbers, and cytoskeleton proteins

inside SMCs were parallel to the direction of the nanoﬁbers. Chitosan

nanoﬁbers and aligned na noﬁbers promoted the attachment of human

osteoblasts and chondrocytes and maintained characteristic morphology

throughout the study [75]. The observed cell orientation on aligned nanoﬁbers

presumably follows the contact guidance theory, which states that cells have a

higher probability of migrating in directions of chemical, structural, and/or

mechanical properties of the substratum [77].

10.2.4 Cell Behavior Toward Nanotopographic Surfaces Created

by Nanoimprinting

Nanoimprint lithography can create ordered nanotopographic features such as

pillars, grooves, and ridges at the required dimensions. In brief, the technique

utilizes a hard mold containing previously deﬁned nanoscale patterns. Wafer

substrates covered by polymer cast under the controlled temperature and

pressure will be imprinted by the hard mold. Hard mold embossing creates a

thickness contrast on the polymer surfa ce with the required nanotopographic

features. It is possible to create multilayer three-dimensional structures using

nanoimprint lithography at comparatively lower cost than electron beam

lithography. However, the present technique also utilizes expensive equipment,

and the process is time consuming. Thus, created ordered nanotopographic

features elicit changes in c ellular functions in vitro [78–80].

Nanopatterned gratings with 350 nm line width, 700 nm pitch, and 350 nm

depth created on poly(methyl methacrylate) and poly(dimethylsiloxane)

showed decreased proliferation of bovine pulmonary artery SMCs than the

ﬂat control [78]. More than 90% of the cells were aligned and assumed an

elongated structure in both cytoskeleton and nuclei on nanopatterned surfaces

as seen in Fig. 10.4 [78]. Also, the polarization of microtubule organizing

centers of the SMCs associated with cell migration showed a preference toward

the axis of cell alignment in an in vitro wound healing assay on nanopatterned

surfaces. Polystyrene surfaces containing nanopillars in the diameter range of

1601000 nm and 1000 nm height were tested as an alternative tissue culture

plate by studying the in vitro behavior of epithelial-like cell line (HeLa) on the

said topography [79]. HeLa cells adhered only to the heads of the nanopillar

sheet with a signiﬁcantly lower number of cells than the control and acquired

274 BIOMEDICAL NANOSTRUCTURES

rounded morphology on nanopillars in comparison to a spread structure on

the ﬂat control. Also, actin molecules appeared to localize to the circumference

of the nanopillar heads, whereas vinculin molecules were distributed

homogeneously in regions away from the nanopillar heads [79]. Thus, the

low adhesion properties exhibited by the cells might lead to alternative tissue

culture plates that do not require conventional trypsin treatment to lift the

cells. Polystyrene nanopatterned surfaces with grooves having two different

depths of 50 and 150 nm with a periodicity of 500  100 nm showed a strong

FIGURE 10.4 Confocal micrographs of F-actin stained SMCs on (a) nanoimprinted on

PMMA, (b) PDMS, and (c) nonpatterned PMMA surfaces (scale bar 50 mm). SEM

micrographs on (d) nonpatterned PMMA and (e) nanoimprinted surfaces show elongated

structures on patterned surfaces. (Reprinted with permission from Reference [78].)

CELL BEHAVIOR TOWARD NANO STRUCTURED SURFACES 275