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Biomaterial Surfaces and Biointerfaces
Surface Science II
April 29, 2013
Prof. Prabhas Moghe
Biomedical Engineering; Chemical & Biochemical Engineering
Rutgers University
1
Areas and Technologies where Surface Properties of
Biomaterials Are Relevant:
2
A Sampling-- can you think of some more? •Development of Biosensors •Point of Care Devices •Biomaterials for Growing Living Cells (Cell Culture) •Biomaterials (e.g. Catheters, Bandages) that Resist Microbial Growth and Infection •Multifunctional Nanoparticles that Bind to Cancer Cells and Cause Ablation/Killing •Hydrogels and Dressings for Wound Healing •Scaffolds for Tissue Regeneration •Vascular Stents that Prevent Blood Vessels from Occluding (Stenosis) •Titanium implants for bone fracture fixation and hip prostheses
SURFACE PROPERTIES OF MATERIALS
cell tissue fluid
and proteins
BIOMATERIAL
• Surface properties determine biomaterial-tissue interface.
• Properties: Biocompatibility
Topography/Roughness
Wettability
Surface Mobility
Crystallinity
Chemical Composition 3
Method Principle Depth analyzed Resolution CostContact
angles
Liquid wetting of materialestimates surface energy
3 – 20 Ao 1 mm Moderate
ESCA /
XPS
X rays causeElectron emission
10 –250 A 10 –1 50um
V.Expensive
AES Electron beam causesAuger e. emission
50 – 100 A 100 A V.Expensive
SIMS Bombarded ions cause surfacesecondary ion emission
10 A – 1 um 100 A V.Expensive
FTIR-
ATR
IR radiation causes excitation 1-5 um 10 um Expensive
STM Quantum tunneling current betnprobe & conducting material
5 A 1 A Expensive
SEM Electron beam causes secondaryelectron emission
5 A 40 A Expensive
CLSM Reflected /fluorescent imagesspatially determined
10 um – 2000um
0.5 um Expensive
Methods to Characterize Biomaterial Surfaces
Other Methods: Quartz crystal microbalance (Measure protein or biomolecule adsorption on materials); Ellipsometry (Measure thickness of biointerface); Surface Plasmon Resonance (Kinetics of binding); Fluorescence Resonance Energy Transfer (Biomolecule-Biomolecule Binding) 4
Surface Contact Energetics
-Molecules exterior to materials are most accessible to adjacent phases as well as incoming cells (Surface Reaction) -There is always a positive energy necessary to create a unit area of surface. Systems reach equilibrium by minimizing this surface area/energy. In solids, this happens by changing the nature of interface to one with lower (lowest) energy. -Energy minimization occurs when groups/chains in a biomaterial (e.g., polymer) rearrange to yield lowest interfacial energy. e.g. hydrogel migrates to/from surface of a graft copolymer exposed to water/dry air.
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Determination of Surface Energetics
• Excess free energy per unit surface area is surface tension
• Young’s Theory of the Spreading of Liquid Droplet:
sv sl
lv
At equilibrium, surface energy sv sl lv cos = +
6
Techniques to measure contact angles
air
Static Drop
Capillary air-bubble
DuNouy Ring or
Wilhelmy plate 7
Dynamic Contact Angle Measurements
r a
ADVANCING
CONTACT ANGLE
RECEDING
CONTACT ANGLE
Dynamic contact analysis is done by increasing or
decreasing the drop volume until the three-phase
boundary moves over the surface.
8
Thermodynamics of Spreading/Adhesion on Materials
Fadh = cs - cl - sl
Interfacial free energy of adhesion = Cell-solid interfacial free
energy - Cell-liquid interfacial free energy - Solid-liquid
interfacial free energy.
If Fadh < 0, adhesion and spreading are energetically favorable
50 100
s [erg. cm-2]
Fadh
and Substratum free energy (wettability) Fadh
Very hydrophobic
substrates 9
CELLS INTERACT WITH PROTEIN-ADSORBED BIOMATERIALS
Cell
Polymer
Proteins
Receptors
10
Discussion of Research Paper 1
Tziampazis, Kohn, and Moghe, Biomaterials 21:511, 2000
11
Our study focused on the role of poly(ethylene glycol) (PEG) in actively regulating the biological responsiveness of protein adsorbed biomaterials. To this end, we designed PEG-variant biomaterials from a family of tyrosine/PEG-derived polycarbonates to present surfaces ranging from low to intermediate levels of PEG concentration, below the PEG level requisite for complete abolition of protein adsorption. We analyzed the e!ect of PEG concentration on the amount, conformation and bioactivity of an adsorbed model protein, fibronectin, and on the attachment, adhesion strength and motility of L929 fibroblasts. Our results demonstrate that low levels of PEG can regulate not only the extent but also the conformation and speci"c bioactivity of adsorbed fibronectin. As the PEG concentration was increased from 0 to 6 mol%, the amount of adsorbed fibronectin decreased linearly yet the fibronectin conformation was altered such that the overall bioactivity of adsorbed fibronectin was uncompromised. We report that the degree of cell attachment varied with PEG concentration in a manner similar to the dependence of "bronectin bioactivity on PEG. In contrast, the nature of cell adhesion strength dependence on PEG paralleled the pattern observed for fibronectin surface concentration. Our studies also indicated that the rate of cell migration was inversely correlated with PEG concentration over a narrow range of PEG concentration. Overall, these results highlight the striking ability of PEG-variant biomaterials to systematically regulate the behavior of adsorbed cell adhesion proteins and, consequently, se!ect cell functions.
Relationship between substrate PEG content and surface hydrophobicity
Tziampazis, Kohn, and Moghe, Biomaterials 21:511, 2000
0
10
20
30
40
50
60
0 2 4 6 8 10
%PEG1k Content
Rec
ed
ing
Co
nta
ct
An
gle
(d
eg
rees
) Unconditioned
Protein conditioning
12
CH2-CH2-C-NH-CH-CH2O O-C
O O
C
O
R
O
O-PEG-O-C
O
1-f f R = CH3CH2
Chemical structure of poly(DTE-co-PEG carbonate)s. In this work, the alkyl group, R, on all
polymers was ethyl (E), and the Mw of PEG blocks was 1000. The molar fraction of PEG units in
the copolymer was varied from 10 mole% down to 0 mole% PEG.
Effect of PEG concentration on the hydrophobicity of PEG-variant biomaterials. Lower receding
contact angles indicate lower hydrophobicity (increased hydrophilicity).
PEG Interfaces Alter Protein Adsorption & Activity
13
0.0
0.10
0.20
0.30
0.40
0.50
0 2 4 6 8 10
Ad
so
rbed
FN
(mg
/cm
2)
PEG Content (%)
DataLinear Fit
R2 = 0.962
**
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 2 4 6 8 10
PEG Content (%)
FN
Bio
ac
tiv
ity
(EL
ISA
Ab
so
rba
nc
e)
0.5
0.8
1.0
1.3
1.5
1.8
2.0
2.3
0 2 4 6 8 10
PEG Content (%)
Sp
ec
ific
Bio
ac
tiv
ity In
de
x
Sp
ecif
ic B
ioa
ctiv
ity
of
Pro
tein
FN
Cel
l B
ind
ing A
ctiv
ity
Figure 3. PEG-mediated regulation of the amount of fibronectin adsorption on PEG-variant
biomaterials. As described in Materials and Methods, adsorbed hpFN was quantified using
radiolabeled FN as a tracer.
Figure 4. Bioactivity of FN adsorbed on PEG-variant biomaterials. A . Cumulative sur face
bioactivity was ev aluated using fibronectin E LISA. B. Specific FN bioactivity index was
computed by normalizing FN bioactivity to adsorbed amount and expressing the values relative to
the specific bioactivity of FN adsorbed on poly(DTE carbonate).
Organization & Conformation of FN
*
*
*
*
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 2 4 6 8
PEG content (%)
Su
rfac
e R
ou
gh
ne
ss
(n
m)
PBS FN
Surf
ace
Rou
ghne
ss In
dex Figure 5. Topographic imaging of polymer surfaces before and after fibronectin adsorption. Fluid-
mode atomic force microscopy was employed to analyze the surfaces in a physiologically relevant
state. Images depict the topography of a square area, 1 µm wide, as would be seen from the front
with a 70 degree pitch. For all images the scale of the Z-axis (height) is 8nm.
Effect on Cell Adhesion Strength
14
*
**
0
20
40
60
80
100
0 2 4 6 8 10
PEG Content (%)
Cell A
ttach
me
nt
(%)
*
*
10
15
20
25
30
35
40
45
0 2 4 6
PEG Content (%)
Ave
rag
e C
ell
Mo
ve
men
t (µ
m/h
)
Figure 7. Extent of L929 cell adhesion on PE G-variant biomaterials. A. Fraction of cells attached
to the biomaterial after one day of culture. Asterisks denote values that differ significantly
(p<0.05) from the cell attachment data in the absence of PEG. B. PEG mediated regulation of cell
adhesion strength. The critical level of shear required to detach 50% of the attached cell popula tion
was used as a single parameter measure of cell adhesion strength.
Discussion
15
Fibronectin Schematic
Moderately Hydrophilic
Strongly Hydrophilic
Cell Binding Domain
Moderately Hydrophobic
Strongly Hydrophobic
poly(DTE-co-PEG carbonate)
PEG (Hydrophilic)DTE (Hydrophobic)
Intermediate PEG
Low or no PEG
High PEG
PEG Microdistribution Regulates Cellular Survival Soft Matter, 2010, 6, 5196–5205
Fig. 1 Chemical composition of PEG-containing polymers and variations
in hydration and protein adsorption due to increasing PEG1k mol%.
(a) The chemical structure of polymers used in this study. Polymers were
composed of two different co-macromers: DTE and PEG (Mw ¼ 1000).
The chemical formula was abbreviated as p(DTE-co-y%PEG1k c). PEG1k
mole percentages used in this study were 5, 8, 10, and 20%. (b) Water
contact angle measurement for 0 and 2 hours of hydration. (c) FBS
adsorption measured by QCM-D on given substrates. The Voigt model
was applied to obtain the adsorbed mass (mg cm�2). m
Fig. 2 Increasing PEG content of polycarbonate polymers correlates
with an increase in oxidation. (a) Measurement of residual H2O2 (mM)
on polymer surfaces. (b) Protein carbonyl formation (nM) generated on
polymer surfaces. *p < 0.05 vs. the condition without the addition of
ebselen on the same polymer type. (c) Intracellular peroxide production
(O.D.) from cells cultured on polymer substrates. *p < 0.05 vs. the
condition with no addition on the same polymer type. †p < 0.05 vs. the
other conditions on the same polymer type. Ebselen was added to
scavenge H2O2. DPI was added to inhibit the activity of NADPH
oxidase.
Fig. 4 Diverging effects of PEG content on attachment and spreading of
SaOS-2 cells. (a) Cell populations and (b) cell spreading on polymer
substrates as measured at 24 h postseeding by light microscopy and image
processes. *p < 0.05 vs. p(DTEc); †p < 0.05 between two test materials
indicated by a line.
Fig. 5 Formation of PEG microdomains and water uptake characteristics
of PEG containing polymers. (a) SANS; (b) SAXS; and (c) water
uptake, Tg, Young’s modulus, and elongation to failure of polymer
samples.
Fig. 6 PEG-induced modulations of cell spreading via integrin a5. (a) Intracellular distribution of GFP–integrin a5 fusion proteins. (b) Protein levels of
integrin a5 quantified by the fluorescence intensity of antibodies raised against integrin a5 on polymer samples both without and with the presence of
ebselen. *p < 0.05 vs. p(DTEc) under the appropriate condition (i.e., with or without ebselen). †p < 0.05 between samples indicated by a line. (c)
Expression of integrin a5 mRNA in cells cultured on polymer samples under different conditions as detected by in situ hybridization. *p < 0.05 vs.
p(DTEc) with no addition. †p < 0.05 vs. the other conditions, on the same polymer type. (d) Relative changes in gene expression of integrin a5 at 24 hours
after seeding on polycarbonate–PEG substrates normalized by gene expression at 4 hours postseeding. A relative expression of 1 indicates no change in
expression between 4 and 24 hours. *p < 0.05 for 10 mol% and 20 mol% PEG1k compared to p(DTEc) and 5 mol% PEG1k.
Applications of PEG Biointerfaces
Plasma-micropatterning of albumin nanoparticles: Substrates for enhanced
cell-interactive display of ligands María Pía Rossi, Jing Xu, Jean Schwarzbauer, Prabhas V. Moghea� Department of Chemical and Biochemical Engineering, Graduate Program in Cellular and Developmental Biology, and
Department of Biomedical Engineering, Rutgers University, 599
Piscataway, New Jersey 08854
�Received 29 August 2010; accepted 11 October 2010; published 11 November 2010� The authors demonstrate a novel, efficient, and widely applicable approach to direct the patterning of ligand-functionalized organic nanoparticles derived from albumin on nonconductive, biodegradable polymeric substrates. In contrast to traditional deposition methods for inorganic nanoparticles, the approach involves oxygen plasma treatment of spatially restricted regions on a nonbiopermissive polymer. Albumin nanoparticles conjugated with a truncated fragment of fibronectin containing the Arg-Gly-Asp domain were successfully patterned and used as templates to elicit adhesion and spreading of human mesenchymal stem cells and fibroblasts. Attachment and spreading of both cell types into the plasma-exposed polymer areas was considerably more pronounced than with the ligand alone. The authors hypothesize that the underlying mechanism is oxygen plasma treatment-induced selective enhancement of ligand exposure from the deposited functionalized nanoparticles, which facilitates ligand receptor clustering at the cell membrane. The results highlight a promising nanoscale approach to modulate ligand presentation and spatially direct cell attachment and phenotypic behaviors. © 2010 American Vacuum Society.
Biointerphases 5(4), 105, December 2010
FIG. 1. Schematic illustrating the microscale plasma-initiated patterning process. �A� A PDMS stamp is placed on the biocompatible, bioresorbable polymer surface and treated in oxygen plasma at a pressure of 660 mTorr for 60–120 s and 50 W. Areas of the polymer exposed to the plasma undergo surface functionalization via the formation of end groups by interaction with the radicals, electrons, and ions in the oxygen plasma. �B� Biofunctional ANPs then preferentially adsorb to the exposed area of the material. �C� By exposing the polymer to the oxygen plasma for 60 s, rotating the stamp by 90°, and exposing the polymer to the oxygen plasma for 60 s again, areas of differentially exposed polymer enabled the differential deposition of nanoparticles for cell attachment.
FIG. 2. �Color online� Biointerfacial characterization of spatially restricted patterning of bioactive albumin nanoparticles. �A� Fluorescence microscopy image of patterned nanoparticles on poly�DTE-co-8% PEG1K carbonate�. Briefly, after plasma-initiated patterning of the substrate with 660 mTorr oxygen at 50 W for 120 s, the nanoparticles were adsorbed onto the surface at 4 °C overnight, then washed, fixed, and stained with mouse antihuman serum albumin as a primary antibody and FITC-conjugated Donkey antimouse secondary antibody for visualization under fluorescence. �B� Atomic force microscopy image of nanoparticle patterns on poly�DTE-co-8% PEG1K carbonate�. While nanoparticle monolayers are observed on one of the stripes �the plasma-exposed area�, minimal nanoparticle adsorption is observed on the other stripes �unexposed or plasma-protected areas�. �C� Scanning electron microscopy image of the nanoparticles patterned on the polymer substrates after plasma treatment. �D� High magnification scanning electron microscopy image of �C�. �E� Higher yet magnification of plasma-treated region shows organization of nanoparticles in �D�.
FIG. 5. �Color online� Human mesenchymal stem cells adhere and organize effectively on spatially patterned albumin nanoparticles. Fluorescent images �green—actin; blue—DAPI� show MSCs patterned with FNf-functionalized albumin nanoparticles ��A� 4�; �B� 20��, compared to those patterned on FNf fragment �control� �ı Cˇ 4̌ ; ı Dˇ 20̌ �, and on positive control, wholelength fibronectin ��E� 4�; �F� 20��. Patterning was sparse with ligand alone. Patterning with the FNf-ANPs is more effective than with FNf alone, yields more contiguous organization than whole-length fibronectin control, and cells clearly spread within the plasma-exposed stripes �ı Cˇ and ı Dˇ �.
Summary
1. Surface contact energetics of materials can be measured using contact angles.
2. The surface energetics of polymeric materials govern protein adsorption and cell adhesion and spreading.
3. Changing the PEG microdistribution within biomaterials can control the bioactive conformation of proteins, which in turn, can effect a qualitative change in the nature of cell adhesion, and a quantitative change in cell responses, including attachment/detachment kinetics leading to cell migration; cell adhesion-based survival or apoptosis.
4. Applications of PEG-based interfaces is in restricting protein adsorption and promoting specific cellular/tissue interactions with “functionalized” bioactive ligands.
