Transcript NSF Grantees Meeting 12/4/07

NIRT: Self-Assembled Nanohydrogels for
Differential Cell Adhesion and Infection Control
Matthew Libera, Woo Lee, Svetlana Sukhishvili, Hongjun Wang, and Debra Brockway
Stevens Institute of Technology, Hoboken, New Jersey 07030
It is a catastrophic problem, because bacteria that colonize an implant surface develop
into biofilms where they are as much as 10,000 times more resistant to antibiotics than
planktonic bacteria. The most effective therapy is to remove an infected implant, cure
the infection, and then pursue a subsequent revision surgery. The consequences to
patient well being and medical cost in this situation are compellingly significant.
Surface Self-Assembled PEGDA Hydrogel Particles
to Control Bacteria/Cell-Biomaterial Interactions
~1 m
At its core, implant infection is a biomaterials problem. While surfaces have
been developed which repel bacterial adhesion – e.g. PEGylated surfaces – these also
repel the eukaryotic cells necessary for the development of a healthy implant-tissue
interface. Instead, surfaces are needed that are differentially adhesive, i.e. that it
promote eukaryotic (e.g. osteoblast) adhesion and proliferation while simultaneously
repelling bacteria. This is a
fundamental biomaterials problem
that remains unsolved.
PLL
Si
Cell-Interactive
nanohydrogels
hierarchically structured
on the surface of a
macroscopically beaded
surface of a modern
orthopaedic implant.
~2 mm
PEG gel particles can deposit
on the PLL modified Si wafer
surface by electrical selfassembly to obtain modified
surfaces with controllable gelparticle density. Lower and
higher particles density surface
were both tested in bacteria and
osteoblast culture.
UV is used to polymerize
PEGDA in the DCM
droplets which obtained by
DCM/water emulsion.
PEG gel-modified surface reduces
short term S.epi adhesion/growth.
Bacteria/Cell/Biomaterial
Interactions
1
~350 m
3
4
Infection Rates
0.8
bare Si wafer
0.7
0.6
PLL deposited Si
wafer
low nanohydrogel
coverage
high nanohydrogel
coverage
pure PEGDA
0.5
0.4
0.3
0.2
0.1
0
 Hips
This project explores a new
mechanism to create differentially
adhesive surfaces. We hypothesize
that heterostructures of nanosized
hydrogels self assembled in 2D
over micrometer length scales will
allow focal contact formation and
subsequent osteoblast adhesion
but prevent bacterial adhesion.
Osteoblast 4 days
culture result
Osteoblast Cytoskeleton Density on Different Substrates
S. epi grow on different substrates
4 types of substrate were studied:
1.bare Si;
2.2 – PLL modifed Si;
3.Gel modified Si (low conc);
4.Gel modified Si (high conc);
2
Lower PEGDA density higher PEGDA density
Ratio of Surface Covered
by Cytoskeleton
Infection occurs in approximately 0.5 – 5% of all hip and knee replacements.
Differentially Adhesive Surfaces Repulsive to Bacteria but Attractive to Eukaryotic Cells
S. epi per. area
Project Overview
0.3 - 1%
0
1 - 4%
4
6
S.epi covered
area percentage
• S. aureus (40%)
• S. epidermis (20%)
0.01
0
PLL layer
low PEGDA
high PEGDA
substrate type
Microfluidic Co-Culture Tool for
Physiologically Relevant
in vitro Evaluation
Infection by
Staphylococcal Biofilms
0.02
bare wafer
bare
PLL
PLL coated Si
lower PEGDA
particles
high PEGDA
5 min adhesion
S.epi (green)
Si substrate (blue)
Biological Framework
An additional component of our work involves continuous hydrogel thin films
deposited using layer-by-layer self assembly. The hydrogels are derived from layerby-layer hydrogen-bonded films stabilized by chemical crosslinking. Specifically,
we have synthesized surface hydrogels by depositing poly(vinyl pyrrolidone)
(PVPON)/ poly(methacrylic acid) (PMAA) multilayers at the surface of precursormodified silicon wafers, followed by crosslinking using carbodiimide chemistry
with addition of ethylene diamine ( EDA) as a crosslinker. The resulting hydrogels
were loaded at pH 7.5 with an antibacterial polypeptide.
NH2
NH2
Stabilization of hydrogel
PVPON
PMAA
acidic pH
acidic pH,
after crosslinking
stabilization
Loading hydrogel with antibacterial
at basic pH
polypeptide JFLO
PMAA gel loaded w/ We have explored adhesion and growth of
PMAA hydrogel
polypeptide JFLO
Staphylococcus Epidermidis bacterial culture
at surfaces coating with JLFO-loaded
hydrogels. We used initial concentration
5x106 colonies/mL in 3% tryptic soy broth
(TSB). We found that bacterial cells adhered
and grew on bare hydrogels (Fig. 1, a).
However, adhesion and growth of S.
Epidermidis to hydrogels loaded with JFLO
a (PMAA) 10EDA
was completely inhibited after 2 and 4 hours.
b (PMAA) 10 EDA + JFLO
S. Epidermidis 4 h
10 μm
Therapeutic Delivery/
Host defense mechanism
S. epidermidis
Confocal images (left) and SEM (right) imaging
shows good osteoblast adhesion and spreading on
surfaces with cell adhesiveness modulated by PEG
gel partyicles on a cell-adhesive PLL surface. cell
spreading on the PEGDA modified surface.
Protein
Conditioning
Osteoblast
Broader Impact: Nanotechnology in High Schools
Implant Material
Goals of the HS Outreach Effort
Device Attributes
• High-throughput
• Time-lapsed visualization
• Cross contamination-free
Bacteria Dispersion in 8-Channel Device
9
10
8
10
Released S. epidermidis (cfu/ml)
Self-Assembled Hydrogel Films for
Controlled Antimicrobial Release
PEGDA particles
(black)
Confocal image
for S.epi growing
on the PEGDA
modified surface.
7
- Expose high school students to
nanotechnology-based research
- Demonstrate societal relevance
- Enhance and modernize topics taught in
standard high school biology and chemistry
10
Attributes of the Modules
6
10
Biomaterial Integration
5
10
- Ease of implementation
in biology and chemistry courses
- Minimal time requirement for implementation
- Contain a hands-on or laboratory activity
- Address National Science
Education Standards (NSES)
4
10
(a)
Osteo 1
Osteo 2
2
Osteo+10 S epi 1
2
Osteo+10 S epi 2
5
Osteo+10 S epi 1
5
Osteo+10 S epi 2
2
10 S epi
5
10 S epi
3
10
2
10
1
10
0
10
-1
(f)
(b)
(g)
(c)
(h)
(d)
10
-2
10
4
6
8
10
12
14
16
18
20
22
24
26
28
10 μm
A small number of opportunistic bacteria (1-1000) pre-inoculated on
Ti alloy surface can significantly damage osteoblasts within one day.
Osteoblast +
102 cfu/ml S. epidermidis
100 m
Year
3
Implement
small pilot
Implement
larger pilot
Revise draft
modules
Finalize
modules
Dissemination
A Dutch-US Student/Faculty Exchange
Objectives
Osteoblast +
105 cfu/ml S. epidermidis
Leverage Stevens’expertise in
biomaterials design, synthesis,
and processing with UMCG
expertise in clinically oriented
physiological assessment
S. Epidermidis 4 h
The figure to the left illustrates the
growth of S. Epidermidis at surfaces of
bare (a) and JFLO-loaded (b) (PMAA)10
EDA -crosslinked hydrogels during
exposure of substrates to TSB after 4
hours.
Year
2
Develop draft
modules
CIESE has nearly 20 years of K-12 curriculum and professional development
expertise in STEM education, and has impacted over 20,000 educators worldwide
Time since inoculation (h)
Osteoblast only
Year
1
(i)
(e)
100 m
Live (green) and dead (red) osteoblasts
100 m
University Medical
Center Groningen (UMCG)
higher PEGDA
particles
Cytoskeleton covered
substrate area is used to
indicate how the cells adhere
and spread. After 4 days
culture, the osteoblast can still
adhere and spread on the gelmodified surfaces.
0.03
Fixation devices
> 15%
e.g. Intramedullary trauma rods
bare Si
Substrate Type
8
time (hr)
S.epi adhesion on 5 types of substrates in 5 min
Knees
2
6 hours
1 day
4 days
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Create international exchange
opportunities for Stevens
undergrads rooted in Stevens
faculty research
Left: Stevens PhD
student Eva Wang
meeting with UMCG
collaborators on flowcell experiments.
Below: Stevens
undergrad Altida
Patimetha working on
her summer 2009 coculture experiment at
UMCG.