NIRT: Self-Assembled Nanohydrogels for Differential Cell Adhesion and Infection Control

Matthew Libera, Woo Lee, Svetlana Sukhishvili, Hongjun Wang, and Debra BrockwayStevens Institute of Technology, Hoboken, New Jersey 07030

Project Overview

Infection occurs in approximately 0.5 – 5% of all hip and knee replacements. 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.

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.

This project explores a new mechanism to create differentially adhesive surfaces. We hypothesize that heterostructures of nanosizedhydrogels self assembled in 2Dover micrometer length scales willallow focal contact formation andsubsequent osteoblast adhesion but prevent bacterial adhesion.

CIESE has nearly 20 years of K-12 curriculum and professional development expertise in STEM education, and has impacted over 20,000 educators worldwide

Infection Rates

Hips 0.3 - 1%

Knees 1 - 4%

Fixation devices > 15% e.g. Intramedullary trauma rods

Infection by Staphylococcal Biofilms

• S. aureus (40%)• S. epidermis (20%)

Differentially Adhesive Surfaces - Repulsive to Bacteria but Attractive to Eukaryotic Cells

~2 mm

~350 m

Cell-Interactive nanohydrogels hierarchically structured on the surface of a macroscopically beaded surface of a modern orthopaedic implant.

~1 m

Broader Impact: Nanotechnology in High Schools

Develop draft modules

Implement small pilot

Implement larger pilot

Revise draft modules

Finalize modules

Dissemination

Year 1

Year 2

Year 3

Attributes of the Modules

- 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)

Goals of the HS Outreach Effort

- Expose high school students to nanotechnology-based research - Demonstrate societal relevance- Enhance and modernize topics taught in standard high school biology and chemistry

Self-Assembled Hydrogel Films for Controlled Antimicrobial Release

Surface Self-Assembled PEGDA Hydrogel Particles to Control Bacteria/Cell-Biomaterial Interactions

An additional component of our work involves continuous hydrogel thin films deposited using layer-by-layer self assembly. The hydrogels are derived from layer-by-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 precursor-modified 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.

We have explored adhesion and growth of 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 was completely inhibited after 2 and 4 hours. (PMAA) 10

EDA

S. Epidermidis 4 h

a

10 μm

b (PMAA) 10 EDA + JFLO

S. Epidermidis 4 h

10 μm

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.

PMAAPVPON

acidic pH acidic pH,after crosslinking stabilization

at basic pH

O NH

+

CH3CH3

NH

C

O

OCH2CH3

CH2CH3CH2CH3 Cl O NH

+

CH3CH3

NH

C

O

OCH2CH3

CH2CH3CH2CH3 Cl O NH

+

CH3CH3

NH

C

O

OCH2CH3

CH2CH3CH2CH3 ClO NH

+

CH3CH3

NH

C

O

OCH2CH3

CH2CH3CH2CH3 Cl O NH

+

CH3CH3

NH

C

O

OCH2CH3

CH2CH3CH2CH3 Cl O NH

+

CH3CH3

NH

C

O

OCH2CH3

CH2CH3CH2CH3 Cl

PMAA hydrogelPMAA gel loaded w/

polypeptide JFLO

Stabilization of hydrogel

Loading hydrogel with antibacterial polypeptide JFLO

NH2NH2

A Dutch-US Student/Faculty Exchange

University Medical Center Groningen (UMCG)

Objectives

Leverage Stevens’expertise in biomaterials design, synthesis, and processing with UMCG expertise in clinically oriented physiological assessment

Create international exchange opportunities for Stevens undergrads rooted in Stevens faculty research

Left: Stevens PhD student Eva Wang meeting with UMCG collaborators on flow-cell experiments.

Below: Stevens undergrad Altida Patimetha working on her summer 2009 co-culture experiment at UMCG.

UV is used to polymerize PEGDA in the DCM droplets which obtained by DCM/water emulsion.

PEG gel particles can deposit on the PLL modified Si wafer surface by electrical self-assembly to obtain modified surfaces with controllable gel-particle density. Lower and higher particles density surface were both tested in bacteria and osteoblast culture.

SiSiPLLPLL

Lower PEGDA density higher PEGDA density

Bacteria/Cell/Biomaterial Interactions

1 2

3 4

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);

S. epi grow on different substrates

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 2 4 6 8

time (hr)

S.

ep

i p

er.

are

a

bare Si wafer

PLL deposited Siwafer

low nanohydrogelcoverage

high nanohydrogelcoverage

pure PEGDA

PEG gel-modified surface reduces short term S.epi adhesion/growth.

Osteoblast 4 days culture result

Osteobl ast Cytoskel eton Densi ty on Di ff erent Substrates

00. 10. 20. 30. 40. 50. 60. 70. 80. 9

1

bare Si PLL coated Si l ower PEGDApart i cl es

hi gher PEGDApart i cl es

Substrate Type

Rati

o of

Sur

face

Cov

ered

by C

ytos

kele

ton

6 hours1 day4 days

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 gel-modified surfaces.

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.

S. epi adhesi on on 5 types of substrates i n 5 mi n

0

0. 01

0. 02

0. 03

bare wafer PLL l ayer l ow PEGDA hi gh PEGDA

substrate type

S.ep

i co

vere

dar

ea p

erce

ntag

e

bare PLL high PEGDAMicrofluidic Co-Culture Tool forPhysiologically Relevant

in vitro Evaluation

Osteoblast

S. epidermidisTherapeutic Delivery/Host defense mechanism

Implant Material

ProteinConditioning

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Osteoblast only Osteoblast + 102 cfu/ml S. epidermidis

100 m 100 m100 m

Osteoblast + 105 cfu/ml S. epidermidis

Live (green) and dead (red) osteoblasts

Biomaterial Integration

A small number of opportunistic bacteria (1-1000) pre-inoculated on Ti alloy surface can significantly damage osteoblasts within one day.

4 6 8 10 12 14 16 18 20 22 24 26 2810-2

10-1

100

101

102

103

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105

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108

109

Re

lea

se

d S

. ep

ide

rmid

is (

cfu

/ml)

Time since inoculation (h)

Osteo 1 Osteo 2

Osteo+102 S epi 1

Osteo+102 S epi 2

Osteo+105 S epi 1

Osteo+105 S epi 2

102 S epi

105 S epi

Bacteria Dispersion in 8-Channel Device Device Attributes• High-throughput• Time-lapsed visualization• Cross contamination-free

Biological Framework