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 07030Project 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: A Major Mode of Orthopaedic Implant Failure

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%)

polycation primer layer

substrate self-assembled nanohydrogels

repulsive adhesive

osteoblastS. epi

Source: Merrill LynchCourtesy of G. GrobeDePuy Orthopaedics

Global Orthopaedic Markets (2005)attachment Polysaccharide

secretionMaturation and biofilm growth

Release of planktonic progeny

Bacterial biofilms form complex and hierarchically structured communities which are as much as 10kx more resistant to antibiotics than planktonic bacterial.

The biofilm cycleadapted from Sauer, Genome Biology, 2003)

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

Multiplexed Microfluidic Methodology forEvaluation of Differential Adhesion

Glass substrate

Objective Lens

Osteoblasts before S. epidermidis inoculation

MC3T3-E1 osteoblast were seeded (1M/ml M-MEM) in a prototype microchannel on a glass slide, cultured at 0.1 l/min flow rate for 24h

CMFDA, pre-stainedC12-reazurin, post-stained

20x 20x20x

S. epidermidis

SYTO9

20x

S. epidermidis, 108 CFU/ml, 5h flow culture in M-MEM

Osteoblasts co-cultured with S. epidermidis for 2h under flow

CMFDA, pre-stained

C12-reazurin/Sytox, post-stained

MC3T3-E1 osteoblasts were seeded (1M/ml) in a microchannel on a glassslide, cultured in M-MEM at 0.1 l/min flow rate for 24h, and then inoculated with 103 CFU/ml S. epidermidis, and cultured for 2h

Prototype device bonded to glass

Devices integrated to agrit-blasted Ti alloy (top), a polished Ti alloy (middle),

and a glass slide (bottom)

Osteoblast

S. epidermidisTherapeutic Delivery/Host defense mechanism

Relevant Biomaterial/Engineered Material

Protein Conditioning

Broader Impact: A Partnership to Introduce Nanotechnology Research in High School Classrooms

Develop draft modules

Implement small pilot

Implement larger pilot

Revise draft modules

Finalize modules

Dissemination

Year 1

Year 2

Year 3

Stevens undergraduate Zareen Mobin working with Ms. Clare Kennedy fromthe Academies@Englewood/Dwight Morrow High School

Dr. Ron Schreck from the Academies@Englewood/Dwight Morrow High Schooland ACS SEED student Ashley Contreras

Attributes of the Module

- 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 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

Nanohydrogel Synthesis and Self Assembly

+

PEGDA, AA, photo initiator and DCM

vortex

Add DI water during stirring

Sonicate for 30 min in darkness

UV for 15 min during sonication

SiSi

- - - - - - - - - - -

SiSi

- - - - - - - - - - -+ + + + + + + + + + +

A Si wafer was used as substrate and hydroxylated by NaOH. It was immersed in PLL solution to get a positive charged PLL primer layer.

The PLL coated wafer was then immersed in a nanohydrogel suspension at pH 7.4 for electrostatic gel deposition.

SiSi

- - - - - - - - - - -+ + + + + + + + + + +

-- - - - - - --

-- - -

-- -

--- -

Electrostatic

Self Assembly Emulsion Polymerization

Deposition Efficiency

Si Si + PLL Si + PLL + nanohydrogel

Reduced S. epi Adhesion