elizabeth i. maurer dr. sharmila m. mukhopadhyay dr. …cecs.wright.edu/~smukhopa/beth.pdfdr. saber...

Elizabeth I. Maurer1,2

Dr. Sharmila M. Mukhopadhyay1

Dr. Saber Hussain2

1Wright State University2Air Force Research Laboratories

Objective: Approach:

Research surface modifications to enhance the function of carbon structures for biological applications

Collaboration between Wright State University and AFRL/RHPB

◦ Materials Engineering background

◦ Bio-interaction of nanomaterials group

Potential use of microcellular hierarchical structure as a scaffolding for biological composites.

Requirements

◦ Non-toxic or biocompatible for in vitro applications

◦ Allow rapid growth of natural tissue on surface

◦ Optimal bond between tissue and scaffold

Previous studies

◦ Favorable growth on uneven surfaces1

◦ Carbon coatings have been shown to be biocompatible2

Goals:

Investigate a porous structure that can facilitate growth of cells that

can easily integrate with surrounding tissue

Determine biocompatibility of functionalized carbon foam with osteoblast cell line

Carbon foam purchased from Koppers Inc.

Pores ~600μm in diameter

Manipulate surface of carbon foam to alter cell function and growth

SiO2

Collagen

Carbon Nanotubes

Mesenchymal

Stem Cell

Hepatocyte

stem cell

Skeletal

muscle

stem cell

Pre-

osteoblastOsteoblast Osteocyte Bone or

cartilage

Mesenchymal

Stem Cell

Hepatocyte

stem cell

Skeletal

muscle

stem cell

Pre-

osteoblastOsteoblast Osteocyte

Mesenchymal

Stem Cell

Hepatocyte

stem cell

Skeletal

muscle

stem cell

Pre-

osteoblastOsteoblast Osteocyte Bone or

cartilage

Biological Structure: Osteoblast

•Derived from the

mesenchymal stem cell.

•Progenitor cell

– Precursor cell

type for bone

•Will differentiate into an

osteocyte to eventually

produce bone or cartilage.

Background of cell line used:

Techniques Used: Materials

◦ SEM (Scanning Electron Microscopy)

Cell Morphology◦ SEM

◦ Fluorescent Microscopy (with cell staining)

Biological◦ MTS assay

To determine cell viability (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium )

Modifications to cellular foam substrate

◦ SiO2 coating

40nm microwave enhanced chemical vapor deposition

HMDSO (Hexamethyldisiloxane) and O2.

◦ Collagen coating

Dip coating process

Sample soaks in collagen solution and left to dry

◦ Carbon Nanotube layer

Chemical vapor deposition(CVD) growth process3

Using ferrocene and xylene in a CVD reactor

UC Control SiO2 Control CC Control CNT Control

Seed 106 cells/ml

Incubate for 72hrs at 37°C in a humidified atmosphere of 5% CO2

in air

Fix and dehydrate cells for imaging

Control - without cells

With cell growth (72 hrs)

Cells grown in culture for 48 hours

UC Control

UC

Before cell growth

After 72hr cell growth

Before cell growthSiO2 Control

SiO2After 72hr cell growth

Before cell growth

CC Control

CC After 72hr cell growth CNT

CNT Control

After 72hr cell growth

Porous structure maintained in all substrates

Cells were seen permeating the foam in each sample

Cells show the similar morphology as those grown in routine cell culture

◦ Cell density changes seen from the surface modifications

CCUC SiO2CNT

Nucleus

Cytoplasm

Seeded cells on foam samples

Incubate for 72hrs at 37°C in a humidified atmosphere of 5% CO2 in air

Fix and stain cells:◦ Fluores at specific wavelengths:

◦ Red – Actin filaments (Alexafluor 555)

◦ Blue – Nucleus (Prolong Gold Reagent with DAPI counterstain)

UCUC CCCCSiO2SiO2CNT

90

93

96

99

102

105

108

1 2 3 4 5

Cell V

iabilit

y (%

Contr

ol)

Nuclear Density (x103 cells/mm2)

CNT

SiO2

Uncoated

Collagen80

100

120

Cell V

iab

ilit

y %

Co

ntr

ol

Control Uncoated Foam

SiO2 Coated Foam Collagen Coated Foam

Carbon Nanotube Coated Foam

MTS◦ No significant decrease in cell viability◦ Increase in cell function in CNT coated foam samples

Cell Density◦ CNT and SiO2 showed highest density◦ Enhanced cell function on CNT coated sample

MTS/Cell Density Study

• MTS solution with

cells for 1 hr

• Absorbance is

recorded at 490nm

• Compared to

control well (no

foam)

Carbon foam was shown to be biocompatible and is an excellent scaffold for growth of natural cells utilizing surface modifications

The microcellular foam/osteoblast composite has the potential for seamless integration into the body

Small variation in cell viability due to coatings:◦ Decrease in cell function of cells due to SiO2

Enhanced growth of cells on carbon nanotube coated foam◦ MTS and cell density study

Further studies: Examine other biomarkers such as:

◦ Alkaline Phosphatase◦ Osteocalcin

Expand to other cell lines◦ Skin, liver, etc.

Surface modification research is being extended to other studies

Sensor Schematic:

Gold nanoparticles attached to carbon

nanotubes on graphite substrate

Graphite

SiO2

CNT

Biomolecule

http://www.marlerblog.com/uploads/image/GP2144.jpg

Bacteria:E.Coli (DH5α)

This project was made possible with support from :◦ Dayton Area Graduate Studies Institute, Wright Patterson Air Force Base

AFMC 711 HPW/RHPB , Wright State University, Ohio Board of Regents(OBOR), and the National Science Foundation.

Dr. Sharmila M. Mukhopadhyay◦ Center for Nanoscale Multifunctional Materials at Wright State University

◦ WSU graduate/PhD students

Dr. Saber Hussain◦ Biological Interaction of Nanomaterials (BIN) group at AFRL

1Schmidt et. al. Journal of Biomedical Materials Research Volume 63, 252 - 261 (2002),

2 A. Grill. Diamond and Related Materials, Volume 12, 166-170 (2003)

3S.M. Mukhopadyhyay, A. Karumuri, I.T. Barney, J. Phys. D, 42, 19 (2009)

Questions?