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