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In Vitro Cartilage Development
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Introduction
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Design of bioactive devices for regenerative
medicine involves controlling many interdependent variables.
Determining how the interplay between material, biochemical,
and mechanical variables guides tissue development requires
the establishment of measurement methods for quantifying cellular
responses in three-dimensional tissue engineering scaffolds.
We are developing such measurement tools for cartilage tissue
engineering in collaboration with top researchers at the National
Institutes of Health and the University of Colorado.
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Experimental Approach
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Cartilage constructs are grown by seeding primary
bovine chondrocytes in photopolymerizable hydrogels. Constructs
are cultured under variable fluid stresses in a microfluidic
bioreactor to promote cell differentiation and matrix production.
Cellular responses are characterized by histological techniques,
optical coherence tomography (OCM), and quantitative and real
time PCR (QRT-PCR).
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| Primary bovine chondrocytes (Dr. Rocky Tuan - NIH/NIAMS) are
seeded into 6mm diameter PEG-Dimethacrylate (PEGDM) hydrogel
cylinders along with cell medium (Dulbecco modified Eagle medium
(DMEM) + 20 % Fetal Bovine Serum (FBS) + 1 % minimum essential
medium vitamin solution + 1 % L-ascorbic acid 2-phosphate +
1% Penicillin / Streptomycin)
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Schematic representation of the hydrogel bioreactor chamber
with insert photo of cell-hydrogel scaffold
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| Hydrogel cylinders bearing chondrocytes are placed in a flow
cell (diagram at right) and medium is passed through the cell
in a pulsitile fashion.
Pulsitile flow parameters such as duty factor, flow rate,
and pulse pressure are varied semi-independently.
Classical histology, along with QRT-PCR and OCM are used
to determine extent and temporal evolution of extracellular
matrix (ECM) production. We are developing spectroscopic imaging
methods that will aid in non-invasive determination of ECM
composition.
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Results
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 Histology
(panels a and b) showed an increase in ECM (sulfated proteoglycan)
production for the dynamically cultured scaffolds as compared
to the static culture scaffolds which may be indicative of
a particular phenotypic stage of differentiation.
Optical Coherence Tomography (panel c and d) showed
evidence of increased ECM production for dynamically cultured
cells.
Quantitative Real-Time PCR (panel e) showed the expression
of genes for collagen type II and aggrecan under dynamic conditions
and only aggrecan under static conditions.
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Future Activities
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Future studies will:
1. Establish a method for quantifying cell deformation during
dynamic loading
2. Quantify flow rates and pressures (e.g., cell deformation
levels) needed to activate the cartilage gene markers for expression.
3. Investigate influence of hydrogel mechanical properties in
transducing effects of fluid stresses.
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Publications
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| Manuscript in preparation
Work presented at
2004 Gordon Research Conference
-Musculoskeletal Biology & Bioengineering
2004 Polymer Network Conference
-Tissue Engineering and Hydrogel Scaffolds
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NIST Contributors
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James Cooper
Lee Ann Bailey
Joy Dunkers
Steve Hudson
Seung-ho Moon
Jean Stephens
Sheng Lin-Gibson
Marc Cicerone*
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Collaborators:
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Roger Li & Rocky Tuan
(NIH/NIAMS)
Kristi Anseth
(University of Colorado )
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