BioE Thesis Defense
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Transcript of BioE Thesis Defense
The Novel Design of a Bioreactor for in vitro Proliferation and Differentiation of Human Mesenchymal Stem Cells
Author: Joshua Salvi, Department of BioengineeringHonors Advisor: Dr. William Hancock, BioengineeringThesis Advisors: Dr. Henry Donahue, Orthopaedics (HY)
Dr. Peter Butler, Bioengineering (UP)
April 16, 2009
Al-Rubeai, et al., 2005; Davies, et al., 2006
Background
650,000 bone allograft transplantationsLow Risk of HIV infection – 1:150,471Failure rate within ten years – 60%
SOLUTION? Tissue-engineered implants
Hypotheses
Nanoscale substrates select for subpopulations of progenitor cells through differentiation into the osteoblastic lineage, as influenced by surface characteristics, including chemistry and topography.
The same progenitor cell lines cultured on three-dimensional calcium phosphate scaffolds will display significantly maintained differentiation potential with continued expansion in vitro compared with two-dimensional substrata and flat controls.
A bioreactor designed with finite element methods will satisfy all the criteria needed for long-term culture on three-dimensional scaffolds with induced biophysical signals.
Fabrication of Two-Dimensional SubstrataPS/PBrS 60/40 w/w – 0.5%, 1.0%, 2.0%
150300
0
nm
150300
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nm
150
300
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0 1 2 34 5
m0 1 2 3
4 5
m0
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34
5
m
PLLA/PS 70/30 w/w – 0.5%, 1.0%, 2.0%, 3.0%
*p<0.05 w/ Flat; #p<0.05 w/ 12 nm; ##p<0.01 w/ 12 nm; N=3
PLLA/PS Response under Oscillating Fluid Flow
*p<0.05 w/ Flat; **p<0.01 w/ Flat; ##p<0.01 w/ 85 nm; N=3
PS/PBrS FACS Analysis of Differentiation Potential(Day 7)
*p<0.05 w/ Flat; **p<0.01 w/ Flat; ##p<0.01 w/ 85 nm; N=3
PS/PBrS FACS Analysis of Differentiation Potential(Day 12)
***p<0.001 w/ 11 nm; N=6
Alkaline Phosphatase Activity
Summary
Hypotheses
Nanoscale substrates select for subpopulations of progenitor cells through differentiation into the osteoblastic lineage, as influenced by surface characteristics, including chemistry and topography.
The same progenitor cell lines cultured on three-dimensional calcium phosphate scaffolds will display significantly maintained differentiation potential with continued expansion in vitro compared with two-dimensional substrata and flat controls.
A bioreactor designed with finite element methods will satisfy all the criteria needed for long-term culture on three-dimensional scaffolds with induced biophysical signals.
3D Scaffold Characterization by SEM
A/B : BD© Calcium PhosphateC/D : BoneMedik© CoralE/F : NaCl-leached PLLA (150-300 μm)G/H : NaCl-leached PLLA (300-500 μm)I/J : NaCl-leached PLLA (500-710 μm)
*p<0.05 w/ PLLA(300-500); **p<0.01 w/ PLLA(300-500); ***p<0.001 w/ PLLA(300-500); +++p<0.001 w/ PLLA(500-710) N=10
Estimated Porosity Values from ImageJ
To be utilized in FEM Analyses
**p<0.01 w/ coral; N=6
Alkaline Phosphatase Activity
Hypotheses
Nanoscale substrates select for subpopulations of progenitor cells through differentiation into the osteoblastic lineage, as influenced by surface characteristics, including chemistry and topography.
A bioreactor designed with finite element methods will satisfy all the criteria needed for long-term culture on three-dimensional scaffolds with induced biophysical signals.
Protocol for Finite Element Analyses
Software: COMSOL MultiphysicsModel: Incompressible Navier-Stokes, Stress-Strain Analysis (Transient)Solver: Time Dependent, Direct (PARDISO)Fluid Flow: Newtonian, “No-slip” at surface
Inlet Pressure: Outlet Pressure:Constants:
Navier-Stokes:
von Mises Stress:
Similar data found for “Cell Left” and “Cell Right”
Effects of Cell Height on Surface Stresses
*: p < 0.05, **: p < 0.01, ***: p < 0.001
Effects of Substrata on Cell Surface Stresses
Utility of FEM in Tissue Engineering
Hypotheses
Nanoscale substrates select for subpopulations of progenitor cells through differentiation into the osteoblastic lineage, as influenced by surface characteristics, including chemistry and topography.
A bioreactor designed with finite element methods will satisfy all the criteria needed for long-term culture on three-dimensional scaffolds with induced biophysical signals.
Design Criteria Design Specifications
The bioreactor must maintain physiologic shear stresses as witnessed in vivo.
Maintain a uniform shear stress throughout the volume of the scaffold at 5, 10, and 20 dynes/cm2.
Standard scaffolds must fit within the bioreactor volume.
Ensure the bioreactor has a variable diameter between 2 and 10 mm.
The bioreactor must withstand oscillating fluid flow conditions.
Determine a symmetrical geometry capable of withstanding 1 Hz oscillations.
Flow must be uniform throughout the scaffold volume.
Simulate the flow profiles throughout the volume of the scaffold to ensure all are greater than zero.
The bioreactor must work for all scaffolds.After determining the relative porosities of each
scaffold, repeat the above analyses with each porosity value.
Bioreactor Design Specifications
Bioreactor FEM Geometry
Tight versus Loose Geometries
Comparison of Various Scaffolds in Loose Geometry
Summary
Hypotheses
Nanoscale substrates select for subpopulations of progenitor cells through differentiation into the osteoblastic lineage, as influenced by surface characteristics, including chemistry and topography.
A bioreactor designed with finite element methods will satisfy all the criteria needed for long-term culture on three-dimensional scaffolds with induced biophysical signals.
Modification of the Classic Approach
Acknowledgments
Dr. Henry DonahueDr. Peter ButlerDr. Jung Yul LimDianne McDonaldDrs. Yue Zhang and Christopher Niyibizi, Jacqueline YanosoDr. Ryan Riddle, Amanda Taylor, Peter Govey
Dr. William HancockDr. Margaret SlatteryCarol Boring
Bioengineering FacultySURIP, Step-Up, BBSI StudentsStudents of Bioengineering
Penn State College of MedicinePenn State College of EngineeringSchreyer Honors CollegeDepartment of Bioengineering
Biomaterials and Bionanotechnology Summer Institute(BBSI)