designing vascularized soft tissue constructs for transport eid 121 biotransport eid 327 tissue...
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Designing Vascularized Soft Tissue Constructs for Transport
EID 121 Biotransport
EID 327 Tissue Engineering
David Wootton
The Cooper Union
Acknowledgement and Disclaimer
This material is based upon work supported in part by the National Science Foundation under Grant No. 0654244
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation
Challenge
Develop a CAD model for printing a hydrogel tissue engineering construct for soft tissue• Vascular template• Sufficient oxygen delivery • Model validation/justification
Learning Objectives
Tissue Engineering (for EID 121) Oxygen Transport
• With oxygen carriers Vascular Anatomy Biomanufacturing for Tissue
Engineering• Bulk Methods• Computer-aided Manufacturing• Organ printing
Overview of Tissue Engineering
Working definition (1988):“The application of the principles and methods of engineering and life sciences toward the fundamental understanding of structure-function relationships in normal and pathological mammalian tissue and the development of biological substitutes to restore, maintain, or improve tissue function.”
Where we are already:•Robust research area•Tissue Engineered Medical Products – several approved•Expansion to biological model systems•Many unsolved challenges remain•Science base is rather weak for engineering (fundamental laws?)
A Famous Picture of TE
Polymer Ear shape
Bovine chondro-cytes
Implant in Nude Mouse
Potential TE Applications
Indication Annual Need, US
Skin - Burns 2,000,000
Bone – Joint Replacement 600,000
Cartilage –Arthritis 400,000
Arteries – bypass grafts 600,000
Nerve and spinal cord 40,000
Bladder 60,000
Liver 200,000
Blood Transfusion 18,000,000
Dental 10,000,000
Tissue Engineering Market Size
Costs of tissue-related disease procedures: $400 B (1993)
70+ companiesAverage $10
M/yearOrgan transplant
waiting lists are growing (doubled in 6 years)
$$
One Famous TE Paradigm
Your Design Challenge
Overcome practical size limit on engineered tissue• Diffusion is not sufficient for
oxygenation in thick tissues Compare 3 Approaches:
1. No flow (diffusion only)
2. Porous scaffold with permeation flow
3. Hydrogel with vascular channels
Design Challenge Example: engineer a 1 cm3 liver tissue construct
• Scaffold + hepatocytes• How will you make the scaffold?• How will you assure oxygenation?• What else do you need to know?
Polysaccarid
Questions for instructor? Discuss in groups of 3
http://licensing.inserm.fr/upload/ 270109_140959_PEEL_U5UFfJ.gif
Polysacchiride scaffold Cell-seeded scaffold
Design Challenge What else do you need to know? Formulate biotransport problem
• Hepatocyte (cell) properties• Oxygen transport properties• Dimensions• Is there a vascular system?
Oxygen Transport References:
• Truskey, Yuan, and Katz. Transport Phenomena in Biological Systems. 2nd Ed., 2009. (Section 13.5)
• RL Fournier. Basic Transport Phenomena in Biomedical Engineering. 2nd ed, 2006. (Ch. 6)
O2 Readily crosses cell membranes Transport Mechanisms: diffusion,
convection Metabolic demand and cell density
control oxygen concentration
Oxygen Diffusion Transport Simplest Approach: diffusion only Use 1D slab for simplicity How deep can O2 penetrate?
tissue
Oxygen Diffusion Transport Half-slab model (thickness 2L, max
concentration on top and bottom) Dissolved O2 in medium via Henry’s Law
22 pOHCO
x
L
0
O2 in blood at 37ºC, H = 0.74 mmHg/mM Typical air pO2 = 140mmHg, CO2 = 190mM
tissue
Oxygen Diffusion Transport O2 uptake rate RO2 or Gmetabolic Expect Michealis-Menten kinetics, e.g.
2
2max
pOK
pOV
mmetabolic
maxVmetabolic
22
2
Oe Rdx
CdD tissue
x
L
0
Usually pO2 >> Km, so ~ zero order:
C = C0 = 190mM
0dx
dCSymmetry:
C = C0 = 190mM
Oxygen Diffusion Transport Diffusion flux = uptake (1-D):
max2
2
Vdx
CdDe
tissue
C = C0 = 190mM
x
L
0
0dx
dCSymmetry:
Effective Diffusivity, De
Uptake rate Cell seeding density, r
Hepatocytes:Vmax = 0.4 nmol/106 cells/secKm = 0.5 mmHgCell diameter = 20 mmDensity up to rcells = 108 cell/cm3
Oxygen:H = 0.74 mmHg/mMDe = 2 x 10-5 cm2/s
C = C0 = 190mM
max2VRO
Oxygen Diffusion Transport Diffusion flux = uptake (1-D):
cellcells 1
max2
2
22 ; VRR
dx
CdD OOe
tissue
x
L
0
Void volume, e Effective Diffusivity, De
Hepatocytes:Vmax = 0.4 nmol/106 cells/secKm = 0.5 mmHgCell diameter d = 20 mmDensity up to rcells = 108 cell/cm3
Oxygen:H = 0.74 mmHg/mMDe = 2 x 10-5 cm2/s
C = C0 = 190mM
0dx
dCSymmetry:
C = C0 = 190mM
Oxygen Diffusion Transport
Work in small groups What is the O2 uptake rate in the
tissue? What is the concentration
distribution? How thick could the construct be? Check vs. following solution
Oxygen DiffusionTransport solution
Uptake rate: Hepatocytes:Vmax = 0.4 nmol/106 cells/secKm = 0.5 mmHgCell diameter d = 20 mmDensity up to rcells = 108 cell/cm3
Oxygen:H = 0.74 mmHg/mMDe = 2 x 10-5 cm2/s
sMcmnmol
M
scells
nmol
cm
cellsVRO /40
/
1
104.010
3638
max2
L
x
L
x
D
LRCC
e
O
21
22
2
02
Solution:
Maximum thickness Set C(L) to zero:
Example gives Lmax = 138 mm How far would you need to reduce cell
density to compensate, for 1 cm construct?
2
0max
2
O
e
R
DCL
Oxygen Diffusion Transport Simplest Approach: diffusion only Use axisymmetric cylinder for
simplicity How deep can O2 penetrate?
Oxygen Diffusion Transport Cylinder model (radius Rc, max
concentration on surface) Dissolved O2 in medium via Henry’s Law
22 pOHCO
O2 in blood at 37ºC, H = 0.74 mmHg/mM Typical air pO2 = 140mmHg, CO2 = 190mM
tissue
rRc
0
Oxygen Diffusion Transport O2 uptake rate RO2 Expect Michealis-Menten kinetics,
2
2max
pOK
pOV
mmetabolic
maxVmetabolic
maxVdr
dCr
dr
d
r
De
tissue
r
Rc
0
Usually pO2 >> Km, so ~ zero order
C = C0 = 190mM
0dr
dCSymmetry:
Oxygen Diffusion Transport Diffusion flux = uptake (axisymmetric):
tissue
C = C0 = 190mM
Symmetry:
Effective Diffusivity, De
Hepatocytes:Vmax = 0.4 nmol/106 cells/secKm = 0.5 mmHgCell diameter = 20 mmDensity up to rcells = 108 cell/cm3
Oxygen:H = 0.74 mmHg/mMDe = 2 x 10-5 cm2/s
maxVdr
dCr
dr
d
r
Dcells
e
0dr
dC
r
Rc
0
Oxygen Diffusion Transport Diffusion flux = uptake (1-D):
cellcells 1
max2
2
22 ; VRR
dx
CdD cellsOOe
tissue
Void volume, e Effective Diffusivity, De
Hepatocytes:Vmax = 0.4 nmol/106 cells/secKm = 0.5 mmHgCell diameter d = 20 mmDensity up to rcells = 108 cell/cm3
Oxygen:H = 0.74 mmHg/mMDe = 2 x 10-5 cm2/s
C = C0 = 190mM
0dx
dCSymmetry:
r
Rc
0
Oxygen Diffusion Transport
Work in small groups What is the O2 uptake rate in the
tissue? What is the concentration
distribution? How thick could the construct be? Check vs. following solution
Oxygen DiffusionTransport solution
Uptake rate: Hepatocytes:Vmax = 0.4 nmol/106 cells/secKm = 0.5 mmHgCell diameter d = 20 mmDensity up to rcells = 108 cell/cm3
Oxygen:H = 0.74 mmHg/mMDe = 2 x 10-5 cm2/s
sMcmnmol
M
scells
nmol
cm
cellsVRO /40
/
1
104.010
3638
max2
22
0
20202
2
10
1
2
14
4)( ;
4
00 ;2
2
2
22
2
2
2
ce
cO
ce
Oc
e
O
re
O
e
O
e
O
R
r
D
RRCC
RD
RCCCRCCr
D
RC
Cdr
dC
r
Cr
D
R
dr
dC
rD
R
dr
dCr
rD
R
dr
dCr
dr
d
Solution:
Oxygen DiffusionTransport solution
Uptake rate: Hepatocytes:Vmax = 0.4 nmol/106 cells/secKm = 0.5 mmHgCell diameter d = 20 mmDensity up to rcells = 108 cell/cm3
Oxygen:H = 0.74 mmHg/mMDe = 2 x 10-5 cm2/s
sMcmnmol
M
scells
nmol
cm
cellsVRO /40
/
1
104.010
3638
max2
Solution:
Maximum thickness Set C(0) to zero:
Example gives Rmax = 195 mm How far would you need to reduce cell
density to compensate, for 1 cm construct?
2
0max
4
O
e
R
DCR
22
0 14
2
ce
cO
R
r
D
RRCC
Checking your learning progress
What is diffusion transport? Diffusion is fast over short
distances, slow over long distances• Why?
How does oxygen uptake reaction affect oxygen penetration into tissue• Dimensionless transport-reaction
parameter (see Krogh cylinder model F)
Class Discussion Time
Q&A about diffusion transport Make suggestions to improve oxygen
transport rate
Oxygen Transport Problem
We can improve transport with flow (convection) through thick direction
Four approaches to consider• Tissue in to spinner flask• Drive permeation flow through pores• Tissue with engineered vascular
channels• Let tissue form vascular system
Oxygen Transport Problem
Spinner flask doesn’t help much• Minimal medium flow due to small pressure
gradients• Best model: diffusion through tissue
Permeation flow• Manufacturing methods needed to control pores• Characterize scaffold media flow• Can scaffold withstand pressure required?• Implantation issue: source of pressure?
Oxygen Transport Problem
Engineered vascular system• How to manufacture?
• Current research subject
• Proposed solutions use computer-aided manufacturing (CAM) and design (CAD)
• What are the mass transport requirements for the vascular system?
Tissue Engineering Manufacturing Overview
How to make tissues more efficiently?
How to improve control of tissue constructs?
Use modern manufacturing methods
Bulk Scaffold Manufacturing Methods
First consider “Bulk” scaffold manufacturing methods
Widely used:• Relatively easy to replicate• Relatively fast
Good control of material biochemical properties
Recipes influence scaffold architectural properties (indirect control)
Bulk Scaffold Manufacturing Examples
Electrospinning Salt Leaching Freeze Drying Phase Separation Gas Foaming Gel Casting
Electrospinning
http://www.centropede.com/UKSB2006/ePoster/images/background/ElectrospinFigure.jpg
Salt Leaching
Agrawal CM et al, eds, Synthetic Bioabsorbable Polymers for Implants, STP 1396, ASTM, 2000
Freeze Drying
Phase Separation
Bulk methods pros and cons+ Relatively fast batch processing
+ Often low investment required
- Non optimal microstructures:• High porosity (required for
connectedness)• Permeability often low (especially foams)• Low strength (eg too low to replace bone)• Modest control of pore shape
Computer-aided manufacturing Top-down control of scaffold
• CAD models• Reverse engineering (from medical
images) Based on existing technology
• Inkjet/bubblejet/laserjet printers• Rapid prototyping machines• Electronics and MEMS manufacturing
Often compatible with bulk methods
Photopatterning Surface Chemistry
Microcontact and Microfluidic Printing
Micromachining, Soft Lithography
Soft Lithography
3D Printing
Spread powder layer Print powder binder
Solid Freeform Fabrication
Make arbitrary shapes Limited resolution Incrementally build
• Layer by layer• Fuse Layers to get 3D part
Several processes including• Fused deposition• Drop on demand• Laser sintering
http://www-ferp.ucsd.edu/LIB/REPORT/CONF/SOFE99/waganer/fig-2.gif
http://www.msoe.edu/rpc/graphics/fdm_process.gif
CAD-based Porogen Method
Mondrinos M et al, Biomaterials 27 (2006) 4399–4408
Current Research on Scaffolds EWOD Video Clips
Live
Dead
Current Research on Scaffolds Drexel, Duke, Cooper Union collaboration Electrowetting tissue manufacturing CAD model Print components
• Hydrogel• Crosslinker• Cells• Growth Factor
Web site:
X-Y Moving Control System
EWOD Microarrays Control System
Hydrogel Microarray Crosslinker
Microarray
Cell Microarray
Growth Factor Microarray
Hydrogel Reservoir
Crosslinker Reservoir
Cell Reservoir
Growth Factor Reservoir
EWOD Microarrays Mounted on X-Y Moving Planar Arm Material
Delivery System
Moving Table
Scaffold
Z Moving Control System Moving Direction
http://www.mem.drexel.edu/zhou2/research/electro-wetting-on-di-electric-printing
Modeling Permeation Flow and Transport (optional)
Goals• Understand design/manufacturing
requirements for porous scaffolds• Predict flow for oxygenation• Predict pressure-flow relationship• Estimate scaffold strength and stiffness
requirements• Relate flow to shear stress on cells
Porous Media Mixture of solid phase and pores
• Fibrous media (mats, felts, weaves, knits)• Particle beds (soils, packed beads)• Foams (open-cell)• Gels
Advantages for tissue engineering• Large surface area for cell attachment • Good mass transport properites
• High surface to volume ratio• Open pores allow media flow
Modeling Vascular Transport
Goals• Understand design/manufacturing
requirements for vascular tissue design• Predict flow for oxygenation• Predict pressure-flow relationship• Estimate scaffold strength and stiffness
requirements• Relate flow to shear stress on cells• Understand/analyze effect of oxygen carriers
Krogh Cylinder Model A simplified model of oxygen transport from capillary to
tissue Named after August Krogh (1874-1949, 1920 Nobel Lauriat;
pronounced “Krawg”) Tissue modeled as cylinders around parallel capillaries
(axisymmetric)
capillary
tissue
ignored
Krogh Cylinder Assumptions Radial diffusion in the tissue is the dominant
mass transfer resistance• Mass transfer in blood and plasma is ignored• Axial diffusion ignored• Improve by modeling plasma layer at vessel wall
Oxygen carrier kinetics are instantaneous• Plasma oxygen at equilibrium with oxygen carriers
Steady state
Krogh Cylinder Equations, 1 Radial Diffusion in tissue:
• PDE
• BC’s
• Solution
Maximum oxygenated radius:r
RV
0 z
R0
L
max22 where, VRR
dr
dCr
dr
d
r cellsOOe
D
0 );()(0
R
wV dr
dCzCRC
2
0
2
0
20 ln2
41)( 2
R
r
R
R
R
r
C
RRCrC V
Vew
Ow D
220
020
0
2
max
max
max
max
4ln2
0)(
VO
ew
V
RR
CR
R
RR
RC
D vz
Krogh Cylinder Equations, 2 Nondimensional Form:
• Solution
)for 0(
ln21
max0
2*2**
**
Rr C
rRR
r
C
CC
w
0
*
0
*
20
42
R
rr
R
RR
C
RR
V
ew
O
D
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
C*
r*
0.01
0.05
0.1
0.15
0.2
0.25
• Example, R* = 0.05
Krogh Cylinder Equations, 2a Nondimensional Form:
• Solution
)for 0(
ln21
max0
2*2**
**
Rr C
rRR
r
C
CC
w
0
*
0
*
20
42
R
rr
R
RR
C
RR
V
ew
O
D
• Example, R* = 0.20
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
C*
r*
0.01
0.1
0.2
0.3
0.4
0.5
Krogh Cylinder Equations, 3 Critical Radius vs. Reaction Rate:
• Relate reaction rate to critical radius:)ln(21
1*2* RR
0
*
R
RR V
1
10
100
1000
10000
0.01 0.1 1 10 100
R0/RV
Hypoxic
OK
Dimensionless Reaction Rate What is the meaning of F? Dimensionless reaction rate ...
• Estimate rate of oxygen uptake in an R0 x L cylinder
• Estimate rate of oxygen diffusion through an R0 x L cylinder
=FUptake Rate
Transport Rate we
O
we
O
C
RR
LRRC
LRR
DD
20
00
20 22~
• Low F is slow uptake, allowing deeper O2 diffusion
• High F is fast uptake, reduced radius for cylinder
Krogh Cylinder Equations, 4 Axial convection:
• Balance oxygen flow in medium/blood with uptake in tissue• Assume C>0 in tissue, average medium velocity vz
TzV CvR2
RV
z
R0
vz
dz
dz
dz
dCCvR T
TzV2• Inflow: • Outflow:
• Tissue uptake: 2
220 OV dzRRR
• Mass Balance:
2
220
22OV
TTzVVzV dzRRRdz
dz
dCCvRCvR
zv
R
R
RCC
v
R
R
R
dz
dC
z
O
VTT
z
O
V
T
2
0
2
1
1
2
20
2
20
Krogh Cylinder Application Apply to hepatocyte TE example:
• Uptake rate
• Inflow oxygen in medium: CB0 = 190 mM
• Want 1 cm thick tissue with 10 um diameter capillaries• What flow velocity vz and channel spacing would work?
• Derive R0max vs. vz based on CBT
(L) > 0
sMVRO /40max2
r
Rc
0 z
R0
L )/1/(75.4110
1/40
1901)10(1
01)(
max
2
0
2
0
0
2220
2
20
scmvmR
cm
v
sM
Mm
LR
vCRR
Lv
R
R
RCLC
z
z
O
zTV
z
O
VTT
2
0
10/21.0 max
m
Rscmvz
vz
Krogh Cylinder Application E.g. to get 200 mm vessel spacing requires about
1 m/s flow speed!
0.1
1
10
100
1000
10000
10 100 1000
v(cm/s)
R0 (m)
Krogh Cylinder Application Check shear stresses and pressure drop required
(assuming fully-developed flow): These are
very high shear stresses!
Want t<2Pa (R0 < 20 mm)
Need shorter vessels or augmented transport
0.1
1
10
100
1000
10000
10 100 1000
t(Pa)
R0 (m)
Oxygen Carriers References
• Truskey, Yuan, and Katz. Transport Phenomena in Biological Systems. 2nd Ed., 2009. (Sections 13.2 – 13.3)
• RL Fournier. Basic Transport Phenomena in Biomedical Engineering. 2nd ed, 2006. (Secitions 6.2 to 6.5, 6.12)
• M Radisic et al, Mathematical model of oxygen distribution in engineered cardiac tissue ...” Am J Physiol Heart Circ Physiol 288: H1278-H1289, 2005.
Water and cell culture media have low O2 capacity Blood has hemoglobin in red blood cells to store
and release O2 Artificial O2 carriers have also been developed as
an alternative to blood transfusion• Perfluorocarbons (PFCs)• Stabilized hemoglobins
Hemoglobin-Oxygen Binding At saturation each Hb binds 4 O2 molecules % saturation vs. O2 partial pressure is nonlinear
mmHg 26
34.2
)(
)(
50
250
2
P
n
pOP
pOS
nn
n
00.10.20.30.40.50.60.70.80.9
1
0 20 40 60 80 100 120 140 160
S
pO2 (mmHg)
Hemoglobin Saturation
RBCs Increase O2 capacity Total blood oxygen concentration:
saturation
M 5111MmmHg/ 74.0
4
2
2
2
S
CH
SHctCH
pOC
Hb
O
HbO
T
0
2,000
4,000
6,000
8,000
10,000
12,000
0 20 40 60 80 100 120 140 160
CBT
(M)
pO2 (mmHg)
50%
45%
40%
20%
0%
Hct
Oxygen content at 100 mmHg and 45% Hct is about 70x higher than in plasma or media
Our TE Application, with RBCs Assume Hct = 40%, pO2 = 140 mmHg
• Oxygen in inflow plasma is still: C = 190 mM
• Inflow total oxygen concentration is CBT = 8200 mM
• Rederive CT equation with nonlinear saturation curve?
r
Rc
0 z
R0
Lvz
)/1/205(110
1/40
82001)10(1
01)(
max
2
0
2
0
0
2220
2
20
scmvmR
cm
v
sM
Mm
LR
vCRR
Lv
R
R
RCLC
z
z
O
zTV
z
O
VTT
2
0
10/004878.0 max
m
Rscmvz
Krogh Cylinder, Blood E.g. to get 200 mm vessel spacing requires about
2 cm/s flow speed
0.001
0.01
0.1
1
10
100
10 100 1000
v(cm/s)
R0 (m)
Krogh Cylinder Application Check shear stresses required (assuming fully-
developed flow, viscosity ~ 0.005 kg/m-s):
These are still rather high shear stresses
Want t<2Pa Spacing ~
50 mm looks feasible
0.1
1
10
100
1000
10 100 1000
t(Pa)
R0 (m)
Krogh Cylinder Application Check pressure required (assuming fully-
developed flow, viscosity ~ 0.005 kg/m-s):
These are low pressures (less than 1 cm H2O for spacing less than 100 mm)0.001
0.01
0.1
1
10
100
10 100 1000
Pinlet
(mmHg)
R0 (m)
Reflection How do RBCs increase blood’s oxygen-
carrying capacity?• Mechanism• Quantitative effect
How do RBCs effect vessel spacing, shear stress, and pressure requirements?
What are the difficulties of using blood to culture tissue?
Perfluorocarbons (PFCs) Synthetic oxygen carriers Not currently FDA approved for human
use (Fluosol-DA-20 was approved 1989 but withdrawn 1994)
Several in clinical trials High oxygen solubility: Henry constant
HPFC = 0.04 mmHg/mM Example (in clinical trials): Oxygent
• Emulsion of 32% PFC
Perfluorocarbons (PFCs) Linear increase in O2 with %PFC and pO2
0
200
400
600
800
1,000
1,200
1,400
1,600
0 50 100 150 200
CT
(M)
pO2 (mmHg)
20%
12%
7%
3%
0%
PFC
Perfluorocarbons (PFCs) PFCs don’t match RBC performance except
at supraphysiologic oxygen pressures
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
9,000
10,000
0 20 40 60 80 100 120 140 160
CBT
(M)
pO2 (mmHg)
20% PFC
12% PFC
7% PFC
3% PFC
0% PFC
45% Hct
Blood,45% Hct
Our TE Application, with PFCs Assume 12.8% PFC (40% Oxygent), pO2 = 160
mmHg• Oxygen concentration with PFCs:
• Inflow CBT = 700 mM
r
RV
0 z
R0
L
)/1/5.17(110
1/40
7001)10(1
max
2
0
0
2220
scmvmR
cm
v
sM
Mm
LR
vCRR
z
z
O
zTV
2
0
10/057.0 max
m
Rscmvz
MmmHg/ 04.0
MmmHg/ 74.0
)1(2
PFC
plasma
PFCplasmaT
H
H
H
PFC
H
PFCpOC
vz
Krogh Cylinder, 12.8% PFC E.g. to get 200 mm vessel spacing requires about
25 cm/s flow speed
0.01
0.1
1
10
100
1000
10 100 1000
v(cm/s)
R0 (m)
Krogh Cylinder, PFCs Check shear stresses required (assuming fully-
developed flow, viscosity ~ 0.001 kg/m-s):
Spacing ~ 30 mm looks feasible
Need to confirm viscosity ...
0.1
1
10
100
1000
10000
10 100 1000
t(Pa)
R0 (m)
Krogh Cylinder, PFCs Check pressure required (assuming fully-
developed flow, viscosity ~ 0.001 kg/m-s):
These are still fairly low pressures
0.01
0.1
1
10
100
1000
10 100 1000
Pinlet
(mmHg)
R0 (m)
Summary of Problem so far
Perfusing liver TE construct is difficult:• High cell demand x high cell density• Large volume (order 1 ml)• Diffusion transport too slow• Culture medium has low oxygen density
Vascular channels and oxygen carriers improve transport
Summary of Problem so far
Perfusing liver TE construct is difficult:• High cell demand x high cell density• Large volume (order 1 ml)• Diffusion transport too slow• Culture medium has low oxygen density
Vascular channels and oxygen carriers improve transport
Summary of Problem so far
Part of our problem was high shear stress at required flow rates
What if we made wider channels, eg 100 mm radius?
Summary of Problem so far Larger channels: larger surface area,
but more MT resistance in vessel Break O2 flow in to steps
O2 convection
diffusion
Uptake reaction
1. Vessel: Convection MT
2. Tissue: Diffusion MT
3. Tissue: Uptake Reaction
Cm
Cw
O2 Flow Steps
Convection MT radial flux
O2 convection
diffusion
Uptake reaction
Cm
Cw
Diffusion MT radial flux VRr
er r
CJ
D
][ wmmr CCkJ
Co Uptake
2
0
20 1
22
R
R
R
RRJ V
V
Or
Radial flux
Convectioncoefficient
Nondimensional Parameters Simplify the problem where possible Use nondimensional parameters to
compare steps, eliminate steps that don’t control O2 delivery • Biot #: convection vs. diffusion MT• Damkohler #: transport vs. reaction rate
Other parameters simplify math• Peclet #: axial vs. radial diffusion• Sherwood #: convection coefficient• Reynolds #: flow regime• Graetz #: convection regime
Mass transport wider channels
Mass transport in flow (eg cylindrical coordinates)
Biot number:
Bi gives relative importance of convection• Bi >> 1, fast convection can be ignored• Bi ~ 1, convection slows transport• Bi << 1, fast conduction can be ignored
r
Cr
rrz
Cu
D
DD)(
)/(ratetransport diffusion tissue
rate transportconvective 0
0
Vm
V
m RRk
RR
kBi
In Our Example
Use lower limit (fully developed MT) convection coefficient, km = 2.182 DV /R V
Assume DV ~ De
e
Vm RRkBi
D)( 0
]1)/[(2)(182.2
00
VeV
Ve RRR
RRBi
DD
E.g. medium, RV = 10 mm, R0 = 20 mm, Bi = 2. Convection plays a significant role.
E.g. with RBCs, 45% HCT, RV = 10 mm, R0 = 50 mm, Bi = 8. Convection is negligible.
Mass transport in wider channels
Mass transport in flow (eg cylindrical coordinates)
Graetz number:
2
2
z
C
r
Cr
rrz
Cu
DD
DD
L
VD
L/v
DGz
z
22 /
timeconvection axial
timediffusion radialr
D = 2RVz
L
R0
vz ReScL
DGz
Small when Pe >>1
Mass transport wider channels
Gz characterizes mass transport regime High Gz (Gz > 20)
• Axial flow faster than radial diffusion• Not all O2 in vessels can reach wall (tissue)
• Mass transport boundary layer forms• Higher convection coefficient
Low Gz (Gz < 20)• Concentration profiles similar shape• “Fully-developed” mass transport• Lower, constant convection coefficient
DL
DvGz z
2
In Our Example
Constant D, others parameters variable Consider L = 1cm, vz= 1cm/s
• Gz < 20:
Model larger vessel diameters or faster velocities with entrance flow model
Or use numerical solver (eg Comsol was used in Radisic et al reference)
mcmscm
scmxcm
v
GzLD
z
20002.0)/1(
)/102)(1(20 25
D
Convection Mass Transport
We’ll see three regimes:• Entry region (boundary layer MT) (Gz > 20)• Fully-developed MT (Gz < 20)• Negligible convective MT resistance (Da << 1)
Analysis assumes • Dilute species• Fully developed flow velocity profile• Steady laminar flow and steady mass transport
With dilute species, heat transfer and mass transfer are analogous (same math)
Convection MT Equations
Definitions
r
RV
0
R0
Lvz
z
z
RV
vz
r
u
L Vessel LengthRV Vessel radiusD Tube Diameter, D = 2RV
R0 Tissue outer radius (1/2 vessel spacing)vz Average axial velocity (flow/XC area)u local axial velocity, u(r)DV Vessel effective diffusivityDe Tissue effective diffusivitykm Convection coefficient, mass transferRO2
Tissue oxygen uptake rate
m Vessel (Effective) Viscosityr Vessel mass densityC Plasma/medium Oxygen concentrationJr Flux of oxygen, in radial direction
Fully Developed Laminar Flow, 1
Steady flow Driven by pressure
difference, pi-po
Laminar flow
z
RV
vz
r
u
Re = Reynolds #
ReDL 05.05.0/
Newtonian fluid• Constant m
Fully Developed
2200Re
forces viscous
forces inertial
Dv
Re z
L
pi po
Fully Developed Laminar Flow, 2
Flow profile is parabolic:
z
RV
vz
r
u
2
8
V
zoi R
Lvppp
Shear stress at the vessel wall:
2)/(12)( Vz Rrvru
Vzw Rv /4t
Pressure drop over vessel length:
Convection MT in FD flow
Assumptions• Steady mass transport• Fast release of O2 from carriers
• Constant O2 uptake rate RO2
• Constant flux of O2 at vessel wall
→ ie no hypoxic zones In vessel
r
Cr
rrz
Cu V
D
Convection MT in FD flow
Constant flux wall boundary condition Assume negligible axial diffusion Boundary condition: Oxygen flux at vessel
wall balances oxygen uptake in tissue
z
R
RRR
r
C
dzR
dzRRR
AR
r
CJ
VV
VO
Rr
V
VO
wall
tissueO
RrVr
V
V
rt constant w 2
222
0
220
2
22
D
D
Convection MT in FD flow
Define mean concentration in the vessel
][ wmmRr
Vr CCkr
CJ
V
D
Oxygen flux at the vessel wall:
Define local convection mass transfer coefficient, km
Az
m uCdAAv
C1
][ wmV
rV
m CCD
ShJ
DkSh
DD
Convection MT in FD flow
We solve the convection MT equation with constant-flux boundary condition to get an equation for the Sherwood number, Sh
Use Sh to relate concentration difference to MT rate at wall
For Fully-developed MT (Gz < 20),
Sh = 4.364
Coupling FD convective MT to diffusion in tissue cylinder
Use Sh to relate concentration difference to MT rate at wall
Use Krogh cylinder solution for tissue MT rate at wall
r
RV
0
R0
Lvz
zCm
CW
C(r)
][ wmV
r CCD
ShJ
D
2
0
20
0
00
20
20
2
0
2
0
20
12
2
22
4
ln24
1
2
22
2
R
R
R
RR
R
R
R
RRR
R
r
rC
RRC
R
r
R
R
R
r
C
RRC
r
r
CJ
V
V
O
V
V
O
Rrew
Owe
V
Vew
Owe
Rrer
V
V
D
D
DD
D
Coupling FD convective MT to diffusion in tissue cylinder
Tissue uptake, balanced to convection MT rate, sets wall concentration “defect”
r
RV
0
R0
Lvz
zCm
Caw
C(r)
2
0
20
2
0
20
13644
12
2
][
2
2
R
R
.
RRC
R
R
ShR
DRRC
Sh
RJC
Sh
DJCC
CCD
ShJ
V
V
Om
V
VV
Om
V
Vrm
V
rma
amV
r
W
W
D
D
DD
D
When is FD convective MT important?
When defect is same magnitude as inlet concentration
Ignore convective MT when
r
RV
0
R0
Lvz
zCm
Cw
C(r)
0
2
2
0
20 1Defect B
V
V
O CR
R
Sh
RR
D
Damkohler Number
The Damkohler #, Da, is a dimensionless parameter comparing reaction rate to transport rate
For FD MT coupled to zero-order oxygen consumption, define
0
2
20
RateTransport
RateReaction
BV
O
CSh
RRDa
D
You can ignore mass transport effects when Da << 1
Reflection: what does this mean?
Da just depends on vessel spacing (tissue radius), diffusivity, uptake rate and inlet (total) blood oxygen concentration
0
2
20
RateTransport
RateReaction
BV
O
CSh
RRDa
D
Why ignore MT when MT rate is high? Because MT resistance matters ... The slow rate controls the overall rate
Developing Mass Transport
Now consider faster flow, Gz < 20• “Developing” concentration profile changes
with axial location z• Faster mass transport (higher Sherwood #)
Reference: Convective Heat and Mass Transfer, Kays WM and Crawford ME, 2nd Ed., 1980, McGraw Hill, Ch. 8, pp 112-114.
Define dimensionless axial position,
2
2
Dv
zz
z
VD
Developing Mass Transport Numerical Solution, Sh(z+) Sh ~ 4.364 when z+ > 0.1
2
2
Dv
zz
z
VD
05
10152025303540
0.0001 0.001 0.01 0.1 1
Sh
z+
Developing Mass Transport Recall concentration “defect”, which
increases with decreasing Sh:
05
10152025303540
0.0001 0.001 0.01 0.1 1
Sh
z+
2
0
20 12
R
R
Sh
RRCC V
V
Omw D
Longer vessels have lower Sh, lower C at wall
Critical calculation is Cw at end of vessel
Note z+(L)= 2/Gz
Including Oxygen Carriers in Convective MT problem
Oxygen carriers complicate analysis But they improve oxygen delivery! Refs:
• M Radisic et al, Mathematical model of oxygen distribution in engineered cardiac tissue ...” Am J Physiol Heart Circ Physiol 288: H1278-H1289, 2005.
• WM Deen, Analysis of Transport Phenomena, 1998, Oxford University Press, pp. 192-194.
Convection with O2 Carriers
More definitionsf Carrier volume fraction or hematocritS Hemoglobin saturation (fraction)Ca Aqueous phase Oxygen concentrationCc Carrier oxygen concentrationCT Total Oxygen concentration (Ca + Cc)K Carrier phase partition coefficient (Cc / Ca)R0 Tissue outer radius (1/2 vessel spacing)vz Average axial velocity (flow/XC area)u local axial velocity, u(r)Da Aqueous phase diffusivityDc Carrier phase diffusivityDVe Effective diffusivity in vessel (relative to Ca)
Convection with O2 Carriers
O2 carrier increases • Total oxygen concentration in the vessel• Effective diffusivity in the vessel
Assume carrier and aqueous phase concentrations are in equilibrium at all times
Choose aqueous phase concentration as independent variable • Caw
= Ctissue at the vessel wall
Write mass conservation in terms of Ca
Convection with O2 Carriers
Total Concentration: PFC suspension: K = Haqueous/HPFC = 20.1
Da = 2.4 x 10-5 cm2/sDc = 5.6 x 10-5 cm2/s
aT CKC ])1(1[
Mass conservation in vessel, FD flow: r
Cr
rrx
Cu aVeT
D
a
caVe
K
DD
DD
and 2
131 where
r
Cr
rrCK
xu aVe
a
D
])1(1[
Convection with O2 Carriers
f is approximately constant (except within skimming layer ~ 1 mm)
For PFCs K and g are constant
Boundary condition
r
Cr
rrx
CuK aVea
D
])1(1[
z
R
RRR
r
C
VVe
VO
Rr
a
V
rt constant w 2
220
2
D
Exercise Derive conservation equation for mean
flow aqueous oxygen concentration Use earlier approach: balance mean
oxygen flow reduction with tissue oxygen consumption
Convection with O2 Carriers
Mean aqueous oxygen concentration conservation equation
Recall axial convection balance result from Krogh cylinder,
Substitute for aqueous concentration
aT CKC ])1(1[
zv
R
R
RCC
z
O
CBTm
2
01
2
20
zv
R
R
R
KCC
z
O
Caam
2
01
])1(1[
12
20
FD Convection with PFCs Let’s look back at Fully-Developed
convective mass transport. What’s different with PFC vs. culture
medium?• Effective diffusivity is different
• Slope of Cm vs. z is reduced
zv
R
R
R
KCC
z
O
Vaam
2
01
])1(1[
12
20
a
caVe
K
DD
DD
where2
131
What about our practical problem?
Shortening vessels would help• Biomimetic approach: Use a branched network
Carry over Cm from parent vessel outlet to daughter vessel inlets
Example: Patrick’s branched structure
L ~ 4mm, D ~ 1mm,
RV ~ 500 mm, R0 ~ 1500 mm
rcells ~ 0.3 x 108 cells/ml