BME 430: Tissue Engineering

Vascularization
Fall 2022
Notes from Homework 4
• Mean: 9.0, Median: 9

• Comments:
• Make sure you define all acronyms in your writing
• Provide clear clinical needs and significance when contextualizing the
study
• Make sure you are being clear as to what is the important finding in
the manuscript (and why!)

• (+) Critiques have gotten a lot better. For some, be sure to provide a
clear rationale behind your intended critique

• Stats from HW1: Mean: 7.5, Median 8.0

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 ANGIOGENESIS

How does this differ from “vasculogenesis”?

“arteriogenesis”?

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Mechanisms of Blood Vessel Growth
• Vasculogenesis
Aggregation of angioblasts or endothelial progenitor cells to
form blood vessels

• Angiogenesis:
Formation of new blood vessels from pre-existing vessels by the
proliferation and migration of differentiated endothelial cells

• Arteriogenesis
Maturation and development of blood vessels from existing
vessels

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Methods of Blood Vessel Creation

Korpisalo and Yla-Herttuala. Integrative Bio, 2010. 5

Stages of Vasculogenesis
1. Differentiation of mesodermal cells to
hemangioblasts
2. Formation of blood islands
3. Differentiation (angioblasts and
hematopoietic stem cells)
4. Blood vessel formation (aggregation
into plexus and primitive capillaries)
5. Lumenization (primitive vessels to
organized capillaries)
Uhrin. Fund of Vasc Bio, 2019.

*Triggered by growth factor gradients

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 Stages of Angiogenesis
1. Endothelial cells (ECs)
sense hypoxic conditions
2. “Tip” ECs lead formation
of new vessel
3. “Stalk” ECs proliferate
and form a lumen
4. “Phalanx” ECs regulate
flow into nascent vessel
5. Pericytes are recruited,
and ECM is secreted

*Triggered by hypoxia
(secretion of VEGF or other
growth factors)
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Bryan and D’Amore. Cell and Mol Life Sci CMLS, 2007.
 Stages of Arteriogenesis
1. Change in shear stress (occlusion?)
2. Endothelial cells (ECs) sense change in
shear
3. ECs secrete factors such as MCP1,
ICAM1, and VCAM1
4. Recruit circulating monocytes
5. Monocytes secrete MMPs and uPA
6. Factors degrade/“loosen” ECM
7. Degradation products stimulate SMC
Persson and Buschmann. Front Mol Neurosci, 2011.
proliferation
*triggered by changes in shear stress (flow)
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Therapeutic Angiogenesis
• Myocardial infarction
• Ischemia resulting from blockages in the left
anterior descending (LAD) artery
• Over 1 billion cardiomyocytes die (ischemia)

• What can we add to repair injury site?

• Add growth factors (VEGF, FGF, etc.)
• Add endothelial cells (or progenitors)
• Add cardiomyocytes or MSCs

• Where should these therapeutics go?

• In blood (systemic)
• Within myocardium
• On the surface (in a patch)
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Therapeutic Angiogenesis
• Systemic delivery of factors/cells is highly
ineffectual (<1% engraftment)
• IM injection results in cells ejecting from
contracting tissue (<5% engraftment)
• Patches keep everything close, but then
need to migrate into damaged tissue

• Successful Outcomes
• Angiogenesis (direct, local stimulatory
effect)
• Paracrine signaling (indirect, angiogenesis
or cell survival)
• Recruitment of circulating EPCs (indirect,
recruited cells then perform angiogenesis)
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Vascularization in Tissue Engineering
• The development of blood
vessels in [a space]

• Many conflate “vascularization”

with:
• Angiogenesis
• Vasculogenesis
• Arteriogenesis

• End goal is to develop tissues

with blood vessels for nutrient
transport and proper
physiologic signaling
Bodies Exhibit
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Strategies for Vascularization
• Cell source and
differentiation
method?
• How do we generate
stable structures?

• How do we generate
adequate hypoxic
environment?
• How to support long
term perfusion?

• Can we support vessel

culture ex vivo?
• Are monocytes critical
for this method (not in
standard culture
methods)?
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In Vivo Models of Vascularization
• Implant scaffold around
native vessel for
vascularization
• -> What vascularization
method?
• Let host deliver appropriate
factors, in correct sequence
• Technical Problems?
• Isolation of material and host
• Immune response
• No control of host response
• Separation of material and host
• Reintegration elsewhere

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 Development of Vascularized Muscle Graft

Shandalov. Methods, 2015. 14

Idealized Vascularization Strategy
• Stepwise expansion of
vascular network (both
size and maturity)

• A key parameter for

vascular maintenance is
shear stress (flow)
• Why?

• Typical shear stress is

~25 dynes/cm2
• How can we estimate shear
stress from flow?

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Decision Points for Vascularization
• Strategies impacted by
end design

• “Biologically
mediated” is least
used strategy

• Major question is cell

source

• Will the architecture

support vessel
formation?
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Biologically Replicated Vascularization Strategies
• Theme: Form new vessels
from initial cell populations

• Spontaneous capillary
formation within constructs
• Can start with endothelial
progenitor cells (EPCs) and
instruct with scaffold/factors
to generate lumens

• Generating a lumen, and

maintaining it over time is
non-trivial

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Shear Forces Lead to Sprouting
• Shear force and growth factor
delivery enhance vascular
sprouting

• Can be con- or counter-

current flow

• Perfusion is essential for EC

health and maintenance
(e.g. normo- vs. hyper-
tensive flow)
Nguyen et al. PNAS, 2013.

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Biologically Mediated Vascularization Strategies
• Theme: Allow host to supply
constructs with blood vessels

• In situ growth of tissue is

critical for proper
organization and integration
of nascent tissue with host

• Issues may arise if

implantation site is not the
final location, need to remove
and reintegrate…

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Biologically Inspired Vascularization Strategies
• Theme: Mimic the
architectural surroundings of
vessels to generate vessels

• Generate vascular shapes (i.e.

long channels, 3D printing
and/or soft lithography)
• Combine motifs from
strategy 1, but physically
constrain system

• Difficulty in maintaining
vessel integrity, not all have
ECs
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Cell Printing Vascular Structures
• 3D print a variety of structures, including constitutive layers of
blood vessels
• Assume that the cells themselves can produce the ECM
necessary for tissue formation (now including fibroblasts as well)

Norotte et al. Biomaterials, 2009.

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 Bringing it All Together

Why do these Processes Matter?

Can we Plan for Diffusion Limitations?

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Mass Transport and Metabolic Demand
• Cell survival requires
• Access to nutrient (oxygen, glucose, amino acids, etc.
• Clearance of products of metabolism (CO2, lactate, urea, etc.)

• Mass transport is defined as the movement of these molecules in and out

of tissue constructs or grafts
• Mediated by fluid flow (convection) (e.g. circulatory system, bioreactors, extracellular
space)
• Mediated by passive diffusion (actual transport across endothelium)
• Driven by pressure gradients (both in flow (e.g. movement or contraction) and mass
transport itself)
• Convection is dominant term when cells are embedded in dense ECM (e.g. bone,
cartilage) while passive diffusion is dominant elsewhere

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Transport Realities in Clinical Grafts
• The maximum distance between a
capillary and a cell is: _____

• Most clinical grafts have a

diffusion distance of > 5 mm,
50 times the maximum diffusion
distance
• This is the origin necrotic cores
(donut effect)
• Need to maximize transport into the
core to maximize wound healing

• Understanding this limitation is

critical for context in discussions Huang et al. J Appl Physiol, 2005.
regarding cellular vs. acellular
approaches to tissue engineering

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Modeling Oxygen Diffusion and Reaction Kinetics

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Modeling Oxygen Diffusion and Reaction Kinetics
• Variables
• CO2 at surface of graft (CO)
• Ccell and distribution in the graft/site
• Rate of oxygen consumption
• Fluid flow within site
• Oxygen diffusion constant
• Cell response to hypoxia

• Little known about balance between

diffusion and consumption of
proteins, peptides, or signaling
molecules
• Diffusion of oxygen is relatively slow
(and consumption is high), transport
of other nutrients is usually more
favorable than oxygen
• Therefore, oxygen is the limiting
factor in cell survival in most tissues
(in vitro or in vivo)

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Estimating Maximum Cell Densities
• Estimate maximum cell density ([Cell]max) by setting Φ2 = 1
• Oxygen consumption averages 4 * 10-17 mol/cell-sec but varies with cell type:
• Hematopoietic stem cells: ~0.47-3.3 * 10-17 mol/cell-sec
• Fibroblasts: ~4-7 * 10-17 mol/cell-sec
• Granulocytes and monocytes: ~0.6-18 * 10-17 mol/cell-sec
• Oxygen diffusion coefficient in tissue is ~2 * 10-5 cm2/sec (at 37 °C)
• Oxygen Concentration (CO) in normal tissues: ~0.07 mM

• [Cell]max for a 2 cm thick graft: ~70,000 cell/cm3

• [Cell]max for a 1 cm thick graft: ~280,000 cell/cm3

• This is 1,000 fold lower than [Cell] in native bone (~5 * 108 cell/cm3) and
100 fold lower than mean [Cell] in a marrow aspirate (~4 * 107 cell/cm3)

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Maximum Cell Density: Issues with Scale-Up
• Inverse square relationship between [Cell]max and
diffusion distance
• Increase graft dimension by a factor of 5 (going from rat
to dog) will decrease [Cell]max by a factor of 25
• Why many cell transplantation methods work in small
animals but fail in larger animals (and clinical trials)

• Additional factors to consider

• Cell delivery systems usually prepared in air (saturated
with oxygen); this will support cell respiration for several
hours after implantation
• Not all implanted cells continue to respire at basal rates
– some cells sensitive to transplantation may die
(necrosis), reducing metabolic demand
• Cell death results in release of products leading to local
inflammation, resulting in increased metabolic demand

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Vascularization: Solution to Transport Issues
1. Release of angiogenic factors
2. Transport of factors to endothelium
receptors
3. Binding of factors to receptors
4. Basement membrane degradation
5. Endothelial proliferation
6. Endothelial migration (sprouting)
7. Extension of new vessels by MMP-
mediated ECM degradation
8. Stabilization of vessels by recruiting
support cells (pericytes and/or
MSCs)

Theme: Same processes occur in

embryogenesis, wound healing, tissue
engineering, and tumor growth

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Vascularization Strategies in Tissue Engineering

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Review for Exam 2

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Exam 2 – Notes and Comments
• General format:
• Definitions
• T/F section
• Multiple choice
• Short answer
• Long(er) essay questions
• General note on “cumulative”
content:
• In general, exam will focus on the
second unit of the course (L 12-20)
• However, there will be overlapping
concepts that may reappear…

• In general, it is better to be succinct, than overly verbose

• Bring a pen!

• Project draft is due at start of class on Monday (11/21)!

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Lecture 12 – The Immune System
• Definitions
• Inflammation • Fibrosis
• Frustrated phagocytosis • Opsonization
• Granulation tissue • MHC
• Foreign body giant cells • Cytokines

• Temporal events of wound healing/biomaterial implantation

• Acute vs. Chronic inflammation
• Neutrophils/Macrophages (and macrophage polarity/function)
• Skin wound healing timeline
• Innate/Adaptive defenses and their interactions
• “Self” vs. “Foreign” and Memory
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Lecture 13 – Biomaterial-Tissue Interactions
• Definitions
• Adsorption
• Absorption
• Langmuir model
• Vroman effect

• 4 transport mechanisms (and how proteins interact with

hydrophobic vs hydrophilic surfaces)
• Temporal events when a biomaterial is implanted
• Protein-substrate/biomaterial interactions
• Complement pathways and membrane attack complex

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Lecture 14 – Other Biomaterial Interactions
• Definitions
• FDPs • Factor XIII
• Thrombin • Embolism
• t-PA (or uPA)

• Hypersensitivity reactions (ACID)

• Coagulation pathways
• Fibrinolysis and fibrin degradation products
• Platelet plugs and thrombosis
• Calcification
• Biofilm formation and mitigation
• Other tissue-biomaterial interactions 35
Lecture 15/16 – Tissue Fabrication/Scaffold Technology
• Definitions
• Off-the-shelf • Bioink
• Scaffold-free • Bioprinting
• Lyophilization • Organ-on-a-chip
• Particulate leaching
• Requirements and ideal properties for tissue fabrication strategies
• For each of the techniques described for tissue fabrication:
• Know what they are
• How they are useful in tissue engineering
• What tissue types (applications) are compatible for which approaches
• General process conditions for each method
• How changing matrix formulation affects tissue formation
• Different types of fabrication methods for scaffolds
• How different approaches can be modular/build in complexity (e.g. layer-by-layer)
• Pros/cons for each approach (e.g. organ-on-a-chip complexity)
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Lecture 17 – Bioreactors
• Definitions
• Bioreactor • Stimulus
• Wolff’s law • Physiologic conditioning
• Automation
• Problems with in vitro culture conditions
• Environment and conditioning affect functionality
• Functions and classifications of bioreactors
• Importance between coupling of stimuli delivery and sensing
• Types of stimuli, and control/design requirements for bioreactors
• Requirements for perfusion systems
• Stretch/mechanical loading on tissue growth/maturity, and biomaterial
properties
• Treatment modalities and bioreactor strategies for electrical stimulation
• Importance of matching critical components to each tissue type (and
themes…)
• How the function of bioreactors is dependent on design requirements
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Lecture 18 – Animal Models
• Definitions
• Humanized mouse • Founder
• Single nucleotide polymorphism • Knockout / Knockin
• INDEL • Floxed gene
• Transgenic animal

• Goals (and varieties) of animal models

• Genetic vs. surgical vs. diet-induced vs. long term disease models
• Advantages/disadvantages of animal models
• Principles of “reverse” (and “forward”) genetic approaches
• Cre-Lox systems (reporter, knockout, knockin…)
• Tet-on / Tet-off systems
• How Cre-Lox and/or Tet-on/off systems can be used
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Lecture 19 – Introduction to Bio-Transport
• Definitions
• Diffusion/Convection • Intima/media/adventitia
• Flux • Hypoxia
• Fick’s first law

• Driving forces behind diffusion, mass transfer, and mass balance

• Assumptions behind Fick’s Law and transport
• Limitations of diffusion
• Estimating diffusion distance, clearance
• Hypoxia realities and cell survival

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Lecture 20 –Vascularization
• Definitions
• Vasculogenesis • Arteriogenesis
• Angiogenesis • Vascularization

• Methods/steps for vasculogenesis/angiogenesis/arteriogenesis

and key driving forces
• For each strategy of vascularization: know steps, key
parameters, themes, limitations, methodologies
• Issues with metabolic demand, maximum cell density, and
scale-up

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Homework 3 and 4 (All 6 papers)
• Clinical need/importance
• Why was the study done?
• What were the authors hoping to achieve?

• Significant findings
• What do they mean?
• How did the authors obtain these results?

• Major critique points

• What wasn’t done well?
• What could have been done in addition?
• How/why?

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