Tissue Engineering

1
Tissue Engineering

• Tissue Engineering is the in vitro development
  (growth) of tissues or organs to replace or
  support the function of defective or injured body
  parts, or the directed management of the repair
  of tissues within the body (in vivo).

• Research is presently being conducted on several
  different types of tissues and organs, including
• Skin
• Cartilage
• Blood Vessels
• Bone
• Muscle
• Nerves
• Liver
• Kidney
• etc. etc. etc.

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Tissue Organization

• Before a tissue can be developed in vitro, first
  we must understand how tissues are organized. The
  basic tenet here is that
• all tissues are comprised of
• several levels of structural hierarchy
• These structural levels exist from the
  macroscopic level (centimeter range) all the way
  down the molecular level (nanometer range)
• there can be as many as 7-10 distinct levels of
  structural organization in some tissues or organs

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Organization of the Tendon
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Organization of the Kidney
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Functional Subunits

• The smallest level at which the basic function of
  the tissue/organ is provided is called a
  functional subunit
• functional subunits are in the order of 100 mm
  (whereas cells are of the order of 10 mm)
• each organ is comprised between 10-100 x 106
  functional subunits
• each functional subunit is comprised of a mixture
  of different cell types and extracellular matrix
  (ECM) molecules
• Separation of the functional subunit into
  individual cohorts (i.e. cells and ECM) leads to
  a loss of tissue function. For this reason, this
  is the scale that tissue-engineering tries to
  reconstruct.
• So, how can the functional subunit be built in
  vitro?

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Microenvironment

• Since cells are entirely responsible for
  synthesizing tissue constituents and assembly of
  the functional subunit, much attention is paid to
  the microenvironment surrounding the cell(s) of
  interest.
• The microenvironment, which can be very different
  depending on the type of cell, is typically
  characterized by the following
• Cellularity
• Cellular Communications
• Local Chemical Environment
• Local Geometry

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Cellularity

• Packing Density
• maximum theoretical packing density is about 1 x
  109 cells/cm3
• cell densities in tissues typically vary between
  10 500 x 106 cells/cm3
• relates to about 100 - 500 cells per
  microenvironment (100 mm)3
• extreme cases, such as cartilage which has 1
  cell per (100 mm)3
• thus its microenvironment is essentially 1 cell
  plus associated ECM
• Cellular Communication
• Cells communicate in three principal ways
• secretion of soluble signals
• cell-to-cell contact
• cell-ECM interactions
• Cellular communication can affect all cellular
  fate processes (migration, replication,
  differentiation, apoptosis) and the method(s) of
  communication used depends, in part, on how the
  cells are packed within the tissue.

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Cellular Communications

• Soluble Signals
• includes small proteins such as growth factors
  and cytokines (15-20 kDa), steroids, hormones
• bind to membrane receptors usually with high
  affinity (low binding constants 10-100 pM)

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Cellular Communications

• Cell-to-Cell Contact
• some membrane receptors are adhesive molecules
• adherent junctions and desmosomes
• other serve to create junctions between adjacent
  cells allowing for direct cytoplasmic
  communication
• gap junctions
• 1.5-2 nm diameter and only allow transport of
  small molecules 1 kDa

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Cellular Communications

• Cell-ECM Interactions
• ECM is multifunctional and also provides a
  substrate that cells can communicate through
• since cells synthesize the ECM, they can modify
  the ECM to elicit specific cellular responses
• cells possess several specialized receptors that
  allow for cell-ECM interactions
• integrins, CD44, etc.
• also a mechanism by with cells respond to
  external stimuli (mechanical transducers)

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Chemical Environment

• Oxygenation
• mammalian cells do not consume oxygen rapidly but
  uptake is large in comparison to the amount in
  blood or culture media
• air-saturated aqueous media (37C) contains only
  21 mM O2
• mammalian cells consume O2 at rate of 0.05-0.5
  mmol/106 cells/hour
• cell cultures for tissue engineering have
  relatively large cell densities (106 cells/mL)
  which results in total O2 depletion in 0.4-4
  hours!
• concentration must be within a specific range
  since oxygenation affects a variety of
  physiological functions
• low O2 concentration ? can retard growth
• high O2 concentration ? can be inhibitory or
  toxic (oxidative stress)
• Metabolism
• typically, there are no transport limitations for
  major nutrients although uptake rate depends on
  their local concentrations
• glucose uptake rate 0.1-0.5 mmol/106 cells/hour
• amino acid uptake rate 1.0-5.0 nmol/106
  cells/hour

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Local Geometry

• Geometry of the microenvironment depends on the
  individual tissue
• needs to be re-created for proper tissue growth
• two-dimensional layers or sheets
• three-dimensional arrangements
• transport issues
• local geometry also affects how cells interact
  with the ECM
• remember, the ECM serves as a substrate for
  cellular communications
• For these reasons, considerable effort has been
  geared at creating artificial ECMs (aka
  scaffolds) to provide the appropriate substrate
  to guide in vitro tissue growth and development.

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Tissue Engineering

• General Paradigm

SJ Shieh and JP Vacanti Surgery 137 (2005) 1-7
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Tissue Engineering Scaffolds

• Scaffold Materials
• synthetic polymers
• poly(lactide) ,poly(lactide-co-glycolide),
  poly(caprolactone).
• foams, hydrogels, fibres, thin films
• natural polymers
• collagen, elastin, fibrin, chitosan, alginate.
• fibres, hydrogels
• ceramic
• calcium phosphate based for bone tissue
  engineering
• porous structures
• permanent versus resorbable
• degradation typically by hydrolysis (except for
  natural materials)
• must match degradation rate with tissue growth
• Chemical and Physical Modifications (synthetic
  materials)
• attachment of growth factors, binding sites for
  integrins, etc.
• nanoscale physical features

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Tissue Engineering Scaffolds
smooth muscle cells on unmodified poly(CL-LA)
elastomer (L) and modified surface having bound
peptide sequence (R)
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Culturing of Cells

• Types of Cell Culture
• monolayer (adherent cells)
• suspension (non-adherent cells)
• three-dimensional (scaffolds or templates)

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Culturing of Cells

• Sterilization Methods
• ultra-violet light, 70 ethanol, steam autoclave,
  gamma irradiation, ethylene oxide gas
• Growth Conditions
• simulate physiological environment
• pH 7.4, 37C, 5 CO2, 95 relative humidity
• culture (growth) media replenished periodically
• Culture (Growth) Media
• appropriate chemical environment
• pH, osmolality, ionic strength, buffering agents
• appropriate nutritional environment
• nutrients, amino acids, vitamins, minerals,
  growth factors, etc.

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Cell Sources

• Since the ultimate goal of tissue engineering is
  to develop replacement tissue (or organs) for
  individuals, the use of autologous cells would
  avoid any potential immunological complications.
• Various classifications of cells used in tissue
  engineering applications
• primary cells
• differentiated cells harvested from the patient
  (tissue biopsy)
• low cellular yield (can only harvest so much)
• potential age-related problems
• passaged cells
• serial expansion of primary cells (can increase
  population by 100-1000X)
• tendency to either lose potency or
  de-differentiate with too many passages
• stem cells
• undifferentiated cells
• self-renewal capability (unlimited?)
• can differentiate into functional cell types
• very rare

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Stem Cells

• Stem cells naturally exist in essentially all
  tissues (especially those that rapidly
  proliferate or remodel) and are present in the
  circulation.
• There are two predominant lineages of stem cells
• mesenchymal
• give rise to connective tissues (bone, cartilage,
  etc.)
• although found in some tissues, typically
  isolated from bone marrow
• hematopoietic
• give rise to blood cells and lymphocytes
• isolated from bone marrow, blood (umbilical cord)
• Stem cells are rare bone marrow typically has
• a single mesenchymal stem cell for every
  1,000,000 myeloid cells
• a single hematopoietic stem cell for every
  100,000 myeloid cells

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Stem Cells (Mesenchymal)
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Stem Cells (Hematopoietic)
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Proliferation versus Commitment
Proliferation
Commitment or Differentiation
Clonal Succession
Stem Cell
Deterministic or Stochastic Succession
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Stem Cells

• Identification
• Stem cells are identified by the expression of
  specific antigens on their surface, for example
• hematopoietic stem cells express CD45, CD34 and
  CD14
• mesenchymal stem cells do not express these
  markers (i.e. CD34-, CD45-, CD14-)
• Selective separation of positive marker cells (in
  a mixed cell population) can be done by several
  techniques (e.g. immunomagnetic methods).
• Characterization and Commitment
• The most common approach to characterize
  multi-lineage- or single lineage-committed stem
  cells is through colony-forming assays
• cells grown under culture conditions that promote
  their proliferation and differentiation
• the clonal progeny of a single progenitor cell
  stay together to form a new colony of mature
  cells
• colony-forming assays are used to
• characterize stem cells from different sources
  (e.g. BM, umbilical cord blood)
• investigate responses to growth factors,
  cytokines and other drugs
• expansion, commitment, etc.
• quality control for collection, processing and
  cryopreservation

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Colony-Forming Units (CFUs)
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Scale Up

• The conditions of the in vivo microenvironment
  are a fine balance between biological dynamics
  and the physiochemical processes that constrain
  them. Thus, the design of cell and tissue culture
  devices must be such that this balance is
  maintained down to about 100 mm the size of the
  tissue microenvironment.
• Several important design challenges
• mass transfer (delivery and removal)
• fluid flow

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Mass Transfer

• The importance of mass transfer in tissue and
  cellular function is often overlooked. The
  diffusional penetration lengths over
  physiological time scales are surprisingly short
  and constrain the in vivo architecture of tissues
  and organs.
• Similar constraints are faces with the
  construction of cell culture devices and it may
  be difficult to provide the appropriate
  mass-transfer rate into a cell bed of
  physiological cell density.
• For any nutrient (O2, glucose, growth factor,
  etc.), there are two primary concerns for
  appropriate delivery
• provided at physiological concentrations
• provided at the same rate it is consumed

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Mass Transfer

• Can estimate the time it takes to deplete a
  nutrient from the media using the following
  relation

• t time until total depletion hours
• C concentration of nutrient mM
• q specific nutrient consumption rate
  mmol/cell/hour
• X number of viable cells per unit volume
  cells/mL
• The product (q X) is the total nutrient
  consumption rate for the particular system and
  this rate must be balanced with the total
  delivery rate to ensure proper cellular function.
• An imbalance between delivery and consumption
  will alter the local nutrient concentration which
  can have adverse affects on cellular function.
• too high or too low can be inhibitory or even
  toxic

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Mass Transfer

• Oxygen
• physiological concentration 5-30 of saturation
  in air, which is 0.2 mM
• specific uptake rate 0.05 1 mmol/106
  cells/hour
• Primary Nutrients (glucose)
• physiological concentration mM range
• specific uptake rate 0.05 0.1 mmol/106
  cells/hour
• Secondary Nutrients (amino acids, growth factors)
• physiological concentration nM mM range
• specific uptake rate 0.01 1.0 nmol/106
  cells/hour
• Waste Products (lactic acid, ammonia)
• physiological concentration negligible
• specific production rate 0.01 0.2 mmol/106
  cells/hour

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Fluid Flow

• The circulatory system provides blood flow to all
  of the microenvironments of the body. Overall,
  the perfusion rate in humans is about 5
  L/min/person.
• This is roughly equivalent to 50 400 mL/min/106
  cells
• different depending on the metabolic activity of
  the specific cell type
• very low in magnitude compared to fermenters
• such low flow can lead to additional problems
  such as surface-tension effects and capillary
  action
• Fluid flow not only provides the delivery of
  nutrients (dissolved gases, glucose, growth
  factors, etc.) but also serves to remove
  inhibitory waste products, cytokines and
  degenerative enzymes
• waste products of metabolism
• carbon dioxide (CO2), lactic acid, ammonia
• inhibitory cytokines
• inflammatory cytokines (e.g. IL-1, IL-10, TNF-a)
• reactive oxygen species (e.g. NO-,O2-, H2O2)
• degenerative enzymes
• matrix metalloproteinases (MMPs), aggrecanases,
  etc.

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Fluid Flow

• Cell culture devices must be uniform down to the
  size of the microenvironment (i.e. 100 mm) which
  can be difficult to achieve.
• The problem here is that during fluid flow there
  is typically a no-slip condition at any solid
  surfaces creating regions of low flow at walls of
  tubing and sides of bioreactors (boundary layer).

These regions of low flow within the boundary
layer can lead to differences in the local
concentration of solutes compared to the mid-line
flow. Differences in solute concentration within
the flow-field results in non-uniform solute flow
which can pose problems during cell culture.
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Fluid Flow

• Some potential problems of non-uniform solute
  flow during cell culture
• impaired mass transfer if boundary layer is
  relatively thick

• solute movement in boundary layer governed by
  diffusion rather than convection
• boundary layer thickness is inversely
  proportional to fluid velocity
• influence cell and tissue growth
• induce or alter cellular migration
• Chemotaxis (directed movement of a cell or
  organism toward (or away from) a chemical source)

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Fluid Flow

• Residence time (the time it takes for a fluid
  particle to leave a control volume under
  steady-state), is defined by

tr residence time min V volume of vessel
(bioreactor) mL F volumetric flow-rate
mL/min
With proper mixing, a single residence time
occurs (i.e. plug flow), however, any
non-uniformity in the flow conditions can then
result in a distribution of residence
times. This may potentially cause problems or
simply lead to excessive variability in cell and
tissue growth.
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Bioreactors

• a) Spinner Flask
• semi-controlled fluid shear
• can produce turbulent eddies which could be
  detrimental
• b) Rotating Wall
• low shear stresses, high mass transfer rate
• can balance forces to stimulate zero gravity
• c) Hollow Fibre
• used to enhance mass transfer during the culture
  of highly metabolic cells
• d) Perfusion
• media flows directly through construct
• e) Controlled Mechanics
• to apply physiological forces during culture

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Bioreactors
35
Tissue Engineering

• Most successes have been limited to avascular or
  thin tissues (lt 200 mm)
• skin, cartilage, cornea
• The most important problems associated with
  thicker or more complex tissues include
• the need for multiple cell types
• the need for the tissue to become vascularized
• vascularization of the 3-D construct is a
  critical and unresolved problem