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Tissue Engineering
Cato T. Laurencin, M.D., PhD
Drexel University
BACKGROUND
Tissue Engineering
The field of tissue engineering can be defined as “the application of the principles and methods
of engineering and the life sciences toward the fundamental understanding of structure-function
relationships in normal and pathological mammalian tissues and the development of biological
substitutes that restore, maintain, or improve tissue function.” A more concise definition is “the
concept of the rational design and fabrication of living tissues and organs for repair and
replacement.” Another description states that through tissue engineering, “new functional living
tissue is fabricated using living cells which are usually associated in one way or another to a
matrix or scaffolding which can be natural, man-made, or a composite of both.” One final
definition for tissue engineering is “the application of biological, chemical, and engineering
principles toward the repair, restoration, or regeneration of living tissues using biomaterials,
cells, and factors alone or in combination.” Although each definition is slightly different, each
share some common elements; the repair or replacement of living tissue, the application of
interdisciplinary principles, and the use of natural and/or synthetic materials. This is the essence
of tissue engineering.
Tissue engineered heart valve using a degradable polymer to support cell growth
The Repair or Replacement of Living Tissue
The current methods of repairing or replacing damaged tissue include autografts, allografts, and
xenografts. Autografts, which involves transplanting tissue from one region of the body to
another, is an ideal solution to the problem because there is no risk of immune response from the
host and no risk of disease transmission from foreign tissue.
Tissue engineered nose and ear constructed by seeding chondrocytes onto a biodegradable matrix
However, the tissue harvesting procedure involves a second surgery, and introduces additional
trauma (donor-site morbidity) and increased risk to the patient from additional surgeries.
Further, there is a limited supply of tissue if further surgeries are necessary. Allografts, which
are tissue donated from similar species but a different host, eliminate the concerns of donor-site
morbidity and limited tissue supply, but introduce the risk of foreign body immune responses
and the risk of disease transmission from donor to host. Xenografts, or transplants of tissue from
an animal to a human is another alternative, but much of the same risks that exist with allografts
are an even greater concern with xenografts. Regardless of the greater availability of allografts
and xenografts over autografts, donated tissue and organs are still very much in demand. In the
United States in 1995 there were approximately 45,000 patients on waiting lists for organ
transplants, with over 3,500 deaths to people on waiting lists the same year. For organs such as
kidney, liver, pancreas, heart and lung, anywhere from 35-71% of patients in need of an organ
transplant do not receive one. As the population of the United States grows older, these numbers
will only increase.
The need for organs is high and constantly increasing, which illustrates the need for tissue engineering
The Application of Interdisciplinary Principles
As previously mentioned, the field of tissue engineering requires the application of principles
from several disciplines.
The design of the scaffold alone involves several engineering
disciplines, depending on the nature of the tissue being engineered. For instance, the design of a
synthetic artery requires knowledge of fluid dynamics to maintain adequate flow through the
artery, and knowledge of mechanical engineering principles to be able to design the stiffness and
compliance of the synthetic artery. Similarly, designing a synthetic ligament also requires some
mechanical engineering knowledge as well as some expertise in textile engineering if the
ligament is braided or woven.
Regardless of the implant, some knowledge of materials
engineering is a necessity to choose appropriate materials and manipulate them as necessary. It
is also not only the material and strength of materials, but the architecture of the implant that is
critical to its success as well. If one is developing a bone scaffold, it is important to have a
porous and interconnected structure, as well as suitable mechanical properties. However, the
interdisciplinary nature of tissue engineering does not stop at engineering principles. In fact, just
as critical to the success of the engineered scaffold are the biological principles that must be
considered. Several of these principles are applied through the use of tissue culture.
Development of specific tests and experiments to determine the viability of a tissue-engineered
implant is of paramount importance. Once a material has been developed for use in the body,
that material must undergo rigorous tests before it can be used in any animal or person. In order
to simulate a situation that is as close to a body without actually implanting it, the new
engineered product needs to be subjected to a variety of examinations that will ensure its
compatibility with live tissue. For this reason, tissue culture techniques are important in the
process of optimizing a material prior to its implantation into a living organism. By developing
more in vitro studies, it is also possible to decrease the need for unnecessary animal studies.
While it is widely recognized that animal studies are necessary, there is a constant effort to
minimize the use of animals, using animal models only when necessary and at a later stage of
material development.
The study of biomedical implants via tissue culture techniques allows the researcher a window
into how the material will interact with the body’s cells upon implantation. By developing
certain organ-specific cell-lines, researchers are able to target their experiments using cells that
are appropriate for their study. Because cells can be cultured from just about any organ, the
potential for developing a viable implant is greatly improved.
The two main types of cell cultures used in experiments include primary and continuous.
Primary cells are taken (isolated) from animals, processed to eliminate all unwanted tissue, and
grown for use in a current study. Continuous cell cultures grow and multiply many times in
culture before they cease to divide. The immortalization of a cell-line involves the formation of
a continuous cell line with a phenotype that remains constant from one generation of cells to the
next; essentially ensuring a usable cell line for many generations and not just a few, as is the case
with the continuous or primary cell lines. Each cell line has its own phenotypic markers and
longevity, so it is important to recognize what type of cell line is in use and which cell line is
most appropriate for the type of material and experiment being utilized.
Primary cells are derived from animals, and can be processed for use in tissue engineering experiments
For any given experiment, cells are generally seeded onto the material and then at a later time
point, the cells or solution or material itself is analyzed for particular markers including cell
growth, adhesion, development, and the response of the material onto which the cells were
seeded. Given the expansiveness of tissue engineering and the resultant variety of cell types and
biomaterials, it is difficult to summarize the methods for analyzing the cell/matrix interaction.
However, certain basic experimental protocols are widely practiced.
Certain characteristics of the cells can be analyzed upon their exposure to a given matrix or
compound. The types of cellular functioning that lend themselves to experimental analysis
include, but certainly are not limited to analysis of DNA and RNA, cellular proteins, cell
signaling pathways, cell-cell interactions, cell proliferation, and production of cell-specific
hormones and other markers. A great number of microbiology techniques are used to analyze
these characteristics; they include Northern Blotting, electrophoresis, and Western Blotting,
along with PCR (Polymerase Chain Reaction) which increases quantities of RNA or DNA for
further study, or simply is used to quantify the original amount of RNA or DNA in sample. A
number of stains can also be used to determine certain cellular products (such as for Calcium in
bone experiments), and labeling proteins or other compounds within the cells helps to determine
cellular activity or cell migration and adherence onto a particular material. The field is very
diverse, and a large number of methods are in use and are still being developed to study the everincreasing variety of new biomaterials.
By developing materials that respond well to appropriate cell types, experimenters are able to
create a material that is closer to a final implantable product, while at the same time reducing the
need for unnecessary in vivo animal surgeries. By optimizing the biomaterials through in vitro
techniques, it also makes the in vivo studies more complete. Behavior of cells in response to a
new biomaterial is studied in great detail prior to that material's implantation into an animal. The
knowledge gleaned from the in vitro work can be used to further enhance the material and create
a new product that is closer to the ultimate goal of being used to help humans.
The Use of Natural and/or Synthetic Materials
The final consideration for tissue engineering is the material used to support the growth of new
tissue. Several different materials have been used in the past as synthetic implants and organs,
but for the majority of them, the underlying importance was longevity, how long will this
implant material continue to do its assigned task.
However, with tissue engineering the
motivation behind material selection is to choose one that will be biocompatible but also will
degrade over time, to leave no foreign matter in the body, but at an appropriate rate to allow the
body to replace the material with its own cells and resume normal function.
Bone Tissue Engineering
A significant amount of research has been conducted in the area of bone tissue
engineering. The need for surgical reconstruction or replacement is often the result of trauma,
pathological degeneration, or congenital deformity of tissue. Reconstructive surgery is based
upon the principle of replacing these types of defective tissues with functioning alternatives. In
the skeletal applications, surgeons have historically used bone grafts. The two main types of
bone grafts currently used are autografts and allografts. An autograft is a section of bone taken
from the patients own body, while an allograft is taken usually from a cadaver. These methods
of grafting have the potential of providing the defect site with an osteoconductive environment.
However, both types of grafts are limited by certain uncontrollable factors. For autografts, the
key limitation is donor site morbidity where the harvest site is commonly a source of pain and
infection. Other considerations include the limited amount of bone available for harvesting. The
main limitations of allografts has been a risk of disease transmission, risks of unreliable
resorption of the graft, and a risk of an immunologic response.
One alternative to bone grafts is the use of porous, biodegradable polymer matrices.
Using the co-polymer poly(lactide-co-glycolide) (PLAGA) and its homopolymers poly(lactic
acid) and poly(glycolic acid), several researchers have fabricated porous matrices for use in bone
replacement applications.
However, the major drawback of these structures has been the
relatively low mechanical strength which may limit their clinical applicability. Synthetic bone
implants should be biomechanically similar to the type of bone they are replacing. Trabecular
bone has a compressive modulus ranging from 0.1 to 2.0 GPa, while the modulus of cortical
bone ranges from 17 to 20 GPa. In combination with this mechanical compatibility, the matrix
should also be porous. A study by Hulbert et al. has shown that bone ingrowth occurs with a
matrix pore size above 150um. The development of a matrix structure for bone repair must
incorporate these factors.
One possible matrix structure is formed by sintering together microspheres made from
PLAGA.
This fabrication method provides a porous, interconnected scaffold onto which
osteoblasts can attach and migrate. The porous structure also permits the transport of nutrients
and waste products to and from the cells. By choosing a relatively amorphous type of PLAGA,
it is possible to heat the microspheres to a temperature that allows them to bond together but
maintain their spherical shape.
Ligament Tissue Engineering
Ligaments are bands or sheets of fibrous connective tissue connecting two or more bones.
The role of the ligament is to augment the mechanical stability of the joints, to guide the joint
motion and to prevent excessive motion. The ligament is composed of 75% collagen fiber
bundles (type I and type II), less than 1% elastin, and 24% proteoglycans and glycoproteins.
Fibroblasts make up 20% of the tissue, and extracellular matrix comprises the other 80%. The
ligament is comprised of collagen bundles grouped into larger bundles called fascicles arranged
into either helical patterns near the ligament-bone junction, or fascicles arranged parallel to the
ligament axis along its length and away from the edges.
To replace a damaged ligament, such as the anterior cruciate ligament (ACL) found in the
knee, a few options exist. Both autografts and allografts are most commonly used to reconstruct
the damaged ligaments. However, success has been limited by complications similar to those of
bone replacements common to autografts and allografts, namely donor site morbidity and risk of
infection, respectively.
In hopes of creating a material that will reestablish normal joint
kinematics with minimal risk to the patient, a number of synthetic alternatives have been
developed. Some materials that have been used to fabricate synthetic ligaments are carbon
fibers, Gore-Tex (polytetrafluoroethylene, or teflon), polypropylene, and polyester (Dacron).
Although the FDA has conditionally approved some of these for ACL replacement, they fall
short of ideal because they lack the mechanical strength and surface properties of natural ACLs.
One alternative to the above mentioned synthetic materials is a degradable polymer such
as the copolymer poly(D,L-lactide-co-glycolide) (PLAGA).
The benefit of using a
biodegradable polymer is that while new fibroblasts (cells responsible for ligament growth) are
attaching and proliferating, the matrix is slowly degrading, allowing for a completely natural
replacement. By braiding PLAGA fiber bundles into a 3-dimensional matrix we are able to
mimic the hierarchical structure of natural ligament tissue and create a certain degree of porosity,
which allows for cellular ingrowth and nutrient and waste transport.
Cartilage Tissue Engineering
Cartilage is composed of chondrocytes that produce and breakdown the extracellular
matrix, which consists of proteoglycans for elasticity, collagen for tear resistance and strength,
and water. Cartilage has no blood supply and no nervous innervation, so it has a limited capacity
for self-repair.
In instances of small non-critical osteochondral defects, or minor cartilage
damage, the cartilage is able to repair itself. However, in the case of partial or complete
thickness damage, the body is unable to completely repair the damaged cartilage.
Cartilage replacement procedures are performed to repair damaged articular cartilage or
cartilaginous facial structures.
A common pathology leading to cartilage replacement is
osteoarthritis, a condition caused by the degeneration of articular cartilage that leads to severe
pain and/or loss of joint mobility.
GENERAL PRINCIPLES
Bone Tissue Engineering Matrix Synthesis
To create an interconnected porous matrix suitable for bone tissue ingrowth, PLAGA will
be put into solution and emulsified in PVA, a surface acting agent (surfactant). The surfactant
forces the polymer solution into small spheres, the size of which depends on the concentration of
polymer solution, the speed at which the polymer solution is spun in the PVA, and the length of
time the polymer solution is spun. As the ratio of polymer to solvent decreases, the resulting
microsphere size decreases as well. As the stirring speed of the PVA solution increases, the
increase in force causes the polymer solution to break into smaller microspheres. As the stirring
time is increased, more of the solvent evaporates from the microspheres, leaving a smaller final
microsphere size. After the microspheres are formed and all of the solvent has evaporated from
the polymer, they will be sintered together to form an intact matrix. The sintering process
involves heating the polymer above its glass transition temperature (Tg), which is the
temperature at which the chains of polymers begin to move and is typical of amorphous
polymers, like PLAGA. By heating the microspheres above the Tg, the polymer chains are
allowed to travel from one microsphere to another, creating a bond between microspheres. This
bond is partially responsible for the matrix’s ideal mechanical characteristics, and helps maintain
an interconnected porous structure.
SEM photograph of sintered PLAGA microspheres (50x)
Ligament Tissue Engineering Matrix Synthesis
The technique of 3-dimensional braiding using PLAGA yarns is used to create a synthetic
biodegradable ligament. The technique of 3-D braiding involves the intertwining of fibers in the
x, y, and z plane, as opposed to 2-dimensional braiding which intertwines fibers in the x and y
plane. By adding the third dimension to the braiding, the resulting matrix has added strength and
surface area onto which newly seeded and formed cells can attach. The technique is similar to
weaving, although maintaining a constant braiding angle is crucial to a predictable matrix
architecture.
3-dimensional braiding set-up
Cartilage Tissue Engineering Matrix Synthesis
The basis of this technique is the generation of an electric field between an oppositely charged
polymer fluid and a collection screen (see figure 4). A polymer solution is added to a glass
syringe with a capillary tip. An electrode is placed on the needle with another connection made
to a copper screen. As the power is increased, the polymer solution becomes charged and is
attracted to the screen. The electrospinning process is driven by the electrical forces on free
charges on the droplet surface or inside the polymer solution. Once the voltage reaches a critical
value, the charge overcomes the surface tension of the droplet and a jet of nanofibers is
produced. As the charged fibers are splayed, the solvent quickly evaporates and the fibers
randomly accumulate on the surface of the collection screen. Using this technique, a nanofibrous
structure with extremely high surface area to volume ratio is created. It is our hypothesis that
this similarity to nature’s dimensional scale and the high surface area to volume ratio of the
nanofibrous structure are conducive to cell attachment and cell proliferation.
Certain experimental parameters such as the concentration of polymer solution or the charge
density on the droplet can influence the fiber diameter and matrix morphology which may or
may not affect cell proliferation.
Electrospinning set-up for nanofibrous matrix processing
MATRIX CHARACTERIZATION
BACKGROUND
Conventional orthopaedic implants such as screws, plates, pins, and rods serve as loadbearing replacements for damaged tissue and are usually composed of a metal or an alloy.
Although these implants are capable of providing rigid bone fixation and stabilization, the large
difference in modulus between bone and the metals used for implants causes problems (elastic
modulus for metal and alloy implants is 100-200 GPa, compared to 17 GPa for cortical bone and
2 GPa for trabecular bone).
Stress shielding occurs when the modulus of the implant is
significantly higher than that of the surrounding bone. In this scenario, the implant bears the
majority of the stress, shielding the tissue from this load. The reduction in stress causes the
density of the bone surrounding the implant to decrease, weakening it and making it prone to
refracture.
This results in improper remodeling of the bone surrounding the implant.
Conversely, an implant that has a modulus lower than bone will cause premature loading of the
healing tissue, resulting in fracture site instability, delayed healing, or a non-union. This is
called stress overloading.
An ideal tissue replacement would be designed to replace a specific tissue. This tissueengineered matrix would have the same mechanical properties as the natural tissue and it would
serve as a scaffold for tissue regeneration.
The optimal matrix/tissue response for a
biodegradable matrix is shown in figure 1. Initially the matrix withstands the majority of the
stress. As the matrix begins to degrade and tissue ingrowth occurs, the newly regenerating cells
are gradually loaded with physiological stress, further simulating tissue regeneration. Eventually
the matrix completely degrades and the regenerated tissue bears the stress.
Mechanical Properties
Bone Formation
Matrix
Degradation
Time (Months)
Optimal Matrix/Bone Response
GENERAL PRINCIPLES
Although various methods can be used to establish the structure-property relationship,
from a tissue engineering perspective scanning electron microscopy (SEM), mechanical testing,
and porosimetry are some of the most informative techniques. Using data obtained from these
types of characterization, matrix design can be modified to produce an optimal structure that
maximizes mechanical properties while maintaining a suitably porous structure.
Scanning Electron Microscopy
By using electrons instead of light to form an image, the scanning electron microscope
can produce images of high resolution, large depth of field, and high magnification. Briefly, a
tungsten filament is heated up to the point where it emits an electron beam. The electron beam is
accelerated through a magnetic lens within an evacuated chamber toward the specimen (see
figure 2) which has been sputter coated with an electrically conductive material (gold is
commonly used). It is critical to have the electron chamber under vacuum, as the tungsten
filament would burn out immediately otherwise, and the electrons passing through would collide
with gas molecules, preventing them from reaching the specimen. When the electron beam
strikes the specimen, secondary electrons are created and collected by a secondary electron
detector that converts the electrons to a voltage and amplifies them. The amplified voltage is
applied to a TV scanner, which expresses the voltage intensity as a spot of light. By displaying
thousands of voltage intensities as different light intensities on the TV scanner, the image is
formed. We will be using an Amray 1830/D4 scanning electron microscope with a Tungsten
electron gun at 20 kV.
Schematic of the insides of a scanning electron microscope.
Mechanical Testing
Mechanical testing encompasses compressive, tensile, and torsional testing. Through the
use of compression and tensile testers (see figure 3), stress/strain data can be determined, from
which a number of mechanical parameters can be determined, such as yield stress, ultimate
stress, fracture stress, and elastic modulus. The stress/strain curve is a measure of the force
applied to a unit area of a sample, and the change in shape resulting from that force. From such
testing, data values of synthetic materials can be compared to normal values of natural tissue for
similarity.
Several different mechanical testing machines can be used to test samples. Here are
examples of two. The KES-G1 Multi-Purpose Tensile Tester has a 5 kg load cell and can be
used to make accurate evaluations of uni-axial tensile and visco-elastic properties on single
fibers, yarns, fabrics, or industrial materials. Compared to conventional tensile testers, the single
fiber tensile tester is able to accurately measure loads as small as 1mg and up to 5kg while
measuring displacements on the micron level.
Single fiber tensile tester.
The 3-D braid and microsphere matrices can be tested using an Instron model 1127 mechanical
tester with a maximum load cell of 25,000 kg, which is clearly inappropriate for single fibers or
delicate materials such as the nanofiber matrix, but better suited for both the 3-D braid and the
sintered microspheres.
Typical Instron material testing system with tensile testing grips.
Porosimetry
Porosimetry analyzes both the porosity and the pore size of the matrix being analyzed. It
is important to note that porosity and pore size, although related, are not the same thing. Porosity
is the measure of open space within a matrix and is computed by dividing the amount of material
in a matrix by the amount of space, or volume, that the matrix occupies. This dimensionless
number is usually expressed as a percentage and can provide critical information about how
much material is contained within a matrix. The amount of material in a matrix can effect
degradation rate and mechanical strength. Pore size is a measure of the distance across the
openings within a matrix, and is important as the spaces within the matrix can permit or restrict
the migration of cells as well as the transport of cell nutrients and waste.
The porosimeter measures the porosity and distribution of pore sizes of a sample matrix
by infusing pressurized mercury into the sample. With large pores, relatively little pressure is
needed to infuse the mercury. However, as the pore size decreases, the necessary pressure
increases. By recording both the pressure necessary to fill the pore with mercury and the volume
of mercury infused, it is possible to measure the pore diameter and the surface area of the
polymer within the matrix. The results give a distribution of pore size in the matrix sample,
which is important data when considering the ideal environments for certain cell types.
CELL CULTURE TECHNIQUES
ISOLATING AND PLATING OSTEOBLASTS
BACKGROUND
Since its origin in the early 1900’s, cell culture has become a vital factor in biomedical
research.
It was developed to provide a model of physiological function in an in vivo
environment. Studies using such techniques encompass broad topics in a variety of fields. Some
areas
of
major
interest
in
cell
culture
include:
INTRACELLULAR ACTIVITY
transcription
protein synthesis
energy metabolism
drug metabolism
ENVIRONMENTAL INTERACTION
infection
drug action
ligand receptor interactions
carcinogenesis
CELL-CELL INTERACTION
metabolic cooperation
cell proliferation
contact inhibition
density limitation of growth
INTRACELLULAR FLUX
hormones
metabolites
signal transduction
membrane trafficking
The cell culture technique is based upon removal of a tissue sample from a donor organism and
subsequent isolation of a particular cell type. Because cell proliferation is often found in such
cultures, propagation of cell lines becomes feasible. Cells are grown in an in vitro setting and
are maintained with nutrients essential for their growth, proliferation, and maturation.
From a tissue engineering standpoint, in vitro techniques are often used to study
biocompatibility of materials and implants.
These studies are efficient for the initial
investigation on the biocompatibility of novel materials.
They have the advantage that
cell/material interactions can be studied without the predominant wound reaction that normally
occurs in vivo. The response of the immune system towards an implanted device often makes
evaluation of a material difficult to ascertain. By using specific cell lines such as fibroblasts
(ligament cells), chondrocytes (cartilage cells), osteoblasts (bone cells), and epithelial cells (skin
cells), the biological response of a specific tissue can be evaluated.
Although cell culture provides researchers with vital biological information, the
techniques must be carried out under strict conditions. Since animal cells grow much less
rapidly than many common contaminants like bacteria, mold, and yeast, it is vital to maintain
aseptic conditions during cell culture experiments. Techniques such as cell isolation, freezing,
thawing, counting, plating, splitting, and maintaining cells are common methods for establishing
cell culture studies. When properly followed, cell culture techniques can provide an efficient and
informative means by which the biological response to materials can be evaluated.
GENERAL PRINCIPLES
Osteoblasts will be extracted from the skulls (calvaria) of newborn rats (pups). The
calvaria of pups are commonly used as sources of osteoblasts for cell culture experiments. After
the cells are harvested from the calvaria, are fed with tissue culture medium and allowed to
proliferate for a couple of weeks, as they are not quite ready for tissue culture studies when first
harvested and require some time to mature. Once mature, they are removed from the tissue
culture flasks and placed onto the matrices formed in the previous lab. Once on the matrices,
they will proliferate and differentiate into mature osteoblasts. The nature in which they grow and
change will give an indication of the success of the matrix as a suitable environment for tissue
regeneration.
CELL CHARACTERIZATION
BACKGROUND
As a cell grows in vivo, it proliferates, differentiates, matures, and expresses different
proteins, growth factors, etc. that contribute to the production of its type. Therefore, it is
imperative that cells grown in vitro express the same behavior as those cells in vivo. To simply
proliferate is not enough. As a measure of the cell’s performance in vitro, there are a number of
assays, or tests, that can be performed to evaluate the cell’s progress. For instance, with bone
cells, after a certain amount of time they begin to produce mineral deposits, a process known as
mineralization. To be able to say that an osteoblast is indeed an osteoblast, it must be confirmed
that this cell is mineralizing. A test that measures the amount of calcium in a sample can be used
to determine the amount of mineralization occurring.
Another marker of normal cellular
development is alkaline phosphatase, a protein produced by normally developing bone cells.
Evidence of AP in cell culture further confirms that the cell is developing normallly. There are
several techniques to measure these proteins, one being fluorescence, which is nice because it is
quick, easy to see, and inexpensive. Alizarin Red is another technique that is good. A visual
check of the growth of cells is good as well. This can be done by SEM if the samples are
pretreated appropriately.