Introduction to Tissue Engineering

Tissue engineering is defined as “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” – Y.C. Fung.
“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” – C. T. Laurencin.
From these definitions, it is clear that tissue engineering has the potential to address the organ failure and tissue loss. This is
because current clinical approaches for restoring the organ or tissue function are organ transplantation, surgical reconstruction,
or use of prostheses. However, each treatment strategy has its own merits and demerits
Biomaterials, cells and growth factors are known as the “Tissue Engineering TRIAD”.

In the first approach cells can be used as therapeutic agents to restore the functional tissue. This approach mainly involves the
isolation of cells from different cell sources (autologous, allogenic; syngenic and xenogenic) and even use of stem cells using a
special technique called stem cell therapy, placing them in the site of interest for improving the tissue function.
The second approach involves the exogenous delivery of growth promoting substances like growth factors using the carriers
(polymeric or lipidemic) can stimulate the endogenous stem/progenitor cells for the specific differentiation thereby replacing
the lost cells or tissues.
The third approach is to use artificial 3-dimentional scaffolds or matrices for the growth of cells where cells can be either
recruited from the host tissue in vivo or seeded in vitro.
Challenges in tissue engineering

1. How do we develop a close approximate biological replica?

2. Should the tissue be produced in vitro implanted in vivo?
3. What type of scaffold should we choose?
4. How do we manufacture?
5. What cells are to be used?
6. Under what conditions can cells expand without affecting its phenotype?
7. What regulators do we use to stimulate the cell proliferation and differentiation?

There are few scientific challenges to construct a tissue or organ using these approaches.
For example, use of cells: limited supply of autologous cells;
requirement of immunosuppressant for allogenic source;
disease transmission associated with the xenogenic source;
ethical considerations with the use of stem cells are the major challenges in case of first approach.
 Tissue Dynamics

All tissues have their own characteristic replacement and production rates of cells and hence considered to be very
dynamic. However the time scale of the tissue dynamics varies with the tissues and is relatively long. Bone marrow is the
most prolific tissue in the human body, which is followed by the lining of the small intestine and epidermis. In all these
three tissues, the cell turn over rate is in order of a few days while in other tissues it is longer. For example, hepatocytes
replacement in the liver takes place for about a year.

Dynamic States of Tissues

There are basically three dynamic states of a tissue which includes tissue homeostasis, tissue repair and tissue formation.

Tissue Homeostasis
It is the normal steady state function of the tissue. Each tissue has distinct function and is necessary to maintain the
steady state function of the body. Some tissues like bone marrow and skin produces cells as their main function, while
glands produce a secreted product. Lungs and kidneys primarily carry out the mass transfer operations while liver act
as biochemical refineries. Muscular hypertrophy and ventricular hypertrophy (hypertrophi – Increase in cell size) are
the examples of how well tissue adapts to maintain homeostatic balance in accordance with the physiological
demands.
• Tissue repair:
Wound healing process is one of the common phenomenon exhibited by a biopsied tissue and the tissue grafts. Immediately
after implantation of the tissue grafts, it initially shows a healing response, which is then followed by the integration of tissue
grafts with the host tissue.
Tissue formation:
Tissue formation involves developmental biology and morphogenesis. Morphogenic process is very important in tissue dynamics
and plays a very important role in tissue reconstruction, repair and engineering.
Homeostasis in highly prolific tissues:
Tissues such as bone marrow, villi of the small intestine and skin are the three different examples for highly prolific tissues and
we will look into each of them in more detail.
a.Bone marrow:
Bone marrow is the most dynamic tissue in the human body. It produces about 40 billion myeloid cells daily all of them are
originated from the adult pluripotent adult stem cells. Bone marrow consists of 500 to 1000 billion cells and it produces many
cells within two or three days. It is the site of hematopoiesis. There are two different types of bone marrow includes red
(vascular) and yellow (fatty) bone marrow. Red bone marrow forms all blood cells except lymphocytes and destruct old RBC. It is
found mainly in hips, skull, ribs, and also ends of long bones. Yellow marrow stores fat and mainly fills the cavities of the bone.
At critical conditions such as any severe blood loss, the yellow marrow can be converted into red marrow. The major function of
bone marrow is the production of mature blood cells. Daily output of mature blood cells from bone marrow is 2.5 billion
erythrocytes; 2.5 billion of platelets; 50-100 billion granulocytes; and huge lymphocytes and monocyte production.
• Villi in the small intestine:
The lining of the small intestine consists of villi that help in the absorption of nutrients. The outer layer of villi consists of
epithelial cells and is highly dynamic. The turn over rate of the cellular contents is about five days approximately. Between villi,
there are epithelial infoldings called cryptus, which are tubular in shape. The production of intestinal epithelial cells occurs in the
crypt.
The bottom of the crypt consists of slowly dividing tissue specific stem cells and there are about 20 stem cells in the crypt. It then
undergoes cell division and the daughter cells that are formed moves up the crypt as it becomes cycling progenitor cell, which
has the cycling time of 12 hours. These cells are also referred to as transit amplifying cells, which then move up the crypt and
differentiate. The mature cells leave the crypt and reach the base of the villi. Then over a period of five days, they move from the
base to the top of the villi where they die and slough off. As you see, the villi consists of tubular infoldings called crypts where the
cells divide, differentiate into mature cells as they move from bottom to top. During their passage they perform the vital
functions like absorption and digestion of nutrients that come from the lumen of the gut.

Skin:
Skin consists of two layers – outer epidermis and inner dermis, which is separated by basal lamina. The connective tissue
dermis is made up of fibroblasts while epidermis is made up of keratinocytes with the least differentiated one located at
the basal lamina. Skin is made up of squamous columnar epithelium and each column is about 30 micrometer in diameter.
The cells that are closer to the basal lamina replicate and since the area available is constant, the cells begin to move up.
As they move up, they differentiate into prickle cells, then to granular cells, which then develop into keratinized squamous
cells that flake off. Thus only the cells that are present near the basal lamina divides and later it differentiates into well-
defined squamous cells. There are about 10-12 cells near the basal lamina that is known as epidermal progenitor cells, which is
responsible for the production of squamous columnar epithelial cells. The turnover rate of the cells is in the order of few weeks.
Tissue repair
Wound healing is a highly coordinated process involving series of cellular events that occurs in a damaged tissue and it varies
with age. Fetal wound healing and adult wound healing differs in the time taken for healing and also the scars that are formed
during the healing process. Fetal wound healing is very fast and results in scarless tissue, while on the other hand, adult wound
healing is slow and results in the scar formation.

Sequences of events that underlie wound healing:

Immediately after the injury to control the bleeding, platelets are activated and adhere to the injured site. They secrete the
storage granules, which help in recruiting more platelets to the site of injury to form thrombus. Release of agents from the
platelets causes vasodilation and increased permeability of blood vessels. The clotting cascade will be then initiated resulting in
the cleavage of fibrinogen to form fibrin plug. The fibrin plug along with the fibronectin forms the provisional matrix. This
provisional matrix helps in the recruitment of inflammatory cells and then the fibroblasts and other accessory cells. During this
time, the epidermal cells begin to proliferate and migrate acrosss the wound.
The next step in the wound healing process is the recruitment of inflammatory cells to the injured site. Neutrophils from the
circulating blood reach the site early and they degranulate and die. Subsequently the number of macrophages at the site
increases thus helps in phagocytosis of cellular debris and other microorganisms. These macrophages are the source of
chemoattractants and mitogens, which induce the migration of endothelial cells and fibroblasts to the injured site followed by
their proliferation. Effective wound healing process requires the invasion of macrophages to the wounded site since it clears the
cellular debris and pathogenic microorganisms. In the epidermal layer, the basal layer is reformed.
The invasion of fibroblasts results in the formation of granulation tissue in the dermis. The granulation tissue mainly consists of
dense population of fibroblasts, macrophages and developing vasculature consisting of ECM components like fibronectin,
collagen and hyaluronic acid. The fibroblasts present in the granulation tissue produce collagen mainly type I and type III, which
helps in increasing the tensile strength of the wound. Myofibroblasts begins to contract and due to the shrinking of the wound
the fluid oozes out. During this time, the epidermal layer gets stratified.
Then the matrix undergoes remodeling which involves synthesis and degradation of ECM components. During remodeling, the
composition of the matrix changes. Previously, collagen III, which is dominant is converted to collagen I. The remodeling process
actually determines the scar formation. Although, the wound is healed at this point of time, lot of chemical and structural
changes takes place within the wound site and may continue for many months till the composition of matrix returns to the
original state.
Engineered Wound Healing:
Tissue engineering strategies are being employed to enhance the wound healing process. For example, people have used a
porous template made up of collagen I and chondroitin-6-sulphate to mimic the extracellular matrix of the dermis of skin. This
template was fabricated with a range of pore sites and degradation rate. First, the time required for the 50% reduction in the
wound area was measured and the pore size in range of 20 and 120 micrometer was found to be optimum to cause delay in
wound contracture thereby improving the wound healing due to the migration of fibroblasts. Usually wound contraction occurs
after the migration of fibroblasts in the provisional matrix and subsequent conversion into myofibroblast. Hence, when the
fibroblast migration is delayed, automatically wound contraction and scar formation is also delayed thus allowing sufficient time
for the wound healing process. Tissue engineering strategies are being employed to enhance the wound healing process. For
example, people have used a porous template made up of collagen I and chondroitin-6-sulphate to mimic the extracellular matrix
of the dermis of skin. This template was fabricated with a range of pore sites and degradation rate. First, the time required for
the 50% reduction in the wound area was measured and the pore size in range of 20 and 120 micrometer was found to be
optimum to cause delay in wound contracture thereby improving the wound healing due to the migration of fibroblasts. Usually
wound contraction occurs after the migration of fibroblasts in the provisional matrix and subsequent conversion into
myofibroblast. Hence, when the fibroblast migration is delayed, automatically wound contraction and scar formation is also
delayed thus allowing sufficient time for the wound healing process. The normal wound healing rate for skin is three weeks and
for peripheral nerves is six weeks. Another alternative for the polymeric scaffolds are the cell-contracted extracellular matrix gels.
It exploits the cell-based contraction for the formation of tissue like constructs. It is found that with increase in cell number, the
contraction increases and with increase in extracellular matrix contraction, the contraction decreases. This cell-contracted
extracellular matrix gels form dermal equivalents and are commercialized for tissue engineered skin product.
Fatal wound healing
Fetal wound healing occurs very rapidly without scar formation while adult wound healing is a very slow process with the
formation of fibrosis and scar at the injured site. Moreover in adult wound healing there is poor reconstitution of epidermal and
dermal tissue at the healed wound site. However, the process of re-epithelization and connective tissue contraction is almost
same in adults and embryo but the mechanism by which they occur is different. In embryo, the gap in the epidermis is closed by
the contraction of actin purse-string, but in the case of adult, the epidermal cells migrate and fill the gap. Myofibroblasts in the
adult wound helps in the contraction of the exposed connective tissue thereby bring the two edges of the wound closer. On the
other hand, in fetal wound healing, embryonic cells help in the contraction of the connective tissue by exerting similar traction
forces.
Another major difference is that in adult wound healing, there is extensive inflammatory response while in embryo there is no
inflammatory response. Fetal wound contains “basket weave” collagen matrix and collagen III as the major ECM component,
while adult wound contains bundles of collagen and fibronectin as the major ECM component. Fetal wounds exhibit low tension
while adult wound exhibit high tension.
Since there is no inflammatory response and minimal scar formation in fetal wound healing, it would be interesting to
understand the mechanism so that they can mimic them in the tissue engineered medical products (TEMPs) for effective wound
healing.

Tissue dynamics as interacting cellular fate processes:

Cell replication – increase in cell number
Cell differentiation – change in gene expression and acquisition of particular function that is specific for the particular cell type
Cell motility – movement of cell into a particular niche Cell apoptosis – programmed cell death
Cell adhesion – binding of the cell to the neighboring cell or the extracellular matrix components
Cellular fate processes

Cell Differentiation
Differentiation is a process in which a cell undergoes phenotypic changes to a specialized cell type. The specialized cell type will
then carry out the physiological function. Thus the process of cell differentiation begins with the lineage commitment followed
by a coordinated series of gene expression events and finally it will differentiate into a new specialized cell.
Differentiation is usually identified by the relative change in the gene expression as gene expression varies with different cell
types. It is actually based on the gene expression and the physiological function of the cell. Lot of technologies have been
developed so far, which enable us to determine the gene that is expressed in a cell at a particular point of time.
This process is called expression profiling and it can be accomplished by the use of microarray technology.
Cell differentiation involves three stages and the first is the change in gene expression, which is followed by the change in
biochemical function and the ultrastructural features. Such differentiation process can be determined either by measuring the
changes in gene expression; changes in the cell function or even measured mathematically. Let us take a simple example to
evaluate the osteoblast differentiation and maturation through the changes in gene expression. There are different phases in
differentiation and maturation such as growth or proliferation phase, matrix maturation phase, matrix mineralization phase
and then apoptosis.
There are proteins such as osteopontin, fibronectin, collagen, and histone expressed higher at the proliferative phase.
Alkaline phosphatase, bone sialoprotein, collagen are expressed higher at the matrix maturation phase.
Osteocalcin, osteopontin, collagenase are the chief markers to confirm the matrix mineralization phase.
In the bone tissue, two major types of cells are present namely bone depositing cells (osteoblast) and bone resorbing cells
(osteoclast). Generally osteocalcin, osteopontin, alkaline phosphatase and collagen are the chief markers to evaluate the
osteoblast differentiation and maturation.
Osteocalcin is a bone gamma carboxyglutamic acid containing protein, marker of bone formation. The major function of this
protein is to build up the inorganic material hydroxyapatite (HA) in a right pattern, which is chief material contributing bone
strength.
The next protein is osteopontin, bone sialoprotein (BSP-1), which is a prominent component of mineralized ECM of bones. Hence,
the over expression of this extracellular structural protein confirms the matrix mineralization phase. Alkaline phosphatase is also
one of the markers of bone formation. This is produced by the osteoblast to promote a high phosphate concentration at
osteoblast surface during mineralization. Thus, by studying the expression profile of different markers at different time points will
determine the differentiation.
Another example is erythropoiesis, which is the process of red blood cells production. Erythropoiesis replaces the decaying
mature red blood cells and it is estimated that 200 million new red blood cells needs to be produced daily in the human adult.
This process involves lot of biochemical and also ultra structural changes.

The cell size, the rates of DNA and RNA synthesis and the protein content change in a progressive and coordinated fashion during
the process. It almost takes 180 hours for the differentiation of pronormoblast to fully mature enucleated erythrocyte and the
cell replication activity is highest at the preprogenitor and progenitor stage but once it reaches the precursor stage, the rate of
cell division ceases. Ultra structural changes that are observed during the process of cell differentiation can be observed by using
fluorescent markers or light microscopy.
Since, there is difference in the surface protein that is expressed during the process of differentiation flow cytometry can be used
to monitor the process of cellular differentiation. Transferrin receptor and glycophorin A are the two surface proteins that are
commonly expressed by the red blood cells. Hence, these proteins can be used to trace erythropoiesis.
While transferrin receptor is needed for the sequestration of iron in hemoglobin, glycophorin A, which is an erythroid specific
surface protein is needed to prevent the aggregation of red blood cells by providing negative charge to them.
Cell Migration
Cell migration plays an important role in all physiological as well as some pathological processes. Cell migration is very important
during organogenesis and embryonic development. It plays a role in tissue-repair response during both wound healing and
angiogenesis. On one hand, the immune system relies on cell migration, while on the other cancer metastasis is characterized
by the cell motility. The above-mentioned are the ones that normally happen in the body. But there are cases where migration of
cells is induced by infusing chemotactic growth factors. For example, bone marrow stem cells can be induced to migrate out of
the marrow into the blood circulation by infusing chemotactic growth factors into the patient.

Based on the stimuli, there are five different modes of cell migration:
1. Chemotaxis - soluble concentration gradient
2. Hypotaxis - insoluble concentration gradient
3. Galvanotaxis - electrical currents
4. Contact guidance - surface topology
5. Contact inhibition - lamellipodium inhibited by neighbor adjacency
Cell migration is a five-step process:
Step 1: Protrusion of membrane lamellopodia.
Step 2: Adhesion to the matrix via integrin receptors.
Step 3: Contraction of the cytoplasm by myosin based motors.
Step 4: Rear release and cell displacement.
Step 5: Recycling of remaining integrins
Step 1: Protrusion of the membrane lamellipodia occurs by the polymerization of actin. In the actin polymerization process,
there is increase in the number of sites for actin polymerization followed by the addition of actin monomers on the filament
growth sites near the membrane. The new sites for actin polymerization may arise either by the uncapping of existing
filaments or the formation of new nucleation sites.

Step 2: The actin cytoskeleton is linked to the extracellular matrix via integrins, which are the cell surface receptors thus forming
specialized complexes. Integrin receptors have the affinity towards certain motifs like RGD that is present in the extracellular
matrix. Upon binding to the ligands (RGD), integrin cross-linking occurs and matrix stiffness is increased. These specialized
complexes are regulated by the rho subfamily of the ras family of GTP-binding proteins but however the mechanisms by which
they exert the effects are not known.

Step 3: There are two forces involved - contractile force and the traction force. Contraction of cytoplasm occurs via myosin-
based motors. However, for a cell to move, the cell must overcome the contraction force provided by the myosin-based motors.
Fibroblast cells can generate a traction force of approximately 2X104 µdynes against the substrate.

Step 4: Rear side release of the integrins and the cell displacement involves both physical and enzymatic processes. Either by
the transmission of physical forces or by regulating the local Ca2+ concentration, asymmetry in the adhesion of cell to the
substrate can be brought out thus breaking the rear side contacts and the cell displacement.

Step 5: The integrins that are released from the rear side is endocytosed into vesicles which is then followed by intracellular
diffusion and is directed towards the cell surface of the leading edge.
 Factors affecting cell migration speed
There are two factors, which affect the cell migration speed is substratum adhesive strength and intracellular contraction force.
Assume when the adhesion is very weak, then obviously intracellular contraction force dominates, thereby preventing the
traction force against the substratum. Therefore, the movement could be very negligible.
Suppose the intracellular contraction force is comparable with the substratum adhesive strength, cells can impart the traction
force against substrate promoting the rear release by the detachment of rear cell-substrate attachment, thereby promoting cell
speed significantly
However, if there is a strong adhesion between cell and substrate, the rear release could not be promoted and hence cell
migration is very negligible. From these it is very clear that the ratio of intracellular contraction force and the substratum
adhesive strength will determine the cell migration speed