Wuolah Free Apuntes Tejidos

Apuntes-Tejidos.
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Crespo_99
Fundamentos de ingeniería de tejidos y medicina

regenerativa
3º Grado en Ingeniería Biomédica
Escuela Politécnica Superior

Universidad Carlos III de Madrid
Reservados todos los derechos.

No se permite la explotación económica ni la transformación de esta obra. Queda permitida la impresión en su totalidad.
CARLOS III UNIVERSITY OF MADRID
Bachelor’s Degree in Biomedical Engineering
Fundamental of tissue engineering and regenerative medicine

CLASS NOTES
David Crespo Acero

February 12, 2021
Supervised by:
Me :)
a64b0469ff35958ef4ab887a898bd50bdfbbe91a-3904786
Contents
1 Basic concepts 7
1.1 Tissue Engineering(TE): Classical Definition . . . . . . . . . . 7
1.2 Tissue Engineering(TE): General Paradigm . . . . . . . . . . . 7
1.3 Regenerative medicine (RM) . . . . . . . . . . . . . . . . . . . 7
1.3.1 Applications . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.2 Research areas . . . . . . . . . . . . . . . . . . . . . . 8

2 Organization of cells into Higher-Ordered structures 9
2.1 Levels of Organizaon . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Why cells differentiate in different size, shape and function? . 9
2.3 Extracellular matrix (Scaffold) . . . . . . . . . . . . . . . . . . 9
2.4 Tissue Families . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.5 Cell lineages plasticity . . . . . . . . . . . . . . . . . . . . . . 10
2.6 Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.7 Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3 Histologic techniques, microscope and virtual microscope 12

3.1 Histology techniques . . . . . . . . . . . . . . . . . . . . . . . 12
3.2 Histology techniques major steps . . . . . . . . . . . . . . . . 12
3.3 Egg paradox . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.4 Virtual Microscope . . . . . . . . . . . . . . . . . . . . . . . . 13
4 Epithelial Tissues 15
4.1 Epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.1.1 Functions . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.2.1 Number of cell layers . . . . . . . . . . . . . . . . . . . 16
4.2.2 Cell shape and size . . . . . . . . . . . . . . . . . . . . 16
4.3 Basement Membrane (BM) . . . . . . . . . . . . . . . . . . . . 17
4.4 The Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.5 Basal Specialization and Epithelial Attachment . . . . . . . . 17
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4.5.1 Basal surface specializations . . . . . . . . . . . . . . . 17
4.5.2 Lateral specializations . . . . . . . . . . . . . . . . . . 18
4.6 Apical Specializations . . . . . . . . . . . . . . . . . . . . . . . 19
4.6.1 Microvilli . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.6.2 Cilia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.7 Glandular epithelia . . . . . . . . . . . . . . . . . . . . . . . . 19
4.7.1 Types of secretion . . . . . . . . . . . . . . . . . . . . . 20
5 Connective Tissues 21
5.1 What is a Connective Tissue? . . . . . . . . . . . . . . . . . . 21
5.1.1 Functions . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.2 Cells of the connective tissue . . . . . . . . . . . . . . . . . . . 22
5.2.1 Other cells of the connective tissue . . . . . . . . . . . 22
5.3 Extracelluar matrix . . . . . . . . . . . . . . . . . . . . . . . . 23
5.3.1 Reticular Fibers . . . . . . . . . . . . . . . . . . . . . . 23
5.3.2 Elastic fibers . . . . . . . . . . . . . . . . . . . . . . . 23

5.3.3 Ground substance . . . . . . . . . . . . . . . . . . . . . 24
5.3.4 Adhesive glycoproteins . . . . . . . . . . . . . . . . . . 24
5.4 Classification of Connective Tisues . . . . . . . . . . . . . . . 24
5.4.1 Cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5.4.2 Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5.5 Connective tissue regeneration . . . . . . . . . . . . . . . . . . 26
5.5.1 Mesenchymal stem cells (MSC) . . . . . . . . . . . . . 26
6 Muscular Tissue 27
6.1 What is muscle . . . . . . . . . . . . . . . . . . . . . . . . . . 27
6.2 Anatomical organization of the muscle . . . . . . . . . . . . . 27
6.2.1 Sarcomere . . . . . . . . . . . . . . . . . . . . . . . . . 28
6.3 How Contraction Occur . . . . . . . . . . . . . . . . . . . . . 29
6.4 Types of muscle . . . . . . . . . . . . . . . . . . . . . . . . . . 29
6.4.1 Skeletal muscle . . . . . . . . . . . . . . . . . . . . . . 29
6.4.2 Smooth muscle . . . . . . . . . . . . . . . . . . . . . . 29
6.4.3 Cardiac muscle . . . . . . . . . . . . . . . . . . . . . . 30
6.5 Muscle: Regeneration vs Repair . . . . . . . . . . . . . . . . . 30
6.5.1 Why Regeneration or Repair? . . . . . . . . . . . . . . 30
6.5.2 How does an organ increase the size?? . . . . . . . . . 31
6.5.3 Smooth muscle . . . . . . . . . . . . . . . . . . . . . . 31
6.5.4 Skeletal muscle . . . . . . . . . . . . . . . . . . . . . . 31
6.5.5 Cardiac muscle . . . . . . . . . . . . . . . . . . . . . . 31

7 Blood and hematopoiesis 32
7.1 Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
7.2 Blood components . . . . . . . . . . . . . . . . . . . . . . . . 32
7.2.1 Erythrocyte . . . . . . . . . . . . . . . . . . . . . . . . 32
7.2.2 Leukocytes - Granulocytes . . . . . . . . . . . . . . . . 33
7.2.3 Leukocytes-Agranulocytes . . . . . . . . . . . . . . . . 34
7.2.4 Platelets (thrombocytes) . . . . . . . . . . . . . . . . . 35
7.2.5 Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
7.3 Immune response . . . . . . . . . . . . . . . . . . . . . . . . . 37
7.4 Bone marrow and hematopoiesis . . . . . . . . . . . . . . . . . 38
7.4.1 Bone marrow . . . . . . . . . . . . . . . . . . . . . . . 38
7.4.2 Regenerative medicine of Blood and Bone Marrow . . . 38
8 Circulatory system 41
8.1 Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
8.1.1 Endocardium . . . . . . . . . . . . . . . . . . . . . . . 41

8.1.2 Myocardium . . . . . . . . . . . . . . . . . . . . . . . . 41
8.1.3 Epicardium . . . . . . . . . . . . . . . . . . . . . . . . 42
8.1.4 Mesothelium . . . . . . . . . . . . . . . . . . . . . . . . 42
8.1.5 Heart valves . . . . . . . . . . . . . . . . . . . . . . . . 42
8.1.6 Diastole-Systole . . . . . . . . . . . . . . . . . . . . . . 42
8.1.7 Serosa vs. Adventitia . . . . . . . . . . . . . . . . . . . 42
8.2 Blood vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
8.2.1 Wall of blood vessels . . . . . . . . . . . . . . . . . . . 43
8.2.2 Arteries . . . . . . . . . . . . . . . . . . . . . . . . . . 43
8.2.3 Veins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
8.2.4 Capillaries . . . . . . . . . . . . . . . . . . . . . . . . . 44
8.2.5 Venules . . . . . . . . . . . . . . . . . . . . . . . . . . 45
8.3 Lymphatic Capillaries . . . . . . . . . . . . . . . . . . . . . . 46
8.4 Why such progress in Tissue Engineering of Blood vessels? . . 46
9 Lymphatic Tissue 47
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
9.2 Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
9.2.1 Fluid balance: Formation of Lymph . . . . . . . . . . . 47
9.2.2 Absorption of fat . . . . . . . . . . . . . . . . . . . . . 48
9.2.3 Inmunity . . . . . . . . . . . . . . . . . . . . . . . . . . 48
9.3 Organs of the lymphatic system . . . . . . . . . . . . . . . . . 48
9.3.1 Lymph nodes . . . . . . . . . . . . . . . . . . . . . . . 48
9.3.2 Spleen . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
9.3.3 Thymus . . . . . . . . . . . . . . . . . . . . . . . . . . 49

9.3.4 Malt (mucose associated lymphoid tissue) . . . . . . . 49
9.4 Tissue Engineering of Lymphatic tissue . . . . . . . . . . . . . 50
9.5 Immune system diseases . . . . . . . . . . . . . . . . . . . . . 50
10 Nervous System 51
10.1 Central nervous system . . . . . . . . . . . . . . . . . . . . . . 51
10.1.1 Ventricular System . . . . . . . . . . . . . . . . . . . . 51
10.1.2 Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
10.1.3 Cells in the Nervous system . . . . . . . . . . . . . . . 51
10.2 Peripheral Nervous System . . . . . . . . . . . . . . . . . . . . 54

10.2.1 Somatic Nervous System (voluntary nervous system) . 54
10.2.2 Autonomic Nervous System . . . . . . . . . . . . . . . 54
10.2.3 PNS - Nerves . . . . . . . . . . . . . . . . . . . . . . . 54
10.2.4 Satellite glial cells . . . . . . . . . . . . . . . . . . . . . 55
10.2.5 Ganglia . . . . . . . . . . . . . . . . . . . . . . . . . . 55
10.3 Nervous Tissue Regeneration . . . . . . . . . . . . . . . . . . . 56
10.4 What are the medical needs? . . . . . . . . . . . . . . . . . . . 56
10.5 Tissue Engineering of the CNS . . . . . . . . . . . . . . . . . . 56
11 Cell Culture 57
11.1 2-D Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . 57
11.1.1 Growth curve . . . . . . . . . . . . . . . . . . . . . . . 57
11.1.2 Primary culture . . . . . . . . . . . . . . . . . . . . . . 57
11.1.3 Cell subculture . . . . . . . . . . . . . . . . . . . . . . 57
11.1.4 Cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . 58
11.2 3-D cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . 58
11.2.1 Types . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
11.3 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
11.4 Biorreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
11.4.1 Types . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
11.4.2 Engineering Parameters in Bioreactor Design . . . . . . 60
11.4.3 Functions . . . . . . . . . . . . . . . . . . . . . . . . . 61
12 Stem Cells I 63
12.1 History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
12.2 Types of Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . 63
12.3 Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
12.3.1 Amniotic fluid-derived stem (AFS) cells . . . . . . . . . 64
12.3.2 Umbilical cord stem cells . . . . . . . . . . . . . . . . . 64
12.4 Limitations to clinical use of ES cells . . . . . . . . . . . . . . 65
12.5 Induced Pluripotent Stem Cells . . . . . . . . . . . . . . . . . 66
12.6 Organ Transplantation and Cell Therapie . . . . . . . . . . . . 66
12.6.1 Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . 67
12.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
13 Stem Cells III 69
13.1 Stem Cell Niche . . . . . . . . . . . . . . . . . . . . . . . . . . 69
13.2 Hematopoietic Stem Cells (HSC) . . . . . . . . . . . . . . . . 69
13.2.1 Conditioning Regimens . . . . . . . . . . . . . . . . . . 69
13.2.2 Complications After HSC Transplantation . . . . . . . 71
13.2.3 HSC Transplant Matching . . . . . . . . . . . . . . . . 71

List of Figures
4.1 Types of epithelium . . . . . . . . . . . . . . . . . . . . . . . . 16

4.2 Surface specializations . . . . . . . . . . . . . . . . . . . . . . 18
4.3 Types of glands . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5.1 Connective tissue elements . . . . . . . . . . . . . . . . . . . . 22

5.2 Cartilage structure . . . . . . . . . . . . . . . . . . . . . . . . 25
6.1 Skeletal muscle structure . . . . . . . . . . . . . . . . . . . . . 28

7.1 Types of granulocytes . . . . . . . . . . . . . . . . . . . . . . . 33
7.2 Types of agranulocytes . . . . . . . . . . . . . . . . . . . . . . 34
7.3 T cell at the right and B cell at the left . . . . . . . . . . . . . 35
7.4 Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
7.5 Hematopoiesis in humans . . . . . . . . . . . . . . . . . . . . . 38
7.6 Autologous transplant . . . . . . . . . . . . . . . . . . . . . . 39
7.7 Allogenic transplant . . . . . . . . . . . . . . . . . . . . . . . 40
8.1 Types of arteries . . . . . . . . . . . . . . . . . . . . . . . . . 43

8.2 Comparison between an artery and a vein . . . . . . . . . . . 45
10.1 Types of neurons . . . . . . . . . . . . . . . . . . . . . . . . . 52

10.2 Synapsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
10.3 Myelinated and unmyelinated nerve conduction . . . . . . . . 55
11.1 Growth curve . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

11.2 Spheroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
11.3 Membrane flask bioreactor . . . . . . . . . . . . . . . . . . . . 61
11.4 Bubble column bioreactor . . . . . . . . . . . . . . . . . . . . 62
12.1 Advantages and disadvantages . . . . . . . . . . . . . . . . . . 64

12.2 Cord blood vs Bone Marrow . . . . . . . . . . . . . . . . . . . 65
13.1 Stem Cell Niches (also Hematopoietic Stem Cell Niche, not
included) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Chapter 1
Basic concepts
1.1 Tissue Engineering(TE): Classical Defini-

tion

An interdisciplinary field that applies the principles of engineering and life
sciences toward the development of biological substitutes that restore, main-
tain, or improve tissue function or a whole organ.
1.2 Tissue Engineering(TE): General Paradigm

The general paradigm in TE is the in vitro cultivation of cells in bioreactors
and then use those cells to construct a cell matrix that will be implanted in
the patient.
1.3 Regenerative medicine (RM)

Process of creating functional tissues to repair or replace tissue or organ
function lost due to age, disease, damage, or congenital defects. We can do
this:
• By stimulating organs to heal themselves (In vivo).
• By growing tissues and organs in the laboratory and safely implant

them when the body cannot heal itself (Ex vivo).
The term RM is often used synonymously with TE, although those in-
volved in RM place more emphasis on the use of stem cells to produce tissues.

1.3.1 Applications
• Therapeutic: tissue is either grown in a patient or outside the patient
and transplanted.
• Diagnosis: tissue is made in vitro and used for testing drug metabolism
and uptake, toxicity, and pathogenicity.
1.3.2 Research areas

• Biomaterials.

• Cells: Including enabling methodologies for the proliferation and dif-
ferentiation of cells, acquiring the appropriate source of cells (autolo-
gous, allogeneic, xenogeneic, stem cells, or genetically engineered cells).
• Biomolecules: Including angiogenic factors, growth factors, differen-

tiation factors...
• Engineering Design Aspects: including 2D cell expansion, 3-D tis-

sue growth, bioreactors, ...
• Biomechanical Aspects of Design: Including properties of native

tissues, mechanical signals regulating engineered tissues, efficacy and
safety of engineered tissues...
• Informatics.
Chapter 2
Organization of cells into

Higher-Ordered structures
2.1 Levels of Organizaon

An individual is formed by different systems, systems are formed by organs
and organs by tissues. Finally, tissues are formed by cells and extracellular
matrix (ECM). The cells in each tissue are highly specialized (differentiated)
to perform the function of that particular tissue.
Cell lineage is the genealogic tree of the tissues, all derived from a
common ancester, which is the zygote. Can be defined as the biological
history of the cells that form the tissues.
Cell divisions can be symmetric or asymmetric.
2.2 Why cells differentiate in different size,

shape and function?
As a results of programming of the pluripotential cells and also environmental
influences (contact with other cells and ECM) cells change the profile of gene
expression. We know that environmental signals activate translation factors
that make the specific genes relevant to a tissue to be expressed.
2.3 Extracellular matrix (Scaffold)

The extracellular matrix is a complex substance formed by fibers (collagen
and elastin) and embedded in a ground substance of glycoproteins, hyaluronic
acid, proteoglycans, glycosaminoglycans, etc.

The ECM has not only a structural function but also provide signals to
the cells of the tissue. These signal may regulate prolifeation, differentiation,
survival, etc.
2.4 Tissue Families
• Epithelia (surface and glandular epithelia)
• Connective Tissue (including cartilage and bone)
• Muscle (smooth, squeletal, cardiac)
• Nervous tissue (Central and Peripheric)
The connective tissues and muscle are often referred as Mesenchymal
tissues.
• Common origin: Mesenchyma is the connective tissue of the embryo

and from this tissue derived all connective tissues and muscle.
• Functional Differences:
Epithelia: Stand on extracellular matrix, polarized, low movili-
ty/high attachment, usually parenchyma.
Mesenchymal: Immersed in extracellular matrix, non-polarized,
high mobility/low attachment, usually stroma.
The parenchyma are the functional parts of an organ in the body. With
some exception these are epithelial tissues. The stroma is the structural and
support tissue of organs. They are mesenchymal tissues (mainly connective
tissues).
2.5 Cell lineages plasticity

There are several exceptions in which epithelial cells may acquire mesenchy-
mal characteristics Epithelial Mesenchymal Transition (EMT) and vice versa
Mesenchymal Epithelial Transition (MET).
• The embryo
• Tissue repair and regeneration
• Pathologic conditions and cancer
• In vivo genetic manipulation
10

2.6 Organs
Organs are the combination of several tissues in order to perform a func-
tion. Most organs are formed by a epithelial parenchyma and a mesenchymal
stroma. The organ regeneration is more complex because require multiple
tissues.
2.7 Systems
Group of organs that work together for one purpose. There is 10 in the
human body.
11

Chapter 3
Histologic techniques,

microscope and virtual
microscope
3.1 Histology techniques

Tecniques carried out to be able to study a specimen under the microscope.
We need to be able to see the organ through a light microscope. Thus,
we need to transform the organ or sample into an histologic slide (It is a
sample of a fixed tissue, very thin (4- 5µm), stained, and mounted in a glass
slide and covered with a very thin lamina of glass called cover slip).
3.2 Histology techniques major steps

1. Fixation: To prevent tissue decay and at the same time preserving the
morphology of cells and tissue structures. The fixatives kill bacteria
and stop cellular enzymes that produce the degradation of the tissue.
Many fixatives are used. By far, the most common is 10% formalin.
2. Dehydration: To embed the tissue in paraffin (hydrophobic). Per-

formed with a gradient of alcohols and organic solvents.
3. Embedding: Embedding the tissue in paraffin at 60o C to give texture

and then cutting them with a special machine called microtome.
4. Sectioning: for the light to pass through the sample. 4-5 µm thick.
12
5. Desparafinization and re-hydration: to color the samble (colorants are
hydrosoluble). Performed with a gradient of alcohols and organic sol-
vents. Important colorants are hematoxilin (for nuclear components,
including hematocromatin and nucleoli) and eosin (for cytoplasmic
components including cytoplasmic granules, extracellular components
including collagen and elastic fibers, muscle fibers and red blood cells),
they include substances to clean the tissue (xylene).
6. Staining: Most tissues naturally do not have any color and; therefore
the microscope does not reveal structures. However, different tissues
or cells have different affinities for certain stains and this allows one to
study the structure of the tissue.
7. Final dehydration: the tissues are covered by hydrophobic resins, same
methodology as before.
8. Covering the slides: to prevent refraction that distort the image and to

protect the preparation.
9. Quality control.
3.3 Egg paradox

Histologic sections can have on average a surface of 1 cm2 in the X and Y
axes but in the Z axis (the thickness of the section) it has only 3-5 um. Thus
the Z is irrelevant when compared with X and Y and therefore we are looking
at a two dimension object. However, we want to interpret the structure of
an organ that is 3D. Sections at different levels provide “apparent” different
structures.
3.4 Virtual Microscope

• Advantages
Virtual slide is in a server and is accessible to multiple pathologist-
s/researchers/ students.
There is a much lower risk in losing or breaking the slide
Allows sharing of information for consultation with other patholo-
gists in any part of the world
Makes large retrospective collections of slides available to every-
body (for example, the Rosai’s collection)
13

Easier to locate for retrospective studies
Reduced costs, particularly for teaching purposes
• Disadvantages
Not yet approved for primary diagnosis
Excellent image quality but still one notch below the actual micro-
scope
Lack of Z axis (scanner focuses automatically in a single plane)
Resistance of some of the old pathologists
14

Chapter 4
Epithelial Tissues
4.1 Epithelia
The epithelium is a tissue composed of a layer(s) of cells of mostly uniform

type, closely bound to one another.
Epithelia covers the outside of the body, inside cavities (mouth, respira-
tory tract, etc) and the lumen of internal organs (stomach, intestine, bladder,
etc).
They are biological walls that separate the “internal milieu” of the body
from the outside. They provide a barrier for surfaces that are or may be in
contact with foreign substances (intestine, respiratory tract). They provide a
barrier to separate fluids with different physicochemical properties (e.g urine,
blood).
Epithelial tissue is always supported by connective tissue (also referred
in this context as stroma). It rests on a specialized structure that separate
and attach to the connective tissue called basement membrane. Epithelia
is polarized (asymmetrical in the vertical axes: one pole is adapted to deal
with the connective tissue and the other with the air).
Epithelia do not have ECM, blood vessels, lymphatic vessels and nerves,
therefore depends on connective tissue for nutrition, oxygenation and inner-
vation.
4.1.1 Functions
• Compartmentalization
• Protection
• Absorption/Selective permeability
15

Figure 4.1: Types of epithelium
• Transport
• Secretion (glandular epithelium)
• Lubrication
4.2 Classification
The outermost layer defines the name of the stratified epithelia (Figure 4.1):
4.2.1 Number of cell layers

• Simple
• Stratified
• Pseudostratified
4.2.2 Cell shape and size

• Squamous
• Cuboidal
• Columnar
16
4.3 Basement Membrane (BM)
A thin, highly organized layer of Extracellular Matrix(ECM) underlying ep-
ithelia with adhesive, barrier and filtering properties.
Its major components are Collagen IV (coarse mesh) and Laminin (fine
mesh), although it also has several other glycoproteins. Lamina Lucida is
formed primarily by laminin and other glycoproteins while Lamina Densa
is formed by Collagen IV.
4.4 The Cytoskeleton

The cytoskeleton is an organized network of three primary protein filaments:
• Microtubules (tubulin): Microtubular structures formed by polymer-

ization of tubulin. In epithelia involved in cilia movement.

• Microfilaments (Actin filaments): Filaments formed by actin and other
contractile proteins. Responsible for cell movement and providing the
mechanical structure to microvilli.
• Intermediate filaments (Tissue Specific): In the epithelia intermediate

filaments are formed by keratins.
The cytoskeleton is essential to attach cells to each other and to attach

the epithelium to the Basement Membrane. Also the cytoskeleton plays a
role in basal, apical and lateral membrane specializations.
4.5 Basal Specialization and Epithelial At-

tachment
Epithelia attach to CT by anchoring the cytoskeleton to the BM (Figure 4.2).
4.5.1 Basal surface specializations

• Hemidesmosomes
• Focal adhesions
17

Figure 4.2: Surface specializations
4.5.2 Lateral specializations

• Tight junctions: Join cells to form impermeable barriers.
• Zonula adherens: Junctional complex that binds adjacent cells to-

gether.
• Macula Adherens (desmosomes): Junctional complex that binds adja-

cent cells together.
• Gap Junctions: Mediate cellular communication allowing the passage

of ions and small molecules between adjacent cells.
18

4.6 Apical Specializations
4.6.1 Microvilli
Apical structures formed by folding of the cell membrane and a core of actin
filaments.
The function of microvilli is to increase the area of the apical surface to
facilitate absorption.
Examples: intestinal epithelium and epithelia of some kidney tubes.
4.6.2 Cilia
Apical structure formed by 9 pairs of microtubules aligned around a central
microtubule pair (9+2). The structure is covered by the cell membrane.
The function is transport of material along the epithelial surface.
Examples: respiratory epithelium moving mucus toward the pharynx and
the epithelium of the oviduct moving the egg toward the uterus.

4.7 Glandular epithelia
In the previous section, we used the term epithelia to refer to the tissues cov-
ering the body or internal cavities. We called this type of epithelia covering
or surface epithelia.
We also call epithelia for tissues forming glands, organs with the function
to secrete a large variety of substances (mucus, enzymes, hormones, etc.)
essential for life.
To differentiate these secretory tissues from the covering epithelia we call
them glandular epithelia (they may be also referred as secretory epithelium).
There are special cases in which epithelial cells have both functions: cov-
ering and glandular.
• Developmentally, glandular epithelia always derive from a covering ep-

ithelia.
• Both types of epithelia (covering and glandular) are layer(s) of cells of

mostly uniform type, closely bound to one another.
• They are essentially devoid of extracellular matrix.
• Both are polarized and have basement membranes.
• They have also similar intercellular attachment mechanisms.
19

Figure 4.3: Types of glands
• In both epithelia the intermediate filaments are keratins.
• Some epithelial cells have both covering and secretory functions.
4.7.1 Types of secretion

• Exocrine: Substances(enzymes, mucus, other proteins) secreted to the
exterior or to an internal cavity by a gland through a ductal structure.
• Endocrine: Substances (hormones) secreted directly into the blood by

ductless gland.
• Paracrine: Substances secreted by cells in a tissue or organ targeting

other cells in close proximity.
• Autocrine:Substances secreted by cells and targeting their own recep-

tors.
20
Chapter 5
Connective Tissues
5.1 What is a Connective Tissue?

Connective Tissues are composed by different cell types, mainly with one

predominant type (the fibroblast) embedded in an abundant Extracellular
Matrix. Abundance of Extracellular Matrix (ECM) is the hallmark of the
connective tissue.
• Carry blood and lymphatic vessels
• Carry nerves
• Cells are not polarized
• Cells are immerse in the ECM (not on top of it, as in the epithelia)
5.1.1 Functions
• Serve as support for Epithelial Tissues (stroma functions)
• Form high resistant structures important for the function of the mus-
cular skeletal system (Ex: tendons, ligaments, etc)
• Provide structural support for organs, connecting and keeping together

the different tissues forming an organ
• Provide the battleground for the defense mechanisms and immunity

and are involved in the repair process
21

Figure 5.1: Connective tissue elements
5.2 Cells of the connective tissue

The main resident cells of the connective tissue are the fibroblasts and fibro-
cytes, specialized in the secretion of collagen and elastin.
Fibroblast are much more than mere factory of ECM. They have influence
in many Fisiologic and pahtologic processes. Among the more important
are repair-regeneration, fibrosis, inflammation and cancer. To perform this
diversity of function fibroblast produces morfologic and physiologic changes
which are known as Fibroblast activation. Fibroblast can be activated by
chemical or mechanical stimulus. Activation means that they are hyperactive
in terms of synthesis and prolferation as well as production of paracrine
signals to epithelia, the inmune system and other cells of the connective
tissues.
5.2.1 Other cells of the connective tissue

• Macrophages (Histiocytes) - Scavenger cells that specialize in phago-
cytosis of invading microorganisms, dead cells, and foreign proteins.
Macrophages are derived from a type of WBC called a monocyte.
Macrophages also play an important role in activating humoral and
cell- mediated immunity - an important defense against microorgan-
22

isms.
• Mast Cells - Cells which are widely distributed in all vascular tissues.
They produce histamine and other compounds which help trigger in-
flammation and allergic responses. Mast cells are thought to be derived
from basophils (?), a type of WBC circulating in the blood.
• Plasma Cells - A cell found in the bone marrow which is derived from
a type of WBC called B Lymphocyte. Plasma cells play a role in the
immune response by synthesizing antibodies (immunoglobulins).
• Leukocytes - White blood cells (WBC’s). This family of cells play an

important role in the inflammatory response, and the defense of the
body against viral, bacterial, fungal, and parasitic infections.
5.3 Extracelluar matrix

5.3.1 Reticular Fibers
Reticular fibers are another form of collagen (Type III). They are arranged
as a loose meshwork of thin fibers providing a supportive scaffolding for the
specialized cells of various organs as well as blood vessels. This function
is somewhat analogous to the frame of a house or the girders supporting a
skyscraper. Reticular fibers are prominent in loose connective tissues and
in structures that are subject to periodic changes in size and volume (e.g.,
lymph nodes and the uterus), or are exposed to physical stresses requiring
some degree of flexibility (e.g., dermis of skin and wall of intestines).
Reticular fiber can not be distinguished by light microscopy. They were
discovered using silver staining.
5.3.2 Elastic fibers

Elastic fibers, form by elastin, are found in those organs and tissues that
are elastic and must stretch or recoil (e.g., arteries, skin, alveoli of lungs,
etc.). Unlike collagen, which is organized into dense fibrous bundles, elastin
is somewhat amorphous and arranged in relaxed cross- linked coils. When
elastic tissue is stretched, the elastin molecule is elongated into a more linear
conformation. When the stretching force is released, the elastin returns to
the more stable random-coil structure. In this respect, elastin fibers function
much like rubber bands.
23

5.3.3 Ground substance
The space between cells and fibers, is filled with the ground substance, a
mixture of Glycosaminoglycans, proteoglycans and adhesive gycoproteins.
5.3.4 Adhesive glycoproteins

Help hold tissue structures together much like mortar binds bricks. One of
these compounds, fibronectin, links the cytoskeleton of cells to collagen fibers
and other elements of the extracellular matrix. Laminin helps hold epithelial
cells to the basement membrane. Adhesive glycoproteins contribute to the

ability of individual muscle fibers to work as a unit by holding them together
when they contract. Other members of the adhesive glycoprotein family play
an important role in the differentiation and growth of cells, development of
blood vessels, and cell movement.
5.4 Classification of Connective Tisues

1. Embryonic: Connective tissues in the embryo. Outside the scope of
this class. Examples: mesenchyma and mucous CT
2. Connective Tissue Proper
Loose (areolar) Connective Tissue: It is abundant in ground sub-
stance and various type of cells (fibroblasts and others) and a moderate
number of fibers. Examples: CT connecting blood vessels with other
tissues, the lamina propia of mucosas. Fills also various spaces in the
body connecting tissues and organs
Dense Connective Tissue: the main component is collagen and or
Elastic fibers. Few cells (fibrocytes) and scarce ground substance. Can
be highly organized forming tendons and ligaments, or in the dermis.
Reticular Tissue: Conective tissue formed by thin fibers of collagen
III
Elastic Connective Tissue: rich in elastic fibers. Some arteries
3. Specialized Connective Tissues
Adipose: White (unilocular, Adipocytes contain a single lipid droplet
giving the white color. It is very rich in capillaries) and Brown (Mul-
tilocular fat cells. Fat is stored in multiple droplets).
Cartilage
Bone
24
Figure 5.2: Cartilage structure

5.4.1 Cartilage
Specialized CT composed of cells and abundant Extracellular Matrix. It is
the only connective tissue that do not have blood vessels or nerves.
Functions:
• Structural functions as part of the skeletal system
• Provide resistance with flexibility (nose, trachea)
• Cartilage is also the skeleton of the fetus and serve as a template for
bone development
• Shock adsorbent
• Form the smooth surface of joints
5.4.2 Bone
Specialized Connective Tissues composed of cells, and abundant calcified Ex-
tracellular Matrix (fibers + ground substance). Bone is too hard to be pro-
cessed with conventional histology techniques. It can be studied by grinding,
making a thin translucent slide or by decalifying (EDTA or acid solutions)
and then using conventional histology processing.
Types:
25

• Travecular bone: Because the travecular bone is formed by a network
of thin lamina and spikes creating a honeycomb structure. The empty
spaces are filled with blood vessels, CT (conective tissue) and bone
marrow.
• Dense bone: Formed by a very organized system containing blood ves-
sels inside of the bone. The unit is the Osteon which contain a vascular
canal (Harvesian Canal) surrounded by concentric layers of bone.
Functions:
• Structural functions as part of the skeletal system
• Protection of the vital organ of the CNS
• Form the rib cage that allows Respiration
• It is the Ca++ reservoir of the body

Cell types:
• Osteocyte: They are the cells that form the bone. They form mono-
layers in the outside of bone masses
• Osteoclast: The function is to reabsorb bone.
5.5 Connective tissue regeneration

5.5.1 Mesenchymal stem cells (MSC)
MSCs are heterogeneous cell populations; to induce bone regeneration effec-
tively and reproducibly, it is important to understand the mechanisms by
which growth factors or cytokines regulate osteoblast differentiation. Cells
with self-renewal capacity
Important properties: Availability, capacity to differentiate into various
tissues, have immunosupresive and immunoevasive capabilities.
26

Chapter 6
Muscular Tissue
6.1 What is muscle

Muscle is a tissue formed by cells that have the capacity to contract upon a

chemical or electrical stimuli
The cells are usually very large cells and are rich of contractile fibers
(actin/myosin)
They do not have ECM except for connective tissue that held the muscle
cells together and anchor them to other organs (ex. bone).
Each cell is surrounded by a basement membrane (similar to the epithe-
lia).
Responsible for the all the movements in the body (voluntary and invol-
untary).
6.2 Anatomical organization of the muscle

The muscle is held together by connective tissue (CT):
• Endomysium: each fiber (cell) is wrapped in this CT.
• Perimysium: surround a bundled of fibers of the same type (fascicles).
• Epimysium: Surround several fascicles, providing the outer layer of the

whole muscle.
The Epimysium continues to form the tendon that joint the muscle to the
bone (through periosteum).
27

Figure 6.1: Skeletal muscle structure
6.2.1 Sarcomere
It is the contractile unit. It is formed by a highly organized arrangement of
actin, myosin and other proteins. Gives the muscle the striated appearance.
Molecular Organization of the Sarcomere

1. Thin filaments: Composed of two G Actin strands and Tropomyosin.
It also contains Troponin, a calcium binding protein. When Troponin
binds to Ca++ it changes conformation, allowing the interaction be-
tween Myosin and Actin
2. Thick filaments: ormed by tail to tail aggregation of Myosin, with the

head projecting outside. The Thick filament are attached to the Z disks
by a very large elastic protein, called Titin.
3. Z discs: It is the site of attachment of thin Filaments and Titin (indirect

attachment of thick fillaments). It is formed by α-actinin, desmin and
other proteins .
28
6.3 How Contraction Occur
Regulated by Ca++ cytosolic concentration, which is controlled by mem-
brane potential. All start with the signal from a nerve. The neuromuscular
junction connects the nerve with the muscle. Adjacent to each sarcomere
there is a structure formed by a T-tubule (transverse tubule) and 2 cys-
terns of the sarcoplasmic reticulum (SR):
• SR accumulate Ca++
• T-tubule deliver the electric impulse from the membrane.
Voltage sensitive proteins connecting T-Tubule and the SR open Ca-Gates

allowing the release of Ca++ in the proximity of the sarcomere and triggering
the Myosin/Actin interaction. Immediately after, Calcium pumps in the SR,
membrane and mitochondria pump the Ca++ out of the cytosol allowing
relaxing. Maintaining the calcium gradient is ATP dependent.

6.4 Types of muscle
6.4.1 Skeletal muscle
The basic unit of the muscle is the fiber, which is actually the muscle cell.
It is originated by fusion of myoblasts (multinucleated sincitium). The fiber
is multinucleated with nuclei located in the periphery adjacent to the plama
membrane. It has characteristic cross-striations caused by the particular
arrangement of actin and myosin (sarcomers).
Its functions are:
• Voluntary movement
• Maintain posture
• Respiratory movements (diaphragm)
6.4.2 Smooth muscle

Single nucleus, centrally located, Fusiform shape, No sarcomers (no stria-
tion), Weak, slow, involuntary contraction, Under the control of autonomous
nervous system, Can maintain tone and perform slow movements, Cell com-
municate by Gap Junctions, allowing coordinate movements
Functions in: wall of GI tract, respiratory tract (bronchi), regulate the
lumen of artery, wall of uterus, wall of bladder, etc.
29

Smooth muscle contraction
The contractile filaments are linked to the membrane in regions called fo-
cal density. There is no linear contraction: Fusiform shape (relaxed) − >
spherical shape (contracted).
They do not have SR or T-tubules (do not need fast diffusion of Ca2+,
it can be obtained from external medium, as movements are slow). They do
have thin and thick filaments.
6.4.3 Cardiac muscle

Forms the contractile mass of the heart and it contracts spontaneusly.
Striations are present but not as evident as in skeletal muscle because the
sarcomeres are not so well aligned.
Unlike the skeletal muscle that binds together by a strong CT scaffold, the
cardiac muscle helds together by strong attachment among cells (intercalated
disks: gap junctions).

Purkinje cells/fibers (modified muscle cells)
Conduction system that consists of modified cells (Purkinje) that do not
contract, but conduct electrochemical signals from the upper chambers of
the heart to the lower chambers.
6.5 Muscle: Regeneration vs Repair

They are two complete different mechanisms:
1. Regeneration: Full recovery morphologically and functionally by pro-

liferation of the same cells that were originally in the organ
2. Repair: The organ is repaired with connective tissue. No functional

regeneration. Fibrosis or scar. It is very efficient but can produce
functional problems.
6.5.1 Why Regeneration or Repair?

• Tissue/organ: Heart never regenerate, Epidermis and GI epithelium
always regenerate.
• Severity of the injury: The larger the lesion, the more likely that will
do repair instead of regenerate.
30

• Type and length of inflammation: Chronic inflammation leads to re-
pair. Acute will regenerate.
I.e Muscle: Can regenerate or repair depending of the type of muscle.
6.5.2 How does an organ increase the size??
Hypertrophy: Same amount of cells, the size of the cell increase. Hyperplasia:
Size of the cell remains the same but the number of cells increase
6.5.3 Smooth muscle

Smooth cells have the greatest capacity to regenerate of all the muscle cell
types. They retain the ability to increase in number by dividing (hyperpla-
sia). Also, they can hypertrophy. Furthermore, they can be cultured and
bioengineered in appropriate scaffolds.

6.5.4 Skeletal muscle
Skeletal muscle contains numerous “satellite cells” underneath the basal lam-
ina. They are mononucleated quiescent cells. When muscle is damaged, they
are stimulated to divide. After dividing, the cells fuse with existing mus-
cle fibres, to regenerate and repair the damaged fibers. The skeletal muscle
fibers themselves, cannot divide. However, muscle fibers can lay down new
protein and enlarge (hypertrophy).
In case of large injuries stem cells from the bone marrow can also differ-
entiate in myoblasts and contribute to muscle regeneration.
6.5.5 Cardiac muscle

This is the most important muscle from the point of view of tissue engineer-
ing. Cardiac muscle can hypertrophy, but there are no equivalent to satellite
cells found in skeletal muscle. Thus when cardiac muscle cells die, they are
not replaced. An injury of the heart (the most common is the infarction)
does not regenerate but repair.
31

Chapter 7
Blood and hematopoiesis

7.1 Blood
Tissue formed by a variety of cells suspended in a fluid media called plasma.
Its function is the transport for gases, nutrients, metabolic products, hor-
mones and cells to different parts of the body.
Blood smear in histology slides, fixed in methanol, stained with Wright’s
staining and allowed to dry (other staining: May-Grünwald Giemsa
(MGG)).
7.2 Blood components

7.2.1 Erythrocyte
Is the red blood cell.
• Biconcave cell of 7-8 m diameter. Lack nuclei and organelles

• Formed by cell membrane, cytoskeleton, large amounts of hemoglobin
(Hb) and a few enzymes
• Hb transports oxygen from the lungs to the tissues. Hb also binds CO2
and carry it from the tissues to the lungs
• Lifespan: around 120 days
• Most common cell in blood with 5x106 per mm3 of blood
• Capacity to alter the lumen of blood vessels
• Lots of pumps and enzymatic activity
32
Figure 7.1: Types of granulocytes
• Very active metabolically
• It need high amount of energy but does not have mitochondria − >
Glycolysis: degradation of glucose with formation of pyruvate and ATP
7.2.2 Leukocytes - Granulocytes

Neutrophils
Most common leukocyte (60-70%). Multi- lobulated nuclei with fine neutral
pink granulation. Life span: ¡ 1week. Function: antibacterial and micro-
phagocytosis. Grain have antibacterial enzymes as well as enzymes that
produce reactive oxygen species (ROS). Also release pro-inflammatory sub-
stances.
33

Figure 7.2: Types of agranulocytes

Eosinophils
Represent 1-6% of leukocytes. Generally bi-lobulated nuclei with visible dark
red granulation. Life span: ¡ 2 weeks. Function: fight parasites and also in-
volved in allergic reactions. Other function is to micro-phagocyte and destroy
antigen-antibody complexes.
Basophils
Low frequency, less than 1% of leukocytes. Nuclei irregular, S shaped with
large blue granulations. Life span: Years. Function: Associated with allergic
reactions and asthma (very similar to mast cells). Binds IgE and releases
histamine and other inflammatory mediators.
7.2.3 Leukocytes-Agranulocytes
Monocytes
Relatively low frequency (1-10%) but is the largest leukocyte (12-20 µm).
Kidney shaped nuclei, fine chromatin and very few or no granulation. Life
span: ¡ 1week. Function: precursor of resident macrophages in tissues and
dendritic cells. Can be rapidly recruited in areas of inflammation (less than
12 hours).
34

Figure 7.3: T cell at the right and B cell at the left

Lymphocytes
There are 20-40% lymphocytes in human blood. They have round dark nuclei
and basophilic cytoplasm. Function: critical cells of the acquired immune
system.
• T-cells (smaller with very little cytoplasm)
• B-cells (larger with more abundant cytoplasm)
Immunohistochemistry allows us to differenciate between these two
cells.
7.2.4 Platelets (thrombocytes)

Cell fragments (enucleated) about 1/5 to 1/3 the diameter of the RBC. There
are 150-300,000 platelets per l blood. Produced in bone marrow by frag-
mentation of megakaryocytes. The life span is 5-9 days. They have a cell
membrane, cytoskeletal elements and different types of granules (coagulating
factors, growth factors,...). Do not have nucleus. Function: blood clotting
and tissue repair
Coagulation cascade
• Tissue factor pathway (extrinsic): The main is to generate a “thrombin
burst”, a process by which thrombin, the most important constituent
35

36
Figure 7.4: Platelets
of the coagulation cascade in terms of its feedback activation roles, is
released very rapidly.
• Contact activation pathway (intrinsic): Begins with formation of the

primary complex on collagen by kininogen (HMWK). Has minor role
in clot formation. Instead, seems to be more involved in inflammation,
and innate immunity.
• Common pathway
7.2.5 Plasma
Liquid component of the blood. 45% of the total blood. Composed by water,
proteins, glucose and other nutrients, electrolytes, hormones and clotting
factors.
Healthy composition of the plasma is very important: Plasma moves out
the blood to the tissues forming the extracellular fluid that provide cells with

nutrients and maintain the electrolytic balance, pH and osmotic pressure. It
is also a reserve of proteins.
Blood serum: plasma without clotting factors (fibrinogen, prothrombin,
Factor VII etc).
Hematocrit: Blood treated with anticlotting factors (Heparin, EDTA)
is spinned down, separating three layers: RBC, Buffy coat (leukocytes and
platelets) and plasma.
7.3 Immune response

There are two types of immunity: innate immunity (rapid response) and
adaptive response (slow response).
Both systems included humoral immunity components and cell-mediated
immunity components:
• Humoral: is mediated by macromolecules found in extracelular fluids

such as secreted antibodies, complement proteins, and certain antimi-
crobial peptides.
• Cell-mediated: does not involve antibodies. Rather, is the activation of

phagocytes, antigen- specific cytotoxic T-lymphocytes, and the release
of various cytokines in response to antigen.
37

Figure 7.5: Hematopoiesis in humans
7.4 Bone marrow and hematopoiesis

7.4.1 Bone marrow
Primary site of hematopoiesis (hematopoietic stem cells), houses stem cells
not only for hematopoietic lineages but also for many other tissues
Located in the intertrabecular spaces of the bones and in the medullar
cavity of the long bones.
• Active bone marrow is called red marrow

• As people or animals age, part of the bone marrow is replaced with
adipose tissue, which is called Yellow Marrow
7.4.2 Regenerative medicine of Blood and Bone Mar-

row
Blood
Transfusion is only a temporary solution. Issues with compatibility (ABO-
RH systems). Problems with infectious diseases (AIDS, Hepatitis). Shortage
of donors is not as serious as in other tissues/organs. Essential for acute
situations. Chronic problems are addressed treating bone marrow.
38

Figure 7.6: Autologous transplant
Replacement of blood is being developed (it is made mainly of soluble

proteins that can carry O2 )
Bone marrow transplant surgery

Cancerous conditions: leukemia, lymphoma, multiple myeloma, myelodys-
plasia. Permanent or long term solution. Autologous or allogenic.
Stem cell therapy

Types: Syngeneic, allogeneic, autologous
Sources: Bone marrow, adipose tissue, blood (apheresis), umbilical cord
blood (after birth), amniotic stem cells are a topic of active research.
39

40
Figure 7.7: Allogenic transplant
Chapter 8
Circulatory system
Composed by the heart and the blood vessels (arteries, veins and capillaries).
The circulatory system is formed by several tissues: Endothelium (special
epithelium), Loose Connective Tissues, Elastic Connective Tissue, Smooth

muscle, Cardiac Muscle and Mesothelium (special epithelium).
8.1 Heart
The heart is covered by three wall layers: epicardium, myocardium and en-
docardium (from outside to inside).
8.1.1 Endocardium
The endocardium is the innermost layer of tissue that lines the chambers
of the heart. Provides protection to the valves and heart chambers. Its
cells are embryologically, morphologically and functionally very similar to
the endothelial cells that line blood vessels. The cardiac endothelium rest on
a thin layer of CT and BM.
8.1.2 Myocardium
Thickest component of the heart wall (cardiac muscle). Rich in capillaries
(BV) coming from the coronary arteries of the pericardium. It has a a fine
collagen network (electrical isolation), that in the atria is more prominent
and separate some of the fibers in bundles.
41

8.1.3 Epicardium
External layer of the heart. Formed by CT and adipose tissue covered by spe-
cialized epithelium (mesothelium). It carries important arteries and nerves.
Forms the visceral layer of the pericardium.
8.1.4 Mesothelium
It is a squamous epithelium covering the peritoneum, pleura and pericardium
(serous membranes). Unlike the endothelium the mesothelium has keratin
intermediate filaments. The luminal surface is covered with microvilli. The
function is to provide a frictionless surface by producing a lubricated smooth
surface.
8.1.5 Heart valves

Formed by CT and muscle and covered by endothelium. Attached to the

heart fibrous skeleton. They have different size (the AV valves being larger)
and shape (bicuspid, tricuspid, mitral).
The AV valves are attached to the wall of the ventricles by a group of
ligaments and muscles. Function: prevent prolapse of the valves rather
than perform the actual closing. The aortic and pulmonary valves do not
have subvalvular structure.
• Atrioventricular Valves: Mitral (left atrioventricular passage), Tricus-

pid (right atrioventricular passage)
• Semilunar Valves: Pulmonary Valve, Aortic Valve
8.1.6 Diastole-Systole
• Diastole is the period of time when the heart relaxes after contraction
• Systole is the contraction of heart chambers, driving blood out of the

chambers.
8.1.7 Serosa vs. Adventitia

External layers of an organ. Main difference: presence of mesothelium in the
serosa (absent in the adventitia).
42

Figure 8.1: Types of arteries
• Serosa allows independent movement of organs. The smooth surface

of the mesothelium and its secretion of a serous lubricant allows that
“freedom”. E.g. heart, gastrointestinal tract’s organs, lungs...
• The adventitia anchors the organ to nearby structures (does not allow
movement). Composed by CT. E.g. blood vessels, organs in pelvic
area and some portions of the esophagus.

8.2 Blood vessels
8.2.1 Wall of blood vessels
• Intima: endothelium, BM and variable amounts of CT.
• Media: elastic tissue and/or muscle (depending of the blood vessel)
• Adventitia: CT that connects (anchor) the blood vessels to adjacent

organs.
8.2.2 Arteries
Thick walls (stand high blood pressure) and plenty of elastic tissue. Thick-
ness and elastic tissue content decrease when the pressure decreases (further
away from the heart). Smaller lumen.
Elastic artery
The tunica media constitutes the major portion of the vessel wall: multiple
elastic lamellae alterned with thin layers of circularly oriented smooth muscle.
Tunica intima (endothelium) is almost attached to the elastic tissue. Tunica
adventitia is mainly loose CT and blood vessels (vasa vasorum).
43

Important role in absorbing the impact of the heart beat (pressure pulses
in systole).
Muscular arteries
% elastic fibers decreases and % smooth muscle increases (abundant in t.
media).
Internal elastic lamina (between media and intima) and a less well defined
external elastic lamina.
Tunica adventitia grades off into the surrounding connective tissue, but

is well defined. Often, most of the wall’s overall thickness.
Arterioles
The arterioles are the smallest artery. Thin muscular walls (usually 1-2 layers
of smooth muscle) and are the primary site of vascular resistance.
Protruding nuclei of the endothelium into the lumen, result of the con-
traction of elastic tissue (arteriole vs venula).
8.2.3 Veins
Low remaining pressure (don’t need thick walls). Offer low resistance and
therefore their lumen is large. Have valves to avoid blood reflux.
8.2.4 Capillaries
Smallest vessels. Single layer (endothelium) for interchanging gasses and
nutrients.
Fenestrated (visceral)
Have pores in their walls, are located wherever immediate movement of mate-
rials is a functional necessity (e.g. endocrine organs, kidney and small intes-
tine). There’s no ”transcytosis” activity, because bulk movement molecules
through the pores is easy. They have diaphragm in fenestrae.
Sinusoidal (discontinuous)
Have a wider lumen, multiple fenestra but also have a discontinuous BM
and adjacent cells do not overlap perfectly but leaves open spaces (e.g. liver,
spleen, lymph nodes).
44
Figure 8.2: Comparison between an artery and a vein

Capillary bed
Network of capillaries supplying an organ. The more metabolically active a
tissue, the more capillaries it will require to supply nutrients.
Pericytes
Cells from the vascular smooth muscle lineage. They apparently conserve
some of the contractile properties of muscle and are embedded in the en-
dothelial BM. They extend along and encircle the endothelial tube. Also,
make focal contacts with them through specialized junctions. Pericytes may
contribute to the mechanical stability of the microvessels:
• by matrix deposition
• by the release, presentation and activation of signals that promote en-
dothelial cell differentiation and quiescence. They may integrate cer-
tain endothelial function of neighboring endothelial cells (Muscle TE
and regenerative medicine).
8.2.5 Venules
They came immediately after the capillaries. They can be easily detected
because they have a large lumen, usually irregular and a very thing wall.
Normally, one layer of smooth muscle. Generally they are paired with arte-
rioles and sometimes a nerve.
45

8.3 Lymphatic Capillaries
Lymphatic system plays a central role in detecting infection, foreign proteins
and prevent spreading of cancer cells. In the GI system, lymphatic vessels
are involved in the transport of fatty acids.
Is not closed and has no central pump. Moves slowly (low pressure due
to peristalsis, valves and skeletal muscles).
8.4 Why such progress in Tissue Engineering

of Blood vessels?
• Morphology of the blood vessel. Well defined layer of homogeneous
tissue
• All the cells of the blood vessels can be culture and manipulated

• Adequate scaffolds and conditions of cultures to maintain differentia-
tion have been developed
46

Chapter 9
Lymphatic Tissue
9.1 Introduction
The lymphatic system is formed by the lymphatic vessels and lymphoid or-

gans (primarily populated by lymphocytes: lymph nodes, thymus, spleen,
etc.). There is a major overlapping between the immune and the lymphatic
systems (but not synonyms).
• Lymphatic system do not include the bone marrow
• Lymphatic tissue has functions which are not related to immunology
9.2 Functions
9.2.1 Fluid balance: Formation of Lymph
Lymphatic vessels
Vessels of lymphatic system are thinner and more permeable than capillaries,
and have valves formed by the same squamous cells, which prevent the back-
flow of fluid
Location of lymphatic vessels

They are in the whole body except CNS, epithelia and cartilage, bone marrow
and teeth.
They grow into larger vessels and eventually converge into two large ves-
sels: Thoracic Duct and the Righ Lymphatic Duct that drain into the
venous system (subclavian veins).
47

The lymph is filtered by the lymph nodes which remove and create im-
munodefenses against any foreign element.
Lymphatic Ducts
As lymph capillaries merge they form larger vessels called lymphatic ducts.
The lymphatic ducts are larger in diameter: have a poorly developed muscle
layer and adventitia.
9.2.2 Absorption of fat

Fat is absorbed by the lacteal (modified Lymphatic capillaries located in the
villi of the small intestine) and mix with the lymph forming the chyle, a
milk-like liquid. The chyle is drained into the venous system through the
thoracic duct.
9.2.3 Inmunity
Lymphatic system plays a central role in detecting infection, foreign proteins
and prevent spreading of cancer cells. Two cells of the lymphoid organs play
a huge role in the immune response: (1) the T-cells which are in charge of the
regulatory function and the cytotoxic function (CTLs) (Cellular Immunity),
(2) the B-cells (plasma cells) which secrete antibodies (Humoral Immunity),
and (3) macrophages and other antigen presenting cells (APC).
9.3 Organs of the lymphatic system

9.3.1 Lymph nodes
The lymph nodes are like centinels, involved in infectious diseases and cancer
(it is the first barrier before metastasis).
9.3.2 Spleen
The functions of the spleen are:
• Filter blood (similar to lymph nodes with lymph)
• Destroy old red blood cells
• Blood and platelet storage
48
• Hematopoyesis in the fetus (in adults there is only production of lym-
phocytes)
We can find inside the spleen two kinds of tissue
• Red pulp: Mechanical filtration of RBC.
• White pulp: Active immune response through humoral and cell- medi-
ated pathways.
9.3.3 Thymus
Is the central lymphoid organ, Divided in two lobes (each one by multiple
lobules divided by loose CT).
Cortex

Very rich in lymphoid cells. Take place earliest events in thymocyte develop-
ment, where T cell receptor gene rearrangement and positive selection takes
place.
Medulla
Epithelium reticulum more prominent and less lymphoid cells (in human con-
tains Hasall’s corpuscules that represent dead epithelial cells). Take place lat-
ter events in thymocyte development: negative selection where lymphocytes
that recognize self-antigens undergo apoptosis.
9.3.4 Malt (mucose associated lymphoid tissue)

Small accumulation of lymphoid tissue (Lymphocytes T and B, macrophages,
plasma cells) located in the GI, respiratory tract, thyroid, breast, etc.
Its function is related to mucosal immunity (similar role to lymph nodes).
The most prominent MALT structures are:
• Tonsils
• Adenoids
• Peyer Patches (intestine)
• Appendix
• Diffuse lymphoid structures in many organs
49

9.4 Tissue Engineering of Lymphatic tissue
The applications are disease modelling, wound healing and therapeutic ap-
plications.
9.5 Immune system diseases
Inmune diseases are being treated with a different approach: Bone marrow
transplant The rationale for this approach is:
• The precursors of all the immunocompetent cells are in the bone mar-
row
• Immune cells in lymphatic system are under continuous renovation.
Therefore, correcting any immunodefficiency in the precursor cells of the

bone marrow will result on the recovery of immune function.
50

Chapter 10
Nervous System
10.1 Central nervous system

The CNS is encased in bone structures (skull and vertebra) because CNS

is not able regenerate and neurons are super fragile. It is also protected
by meninges that act as a cushion between the bone and the fragile neural
structures (dura mater, arachnoid and pia mater). Finally the CNS has a
system of cavities filled with fluid which also serve as additional protection.
10.1.1 Ventricular System

A system of connected cavities in the CNS filled with CSF (cerebrospinal
fluid) which is produced in the Choroid plexus in lateral ventricles. Its func-
tion is the mechanical protection and detoxification. CSF fluid is essentially
a plasm filtration. It moves to the different compartments and eventually
is re-absorbed by villi cells into the vein system. Major compartments are:
lateral ventricles, third and forth ventricule, cerebral aqueduct...
10.1.2 Brain
The brain is divided in gray and white matter (differ in composition). Grey
matter contains the cell bodies, dendrites and axon terminals of neurons, so
it is where all synapses take place. White matter is made of axons connecting
different parts of grey matter to each other.
10.1.3 Cells in the Nervous system

The nervous tissue is composed by neurons (conducting cells) and supporting
cells.
51

Figure 10.1: Types of neurons
• Neurons: Cells specialized to receive and conduct electrical impulses.

Are arranged as communication network. They form both the CNS
and PNS.
• Supporting cells: Non-conducting. Physical contact with neuron. Pro-

vide physical support. Provide electric isolation. Provide nutrition and
metabolic support
Anatomy of the Neuron

Neurons are very large cells with large nucleus and prominent nucleolus.
They have long processes (extension of cytoplasm): axon and dendrites.
There is only one axon per neuron. It conduct signal from the body to
the periphery. Dendrites may be multiple. They receive stimuli and bring
them to the body of the neurons.
Types of neurons
Axons and Dendrites
• Extensions of the cytoplasm Surrounded by cell membrane
• Major structural component: microtubules (also microfilaments)
52
Figure 10.2: Synapsis
• They have mitochondria
• The only major difference is the direction of conduction.
Synapsis
Structure that permits a neuron to pass an electrical or chemical signal to
another neuron or to the target effector cell. Axons may have myelin or not.
Glia of the CNS

The CNS does not have CT to provide structure and nutrition...so, glial cells
take many of the function of CT in other organs. 90% of the cells of the CNS
are glial and only 10% are neurons.
CNS Glial Cells:
• Astrocytes: Star shaped, small nucleus, most common glia of the CNS.
Very rich in dendritic extension of the cytoplasm. Functions: Struc-
tural, Glycogen reserve buffer and metabolic support, Transmitter up-
take and release and modulation of synaptic, Regulation of ion in the
53

extracellular space, Vasomodulation (of blood flow in the brain), Pro-
motion of the myelination activity of oligodendrocytes, Nervous system
repair: Cognitive functions?
• Oligodendrocytes: Type of neuroglia whose main functions are to pro-
vide support and insulation to axons in the CNS (equivalent to the
function performed by Schwann cells in the PNS). Create myelin sheath
(each forms one segment of myelin for several adjacent axons).
• Microglia: Act as the first and main form of active immune defense
in the CNS. Microglia are key cells in overall brain maintenance—they
are constantly scavenging the CNS for plaques, damaged or unnecessary
neurons and synapses and infectious agents. Microglia are extremely
sensitive to even small pathological changes in the CNS.
• Ependymal cells: The ependyma is made up of ependymal cells called
ependymocytes, a type of glial cell. These cells line the CSF-filled
ventricles in the brain and the central canal of the spinal cord. These

are nervous tissue cells with a ciliated simple columnar shape, much like
that of some mucosal epithelial cells. Have key role in CSF production
and have cilia and microvilli on their surfaces to circulate and absorb
CSF.
10.2 Peripheral Nervous System

10.2.1 Somatic Nervous System (voluntary nervous sys-
tem)
Voluntary control of body movements via skeletal muscles: (1)Sensory nerves
(afferent): carry messages to the CNS and (2) Motor (efferent) nerves: carry
messages from the CNS to the skeletal muscles of the body.
10.2.2 Autonomic Nervous System

Carries messages between the CNS and the internal organs. Acts largely
unconsciously and regulates body functions such as the heart rate, digestion,
respiratory rate, pupillary response, urination, and sexual arousal.
10.2.3 PNS - Nerves

A nerve is an enclosed, cable-like bundle of axons. Axon are wrapped by
Schwann Cells which are the equivalent of oligodendrocytes. Each peripheral
54

Figure 10.3: Myelinated and unmyelinated nerve conduction

nerve is covered externally by: the epineurium (CT), perineurium (layer of
flat cells forming a complete sleeve) and endoneurium (surrounding each
fiber). Peripheral nerves are richly supplied with blood.
Myelinated and Unmyelinated Nerves
10.2.4 Satellite glial cells

Cover the surface of nerve cell bodies in sensory, sympathetic, and parasym-
pathetic ganglia.
Its functions are:
• Control over the microenvironment of sympathetic ganglia
• Supply nutrients to the surrounding neurons and also have some struc-
tural function (similar to astrocytes)
• Act as protective, cushioning cells
10.2.5 Ganglia
Tissue mass composed mainly of neuron bodies and dendrites. Neurons are
surrounded by satellite cells. Provide relay points and intermediary connec-
tions between different neurological structures in the body (PNS and CNS).
In PNS, there are two major groups of ganglia:
55

• Dorsal root ganglia (spinal ganglia): contains the cell bodies of sensory
(afferent) nerves
• Autonomic ganglia (sympathetic and parasympathetic): contains the

cell body of autonomic nerves.
10.3 Nervous Tissue Regeneration

At the CNS there are no regeneration. Some neuron regeneration has been
shown in very discrete areas of the brain (exception to the rule).

At the PNS ganglia neurons do not regenerate. Periferal nerves regenerate
(unless the trauma is very extensive and tissues are very damaged).
10.4 What are the medical needs?

CNS: Neuronal degeneration by trauma, cardio-vascular accident, degenera-
tive diseases (E.g. Alzheimer). Spinal cord trauma: common in car accident
(as axons do not regenerate in the CNS, it disconnects the brain from the
PNS...)
PNS: Lack of regeneration of nerves due to the extent of the damage
10.5 Tissue Engineering of the CNS

Some current approaches:
• Implantation of stem cells in the injury site
• Bone marrow transplantation (precursors for other lineages and stromal

cells).
• Pharmacological approach (paliative or attempts to cure) (systemic or

in situ): morphogens, growth factors, antiinhibitors of myelinization
(in situ)
• Implantation of neural tissue developed ex vitro. Multiple scaffolds

have been developed (support neuronal growth and differentiation)
56
Chapter 11
Cell Culture
11.1 2-D Cell culture

11.1.1 Growth curve

• Lag/latency phase: substratum fixation
• Exponential phase: cell division
• Confluence (% of space occupied by cells): saturation of cells because

of (1) the lack of culture medium or because of (2) contact inhibition.
• Senescence: death
11.1.2 Primary culture

1. Biopsy
2. Tissue isolation
3. Dissecation/disaggregation
4. Cell culture in a flask
11.1.3 Cell subculture

When the culture has high confluence we ressed a primary culture to increase
the amount of cells and to avoid that culture medium completely depletes.
1.
57

Figure 11.1: Growth curve
11.1.4 Cell lines

Obtained by subculturing and later culture selection or from tumoral tissues.
• Adherent: HeLa (epithelial cells)
• Suspension: Jurkat (linphocytes)
• Mirar: BAE-1 (endothelial), Hep-2 (fibroblasts)
11.2 3-D cell culture

Complex interactions with matrix (signaling)
11.2.1 Types
• Spheroids (Scaffold-free platforms): used in cancer and stem cells re-
search
• Scaffolds or 3D matrices: different porosities, permeabilities and me-

chanical characteristics
58

Figure 11.2: Spheroids
2D culture 3D culture
Develops one type of cell More than one cell type
Does not mimic real tissue interactions (lack of 3rd dimension Has the 3rd dimension
Not similar to real organ structure More similar to a real organ
Induces forced polarization of cells Better aproximation to real organ responses
Table 11.1: 2D vs 3D
• Gels/hydrogels: soft tissue-like stiffness and aim to mimic the ECM.
• Skin equivalents: supported on a fibrin-based hydrogel.

11.3 Applications
• Cancer
Growing Cells in 3D alters proliferation and cell morphology
Growing cells in 3D reveals a more realistic drug response.
Growing cells in 3D captures phenotypic heterogeneity.
Growing cells in 3D changes gene expression and cell behavior.
Growing cells in 3D mimics the tumor microenvironment.
• Stem cells
Generating Embryoid Bodies (EB): 3D aggregates of Pluripotent
Stem Cells(PSCs) that resemble an embryo, meaning they contain cells
from all 3 germ layers (ectoderm, endoderm, and mesoderm). Method:
Inhibiting PSCs from sticking to a surface and promoting E-cadherin
dependent adherence to each other.
• Bioprinters: An alternative for getting 3D culture more homogeneous

or nearer to reality.
3-D human skin
59

3-D human bone: The printer’s “ink” consists of a polymer called
polylactic acid and a gel-like substance called alginate. The polylactic
acid provides the hard, mechanical strength of bone, while the alginate
acts as a cushioning material for the cells. The printed product can be
implanted in the body, where the scaffold will degrade and be replaced
by new bone.
11.4 Biorreactors

Device that attempts to simulate a physiological environment in order to
promote ex vivo (in vitro) cell or tissue growth. Control and manipulation
of the culture environment is as important as cells and scaffold issues.
BRs must provide a biochemical environment which is controlled by nu-
trient transfer and a biomechanical environment.
11.4.1 Types
• Static: Unenforced power input. Not homogeneous distribution of nu-
trients.
Membrane flask bioreactor: Separate the cell cultivation area from
the media chamber. Upper membrane allows continuous nutrient dif-
fusion and waste elimination while lower membrane provides direct
oxygenation and gas exchange.
• Dynamic: move the medium (Driven by External Elements or driven

by Internal Elements). Uniformly distribution of nutrients
Bubble column bioreactor: Composed of a cylindrical vessel fitted
with a gas sparger.
11.4.2 Engineering Parameters in Bioreactor Design

• Mixing: Needed to mix nutrients and to keep the medium homoge-
neous. Also helps oxygenation.
High power consumption
Damage to cells by excessive speed
• Heat production: They can be refrigerated with an external jacket

or, for very large vessels, with internal coils to maintain a constant
temperature.
60
Figure 11.3: Membrane flask bioreactor

• Scale up
Problems: Mixing times, oxygen transfer, heat transfer, power in-
put, aeration rates, product quality, foaming, by-product formation.
11.4.3 Functions
• Cell seeding of 3D scaffolds
Static seeding: Inhomogeneous since gravity may not suffice for the
cells to penetrate throughout the scaffold pores
Dynamic seeding: perfusion seeding (perfusion of a cell suspension
through the pores of a 3D scaffold)
• Maintance of a controlled culture enviroment
• Physical conditioning of cells/scaffold constructs

Physical forces: hydrodynamic/hydrostatic (shear stress, differen-
tial pressure), mechanical (tension, compression), and electrical
Introduction of liquids and gases, and removal of wastes
61

62
Figure 11.4: Bubble column bioreactor

Chapter 12
Stem Cells I
Stem cells are unspecialized cells that give rise to more than 250 special-
ized cells in the body. They serve as the body’s repair system, replicating
themselves over and over again.

12.1 History
12.2 Types of Stem Cells
• Early stem cells
Totipotent stem cells: Each cell can develop into a new individual
(1-3 days embryos)
Pluripotent(embryonic)stem cells: potential to become all the adult
specialized cell types (embryos)
• Mature stem cells

Multipotent(determined)stemcells: can produce some, but not all,
of the specialized cell types (Undifferentiated cells in fetal, neonatal,
and adult organisms)
12.3 Sources
• Embryonic stem cells: Blastocyst, in vitro fertilization, therapeutic
clonning (somatic cell nuclear transfer: Transfer of nucleus from a dif-
ferentiated cell into an enucleated egg)
63

Figure 12.1: Advantages and disadvantages
• Adult stem cells: Fetus (amniotic fluid, chorionic villi, placenta, umbil-
ical cord) and adult (bone marrow, fat, peripheral blood, hair follicles,
gastrointestinal, placenta, skeletal muscle, brain, cord blood...)
12.3.1 Amniotic fluid-derived stem (AFS) cells

• Isolated from amniotic fluid and cytogenetics lab cultures by immunos-
election for c-Kit (CD117).
• Extensive culture without apparent senescence
• Normal karyotype, long telomeres
• Non-tumorigenic (in mice)
12.3.2 Umbilical cord stem cells

• Isolated prior to/immediately following birth
• Hematopoietic stem cells (Majority)
64
Figure 12.2: Cord blood vs Bone Marrow

• Transplanted UCSC provide benefit in cardiac function recovery after
acute myocardial infarction in rats
Limitations of Cord Blood Stem Cells

• Limitations of cord blood stem cells for allogeneic transplantation
Delayed platelet engraftment
Delayed neutrophil engraftment
• Limitations of cord blood as a stem cell source

Limited amount of source material
Same donor not available for repeated dose
• Potential unknown genetic mutations could result in recurrence of dis-

ease in cases of auto-transplant
12.4 Limitations to clinical use of ES cells

• Tumorigenicity
Undifferentiated ES cells injected ectopically (i.e. not in uterus)
cause tumors at high rates
65

Lineage-restricted ES cells (differentiated in culture into determined
stem cells) form tumors at reduced rates
• Lineage restriction requires multiple signals and precise timing

Hormones, extracellular matrix, cell contact
Mechanisms still poorly understood
• Separation of determined stem cells from residual ES cells must be

efficient
• Immune rejection of transplanted cells by recipient

ES-derived cells will mature to cells that express donor’s histocom-
patibility antigens
May require life-long use of immunosuppressive drugs
Need new technologies to induce immune tolerance

12.5 Induced Pluripotent Stem Cells
2006- Yamanaka’s group in Japan determines a method to push differentiated
fibroblasts backwards in time to make ESC-like cells termed induced pluripo-
tent stem cells by expression of 4 transcription factors. Original transcription
factors used included Oct4, Sox2, Fbx15 (Klf4), and c-Myc.
Advantages:
• Generate disease-specific iPS to study the etiology of disease
• Generate patient-specific iPS cells as potential therapy (immune matched)
• Potentially circumvents ethical issues of human ESC research
12.6 Organ Transplantation and Cell Thera-

pie
In conventional organ transplantation there is one donor and one recipient,
while in stem cell therapy cells are cultured and serve as tissue for many
recipients.
66

12.6.1 Cell Therapy
Cell therapy describes the process of introducing new cells into a tissue in
order to treat a disease (promoting healing and regeneration, and/or devel-
oping external bioartificial organs that will buy time for the body to repair
itself). There are many potential forms of cell therapy:
• The xenotransplantation of non-human cells that are used to produce

a needed substance.
• The transplantation of mature, functional cells.
• The transplantation of stem cells that are autologous (from the patient)
or allogeneic (from another donor).
• The application of modified human cells that are used to produce a

needed substance.

• Increasingly, mesenchymal stem cells are being proposed as agents for
cell-based therapies, due to their plasticity, established isolation proce-
dures, and capacity for ex vivo expansion.
Established therapies based on determined stem cells

• Hematopoieticstemcell transplantation
Restores blood-forming cells in cancer patients or individuals lack-
ing immune system cells
Stem cells obtained from bone marrow, peripheral blood, or umbil-
ical cord blood
Self or matched donor
• Tissue-engineered skin: used to treat serious skin diseases
• Bone and cartilage repair
• Mesenchymal Stem Cell (MSC) transplantation for cardiac, bone, car-

tilage, GVHD, ulcers, acute and chronic organ failure repair, etc.
• ACT Stargardt’s Macular Dystrophy and Dry Age-Related Macular

Degeneration
67

12.7 Conclusions
• Unlimited source of stem cells from biological waste
• Collection of cord stem cells is painless and amniocentesis is a routine

procedure
• Collection of cord stem cells is risk free to mother and baby
• Fetal determined stem cells have a greater ability to differentiate into

other cell types (more plasticity)

• These cells have longer growth potential (longer telomeres) and have
been shown to have a greater rate of engraftment
• Cord blood stem cells are much more tolerant to HLA tissue mismatch-
ing than bone marrow therefore leading to lower rate of GVHD
• Fetal determined stem cells are not exposed to the toxins and radiations
(less potential of malignant mutations)
• Cord blood stem cells are being used in the treatment of 40 medical
conditions with over 72 potential disease targets
68
Chapter 13
Stem Cells III
13.1 Stem Cell Niche

Stem cell niche refers to the microenvironment where stem cells are found,

which interacts with stem cells to regulate cell fate. They are located in
hypoxic areas.
It can refer to the in vivo or in vitro stem cell microenvironment.
13.2 Hematopoietic Stem Cells (HSC)

13.2.1 Conditioning Regimens
• Myeloablative
The bone marrow is ablated (destroyed) with high dose-levels that
cause minimal injury to other tissues. This treatment also causes an
immunosuppressive effect that prevents rejection of the HSC by the pa-
tient’s immune system. The post-transplant prognosis often includes
acute and chronic graft-versus-host disease (GVHD) that may be life-
threatening. However, in certain leukemias this can coincide with pro-
tection against cancer relapse owing to the graft- versus- tumor-effect
(GVTE).
• Non-Myeloablative
Also termed reduced-intensity conditioning (RIC), uses doses of
chemotherapy and radiation too low to eradicate all the bone marrow
cells of the recipient. Instead, non-myeloablative transplants run lower
risks of serious infections and transplant- related mortality while relying
upon the GVTE to resist the inherent increased risk of cancer relapse.
69

Figure 13.1: Stem Cell Niches (also Hematopoietic Stem Cell Niche, not
included)
70

13.2.2 Complications After HSC Transplantation
• Infection -¿ Sepsis
• Graft-versus-host-disease (GVHD)
• Veno-occlusive Disorders
• Mucositis
13.2.3 HSC Transplant Matching

• The HLA genes fall in two categories (Type I and Type II). In gen-
eral, mismatches of the Type-I genes (i.e. HLA-A, HLA-B, or HLA-C)
increase the risk of graft rejection.
• A mismatch of an HLA Type II gene (i.e. HLA-DR, or HLA-DQB1)

increases the risk of graft-versus-host disease.

• Race and ethnicity are known to play a major role in donor recruitment
drives, as members of the same ethnic group are more likely to have
matching genes, including the genes for HLA.
71


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Fundamentos de ingeniería de tejidos y medicina

Escuela Politécnica Superior

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Bachelor’s Degree in Biomedical Engineering

Fundamental of tissue engineering and regenerative medicine

David Crespo Acero

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3 Histologic techniques, microscope and virtual microscope 12

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13 Stem Cells III 69

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4.1 Types of epithelium . . . . . . . . . . . . . . . . . . . . . . . . 16

5.1 Connective tissue elements . . . . . . . . . . . . . . . . . . . . 22

6.1 Skeletal muscle structure . . . . . . . . . . . . . . . . . . . . . 28

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8.1 Types of arteries . . . . . . . . . . . . . . . . . . . . . . . . . 43

10.1 Types of neurons . . . . . . . . . . . . . . . . . . . . . . . . . 52

11.1 Growth curve . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

12.1 Advantages and disadvantages . . . . . . . . . . . . . . . . . . 64

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1.1 Tissue Engineering(TE): Classical Defini-

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1.2 Tissue Engineering(TE): General Paradigm

1.3 Regenerative medicine (RM)

• By stimulating organs to heal themselves (In vivo).

• By growing tissues and organs in the laboratory and safely implant

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1.3.2 Research areas

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• Biomolecules: Including angiogenic factors, growth factors, differen-

• Engineering Design Aspects: including 2D cell expansion, 3-D tis-

• Biomechanical Aspects of Design: Including properties of native

Organization of cells into

2.1 Levels of Organizaon

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2.2 Why cells differentiate in different size,

2.3 Extracellular matrix (Scaffold)

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2.5 Cell lineages plasticity

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3.1 Histology techniques

3.2 Histology techniques major steps

2. Dehydration: To embed the tissue in paraffin (hydrophobic). Per-

3. Embedding: Embedding the tissue in paraffin at 60o C to give texture

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3.3 Egg paradox

3.4 Virtual Microscope

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