Biomaterials /certified fixed orthodontic courses by Indian dental academy

THE EXTRACELULAR
MATRIX,
CELL-BIOMATERIAL
INTERACTIONS
INDIAN DENTAL ACADEMY
Leader in continuing dental education
www.indiandentalacademy.com

1.

TISSUE RESPONSE TO INJURY
A. Inflammation and Repair
B. Cell Regenerative Capacity
C. Extracellular Matrix Remodeling

2. CELL/TISSUE-BIOMATERIALS INTERACTIONS
3. TECHNIQUES FOR ANALYSIS OF CELLS AND TISSUES
A. Light Microscopy

a.
b.
c.
d.
e.
f.

B.
C.
D.
E.

Tissue Sample
Fixation
Dehydration&Embedding
Sectioning
Staining
Special Staining

Electron Microscopy
Three-Dlmenslonal Interpretation
Artifacts
Artifacts Identification, Genotyping, and Functional Assessment
of Cells, Including Synthetic Products,In Cells or Tissue
Sections

1.A.Inflammation and Repair

Inflammation and repair follow cell and tissue injury induced by
various exogenous and endogenous stimuli.
Inflammation is a protective response that eliminates (i.e.,
dilutes, destroys, or isolates) the cause of the injury (e.g.,
microbes or toxins) and disposes of both the necrotic cells
and tissues that occur as a result of the injury.


炎滲液

Pathways of reparative responses after acute inflammatory injury .

The inflammatory response initiates the process that heals
and reconstitutes the normal tissue.
During the reparative phase, the injured tissue is
replaced by native parenchymal cells, or by filling up the
defect with fibroblastic scar tissue, or both.
The outcome depends:
Restoration of normal structure and function
(1) tissue injury is transient or short-lived
(2) tissue destruction is small
(3) the tissue is capable of regeneration
Scarring results
the injury is extensive or occurs in tissues that
do not regenerate.

( A ) Initial contact of
cell with solid substrate .
( B ) Formation of
bonds between cell
surface receptors
and cell adhesion
ligands .
( C ) Cytoskeletal
reorganization with
progressive spreading of the
cell on the substrate for
increased attachment
strength .

An abscess 膿腫 is the outcome when an infection cannot be
eliminated, the body "controls" the infection by creating a wall
around it.
Inflammation processes:
@ Acute inflammation
@ Chronic inflammation
@ Scarring

@ Acute inflammation
The immediate and early response to injury, of
relatively short duration, is characterized by fluid and
plasma protein exudation into the tissue, and by
accumulation of neutrophils.

@ Chronic inflammation
•

•

•

This phase is manifested with concurrent tissue
destruction, and can evolve into repair involving
fibrosis and new blood vessel proliferation.
A special type of inflammation characterized by
activated macrophages and often multinucleated giant
cells is called a granuloma.
The pattern occurs where the inciting agent is not
removable, including the foreign body reaction, a
characteristic inflammatory reaction to the implantation
of a biomaterial.

@ Scarring
Scarring occurs as a composite of three sequential
processes:
(1) formation of new blood vessels (angiogenesis )
(2) deposition of collagen (fibrosis)
(3) maturation and remodeling of the scar (remodeling)
The early healing tissue rich in new capillaries and
proliferation of fibroblasts is called granulation tissue.

Inflammation is also associated with the
release of chemical mediators from plasma,
cells, or extracellular matrix, which regulate
the subsequent vascular and cellular events
and may modify their evolution.


The chemical mediators of inflammation:
vasoactive amines ( 血管活性胺 )
arachidonic acid metabolites ( 花生四烯酸代謝產物 )
源
自
細
胞

in the cyclooxygenase( 環加氧黴 ) pathway
( the prostaglandins, PG 前列腺素
and the lipoxygenase( 脂質加氧黴 ) pathway
( the leukotrienes, LT 白細胞三烯
platelet-activating factor
lysosomal granules of inflammatory cells

源
自
血
漿

nitric oxide

Polypeptide growth factors cytokines

plasma proteases ( of the coagulation 凝血 , fibrinolytic 纖溶
, kinin 激汰 , and complement 補體四個系統 )

Growth factors may act by endocrine (systemic), paracrine
(stimulating adjacent cells) or autocrine (same cell carrying
receptors for their own endogenously produced factors)
mechanisms.

Endocrine 釋放出的 factor 經由血液運送到身體其他較遠的細胞接收

Paracrine 釋放出的 factor 經由擴散由周圍不同類型細胞的 receptor 接收

Autocrine 釋放出的 factor 由同一細胞或周圍同種細胞的 receptor 接收


1.B.Cell Regenerative Capacity
Most types of cell populations can undergo turnover, but
the process is highly regulated, Rates of proliferation are
different among various cell populations and are frequently
divided into three categories:
(1) renewing (also called labile)
cells have continuous turnover, with proliferation
balancing cell loss that accrues by death or physiological
depletion;
(2) expanding (also called stable)
cells, normally having a low rate of death and replication,
retain the capacity to divide following stimulation;
(3) static (also called permanent)
cells not only have no normal proliferation, but have lost
their capacity to divide.

1.C.Extracellular Matrix Remodeling
The maintenance of the extracellular matrix requires
constant collagen remodeling, itself dependent on
continued collagen synthesis and collagen
catabolism.
Turnover of the extracellular matrix is a unique
biological problem because of the high collagen
content of most extracellular matrix structures and the
resistance of these triple helical molecules to the
action of most proteases.

Connective tissue remodeling, is a highly organized process
that involves the selective action of a group of related
proteases that collectively can degrade most components of
the extracellular matrix.
These proteases are known as the matrix metalloproteinases
(MMPs 間質金屬結合蛋白脢 ). Subclasses include the
interstitial collagenases(MMP-1,MMP-13), stromelysins(MMP3,MMP-10), and gelatinases(MMP-2).
MMP 家族是一群含有鋅和鈣離子依賴性的蛋白酶，它們能分解大多數的 ECM ，
人類約有二十三種 MMP ，如膠原蛋白酶（ collagenases) ， MMP-l 和 MMP13 等屬此群，此群主要是切開第一、三和四型 collagen ；另一個家族為
Gelat inases (MMP-2 和 MMP -9) ，此族成員可裂解 laminin 、 f ibr onect in 和
第四型 collagen ，而第四型 collagen 為基底膜最重要的內容物；
St r omelysills ，如 MMP-3 和 MMP-10 等屬此家族 .


Enzymes that degrade collagen are synthesized by
macrophages, fibroblasts, and epithelial cells.
Collagenases are specific for particular types of collagens,
and many cells contain two or more different such enzymes.
Particularly important in tissue remodeling are myofibroblasts,
a particular phenotype of cells that show both features of
smooth muscle cells (contractile proteins such as a-actin) and
features of fibroblasts (rough endoplasmic reticulum in which
proteins are synthesized).

These cells may also be responsible for the production of
tissue forces during remodeling, thereby regulating the
evolution of tissue structure according to mechanical
requirements.
Evidence suggests that growth factors and hormones
(autocrme, paracrine, and endocrine) are pivotal in
orchestrating both synthesis and degradation of ECM
components.


Cytokines such as TGF-,8, PDGF, and IL-1 clearly play an
important role in the modulation of collagenase and TIMP
expression.
MMP enzymatic activities are regulated by tissue inhibitors of
metalloproteinases (TIMPs), which are especially important
during wound repair.
Turnover of the extracellular matrix is mediated by an excess
of MMP over TIMPs activity. Distortion of the balance between
matrix synthesis and turnover may result in altered matrix
composition and amounts.


2.CELL/TISSUE-BIOMATERIALS INTERACTIONS
Cell interactions with the external
environment are mediated by receptors in
the cell membrane, which interact with
proteins and other ligands that adsorb to the
material surface from the surrounding
plasma and other fluids (Lauffenburger and
Griffith, 2001).
.


2.CELL/TISSUE-BIOMATERIALS INTERACTIONS
•

Cell adhesion triggers multiple functional biochemical
signaling pathways within the cell. Most tissue-derived
cells require attachment to a solid surface for viability,
growth, migration, and differentiation.

•

Following contact with tissue or blood, a bare surface
of a biomaterial is covered rapidly (usually in seconds)
with proteins that are adsorbed from the surrounding
body fluids


2.CELL/TISSUE-BIOMATERIALS
INTERACTIONS
•

•

The chemistry of the underlying substrate (particularly
as it affects wettability and surface charge) controls the
nature of the adherent protein layer.
Cell adhesion to biomaterials is mediated by
cytoskeletally associated receptors in the cell
membrane, which interact with cell adhesion proteins
that adsorb to the material surface from the
surrounding plasma and other fluids (Fig. 12)
(Saltzman, 2000).

Fig 12
( A ) Initial contact of
cell with solid substrate .
( B ) Formation of
bonds between cell
surface receptors
and cell adhesion
ligands .
( C ) Cytoskeletal
reorganization with
progressive spreading of the
cell on the substrate for
increased attachment
strength .

Cell binding to the extracellular matrix through specific cellsubstratum contacts is critical to cell-growth control through
mechanical forces mediated through associated changes in cell
shape and cytoskeletal tension (Ingber, 2002).
Focal adhesions are considered to represent the strongest
such interactions. They comprise a complex assembly of intraand extracellular proteins, coupled to each other through
transmembrane integrins.
Cell-surface integrin receptors promote cell attachment to
substrates, and especially those covered with the
extracellular proteins fibronectin and vibronectin.
These receptors transduce biochemical signals to the nucleus
by activating the same intracellular signaling pathways that are
used by growth factor receptors.

(A) Schematic diagram
showing the initial pattern
design containing differentsized square adhesive
islands and Nomarski (DIC)
views of the final shapes of
bovine adrenal capillary
endothelial cells adherent
to the fabricated substrate.
Importantly, as the bead
diameter was decreased to
10 um, cells became more
rounded, and the apoptotic
index increased to match
that in nonadherent cells
(Fig. 13).

( B ) Apoptotic index
(percentage of cells
exhibiting positive TUNEL
staining) and DNA
synthesis index
(percentage of nuclei
labeled withs 5bromodeoxyuridine) after
24 hours, plotted as a
function of the projected
cell area. Data were
obtained only from islands
that contained single
adherent cells; similar
results were obtained with
circular or square islands
and with human or bovine
endothelial cells.

( C ) Fluorescence micrograph
of an endothelial cell spread
over a substrate containing a
regular array of small circular
ECM islands separated by
nonadhesive regions created
with a microcontact printing
technique. Yellow rings and
crescents indicate
colocalization of vinculin
(green) and F-actin (red) within
focal adhesions that form only
on the regulatory spaced
circular ECM islands .

The more cells spread, the higher their rate of proliferation.
(Fig. 13) (Chen et al., 1997).
Cells spread to the limits of the islands containing a
fibronectin substrate; cells on circular islands were
circular while cells on square islands became square in
shape and had 90°corners. When the spreading of the
cells was restricted by small adhesive islands (10-30 μm),
proliferation was arrested, whereas larger islands (80μm)
permitted proliferation. (Fig 13 A)

This confirmed that the ability to proliferate depended
directly on the degree to which the cells were allowed to
distend physically, and not on the actual surface area of
substrate binding. Thus, cell distortion is a critical
determinant of cell behavior.
Interactions of cells with ECM differ from those with soluble
regulatory factors owing to the reciprocal interactions
between the ECM and the cell's actin cytoskeleton (Ingber,
2003).


Thus, the properties of the nature and
configuration of the surface-bound ECM on a
substrate and the properties of the substrate
itself can regulate cell-biomaterials interactions.
The key concept is that a biomaterial surface can
contain specific chemical and structural
information that controls tissue formation, in a
manner analogous to cell-cell communication
and patterning during embryological
development.

Employ biomaterials with surfaces designed to stimulate
highly precise reactions with proteins and cells at the
molecular level.
The binding domains of the extracellular matrix (ECM)
environment can be mimicked by a multifunctional celladhesive surface created by specific proteins, peptides,
and other biomolecules immobilized onto a material.


The prototypical binding site present in the adhesive proteins
fibronectin and vitronectin is the three amino acid sequence
arginine-glycine-aspartic acid (RGD) which binds to a specific type
of integrin receptors on the cell surface (see Fig. 3C)


This sequence supports the adhesion and spreading of
human endothelial cells but not smooth muscle cells,
fibroblasts, or blood platelets (Hubbell, 1999).
Moreover, cellular responses induced can vary with the
surface density of RGD peptides immobilized (Koo et al.,
2002).


Through selection of ligands, surfaces can be designed
1. to reduce protein and cell adhesion,
2. to prevent coagulation,
3. to encourage endothelial cell attachment and retention,
4. to promote capillary infiltration, and
5. to prevent excessive smooth-muscle proliferation and
collagen production.
This manipulation of cell-integrin interactions with engineered
ligands on synthetic biomaterials could improve function in
existing applications such as the healing of vascular grafts.
A particularly exciting and active area is the use of chemically
patterned surfaces to control cell behavior by creating adhesive
and nonadhesive regions and perhaps even chemical gradients.

By varying the size and chemistry of the various regions, and
thereby the type, architecture, directional migration, and function
of cells, a sort of two-dimensional organ can be grown.

This strategy has been used to "engineer" constructs of hepatic
tissue in which hepatocytes and endothelial cells self-sort to form
endothelium-lined liver cell plates (Kim et al., 1998).


Different levels of type 1 collagen coating on a culture dish result
in different organization of endothelial cells and hepatocytes . High
collagen levels cause both cell types to spread across the
substratum (left) .

hepatocytes
endothelial cells

On intermediate collagen levels, endothelial cells form a layer on
the substratum whereas hepatocytes form a layer on top of the
endothelial cells (center) .

hepatocyte

Low levels of collagen result in an inner layer of hepatocyte
aggregate surrounded by endothelial cells (right) .

A key challenge in tissue engineering is to understand
quantitatively cells respond, and to control nonspecific
interactions.
In addition, special relationships may accrue 產生 for
biodegradable polymers, since the polymer disappears as
functional tissue regenerates.
Thus, polymer degradation may yield a dynamic surface whose
chemistry might be unpredictable,
but could possibly be manipulated to provide an additional level of
control over cell interactions.
Moreover, covalently immobilized growth factor, can retain its
biological activity, and potentially DNA delivered upon a
biomaterial surface can be efficiently taken up by cells and the
encoded gene expressed in a wound-healing environment (Swindle
etal., 2001; Richardson etal., 2001).


Topography has also been studied for its effect on cell behavior,
including depth and width of groove, and roughness.
Surface texture influences cell behavior, including
1. adhesion and movement attachment,
2. spreading area,
3. proliferation,
4. orientation of cells to the topography,
5. biochemical activity, and
6. neurite (nerve) growth.
Moreover, fibroblasts, neurons, and other cells will orient along
fibers, ridges, and grooves with potential therapeutic application in
nerve regeneration, and texture has been shown to influence
macrophage spreading and fibroblast growth.

3.TECHNIQUES FOR ANALYSIS OF CELLS AND
TISSUES
Gross examination
Overall specimen configuration ; many diseases and processes
can be diagnosed at this level
Light microscopy (LM)
Study overall microscopic tissue architecture and cellular structure
; special stains for collagen , mucin , elastin , organisms , etc . are
available

Transmission electron microscopy (TEM)
Study ultrastructure ( fine structure and identify cells and their
organelles and environment

Scanning electron microscopy ( SEM )
Study topography and structure of surfaces
Enzyme histochemistry
Demonstrate the presence and location of enzymes in gross or
microscopic tissue sections
Immunohistochemistry
Identify and locate specific molecules, usually proteins, for
which a specific antibody is available


In situ hybridization
Localizes specific DNA or RNA in tissues to assess tissue identity
or recognize a cell gene product
Microbiologic cultures
Diagnose the presence of infectious organisms

Morphometric studies
Quantitate the amounts, configuration, and distribution of specific
structures

Chemical, biochemical, and spectroscopic analysis
Assess concentration of molecular or elemental constituents

Energy –dispersive X -ray analysis ( EDXA )
Perform site-specific elemental analysis on surfaces

Autoradiography (at LM or TEM levels)
Locate the distribution of radioactive material in sections

3.A.Light Microscopy

Key features of tissue processing for examination by light and
electron microscopy


3.A.a.Tissue Sample

The tissue is obtained by surgical excision (removal), biopsy
(sampling), or autopsy (postmortem examination). A sharp
instrument is used to remove and dissect the tissue to avoid
distortion from crushing. Specimens should be placed in fixatives
as soon as possible after removal.


Fixation

To preserve the structural relationships among cells, their
environment, and subcellular structures in tissues, it is necessary
to cross-link and fix the tissue in a permanent state. Fixative
solutions prevent degradation of the tissue when it is separated
from its source of oxygen and nutrition by coagulating proteins.
This prevents cellular hydrolytic enzymes, which are released
when cells die, from degrading tissue components and spoiling
tissues for microscopic analysis.
Fixation also immobilizes fats and carbohydrates, reduces or
eliminates enzymic and immunological reactivity, and kills
microorganisms present in tissues.

4% formaldehyde( 甲醛 ) solution is the routine fixative in pathology
for light microscopy. For TEM and SEM, glutaraldehyde( 戊二醛 )
preserves structural elements better than formalin.

Adequate fixation in formalin and/or glutaraldehyde requires tissue
samples less than 1.0 and 0.1 cm, respectively. For adequate
fixation, the volume of fixative into which a tissue sample is placed
should generally be at least 5 to 10 times the tissue volume.


3.A.c Dehydration and Embedding
In order to support the specimen during sectioning, specimen
water must be replaced by paraffin wax or other embedding
medium, such as glycol methacrylate ( 乙二醇甲基丙烯酸酯 ). This is
done through several steps, beginning with dehydration of the
specimen through increasing concentrations of ethanol. However,
since alcohol is not miscible with paraffin (the final embedding
medium), xylol (an organic solvent) is used as an intermediate
solution.
Following dehydration, the specimen is soaked in molten paraffin
and placed in a mold larger than the specimen, so that tissue
spaces originally containing water, as well as a surrounding cube,
are filled with wax.
The mold is cooled, and the resultant solid block containing the
specimen can then be easily handled.

Sectioning

Tissue specimens are sectioned on a microtome. The shavings
are picked up on glass slides. Sections for light microscopic
analysis must be thin enough to both transmit light and avoid
superimposition of various tissue components.

If thinner sections are required for TEM analysis, a harder
supporting (embedding) medium (usually epoxy plastic) and a
correspondingly harder knife (usually diamond) are used.


the conventional paraffin technique requires overnight processing,
frozen sections can be used to render an immediate diagnosis
In this method, the specimen itself is frozen, so that the solidified
internal water acts as a support medium, and sections are then
cut in a cryostat ( 冷凍切片機 ). Although frozen sections are
extremely useful for immediate tissue examination, the quality of
the appearance is inferior to that obtained by conventional fixation
and embedding methods.


Staining

Tissue components have no intrinsic contrast and are of fairly
uniform optical density. Therefore, in order for tissue to be visible
by light microscopy, tissue elements must be distinguished by
selective adsorption of dyes (Luna, 1968).

Since most stains are aqueous solutions of dyes, staining requires
that the paraffin in the tissue section be removed and replaced by
water (rehydration).


The stain used routinely in histology involves sequential
incubation in the dyes hematoxylin ( 蘇木素 ) and eosin ( 伊紅 )
(H&E). Hematoxylin has an alkaline (basic) pH that stains bluepurple; substances stained with hematoxylin typically have a net
negative charge and are said to be "basophilic" (e.g., cell nuclei
containing DNA).
Substances that stain with eosin, an acidic pigment that colors
positively charged tissue components pink-red, are said to be
"acidophilic" or "eosinophilic" (e.g., cell cytoplasm, collagen).


Electron Microscopy

Sections are stained with salts of heavy metals (osmium, lead,
and uranium), which react differentially with different structures,
creating patterns of electron density that reflect tissue and cellular
architecture.


Electron Microscopy


Three-Dimensional Interpretation


Artifacts

Artifacts are unwanted or confusing features in tissue sections that
result from errors or technical difficulties in obtaining, processing,
sectioning, or staining the specimen.

The most frequent and important artifacts are autolysis, tissue
shrinkage, separation of adjacent structures, precipitates formed
by poor buffering or by degradation of fixatives or stains, folds or
wrinkles in the tissue sections, knife nicks, or rough handling of
the specimen.

Identification, Genotyping, and Functional Assessment of Cells,
Including Synthetic Products, In Cells or Tissue Sections
Cellular apoptosis and proliferation can be quantified (Watanabe et
al., 2002). Immuno-histochemical markers allow detection of
proteins that are highly expressed in a tissue section.
Tissue micro-assays permit the comparative examination of
potentially hundreds of individual specimens in a single paraffin
block. In addition, laser-assisted micro-dissection techniques
permit isolation of individual or a homogenous population of cells
on selected cell populations under direct visualization from a
routine histological section of complex, heterogeneous tissue
(Eitoum etal., 2002).

Very exciting new imaging technology, termed molecular imaging,
may permit analysis of viable and in vivo tissues (Stephens and Allan,
2003; Weissleder and Ntziachristos, 2003; Webb et al., 2000).

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