CELL BIOLOGY

EXTRA CELLULAR
MATRIX
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Drs. Sofy PERMANA, MSc.,DSc.

TOPICS=
Matriks Ektraseluler
Structures ,
Organization & Functions,
Wound healing

Introduction
This lecture introduces the materials lying
outside the cell, known collectively as the
extracellular matrix (ECM).
There is no one matrix though, with different
tissues having their own specific ECM, which
may be dynamic or static in structure.
In particular the ECM has significant roles in
normal tissue development, function and
disease.
This matrix is manufactured by cells, secreted
and modified outside the cell by several
different enzymes

History
related to cell junctions
research finding

1978 Basal lamina instructs innervation Joshua

Sanes and Jack McMahan show that
regenerating nerve axons take their cues for new
synapse formation from the extracellular matrix
(ECM) of muscle cells and not from the muscle
cells themselves

This first lecture introduces the ECM

and describes the major fiber (fibre)
and matrix components,
the second lecture will cover the major
ECM glycoproteins and
experimental studies of ECM
function.

ECM Function
*Support for cells
*Pattern of ECM regulates
-polarity
-cell division
-adhesion
-motility
*Development
-migration
-differentiation
-growth factors

ECM Features
*stable and able to
be reorganised
*different for
different tissues

HOW THE EXTRACELLULAR

MATRIX COULD, IN
PRINCIPLE, PROPAGATE
ORDER FROM CELL TO
CELL WITHIN A TISSUE
For simplicity, the figure
represents a hypothetical
scheme in which one cell
influences the orientation of
its neighboring cells. It is
more likely, however, that
the cells would mutually
affect one another's
orientation.

ECM Structure
*Glycoproteins
*Fibers
-Collagen- main fibers
-Elastin
*Hydrated Matrix
-Proteoglycans
-high carbohydrate
*Adhesive
-Laminin
-Fibronectin

Shapes and Sizes ECM molecules

THE COMPARATIVE SHAPES AND SIZES OF SOME OF THE

MAJOR EXTRACELLULAR MATRIX MACROMOLECULES
Protein is shown in green, and glycosaminoglycan in red.

Collagen
*tensile strength and elasticity
-Tendons
-Cartilage
-Bone
*half total body proteins (by weight)

Collagen Components
*Insoluble glycoprotein
*protein + carbohydrate
-Protein
high glycine
high proline
hydroxylysine
hydroxyproline
(gly-X-Y)n
-Carbohydrate
glucose
galactose

Collagen Structure
Collagen Protein
3 polypeptide (a) chains
left hand helix, forms fibers
many different (vertebrate) collagens by
different combinations of a-chains
Type I, II, III
main fibers, flexible
Type I
bone, skin, tendons
90% of all collagen

 Type II
cartilage

Collagen Fibers
Type I, II, III cross striated
-e.g. tendons - type I fibrils, have a 67nm period striations and are oriented
longitudinally (direction of the stress)
-showing overlapping packing of
individual collagen molecules
-reticular fibres type III, support
individual cells

Type IV fine unstriated

sheet-like supportive meshwork
mature basal laminae
tracks for embryonic migration
barriers for cell migration
Type V-XII
smaller diameter fibers than I-III
no striations
Collagen Fibers Collagen Non-striated

Collagen Interactions
Collagen fibril types can interact with a
variety of non-fibrous collagen types
(microfiber)
fibrous collagenstypes I, II, III, and V
Cartilage - types II (fiber) and IX
collagen microfibrils
Tendons - type I fibrils bound and
linked by type VI microfibrils.

INTERACTIONS OF FIBROUS AND NONFIBROUS

COLLAGENS
(a) Association of types II and IX collagen in a cartilage matrix. Type II forms
fibrils similar in structure to type I, with a similar 67-nm periodicity,
though smaller in diameter. Type IX contains two long triple helices
connected at a flexible kink. At this point a chondroitin sulfate chain is
linked to the 2(IX) chain. Type IX collagens are bound at regular
intervals along type II fibrils, with an N-terminal nonhelical domain of
type IX projecting outward. It is thought that these domains bind the
collagen fibrils to the proteoglycan-rich matrix. (b) Organization of the
major fibrous components in the extracellular matrix of tendons. Type I
fibrils, with their characteristic 67-nm period, are all oriented
longitudinally, that is, in the direction of the stress applied to the tendon.
The fibrils are coated with an array of proteoglycans, as shown in blue on
the right-hand fibril. Type VI fibrils bind to and link together the type I
fibrils. Type VI collagen consists of thin triple helices, about 60 nm long,
with globular domains at either end. The globular domains of several type
VI molecules bind together, giving a beads-on-a-string appearance to the
type VI fibril. [Part (a) after L. M. Shaw and B. Olson, 1991, Trends
Biochem. Sci. 18:191; part (b) after R. R. Bruns et al., 1986, J. Cell Biol.
103:393.]

Collagen Type Functions

Collagen Type I - skin, tendon, vascular,
ligature, organs, bone (main
component of bone)
Collagen Type II - cartilage (main
component of cartilage)
Collagen Type III - reticular fibers with
type I.
Collagen Type IV - forms bases of cell
basement membrane

Collagen Synthesis
Endoplasmic Reticulum
*mRNA attached to ER
*protein synthesized into ER lumen
*cotranslational and post-translational
modifications
*3 proto-a-chains form soluble
procollagen
*moved to golgi apparatus

Collagen Synthesis
Golgi Apparatus
*packed into secretion vesicles
*fuse with membrane

Collagen
Synthesis
Outside Cell
*procollagen
processed
by enzymes
outside cell
*assemble into
collagen
fibers
*collagen fibrils form

Collagen Synthesis
Golgi Apparatus
*packed into secretion vesicles
*fuse with membrane
Outside Cell
*procollagen processed by enzymes
outside cell
*assemble into collagen fibers
*collagen fibrils form lateral
Interactions of triple helices

THE INTRACELLULAR AND EXTRACELLULAR EVENTS IN

THE FORMATION OF A COLLAGEN FIBRIL

(A) Note that collagen fibrils

are shown assembling in
the extracellular space
contained within a large
infolding in the plasma
membrane. As one
example of how collagen
fibrils can form ordered
arrays in the extracellular
space, they are shown
further assembling into
large collagen fibers,
which are visible in the
light microscope. The
covalent cross-links that
stabilize the extracellular
assemblies are not shown.

THE INTRACELLULAR AND EXTRACELLULAR EVENTS IN

THE FORMATION OF A COLLAGEN FIBRIL

(B) Electron micrograph of a

negatively stained collagen
fibril reveals its typical
striated appearance. (B,
courtesy of Robert Horne.)

Collagen Diseases - Excess

fibrosis
lung- pulmonary fibrosis
overproduction of collagen I
liver- over consumption of alcohol
arteries- atherosclerosis

Collagen Diseases - Insufficient

Ehlers-Danlos syndrome
rubber-man
skin and tendons easily stretched
contortionists often suffer from this
disease

Collagen Diseases - Insufficient

Osteogenesis imperfecta
brittle-bone syndrome
mutation in Type I procollagen
fail to assemble triple helix
degrade imperfect collagen
Leads to fragile bones

 Scurvy
dietary Vitamin C
deficiency
needed for hydroxylation
Proline ->
Hydroxyproline
form too few hydrogen
bonds in collagen
skin, bone, teeth
weakness and
malformation
blood vessels weakened,
bleeding

 Atopic dermatitis (AD)

chronic inflammatory skin disorder and a
major manifestation of allergic disease
mutation in collagen XXIX (COL29A1)
gene

Elastin Elastic Fibers Skin

elastin and elastic fibres
uncoils into an extended conformation
when the fiber is stretched
recoils spontaneously as soon as the
stretching force is relaxed
Elastic fibers are composed of a core of
cross-linked elastin embedded within a
peripheral mantle of microfibrils.

Elastic Fibers Skin

Microfibrils
may regulate assembly and organization of elastic
fibers by acting as a scaffold
guiding tropoelastin deposition
aggregates of threadlike filaments
periodically spaced globular domains (beads)
connected by multiple linear arms
beaded structure is parallel fibrillin monomers
aligned head-to-tail
fibulin-5 induces elastic fiber assembly and
maturation by organizing tropoelastin and
cross-linking enzymes onto microfibrils

Microfibrils

Elastin Structure
elastic fiber synthesis
protein Mr 64 to 66 kDa
composed of the amino acids glycine, valine, alanine, and
proline
cross-linked tropoelastin monomers
first secreted as soluble precursors (tropoelastin)
assembly and crosslinking of tropoelastin monomers
form insoluble elastin matrix into functional fibres
lysine residues in the cross-linking domain of secreted
tropoelastin rapidly cross-linked (both inter- and
intra-molecularly by lysyl oxidase)
hydrophobic segments - elastic properties
-helical segments (alanine- and lysine-rich) - form
cross-links between adjacent molecules

Elastin Structure
elastic fiber synthesis

Elastin Function
structural integrity and function of tissues
requiring reversible extensibility or
deformability
high levels in tissues that require elasticity
lung, skin, major blood vessels
Elastin Disorders
Williams Syndrome
hyaluronan-binding proteins

Proteoglycans
consist of protein (~5%) and polysaccharide
chain (~95%)
form a gel to embed the fibril network
Golgi apparatus - GAG disaccharides are
added to protein cores to form
proteoglycans
10% by weight but fill most of space
unbranched polysaccharide chains
disaccharide subunits
amino sugar

Proteoglycans
PROTEOGLYCANS
IN THE EXTRACELLULAR
MATRIX OF RAT
CARTILAGE

The tissue was rapidly frozen at -196C, and fixed and stained while still
frozen (a process called freeze substitution) to prevent the GAG chains
from collapsing. In this electron micrograph, the proteoglycan molecules are
seen to form a fine filamentous network in which a single striated collagen
fibril is embedded. The more darkly stained parts of the proteoglycan
molecules are the core proteins; the faintly stained threads are the GAG
chains. (Reproduced from E.B. Hunziker and R.K. Schenk, J. Cell Biol.
98:277282, 1985. The Rockefeller University Press.)

Glycosaminoglycans (Gags)
Five types
Hyaluronan (or hyaluronic acid) main
glycosaminoglycan in connective tissue
high molecular weight (~ MW 1,000,000 )
length of about 2.5 m hyaluronan
"backbone" for the assembly of other
glycosaminoglycans

Hyaluronan is also a major component of

the synovial fluid, which fills joint
cavities, and the vitreous body of the
eye.
Other 4 major glycosaminoglycans
chondroitin sulphate, dermatan sulphate,
keratan sulphate and heparan sulphate
(UK sulphate, US sulfate)
attach through core and link proteins to
hyaluronic acid backbone

Proteoglycan Function
trap water
resistant to compression
return to original shape
occupy space
link to collagen fibers
form network
in bone combined with calcium hydroxyapatite, calcium
carbonate
Development
produce a cell-free space
for cell proliferation and migration into
heart, cornea
Adult
in areas of compression
tissues, joints

Hyaluronan Synthesis differs from other GAG synthesis

synthesized at plasma membranes nascent chains directly
extruded into ECM

superfamily of hyaluronan-binding proteins. These proteins contain one or more

link modules that bind to hyaluronan. Like many extracellular matrix proteins, the
link module superfamily members contain various subdomains, depicted by the
following annotated symbols: (IG) immunoglobulin-like domain; (EGF) epidermal
growth factorlike domain; (CLECT) C-type lectin domain; (CCP) complement
control protein module; (Link) hyaluronan-binding module; (CUB) domain found in
some complement proteins, peptidases, and bone morphogenetic protein; (FAS)
domain found in fasciclin I family of proteins. (See the SMART database at EMBL
for additional information on these domains:

Hyaluronan Synthesis
differs from other GAG synthesis
synthesized at plasma membranes
nascent chains directly extruded into ECM
Cell adhesion
embryonic migration

Proteoglycan- Disease
Mucopolysaccharidosis type I (MPS I) - Hurler disease
lysosomal storage disease, is associated with an altered
elastic matrix
excess heparan sulphate and dermatan sulphate
Cancer development
altered types and kinds of proteoglycans formed by
cells
normal cells -> malignant
Arthritis
Cartilage breakdown (cartilage erosion)
chondrocytes elicit a catabolic response which exceeds
anabolism of new matrix molecules
Degrade proteoglycan (aggrecan)
Also a mouse model generates antibodies to

Wound Healing

Introduction

Basis of repair of tissues

Enables surgical treatment
May take advantage for our benefit
Complications
Modification and Enhancement

Phases of Wound Healing

1. Vascular and inflammatory phase
2. Reepithelization
3. Granulation tissue formation
4. Fibroplasia and matrix formation
5. Wound contraction
6. Neovascularization
7. Matrix and collagen remodelling

Vascular
Initial vasoconstriction (5-10
min) then vasodilation
(persistent)
Exposure of subendothelial
von Willebrand / factor VII,
and fibrillar collagen
platelet plug
Hageman factor (XII)
initiation of clotting cascade
and fibrin clot formation

Clotting Cascade
Intrinsic Pathway

Extrinsic Pathway

Surface Contact
Collagen
FXII activator
F XII

Tissue/Cell Defect

F XIIa
F XI

F IXa

Ca2+

F VII

F III (Tissue
Thromboplastin)

F VIIIa
Platelet Factor 3

Factor F X

Ca2+

Factor F Xa
F Va

Prothrombin I
F XIIIa
Crosslinked
Fibrin Meshwork

Ca2+

F XIa

Ca2+

F IX
F VIII

F VIIa

Ca2+

Ca2+

Factor F X

FV

Thrombin

F XIII
Fibrin
polymers

Fibrin
monomers

Fibrinogen

Inflammatory

Platelets

derived growth factor (PDGF), proteases and vasoactive substances such as serotonin and
histamine

Polymorphonuclear leukocytes
Macrophages (replace PMNs after 5 days)
Fibroblasts (recruited by chemotactic factors released by the above cells)

Reepithelization
Migration (wound edges, hair follicles,
adnexa)
Proliferation (48-72 hours)
Sutured wounds have a layer of keratinocytes
within 24-48 hours

Keratinocytes
Fibronectin

Cross links to fibrin matrix/scaffold for keratinocyte adhesion and

migration
Functions as an early component of the extracellular matrix.
Binds to collagen and interacts with matrix glycosaminoglycans.
Has chemotactic properties for macrophages, fibroblasts and endothelial
and epidermal cells.
Promotes opsonization and phagocytosis.
Forms a component of the fibronexus.
Forms scaffolding for collagen deposition

Collagenases and neutral proteases debridement

Plasminogen activator clot dissolution
Type V collagen
Requires moisture for epithelial migration

Granulation
Highly vascular network of
glycoproteins, collagen and
glycosaminoglycans
Fibroblasts

collagen
Elastin
Fibronectin
Sulfated and non-sulfated
Glycosaminoglycans
Proteases

Inflammatory cells

Fibroplasia

Fibroblasts
Mainly Type III collagen first
Replaced by type I and II collagen
Hydroxylation of proline and lysine

Iron, copper, vitamin C

Crosslinkage

Contraction
Myofibroblasts
Fibronexus (Singer)

Connections between intracellular actin

microfilaments and extracellular collagen,
fibronectin, and between myofibroblasts
Transmits force along entire network
Centripetal contraction

Neovascularization
Fibronectin
Macrophage derived angiogenic factor
Endothelial migration

Wound Remodeling
Increased tensile strength
Decreased bulk, and
erythema
Replacement of
fibronectin by collagen
Dehydration

Promotes further
crosslinkage of collagen
Reorientation of collagen to
parallel skin collagen.

Local Factors

Infection
Technique (wound edge ischemia)
Hematoma
Foreign body reaction
Tissue ischemia
Topical medications and dressings

Systemic Factors
Deficiency states

Insulin
Protein (nitrogen balance)
Vitamins
A slower re-epithelization
C Collagen
K clot formation

Trace minerals and elements

Zinc, copper, iron, manganese

Systemic Factors
Medications

Glucocorticoids
Anticoagulants
Antineoplastic drugs
Cyclosporin A
Colchicine
Penicillamine
Zinc sulfate (high doses)
Beta amino proprionitrile

Growth Factors

Epidermal growth factor

Macrophage derived growth factor (MDGF)
Platelet derived growth factor (PDGF)
Thrombin
Insulin
Lymphokines

PDGF

Chemotaxis, Fb, angiogenesis contraction

TGF-alpha

Keratinocyte and Fb activation

TGF-beta

Fibroplasia, angiogenesis, Beta-3 has some

antiscarring effects

EGF

Keratinocytes

FbGF

Fb and keratinocyte , angiogenesis

KGF

Migration, differentiation, proliferation

TNF

Macrophage activation

IL-1,2,6,8
IFN-alpha, beta,
delta

Macrophage activation

VEGF

Angiogenesis, vascular permeability

IGF-I

Reepithelization and granulation

Thromboxane A2

Vasoconstriction

Plasminogen activator inhibitor -1

Found to be elevated in Keloid scars
PAI-1 -/- knockout mice show accelerated
wound healing after cutaneous injury
PAI-1 seems to regulate fibrinolytic and
proteolytic activity during the replacement
of fibrin by collagen.
PAI-1 is upregulated in cultured fibroblasts
in a hypoxic environment

Metalloproteinases & Tissue

Inhibitor of Metalloproteinases
Regulatory role in fibroblasia and scarring

Found in high concentrations in fetal wounds

MMP/TIMP is higher in scarless fetal wounds
TGF-beta decreased the MMP/TIMP ratio by
increasing TIMP
May promote more rapid epithelization

TGF Beta-1
Higher concentrations and exaggerated
response in keloid fibroblasts
When added to fetal wounds thicker scars
made.

Silicone
Decreases TGF-Beta-2, and contraction of
RAFT-fibroblast cultures (Kuhn et. al.)
Increased bFGF levels in cultured
fibroblasts (HanasonoKoch)
Anecdotal evidence

References

Silvia Wagner, PhD et. al. Comparison of inflammatory and systemic sources of growth factors
in acute and chronic human wounds, Wound Repair and Regeneration Volume 11 Issue 4 Page
253 - July 2003 doi:10.1046/j.1524-475X.2003.11404.x
Deodhar AK, Rana RE. Surgical physiology of wound healing: a review. J Postgrad Med [serial
online] 1997 43:52-6 http://www.jpgmonline.com/article.asp?issn=00223859;year=1997;volume=43;issue=2;spage=52;epage=6;aulast=Deodhar
Ziv PM et. al., Matrix Metalloproteinases and the ontogeny of scarless repair: the other side of the
wound healing balance, Plastic and Reconstructive Surgery 110(3):801-11 2002
Bullard KM et. al. Fetal wound healing: Current Biology, World J. Surg 27: 54-61, 2003
Singer AJ, Clark RAF, Cutaneous Wound Healing, NEJM 341(10) 738-46; 2001
Saulis AS, Mogford JH, Mustoe TA.Related Articles, Links Effect of Mederma on hypertrophic
scarring in the rabbit ear model.
Plast Reconstr Surg. 2002 Jul;110(1):177-83; discussion 184-6.
Hanasono MM, Lum J, Carroll LA, Mikulec AA, Koch RJ.Related Articles, Links The effect of
silicone gel on basic fibroblast growth factor levels in fibroblast cell culture.
Arch Facial Plast Surg. 2004 Mar-Apr;6(2):88-93.

Wound healing

Phases of healing

Early
Intermediate
Late
Terminal

Early wound healing events

Hemostasis

Platelet aggregation
Intrinsic and extrinsic coagulation cascade
Thrombin, fibrin
Vasoconstriction

Early wound healing events

Inflammation
Vasodilatation
Increase in vascular permeability
Chemotaxis
Cellular response

Early wound healing events

Homeostasis
Neutrophils
48-72h- macrophages
5-7 days- few inflammatory cells.

Intermediate wound healing events

Mesenchymal cell chemotaxis and
proliferation
Angiogenesis
Epithelisation
2-4 days after injury
Mediated by cytokines

Intermediate wound healing events

Mesenchymal cell chemotaxis and
proliferation
Fibroblasts- migration and proliferation
Smooth muscle
Angiogenesis- reconstruction of vasculature
Stimulate: High lactate, acidic Ph, low O2
tension
Endothelial cell migration and proliferation

Intermediate wound healing events

Epithelisation
Partial thickness- Cells derived from wound edges
and epithelial appendages.
Incisional wound: cellular migration over less then
1 mm. Wound sealed in 24-48h.

Cellular detachment
Migration
Proliferartion
differentiation

Late wound healing events

Collagen synthesis
3 helical polypeptide chains
Lysine and proline hydroxylation
Required for cross-linking

Late wound healing events

Collagen synthesis
3-5 days post injury
Primarily by fibroblasts
Maximum synthesis rate 2-4 weeks
Declines after 4 weeks
Type 1 collagen most common ( 80-90% of skin
collagen)
Type 3- seen in early phases of wound healing

Wound contraction
Centripetal movement of the wound edges toward
the center. ( 0.6-0.7 mm/day)
Begins at 4-5 days
Maximal contraction 12-15 days
Trivial component in closed incisional wounds,
significant for closure of open wounds
Rate- depends on tissue
Circular wounds- slower closure but avoid
stenosis

Wound contraction
Mechanism- cell mediated processes, not
requiring collagen synthesis
Myofibroblasts- fibroblasts with
myofilaments in cytoplasm
Appear in wound day 3-21
Located in periphery- pull wound edges
together.
Contractures- contraction across joint
surface

Terminal wound healing events

Remodeling- turnover of collagen. Type 3
replaced by type 1
Day 21- net accumulation of wound collagen
becomes stable
Wound bursting strength- 15% of normal.
Week 3-6- greatest rate of increase
6 weeks- 80-90% of eventual strength.
6 months maximum strength ( 90% ). Process
continues for 12 months

Cytokines and growth factors

Primary mediators in wound healing.

Endo, para, auto, intracrine function
EGF
FGF
PDGF
TGF

Growth factors in wound healing

Which of the following is primarily responsible

for tensile strength in a healing wound 4 days
after injury?

Collagen
Elastin
Fibrin
Fibronectin
Hyaluronic acid

Which of the following is primarily responsible

for tensile strength in a healing wound 6 weeks
after injury?

Myofibroblasts
Fibrin
Fibronectin
Collagen
Collagen cross linking

Local factors affecting wound healing

Infection
foreign body/ necrotic tissue, hematomas
local/ systemic factors
type of surgery

Local factors affecting wound healing

Hypoxia and smoking

O2 delivery necessary for cellular
respiration and hydroxylation of proline and
lysine
Smoking- vasoconstriction, atherosclerosis,
carboxyhemoglobin.

Local factors affecting wound healing

Radiation
Collagen synthesized to abnormal degreefibrosis
Fibrosis of vessels- (media)-occlusion
Thinned epidermis, pigmentation
Limited access of inflammatory cells and
cytokines- impaired healing
Damage to fibrocytes and keratinocytes.

Systemic factors
Malnutrition
Limited AA supply for collagen synthesis
Consumption of proteins d/t CHD and fat
deficiency.
Vit C deficiency- diminished hydroxylation of
lysine and proline,
Vit D- impaired bone healing
Zinc- inhibition in cellular proliferation and
defficient granulation tissur formation

Normal healing is accelerated by which

of the following?

VitC
VitA
Zinc
Increased local oxygen tension
Scarlet red

Systemic factors
Cancer
Cachexia, anorexia
Altered host metabolism.
Protein catabolism
Abnormal inflammatory cell response

Systemic factors
Old Age
Diabetes
Impaired healing ( decreased chemotaxis
and phagocyte function )
Risk of infection

Systemic factors
Steroids, immunsuppression
Inhibits all aspects of healing process
Impaired cellular function, deficiency in
inflammatory cell function, cytokine
production, fibroblast proliferation
All effects ( except contraction ) reversed
by Vit A.

Hypertrophic scars and kelloids kelloids

Excessive healing processes- increase in net
collagen synthesis raised thickened scar
Keloid- Extension beyond wound margin, familial,
may develop up to 1 year, rarely subside
Hypertrophic scar- Confined to wound margin,
light skinned, early after injury, may subside,
cause contractures
Tx- excision, steroid injection, pressure garments,
radiation tx

Types of wound closure

Primary closure
Approximation of acutely disrupted tissue
with sutures, staples or tape

Types of wound closure

Delayed primary closure
Approximation of wound margin delayed
for several days
Prevents wound infection in cases of
contamination/foreign bodies/tissue trauma
Less bacterial colonization in open wound
Normal healing progress occurs

Types of wound closure

Secondary wound closure
Open wound margins approximate by
biologic contraction

 If a patient requires reoperation 1 month after a

midline abdominal incision which of the following
promotes the most rapid gain in strength of the
new incision

Separate transverse incision

Midline scar is excised with a 1 cm margin
Midline incision reopended without scar excision
Rate of strength ganed is not effected by incision
technique

Thank You