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The HSE demonstrated a multilayered and differentiated
epidermis with all epidermal layers including stratum
corneum.
.
Schematic of three-dimensional tissue
construction.
(A) A thin, acellular layer of collagen is first
constructed; it provides an attachment substrate
for the cellular collagen.
(B) A collagen gel embedded with human dermal
fibroblasts is layered onto the acellular layer.
(C) While submerged in medium for 7 days,
dermal fibroblasts remodel the collagen matrix,
causing it to contract away from the walls of the
insert. The contracted collagen forms a plateau.
(D) Keratinocytes are then added to the center of
the plateau of contracted collagen and allowed
to attach to the collagen to create a monolayer that
will form the basal layer of the tissue.
(E) Tissues are raised to an air-liquid interface
to initiate stratification. Keratinocytes stratify and
differentiate and form a suprabasal layer that
mimicsin vivo skin both morphologically and
biochemically.
(F) Further exposure to the air-liquid interface
and additional feedings with cornification medium
results in an increase in the
thickness of the spinous and cornified layers of the
tissue.
FABRICATION OF THREE-DIMENSIONAL MODEL OF
HUMAN SKIN CANCER
The tissue microenvironment is defined by the complex network of
intercellular interactions that are mediated by physical attachment (as in direct
cell-cell or cell-extracellular matrix interactions) and by biochemical signals
mediated by soluble molecules (Hagios et al., 1998).
The adhesive cues in 3D tissues and is required to maintainn normalization of
tissue architecture and homeostasis.
Cancer is a disease of altered tissue architecture and that neoplastic
progression is a consequence of abnormal interactions between tumor cells
and their tissue microenvironment.
(As a result, it is essential to study the impact of the tissue microenvironment
on cancer progression in human tissues that incorporate 3D tissue context so that
they faithfully mimic their in vivo counterparts. Monolayer, 2D culture systems do
not generate the spatially organized, 3D structures that are seen in vivo and have
been of limited use in studying complex cellular responses)
To recapitulate the precancerous stages of progression of skin
cancer, 3D tissue models must be constructed to reflect alterations in
cell-cell interactions that could propel the tissue towards
a neoplastic fate (Margulis et al., 2005). This has been accomplished
by developing tissue models in which the fate of potentially
malignant cells can be mapped by mixing normal keratinocytes with
genetically-marked (with β-galactosidase) tumor cells at varying
ratios to mimic precancerous conditions.
Carcinoma cell lines can be grown as pure cultures at an air-liquid interface to simulate a
carcinoma in situ, or they can be grown as mixtures with normal keratinocytes to mimic an
earlier stage of epithelial dysplasia. Previous studies with different SCC cell lines indicate that
tumor cells may either be maintained as individual cells within the tissue (Javaherian et al.,
1998), or they may undergo intraepithelial expansion (Vaccariello et al., 1999).
LA
GUARIGIONE
DELLE
FERITE
E’
UN
PROCESSO
COMPLESSO
CHE
COMPRENDE DIVERSE FASI E RICHIEDE L’INTERVENTO COORDINATO DI
NUMEROSI TIPI DI CELLULE.
LE DIVERSE FASI DEL PROCESSO DI GUARIGIONE
Formazione della cicatrice
Il processo di riparazione avviene in tre
stadi.
Dopo una iniziale fase acuta che comporta
formazione del coagulo e quindi invasione
di cellule infiammatorie all’interno
dell’area danneggiata, vi è una fase
proliferativa durante la quale si forma
matrice tissutale connettivale molto
vascolarizzata (neo-angiogenesi).
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
DURANTE LE PRIME FASI DEL PROCESSO
DI
GUARIGIONE,
IL
TESSUTO
DI
GRANULAZIONE E’ CARATTERIZZATO DA
UN’INTENSA ATTIVITA’ ANGIOGENETICA.
I CAPILLARI NEOFORMATI SONO DESTINATI
A SCOMPARIRE NELLE FASI SUCCESIVE DI
FORMAZIONE DELLA CICATRICE (FASE
FIBROGENETICA).
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
• 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
FABRICATION OF THREE-DIMENSIONAL WOUND
HEALING MODEL OF
HUMAN SKIN
In vitro studies of wound re-epithelialization have often been
limited by their inability to
simulate wound repair as it occurs in humans. For example,
wound models using skin explants or monolayer, submerged
keratinocyte cultures demonstrate limited stratification, partial
differentiation, and hyperproliferative growth culture systems
have been helpful in studying keratinocyte migration in response
to wounding. However, they have been of limited use in studying the complex
nature of keratinocyte response during wound repair, as these cultures do not provide the
proper tissue architecture to study the in vivo wound response
HSE can be adapted to study wound repair in human epithelium
simulating the chronology of events that occur during re-epithelialization
in human skin and allowing direct determination of the key response
parameters of wounded epithelium including cell proliferation, migration,
differentiation, growth-factor response, and protease expression.
(A) A wound is
generated through the
full thickness of a
human skin equivalent
(HSE) and the excised
tissue is removed.
(B) The wounded tissue
is placed on a second,
contracted collagen gel.
(C) Keratinocytes
undergo migration to
close the wound gap.
(D) Keratinocytes have
restored epithelial
integrity, have
closed the wound gap,
and undergo
stratification.