B. Conjunctiva synthesis in
vivo (regeneration)
1. Structure and function of conjunctiva.
2. Clinical effects of conjunctival scarring.
3. Anatomically well-defined defect.
4. Synthesis of conjunctival stroma,
followed by re-epithelialization.
article handed out
Inhibition of Conjunctival Scarring and Contraction
by a Porous Collagen-Glycosaminoglycan Implant
Hsu et al., in Investigative Ophthalmology
and Visual Science 41:2402-2411
1. Structure and function of
the conjunctiva
The conjunctiva covers the exposed part of
the sclera (opaque part of eye) and the
inner surface of the eyelids.
The epithelial tissue is stratified and
contains goblet cells in the surface layers.
Goblet cells synthesize and secrete
mucus that contributes to the protective
and lubricating layer on the exposed
surface of the eye.
Underneath the epithelial tissue is the
conjunctival stroma, a loose vascular
supportive tissue.
2. Clinical effects of
conjunctival scarring
? Spontaneous healing of deep wounds in
the conjunctiva occurs by contraction,
scar formation and re-epithelialization.
? Conjunctival scarring is common endpoint
for several opthalmic disorders, resulting
from infection (trachoma), traumatic
(chemical burns) and surgical (pterygium)
causes.
Anatomy of the conjunctiva
Fornix
Eyelid
Cornea
Sclera
Epithelium
Substantia
Propria
Conjunctival
stroma
3. Anatomically well-defined
defect
Defect: Excise complete epithelia and
stroma.
Spontaneous healing: Full-thickness
defect in conjunctiva heals by
contraction and scar synthesis.
Conjunctiva wound model
Conjunctiva
Tenon’s capsule
Sclera
Sclera
Scaffold DRT
Sutures
4. Synthesis of conjunctival
stroma, followed by re-
epithelialization
Sequential synthesis (as for skin):
Synthesize stroma using dermis regeneration
template (DRT).
Epithelial tissue spontaneously synthesized over
new stroma.
Effect of DRT on contraction kinetics of conjunctival defect. It is
experimentally convenient to study contraction of the fornix,
a tissue attached to the conjunctiva.
ungrafted
grafted with scaffold DRT
30
% Fornix Shor
tening
15
0
15 300
Days
Hsu et al., 2000
14 days
epithelialization
(red)
of scar (blue)
ungrafted
Image removed due to copyright considerations.
See Figure 6 in [Hsu 2000]
Image removed due to copyright consi erations.
See Figure 6 in [Hsu 2000]
epithelialization
(red)
of incipient
new dermis
(blue)
grafted with DRT
Test of synthesis of conjunctival stroma
(use microscope polarizing stage to study orientation of
collagen fibers)
Images removed due to copyright considerations.
See [Hsu 2000]
I r d due t c right c iderations.
See [Hsu 2000]
synthesized
conjunctival stroma
conjunctival scar
normal conjunctiva
C. Nerve synthesis in vivo
(regeneration) (Ch. 6)
1. Structure of peripheral nerve.
2. Experimental parameters for study of
induced regeneration.
3. Synthesis of myelinated axons and BM
(nerve fibers)
4. Evidence (?) of synthesis of an
endoneurium.
5. Synthesis of a nerve trunk (including
summary of kinetics of synthesis).
6. Comparative regenerative activity of
various reactants.
Nervous system =
central nervous
system (CNS) +
peripheral nervous
system (PNS)
Image removed due to copyright considerations.
Image removed due to copyright consi erati ns.
Nervous System
Chamberlain et al., 1998
CNS PNS
Image removed due to copyright considerations.
Image removed due to copyright consi erations.
1. Structure of a peripheral
nerve. I
Nerve fibers comprise axons wrapped in a
myelin sheath, itself surrounded by BM
(diam. 10-30 μm in rat sciatic nerve).
Axons are extensions (long processes) of
neurons located in spinal cord. They
comprise endoplasmic reticulum and
microtubules.
1. Structure of a peripheral nerve.
II
Myelinated axons (diam. 1-15 μm) are
wrapped in a myelin sheath;
nonmyelinated axons also exist. They are
the elementary units for conduction of
electric signals in the body. Myelin formed
by wrapping a Schwann cell membrane
many times around axon perimeter. No
ECM inside nerve fibers.
Myelin sheath is a wrapping of Schwann cell
membranes around certain axons.
1. Structure of a peripheral nerve.
III
Nonmyelinated axons (diam. <1 μm) function
in small pain nerves. Although surrounded
by Schwann cells, they lack myelin
sheath; Schwann cells are around them
but have retained their cytoplasm.
Basement membrane (tubular) encases the
myelin sheath. Structure similar to that of
skin BM.
1. Structure of a peripheral nerve.
IV
Nerve fibers are embedded in endoneurium:
a delicate packing of loose vascular
supporting tissue that is rich in collagen
fibers. Definitely ECM!
Many nerve fibers with their associated
endoneurium are packed in a collagenous
layer, the perineurium. This forms a
fascicle.
Multifascicular nerves encased in a
collagenous layer, the epineurium.
Cylindrical symmetry of peripheral
nerve structure
Summary of nerve trunk structure
proceeding radially from the center out:
[axon ? myelin sheath ? BM] ?
endoneurium ? perineurium ?
epineurium.
[ …. ] = “nerve fiber”
Rat sciatic nerve
(nerve trunk).
One fascicle.
Several thousand
axons.
Image removed due to copyright considerations.
See Figure 10.7 (upper left) in Yannas, I. V. Tissue and Organ
Regeneration in Adults. New York: Springer-Verlag, 2001.
Image removed due to copyright consi erati ns.
See Figure 10.7 (upper left) in Yannas, I. V. Tissue and Organ
Regeneration i Adults. New York: Springer-Verl g, 2001.
(idealized)
nerve trunk
nerve fiber
Rat Sciatic Nerve Cross Section
Individual Axon
Longitudinal view of nerve fiber
Image removed due to copyright considerations.
Image removed due to copyright consi erati ns.
Chamberlain, 1998
2. Experimental parameters
for study of regeneration
A. Anatomically well-defined defect
– Designate experimental volume
– Delete nonregenerative tissue(s)
– Anatomical bounds
– Containment of exudate
B. Timescale of observations
– Short-term (<20 wk) and long-term (>20 wk)
assays
The tissue triad in skin and nerves
Replace w/ Artist redraws
Regenerative similarity of tissues in skin and nerves
Skin
The epidermis is a regenerative tissue. After
excision, it regenerates spontaneously.
Reversible injury. No contraction.
The dermis is a nonregenerative tissue in the
adult. After excision, it does not regenerate
spontaneously. Irreversible injury.
Contraction occurs with scar formation.
Skin
spontaneous healing of full-thickness
skin excision by contraction and scar
formation
The injured myelin sheath regenerates
spontaneously
Myelin sheath
Axoplasm
Injured axoplasm and myelin.
BM and endoneurium intact.
Regenerated
myelin
Neuroma formation. The endoneurium
does not regenerate.
Transected nerve.
Both myelin and
endoneurium are
severely injured.
Neuroma forms
at each stump by
contraction and
scar formation.
Intact nerve fiber
Image removed due to copyright considerations.
See Figure 2.5 in [Yannas].
Image removed due to copyright consi erati ns.
See Figure 2.5 in [Yannas].
healing
Spontaneously
healed nerve fiber
2. Experimental parameters (cont.)
C. Assays of configuration
– Correction for experimental gap length.
– Correction for animal species.
– Critical axon elongation, L
c
. Relation to
defect closure rule.
– Shift length, ?L. Characterization of
devices.
– Long-term: fidelity of regeneration.
C. Assays of configuration (cont.)
Use corrected values of frequency of
reinnervation (%N) across tubulated gaps.
This correction allows comparison of %N
data from studies with different gap
lengths and different species.
Critical axon elongation, L
c
, the gap length
above which %N drops below 50% (or the
gap length where the odds of rein-
nervation are even). Data from several
investigators have shown that L
c
= 9.7 ±
1.8 mm for the rat sciatic nerve and 5.4 ±
1.0 mm for the mouse sciatic nerve.
L
c
= 9.7 ± 1.8
mm for the
rat sciatic
nerve and
5.4 ± 1.0 mm
for the mouse
sciatic nerve
Characteristic
curve defines
critical
axon
elongation, L
c
,
at %N = 50%
Image removed due to copyright considerations.
See Figure 6.1 in [Yannas].
Image removed due to copyright consi erations.
See Figure 6.1 in [Yannas].
Data from rat
and mouse
superpose when
plotted vs.
reduced length,
L/L
c
Use single
data point
to
determine
L
c
for
unknown
device
Image removed due to copyright considerations.
See Figure A.1 in [Yannas].
Image removed due to copyright consi erati ns.
See Figure A.1 in [Yannas].
Relation between L
c
, ?L and C, S, R terms in defect closure rule
Image removed due to copyright considerations.
See Table 6.2 in [Yannas].
Image removed due to copyright consi erati ns.
e 6.2 in [Yannas].
2A. Synthesis of myelinated
axons
[NB: Neuron in culture provides
spontaneous outgrowth of axons that
serve as “substrate” for synthesis of
myelin and BM. Schwann cells also
obtained in culture from a neuron.]
A myelin sheath around axons has been
synthesized in vitro in the presence of
Schwann cells, with or without presence
of an ECM component.
2B. Synthesis of nerve BM
A BM has been synthesized in vitro in
presence of neurons and Schwann cells.
However, neurons were not required to be
present when fibroblasts were cultured
with Schwann cells.
Even fibroblasts not required when laminin
added to neuron-free Schwann cell
culture.
3. Evidence (?) for synthesis
of an endoneurium
Structure. Endoneurial microenvironment
surrounding each nerve fiber comprises blood
vessels coursing through space filled with fluid
and thin collagen fibers (51-56 nm diam.). Fluid
outside blood vessels is maintained under
small, positive hydrostatic pressure.
Endoneurial blood vessels comprise cells that
are bound by tight junctions and constitute a
permeability barrier.
Function. Endoneurial environment protects
nerve fibers from changes in ionic strength and
from pathogens in blood vessels that might
modify conductivity (“blood-nerve barrier”).
Endoneurium
Image removed due to copyright considerations.
See Figure 6.2 (top) in [Yannas].
Image removed due to copyright consi erations.
See Figure 6.2 (top) i [Yannas].
Evidence (?) for synthesis of
endoneurium (cont.)
In vitro. No evidence for synthesis of
endoneurial stroma.
In vivo. Nerve trunks have been synthesized
but endoneurial structures have rarely been
studied. Is endoneurium present? Using
silicone tube to bridge 10 mm gap between
stumps did not yield a functional
endoneurium. Occasionally, collagen fibers,
25-35 nm, or even 40 nm, reported outside
BM, were smaller than endoneurial collagen
fibers.
5. Synthesis of a nerve
trunk (including kinetics)
Structure. A nerve trunk comprises one or more
fascicles. Each fascicle comprises several
thousand nerve fibers. If monofascicular, it is
covered by perineurium; if multifascicular, it is
covered by epineurium. A fascicle comprises
the perineurium with its bundle of thousands
of nerve fibers. Some nerves comprise many
fascicles, each with its own perineurial
sheath; these fascicles are wrapped in a
collagenous tissue, the epineurium.
Function. Conducts strong nerve signals (about
10 mV) at 70 m/s.
Tubulation model.
Gap length variable.
Image removed due to copyright considerations.
See Figure 6.2 (top) in [Yan
Image removed due to copyright consi erations.
Tubulation model.
Gap length variable.
Image removed due to copyright considerations. Diagram of implant configuration.
A look inside the gap
sequence:
Schwann cells +
Fibroblasts >
Nonmyel.
Axons >
Blood vessels >
Myel. axons
Image removed due to copyright considerations.
See Figure 10.6 in [Yannas].
Image removed due to copyright consi erations.
See Figure 10.6 i [Yannas].
axon elongation→
proximal
stump
distal
stump
Columns of
Schwann
cells form
even in
absence
of axons
Image removed due to copyright considerations.
See Figure 10.8 in [Yannas].
Image removed due to copyright consi erations.
See Figure 10.8 i [Yannas].
Contractile cell zone surrounds regenerating
nerve
contractile cells
original stump surface
regenerated nerve
Image removed due to copyright considerations.
Image removed due to copyright consi erations.
Spilker and Seog, 2000
Cell capsule around
regenerated nerves
4-mm gap
Normal
rat
sciatic
nerve
Image removed due to copyright considerations.
See Figure 10.7 in [Yannas].
Image removed due to copyright consi erations.
See Figure 10.7 i [Yannas].
8-mm gap
Regenerated
across
0-mm gap
8 weeks
Image removed due to copyright considerations.
See Figure 6.4 in [Yannas].
Image removed due to copyright consi erations.
See Figure 6.4 in [Yannas].Regenerated
nerves
typically
comprise several
minifascicles
39 weeks
article by Chamberlain et al. handed out
Chamberlain, L.J. et al. “Collagen-GAG Substrates Enhances the Quality of Nerve
Regeneration through Collagen Tubes up to Level of Autograft.”
Experimental Neurology 154: 315-329 (1998)
ain, L.J. et al. “Coll ubstrates Enhances the Quality of Nerve
neration thro h Coll s u to Level of Autograft.”
Experimental Neurol y 154: 315-329 (1998)
KINETICS OF
NERVE
SYNTHESIS
30 weeks
Image removed due to copyright considerations.
See Figure 6.5 in [Yannas].
Image removed due to copyright consi erations.
See Figure 6.5 in [Yannas].
60 weeks
Normal
Y-axis: Amplitude
(strength) of
transmitted
electric signal
X-axis: Time
following
stimulation
(at 0 ms)
Electrophysiological
behavior of normal
(light line)
and regenerated nerve
(dark line)
Regenerated nerve is
weaker (lower peak
amplitude) and slower
(delayed peaking)
Image removed due to copyright considerations.
See Figure 6.6 in [Yannas].
Image removed due to copyright consi erati ns.
See Figure 6.6 in [Yannas].
6. Comparative Rregenerative
activity of various devices
(Table 6.1, pp. 147-8)
What does each of these device features
contribute to the quality of regeneration?
Compare values of L
c
and ?L.
? Tubulation
? Tube wall composition
? Tube wall permeability
? Fillings: Schwann cells, solutions of
proteins, gels based on ECM components,
insoluble substrates
Device components
PNS
regeneration.
Length shift,
?L, measures
regenerative
advantage of
device
relative to
silicone
tube standard.
e.g., ?L>0 is
better than
standard.
Tubulation
Tube wall
Image removed due to copyright considerations.
See Table 6.1 in [Yannas].
Image removed due to copyright consi erations.
e 6.1 in [Yannas].
Tube wall
permeability
Filling: Schwann
cells
Filling: protein
solutions
Filling: ECM-based
gels
Filling: insoluble
substrates
PNS
regeneration
(cont.).
Length
shift, ?L,
measures
regenerative
advantage of
device
relative to
silicone
tube
standard.
e.g., ?L>0 is
better than
standard.
Filling: insoluble
substrates
Image removed due to copyright considerations.
See Table 6.1 in [Yannas].
Image removed due to copyright consi erations.
e 6.1 in [Yannas].
Tubulation. Tube wall composition
and permeability
? Bridging the two stumps with a tube,
almost any kind of tube, greatly improves
quality of regeneration.
? Tube wall composition is critically
important. Silicone tubes are greatly
inferior to collagen tubes.
? Increase of cell (but not protein)
permeability improved quality.
Silicone tube
Partly
regenerated
rat sciatic
nerve.
Tubulated
in silicone
tube.
cross-section
shows thick
sheath
of contractile
cells
Image removed due to copyright considerations.
See Figure 4.5 in [Yannas].
Image removed due to copyright consi erations.
See Figure 4.5 in [Yannas].
Silicone tube
near original
proximal
stump
Contractile cells
(brown)
ensheathe
regenerating
stump
of transected rat
sciatic nerve
Image removed due to copyright considerations.
See Figure 4.6 in [Yannas].
Image removed due to copyright consi erati ns.
See Figure 4.6 in [Yannas].
near original
distal stump
Tube fillings
? Schwann cells, growth factors (aFGF and
bFGF) and several insoluble substrates
increased quality of regeneration,
sometimes greatly.
? NGF had no effect.
? Gels based on ECM components
(collagen, fibronectin, laminin) had no
effective or impeded regeneration.
Regeneration across a 10 mm gap bridged by a silicone tube
filled
unfilled
Image removed due to copyright considerations.
See Fig. 10 in Yannas, I. V. "Biologically Active Analogues of the
Extracellular Matrix: Artificial Skin and Nerves."
Angew. Chem. Int. Ed. Engl. 29 (1990) 20-35.
Effect of degradation rate of tube
filling based on a porous ECM
analog (NRT)
? Undegraded ECM analog physically
impeded axon elongation.
? Optimal quality of regeneration
obtained with ECM anlog that
degraded at an intermediate rate.
Effect of pore
diameter and
degradation
rate on
inverse
conduction
velocity
(latency)
Image removed due to copyright considerations.
See Figure 10.9 in [Yannas].
Image removed due to copyright consi erations.
See Figure 10.9 i [Yannas].
1. pore structure (ligand density)
2. macromolecular
structure (ligand
duration)
Structural features of ECM analogs used as
tube fillings in nerve regeneration
3. chemical composition (ligand identity)
4. orientation of pore channel axes
Image removed due to copyright considerations.
See Figure 6.2 (top) in [Yan
Image removed due to copyright consi erations.
Image removed due to copyright considerations.
See Figure 6.2 (top) in [Yan
Image removed due to copyright consi erations.
Long-term electro-
physiological
properties of
various
regenerated nerves
Image removed due to copyright considerations.
See Tables 6.1 and 6.3 in [Yannas].
Image removed due to copyright consi erations.
es 6.1 and 6.3 in [Yannas].
Summary of results
? Tube presence was essential
? Tube wall composition: collagen > degradable
synthetic polymer > silicone.
? Tube wall permeability: cell-permeable > protein
permeable > impermeable.
? Tube fillings:
--- suspensions of Schwann cells
--- solution of either aFGF or bFGF (not NGF!)
--- crosslinked ECM networks > ECM gels
--- thin polymeric filaments oriented along tube axis
--- highly porous, insoluble ECM analogs with
appropriately small pore diameter, axial orientation of
pore channel axes and critically adjusted degradation
rate.