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From 1 Ophthalmic Plastic and Orbital Surgery, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston; and the 2 Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge.
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Abstract |
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METHODS. A 7-mm trephine was used to produce lesions of the bulbar conjunctiva down to the level of the bare sclera. Full-thickness removal of the conjunctiva and Tenon’s capsule created a reproducible wound bed. Wounds either remained ungrafted (control) or were grafted with CG matrix. In previous studies, this CG matrix has induced partial regeneration of the dermis in the human, the swine, and the guinea pig. Healing of the conjunctival epithelium and underlying stroma was evaluated by histology, immunohistochemistry, and measurement of wound contraction kinetics.
RESULTS. By 28 days, ungrafted wounds had closed by contraction (26.4% ± 5.0% fornix shortening) and the formation of scarlike tissue comprising an aligned array of dense collagen populated with occasional fibroblasts. Grafting of identical defects with CG copolymer matrix resulted in inhibition of wound contraction (6.8% ± 3.2% fornix shortening) and the formation of a tissue that resembled normal conjunctival stroma, being composed of a loose network of collagen fibers and fibroblasts. Contractile fibroblasts (myofibroblasts) were identified at the edge of both ungrafted and grafted wounds during the period of active contraction. Both ungrafted and grafted wounds were completely re-epithelialized by 28 days.
CONCLUSIONS. Implantation of CG copolymer matrix drastically reduced contraction and promoted the formation of a nearly normal subconjunctival stroma.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionConclusionReferences |
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Conjunctival scarring is a final common pathway for a myriad of ophthalmic disorders, representing a broad range of causes including infectious (e.g., trachoma), immunologic (e.g., Stevens–Johnson syndrome, ocular cicatricial pemphigoid), traumatic (e.g., chemical burns), and postsurgical (pterygium, glaucoma filtering procedures, anophthalmic socket). Prevention and modulation of conjunctival scarring could favorably impact each of these disorders.
Substantial evidence indicates that wound contraction in connective
tissues is cell mediated3
4
and that the active cell type
is a modified contractile fibroblast, the
myofibroblast.5
6
Myofibroblasts contain the contractile
apparatus of smooth muscle cells, 
-smooth muscle actin (SMA), and
can be identified by immunohistochemical staining with antibodies to
-SMA.7
Contractile fibroblasts have been identified in
contracting wounds in the skin,6
cornea,8
and
conjunctiva.9
10
Myofibroblasts form intercellular and
cell-to-stroma connections, which serve to transmit the contractile
force and impart some degree of organization to surrounding collagen
fibers.11
The formation of scar occurs by the deposition, maturation, and organization of newly synthesized collagen. Fibroblasts, the major source of collagen, become oriented in the wound bed and synthesize collagen fibers in a direction parallel to their orientation.12 The uniaxial orientation of collagen in scar tissue is different from the random alignment of collagen fibers in normal connective tissues.13
Previous studies in this laboratory have demonstrated that a highly porous chemical analogue of extracellular matrix, composed of type I collagen and chondroitin 6-sulfate (CG copolymer matrix), induces partial regeneration of the injured dermis in the human,14 15 the swine,16 and the guinea pig.17 18 Implantation of the CG copolymer in these models resulted in the inhibition of wound contraction and the regeneration of a nearly physiological dermis. Inhibition of dermal wound contraction by CG matrices may be simply interpreted as the interruption of alignment of contractile myofibroblasts and collagen fibers in the wound bed by the randomly oriented pore walls of the matrix.19
In the present study, we attempted to modulate the healing process
after wounding of the rabbit conjunctiva by implanting a porous CG
copolymer graft. We hypothesized that the porous CG copolymer would
reduce scarring and inhibit wound contraction and shortening of the
fornix. We established an anatomically constant wound model for
examining the healing of full-thickness conjunctival wounds. Healing
was evaluated by histology (hematoxylin and eosin [H&E], Masson’s
trichrome, polarized light, and -SMA immunohistochemistry) and by
the kinetics of wound contraction as measured by shortening of the
conjunctival fornix at the location of the wound.
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Methods |
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Surgical Procedure
All investigations conformed to the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research and were approved by the
Massachusetts Institute of Technology Committee on Animal Care.
Twenty-one female New Zealand albino rabbits weighing between 2.5 and
3.5 kg were anesthetized by intramuscular injection of ketamine (35
mg/kg) and xylazine (5 mg/kg). The eyelids were held open during
surgery by a speculum, and a surgical microscope was used during all
procedures. Two circular lesions were formed on the right eye of each
animal, located at the 10 o’clock (superior lateral) and 8 o’clock
(inferior lateral) positions at a distance of 2 mm away from the
corneal–scleral limbus (Fig. 1)
. A 7.0-mm diameter vacuum trephine (Katena, Denville, NJ) was used to
completely remove the conjunctival epithelium, substantia propria, and
the Tenon’s capsule down to the level of the bare sclera, taking care
to avoid damage to the underlying sclera. Occasionally, the trephine
did not completely sever the Tenon’s capsule. In these cases, Vannas
scissors were used to remove the remaining tissue. Slight retraction of
the wound edges resulted in a final wound diameter of approximately 8
mm. One of the two conjunctival wounds remained ungrafted without
further manipulation and served as a control. The remaining wound on
the same eye was grafted with a disc of CG copolymer 8.0 mm in diameter
and 2.0 mm thick, by using eight interrupted 8-0 polyglactin sutures
(Fig. 1)
. The location of the grafted wound and ungrafted control was
alternated among animals between the superior lateral and inferior
lateral positions. Topical 0.5% erythromycin ophthalmic ointment
(Bausch & Lomb, Tampa, FL) was applied immediately after surgery and
once daily for 1 week after the operation.
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The depth of the conjunctival fornix above each wound was measured immediately after surgery and at postoperative days 1, 7, 14, 21, and 28. Each animal was anesthetized, and a blunt probe was inserted into the fornix at the 8 o’clock and 10 o’clock positions directly above each wound. The probe depth in the fornix was determined by reading scale markings on the surface of the probe. The percentage of fornix shortening was calculated at any given time as the percentage decrease in fornix depth from the fornix depth measured immediately after surgery (day 0).
Histologic and Immunohistochemical Staining
Groups of rabbits were killed on day 1 (n = 4), day 7
(n = 4), day 14 (n = 5), and day 28 (n
= 6) for histologic examination. Intact eyes including eyelids
were removed en bloc by orbital exenteration and fixed by immersion in
4% formaldehyde overnight. The portion of each eye that included
either the grafted or ungrafted wound site, including the underlying
scleral bed, was dissected and embedded in paraffin. Sections were cut
on a microtome at 7 µm and stained with H&E for general cell
morphology and Masson’s trichrome to assess collagen deposition and
remodeling (reorganizing). Identification of specific cell types was
based on cell morphology observed in sections stained with H&E.
Polymorphonuclear neutrophils (PMNs) were identified by characteristic
multilobed nuclei and fine cytoplasmic granules. Eosinophils were
characterized by bilobed or multilobed nuclei and larger eosinophilic
granules. Basophils possessed less lobulated, pale nuclei with
cytoplasmic granules that were less numerous and variable in size.
Fibroblasts were characterized by elongated morphology and oval nuclei.
Slides processed for histology were also viewed under cross-polarizing
filters to visualize the organization and alignment of collagen fibers.
Additional tissue sections were stained with an antibody for -SMA to
identify cells with contractile potential (myofibroblasts). Briefly,
sections were deparaffinized, rehydrated, washed three times (5 minutes
each) with PBS, and blocked with 10% normal goat serum (Gibco,
Gaithersburg, MD). A monoclonal antibody for -SMA (Sigma, St. Louis,
MO) was used at a dilution of 1:400. To verify the specificity of the
primary antibody, adjacent tissue sections were similarly processed
except for the replacement of the primary antibody with nonimmune mouse
serum. Slides were then incubated with biotinylated goat anti-mouse IgG
(Sigma) diluted 1:400, reacted with avidin-biotin complex (Vector,
Burlingame, CA), and developed with AEC chromogen (Sigma), which yields
a red-brown color indicating positive stain.
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Results |
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-SMA was
negative in the wound bed of both control and grafted animals; however,
the smooth muscle cells and/or pericytes of blood vessels and the
ciliary muscle of the adjacent tissue stained positively. Few
fibroblasts were present in the wound bed, and deposition of new
collagen was not detected.
Day 7
A hypercellular condition was noted in ungrafted wounds at day 7,
consisting predominantly of inflammatory cells with occasional
elongated cells resembling fibroblasts. The inner one third to one half
of the grafted CG copolymer adjacent to the sclera was infiltrated by
inflammatory cells and fibroblasts from the wound bed (Fig. 2) . A thin epithelial layer had almost completely covered ungrafted
control wounds by 7 days. A few epithelial cells with prominent
secretory vesicles, morphologically resembling goblet cells, were
present. Only the edges of the grafted wounds were covered with a thin
layer of epithelium. The CG copolymer graft was partially degraded at 7
days, and the remaining material (approximately 70% of initial area)
was grossly visible in the wound bed. Although some degradation of the
peripheral edges of CG copolymer grafts was observed at this time, the
pore structure of the remaining graft material remained intact, as
observed in histologic sections (Fig. 2)
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-SMA revealed that
elongated cells with fibroblastic morphology stained positive for
-SMA at the edge of ungrafted wounds. In grafted wounds, cells at
the wound edge as well as cells that had infiltrated the matrix stained
for -SMA.
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Day 14
The epithelial layer above ungrafted wounds was slightly thicker
than that observed at day 7. Re-epithelialization over the CG copolymer
was complete by 14 days, and a few gobletlike cells were observed near
the wound margin. Degradation of the CG copolymer grafts was more
complete at day 14 (approximately 45% of initial area by gross
observation) than at day 7.
At day 14, ungrafted wounds continued to be populated by inflammatory
cells and fibroblasts, with the fibroblast becoming the more abundant
cell type. Grafted wounds exhibited a hypercellular condition, with
PMNs persisting in the regions of undegraded matrix. At postoperative
day 14, closure of ungrafted wounds by contraction continued, evidenced
by forniceal shortening (23.3% ± 3.3%, Table 1
and Fig. 3 ). Grafted
wounds exhibited significantly less contraction (P <
0.001) than ungrafted wounds, evidenced by comparatively decreased
fornix shortening (6.9% ± 2.4%, Table 1
and Fig. 3
). Staining for
-SMA was positive at the wound edge and in the middle of ungrafted
wounds (Fig. 4)
. Positive-stained cells in ungrafted wounds were predominantly aligned
parallel to the sclera surface. Staining for -SMA in grafted wounds
was positive at the wound edge and within undegraded CG copolymer (Fig. 4)
. -SMA–positive cells in grafted wounds displayed a more random
orientation, after the irregular contour of the matrix pore walls.
Staining was negative in regions where degradation of the CG copolymer
was complete (Fig. 5)
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Day 28
Clinical observation of ungrafted animals at 28 days showed an
apparent thinning and depression of the conjunctiva at the wound site.
A slight increase in conjunctival opacity was also observed. In two of
six ungrafted animals, there was notable distortion of the intact
conjunctiva surrounding the ungrafted wounds. Symblepharon was not
present in either ungrafted or grafted wounds. Matrix-grafted wounds
did not exhibit thinning or distortion of the conjunctiva, but a slight
increase in opacity at the wound site was observed at day 28.
In contrast to earlier time points, day 28 ungrafted wounds exhibited hypocellular appearance and no evidence of a persisting acute inflammatory response. The CG copolymer in grafted wounds was almost completely degraded (approximately 5% of initial area) and was barely distinguishable in the wound bed. Acute inflammatory cells were identified at day 28 in the small regions of undegraded CG copolymer. Both ungrafted and grafted wounds were completely covered by an epithelial layer 2 to 3 cells thick. A few gobletlike cells were present above both peripheral and central regions of the wounds in both groups.
Neither ungrafted nor grafted 28-day-old wounds showed significant
forniceal contraction in addition to that observed at day 14
(P > 0.6). At 28 days, ungrafted wounds showed 26.4%
± 5.0% fornix shortening, whereas grafted wounds showed 6.8% ±
3.2% fornix shortening (Table 1
and Fig. 3
). The difference between
groups was statistically significant (P < 0.01). At
day 28, -SMA staining was negative in the middle and edge of
ungrafted wounds with the exception of pericytes and the smooth muscle
cells of blood vessels. Staining for -SMA was no longer observed at
the edges of grafted wounds but remained positive within undegraded
regions of CG copolymer. Staining was negative in regions of degraded
CG copolymer, which represented the majority of the grafted wound bed.
Collagen fibers in day 28 ungrafted wounds retained their linear alignment, but were more densely packed than those in day 14 wounds (Fig. 2) . The dense, aligned collagen displayed intense birefringence when viewed using polarized light (Fig. 7) . Fibers tended to be highly aligned between the conjunctival epithelium and the sclera surface. In contrast, collagen fibers in grafted wounds were more randomly oriented (Fig. 2) and exhibited only sparse, discontinuous birefringence (Fig. 7) . The density and organization of collagen in the grafted wounds was similar to that of the subepithelial connective tissue (stroma) of the normal conjunctiva (Fig. 7) .
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionConclusionReferences |
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The purpose of this study was to examine spontaneous healing of the damaged conjunctiva and to attempt to modify the wound healing response by implantation of a porous CG copolymer matrix. Previous studies have demonstrated that a similar implant inhibited wound contraction and prevented scar formation in animal models of dermal wound healing,16 17 18 as well as in the human.14 15 In the present study, we focused on four aspects of wound healing: inflammation, re-epithelialization, wound contraction, and the deposition and organization of collagen in the wound bed.
Inflammatory and Immune Responses
Cells that are characteristic of an acute inflammatory response
were present in both ungrafted and grafted wounds at days 1 and 7.
Acute inflammatory cells subsided by day 14 in ungrafted wounds but
persisted until day 28 in regions of undegraded CG copolymer in grafted
wounds. Because these cells were not present in regions where the
matrix had degraded, it is expected that the inflammatory response
would have eventually subsided when the CG material had been completely
resorbed.
No excessive accumulation of plasma cells or lymphocytes was identified in the vicinity of the CG copolymer at any time point (data not shown), indicating that there was no immune rejection of the bovine collagen or the chondroitin 6-sulfate components of the CG copolymer. The CG copolymer has not elicited persistent acute inflammatory response or immune rejection when implanted into a number of tissues, including skin,16 17 18 peripheral nerve,35 and spinal cord.36
Re-epithelialization
At 7 days after surgery, re-epithelialization of ungrafted wounds
was nearly complete, whereas only the edges of grafted wounds were
covered with epithelium. The more prominent contraction of ungrafted
wounds had decreased the wound size and may have allowed
re-epithelialization to occur in a shorter time. In addition, the
irregular surface of the porous CG copolymer may not have provided a
favorable substrate for the migration of epithelial cells over the
graft. By 28 days after surgery, a complete epithelial layer that
contained gobletlike cells covered both ungrafted and grafted wounds.
Our findings concerning the time scale of re-epithelialization of
ungrafted control wounds are consistent with other studies in which
smaller, 4-mm diameter, full-thickness conjunctival wounds were
completely re-epithelialized after 7 days and contained goblet cells
after 14 days.37
Wound Contraction
In the present study, partial closure of ungrafted and grafted
conjunctival wounds occurred by contraction during the period when
myofibroblasts were present in the middle of the wound and at the wound
edge.
Our data indicate that contraction (as measured by forniceal
shortening) of both ungrafted and grafted wounds was most active during
the period up to 14 days after surgery (Fig. 3)
, reaching an apparent
plateau thereafter. We observed the presence of myofibroblasts at the
edges of ungrafted and grafted wounds at days 7 and 14. Myofibroblasts
were no longer present at the wound edge in both groups by day 28, with
the exception of a few labeled cells in the undegraded matrix in
grafted wounds. The correlation between the active period of wound
contraction (between days 1 and 14) and the presence of
-SMA–positive fibroblasts in both ungrafted and grafted wounds
lends evidence to the hypothesis that myofibroblasts actively
participate in conjunctival wound contraction. The appearance and
disappearance of myofibroblasts have been shown by other investigators
to coincide with the active phase and cessation of contraction during
healing of skin wounds.6
38
39
Similar results were
observed in a model of contraction of glaucoma filtering blebs, where
myofibroblasts were identified in the bleb peripheral margin 10 days
after surgery.10
There is also substantial evidence that
fibroblasts that do not express the -SMA-positive phenotype may be
involved in wound contraction.40
41
These findings provide
a possible explanation for the day 1 forniceal shortening of ungrafted
wounds (Fig. 3)
before -SMA staining was observed.
Compared with the ungrafted group, the presence of the CG copolymer in grafted wounds significantly reduced wound contraction, evidenced by statistically significant differences in fornix shortening at days 7, 14, 21, and 28 (P < 0.05 for all time points). The presence of the CG copolymer may have inhibited contraction of grafted wounds during the early stages when a significant amount of undegraded CG copolymer matrix was present in the wound bed. Previous studies of the inhibition of wound contraction by CG copolymer matrix in a dermal wound model have led to the hypothesis that the random porous structure CG copolymer interrupts the continuity of myofibroblasts in the wound, thus preventing contraction.19
Collagen Deposition and Organization
Numerous investigators have studied the role of myofibroblasts in
contraction and alignment of collagen during healing of various tissue
types including skin,42
43
cardiac muscle,44
tendon,45
and conjunctiva.10
The pattern of
collagen deposition as well as the subsequent cross linking and
remodeling of collagen are factors that may determine the final
collagen configuration and the functional characteristics of repair
tissue or scar.46
In this study, initial deposition of new
collagen in the ungrafted wound was found at day 7. At day 28, mature
subepithelial fibrous scar was identified in ungrafted wounds by its
dense linear alignment of collagen47
and its hypocellular
condition.48
49
It is interesting to speculate that over
the time course of this study myofibroblasts may have been involved in
imparting such a high degree of organization to the collagen fibers.
Dermal scar, which is composed of highly aligned collagen fibers, has
mechanical properties distinctly different from those of normal
dermis.50
We would therefore expect that the randomly
oriented collagen fibers of the matrix-grafted wounds would exhibit
mechanical behavior dissimilar to that of aligned collagen fibers of
scar tissue.
We observed that fibroblasts within the CG copolymer matrix tended to adhere to the randomly oriented pore walls and thus adopt a random orientation (Figs. 2 4) . In other models of wound healing, fibroblasts deposit collagen in a direction parallel to their alignment.12 It is not surprising that the random orientation of collagen fibers in day 28 matrix-grafted wounds reflects the random orientation of cells in the matrix pores at earlier time points. The open pore structure of the CG copolymer provided space for fibroblast ingrowth, and the irregular geometry of the pore walls may have disrupted the linear pattern of collagen deposition by fibroblasts.
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Conclusion |
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Footnotes |
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Supported in part by an unrestricted grant from the Research to Prevent Blindness Foundation.
Submitted for publication December 2, 1999; revised February 15, 2000; accepted February 28, 2000.
Commercial relationships policy: N.
Corresponding author: Correspondence to: Ioannis V. Yannas, Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 3-332, Cambridge, MA 02139. yannas{at}mit.edu
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
4 for all groups). For both wounds, most forniceal shortening
occurred during the first 14 days, with little additional change
occurring thereafter. Ungrafted wounds exhibited significantly more
shortening than grafted wounds at days 7, 14, 21, and 28.
