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Received: 25 July 2016 Revised: 15 December 2016 Accepted: 29 March 2017
DO
I: 10.1002/term.2434
R E S E A R CH AR T I C L E
Bilayer silk fibroin grafts support functional oesophageal repairin a rodent model of caustic injury
FIGURE 1 Characterization of alkali oesophageal injury and oesophageal stenosis. (a) Photomicrographs of isolated oesophageal segment prior toand 2 days following transient exposure to 40% NaOH. Scale bars = 7 mm. (b) representative cross‐sections of injured oesophageal segmentdescribed in (a) following MTS staining (top row) and IHC analyses (bottom row). Top row: Scale bars = 1.25 mm for gross and 400 μm for magnifiedviews. Bottom row: Respective marker expression is displayed in red (Alexa Fluor 594 labelling) and blue denotes DAPI nuclear counterstain. Scalebars = 200 μm. (c) representative three‐dimensional images acquired by μ‐CT analysis of injured oesophagus described in (a) following contrastagent (red) gavage. Injured site denoted in yellow with blue labels denote proximal/distal marking sutures of isolated segment. E = oesophagus;S = stomach. (d) quantification of luminal oesophageal cross‐sectional areas from the central region of the injury site (ST) described in (c) ornoninjured reference point adjacent toT7 (*) = p < 0.05 in comparison toT7 control region [Colour figure can be viewed at wileyonlinelibrary.com]
4 ALGARRAHI ET AL.
(11/12) with mortality from obstruction secondary to fur ingestion
noted in one replicate at 1 month following grafting. None of the
surviving rats in either group displayed clinical symptoms of dysphagia
over the course of the study period. In addition, both experimental
cohorts were capable of solid food consumption after a 3‐day liquid
diet and exhibited comparable degrees of weight gain by 2 months
postoperatively (Figure 2A). Global tissue evaluations at 2 months
postoperatively of both nondiseased and injured implant groups
revealed prominent host tissue ingrowth throughout the original graft
sites with no evidence of significant axial contraction between proxi-
mal and distal marking sutures (Figure 2B). Endpoint μ‐CT evaluations
of repaired injury sites revealed preservation of organ continuity with
no evidence of anatomical anomalies (Figure 2C). In comparison to
baseline levels prior to scaffold implantation, repaired diseased
conduits displayed a significant 4.5‐fold increase in luminal cross‐
sectional area, which was statistically similar to nonsurgical controls
(Figure 2D). These results highlight the ability of BLSF grafts to
promote de novo tissue formation and restore function at caustic
oesophageal injury sites.
Constructive tissue remodelling and host tissue responses were
temporally evaluated in damaged oesophagi following BLSF matrix
implantation by MTS analysis (Figure 3). At 1 week postoperatively, a
fibrovascular scar populated by mononuclear inflammatory cells as well
as myofibroblasts was evident at graft sites and lined by a stratified
squamous, keratinized epithelium. The de novo oesophageal wall
contained residual scaffold fragments with putative sites of macro-
phage phagocytosis located around the perimeters. Invasion of host
skeletal muscle fibres and smooth muscle bundles were localized at
the peripheral boundaries of the neotissues. At 1 months postimplan-
tation, an ECM‐rich lamina propria had developed and both the de
novo muscularis mucosa and muscularis externa had further integrated
into the graft region. In addition, organization of the muscularis externa
into circular and outer longitudinal skeletal muscle layers was apparent
at the edges of the neotissues; however, fibrosis still persisted toward
internal areas. The de novo muscularis externa at 2 months following
repair of injured sites demonstrated a qualitative increase in skeletal
muscle density within the central regions of the graft site in compari-
son to early timepoints. Areas of fibrosis had also diminished at this
stage of regeneration and no chronic inflammatory reactions were
noted. The structural architecture of neotissues generated in the set-
ting of caustic damage was qualitatively similar to the nondiseased
repair group at 2 months postoperatively. However, maturation of
the muscularis mucosa and muscularis externa compartments in both
these cohorts was notably underdeveloped in respect to nonsurgical
controls. Finally, the temporal stages of wound healing encountered
during repair of caustic injury sites with BLSF scaffolds were found
to mimic the regenerative responses previously observed in our
nondiseased model of oesophagoplasty (Algarrahi et al., 2015).
FIGURE 2 Evaluation of neotissue formation and oesophageal function in injured and repaired groups. (a) body weight evaluations of nondiseased(BLSF‐ND) and diseased (BLSF‐ST) animals over the course of the 2 months of scaffold implantation period. Means ± standard deviation per datapoint. (b) Neotissues present within the original graft sites at 2 months postoperatively. Proximal/distal and lateral marking sutures are respectivelydesignated by red and black arrows. (c) representative three‐dimensional μ‐CT images of oesophagi 2 days after NaOH exposure (ST) and in BLSF‐ND and BLSF‐ST groups at 2 months postoperatively detailed in (b) following contrast agent (red) gavage. Repaired sites in nondiseased anddiseased groups are coded in yellow while proximal/distal marking sutures are colored in blue. E = oesophagus. S = stomach. (d) quantification ofluminal oesophageal cross‐sectional areas in yellow regions described in (c) and in nonsurgical controls (NSC). (*) = p < 0.05 in comparison to allother groups [Colour figure can be viewed at wileyonlinelibrary.com]
FIGURE 3 MTS analyses of constructive remodelling at implant sites in experimental cohorts. (first row) Photomicrographs of gross oesophagealcross‐sections stained with MTS from nondiseased (BLSF‐ND) and diseased (BLSF‐ST) groups containing original graft site (bracketed) as well asnonsurgical controls (NSC). Scale bars = 1.25 mm. (second row) magnification of global regenerative areas (RA) bracketed in first row or the nativetissue in NSC. Scale bars = 400 μm. (third row) magnified boxed green area from second row profiling muscularis externa development. (fourth row)magnified boxed red area from second row detailing epithelial formation. Scale bars for third and fourth rows = 100 μm [Colour figure can beviewed at wileyonlinelibrary.com]
ALGARRAHI ET AL. 5
IHC (Figure 4A) and parallel histomorphometric (Figure 4B)
evaluations were executed on experimental groups to characterize
further the phases of regeneration during repair of damaged regions
and compare the degree of tissue maturation achieved between
diseased and nondiseased counterparts. Pan‐CK+ epithelia were first
observed spanning reconstructed injury sites at 1 week following
FIGURE 4 Immunohistochemical and histomorphometric assessments of neotissues and controls. (a) Photomicrographs of protein expressionpatterns in repaired, nondiseased (BLSF‐ND) and diseased (BLSFST) graft sites as well as nonsurgical controls (NSC) of epithelia markers, pan‐CK, CK14, CK4, FG; endothelial and innervation markers, CD31 and SYP in mucosa; and contractile muscle markers, α‐SMA and MYH in muscularismucosa and muscularis externa, respectively. Arrowheads indicate SYP+ boutons. For all panels, respective marker expression is displayed in red(Alexa Fluor 594 labelling), green (Alexa Fluor 488 labelling), or white (Alexa Fluor 647 labelling) and blue denotes DAPI nuclear counterstain. For allpanels, scale bars = 200 μm. (b) Histomorphometric analyses of marker expression in regenerated tissues as well as NSC. (*) = p < 0.05 incomparison to respective NSC. (#) = p < 0.05 in comparison to all other groups [Colour figure can be viewed at wileyonlinelibrary.com]
6 ALGARRAHI ET AL.
matrix grafting. By 2 months postoperatively, no significant differences
in the extent of pan‐CK+ epithelia were noted between implant groups
or nonsurgical controls. In addition, distinct epithelial subpopulations
were present in all experimental groups and consisted of CK14 + basal
cells, polygonal CK4+ suprabasal cells, and flattened FG+ superficial
cells. Regenerated vascular networks containing vessels lined with
CD31+ endothelial cells were found in both nondiseased and diseased
graft sites at all examined timepoints. The mean vessel density was
significantly higher in consolidated tissues from all groups in respect
to nonsurgical control levels indicating an ongoing stage of tissue
FIGURE 5 Ex vivo contractility and relaxation behaviours in controls and repaired oesophageal segments. Contractile responses to (a) electric fieldstimulation (2–25 Hz) and (b) KCl (80 mM) in circular oesophageal rings from injured sites 2 days after NaOH exposure (ST), repaired segments fromnondiseased (BLSF‐ND) and diseased (BLSF‐ST) groups at 2 months postoperatively, as well as nonsurgical controls (NSC). Inset in (b) reflectsmagnification of injured and experimental responses. (c) relaxation activities in response to isoproterenol (10 μM) in cohorts detailed in (a)precontracted with carbachol (3 μM). Means ± standard errors per data point. (*) = p < 0.05 in comparison to ST group [Colour figure can be viewedat wileyonlinelibrary.com]
ALGARRAHI ET AL. 7
groups at 2 months postoperatively compared to nonsurgical control
levels. Characterization of the regenerated muscularis externa in
neotissues demonstrated the extent of MYH+ skeletal muscle in
diseased and nondiseased cohorts had respectively achieved 67%
and 76% of control values by 2 months of scaffold implantation. In
contrast to other tissue components, animals subjected to caustic
injury displayed a reduced capacity to support regeneration of the
muscularis mucosa in respect to noninjured subjects. The density of
α‐SMA+ smooth muscle bundles in this compartment was significantly
lower in the diseased setting reflecting 33% of nonsurgical control
levels by 2 months postoperatively, while nondiseased counterparts
were capable of supporting 67%. Taken together, these data
demonstrate the ability of BLSF grafts to promote the formation of
innervated, vascularized oesophageal tissues at sites of caustic injury;
however, the propensity for muscularis mucosal regeneration is muted
in comparison to nondiseased microenvironments.
Peristaltic waves generated from radially symmetrical contraction
and relaxation of oesophageal circular muscle is an essential mecha-
nism for propagation of food bolus through the digestive tract.
Contractile behaviours in repaired conduits from each implant group
as well as nonsurgical controls and injured segments prior to scaffold
implantation were assessed in ex vivo organ bath studies following
stimulation with EFS (Figure 5A) and KCl (Figure 5B). In addition,
relaxation properties of experimental groups precontracted with car-
bachol were analysed following isoproterenol treatment (Figure 5C).
Alkali injury to oesophageal tissues significantly decreased both con-
tractile and relaxation responses to all stimuli tested in comparison to
nonsurgical controls. These results are consistent with our histological
findings demonstrating extensive caustic damage to oesophageal mus-
cular components following transient exposure to NaOH. Following
2 months of BLSF matrix grafting, both repaired nondiseased and
diseased conduits demonstrated the ability to contract in response to
EFS and KCl stimulation to similar extents. The magnitude of KCl and
EFS‐induced force generation in these groups was substantially higher
than in injured controls, but less than nonsurgical counterparts, sug-
gesting partial recovery of contractile machinery elicited by membrane
depolarization as well as neuronal input, respectively. Parallel analysis
of relaxation patterns in experimental groups demonstrated similar
trends as observed for contractile responses.
4 | CONCLUSIONS
The data presented in this study demonstrate the ability of BLSF grafts
to promote functional repair of caustic injury sites in a rat model of
onlay oesophagoplasty. Reconstruction of damaged tissues with BLSF
also supported through the vision and generosity of the Rainmaker
Group in honor of Dr Alan Retik, MD. We also acknowledge Dr Bryan
Sack, MD and Mr Kyle Costa BSc for technical support.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
AUTHOR CONTRIBUTIONS
JRM and KA designed and conceptualized this study. Acquisition of
data was carried out by KA, DF, VC, XY, AS, SA, FMS and MPS.
Analysis and interpretation of data was done by JRM, KA, DF, VC,
AS and MPS. Manuscript was drafted by JRM, KA, VC and, MPS, while
VC, MPS, CRE and JRM performed critical revision of the manuscript
for important intellectual content. Statistical analysis was done by
KA, VC and MPS. Funding was obtained by JRM and CRE. The entire
study was supervised by JRM.
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How to cite this article: Algarrahi K, Franck D, Savarino A, et
al. Bilayer silk fibroin grafts support functional oesophageal
repair in a rodent model of caustic injury. J Tissue Eng Regen