10.14639/0392-100X-N0566
Chronic rhinosinusitis with nasal polyposis is a disease characterised by a mechanical dysfunction that facilitates anomalous growth of the nasal mucosa, due to its unique remodelling process. The development of procedures capable of interfering with this process is thus of the utmost importance. This study deals with the evaluation of the effects of cross-linking upon the nasal polyp tissue extracellular matrix. Six patients undergoing surgery for polypectomy were selected. The riboflavin/UVA collagen cross-linking technique was applied to the surface of epithelialised and de-epithelialised resected polyps of the intervention group. The control group polyps were not submitted to cross-linking procedures. Ultrathin polyp tissue sections (8 μm) were processed for immunofluorescence with mouse anti-type I collagen antibody and AlexaFluor 488 conjugated secondary antibody plus DAPI counterstaining, and analysed by confocal microscopy. The effect of riboflavin/ UVA collagen cross-linking was visible on confocal fluorescence microscopy. Quantitative morphology was associated with fluorescence imaging analysis, and pixel density and brightness were evaluated. The surface of treated polyps exhibited a higher density of collagen fibres compared to control polyps, as could be observed both visually and through objective measurements of the fluorescent regions. The effect was enhanced on the surface of the de-epithelialised polyps. The higher density of collagen fibres exhibited by the deepithelialised treated polyps demonstrates the feasibility of this technique in interfering with the remodelling process and the mechanical dysfunction found in chronic rhinosinusitis with nasal polyposis.
application/xml
1827-675X
566
6
en
Luiz Carlos Gregório
https://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
sinusitis
Structural changes in the extracellular matrix after cross-linking of nasal polyp tissue
Text.Serial.Journal
https://www.actaitalica.it/
40
Rhinology
2021-01-21
2019-12-11
2021-01-21
2021-01-21
1
Luiz Carlos Gregório
2020
10.14639/0392-100X-N0566
ENT Research Laboratory, Department of Otolaryngology-Head and Neck Surgery, Federal University of Sao Paulo, Sao Paulo, Brazil
1827-675X
6
sinusitis
en
Structural changes in the extracellular matrix after cross-linking of nasal polyp tissue
https://www.actaitalica.it/article/download/566/495
40
ACTA OTORHINOLARYNGOLOGICA ITALICA 2020;40:426-434; doi: 10.14639/0392-100X-N0566




Rhinology


Structural changes in the extracellular matrix
after cross-linking of nasal polyp tissue
Cambiamenti strutturali della matrice extracellulare dopo cross-linking del tessuto
polipoide nasale
Nathalia Coronel1*, Rogério Pezato1*, Jônatas Bussador do Amaral1, Leandro Ticlia2, Thiago Freire Pinto Bezerra3,
Lígia Ferreira Gomes2, Juliana Dreyfuss4, Wallace Chamon5,6, Luiz Carlos Gregório1
1
  ENT Research Laboratory, Department of Otolaryngology-Head and Neck Surgery, Federal University of Sao Paulo, Sao Paulo,
Brazil; 2 Laboratory of Microrheology and Molecular Physiology, Institute of Physics, University of São Paulo-USP, São Paulo, SP,
Brazil; 3 Department of Otolaryngology, Federal University of Pernambuco-UFPE, Recife, PE, Brazil; 4 Department of Biochemistry,
Federal University of São Paulo-Unifesp, São Paulo, SP, Brazil; 5 Department of Ophthalmology and Visual Sciences, Federal
University of São Paulo-Unifesp, São Paulo, SP, Brazil; 6 Department of Ophthalmology and Visual Sciences, College of Medicine,
University of Illinois at Chicago, Chicago, IL, United States
*
  N. Coronel and R. Pezato contributed equally to this work.


SUMMARY
Chronic rhinosinusitis with nasal polyposis is a disease characterised by a mechanical
dysfunction that facilitates anomalous growth of the nasal mucosa, due to its unique re-            Received: December 11, 2019
modelling process. The development of procedures capable of interfering with this process           Accepted: August 4, 2020
is thus of the utmost importance. This study deals with the evaluation of the effects of
cross-linking upon the nasal polyp tissue extracellular matrix. Six patients undergoing sur-        Correspondence
gery for polypectomy were selected. The riboflavin/UVA collagen cross-linking technique             Rogério Pezato
was applied to the surface of epithelialised and de-epithelialised resected polyps of the           ENT Research Lab. Department of Otorhinolaryn-
intervention group. The control group polyps were not submitted to cross-linking proce-             gology - Head and Neck Surgery, Federal Universi-
dures. Ultrathin polyp tissue sections (8 μm) were processed for immunofluorescence with            ty of Sao Paulo. Rua Pedro de Toledo 781, 1o andar,
mouse anti-type I collagen antibody and AlexaFluor 488 conjugated secondary antibody                sala 3 - Vila Clementino - São Paulo - SP - Brazil
                                                                                                    CEP 04039-032
plus DAPI counterstaining, and analysed by confocal microscopy. The effect of riboflavin/
                                                                                                    Tel. +55 11 5576-4848 PABX number:17267
UVA collagen cross-linking was visible on confocal fluorescence microscopy. Quantita-               E-mail: pezatobau@gmail.com
tive morphology was associated with fluorescence imaging analysis, and pixel density and
brightness were evaluated. The surface of treated polyps exhibited a higher density of col-
                                                                                                    Funding
lagen fibres compared to control polyps, as could be observed both visually and through             This work was supported by Coordenação de
objective measurements of the fluorescent regions. The effect was enhanced on the surface           Aperfeiçoamento de Pessoal de Nível Superior
of the de-epithelialised polyps. The higher density of collagen fibres exhibited by the de-         (CAPES) PROAP
epithelialised treated polyps demonstrates the feasibility of this technique in interfering
with the remodelling process and the mechanical dysfunction found in chronic rhinosinusi-           Conflict of interest
tis with nasal polyposis.                                                                           The Authors declare no conflict of interest.
KEY WORDS: paranasal sinus disease, chronic disease, rhinosinusitis, chronic rhinosinusitis,
sinusitis                                                                                             How to cite this article: Coronel N, Pezato R, do
                                                                                                      Amaral JB, et al. Structural changes in the extra-
                                                                                                      cellular matrix after cross-linking of nasal polyp
RIASSUNTO                                                                                             tissue. Acta Otorhinolaryngol Ital 2020;40:426-
La rinosinusite cronica con poliposi nasale è una malattia caratterizzata da una disfun-              434. https://doi.org/10.14639/0392-100X-N0566
zione meccanica che facilita la crescita anomala della mucosa nasale, a causa del suo
peculiare processo di rimodellamento. Questo studio si propone di valutare gli effetti del          © Società Italiana di Otorinolaringoiatria
cross-linking sulla matrice extracellulare del tessuto del polipo nasale. Sono stati sele-          e Chirurgia Cervico-Facciale
zionati sei pazienti sottoposti ad intervento chirurgico di polipectomia. È stata applicata
la tecnica del cross linking del collagene con riboflavina / UVA alla superficie dei polipi                        OPEN ACCESS
resecati epitelizzati e disepitelizzati del gruppo di pazienti sottoposti ad intervento. I polipi   This is an open access article distributed in accordance with
del gruppo di controllo non sono stati sottoposti a procedure di cross linking. Le sezioni          the CC-BY-NC-ND (Creative Commons Attribution-Non-
ultrasottili (8 μm) di tessuto polipoide sono state analizzate mediante immunofluorescenza          Commercial-NoDerivatives 4.0 International) license. The
                                                                                                    article can be used by giving appropriate credit and mentio-
con anticorpi anti collagene di tipo I di topo e anticorpi secondari coniugati AlexaFluor           ning the license, but only for non-commercial purposes and
488 con controcolorazione DAPI e analizzate mediante microscopia confocale. L’effetto               only in the original version. For further information: https://
del cross-linking è risultato visibile al microscopio confocale a fluorescenza. La morfologia       creativecommons.org/licenses/by-nc-nd/4.0/deed.en




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quantitativa è stata associata all’analisi di imaging in fluorescenza e sono state valutate la densità e la luminosità dei pixel. La superficie dei
polipi trattati mostrava una maggiore densità di fibre di collagene rispetto ai polipi di controllo, come è stato possibile osservare sia visiva-
mente che attraverso misurazioni oggettive delle regioni fluorescenti. L’effetto è stato potenziato sulla superficie dei polipi disepitelizzati. La
maggiore densità di fibre di collagene esibita dai polipi trattati disepitelizzati dimostra la fattibilità di questa tecnica per interferire con il
processo di rimodellamento e affrontare la disfunzione meccanica riscontrata nella rinosinusite cronica con poliposi nasale.
PAROLE CHIAVE: malattia dei seni paranasali, patologia cronica, rinosinusite, rinosinusite cronica, sinusite



Introduction                                                               was associated with a decrease in polyp volume and milder
                                                                           nasal-obstruction symptoms 9. Considering the close inter-
Chronic rhinosinusitis with nasal polyposis (CRSwNP) is                    dependence between the IHP response and ECM composi-
characterised by chronic inflammation of the nasal mucosa                  tion, the remodelling process is an essential target in ap-
with an altered remodelling process, which affects extra-                  proaching the mechanical dysfunction of CRSwNP.
cellular matrix (ECM) formation and facilitates mechanical                 It is reasonable to speculate that not only would changes
dysfunction and oedema formation 1.                                        in ECM composition be capable of altering the mechanical
The pathogenesis of CRSwNP is still not fully under-                       properties of the tissue, but also that structural changes in
stood 2,3; however, its foundation is the aforementioned                   the existing ECM could affect tissue mechanical properties.
chronic inflammatory process, characterised by a major im-                 In ophthalmology, the riboflavin/UVA cross-linking tech-
balance in immunomodulation which increases inflamma-                      nique has been used to alter ECM conformation and thus
tory cells (such as dendritic cells, eosinophils, and T helper             prevent the progression of corneal bulging in keratoconus,
cells) and decreases immunoregulatory cells (such as regu-                 a disease characterised by derangement of ECM deposition,
latory T cells), as well as an impaired immunomodulation                   which facilitates change in the shape of the cornea 10-12. In
of mesenchymal stem cells 4,5.                                             the same way, the strengthening of the structures that com-
Studies on the mechanical forces involved in nasal polyp                   pose the ECM in nasal polyp mucosa might impact the re-
development have been conducted in an attempt to at least                  modelling process and act on the biophysics mechanisms
partly elucidate the pathophysiology of CRSwNP 1,6,7.                      involved in the nasal polyp development 13.
Changes in the mechanical response of the nasal mucosa                     In this context, we evaluated the effect of this cross-linking
have been observed in patients with CRSwNP compared                        technique upon polyp stromal collagen in patients with
to the healthy mucosa of the middle meatus, with a loss                    CRSwNP.
of effectiveness in adequately increasing the interstitial hy-
drostatic pressure (IHP) in response to water inflow during
the inflammatory process 1.                                                 Materials and methods
This discovery provides a new perspective for biome-                       This study was approved by the University of São Paulo
chanical management of CRSwNP 1 beyond treatment of                        Research Ethics Committee, protocol number 2905802,
the inflammatory process (corticosteroids, non-steroidal                   and conducted in accordance with the provisions of the
anti-inflammatory drugs, leukotriene receptor antagonists,                 Helsinki Declaration.
anti-immunoglobulin antibodies and biological agents that                  Histological sections of nasal polyp tissue from 6 patients
modulate the inflammatory environment) 8.                                  with NP, diagnosed according to the European Position
Within this context, the study of the mechanical dysfunc-                  Paper on Rhinosinusitis and Nasal Polyps (EPOS) crite-
tion of CRSwNP has become increasingly relevant. It has                    ria, were evaluated 8. Patients between 18 and 60 years of
previously been shown that ECM composition interferes                      age, non-steroidal anti-inflammatory tolerant, non-smokers
with the biomechanical response of nasal mucosa. Nasal                     were selected. The exclusion criteria were degenerative or
mucosa with fibrosis (synechiae) and healthy nasal mid-                    autoimmune diseases, cystic fibrosis, pregnancy and topi-
dle meatus mucosa produce similar mechanical responses,                    cal or systemic corticosteroid therapy within the 4 weeks
suggesting that changes in ECM composition may contrib-                    preceding surgery for removal of nasal polyps.
ute to prevent oedema formation and, potentially, have an
impact on CRSwNP treatment 7.                                              Application of the cross-linking technique
Recent studies have confirmed that increased IHP contrib-                  Immediately after resection, polyp fragments were prepared
utes to a reduction of nasal polyps. In one investigation,                 and divided into two groups: control and intervention. The
the authors increased this pressure by imparting continu-                  control group received no intervention. In the intervention
ous positive airway pressure (CPAP) in nasal polyp tissue.                 group, riboflavin/UVA cross-linking was performed on the
Such indirectly increased interstitial hydrostatic pressure                epithelial surface of the polyps of patients 1, 2, 3, 4 and 5.



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In patient 6, the polyp was cut in half and the treatment ap-                          tions were obtained longitudinally (perpendicular to the ex-
plied to the de-epithelialised cut surface (Fig. 1).                                   posed surface), so that all slides included the UVA-exposed
In the intervention group, a photosensitising solution con-                            area and the underlying stroma.
taining 0.1% riboflavin-5-phosphate and 20% dextran
T-500 was applied to the surface of the polyp every 5 min-                             Eosinophil count
utes for 30 minutes to ensure saturation of the polyp stroma                           Part of the polyps was fixed using 10% acetaldehyde, main-
with riboflavin. After 30 minutes, UVA radiation (365 nm)                              tained for 24 hours at room temperature, and then preserved
was applied 45 mm from the polyp surface, for 30 minutes,                              in 70% alcohol for analysis. The previously fixed nasal tis-
using a solid-state device (X-Link, Opto Eletrônica, São                               sue portions were embedded in paraffin and 4-μm-thick
Carlos, Brazil) with a surface irradiance output of 3 mW/                              sections were obtained using a microtome. All sections
cm2 (Fig. 1). The energy applied was 5.375 J/cm2. Surface                              were affixed onto Superfrost Plus glass slides (Menzel Gla-
irradiation was ensured by continuous, microprocessor-                                 ser, Braunschweig, Germany) and dried at 60°C for a few
based, automated monitoring by the X-Link device, which                                hours. For deparaffinisation, slides were washed succes-
uses an internal power meter. After application of the cross-                          sively in xylene (3 times for 10 minutes), 100% ethanol (2
linking technique, polyps were frozen at -80°C.                                        times for 5 minutes), 90% ethanol (2 times for 5 minutes)
Ultrathin cryostat sections (8 μm, -21°C) of treated and un-                           and 70% ethanol (2 times for 5 minutes). The nuclei were
treated polyps were obtained and analysed.                                             stained with alum haematoxylin (Lillie-Mayer’s solution)
In the first four patients, sections were made parallel to the                         for 5 minutes and rinsed in running tap water. Differen-
surface exposed to riboflavin/UVA. In patients 5 and 6, sec-                           tiation was performed with 0.3% acid alcohol, sections
                                                                                       were rinsed again in running tap water and, subsequently,
                                                                                       in Scott’s tap water substitute (sodium hydrogen carbonate
                                                                                       10 g, magnesium sulphate 100 g, distilled water 5 L).
                                                                                       After rinsing in tap water, sections were stained with eosin
                                                                                       solution (1% eosin Y 400 mL, 1% aqueous phloxine 40 mL,
                                                                                       95% alcohol 3100 mL and glacial acetic acid 16 mL) for
                                                                                       2 minutes, dehydrated, and cleared.
                                                                                       Histological examination was performed by a pathology
                                                                                       expert through a Leica DM2000 binocular microscope at
                                                                                       400x magnification. The absolute number of eosinophils
                                                                                       per high-power field (HPF) was counted in an average of 10
                                                                                       independent fields of view selected from the most inflamed
                                                                                       area of tissue.

                                                                                       Processing for immunofluorescence
                                                                                       The slides were washed with phosphate-buffered saline
                                                                                       (PBS), covered with 2.5% PBS and albumin solution, and
                                                                                       stored in a moisture chamber under refrigeration. After 2
                                                                                       hours, they were incubated with primary antibody (anti-
                                                                                       type I collagen, Calbicochem, Darmstadt, Germany) at
                                                                                       1:50 concentration in PBS containing 1% bovine serum
                                                                                       albumin and 0.1% saponin.
                                                                                       The next day, the slides were again rinsed with PBS and
                                                                                       incubated with secondary antibody (AlexaFluor 488 con-
                                                                                       jugated anti-mouse IgG, Molecular Probes, Carlsbad, CA)
                                                                                       at 1:300 concentration for 30 minutes. Next, slides were
                                                                                       washed with ice-cold PBS and incubated with 1:2500 DA-
                                                                                       PI (4,6-diamidino-2-phenylindole dihydrochloride, Molec-
                                                                                       ular Probes) in a PBS solution containing 0.1% saponin, for
Figure 1. Polyp and area of riboflavin/UVA application. (A) In patients 1, 2, 3,
                                                                                       30 minutes, to ensure nuclear staining. Finally, slides were
4, and 5: on the epithelialised surface of the polyp. (B) In patient 6: polyp cut in   washed again with PBS and mounted with Fluoromount-
half and treatment applied on the de-epithelialised cut surface.                       G liquid medium (Electron Microscopy Sciences, Hatfield,



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PA) and, after 12 hours, were sealed with clear nail polish         ity. Edges, in our context, represent the contour of whole
and stored at -80°C.                                                collagen fibres.
For each patient, one intervention slide and one control            We applied the LBP method only to the regions of in-
slide were set aside for use as negative controls. These            terest of the confocal photomicrographs. Data were
slides were not treated with primary antibodies, for pur-           obtained in the area juxtaposed to the surface to which
poses of comparison with control-group polyps.                      the cross-linking agents were applied, at a depth of
All slides were visualised and photographed using a Leica           105 μm (Figs. 2A-C), and in the control polyps, in the
TCS SP8 confocal laser scanning microscope.                         corresponding area also 105 μm in depth (Figs. 2D-F).
                                                                    Finally, a histogram of the decimal values attributed to
Comparison of images after tissue immunofluorescence                each pixel group was generated, representing the texture
Images of sections of epithelialised and non-epithelialised         pattern associated to the digital images of clinical speci-
polyps were first compared visually, with analysis focus-           mens.
ing on the surface region to which riboflavin/UVA was ap-        2. Analysis of luminous intensity distribution
plied and the corresponding region on the control polyp.            Analysis of luminous intensity distribution specifically
Collagen fibres were stained green, while the nuclei of the         evaluates pixels associated with greater brightness. In
epithelial cells were stained blue.                                 our photomicrographs, brighter areas (i.e., those with
                                                                    greater luminosity) represent collagen fibres.
Objective assessment of images                                      Brightness values in a monochrome image range from 0
The images obtained by confocal microscopy were also                to 255, where 0 represents the darkest hue and 255, the
examined for two objective parameters: tissue texture,              lightest. Bright pixels were defined as those with a hue
through pixel encoding by the local binary pattern (LBP)            exceeding a given threshold T, where 0 ≤ T ≤ 255. One
method and luminous intensity distribution.                         threshold value was attributed to the whole set of digi-
1. Analysis of tissue texture by pixel encoding                     tal images under study. Subsequently, it was calculated
   LBP is a method used in the field of computer vision for         which percentage of the whole image pixels belong to
   texture analysis of monochrome digital images. In the            the bright part of the image, revealing the extension of
   context of a digital image, the texture of each pixel is         the image area occupied by collagen fibres. This strategy
   defined by the local variations in intensity of the near-        allowed collagen fibre areas of control and intervention
   est neighbouring pixels. When applied to an image, LBP           images to be compared, as the control and intervention
   compares the intensities of a central pixel with those of        images were acquired under the same conditions.
   its 8 neighboring pixels, assigning a value of 1 when the
   intensity of a neighboring pixel is greater than or equal
                                                                 Results
   to the intensity of the central pixel and 0 otherwise. This
   generates a numeric representation that consists of a se-     Eosinophils per High-Power Field (HPF)
   quence of 8 binary digits (0 or 1), which represents the      All 6 polyps analysed presented more than 5 eosinophils per
   characteristics of the central pixel. This sequence of 8      HPF, characterising the eosinophilic endotype of CRSwNP.
   binary digits is then expressed as its decimal equivalent.
   The 8-digit binary sequence can generate up to 256 dif-       Tissue immunofluorescence microscopy
   ferent local patterns, some of which can be considered        Microscopically, a 105 μm-thick fluorescent line was vis-
   equivalent because they represent the same texture pat-       ible on the treated surface of epithelialised (Figs. 3A-B)
   tern.                                                         and de-epithelialised (Figs. 3C-D) polyps, stratified into a
   All binary sequences representing the same texture were       superficial zone with more organised collagen fibres and a
   grouped into the same set, known as the LBP code set.         deeper zone with less organised collagen fibres. The dif-
   From this perspective, up to 10 sets of LBP codes could       ferences between the control and intervention groups, con-
   be created, which were denoted by numbers 0 to 9. Each        cerning the organisation of collagen fibres were even more
   of these codes was associated with a specific image fea-      evident in the de-epithelialised polyps.
   ture. For example, codes 2 and 6 were associated with
   half-edges, while codes 3 and 5 were associated with          Analysis of tissue texture by pixel encoding
   dark and light edges, respectively. Half-edges, corre-        Among the controls of the epithelialised polyp group, the
   sponding to discontinuous edges which do not exceed           most frequent LBP profile observed was code 4 (Fig. 4A),
   2 pixels in length, represent collagen fibres that were       corresponding to the edges of collagen fibres. Edge detec-
   photographed only partly or present any loss of continu-      tion occurred due to the contrast of the bright collagen fi-



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N. Coronel et al.




Figure 2. Area of LBP analysis in a representative treated polyp (patient 6). (A) Histological section to which cross-linking was applied. (B) In pink, demarcated
area at a depth of 105 μm from which LPB codes were obtained. (C) Colour representation of LBP codes in the previously demarcated area. Area of LBP analysis
in a representative control polyp (patient 6). (D) Histological section of the polyp. (E) In pink, demarcated area at a depth of 105 μm from which LPB codes were
obtained. (F) Colour representation of LBP codes in the previously demarcated area.


bres with dark areas in which fibres were absent. The pres-                        In both situations, the pattern of distribution of LBP code
ence of edges implied a reference level for the control fibre                      densities after UVA exposure has changed; density of code
densities in the epithelialised polyp tissues. The exposure                        4 decreased in the treated polyps, which corresponds to an
of the epithelialised polyp tissues to riboflavin/UVA in-                          increase in collagen fibre concentration.
duced a distinct LBP profile. The lowering of code 4 rela-
tive frequencies and the higher frequencies for code 8 cor-                        Luminous intensity distribution
responded to the bright hue of the labeled areas, denoting a                       Figure 5 shows a plot of tissue luminous intensity distribu-
greater concentration of collagen fibres in which individual                       tion for individual patients. It was demonstrated a higher
edges were less observable.                                                        luminous intensity after UVA applications, which corre-
Among the de-epithelialised polyps (Fig. 4B), code 4 was                           sponds to greater collagen fibre densities in the analysed
also the most frequent LBP profile among the control pol-                          areas.
yps, while the UVA-exposed de-epithelialised polyp had its
highest peak on code 9.
The tissue texture analysis demonstrated that both the epi-
                                                                                   Discussion
thelialised and the de-epithelialised polyps increased col-                        Studies of tissue biomechanics in NP have led to a progres-
lagen fibre concentration.                                                         sive paradigm shift by revealing that the development of



430
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Figure 3. Photomicrograph of epithelialised nasal polyp section stained with anti-type I collagen antibody. (A) Control polyp, with rather disorganised collagen
fibres (green) on the cut surface. (B) Intervention polyp exposed to riboflavin/UVA cross-linking of the epithelialised surface, with greater organisation of collagen
fibres in the superficial stroma (above and to the right of the white line). Photomicrograph of de-epithelialised nasal polyp section stained with anti-type I collagen
antibody. (C) Control polyp, with rather disorganised collagen fibres (green) on the cut surface. (D) Intervention polyp exposed to riboflavin/UVA cross-linking of the
de-epithelialised surface, with highly organised collagen fibres in the superficial stroma (arrow). Sup. Str.: superficial stroma; Deep Str.: deep stroma.




                                                                                                                                                                 431
N. Coronel et al.




Figure 4. Histogram of LBP code density in epithelialised polyp tissue. In      Figure 5. Mean luminous intensity curves of epithelialised (A) and de-epithe-
the control polyp, the peak (blue) occurs on LBP code 4, which represents       lialised polyps (B). Blue represents control sections, and red, the intervention
greater presence of pixels corresponding to edges. In the polyp exposed to      (treated) sections.
cross-linking, the peak occurs on code 8, which represents uniform green-
coloured regions, corresponding to immunostained collagen fibres (A). His-
togram of LBP code density in de-epithelialised polyp tissue. In the control
polyp, the sole peak occurs on code 4, which represents a greater presence      At the cellular level, ontological genetic analysis has shown
of edge-associated pixels, whereas in the polyp exposed to cross-linking, the   that, in mesenchymal stem cells derived from nasal polyps,
peak occurs on code 9 (B).                                                      genes related to the ECM remodelling process are differen-
                                                                                tially modulated when compared to those of bone marrow-
                                                                                derived mesenchymal stem cells 5.
this condition is based on two processes: not only chronic                      It has also been demonstrated that fibrotic tissue found in
inflammation, but also mechanical dysfunction. Within                           the nasal mucosa (synechiae) and the mucosa of the middle
this context, the present study aimed to induce changes in                      nasal meatus have similar mechanical properties. Moreo-
the structure of the polyp ECM with a view to potentially                       ver, it is suggested that the fibrotic modification of the bio-
modify the mechanical dysfunction found in polyp tissue.                        mechanical properties of the nasal mucosa of patients with
This was the first investigation to evaluate a method ca-                       CRSwNP, a tissue repair phenomenon in which increased
pable of altering the shape and density of collagen in the                      collagen concentrations play a predominant role, could in-
ECM of nasal polyps. For this purpose, we used the ribofla-                     terfere in the altered remodelling process found in polypoid
vin/UVA collagen cross-linking process. This method was                         tissue 7.
chosen because it is already in clinical use in ophthalmolo-                    Collagen fibres provide tensile strength to tissues. In living
gy and has proven efficient in modifying the natural history                    tissue, these fibres are stabilised through the formation of
of keratoconus, a disease characterised by derangement of                       intermolecular covalent bonds, mediated by lysyl oxidase
ECM deposition, which facilitates change in the shape of                        enzymes 16. The intensity of this stabilisation can be in-
the cornea 10-12,14.                                                            creased artificially by application of UVA radiation directly
In CRSwNP, the composition of the ECM is different from                         to tissue, without altering the composition of the underly-
that of normal nasal tissue, especially regarding collagen,                     ing ECM, through the cross-linking technique 12.
which is present at lower concentrations 15. This process of                    Previous studies performed on the porcine cornea have
altered tissue remodelling in NP has direct effects on the                      shown that riboflavin acts as a photosensitising agent and,
biomechanical properties of the polypoid tissue, facilitat-                     when irradiated by UVA, leads to the formation of free
ing tissue growth and oedema 1.                                                 radicals that create new chemical bonds within the corneal



432
                                                                                                     Cross-linking effects in nasal polyp tissue




stroma. This procedure increases the diameter of the colla-      the growth of polypoid tissue, as our study showed that,
gen fibres and establishes stronger interfibrillar attachment,   although the method was less effective in epithelialised tis-
increasing the biomechanical stability of the exposed tis-       sue, it still had an effect on the extracellular matrix struc-
sue 10-12,17.                                                    ture of the nasal polyp. Prospective clinical studies are
Based on this method, we employed the same protocol for          needed to evaluate the applicability of this procedure in the
riboflavin/UVA exposure used in ophthalmology for kera-          treatment of CRSwNP.
toconus treatment in order to evaluate whether the same
changes that occur in the corneal ECM can be induced in          Conclusions
nasal polyp tissue 18.
In the images obtained from the epithelialised polyp, there      The results of this study show that riboflavin/UVA cross-
was a slight increase in fibre density up to 105 μm below        linking promotes changes in the structural conformation of
the epithelium, i.e., the depth reached by UVA radiation.        collagen fibres and increases their density on the exposed
Increased fibre density data were corroborated by objective      surface of nasal polyp tissue.
analysis of the pixel patterns and brightness of images.
The increase in collagen fibre density was greater in the        References
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linking 17,18.                                                   3
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Objective analysis of pixel patterns and luminosity in the            filing suggests epithelial dysfunction in polypoid chronic rhinosi-
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tion and provides reliable data for comparison, showing a        4
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These results also provide an opportunity for further inves-          imbio.2014.05.004
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                                                                 5
                                                                      Oliveira PWB, Pezato R, Agudelo JSH, et al. Nasal polyp-derived
                                                                      mesenchymal stromal cells exhibit lack of immune-associated mole-
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                                                                      do Amaral JB, Bloise AC, França CN, et al. Alterations in cellular
mechanical properties of nasal polypoid tissue.                       force parameters and cell projections in nasal polyps-derived fibro-
                                                                      blasts. Auris Nasus Larynx 2020;47:98-104. https://doi.org/10.1016/j.
If the tissue changes observed after exposure to the cross-           anl.2019.06.007
linking technique are shown to have a direct effect on the       7
                                                                      Gregório L, Pezato R, Felici RS, et al. Fibrotic tissue and middle tur-
biomechanics of nasal polypoid tissue, this would open                binate exhibit similar mechanical properties. is fibrosis a solution in
new avenues for the treatment of nasal polyposis, no longer           nasal polyposis? Int Arch Otorhinolaryngol 2017;21:122-5. https://
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based on anti-inflammatory agents or surgery alone, but          8
                                                                      Fokkens WJ, Lund VJ, Hopkins C, et al. European Position Paper
rather by directly altering the mechanical properties of the          on rhinosinusitis and nasal polyps 2020. Rhinology 2020;58(Suppl
affected tissue.                                                      S29):1-464.
This technique has the potential for immediate application       9
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after polypectomy in patients with a history of recurrent             pact of positive airway pressure in nasal polyposis? An experimen-
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                                                                 12
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                                                                      man and porcine corneas after riboflavin-ultraviolet-A-induced
Within this context, riboflavin/UVA cross-linking might               cross-linking. J Cataract Refract Surg 2003;29:1780-5. https://doi.
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