Dextranomer/Hyaluronic Acid Copolymer Implant for ... · a nonanimal, stabilized hyaluronic acid...

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Dextranomer/Hyaluronic Acid Copolymer Implant for Vesicoureteral Reflux: Role of Myofibroblast Differentiation Salvatore Arena,* Carmine Fazzari, Alessandra Implatini, Santo Torre, Daniela Villari, Francesco Arena and Vincenzo Di Benedetto From the Department of Pediatric Surgery, Unit of Pediatric Surgery, Vittorio Emanuele Hospital, University of Catania, Catania (SA, AI, VDB) and Department of Human Pathology, Unit of Histopathological Diagnosis (CF, ST, DV), and Department of Pediatric Medical and Surgical Sciences, Unit of Pediatric Surgery (FA), Policlinico, University of Messina, Messina, Italy Purpose: Dextranomer/hyaluronic acid implantation is associated with a gran- ulomatous inflammatory reaction, replaced by fibrosis. Appearance of myofibro- blasts is considered a crucial event in fibrosis, and CD68 positive cells and other factors are implied in their activation. Mast cells are a source of these factors and tryptase can induce fibroblast to express -smooth muscle actin, which is char- acteristic of myofibroblasts. We evaluated histological changes in refluxing ure- ters treated with dextranomer/hyaluronic acid and immunolocalized CD68 posi- tive cells, tryptase mast cells and myofibroblasts. Materials and Methods: We performed histological, histochemical and immuno- histochemical analyses in 22 refluxing ureters treated with dextranomer/hyal- uronic acid in comparison with 17 refluxing ureters who underwent ureteral reimplantation but did not receive endoscopic bulking agent. We used CD68 antibody for monocytes/macrophages and epithelioid cells, mast cell tryptase mouse antibody for mast cells, and -smooth muscle actin and vimentin antibodies for myofibroblasts. The area of the ureteral lumen in dextranomer/ hyaluronic acid treated and untreated ureteral endings was measured. Results: Sirius red documented a major grade of histological lesions in dex- tranomer/hyaluronic acid treated refluxing ureters. CD68 and tryptase mast cell staining showed a significant enhancement of positive cells in dextrano- mer/hyaluronic acid treated refluxing ureters. Immunostaining for -smooth muscle actin and vimentin displayed a myofibroblastic invasion in dextrano- mer/hyaluronic acid. Measurement of surface in treated refluxing ureters was significantly less than in untreated refluxing ureters. Conclusions: Our data documented a recruitment of CD68 and tryptase positive cells, abnormal accumulation of collagenous stroma and successive extracellular matrix remodeling through differentiation of myofibroblasts. Myofibroblasts might provoke tissue contraction, decreasing the ureteral diameter and modify- ing the ureteral length-to-diameter ratio, preventing urine reflux. Key Words: CD68 antigen, human; dextranomer-hyaluronic acid copolymer; endoscopy; mesangial cells; vesico-ureteral reflux Abbreviations and Acronyms -SMA -smooth muscle actin Dx/HA dextranomer/hyaluronic acid copolymer ECM extracellular matrix HPF high power field RU refluxing ureteral ending SD standard deviation VUR vesicoureteral reflux Submitted for publication August 29, 2008. * Correspondence: Department of Pediatric Surgery, Unit of Pediatric Surgery, Vittorio Eman- uele Hospital, University of Catania, Via Plebis- cito n. 628, CAP 95124, Catania, Italy (telephone: 00390957436501; FAX: 00390957435324; e-mail: [email protected]). ENDOSCOPIC injection of a bulking agent was pioneered in 1981 as an alternative to conservative medical treatment and surgical ureteral re- implantation in patients with vesi- coureteral reflux. 1 Different sub- stances have been investigated, and polytetrafluoroethylene, silicone and cross-linked bovine collagen have been well studied. Concerns re- 0022-5347/09/1816-2695/0 Vol. 181, 2695-2701, June 2009 THE JOURNAL OF UROLOGY ® Printed in U.S.A. Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION DOI:10.1016/j.juro.2009.02.059 www.jurology.com 2695

Transcript of Dextranomer/Hyaluronic Acid Copolymer Implant for ... · a nonanimal, stabilized hyaluronic acid...

Dextranomer/Hyaluronic Acid Copolymer Implant for

Vesicoureteral Reflux: Role of Myofibroblast Differentiation

Salvatore Arena,* Carmine Fazzari, Alessandra Implatini, Santo Torre,Daniela Villari, Francesco Arena and Vincenzo Di BenedettoFrom the Department of Pediatric Surgery, Unit of Pediatric Surgery, Vittorio Emanuele Hospital, University of Catania, Catania (SA, AI, VDB)and Department of Human Pathology, Unit of Histopathological Diagnosis (CF, ST, DV), and Department of Pediatric Medical and SurgicalSciences, Unit of Pediatric Surgery (FA), Policlinico, University of Messina, Messina, Italy

Purpose: Dextranomer/hyaluronic acid implantation is associated with a gran-ulomatous inflammatory reaction, replaced by fibrosis. Appearance of myofibro-blasts is considered a crucial event in fibrosis, and CD68 positive cells and otherfactors are implied in their activation. Mast cells are a source of these factors andtryptase can induce fibroblast to express �-smooth muscle actin, which is char-acteristic of myofibroblasts. We evaluated histological changes in refluxing ure-ters treated with dextranomer/hyaluronic acid and immunolocalized CD68 posi-tive cells, tryptase mast cells and myofibroblasts.Materials and Methods: We performed histological, histochemical and immuno-histochemical analyses in 22 refluxing ureters treated with dextranomer/hyal-uronic acid in comparison with 17 refluxing ureters who underwent ureteralreimplantation but did not receive endoscopic bulking agent. We used CD68antibody for monocytes/macrophages and epithelioid cells, mast cell tryptasemouse antibody for mast cells, and �-smooth muscle actin and vimentinantibodies for myofibroblasts. The area of the ureteral lumen in dextranomer/hyaluronic acid treated and untreated ureteral endings was measured.Results: Sirius red documented a major grade of histological lesions in dex-tranomer/hyaluronic acid treated refluxing ureters. CD68 and tryptase mastcell staining showed a significant enhancement of positive cells in dextrano-mer/hyaluronic acid treated refluxing ureters. Immunostaining for �-smoothmuscle actin and vimentin displayed a myofibroblastic invasion in dextrano-mer/hyaluronic acid. Measurement of surface in treated refluxing ureters wassignificantly less than in untreated refluxing ureters.Conclusions: Our data documented a recruitment of CD68 and tryptase positivecells, abnormal accumulation of collagenous stroma and successive extracellularmatrix remodeling through differentiation of myofibroblasts. Myofibroblastsmight provoke tissue contraction, decreasing the ureteral diameter and modify-ing the ureteral length-to-diameter ratio, preventing urine reflux.

Key Words: CD68 antigen, human; dextranomer-hyaluronic acid copolymer;

Abbreviations

and Acronyms

�-SMA � �-smooth muscle actin

Dx/HA � dextranomer/hyaluronicacid copolymer

ECM � extracellular matrix

HPF � high power field

RU � refluxing ureteral ending

SD � standard deviation

VUR � vesicoureteral reflux

Submitted for publication August 29, 2008.* Correspondence: Department of Pediatric

Surgery, Unit of Pediatric Surgery, Vittorio Eman-uele Hospital, University of Catania, Via Plebis-cito n. 628, CAP 95124, Catania, Italy (telephone:00390957436501; FAX: 00390957435324; e-mail:[email protected]).

endoscopy; mesangial cells; vesico-ureteral reflux

ENDOSCOPIC injection of a bulkingagent was pioneered in 1981 as analternative to conservative medicaltreatment and surgical ureteral re-

implantation in patients with vesi-

0022-5347/09/1816-2695/0THE JOURNAL OF UROLOGY®

Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION

coureteral reflux.1 Different sub-stances have been investigated, andpolytetrafluoroethylene, siliconeand cross-linked bovine collagen

have been well studied. Concerns re-

Vol. 181, 2695-2701, June 2009Printed in U.S.A.

DOI:10.1016/j.juro.2009.02.059www.jurology.com 2695

DEXTRANOMER/HYALURONIC ACID IMPLANT FOR VESICOURETERAL REFLUX2696

garding the safety and efficacy of these agentshave precluded their widespread use.

Dx/HA, approved by the Food and Drug Admin-istration in 2001 for the treatment of VUR, wasthe first endoscopic agent available for this indi-cation in the United States. This endoscopic bulk-ing agent consists of dextranomer (cross-linkeddextran) microspheres 80 to 250 �m in diameter ina nonanimal, stabilized hyaluronic acid gel. Thephysicochemical properties of Dx/HA make it idealfor treating VUR because it is biocompatible, bio-degradable and nonmigratory, exhibits no signs ofmutagenesis and has a good safety profile.2,3 Dex-tranomer, which is the main bulking agent, is onlyslowly degraded by hydrolysis, whereas the hyal-uronic acid matrix acts primarily as a transportmedium and disappears from the body within sev-eral weeks. After submucosal injection the dextra-nomer microspheres stimulate collagen synthesisand fibroblast ingrowth in the degrading hyal-uronic acid matrix, consolidating the implantwithin the bladder wall through endogenous tis-sue augmentation.

It is reported that Dx/HA treatment is associ-ated with a granulomatous inflammatory reaction,as demonstrated by multinucleated giant cellsthat are subsequently replaced by fibrosis.2 Fromthe biological point of view some fibroblasts ex-press features of smooth muscle differentiation.These smooth muscle-like fibroblasts, referred toas myofibroblasts,4 can be identified by certaincharacteristic features of the cytoskeleton, partic-ularly the expression of �-SMA.5 It is known thatmyofibroblasts have a major role in inflammatoryresponses through their production of growth fac-tors, cytokines and soluble mediators. Appearanceof myofibroblasts is considered a critical event inthe evolution of fibrosis, and activated macro-phages and other specific factors are implied intheir activation.6,7 Mast cells are a potentialsource of some of those factors, and in particularmast cell tryptase can induce normal human der-mal fibroblasts to express �-SMA, a distinguishingcharacteristic of differentiated myofibroblasts.8

Moreover, interactions of mast cells with theirECM are crucial events for tissue specific migra-tion, localization and function, since mast cellscirculate as immature precursor cells and home totissue adhering to hyaluronic acid coated surface.9

We evaluated the histological changes in reflux-ing ureters treated with Dx/HA, with respect to im-munolocalized CD68 positive cells, tryptase mastcells and myofibroblasts. We also estimated theirimportance in the biological mechanism of action of

Dx/HA.

MATERIALS AND METHODS

We performed retrospective histological, histochemicaland immunohistochemical analyses of 15 patients (9 boysand 6 girls, age 18.1 � 5.5 months) with grade III orgreater VUR who received 1 implant of 0.7 to 0.9 mlDx/HA, for a total of 22 RUs. The patients had persistentreflux 94 to 115 days (102 � 8) after initial unsuccessfulendoscopic treatment, defined as persistent grade III orgreater VUR on voiding cystourethrography, and under-went ureteral reimplantation using the Cohen technique.VUR was recorded as grade III in 4 RUs, IV in 12 and V in8. For comparison 11 controls (7 boys and 4 girls, mean �SD age 20.4 � 4.9 months) were enrolled in the study, fora total of 17 RUs affected by grade III or greater VUR.Controls underwent ureteral reimplantation using theCohen technique but did not receive any type of endo-scopic bulking agent. In controls VUR was recorded asgrade III in 3 RUs, IV in 9 and V in 5. Indications foropen surgery were poor parental compliance with treat-ment and reflux nephropathy. The control group wasmatched with the Dx/HA treated group for age, genderand reflux grade. The duration of antibiotic prophylaxiswas similar in the treated and control groups.

We used colored sutures inserted into the distal ure-teral endings to identify the distal part of the ureters thatwere used for the histological, immunohistochemical andmorphological evaluations. The resected ureteral seg-ments (about 3 mm) were fixed in 10% neutral formalinand embedded in paraffin. Paraffin blocks were labeledwith a numerical code to ensure blinding to clinical data,and 4 �m thick sections were processed for routine histo-logical analysis, and stained with hematoxylin and eosin.

For immunohistochemical evaluation parallel paraffinsections were heated in citrate buffer (antigen retrievalbuffer, pH 6.0, Dako, Glostrup, Denmark) for 30 minutesin a high-powered microwave oven (400 W) to retrieve theantigens. After rinsing in phosphate buffered saline (pH7.4) endogenous peroxidase activity was blocked in 3%H2O2 for 5 minutes. For immunohistochemical stainingwe used CD68 antibody (clone KP1, Dako) diluted 1:150for identification of monocytes/macrophages and epithe-lioid cells, mast cell tryptase mouse antibody (clone AA1,Abcam Inc, Cambridge, Massachusetts) diluted 1:50 foridentification of mast cells, and �-SMA (clone 1A4, Dako)and vimentin antibodies (clone V9, Dako) diluted 1:100 foridentification of myofibroblasts. CD68, �-smooth muscleactin and vimentin antibodies were incubated overnightat 4C, while mast cell tryptase antibody was incubated atroom temperature for 30 minutes.

After being rinsed in phosphate buffered saline theslides were reincubated for 10 minutes at room tempera-ture with a biotinylated link universal secondary antibody(LSAB®� System-HRP, Dako, Carpinteria, California)and developed in 3.3=-diaminobenzidine-HCl (VIP, SK4600m, Vector® Laboratories, Burlingame, California). Aweak hematoxylin nuclear counterstaining was then ap-plied, and the sections were dehydrated and mounted.Parallel negative controls were processed by omitting theprimary antibody incubation. For histochemical analysisafter dewaxing and hydration parallel sections of the spec-

imens were stained with sirius red for collagen stains.

DEXTRANOMER/HYALURONIC ACID IMPLANT FOR VESICOURETERAL REFLUX 2697

For the morphometric analysis the findings were as-sessed under light microscopy by 3 independent observerswho were unaware of the sample origin, using a computerassisted semiquantitative method. The density of CD68positive cells, mast cell tryptase positive mast cells andmyofibroblasts was graded in Dx/HA treated and un-treated RUs. This grading was carried out by evaluating10 HPFs of 0.152 mm2, at 400 times magnification.

The criteria to assess ureteral wall derangement wasscored on the scale 0—absent, I— mild (25% or less of eachHPF), II—moderate (26% to 50%), III—severe (51% to75%) and IV—extremely severe (greater than 75%) basedon a partly absent muscular coat and replacement of mus-cle fibers by connective tissue or enhanced extracellularmatrix.10 After histological image capture using a digitalcamera the ureteral diameters, area of the ureteral lumen,and mesenchymal/muscular thickness in untreated andDx/HA treated ureteral endings was measured at least 10times in each specimen using modular image acquisitionsoftware.

The quantitative values are expressed as mean and SD.Nonparametric t test was used to compare the ureteraldiameters, mesenchymal/muscular thickness of RUs, areaof ureteral lumen, and density of CD68 positive cells, mastcell tryptase cells and myofibroblasts in Dx/HA treatedand untreated RUs. Statistical significance was acceptedat p �0.05.

RESULTS

Histological and histochemical observations in un-treated RUs revealed a stellate or ovoid lumenwith transitional epithelium underlying the lam-ina propria, with a various extent of collagenousstroma among the smooth muscle layer and singlebundles. Such changes were quantitatively ex-pressed as score IV in 9 of 17 RUs (53%), III in 7(41%) and II in 1 (6%, fig. 1). In contrast, in Dx/HAtreated RUs Dx/HA was macroscopically observedas small spherical masses whose contents eitherwere clear or contained amorphous material notall of the same size, with variations in the diam-eter of the spherical mass. On light microscopy aninflammatory infiltrate was evident that wasmainly composed of mononuclear cells, lympho-cytes and macrophages.

Granuloma formation and foreign body type giantcells surrounding the microspheres were observed inall specimens. Capillary growth was seen in 10 of 22Dx/HA treated RUs (45%). Calcification reactionwas not observed. A fibrous tissue accumulation waspresent around the pseudo-encapsulation and ex-tended between the individual dextranomer pearlsat different stages of resorption (fig. 2).

Histochemical changes were quantitativelyscored as IV in 18 of 22 treated RUs (82%) and IIIin 4 RUs (18%, fig. 3). CD68 staining showed pos-itive cells in untreated (mean � SD 33.7 � 7.8 per

HPF) and Dx/HA treated RUs (more than 200 per

HPF, fig. 4). Mast cell tryptase immunostainingdemonstrated an average � SD cells per HPF of19.6 � 6.7 in untreated RUs and 74.7 � 19.6 inDx/HA treated RUs (fig. 5). In parallel sectionsimmunostaining for �-SMA and vimentin dis-played the absence of myofibroblasts in untreatedRUs, while myofibroblastic invasion (cells positivefor both antibodies) was a prominent finding inDx/HA treated RUs, in particular between dextra-nomer microspheres (fig. 6).

Mean � SD ureteral diameter was 4.43 � 0.61mm2 in untreated RUs, 3.76 � 0.37 mm2 in Dx/HAtreated RUs and 4.80 � 3.38 mm2 on the side ofchronic inflammatory reaction (tables 1 and 2). Inuntreated and Dx/HA treated RUs mean � SD mes-enchymal/muscular thickness was 0.88 � 0.16 mm2

and 0.71 � 0.14 mm2, respectively, and mean � SDureteral surface was 0.19 � 0.03 mm2 and 0.11 �0.04 mm2, respectively.

Statistical examination revealed a significant dif-ference in lesion grade and a highly significant dif-ference in density of CD68 positive and mast celltryptase positive results between untreated andDx/HA treated RUs (p �0.05). Morphometric analy-sis documented a significant difference in ureteraldiameters, in mesenchymal/muscular thickness andin the ureteral area between untreated and Dx/HA

Figure 1. Sirius red stain shows grade II lesion in untreated RUs.Reduced from �200.

treated RUs (p �0.05). Moreover, t test on ureteral

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diameters, mesenchymal/muscular thickness andureteral area between untreated and Dx/HA treatedRUs of the same histological grade of lesions showeda significant statistical difference (p �0.05).

DISCUSSION

Creation of a prominent physical bulge in the blad-der wall below the ureteral orifice, which increasesthe submucosal length of the ureter, is generallythought to underlie the effectiveness of endoscopictreatment of VUR.2 However, it is supposed thatcreation of a fixation point for the ureter may be amore important result of the treatment. Dx/HA co-polymer is reported to be an ideal substance forsubureteral injection in children. In fact, it is a bio-degradable material and does not cause allergic re-actions because of the lack of freely circulating dex-tran molecules.11 Moreover, the particle size ofgreater than 80 �m also eliminates the risk of dis-tant particle migration because it is impossible formacrophages to transport the dextranomer parti-cles.

The inducement by Dx/HA of a mild inflamma-tory reaction resulting in a fixation point for theureter within the bladder (similar to that achievedby surgical reimplantation) is likely to be partic-ularly important.2,3 At the site of inflammationfibroblasts are able to undergo a range of pheno-typic conversions between distinct but related celltypes. Fibroblasts with a contractile phenotype,

Figure 2. Light microscopy of intense periureteral giant cellreaction encapsulating dextranomer microspheres. H & E, re-duced from �200.

namely myofibroblasts, are essential for the syn-

thesis of the collagen rich scar and for providingthe force for contraction.12

Our immunohistochemical investigation of RUstreated with Dx/HA documented the recruitmentof numerous myofibroblasts around the dextrano-mer particles. Our data demonstrate a chronicinflammatory reaction of the foreign body inagreement with the findings of Stenberg et al,2,3

with a high density in CD68 positive cells, smoothmuscle bundle derangement and subsequent in-terstitial fibrosis after subureteral injection of Dx/HA. The presence of a significant increase in CD68positive cells in RUs was described by Oswaldet al.13 It is known that CD68 positive cells gen-erate cytokines, induce an increase in collagenousstroma and have a particular affinity for collagenrich regions, facilitating their accumulation.13 Ab-normal accumulation of fibrillar collagen and suc-cessive ECM remodeling could explain the tissuecontraction in RUs, modifying the ureteral length-to-diameter ratio, implying healing of VUR.13

ECM remodeling is perpetuated by CD68 positivecells by secretion of collagenases.14 We noted asignificant enhancement of CD68 positive cells inDx/HA treated RUs compared to untreated RUs.

It is known that phenotypic transdifferentia-tion of fibroblast to myofibroblast also occurs inresponse to modification of the composition of ECM,

Figure 3. Sirius red stain demonstrates grade IV lesion in Dx/HA

treated RUs. Reduced from �200.

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and the presence of CD68 positive cells, whichpromote ECM hydrolysis, could have a crucial rolein developing differentiated myofibroblasts byneoexpression of �-SMA.2,15 A giant cell inflam-matory reaction replaced by fibrosis as a conse-quence of Dx/HA injection has been described byvarious authors and is considered a mechanism ofaction of Dx/HA.2,3,11 Moreover, myofibroblastshave a crucial role in the contraction and remod-eling of tissue.4,15 Thus, the differentiation ofmyofibroblasts in Dx/HA treated RUs obviouslyprovokes a contraction of the ureteral surface. Onemight assume that the reduction of ureteral sur-face is due to the physical bulking of the materialitself, independent of the inflammatory reaction.We did not note any imprinting of the bulkingagent in the ureteral lumen, but we observed ahomogeneous reduction of the ureteral surface. Inaddition, we did not note any myofibroblasts in

Figure 4. CD68 immunostaining reveals low density positive celRUs (b). Reduced from �400.

Figure 5. Mast cell tryptase antibody staining shows mastocytes in

untreated RUs, while a significant enhancementwas observed in Dx/HA treated RUs. The presenceof myofibroblasts probably represents an impor-tant event in Dx/HA treated RUs, and their differ-entiation might justify ureteral wall contractionwith the subsequent reduction of the ureteral lu-men.

Statistical evaluation comparing untreated andDx/HA treated RUs of the same histological gradeof lesions revealed a significant reduction of theureteral lumen in treated RUs. Therefore, the re-duction of ureteral surface cannot be ascribed onlyto abnormal accumulation of collagenous stroma,and a myofibroblast role is conceivable. Further-more, myofibroblast differentiation leads to accu-mulation of hyaluronic acid as a consequence ofeither an increase in hyaluronic acid synthesis ora decrease in its degradation.12 The release of lowweight hyaluronic acid fragments has immuno-

treated RUs (a) and high density positive cells in Dx/HA treated

ls in un

untreated (a) and Dx/HA treated RUs (b). Reduced from �400

DEXTRANOMER/HYALURONIC ACID IMPLANT FOR VESICOURETERAL REFLUX2700

stimulatory, proinflammatory and angiogenicproperties, and is known to stimulate inflamma-tion via a cell adhesion molecule, with the result-ant complex stimulating mast cell adhesion.9,12,16

Mast cells release a number of active and che-motactic mediators involved in the development ofinflammation and tissue repair,17 among which istryptase. Tryptase is a serine protease unique tomast cells. The induction produced by tryptase hasclear positive effects on �-SMA expression and stim-ulates contraction of a collagen matrix—character-istics of differentiated myofibroblasts.8 Our datashow a significant recruitment of tryptase positive

Figure 6. Stains in parallel sections using vimentin (a) and �-SMaround dextranomer microspheres. Reduced from �400.

Table 1. Ureteral dimensions in Dx/HA treated patients

Pt No.Ureteral

Area (mm2)Ureteral

Diameter (mm)

Ureteral Diameter onSide With ChronicInflammation (mm)

Mesenchymal/Muscular

Thickness (mm)

1 0.09 3.45 5.01 0.622 0.12 3.65 5.10 0.653 0.13 4.01 5.08 0.704 0.15 4.10 5.40 0.845 0.07 3.57 4.82 0.546 0.08 3.45 4.97 0.647 0.09 4.09 4.87 0.518 0.12 4.32 5.04 0.649 0.15 3.40 4.80 0.75

10 0.11 3.98 4.75 0.6011 0.16 3.45 4.20 0.6412 0.07 3.65 4.45 0.4513 0.20 4.54 5.57 0.6114 0.06 3.78 4.61 0.7415 0.09 3.68 4.30 0.8516 0.15 4.56 5.45 0.9117 0.08 3.32 4.65 0.6518 0.09 3.45 4.61 1.0119 0.12 3.68 4.41 0.7420 0.11 3.70 4.42 0.7821 0.14 3.41 4.34 0.85

22 0.09 3.54 4.65 0.94

mast cells in refluxing ureters treated with Dx/HA,and thus their role in differentiation of myofibro-blasts is conceivable. Fibroblast to myofibroblastmodulation represents a crucial step in wound gran-ulation, tissue contraction and production of connec-tive tissue.18

Incorporation of �-SMA into stress fibers signifi-cantly augments the contractile activity of fibroblas-tic cells, and is a hallmark of the contraction phaseof connective tissue remodeling.15 The high densityof myofibroblasts in Dx/HA treated RUs suggeststheir involvement. In fact, the activity of myofibro-blasts in the Dx/HA treated ureter might promote atissue contraction that also synthesizes increasedlevels of ECM components and matrix degradingproteases, promoting fibrosis and tissue remodel-ing.4 Our morphological analysis demonstrated asignificant reduction in mesenchymal/muscular

ntibodies demonstrate high density of myofibroblasts (arrows)

Table 2. Ureteral dimensions in controls

Pt No.Ureteral

Area (mm2)Ureteral

Diameter (mm)Mesenchymal/Muscular

Thickness (mm)

1 0.20 4.54 0.822 0.22 4.35 0.953 0.19 3.45 0.854 0.22 5.02 0.705 0.19 4.05 1.026 0.17 3.87 0.847 0.25 5.54 0.978 0.26 5.30 1.019 0.16 3.54 0.72

10 0.15 3.75 0.6411 0.18 4.20 0.7012 0.19 4.56 0.8713 0.17 4.02 0.9414 0.15 4.31 0.7015 0.22 4.80 1.2116 0.20 4.95 1.10

A (b) a

17 0.18 5.00 0.94

DEXTRANOMER/HYALURONIC ACID IMPLANT FOR VESICOURETERAL REFLUX 2701

thickness and ureteral surface, and a significantenhancement of collagenous stroma in RUs treatedwith Dx/HA compared to untreated RUs. The lengthof the intravesical ureter relative to its diameterseems to be a crucial point supporting the “passive”reflux defense mechanism.10,19 Thus, the standardratio of 5:1 for ureteral reimplantation surgeryseems necessary. In this way the rationale of theendoscopic approach using Dx/HA is not only to aug-ment and elongate the intramural part of the ureter,but also to promote contraction of the distal ureterthrough myofibroblastic differentiation, reducingthe ureteral surface and improving the “passive”antireflux mechanism.

A limitation of this study is that we only exam-ined children in whom the Dx/HA injection hadfailed. However, our histological evidence is inagreement with previous series2,3 and, therefore, a

myofibroblast differentiation in chronic inflamma-

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CONCLUSIONS

Our data reveal that Dx/HA injection in RUs evokesa granulomatous inflammatory reaction, the recruit-ment of CD68 and tryptase positive cells with ab-normal accumulation of collagenous stroma, andsuccessive ECM remodeling through myofibroblastdifferentiation. Moreover, myofibroblasts might pro-voke tissue contraction, decreasing the ureteral di-ameter and modifying the ureteral length-to-diame-ter ratio, thus preventing urine reflux.

ACKNOWLEDGMENTS

Prof. Antony Bridgewood completed the English re-

vision of the manuscript.

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