Endoscopic Treatment with Dextranomer-Hyaluronic Acid for Vesicoureteral Reflux: Histological Findings

0022-5347/03/1693-1109/0 THE JOURNAL OF UROLOGY® Copyright © 2003 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 169, 1109 –1113, March 2003 Printed in U.S.A.

DOI: 10.1097/01.ju.0000053013.49676.89

ENDOSCOPIC TREATMENT WITH DEXTRANOMER-HYALURONIC ACID FOR VESICOURETERAL REFLUX: HISTOLOGICAL FINDINGS A. STENBERG,* E. LARSSON

AND

¨ CKGREN* G. LA

From the Section of Urology, University Children’s Hospital and Department of Pathology, Uppsala University Hospital, Uppsala, Sweden

ABSTRACT

Purpose: Dextranomer-hyaluronic acid co-polymer is the first endoscopic bulking agent approved for vesicoureteral reflux in the United States. We evaluated the histopathological changes associated with this treatment in children with vesicoureteral reflux. Materials and Methods: Children 1 to 11 years old in whom treatment with dextranomerhyaluronic acid co-polymer for grades III or greater vesicoureteral reflux had failed were eligible for the study. Failure was defined as persistent vesicoureteral reflux on voiding cystourethrography done approximately 3 months after implantation. At ureteral reimplantation the implant and surrounding ureteral tissue were resected and fixed for histopathological analysis. Tissue sections (4 to 5 ␮m.) were stained for routine histology and examined under a light microscope. Patients with a similar grade of vesicoureteral reflux who had not undergone endoscopic treatment served as the control group. Results: The study population comprised 23 patients with vesicoureteral reflux, of whom 13 with a mean age of 2 years 8 months at diagnosis underwent 1 to 3 treatments with dextranomerhyaluronic acid co-polymer. The remaining 10 patients with a mean age of 1 year 10 months at diagnosis did not receive the bulking agent before ureteral reimplantation. The implant remained in situ 13 to 39 months (mean 22). On ureteral reimplantation the implant was located at the site of injection in 12 of the 13 patients. Histologically a granulomatous inflammatory reaction indicated by giant cell infiltration was observed at the implantation site. At ureteral reimplantation 9 implants were pseudo-encapsulated. Calcification was present in 9 ureters, while the eosinophil count was greater than 5 cells per 0.125 mm.2 in 7 ureters treated with dextranomer-hyaluronic acid co-polymer. Mast cell infiltration was similar in the treatment and control groups. Conclusions: Endoscopic treatment with dextranomer-hyaluronic acid co-polymer for vesicoureteral reflux is associated with a granulomatous reaction of the giant cell type, inflammatory cell infiltration and implant pseudo-encapsulation. They are typical histological findings associated with implantation of a foreign material. Dextranomer-hyaluronic acid co-polymer remains safe and effective for vesicoureteral reflux in children. KEY WORDS: vesico-ureteral reflux, bladder, prostheses and implants, hyaluronic acid, dextrans

Vesicoureteral reflux, which is characterized by retrograde urine flow from bladder to kidney, affects approximately 1% of the pediatric population and it is commonly associated with urinary tract infections.1 Moderate to severe reflux is a predisposing factor for pyelonephritis,2 while renal scarring arising from persistent pyelonephritic infection may cause long-term renal impairment and renin mediated hypertension.3 Open surgery to reimplant the ureters has traditionally been the only treatment option for moderate to severe vesicoureteral reflux. Since the 1980s, various endoscopic bulking agents, such as polytetrafluoroethylene, silicone, collagen and autologous chondrocytes, have been used to treat reflux. The rationale for this approach is to augment and elongate the intramural part of the ureter by injecting a bolus into the bladder wall below the ureteral orifice, thereby, preventing urine reflux into the ureter. However, efficacy and/or safety issues are a concern with these bulking agents. Collagen has poor long-term efficacy,4 while silicone and polytetrafluoroethylene particles migrate from the injection site and form granulomas.5, 6 Dextranomer-hyaluronic acid co-polymer, which was recently approved by the Food and Drug Administration for

vesicoureteral reflux, is the first endoscopic agent available for this indication in the United States. This novel endoscopic bulking agent consists of dextranomer (cross-linked dextran) microspheres 80 to 250 ␮m. in diameter in a gel of nonanimal, stabilized hyaluronic acid. The physicochemical properties of dextranomer-hyaluronic acid co-polymer make it ideal for treating vesicoureteral reflux because it is biocompatible, biodegradable, nonmigratory, shows no signs of mutagenesis and has a good safety profile.7, 8 Dextranomer, which is the main bulking agent, is only slowly degraded by hydrolysis, whereas the hyaluronic acid matrix acts primarily as a transport medium and disappears from the body within several weeks. After submucosal injection the dextranomer microspheres stimulate collagen synthesis and fibroblast ingrowth into the degrading hyaluronic acid matrix, consolidating the implant within the bladder wall through endogenous tissue augmentation.8 Experimental animal studies indicate that dextranomer-hyaluronic acid co-polymer evokes a granulomatous inflammatory reaction of the foreign body type after submucosal/subureteral injection but causes no tissue necrosis or calcification.8 To our knowledge we present the first study of the histopathological effects of dextranomerhyaluronic acid co-polymer in patients with vesicoureteral reflux.

Accepted for publication October 4, 2002. * Financial interest and/or other relationship with Pharmacia, Ferring and Q-Med. 1109

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DEXTRANOMER-HYALURONIC ACID FOR VESICOURETERAL REFLUX MATERIALS AND METHODS

We performed a retrospective histopathological analysis of patients who received 1 or more implants of dextranomerhyaluronic acid co-polymer for vesicoureteral reflux between February 1996 and April 1999. Patients comprised a subset from a large-scale efficacy study of the efficacy and safety of dextranomer-hyaluronic acid co-polymer in 221 who were 0 to 15 years old for 2 to 7.5 years (mean 5) after implantation.7 Inclusion criteria for the efficacy study included dilating, unilateral or bilateral, grades III or greater vesicoureteral reflux upper urinary tract infection at least 1 year previously, 2 or more diagnoses of reflux on voiding cystourethrography 6 months or less apart and normal creatinine. Efficacy was assessed by voiding cystourethrography 3 and 12 months, and 5 years after co-polymer treatment. Patients with persistent reflux underwent a second or third implantation, or open surgery 3 months or greater after initial treatment. Patients who underwent ureteral reimplantation after unsuccessful endoscopic treatment failure, defined as persistent grades III or greater reflux on voiding cystourethrography, were eligible for histopathological analysis. For the purpose of comparison a historical control group of patients of similar age from the 1990s was included, comprising children with vesicoureteral reflux who underwent ureteral reimplantation but did not receive any type of endoscopic bulking agent. The control group was not matched with the treatment group for age, gender or reflux grade. The duration of antibiotic prophylaxis with nitrofurantoin and trimethoprim was similar in the treatment and control groups. Before ureteral reimplantation cystoscopic bladder examination was performed to assess the position of the implant. The bladder was opened and the distal part of the ureter was dissected free from the surrounding tissue and implant. The ureterovesical junction and distal ureter, including the implant with its surrounding tissue, were resected and sent for pathological evaluation. The ureter was reimplanted according to the Cohen technique. Distal ureteral tissue blocks were fixed in 4% phosphate buffered formaldehyde. Tissue sections (4 to 5 ␮m. thick) were processed for routine histology and stained with hematoxylin and eosin, Van Gieson and Picro-Sirius stains for extracellular matrix, periodic acid-Schiff (PAS) for dextranomer, von Kossa stain for calcium and Alcian blue, pH 2.2, for mast cells. Stained ureteral sections were evaluated by light microscopy for fibrosis, calcification, leukocytes, dextranomer pearls and encapsulation of the dextranomer-hyaluronic acid co-polymer implant. Under high power magnification (400⫻) the number of eosinophils and mast cells was assessed in a field of view of 0.125 mm.2. Sections were scanned to identify fields of view with a high number of eosinophils and mast cells. Eosinophil and mast cell counts were determined in fields with the maximum number of such cells. RESULTS

A total of 13 patients in whom endoscopic treatment failed with the currently marketed formulation of dextranomerhyaluronic acid co-polymer underwent open surgery for ureteral reimplantation. Ten randomly selected patients with reflux who had undergone ureteral reimplantation without previous endoscopic treatment served as the control group. The treatment group comprised 9 girls and 4 boys with a mean age of 2 years 8 months (range newborn to 7 years 2 months) at diagnosis. The control group consisted of 6 boys and 4 girls with a mean age of 1 year 10 months (range 1 month to 5 years, table 1). Eight of the 13 patients (62%) in the treatment group had bilateral vesicoureteral reflux and, thus, 21 ureters were implanted with dextranomer-hyaluronic acid co-polymer. A similar proportion of patients in the control group had bilateral vesicoureteral reflux. Mean patient age at reimplantation was

TABLE 1. Baseline demographic and clinical characteristics of treatment and control groups Pt. No. — Sex

Age at Diagnosis (yrs., mos.)

Lt./Rt. Reflux Grade at Diagnosis*

Treatment group 7, 2 I/IV 0, 4 V/0 Newborn 0/IV 1, 9 IV/IV 1, 10 IV/III 6, 0 III/0 6, 6 IV/IV 0, 5 IV/III 3, 5 IV/IV 1, 5 IV/0 2, 5 III/II 1, 8 III/IV 3, 6 IV/III Control group 1 —F 5, 0 III/0 2 —F 0, 1 III/III 3 —F 0, 6 V/(III)† 4 —M 3, 6 III/IV 5 —M 1, 0 III/IV 6 —M 0, 3 IV/III 7 —F 2, 0 III/0 8 —M 3, 0 0/III 9 —M 3, 0 V/0 10 — M 1, 0 II†/III * Defined by voiding cystourethrogram. † No reimplantation. 1 2 3 4 5 6 7 8 9 10 11 12 13

—M —F —M —F —F —F —F —F —F —M —F —M —F

Age at Ureteral Reimplantation (yrs., mos.) 9, 2, 1, 3, 4, 7, 10, 2, 5, 2, 5, 4, 4,

8 11 7 10 0 10 4 1 4 6 4 9 9

5, 6, 1, 4, 1, 1, 3, 7, 3, 3,

6 5 3 5 8 3 0 5 5 6

marginally higher in the treatment group than in the control group (5 years, range 1 year 7 months to 9 years 8 months versus 4 years, range 1 year 3 months to 7 years 5 months). Before ureteral reimplantation 7 patients in the copolymer group underwent 2 endoscopic treatments, while 5 received a single treatment and the remaining 1 required 3 treatments (table 2). As determined by voiding cystourethrography, treatment efficacy was assessed within 3 months in 8 cases and in the remaining evaluation was done within 6 months (table 2). The implant remained in situ 13 to 39 months (mean 22) and it was still evident at ureteral reimplantation in 12 of the 13 patients (table 3). Distal ureter. The global histological picture at the site of the distal ureter in patients who received dextranomerhyaluronic acid co-polymer showed granulomatous inflammation with multinucleated giant cells and other inflammatory cells, mainly lymphocytes and plasma cells (parts A and B of figure). Fibrosis and mast cell infiltration were observed in all cases and no difference in either parameter was evident in the treatment and control groups. Fibrosis was present around the pseudocapsule and extended between the individual dextranomer pearls (part C of figure). Mast cells were abundant in areas of fibrosis in each group (tables 3 and 4, and part D of figure). Implantation site. The central area of the implant material contained pearls of dextranomer at different stages of resorption and the implant was frequently surrounded by a fibrotic pseudocapsule, that is fibrous tissue accumulation (parts E to G of figure). Evidence of calcification was provided by small granular precipitates, primarily among the dextranomer pearls, in patients who received dextranomer-hyaluronic acid co-polymer but not in controls (tables 3 and 4, and part H of figure). Eosinophils were abundant between dextranomer pearls in some implant areas but not in others (table 3 and part I of figure). They were largely absent in control tissue. Of the 21 ureters implanted with co-polymer 7 had an eosinophil count of greater than 5/0.125 mm.2 field of view (range 6 to 70). Four of these ureters were in patients who underwent treatment for bilateral vesicoureteral reflux but the eosinophil count in the contralateral ureter was appreciably lower at 2 to 5 cells per 0.125 mm.2 field of view.

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DEXTRANOMER-HYALURONIC ACID FOR VESICOURETERAL REFLUX

TABLE 2. Schedule of dextranomer-hyaluronic acid co-polymer treatment, voiding cystourethrography and ureteral reimplantation in the treatment group Voiding Cystourethrogram 1* Pt. No.

Age (yrs., mo.)

Lt./Rt. Grade

Voiding Cystourethrogram 2 Age (yrs., mo.)

Lt./Rt. Grade

Treatment

Age at Reimplantation (yrs., mo.)

1† 8, 1 O/III 8, 8 O/III Yes 9, 2 1, 4 V/O No 2, 3 0, 3 O/III No 1, 4 1, 11 III/III No 3, 5 1, 10 III/II 2, 3 III/II Yes 4, 6 6, 4 III/O 7, 0 III/O Yes 7, 7 7, 1 O/O 9, 7 III/IV No 10, 8 0, 8 III/O‡ 1, 6 III/III‡ Yes 2, 9 3, 9 II/IV‡ 4, 6 O/III‡ Yes 5, 10 1, 5 III/O 1, 11 III/O‡ Yes 2, 11 2, 8 III/O‡ 3, 11 III/O‡ Yes 5, 12 2, 1 O/IV‡ 4, 7 IV/IV‡ No 4, 13 3, 6 III/O 4, 1 IV/IV Yes 4, * Dextranomer-hyaluronic acid treatment was done in all patients. † Third voiding cystogram showed grades O and III reflux on the right and left sides, respectively, at age 9 years 8 months. ‡ Voiding cystourethrogram done within 4 to 6 months of treatment.

8 11 7 10 0 10 4 1 4 6 4 9 9

Mos. Since First Implant 19 19 16 23 26 18 39 17 19 13 32 32 15

TABLE 3. Histological findings at the implantation site in patients who received 1 or more dextranomer-hyaluronic acid co-polymer implants for vesicoureteral reflux Pt. No.

Implant

1 Yes 2 Yes 3 Yes 4(rt./lt.) Small/yes 5(rt./lt.)* Yes/yes 6 Yes 7(rt./lt.) Yes/yes 8(rt./lt.)* Yes/yes 9 Yes 10 Yes 11(rt./lt.) Yes†/yes‡ 12(rt./lt.) No†/no‡ 13(rt./lt.) Yes†/yes‡ * Two samples not separated. † One treatment. ‡ Two treatments.

Fibrous Pseudocapsule No Yes Yes/yes –/Yes Yes Yes/yes Yes No/no No/no Yes/no

Pseudo-encapsulation was observed around 9 implants and calcification was present in 9 ureters treated with co-polymer (table 3). Calcification and pseudo-encapsulation were not noted in the control group (table 4). DISCUSSION

The results of this study indicate that dextranomerhyaluronic acid co-polymer treatment for vesicoureteral reflux is associated with a granulomatous inflammatory reaction, as shown by multinucleated giant cells. Initially the inflammatory reaction is an active and cellular process that is subsequently replaced by fibrosis. Calcification, pseudoencapsulation, and infiltration of fibroblasts and inflammatory cells are also observed at the implantation site. A similar inflammatory reaction associated with subcutaneous and submucosal administration of dextranomer-hyaluronic acid co-polymer has previously been reported in animal studies.8 This typical inflammatory reaction is associated with injection of a foreign material. For example, scleral sutures of silicone, polytetrafluoroethylene and hydrogen are also associated with giant cell infiltration, encapsulation and granuloma formation.9 Based on the evidence that giant cells appear to promote hydrogel hydrolysis9 it was postulated that the inflammatory reaction associated with dextranomerhyaluronic acid co-polymer treatment is benign and except for promoting hydrolysis of the implant it results in little or no long-term detrimental effect. We also identified focal areas of a high eosinophil count in some patients treated with the co-polymer, which may suggest a hypersensitivity reaction to 1 or more of the constitu-

No. Eosinophils/ 0.125 Mm.2

No. Mast Cells/ 0.125 Mm.2

Calcification

5 1 2 6/3 4/2 6 10/3 24/3 26 70 1/4 3/1 5/18

18 8 16 8/16 8/11 18 11/12 24/7 20 11 11/16 14/14 8/18

No No Yes Minor/yes No/yes No No/no Yes/yes Yes Yes No/no No/no Yes/no

ents of the copolymer. Nevertheless, in relative terms these eosinophil counts were low, particularly compared with those reported in patients with allergic nasal polyps. Free hyaluronic acid is known to stimulate inflammation via a cell adhesion molecule10 and the resultant complex stimulates mast cell adhesion,11 fusion of granulocytic phagocytes12 and eosinophil activation.13 However, the hyaluronic acid used in the current study was cross-linked and, therefore, no free hyaluronic acid should have been available to elicit an immunological reaction. The cross-linking of hyaluronic acid results in a polymer with a molecular weight of several orders of magnitude higher than that of free hyaluronic acid and high molecular weight forms of hyaluronic acid are less likely to stimulate eosinophilia.10 Furthermore, this form of hyaluronic acid is synthesized from bacteria under controlled conditions and it can be expected to be free of the hypersensitivity reactions associated with hyaluronic acid derived from animal sources.14 Metabolism of dextranomer-hyaluronic acid co-polymer into free constituents is an unlikely explanation of the high eosinophil count because the compound is hydrolyzed to carbon dioxide and water. Furthermore, the implant was still evident at the injection site in 12 of the 13 treated patients and they remained in situ up to 2 years 8 months, indicating little or no hydrolysis in this period. Similarly degradation of hyaluronic acid by free radicals, which is an established inflammatory pathway,15 is unlikely to have occurred in the current study because high molecular weight hyaluronic acid co-polymer has been shown to scavenge free radicals.16 Dextrans are known to elicit an eosinophilic inflammatory

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DEXTRANOMER-HYALURONIC ACID FOR VESICOURETERAL REFLUX

Representative histopathological findings of dissected distal ureter, including dextranomer-hyaluronic acid co-polymer implant. A, granulomatous reaction with multinucleated giant cells within implant, which is surrounded by fibrotic pseudocapsule. H&E, reduced from ⫻10. B, typical giant cells at high magnification. H&E, reduced from ⫻40. C, fibrotic bands (red areas) surround pseudocapsule and extend between individual dextranomer pearls (yellow areas). Piero-Sirius stain, reduced from ⫻10. D, section of fibrotic ureteral wall shows mast cells (arrows). Alcian blue PAS at pH 2.2, reduced from ⫻20. E, cross-section of distal ureter (left side) and implant (right side) reveals central degeneration of dextranomer pearls. H&E, reduced from ⫻2. F, cross-section of distal ureter and implant demonstrates persistent dextranomer pearls. PAS, reduced from ⫻2. G, accumulation of fibrotic tissue around muscle fibers in ureter and within pseudocapsule surrounding implant. Picro-Sirius stain, reduced from ⫻2. H, calcification (dark brown and black areas) within implant. von Kossa stain, reduced from ⫻40. I, isolated eosinophils (arrows) between dextranomer pearls. H&E, reduced from ⫻20.

Few studies have shown evidence of calcification associated with endoscopic implants. In the current series calcification developed in several patients, mainly within the dextranomer-hyaluronic acid co-polymer bolus. The reason for the calcification is unclear but it may have been due to local hypercalcemia or precipitation of calcium salts from the dextranomer-hyaluronic acid formulation. Alternatively it may have been the result of a local inflammatory reaction or microbial infection.19 Whatever its genesis to our knowledge the long-term consequences of calcification are unknown. Fibrosis and mast cells were evident in similar proportions in the treated and control groups, suggesting that these cellular responses are a pathophysiological feature of vesicoureteral reflux rather than a specific response to the co-polymer implant. This finding was not unexpected because mast cells release a number of active and chemotactic mediators involved in the development of inflammation and tissue repair.20 A possible limitation of this study is the fact the control group was not age or gender matched against the treatment group. However, matching was considered unnecessary because histological findings in the control group were similar and there was no evidence of a granulomatous reaction in these patients. The lack of a random cell count is another potential weakness of this study, although the process of cell counting was consistent among patients. A nonrandom approach may explain the high eosinophil counts in some patients, particularly those with a lower eosinophil count in the contralateral ureter. The lack of an implant in the control group is another study limitation because a high eosinophil counts may have been associated with an inert implant, thereby, lending support to our suggestion that eosinophilia is associated with foreign material implantation. However, it was considered unethical to treat patients in this way who had moderate to severe vesicoureteral reflux and required ureteral reimplantation. CONCLUSIONS

TABLE 4. Histological findings in controls in whom ureters were reimplanted without previous dextranomer-hyaluronic acid copolymer treatment Pt. No.

Eosinophils/ 0.125 Mm.2

Mast Cells/ 0.125 Mm.2

1 1 2 1 3 1 4 2 5 1 6 1 7 1 8 1 9 1 10 1 There was no pseudocapsule or calcification in any patient.

23 20 21 25 16 10 10 16 23 18

reaction,17 although since the introduction of hapten inhibition, dextrans have become one of the safest plasma substitutes. The dextranomer microspheres used in this study were cross-linked and, thus, would have been unable to stimulate an inflammatory response. Therefore, it is unlikely that dextranomer-hyaluronic acid co-polymer or its constituents were responsible for the high eosinophil count. The fact that a high eosinophil count was not observed at the 2 implantation sites in patients who underwent bilateral treatment supports this suggestion. Furthermore, there were no obvious macroscopic signs of an allergic reaction, such as erythema and swelling at the implantation site, in patients with a high eosinophil count. In addition, eosinophilia has been reported after treatment with collagen,18 which may suggest that implantation of a foreign material initiates such a response.

The data in this study show that endoscopic implants of dextranomer-hyaluronic acid co-polymer are associated with an inflammatory reaction of the giant cell type, inflammatory cell infiltration and fibrotic pseudo-encapsulation of the implant. These histological findings are not unexpected and are frequently associated with implantation of a foreign material. The long-term effects of the implant on the ureteral wall are unknown but the implant may completely hydrolyse and leave a small indolent scar free of inflammation. The implant in the current study was surgically removed because patients experienced recurrent vesicoureteral reflux. No safety issues, such as pain or discomfort, were related to the implant. Therefore, dextranomer-hyaluronic acid co-polymer was well tolerated and remains a safe and effective bulking agent for vesicoureteral reflux. REFERENCES

1. Medical versus surgical treatment of primary vesicoureteral reflux. International Reflux Study Committee. Pediatrics, 67: 392, 1981 2. Goldman, M., Bistritzer, T., Horne, T., Zoareft, I. and Aladjem, M.: The etiology of renal scars in infants with pyelonephritis and vesicoureteral reflux. Pediatr Nephrol, 14: 385, 2000 3. Jacobson, S. H., Hansson, S. and Jakobsson, B.: Vesico-ureteric reflux: occurrence and long-term risks. Acta Paediatr, suppl., 88: 22, 1999 4. Haferkamp, A., Mohring, K., Staehler, G., Gerner, H. J. and Dorsam, J.: Long-term efficacy of subureteral collagen injection for endoscopic treatment of vesicoureteral reflux in neurogenic bladder cases. J Urol, 163: 274, 2000 5. Dewan, P. A., Hoebeke, P., Hall, H. E., Chow, C. W., Edwards, G. A. and Terlet, J.: Migration of particulate silicone after ureteric injection with silicone. BJU Int, 85: 557, 2000

DEXTRANOMER-HYALURONIC ACID FOR VESICOURETERAL REFLUX 6. Malizia, A. A., Jr., Reiman, H. M., Myers, R. P., Sande, J. R., Barham, S. S., Benson, R. C., Jr. et al: Migration and granulomatous reaction after periurethral injection of polytef (Teflon). JAMA, 251: 3277, 1984 7. Lackgren, G., Wahlin, N., Skoldenberg, E. and Stenberg, A.: Long-term followup of children treated with dextranomer/hyaluronic acid copolymer for vesicoureteral reflux. J Urol, 166: 1887, 2001 8. Stenberg, A. M., Larsson, E., Lindholm, A., Ronneus, B., Stenberg, A. and Lackgren, G.: Injectable dextranomer-based implant: histopathology, volume changes and DNA analysis. Scand J Urol Nephrol, 33: 355, 1999 9. D’Hermies, F., Korobelnik, J. F., Meyer, A., Chauvaud, D., Pouliquen, Y. and Renard, G.: Experimental encircling scleral buckle with silicone and hydrogel: histopathologic and comparative study of 26 rabbit eyes. Retina, 19: 148, 1999 10. Ohkawara, Y., Tamura, G., Iwasaki, T., Tanaka, A., Kikuchi, T. and Shirato, K.: Activation and transforming growth factorbeta production in eosinophils by hyaluronan. Am J Respir Cell Mol Biol, 23: 444, 2000 11. Fukui, M., Whittlesey, K., Metcalfe, D. D. and Dastych, J.: Human mast cells express the hyaluronic-acid-binding isoform of CD44 and adhere to hyaluronic acid. Clin Immunol, 94: 173, 2000 12. Vignery, A.: Osteoclasts and giant cells: macrophagemacrophage fusion mechanism. Int J Exp Pathol, 81: 291, 2000

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13. Hamann, K. J., Dowling, T. L., Neeley, S. P., Grant, J. A. and Leff, A. R.: Hyaluronic acid enhances cell proliferation during eosinopoiesis through the CD44 surface antigen. J Immunol, 154: 4073, 1995 14. Micheels, P.: Human anti-hyaluronic acid antibodies: is it possible? Dermatol Surg, 27: 185, 2001 15. Li, M., Rosenfeld, L., Vilar, R. E. and Cowman, M. K.: Degradation of hyaluronan by peroxynitrite. Arch Biochem Biophys, 341: 245, 1997 16. Presti, D. and Scott, J. E.: Hyaluronan-mediated protective effect against cell damage caused by enzymatically produced hydroxyl (OH.) radicals is dependent on hyaluronan molecular mass. Cell Biochem Funct, 12: 281, 1994 17. Van Wyck, D. B., Cavallo, G., Spinowitz, B. S., Adhikarla, R., Gagnon, S., Charytan, C. et al: Safety and efficacy of iron sucrose in patients sensitive to iron dextran: North American clinical trial. Am J Kidney Dis, 36: 88, 2000 18. Frey, P., Lutz, N., Berger, D. and Herzog, B.: Histological behavior of glutaraldehyde cross-linked bovine collagen into the human bladder for the treatment of vesicoureteral reflux. J Urol, 152: 632, 1994 19. Brockhurst, R. J., Ward, R. C., Lou, P., Ormerod, D. and Albert, D.: Dystrophic calcification of silicone scleral buckling implant materials. Am J Ophthalmol, 115: 524, 1993 20. Krishnaswamy, G., Kelley, J., Johnson, D., Youngberg, G., Stone, W., Huang, S. K. et al: The human mast cell: functions in physiology and disease. Front Biosci, 6: D1109, 2001