Original Paper Audiology Neurotology Audiol Neurotol 2016;21:254–260 DOI: 10.1159/000447623 Received: March 3, 2016 Accepted after revision: June 15, 2016 Published online: September 2, 2016 Safety and Efficacy of Chitosan-Dextran Hydrogel in the Middle Ear in an Animal Model Selin Ünsaler a Bora Başaran b Şule Öztürk Sarı c Eyüp Kara d Kemal Değer b Peter John Wormald e a Patnos State Hospital, Ağrı, and Departments of b Otolaryngology, Head and Neck Surgery and c Pathology, Istanbul University Faculty of Medicine, and d Department of Audiology, Istanbul University Faculty of Health Sciences, Istanbul, Turkey; e Department of Surgery, Otolaryngology, Head and Neck Surgery, Adelaide University, Adelaide, S.A., Australia Abstract Objectives: To investigate the efficacy of chitosan-dextran hydrogel (CDH) in preventing postoperative adhesions between the tympanic membrane (TM) and intratympanic structures, and to evaluate its ototoxicity in an animal study. Methods: In the first step, ototoxicity was evaluated with 7 male albino guinea pigs (GPs) via auditory brainstem responses (ABR) before and 4 weeks after unilateral intratympanic injection of CDH and saline solution contralaterally. In the second step, 12 GPs underwent bilateral ear surgery. The middle ear (ME) mucosa was abraded, and the cavity was filled with CDH on one side and packed with Gelfoam on the contralateral side. A control group of 6 GPs underwent the same procedure except that no material was applied in the ME. The animals were euthanized at the end of the 7th week, and otomicroscopic findings were noted and the temporal bones harvested for the histologic examination. The findings were scored and compared. Results: There was no statistically significant difference between the pre- and postopera- © 2016 S. Karger AG, Basel 1420–3030/16/0214–0254$39.50/0 E-Mail karger@karger.com www.karger.com/aud tive ABR thresholds. In the otomicroscopic findings, the most prominent difference between the two groups was the presence of retraction of the TM in the Gelfoam group. The histopathologic findings revealed a higher degree of inflammation in the Gelfoam group compared with the CDH group. Conclusion: This study demonstrated that CDH has no ototoxic effects in GPs. Its use as an ME packing material revealed significantly less TM retraction and inflammatory reaction compared with Gelfoam. © 2016 S. Karger AG, Basel Introduction The aims of chronic otitis media surgery are disease eradication and reconstruction of the hearing mechanism. For a successful surgical result, the maintenance of a well-aerated middle ear (ME) cavity with sufficiently mobile ossicles is required. Intratympanic adhesions in the postoperative period may impair the surgical and audiologic outcomes by adhering the tympanic membrane (TM) to the promontory and fixation of the ossicles. This is commonly encountered when tympanic mucosa has to be removed during surgery. Selin Ünsaler, MD Patnos State Hospital Patnos Devlet Hastanesi TR–04500 Patnos, Ağrı (Turkey) E-Mai selinunsaler @ hotmail.com Downloaded by: Istanbul Universitesi 194.27.138.250 - 1/13/2017 7:27:20 AM Key Words Chitosan-dextran hydrogel · Middle ear · Anti-adhesive properties · Ototoxicity Efficacy of Chitodex in the Middle Ear vestigate the efficacy of CDH as an ME packing material in vivo and to evaluate its ototoxicity in an animal study. The CDH used in this study was supplied by Chitogel Ltd. (New Zealand). Materials and Method This prospective blinded randomized controlled study was conducted in the animal laboratory of Istanbul University Institute of Experimental Medicine with the approval of the Animal Research Ethics Committee of Istanbul University (Ref. No. 2013/108). Twenty-seven female guinea pigs (GPs) that weighed between 700 and 850 g were used. All procedures were performed under general anesthesia induced by using 5 mg/kg xylazine (Rompun) and 350 mg/kg ketamine HCl (Ketalar) via intramuscular injection. The study was designed in two steps; the first was the investigation of ototoxicity of CDH. For this purpose, 7 GPs underwent audiologic evaluation using auditory brainstem responses (ABRs), after which they received unilateral intratympanic injections of CDH and saline solution contralaterally in the control ear. Following a period of 4 weeks, they were reevaluated using ABRs, and the results of the pre- and postinjection periods were compared. Having shown that CDH had no ototoxic effect on the GP, the study proceeded to the second step. In the second step, 14 GPs underwent surgery on both ears, and at the end of the procedure, CDH was applied in one ear and Gelfoam in the contralateral ear. Two animals were lost before completion of the surgery due to anesthetic complications. Six other animals underwent surgery unilaterally but no material was applied in the ME at the end of the procedure; these animals were considered as the control group. After a follow-up period of 7 weeks for each animal, the findings on microscopic examination were noted. All 18 GPs were euthanized in an orderly fashion by induction of overdose anesthetics. Following decapitation, the temporal bones were prepared for the histopathologic examination. Step 1: Ototoxicity Assessment Audiologic Evaluation – ABR The ABRs were recorded using an ICS CHARTR EP 200 (GN Otometrics A/S, Denmark) system. The stimuli were as follows: click, 1-kHz tone burst and 8-kHz tone burst. Beginning from 70 dB nHL and decreasing gradually by 20 dB nHL, the thresholds were assessed by descending until 10 dB nHL. In GPs, wave III is the most prominent and stable wave recorded on ABR [Otto et al., 1988]. Therefore, the level of the lowest stimulus at which wave III could be detected was accepted as the threshold. The tests were done before the intratympanic injection and 4 weeks after the injection using the same recording parameters. Intratympanic Injection The TM was penetrated at the anterosuperior quadrant, and the ME cavity was filled with CDH on one side and with 0.9% saline solution on the contralateral side until it was observed that the cavity was full behind the transparent TM; the filling volume was approximately 0.2 ml. Audiol Neurotol 2016;21:254–260 DOI: 10.1159/000447623 255 Downloaded by: Istanbul Universitesi 194.27.138.250 - 1/13/2017 7:27:20 AM ME packing with various kinds of materials is commonly used to support the TM and ossicular grafts after reconstruction. An ideal ME packing material should be biocompatible, nonototoxic and absorbable. It should provide support to TM grafts and the ossicular chain, prevent adhesion formation, promote ME wound healing and hemostasis. To date there is no such material [Shen et al., 2011]. The packing material most commonly used in otologic surgery is Gelfoam (Pharmacia and Upjohn Company, Mich., USA), an absorbable, gelatinbased sponge material. It has been implicated in adhesion formation [Schuknecht, 1962; Hellström et al., 1983]. In order to prevent this complication, different agents have been tried to be applied in combination with Gelfoam [Wiesenthal and Garber, 1999; Bahadir et al., 2003]. Numerous materials have also been investigated to be used instead of Gelfoam such as hyaluronan-based products [Laurent et al., 1988; Bagger-Sjoback et al., 1993; Angeli et al., 2007] and cellulose derivatives [Jang et al., 2008a; Antonelli et al., 2010]. However, none have been shown to fulfill the requirements of the ideal packing material. Chitosan-dextran hydrogel (CDH) has been developed as a postoperative dressing to be used in endoscopic sinus surgery, and has been used in animal [Athanasiadis et al., 2008; Medina et al., 2012] and human trials [Valentine et al., 2010]. In these studies, it significantly reduced the number of adhesions and exhibited excellent hemostatic, mucoadhesive, and antimicrobial properties [Aziz et al., 2012]. Chitosan is a linear-polysaccharide-containing randomly distributed β-1,4-linked Dglucosamine and N-acetylglucosamine residue, and it is produced by deacetylation of chitin, which is the main component of the cell walls of fungi and the exoskeletons of arthropods such as crustaceans (e.g. crabs, lobsters, and shrimps) and insects [Lu et al., 2008; Li et al, 2014]. Chitosan can be chemically modified with the addition of a large negatively charged group, for example by carboxymethylation [Wang et al., 2013] or succinylation [Liu et al., 2009], as preferred in the formulation used in this study, to improve its solubility at a neutral pH. Furthermore, it must be cross-linked to macromolecules to slow its biodegradation and improve its stability. Dextran, a natural bacterial polysaccharide comprised of α-1,6-linked D-glucopyranose residues, was chosen for its ability to serve as a macromolecular crosslinker for polymers containing free amino groups [Draye et al., 1998]. CDH is being used on human paranasal sinuses, but it has never been tried in the ME. This study is aimed to in- Microscopic and Otoendoscopic Examination After a follow-up period of 7 weeks, the findings of the microscopic examination were noted as follows: persistence of perforation, existence of hyperemia and retraction of the TM. Afterwards, each GP was euthanized by induction of an anesthetic overdose. Following decapitation and excision of the auriculae and the external ear canal, an otoendoscopic view of the TM was recorded using a 2.7-mm 30-degree endoscope. Histopathologic Examination After the endoscopic examination, the neurocranium was excised, fixed in 10% formaldehyde, and decalcified in fast decalcifier solution for 48 h. The temporal bones were sliced horizontally with 4-mm thickness and embedded in paraffin wax. The histologic sections with 2-μm thickness were stained with hematoxylin and eosin. A blinded histologic evaluation was performed by the pathologist. The parameters noted and scored by an observer who was blinded to the intervention were as follows: • The degree of thickening of the TM • The degree of thickening of the tympanic mucosa • The degrees of the following inflammation indicators in each of the TM and the tympanic mucosa: fibroblastic activity, vascular proliferation, fibrosis Scorings were: none = 0; mild = 1; moderate = 2; severe = 3. Statistical Analysis The statistical analysis was performed using SPSS version 21.0 (SPSS Inc., IBM, Chicago, Ill., USA). The Wilcoxon test was used in the evaluation of the audiologic results. Otomicroscopic findings were compared between the 3 groups using Fisher’s test, whereas the Mann-Whitney test was used to compare the histopathologic findings of the 3 groups. Results Audiologic Results There was no statistically significant difference between the thresholds recorded in the preinjection ABR and those in the postinjection ABR for either CDH sides or control sides (table 1). In the CDH-applied ears, the latency of wave III in the postinjection ABR was found to be greater (0.08 ms lon256 Audiol Neurotol 2016;21:254–260 DOI: 10.1159/000447623 Table 1. The thresholds (dB) in the preoperative and postoperative ABRs and the statistical analysis results CDH Control click 6 kHz 8 kHz click 6 kHz 8 kHz Preoperative Postoperative Z thresholds thresholds value p value 14.3 ± 11.3 15.7 ± 13.1 12.9 ± 7.6 12.9 ± 7.5 12.9 ± 7.5 12.9 ± 7.6 0.41 0.13 0.65 0.31 0.41 0.31 13.6 ± 9.5 14.3 ± 9.3 14.3 ± 11.3 16.4 ± 11.1 19.3 ± 12.7 10.7 ± 1.9 –0.816 –1.511 –0.447 –1.000 –0.816 –1.000 Table 2. The latency values of wave III in the preoperative and postoperative ABRs CDH Control preoperative postoperative preoperative postoperative Latency, ms t value p value 3.40 ± 0.15 3.48 ± 0.18 3.34 ± 0.16 3.40 ± 0.11 3.33 0.016 2.27 0.065 Table 3. Otomicroscopic results Persisted TM Retraction Hyperemia Effusion in perforation of the TM of the TM the ME Gelfoam CDH Control p value 2/12 3/12 – 0.99a 7/12 1/12 – 0.027b 6/10 3/9 3/6 0.36a 3/10 1/9 – 0.58a a No statistically significant difference between Gelfoam group and CDH group. b Statistically significant difference between Gelfoam group and CDH group. ger) than that measured in the first ABR (table 2). This difference was statistically significant (p = 0.016); however, a lengthening of 0.08 ms of the latency value was not considered clinically significant. There was no such difference in the control ears. Otomicroscopic Results With the exception of 2 ears with CDH, 1 ear with Gelfoam and bilateral ears in one animal, which had bilateral suppurative infections, the perforations had healed in most of the ears. Ünsaler/Başaran/Öztürk Sarı/Kara/Değer/ Wormald Downloaded by: Istanbul Universitesi 194.27.138.250 - 1/13/2017 7:27:20 AM Step 2: Evaluation of the Antiadhesive Property of CDH Surgical Procedure In order to decrease the surgical morbidity and perform a sham operation on both the TM and ME, an endaural approach was undertaken. First a canalplasty was performed to enlarge the external ear canal. Having obtained better exposure of the TM, a large myringotomy with removal of one fourth of the TM was performed. The tympanic mucosa on the medial wall of the ME cavity was stripped off with an elevator without damage to the cochlea. Twelve GPs underwent this procedure bilaterally, at the end of which CDH was applied in one ear while Gelfoam was applied in the contralateral ear. The 6 GPs of the control group received no material in the ME. 1 2 Fig. 1. The otoendoscopic view of the left ear (Gelfoam side) of GP 4. The TM is retracted towards the cochlea (blue star). There is a deep retraction pouch (arrow) located anteriorly. Black star = Malleus. Fig. 2. The otoendoscopic view of the right ear (CDH side) of GP 4. The TM is normal; the perforation (white star) occurred during postmortem dissection. Black star = Malleus. Table 4. Histopathologic findings of the middle ear mucosa and TM Fibroblastic activity Vascular proliferation Fibrosis Thickness of the mucosa none mild moderate severe none mild moderate severe none mild moderate severe none mild moderate severe CDH (n = 12) Gelfoam (n = 12) Control (n = 6) MEM TM MEM TM MEM TM 3 4 5 – 4 3 3 2 6 6 – – 3 4 4 1 4 5 3 – 5 2 4 1 6 4 2 – 4 3 5 – – – 3 9 – 1 6 5 0 3 5 4 – 0 5 7 1 1 6 4 1 3 4 4 – 3 7 2 – 3 6 3 – 5 1 – – 5 1 – 5 1 – – – 5 1 – 1 4 1 – 1 4 1 – 1 4 1 – – 5 1 – The most prominent difference between the two sides was the high rate of retraction in the ears with Gelfoam (table 3). In 7 animals, the TMs were retracted towards the cochlea on the Gelfoam sides (fig. 1); there was no retraction on their contralateral ears treated with CDH (fig. 2). The retracted TMs were opaque, and there was an accumulation of keratin debris in the retraction pockets in 2 ears (fig. 2). There was retraction of the TM on the CDH side in only 1 animal, but no retraction on the Gelfoam side. Four animals had no retraction on either side. No retraction was observed on the TMs of animals in the control group. In the statistical analysis, the difference between Gelfoam sides and CDH sides was found to be significant (p = 0.02). There was also a significant differ- ence between the control ears and ears with Gelfoam (p = 0.03), but there was no significant difference between the control ears and ears with CDH (p = 0.99). The comparisons between the 3 groups in terms of the presence of hyperemia of the TM and effusion in the ME were made after excluding 2 animals with persistent perforation. The results are shown in table 3. There was no significant difference between the 3 groups regarding hyperemia (p = 0.36; p = 0.62; p = 0.62) or effusion (p = 0.58; p = 0.11; p = 0.52). Efficacy of Chitodex in the Middle Ear Audiol Neurotol 2016;21:254–260 DOI: 10.1159/000447623 Histopathologic Results The histopathologic results (table 4) were consistent with the otomicroscopic results. When the degree of fi257 Downloaded by: Istanbul Universitesi 194.27.138.250 - 1/13/2017 7:27:20 AM MEM = Middle ear mucosa. Scoring: none (0), mild (+1), moderate (+2), severe (+3). Fig. 3. The increase in thickness of the TM and adhesion site (blue Fig. 4. The increase in thickness of the TM (+3), fibrosis (+3), the arrow) to the cochlea (black asterisk); the exudate in the tympanic cavity (red asterisk) and the increase in thickness of the mucosa at the site of abrasion (black arrow). The external ear canal is indicated by a blue asterisk. HE. ×2. increase in vascularization (+3) and keratin debris in a retraction pocket. HE. ×10. broblastic activity, vascular proliferation, fibrosis, and thickness of the TM and ME mucosa were scored, the scores were found to be higher in ears with TMs observed to be opaque and retracted (fig. 3), but lower in ears with transparent and bright TMs in the otomicroscopic examination. GPs with scores ≥2 on the CDH side also had high scores on the contralateral sides. With the exception of 2 GPs that had higher scores on the CDH side, the scores were generally higher on the Gelfoam side. It should be noted that there were persistent perforations on the CDH sides in those 2 animals. The fibroblastic activation as well as the vascular proliferation and fibrosis in the tympanic mucosa were significantly greater in ears with Gelfoam than in ears with CDH (p < 0.001; p = 0.017; p < 0.001). The tympanic mucosa was significantly thicker in ears with Gelfoam than in ears with CDH (p = 0.001). There was no difference between the ears with CDH and the control group in either of the parameters (p = 0.87; p = 0.99; p = 0.18; p = 0.83), whereas all parameters were significantly greater in ears with Gelfoam than in the control group (p < 0.001; p = 0.003; p = 0.001; p = 0.001). The fibroblastic activation and fibrosis of the TM were significantly greater in ears with Gelfoam than in ears with CDH (p = 0.004; p = 0.001). The TMs were found to be significantly thicker on the Gelfoam sides (p = 0.02; fig. 4). There was no statistical difference in the degree of vascular proliferation (p = 0.06). There was no significant difference between the ears with CDH and the control group in either of the parameters (p = 0.79; p = 0.96; p = 0.31; p = 0.92), but there was a statistically significant difference between the ears with Gelfoam and the control group in all parameters for TM (p = 0.01; p = 0.05; p = 0.01; p = 0.02). No remnant of packing material was detected in either ear, which shows that both were absorbed before the seventh week. Audiol Neurotol 2016;21:254–260 DOI: 10.1159/000447623 This study has shown that CDH is well tolerated in the ME cavity in an animal model without change in postoperative ABR values and aids healing when compared with the most commonly used material, Gelfoam. An ideal ME packing material should be biocompatible, cause minimal inflammation, be absorbable, nonototoxic, malleable, and structurally stable. It should provide support to TM grafts and the ossicular chain, promote ME wound healing and hemostasis, and prevent adhesions and fibrosis [Shen et al., 2011]. Gelatin sponge is actually the most commonly preferred absorbable ME packing material. Despite its advantages and popularity, it is known to cause a certain degree of fibrosis, adhesion, and TM retraction [Joseph, 1962; Hellström et al., 1983; Liening et al., 1997]. In animal studies, it was shown that in areas denuded of muÜnsaler/Başaran/Öztürk Sarı/Kara/Değer/ Wormald Downloaded by: Istanbul Universitesi 194.27.138.250 - 1/13/2017 7:27:20 AM 258 Discussion showed no difference in the ABR thresholds before and 4 weeks after intratympanic injection of CDH, which indicates that CDH has no ototoxic effect on GPs. The delay in the mean latency by 0.08 ms on the CDH sides [before injection (3.40 ms) and after injection (3.48 ms)] was statistically significant but was not considered clinically significant because it was shorter than 0.20 ms. The delay seen in wave III latency, which was greater in the CDH ears than in control ears [before injection (3.34 ms) and after injection (3.40 ms)], may be due to the persistence of CDH in the ME cavity at the end of the fourth postoperative week and consequent conductive hearing loss. However, this is a speculation because no tympanotomy or histopathologic examination was performed in this period. In the otomicroscopic examination, the most striking result was that TMs were retracted on the Gelfoam side in 7 of the 12 animals, whereas there was no retraction on the CDH side of the same animals and that TMs on the CDH sides were more transparent. These findings show that CDH provides a better supporting surface and antiadhesive effect on the TM when compared with Gelfoam. In humans, the first postoperative results in terms of TM healing and audiometric testing are maintained during the postoperative 6–8 weeks’ follow-up control. Here we present the postoperative 7 weeks’ results, which were promising, and a further study to investigate late postoperative changes should be conducted; it might be a clinical study as well. The anti-inflammatory effect of CDH in the middle ear was investigated by histopathologic examination. The findings revealed that the fibroblastic activity, fibrosis, and the thickness of both the TM and the tympanic mucosa were significantly less on the CDH sides compared with the Gelfoam sides. This indicates that the inflammatory response to CDH is less than that to Gelfoam. There was no significant difference between the CDH group and the control group, which shows that CDH can be preferred in cases that require packing material to support the TM grafts and the ossicles. The results of this study showed that all the histologic parameters (fibroblastic activity, vascular proliferation, fibrosis and thickness of both the tympanic mucosa and TM) of the Gelfoam group were significantly worse than those of the control group, and this supports the conclusions of Hellström et al. [1983] and Joseph [1962] who suggested that Gelfoam may be detrimental to healing in the presence of tympanic mucosal damage. The final mucosal changes in a later postoperative time may be investigated in a longer-lasting animal study. Efficacy of Chitodex in the Middle Ear Audiol Neurotol 2016;21:254–260 DOI: 10.1159/000447623 259 Downloaded by: Istanbul Universitesi 194.27.138.250 - 1/13/2017 7:27:20 AM cosa, Gelfoam was coated by fibrin and infiltrated by fibroblasts and inflammatory cells, which led to the formation of granulation tissue giving rise to fibrosis [Joseph, 1962]. It was mentioned that its gelatinous stroma probably functioned as a scaffold for connective tissue hyperplasia for some time before it was absorbed. Therefore, it was suggested not to use Gelfoam on the areas of injured mucosa [Joseph, 1962; Hellström et al., 1983]. However, it is a common finding to encounter unhealthy tympanic mucosa, which can involve granulation tissue and metaplasia, and in such cases, the mucosa must be excised to eradicate the disease [McGhee and Dornhoffer, 1999]. In the literature, there are numerous studies where different agents were tried to overcome the problem of adhesion formation in the ME cavity; nonetheless, no material has yet gained popularity because of the disadvantages of each. Gelfilm was suggested [Holzer, 1973; Falbe-Hansen and Tos, 1975] instead of Gelfoam because it causes less adhesion owing to its nonporous structure [McGhee and Dornhoffer, 1999], but it has the disadvantage of application because of its hardness [Shen et al., 2011]. Hyaluronic acid was proved to be safe [Laurent et al., 1988; Bagger-Sjoback et al., 1993; Angeli et al., 2007; Jang et al., 2008b] in the ME, and various modified forms were tried to improve its stability such as its esterified form, Merogel [Martini et al., 2000]. However, in one study, the amount of Merogel was mentioned to be insufficient in the ME in the second postoperative week and to be inadequate for dividing into small pieces and was accused of causing a reversible increase in the ABR thresholds in the postoperative sixth week [Park et al., 2006]. The carboxylated form of hyaluronic acid, Carbylan SX, was suggested to be better [Park et al., 2006]. Other materials consisting of hyaluronic acid and carboxymethyl cellulose are Seprafilm [Jang et al., 2008b] and Sepragel [Angeli et al., 2007]. These have also been tried in the ME but lost popularity after carboxymethyl cellulose had been shown to cause an irreversible increase in the ABR thresholds after postoperative week 8, thought to be due to sensorineural hearing loss [Antonelli et al., 2010]. The use of hydrogels as agents aiding postoperative healing has increased in the last decade owing to their water content, flexibility, and ability to mimic living tissue and to fit the topography of the surgical site [Peppas et al., 2000]. Chitosan and dextran, which are both naturally derived polysaccharides, are in vivo biodegradable and biocompatible [Khor, 2002; Shkurupiy et al., 2008; Cabral et al., 2014]. The results obtained in this study Disclosure Statement Conclusion This study has shown that CDH is safe and effective in the ME of GPs. The next step is to evaluate its use in the ME in human trials. In addition, the use of CDH as a packing material in the mastoid cavity to support epithelization after mastoidectomy should be investigated. There is no conflict of interest. P.J.W. is part of a consortium that has patented the use of Chitogel in ENT. References 260 Holzer F: The fate of gelatin film in the middle ear. Arch Otolaryngol 1973;98:319–321. 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