Journal of Biomaterials Science, Polymer Edition
ISSN: 0920-5063 (Print) 1568-5624 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsp20
Novel transparent collagen film patch derived from duck’s feet for tympanic membrane perforation Soo Hyeon Kim, Ho Jun Lee, Ji-Chul Yoo, Hyun Jung Park, Ju Yeon Jeong, Ye Been Seo, Md. Tipu Sultan, Soon Hee Kim, Ok Joo Lee & Chan Hum Park To cite this article: Soo Hyeon Kim, Ho Jun Lee, Ji-Chul Yoo, Hyun Jung Park, Ju Yeon Jeong, Ye Been Seo, Md. Tipu Sultan, Soon Hee Kim, Ok Joo Lee & Chan Hum Park (2018) Novel transparent collagen film patch derived from duck’s feet for tympanic membrane perforation, Journal of Biomaterials Science, Polymer Edition, 29:7-9, 997-1010, DOI: 10.1080/09205063.2017.1374031 To link to this article: https://doi.org/10.1080/09205063.2017.1374031
Accepted author version posted online: 01 Sep 2017. Published online: 07 Sep 2017. Submit your article to this journal
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Journal of Biomaterials Science, Polymer Edition, 2018 VOL. 29, NOS. 7-9, 997–1010 https://doi.org/10.1080/09205063.2017.1374031
Novel transparent collagen film patch derived from duck’s feet for tympanic membrane perforation Soo Hyeon Kima#, Ho Jun Leea,b#, Ji-Chul Yooc, Hyun Jung Parka, Ju Yeon Jeonga, Ye Been Seoa, Md. Tipu Sultana, Soon Hee Kima, Ok Joo Leea and Chan Hum Parka,b a
School of Medicine, Nano-Bio Regenerative Medical Institute, Hallym University, Chuncheon, Republic of Korea; bDepartment of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, Chuncheon Sacred Heart Hospital, Hallym University, Chuncheon, Republic of Korea; cCentral Research Institute, SewonCellontech Co., Ltd, Wooyoung Techno Center, Seoul, Republic of Korea
To increase healing rate of tympanic membrane (TM) perforations, patching procedure has been commonly conducted. Biocompatible, biodegradable patching materials which is not limited across cultures is needed. The authors evaluated the effectiveness of novel transparent duck’s feet collagen film (DCF) patch in acute traumatic TM perforation. This procedure was compared with spontaneous healing and paper patching. Cell proliferation features were observed in paper and DCF patches. Forty-eight TMs of 24 rats were used for animal experiment, perforations were made on each TMs, and divided into three groups according to treatment modality. Sixteen were spontaneously healed, 16 were paper patched and 16 were DCF patched. The gross and histological healing results were analyzed. Both paper and DCF patch showed no cytotoxicity, but cell proliferations were more active in DCF than paper in early stage. In animal study, the healing of TM perforations were completed within 14 days in all three groups, but found to be faster in DCF patch group than paper patch or spontaneous healing group. The DCF patches were transparent and size of DCF patches were gradually decreased, so there were no need to remove the DCF patches to check the wound status or after the completion of healing. According to this result, authors concluded that DCF patch is transparent, biocompatible and biodegradable material, and can induce fast healing in acute traumatic TM perforations.
Received 10 May 2017 Accepted 28 August 2017 KEYWORDS
Tympanic membrane perforation; myringoplasty; duck’s feet collagen; collagen patch
Introduction There are many circumstances that can induce acute traumatic tympanic membrane (TM) perforations these days; physical trauma or barotrauma induced by sports activity, airplane, underwater activity and so on. Accordingly, acute traumatic TM perforation is common clinical situation. If it is not properly healed, it could cause a conductive hearing loss or a middle ear infection. There have been many articles regarding treatment of acute traumatic CONTACT Chan Hum Park [email protected]
, [email protected]
# The first two authors contributed equally to this paper as the first author. © 2017 Informa UK Limited, trading as Taylor & Francis Group
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TM perforation. Conservative management inducing spontaneous healing resulted in high rates of complete healing in many situations [1–3]. But in relatively severe specific situations like followings; large perforations, multi-directional lacerations and so forth, the clinicians should pay more attention and better execute patching procedure to facilitate the healing process and increase the healing rate. Paper patch has been used widely for TM patching procedure. This material has several merits and demerits. It is cheap, easy to obtain and easy to make. But this material itself is not adhesive, so it is sometimes detached from the perforated TM and displaced. Paper is not a biocompatible, so there is possibility of foreign body reaction or inflammation during healing process. Paper is not a biodegradable material, so it has to be removed after healing process is completed. Removal of paper patch from external auditory canal is generally not difficult, but in children or poorly cooperative patients, it is not easy. To overcome these demerits of paper patch, the authors have been investigated and developed the new patch materials for TM perforations. The first one was silk fibroin patch which was made for acute and chronic tympanic membrane perforation [4–6]. This silk fibroin patch (derived from Bombyx mori silk worms) was found to have similar or better result comparing with conventional paper patching or perichondrium myringoplasty, and showed biocompatibility and biodegradability . The next one was a collagen patch. Collagen biomaterials have been reported that it is advantageous for cell seeding and attachment, and will be degraded after the completion of wound healing [8–11]. There were enormous amount of duck’s feet disused as an animal by-product in Korea. Therefore we have chosen duck’s feet as a source of collagen. Additional advantage of collagen materials derived from duck’s feet is possibility of distribution even in Arabic or Hindu communities unlike collagen materials derived from bovine or porcine origin. The authors have developed sponge type collagen material and collagen/silk hybrid material, and confirmed its biocompatibility and biodegradability [8,9]. The demerit of these collagen materials was opacity. The authors thought if these materials were transparent, we could see the wound healing process through the patch. After the investigation, authors found the fabrication process of transparent film type collagen biomaterial. In the present study, we evaluated the effectiveness of transparent Duck’s feet Collagen Film (DCF) patch in the acute traumatic TM perforation. It was compared with spontaneous healing, and paper patch. We hypothesized that DCF patch has advantages over spontaneous healing and conventional paper patch in treatment of acute traumatic TM perforation.
Materials and methods Fabrication of transparent duck’s feet collagen film patch DCF was manufactured in SewonCellontech (Seongsu-dong, Seoul, Korea), and the schema of the process is described in Figure 1. Duck’s feet were obtained from Joowonori (Jincheongun, Chungcheongbuk-do, Korea), and washed with tap water for 24 h at room temperature to remove the dust and blood. Then, 0.5M NaOH was added to the sample at a ratio of 3:1 (NaOH: sample) to remove the fat. And then, 5% citric acid was added to the sample at a ratio 3:1 (citric acid: sample) and stirred for 72 h at 4 °C. After the acid treatment was finished, duck’s feet was removed from the solution and the remaining solution was centrifuged for 15 min at 4 °C, 12,000 rpm. The supernatant of the centrifuged solution was
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Figure 1. The schema of the fabrication of duck’s feet collagen film patch using freeze drying method and ultraviolet cross linking technique.
collected and precipitation reaction was elicited with 100% EtOH. Precipitated collagen was harvested by centrifuging the sample for 5 min at 4 °C, 3500 rpm. The pellet of duck’s feet collagen was harvested and lyophilized in a freeze dryer (Genesis 25-LE, Virtis) at 20 mmHg, −40 °C. Duck’s feet collagen pellet was dissolved in 0.5M acetic acid solution to make a concentration of 0.6~1% weight/volume. And then bubbles were removed with negative pressure. This bubble-free duck’s feet collagen solution was poured into the Petri dishes (1 mL/cm2), and irradiated with ultraviolet rays at intensity of 0.35~0.38 mW/cm2 for 24 h. And then, this solution was dried up at room temperature. Finally, fabricated duck’s feet collagen film was punched into 6 mm sized round shape pieces. Fabrication of paper and porcine collagen film patches Paper patches were made with commercially available cigarette paper. It was designed the same as DCF patch using 6 mm-sized round shape punch, and sterilized with ethylene oxide gas before experiment . To confirm whether the DCF have the common collagen physical property like other collagen materials, and to compare the physical property of DCF with collagen material from another source, pre-existed, more popular collagen material, porcine collagen was made into Porcine Collagen Film (PCF) and it was compared with DCF. It was manufactured in the same company, SewonCellontech (Seongsu-dong, Seoul, Korea). This company made PCF using porcine skin, with the same manufacturing procedures of DCF. Detailed manufacturing information of PCF was not provided from the company, but porcine collagen solution manufacturing method and porcine collagen separation method of this company is registered and can be searched in Korea Intellectual Property Right Information Service (www.kipris.or.kr).
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Morphological analyses The gross morphology and transparency of DCF were observed. Low vacuum scanning electron microscope (LV-SEM, S-3500N, Hitachi, Tokyo, Japan) was used to observe the surface and cross-sectional morphology of the DCF. DCF was processed with 10 nm layer of silver/palladium at 15 mA discharge current with an Ion Sputter (1010, Hitachi, Tokyo, Japan) for 120 s. The microphotographs were taken at magnifications of 1000 at an accelerating voltage of 10 kV. FTIR spectroscopy Using a FT-3000 spectroscopy (BIO-RAD, Seoul, Korea), the attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra of the DCF and PCF were analyzed and compared. Number of scans were 4, resolution was 4 cm−1, and scan range was 400~4000 cm−1. Measurement of tensile strength To evaluate the tensile property, DCF and PCF were designed to be 10 mm in length, 2.5 mm in width, and 50 μm in thickness. Qmesys Universal Testing Machine (QM100S, Qmesys, Korea) was used for the measurement of tensile strength. With a crosshead speed of 0.5 mm/min, tensile strength of DCF and PCF were measured (n = 3, both) and compared. Tensile strength of the collagen membrane samples were determined by computing the maximum stress. Cytotoxicity test using CCK-8 assay NIH3T3 fibroblasts from ATCC (Manassas, VA, U.S.A.) were obtained. Dulbecco’s Modified Eagle’s Medium (DMEM) from Welgene, Fresh Media™ (Dalseo-gu, Daegu, Korea) were purchased. Ten percent of fetal bovine serum and 1% of antibiotics/antimycotics and trypsin were supplemented in this DMEM. Cell counting kit, CCK-8, from Enzo Life Sciences (Farmingdale, NY, U.S.A.) was used for cytotoxicity test. NIH3T3 fibroblasts were cultured on the paper and DCF patches (n = 3, both). The activity of enzyme of the cells was evaluated using CCK-8 assay. After 2 sub-cultures using DMEM, NIH3T3 fibroblasts which were stored in liquid nitrogen were carefully raised. Two hundred microliter of 2 × 104 NIH3T3 fibroblast cells were seeded on paper and DCF patches. These cells were suspended in a culture plate and incubated in 5% of CO2 and 37 °C of temperature. After the 1, 5, and 7 days of incubation, the contents of wells were sucked out and CCK-8 assay was carried out by adding 20 μL of CCK-8 reagent to each patches. It was incubated for 2 h in the temperature of 37 °C, and then 100 μl of incubated cell suspension from each patches were transferred to 96-well plate. Absorbance of the supernatants at 450 nm was analyzed using a plate reader (Molecular Devices, SpectraMax®Plus 384, Sunnyvale, CA, U.S.A.). The proliferation of the cells was assessed by comparing the absorbance of control well containing CCK-8 solution to that of sample solutions.
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Application of patch materials in study animal This animal study was approved by the institutional review board of Hallym University. Forty-eight TMs of twenty-four adult male Sprague-Dawley rats weighing about 300 g were used. All the surgical experiments were conducted under general anesthesia using 75:25 mixture of nitrous oxide and oxygen containing 3% of isoflurane (Air Liquid America, Houston, TX). After general anesthesia, a rodent respirator (Harvard Apparatus, SouthNatick, MA) were used for ventilation. 1.8 mm sized perforations were made on pars tensa of each TMs using a hemisphere-shaped red-hot needle under otomicroscope (Opmi 1, Zeiss, Germany). Study rats were divided into three groups. Each Sixteen TMs of eight rats were assigned to act as the spontaneous healing group, the paper patch group and the DCF patch group respectively. Using a otomicroscope, paper patches and DCF patches were applied onto the perforated TM to cover the perforation and perforation margin. To maintain the patches in place after application, antibiotic ointment was plastered onto the paper patches because paper is not a adhesive material, and DCF was immersed with saline solution. After patch applications, study animals were kept in separate cages, Food and water were freely provided. To prevent the infection, two drops of antibiotic otic solution (Tarivid otic solution®, ofloxacin 3 mg/cm3; Daiichi Sankyo, Tokyo, Japan) were applied into the external auditory canals during the first 3 days. Two mice of each groups were sacrificed at 1, 3, 7, and 14 days after making perforations, and then four TMs of these two mice of each group were examined. Using an otoendoscope, the photographs of each TMs were taken before and after making perforation and daily until the perforations were closed. To compare the healing speed of each group, the percentage of healed perforation area was measured. Paper patches and DCF patches was removed and the remaining perforation size were measured. The calculated area of the healed portion was compared with the pre-treatment TM perforation size using the InnerView™ software (InnerView, Bundang-gu, Korea). To observe how healing processes go along as time passes, histopathological evaluation was performed. They were decapitated under general anesthesia, cranial vault and brain were removed, and external ears were subsequently separated at the osteocartilaginous junctions. The temporal bone was identified, and the tympanic bulla was isolated and fixed in 10% of formaldehyde solution. The specimens were decalcified for 3 days in an EDTA solution, and then dehydrated, embedded in paraffin (Shandon Citadel 1000, Thermo Electron Corp., Walthan, MA). Then, the specimen was bisected through the perforation, perpendicular to the handle of the malleus. The resulting paraffin block was sliced into 5-μm-thick sections, and hematoxylin-eosin (H&E) staining was performed.
Results Morphology of the DCF patch Figure 2 shows the gross appearance of the DCF patch, which was made in a size of 6 mm, and thickness of 55.5 ± 10.9 μm. This DCF patch was so transparent that we could see through the patch. The surface and cross-sectional LV-SEM image of fabricated DCF patch is shown in Figure 3. The surface was smooth, and unlike our previous sponge type collagen patch , pore structure was not definite. Cross-sectional image shows its homogeneity. When TM is perforated, the margins of the perforation are usually inverted or everted by the explosive or implosive force, and this inversion or eversion of the margin can be resulted
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Figure 2. The gross appearances of duck’s feet collagen film patch.
Figure 3. The surface (left, A) and cross-sectional (right, B) LV-SEM micrographs of duck’s feet collagen film patch.
in the incomplete healing of the wound or tympanosclerosis. In this situation, treating the perforated margin, and applying this smooth and homogeneous scaffold might facilitate the wound regeneration by leading and supporting the regenerative cells from perforated margin grow into the desirable direction along this scaffold. And because this material is transparent, we could see inside wound healing process through the patch unlike other patch materials like paper. FTIR spectroscopy The FTIR spectroscopy of the DCF and PCF is showed in Figure 4. Spectra of DCF and PCF were similar and showed the typical absorption bands of native collagens [12–16]. The FTIR spectra of PCF showed strong peaks at 1629, 1542 and 1236 cm−1 in the amide I, II and III bands. DCF showed strong peaks at 1627, 1533 and 1237 cm−1 in the amide I, II and III bands. These bands showed no apparent band shift compared to the spectra of native collagen. According to this FTIR spectroscopy, authors could assume that DCF
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Figure 4. FTIR analysis of duck’s feet collagen film and porcine collagen film.
Figure 5. Tensile strength of duck’s feet collagen film and porcine collagen film according to the methods. Young’s modulus was 0.655 ± 0.163 in DCF and 4.43 ± 0.799 in PCF.
and PCF have structural similarity, and both of the collagen films are structurally similar to native collagens. Measurement of tensile strength The tensile strength of DCF and PCF was measured and the result was shown in Figure 5. The Stress (kgf/mm2)–Strain (%) value of PCF was 21.43 and 18.74 respectively, and its value for DCF was 4.18 and 32.34 respectively. The Young’s modulus was 0.655 ± 0.163 in DCF and 4.43 ± 0.799 in PCF. According to this results, deformation rate of DCF was higher than PCF, and angle of inclination was lower than PCF which mean DCF is more elastic and tougher
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than PCF. Breaking strength (stress) of PCF was higher than DCF and deformation rate of PCF was lower than DCF which means PCF was harder but brittler than DCF. Cytotoxicity test by CCK-8 assay The results of the CCK-8 assay at 1, 5, 7 days of incubation is showed in Figure 6. The NIH3T3 fibroblasts were continually proliferated, so we could assume that both of the paper and DCF patches provided a favorable environment to NIH3T3 fibroblasts. Authors observed no cytotoxic effect from each patches. DCF patch showed better cell growth at day 1 and 5 and reached to cell saturation faster than paper patch. Paper patch showed better cell growth at the Day 7, but this result may attribute to degrading property of collagen film patches. Gross inspection of perforated TMs Figure 7 shows the endoscopic findings of tympanic membrane of study animals. There were no evidence of inflammation or infection at any time point of observation. The DCF patches were better attached to the tympanic membrane perforation area compared to the paper patch which was frequently detached and migrated between day 3 and day 7. The paper patch had to be removed manually from TM to check the healing process, because it is not a biodegradable or transparent material. But in contrast to the paper patch, DCF patch did not have to be removed from TM because it is transparent and biodegradable material. DCF patch showed notable degradation and shrinkage at Day 14. The healing rate of the perforated TM was measured and it was shown in Figure 8. At day 7, DCF patch group showed complete healing of perforated TM. And paper patch group showed near complete healing, but control group showed incomplete healing and remnant perforation. At day 14, all three groups showed complete healing of the perforated TMs. The mean percentage of healed area of control group, paper patch group and DCF patch group were 23, 26, and 35% respectively at 3 days after treatment, and 76, 96, and 100% respectively
Figure 6. Cell proliferation in paper patch and duck’s feet collagen film patch by CCK-8 assay at 1, 5 and 7 days.
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Figure 7. The endoscopic findings of the tympanic membrane. On day 7, perforations of the tympanic membrane still remained in the control group, the paper patch group showed near complete healing, and the duck’s feet collagen film patch group showed complete healing of the perforation. All three groups showed complete healing of the tympanic membrane perforation on day 14.
at 7 days after treatment. At day 14, all three groups showed complete healing. This result shows that, patching procedure with paper or DCF patch induce more rapid healing process compared to control group, and DCF patch may induce more rapid healing process than paper patch especially in early healing process. Histological investigations Figure 9 shows the hematoxylin-eosin (H&E) stain of the each group, at different time point. Until day 3, perforated TM was still observed in all three groups. Paper and DCF patches were well attached to the appropriate site of perforated TM. On Day 7, spontaneous healing group showed eversion of the perforated margin which could cause delayed or incomplete healing. In paper patch group, partial detachment of patch from TM was observed, and remarkable inflammation and epithelial hyperplasia on the perforated margin which could cause tympanosclerosis or TM thickening was identified. In DCF patch group, patch was still attached to perforation site and showed complete healing of TM. There were less inflammation or epithelial hyperplasia in DCF patched TM comparing with paper patched TM. On Day 14, all three group showed complete healing of TM. Regenerated TM was thicker in paper patch group than DCF patch group.
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Figure 8. The measurement results of the healed area of the tympanic membrane perforation. The healing process of the duck’s feet collagen film patch group was faster than that of the paper patch and control groups.
Figure 9. H&E staining findings of the three groups. Some portion of perforated tympanic membrane margin showed eversion in the control group (Day 7). Thickening of the perforation margin was observed in paper patch group (Day 7, 14). Cell migration to the collagen scaffold was observed in the duck’s feet collagen film patch group (Day 7).
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Discussion TM is about 0.1 mm thin grayish semi-transparent flat membrane consisted of three layers; skin layer, lamina propria and mucosal layer [17,18]. It transmits outside sound energy into the inner ear via ossicular chains by vibration. TM also acts a role on the protection of the middle ear by obstructing external auditory canal on the border of external and middle ear. Thanks to TM, outside foreign materials cannot enter into the middle ear cavity. When tympanic membrane is disrupted and perforated, conductive hearing deficit is occur. If the perforation is large, hearing deficit can reach about 30 dB . And if middle ear cavity is opened to outside, there is a chance of infection. So when tympanic membrane is perforated, doctors should pay attention to maximize the healing process, and minimize the sequelae. TM patching procedures has been globally performed to induce rapid and accurate healing process. Paper patch has been widely used, but because this material is not biocompatible, there were a chance to develop a foreign body reaction or infections. Also, because paper patch is not biodegradable, it had to be removed after healing is complete, for this reason, it is not appropriate for infants, children and poorly cooperative personnel. There has been many efforts to develop a new patch material to overcome these demerits of paper patch. Some authors have reported a surgical outcomes of patching procedure using variety of patch materials; silk fibroin, collagen, chitosan, calcium alginate [4–7,9,20]. Our institute have developed silk fibroin patch, and reported its effectiveness on the treatment of acute and chronic tympanic membrane perforation [4–7]. Collagen materials has been widely used in regenerative medical fields, and the senior author chose duck’s feet as a source of collagen to utilize the animal by-product into useful biomaterial. Collagen materials derived from duck’s feet could be used even in Arabic or Hindu communities who are prohibited to use porcine or bovine materials. In the previous study, our institute have evaluated the characteristics, structures and properties of sponge type duck’s feet collagen patch and duck’s feet collagen/silk hybrid biomaterial, and confirmed its biocompatibility via animal study [8,9]. In the present study, transparent film type duck’s feet collagen patch was applied on the perforated TM of the study animals and the treatment outcomes were compared with spontaneous healing and paper patch. DCF patch showed good biocompatibility and appropriate biodegradability when it was applied on animal’s perforated TM. FTIR spectroscopy and measurement of tensile strength showed profiles of duck’s feet collagen film material. Duck’s feet collagen film was structurally similar with porcine collagen film as well as other native collagen materials, and the treatment outcome was comparable with previous study [21–25] This suggest that duck’s feet collagen material could be a good alternative to porcine collagen material or other collagen materials. And different tensile strength profile of DCF and PCF suggest the future user what to choose according to their clinical situation. Water solubility of DCF was not measured in this study, and there is no report regarding water solubility of this kind of collagen film in our best knowledge. Every kind of collagen biomaterials lose their weight in the water as time goes by. But cross-linking technique used in collagen film manufacturing would decrease the water solubility than collagen material without cross-linking technique. There is a report regarding mechanical properties and solubility in water of starch-collagen composite films. They reported that water solubility of collagen film was decreased depending upon the amylase content and starch concentration [26,27]. In the future study, it would be better to perform a water solubility test to get more detailed specification of this DCF patch material.
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There were limitations in this study. First, the small number of study animal made the statistical analyses less meaningful. But we performed this experiment to observe the gross and histologic findings within the limited resources. Second, the treatment outcome of the study groups, including spontaneous healing group, showed no difference in eventual healing rate, but repair speed or cell proliferation in early stage after tympanic perforation. Nevertheless, as we can see in Figure 9 (control, 7th day), eversion of the perforation margin is not rare in spontaneous healing of tympanic membrane perforation. Patch materials act like scaffold, and can decrease the rate of margin eversion or inversion. If the number of the study animals was larger than present study, authors think the rate of healing would different between spontaneous group and patched group. Third, animal study was conducted only in non-complicated simple acutely perforated TM. In this condition, as mentioned before, most of the perforated TM heals within 2 weeks without any treatment. Present study was more focused on the evaluation of mechanical property, biocompatibility of DCF, and histologic change when it was applied on the study animals’ TM, so animal study on clinically important other situations like complicated perforation; multidirectional flapping laceration, large perforation or chronic perforation were not evaluated. There were study regarding the efficacy of other patch material when applied in chronic TM perforation showing good efficacy [5,28]. But efficacy of patching procedure using relatively rapid biodegradable patch material like DCF on chronic TM perforation was not reported yet. There are reports on creating chronic TM perforation animal models [29–35], and in the future investigations, authors think it would be meaningful to perform investigation on efficacy of DCF patch when applied in chronic TM perforation as well as complicated acute TM perforations. Fourth, in the present animal study, possible ototoxicity and hearing benefit from healing of TM perforation was not evaluated for each group. There are reported methods to evaluate the hearing levels of study animal [36,37], and in the future study, pre-treatment hearing level and post treatment hearing gain would better to check to evaluate possible ototoxicity and hearing gain for each study group.
Conclusion In this study, authors concluded that this novel transparent DCF patch has biocompatibility and appropriate biodegradability. And it has advantage on rapid healing, better gross and histological recovery than spontaneous healing and conventional paper patching. So this material could be a good option for TM patching procedure, and the authors also expect that this patch material can be used in variety of communities without religious concerns.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was supported by the Hallym University Research Fund and Technology Commercialization Support Program of Ministry for Food, Agriculture, Forestry and Fisheries of Korea [814005-03-3-HD030].
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