First-in-Human Application of Decellularized Human Amniotic Membrane Hydrogel as an Injectable Intraocular Scaffold for Large Idiopathic Full- Thickness Macular Hole Closure: Four-Month Clinical Outcomes

First-in-Human Application of Decellularized Human Amniotic Membrane Hydrogel as an Injectable Intraocular Scaffold for Large Idiopathic Full- Thickness Macular Hole Closure: Four-Month Clinical Outcomes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First-in-Human Application of Decellularized Human Amniotic Membrane Hydrogel as an Injectable Intraocular Scaffold for Large Idiopathic Full- Thickness Macular Hole Closure: Four-Month Clinical Outcomes Jakkrit Juhong, Hathaichanok Impheng, Mukdawan Sukhang, Auemphon Mordmuang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9176578/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background: Large idiopathic full-thickness macular holes (FTMH) with minimum diameter >400 μm carry surgical success rates of approximately 56% with standard vitrectomy and gas tamponade. Novel biologic scaffolds have improved closure rates but are limited by intraoperative handling challenges. We report the first clinical application of thermosensitive injectable decellularized human amniotic membrane (dAM) hydrogel as an intraocular scaffold for large FTMH, with four-month anatomical, functional, and electrophysiological outcomes. Methods: A 62-year-old man with a Gass stage 4 idiopathic FTMH (minimum diameter 572 μm) and preoperative BCVA of 20/200 underwent combined phacoemulsification and 25-gauge pars plana vitrectomy (PPV) with ILM peeling. Following fluid-air exchange, dAM hydrogel (10 mg/mL, thermosensitive, prepared by detergent decellularization and pepsin solubilization) was injected via a 25-gauge soft-tip cannula, followed by SF6 gas tamponade. Pre-clinical biocompatibility was confirmed by CCK-8 assay and transwell invasion assay prior to clinical use. Results: At one month, SD-OCT confirmed complete Type 1 anatomical closure and BCVA improved to 20/80. At four months (day 121), BCVA further improved to 20/40+1 (logMAR 0.30), with no metamorphopsia, IOP 12.7 mmHg, and sustained Type 1 closure with progressive ellipsoid zone (EZ) reconstitution on OCT. Multifocal ERG (103 segments) confirmed measurable foveal P1 responses (Ring 1: 34.19 nV/deg²) with normal central N1 implicit time (40.2 ms; normal range 36.8–46.4 ms). No intraocular adverse events occurred at any time point. Conclusions: Injectable dAM hydrogel achieves stable Type 1 FTMH closure with progressive visual and electrophysiological recovery over four months. mfERG evidence of preserved foveal photoreceptor function supports this approach as a feasible and safe biologic scaffold for large FTMH repair. Prospective comparative evaluation is ongoing (ClinicalTrials.gov: NCT06433284). macular hole amniotic membrane hydrogel decellularized ECM vitrectomy ILM peeling mfERG intraocular scaffold first-in-human Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Idiopathic full-thickness macular hole (FTMH) affects approximately 33 per 10,000 individuals older than 55 years.¹ While standard 25-gauge pars plana vitrectomy (PPV) with internal limiting membrane (ILM) peeling and gas tamponade achieves closure rates exceeding 90% for holes with minimum diameter (MD) 400 µm.²³ Adjunctive techniques for large FTMH include the inverted ILM flap (IFT)⁴ and the human amniotic membrane (hAM) plug.⁵ The hAM plug leverages the anti-inflammatory,⁸ anti-fibrotic,⁹ and low-immunogenicity properties of amniotic tissue, achieving 100% anatomical success at six months.⁵ However, intraoperative handling of solid hAM sheets is challenging: sub-millimetre trimming is imprecise, tissue loss through vitrectomy ports is possible, and precise intravitreal placement is technically demanding.⁶ Converting decellularized amniotic membrane (dAM) into a hydrogel form addresses these limitations. Decellularization preserves the extracellular matrix (ECM) scaffold—collagen, laminin, fibronectin, and bioactive growth factors—while removing immunogenic cellular components.⁷ The resulting thermosensitive hydrogel is injectable through standard 25-gauge cannulas, conforms to the geometry of large holes, and gels in situ at 37°C. Although dAM hydrogels have been explored in wound healing, cardiac, and cartilage repair, intraocular application has not been previously reported.⁷ We describe the preparation, pre-clinical characterization, and first-in-human clinical application of injectable dAM hydrogel for large FTMH, with four-month anatomical, functional, and electrophysiological outcomes. Case Presentation Patient and Preoperative Assessment A 62-year-old man with a history of diabetes mellitus (DM) and hypertension (HT) presented to the vitreoretinal clinic at Walailak University Hospital with progressive central visual distortion and decreased acuity in the right eye. Preoperative BCVA was 20/200 (logMAR 1.0) in the right eye with pinhole improvement to 20/125, and 20/80 in the left eye. IOP by iCare rebound tonometry was 9.7 mmHg (OD) and 10.5 mmHg (OS). Axial length was 23.0 mm. SD-OCT (Heidelberg Spectralis, Heidelberg Engineering, Germany) confirmed a Gass stage 4 idiopathic FTMH with MD 572 µm, prominent intraretinal cystic changes, and a complete posterior vitreous detachment (PVD) (Fig. 1 A). Ultra-wide-field fundus photography (Optos Daytona) showed no peripheral retinal pathology (Fig. 3 A). Dilated fundus examination confirmed no diabetic retinopathy in either eye. Written informed consent was obtained in accordance with Walailak University IRB No. WUEC-24-331-01. dAM Hydrogel Preparation and Pre-Clinical Characterization Human amniotic membranes were obtained from donated placentas following elective cesarean sections at Walailak University Hospital, with full informed consent and serological screening for HIV, hepatitis B, hepatitis C, and syphilis. Membranes were aseptically separated from the chorion, washed with PBS containing 5% penicillin/1% streptomycin, and enzymatically decellularized using 10% trypsin followed by extensive PBS washing. Sterilization was performed with peracetic acid. Membranes were stored at − 80°C until use. For hydrogel preparation, dAM was mechanically homogenized in PBS and solubilized with pepsin (1 mg/mL in 0.1 M HCl) at 4°C for 48–72 hours, neutralized to pH 7.4 with 1 M NaOH, and adjusted to 10 mg/mL. The hydrogel undergoes thermosensitive gelation at 37°C, forming a stable scaffold in situ. Pre-clinical biocompatibility was evaluated using the CCK-8 assay with RPE-1 cells cultured on dAM hydrogel for 24 and 48 hours, with Corning® Matrigel® Matrix as a non-toxic reference. Surface cell viability was 59.61 ± 1.97% at 24 h and 56.40 ± 5.96% at 48 h, with no significant difference from Matrigel (64.59 ± 5.89%; p = ns). A transwell invasion assay confirmed active RPE-1 migration into the scaffold, indicating cell ingrowth rather than cytotoxicity, supporting biocompatibility prior to clinical use. Surgical Technique Surgery was performed on 5 November 2025 under retrobulbar anesthesia. Combined phacoemulsification (IOL AR40e, 18.0 D) and 25-gauge PPV were performed. The ILM was stained with 0.05% Heavy Brilliant Blue G dye (Ocublue Plus, Aurolab, India) mixed with 10% dextrose (1:2) under BSS and peeled circumferentially for ~ 2 disc diameters using 25-gauge end-gripping forceps (Grieshaber Asymmetrical Forceps, Alcon). Following complete ILM peeling and fluid-air exchange, dAM hydrogel was slowly injected into and over the macular hole via a 25-gauge soft-tip cannula (Alcon) through a valved trocar until the hydrogel filled the hole extending ~ 1 disc diameter from the fovea (Figs. 2 A– 2 B). One milliliter of pure SF6 gas was then injected. Sclerotomies were sutured with Vicryl 8 − 0 and the patient was placed prone. Total operative time was approximately 45 minutes. Postoperative Course and Outcomes The patient maintained strict face-down positioning for 5 days postoperatively and received standard topical antibiotic and steroid drops. One-week follow-up (13 November 2025) No intraocular inflammation, hypopyon, or fibrinous reaction was observed; gas occupied approximately 70% of the vitreous cavity. One-month follow-up (8 December 2025) BCVA improved to 20/80 (logMAR 0.60), a 2-line Snellen gain. IOP was 16.4 mmHg. SD-OCT demonstrated complete Type 1 anatomical closure with restored foveal contour (Fig. 1 B). No residual subretinal fluid, intraretinal cysts, or epiretinal membrane was observed. The dAM hydrogel was no longer visible on OCT, consistent with ECM biodegradation. Four-month follow-up (16 March 2026; day 121) The patient reported no metamorphopsia. BCVA further improved to 20/40 + 1 (logMAR 0.30), a cumulative 3-line Snellen gain. IOP was 12.7 mmHg. Anterior segment examination showed clear cornea, no anterior chamber cells, and an in-place IOL. SD-OCT (Fig. 1 C) confirmed sustained Type 1 closure with progressive EZ reconstitution and normal foveal contour. Ultra-wide-field fundus photography (Fig. 3 C) confirmed no peripheral complications. Clinical and functional outcomes across all time points are summarized in Table 1 . Multifocal ERG mfERG (RETI-scan, 103 segments, Roland Consult, Germany; 28° view angle, DTL electrode, pupil 7 mm dilated) was performed at 4 months (Fig. 4 ). P1 amplitudes were reduced across all six rings relative to normative values, expected following a large chronic FTMH. Critically, the Ring 1 (foveal) N1 implicit time (40.2 ms) was within the normal range (36.8–46.4 ms), confirming preserved foveal photoreceptor signal conduction at the former macular hole site. Measurable responses were present across all rings with no electrophysiological evidence of intraocular hydrogel toxicity. Summary of Clinical and Functional Outcomes Table 1 Summary of clinical and functional outcomes. Parameter Preoperative 1 Month 4 Months BCVA (Snellen / logMAR) 20/200 / 1.0 20/80 / 0.60 20/40 + 1 / 0.30 IOP OD (mmHg) 9.7 16.4 12.7 Macular hole status Open (MD 572 µm, Gass 4) Type 1 closure Type 1 closure (maintained) Ellipsoid zone (OCT) Disrupted Improved Progressive recovery Foveal contour (OCT) Absent Restored Normal Metamorphopsia Present Absent Absent AC cells N/A None None dAM hydrogel on OCT N/A Not visible Not visible mfERG R1 P1 (nV/deg²) N/A N/A 34.19 mfERG R1 N1 implicit time (ms) N/A N/A 40.2 (normal 36.8–46.4) Adverse events None None None Discussion To our knowledge, this is the first reported use of an injectable dAM hydrogel as a surgical scaffold for large idiopathic FTMH closure, with prospective follow-up to four months. The technique achieved sustained complete Type 1 anatomical closure at both one and four months, with progressive BCVA improvement from 20/200 to 20/40 + 1 (a cumulative 3-line Snellen gain) and objective electrophysiological confirmation of photoreceptor viability by mfERG, without any intraocular adverse events. The biological rationale is grounded in the established properties of the amniotic ECM. Decellularization preserves structural proteins (collagen I, III, IV; laminin; fibronectin) and bioactive molecules—including anti-inflammatory⁸ and anti-fibrotic⁹ factors—that promote cell migration, proliferation, and tissue regeneration.⁷ The hydrogel form confers critical intraocular advantages: injectability through 25-gauge cannulas, precise in-hole delivery, uniform gap-filling regardless of hole geometry, and thermosensitive in-situ gelation at 37°C. The four-month mfERG findings provide objective functional corroboration absent from prior FTMH biologic scaffold reports. The foveal N1 implicit time of 40.2 ms—within the normal range of 36.8–46.4 ms—indicates that surviving foveal photoreceptors retain adequate response kinetics. The absence of electrophysiological deterioration provides direct in vivo evidence against dAM hydrogel-related intraocular toxicity, corroborating the pre-clinical CCK-8 biocompatibility data. Compared with the inverted ILM flap, dAM hydrogel injection avoids meticulous flap manipulation and iatrogenic foveal trauma risk. Unlike the solid hAM patch, the hydrogel is delivered through the standard surgical workflow without additional instruments. The biodegradation of the hydrogel—confirmed by its absence on OCT at one month—is a desirable property for any intraocular biomaterial. This report has inherent limitations as a single-case observation without a preoperative mfERG baseline. The absence of baseline electrophysiology limits direct functional comparison; however, the postoperative mfERG findings are interpretable in the context of expected deficits from a large chronic FTMH. Whether dAM hydrogel offers advantages over the inverted ILM flap is the subject of an ongoing prospective RCT (MACROHOLE trial; ClinicalTrials.gov: NCT06433284; Walailak University IRB No. WUEC-24-331-01) comparing both techniques in idiopathic FTMH with MD 400–1500 µm. Conclusions Injectable dAM hydrogel achieves stable Type 1 FTMH closure with progressive visual improvement to 20/40 + 1 and electrophysiological recovery over four months. mfERG evidence of preserved foveal photoreceptor function without intraocular adverse events supports this approach as a feasible and safe biologic scaffold for large FTMH repair and justifies prospective comparative evaluation. Declarations Ethics approval and consent to participate: This study was conducted in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Board of Walailak University (IRB No. WUEC-24-331-01). Written informed consent was obtained prior to all study procedures. Consent for publication: Written informed consent for publication of clinical data and all accompanying images was obtained from the patient. A copy of the signed consent is available to the Editor-in-Chief upon request. Availability of data and materials: The datasets supporting the conclusions of this article are included within the article. Deidentified clinical data are available from the corresponding author upon reasonable request. Competing interests: The authors declare no competing financial interests. A utility model patent application relating to the dAM hydrogel preparation method has been filed with the Thai Department of Intellectual Property. Funding: Supported by the Medical Council of Thailand Research Grant (2567). The funding body had no role in study design, data collection, analysis, interpretation, or manuscript preparation. Authors’ contributions: JJ: conceptualization, surgical procedure, clinical data collection, manuscript drafting. HI: dAM hydrogel preparation, pre-clinical biocompatibility testing, manuscript revision. MS: clinical data collection, figures. AM: project supervision, hydrogel development, critical manuscript revision. All authors read and approved the final manuscript. Acknowledgements: The authors thank the staff of the Ophthalmology Unit and Research Facility at Walailak University Hospital for technical support. Clinical trial registration: ClinicalTrials.gov: NCT06433284 (MACROHOLE trial). Registered April 2024. References Bikbova G, Oshitari T, Baba T, Yamamoto S, Mori K. Pathogenesis and management of macular hole: review of current advances. J Ophthalmol. 2019;2019:3467381. Rossi T, Gelso A, Costagliola C, Trillo C, Ferrari A, Corbo G, et al. Macular hole closure patterns associated with different internal limiting membrane flap techniques. Graefes Arch Clin Exp Ophthalmol. 2017;255(6):1073–1078. Caporossi T, De Angelis L, Barca F, Rizzo S. Long-term outcomes of large macular holes treated with inverted internal limiting membrane flap technique. Retina. 2020;40(7):1295–1302. Michalewska Z, Michalewski J, Adelman RA, Nawrocki J. Inverted internal limiting membrane flap technique for large macular holes. Ophthalmology. 2010;117(10):2018–2025. Rizzo S, Caporossi T, Tartaro R, Finocchio L, Franco F, Barca F, et al. A human amniotic membrane plug to promote retinal breaks repair and recurrent macular hole closure. Retina. 2019;39(Suppl 1):S95–S103. Zhang H, Li Y, Chen G, Han F, Jiang W. Human amniotic membrane graft for refractory macular hole: a single-arm meta-analysis and systematic review. J Fr Ophtalmol. 2023;46(3):276–286. Kafili G, Niknejad H, Tamjid E, Simchi A. Amnion-derived hydrogels as a versatile platform for regenerative therapy: from lab to market. Front Bioeng Biotechnol. 2024;12:1358977. Fitriani N, Wilar G, Narsa AC, Mohammed AFA, Wathoni N. Application of amniotic membrane in skin regeneration. Pharmaceutics. 2023;15(3):748. Mao Y, Hoffman T, Wu A, Fealk A, Bhowmick S, Vasile D, et al. Endogenous viable cells in lyopreserved amnion retain differentiation potential and anti-fibrotic activity in vitro. Acta Biomater. 2019;94:330–339. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 29 Mar, 2026 Reviewers agreed at journal 29 Mar, 2026 Reviewers agreed at journal 27 Mar, 2026 Reviewers agreed at journal 27 Mar, 2026 Reviewers invited by journal 25 Mar, 2026 Editor assigned by journal 25 Mar, 2026 Submission checks completed at journal 24 Mar, 2026 First submitted to journal 20 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9176578","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611938481,"identity":"50d2136c-4448-4429-a71e-60fb2518ccea","order_by":0,"name":"Jakkrit Juhong","email":"","orcid":"","institution":"Walailak University","correspondingAuthor":false,"prefix":"","firstName":"Jakkrit","middleName":"","lastName":"Juhong","suffix":""},{"id":611938489,"identity":"7d185d81-6fb3-44b6-b243-46e222d804ef","order_by":1,"name":"Hathaichanok Impheng","email":"","orcid":"","institution":"Naresuan University","correspondingAuthor":false,"prefix":"","firstName":"Hathaichanok","middleName":"","lastName":"Impheng","suffix":""},{"id":611938494,"identity":"b8a149c7-f6c8-472e-b001-6b7f1f85181b","order_by":2,"name":"Mukdawan Sukhang","email":"","orcid":"","institution":"Ministry of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Mukdawan","middleName":"","lastName":"Sukhang","suffix":""},{"id":611938495,"identity":"3e4b58d7-640c-42d4-8891-6d3ede634a21","order_by":3,"name":"Auemphon Mordmuang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYLACxgYI9QDKZyNaC7MBhMtMvBY2CaK08M9IPrq5cIdNHoPY4WPVvG0M8vwN/Mce4NMicSMt7fbMM2nFDNJABlCL4YwDzOwGeK25kWMGVHk4sUEayMhtY2DcAHSYBD4d8jfyv8G1FAO12BPUYnAjhw2uhRmoJZGgFsMzz8xuz2xLS2yTTkuW/nNOInnGYWYzvFrkjic/u13YZpPYL5188OOMMhvb/vbGZ3i1gAAziIDGhQSUS4yWUTAKRsEoGAU4AQA0vEMK0omrLgAAAABJRU5ErkJggg==","orcid":"","institution":"Walailak University","correspondingAuthor":true,"prefix":"","firstName":"Auemphon","middleName":"","lastName":"Mordmuang","suffix":""}],"badges":[],"createdAt":"2026-03-20 08:11:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9176578/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9176578/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105574022,"identity":"8baa5188-eac5-4676-a868-93b1fcc49385","added_by":"auto","created_at":"2026-03-27 13:33:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":323866,"visible":true,"origin":"","legend":"\u003cp\u003eSD-OCT (Heidelberg Spectralis) timeline. (A) Preoperative: Gass stage 4 full-thickness macular hole with minimum diameter 572 μm and prominent intraretinal cystic changes. (B) One-month postoperative: complete Type 1 closure with restoration of foveal contour; dAM hydrogel no longer visible. (C) Four-month postoperative: maintained Type 1 closure with progressive ellipsoid zone recovery. Scale bar = 200 μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9176578/v1/78c5ad3d5b0b931334ed5019.png"},{"id":105574446,"identity":"f985b41c-2cea-4277-a64d-0a7a18290c34","added_by":"auto","created_at":"2026-03-27 13:34:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":357816,"visible":true,"origin":"","legend":"\u003cp\u003eIntraoperative photographs. (A) dAM hydrogel injection via 25-gauge soft-tip cannula following ILM peeling and fluid-air exchange; hydrogel extends approximately 1 disc diameter from the fovea. (B) Macula after hydrogel placement, with the dAM scaffold in situ prior to SF6 instillation.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9176578/v1/47e04a57229a5c471e96186b.png"},{"id":105574721,"identity":"675bc9b4-254a-44bb-b270-ccf903efe172","added_by":"auto","created_at":"2026-03-27 13:35:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":378777,"visible":true,"origin":"","legend":"\u003cp\u003eUltra-wide-field fundus photography (Optos Daytona). (A) Preoperative: macular hole visible in the right eye; no peripheral retinal pathology. (B) One-month postoperative: macular hole resolved; flat, attached retina. (C) Four-month postoperative: maintained closure with well-attached retina and no peripheral complications.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9176578/v1/5f4afa94aa67992178255cdb.png"},{"id":105574230,"identity":"bf83ee1f-1ceb-4c10-81e2-0f6d73a3ba5e","added_by":"auto","created_at":"2026-03-27 13:34:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":484746,"visible":true,"origin":"","legend":"\u003cp\u003eMultifocal ERG (mfERG; RETI-scan, 103 segments, Roland Consult) at four months (16 March 2026), right eye. Amplitude topographic map (upper left) and 3D plot (lower left) show a discernible foveal peak (Ring 1: 34.19 nV/deg²). Ring trace array (upper right) demonstrates measurable P1 responses across all six eccentricity rings. The central N1 implicit time (40.2 ms) is within the normal range (36.8–46.4 ms), confirming preserved foveal photoreceptor signal conduction at the former macular hole site. All ring amplitudes are reduced relative to normative values, consistent with expected deficits from a large chronic preoperative macular hole.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9176578/v1/c73e04d74ec091bab250ba69.png"},{"id":105576025,"identity":"03014c34-ec3e-48d6-bd34-d8ec461fb9be","added_by":"auto","created_at":"2026-03-27 13:42:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2376372,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9176578/v1/4eb20db6-bad1-4bfe-b3db-92f281566f00.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"First-in-Human Application of Decellularized Human Amniotic Membrane Hydrogel as an Injectable Intraocular Scaffold for Large Idiopathic Full- Thickness Macular Hole Closure: Four-Month Clinical Outcomes","fulltext":[{"header":"Background","content":"\u003cp\u003eIdiopathic full-thickness macular hole (FTMH) affects approximately 33 per 10,000 individuals older than 55 years.\u0026sup1; While standard 25-gauge pars plana vitrectomy (PPV) with internal limiting membrane (ILM) peeling and gas tamponade achieves closure rates exceeding 90% for holes with minimum diameter (MD)\u0026thinsp;\u0026lt;\u0026thinsp;400 \u0026micro;m, success drops to approximately 56% for large holes with MD\u0026thinsp;\u0026gt;\u0026thinsp;400 \u0026micro;m.\u0026sup2;\u0026sup3;\u003c/p\u003e \u003cp\u003eAdjunctive techniques for large FTMH include the inverted ILM flap (IFT)⁴ and the human amniotic membrane (hAM) plug.⁵ The hAM plug leverages the anti-inflammatory,⁸ anti-fibrotic,⁹ and low-immunogenicity properties of amniotic tissue, achieving 100% anatomical success at six months.⁵ However, intraoperative handling of solid hAM sheets is challenging: sub-millimetre trimming is imprecise, tissue loss through vitrectomy ports is possible, and precise intravitreal placement is technically demanding.⁶\u003c/p\u003e \u003cp\u003eConverting decellularized amniotic membrane (dAM) into a hydrogel form addresses these limitations. Decellularization preserves the extracellular matrix (ECM) scaffold\u0026mdash;collagen, laminin, fibronectin, and bioactive growth factors\u0026mdash;while removing immunogenic cellular components.⁷ The resulting thermosensitive hydrogel is injectable through standard 25-gauge cannulas, conforms to the geometry of large holes, and gels in situ at 37\u0026deg;C. Although dAM hydrogels have been explored in wound healing, cardiac, and cartilage repair, intraocular application has not been previously reported.⁷\u003c/p\u003e \u003cp\u003eWe describe the preparation, pre-clinical characterization, and first-in-human clinical application of injectable dAM hydrogel for large FTMH, with four-month anatomical, functional, and electrophysiological outcomes.\u003c/p\u003e"},{"header":"Case Presentation","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePatient and Preoperative Assessment\u003c/h2\u003e\n \u003cp\u003eA 62-year-old man with a history of diabetes mellitus (DM) and hypertension (HT) presented to the vitreoretinal clinic at Walailak University Hospital with progressive central visual distortion and decreased acuity in the right eye. Preoperative BCVA was 20/200 (logMAR 1.0) in the right eye with pinhole improvement to 20/125, and 20/80 in the left eye. IOP by iCare rebound tonometry was 9.7 mmHg (OD) and 10.5 mmHg (OS). Axial length was 23.0 mm.\u003c/p\u003e\n \u003cp\u003eSD-OCT (Heidelberg Spectralis, Heidelberg Engineering, Germany) confirmed a Gass stage 4 idiopathic FTMH with MD 572 \u0026micro;m, prominent intraretinal cystic changes, and a complete posterior vitreous detachment (PVD) (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Ultra-wide-field fundus photography (Optos Daytona) showed no peripheral retinal pathology (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Dilated fundus examination confirmed no diabetic retinopathy in either eye. Written informed consent was obtained in accordance with Walailak University IRB No. WUEC-24-331-01.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003edAM Hydrogel Preparation and Pre-Clinical Characterization\u003c/h3\u003e\n\u003cp\u003eHuman amniotic membranes were obtained from donated placentas following elective cesarean sections at Walailak University Hospital, with full informed consent and serological screening for HIV, hepatitis B, hepatitis C, and syphilis. Membranes were aseptically separated from the chorion, washed with PBS containing 5% penicillin/1% streptomycin, and enzymatically decellularized using 10% trypsin followed by extensive PBS washing. Sterilization was performed with peracetic acid. Membranes were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e\n\u003cp\u003eFor hydrogel preparation, dAM was mechanically homogenized in PBS and solubilized with pepsin (1 mg/mL in 0.1 M HCl) at 4\u0026deg;C for 48\u0026ndash;72 hours, neutralized to pH 7.4 with 1 M NaOH, and adjusted to 10 mg/mL. The hydrogel undergoes thermosensitive gelation at 37\u0026deg;C, forming a stable scaffold in situ.\u003c/p\u003e\n\u003cp\u003ePre-clinical biocompatibility was evaluated using the CCK-8 assay with RPE-1 cells cultured on dAM hydrogel for 24 and 48 hours, with Corning\u0026reg; Matrigel\u0026reg; Matrix as a non-toxic reference. Surface cell viability was 59.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97% at 24 h and 56.40\u0026thinsp;\u0026plusmn;\u0026thinsp;5.96% at 48 h, with no significant difference from Matrigel (64.59\u0026thinsp;\u0026plusmn;\u0026thinsp;5.89%; p\u0026thinsp;=\u0026thinsp;ns). A transwell invasion assay confirmed active RPE-1 migration into the scaffold, indicating cell ingrowth rather than cytotoxicity, supporting biocompatibility prior to clinical use.\u003c/p\u003e\n\u003ch3\u003eSurgical Technique\u003c/h3\u003e\n\u003cp\u003eSurgery was performed on 5 November 2025 under retrobulbar anesthesia. Combined phacoemulsification (IOL AR40e, 18.0 D) and 25-gauge PPV were performed. The ILM was stained with 0.05% Heavy Brilliant Blue G dye (Ocublue Plus, Aurolab, India) mixed with 10% dextrose (1:2) under BSS and peeled circumferentially for ~\u0026thinsp;2 disc diameters using 25-gauge end-gripping forceps (Grieshaber Asymmetrical Forceps, Alcon).\u003c/p\u003e\n\u003cp\u003eFollowing complete ILM peeling and fluid-air exchange, dAM hydrogel was slowly injected into and over the macular hole via a 25-gauge soft-tip cannula (Alcon) through a valved trocar until the hydrogel filled the hole extending\u0026thinsp;~\u0026thinsp;1 disc diameter from the fovea (Figs. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). One milliliter of pure SF6 gas was then injected. Sclerotomies were sutured with Vicryl 8\u0026thinsp;\u0026minus;\u0026thinsp;0 and the patient was placed prone. Total operative time was approximately 45 minutes.\u003c/p\u003e\n\u003ch3\u003ePostoperative Course and Outcomes\u003c/h3\u003e\n\u003cp\u003eThe patient maintained strict face-down positioning for 5 days postoperatively and received standard topical antibiotic and steroid drops.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOne-week follow-up (13 November 2025)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo intraocular inflammation, hypopyon, or fibrinous reaction was observed; gas occupied approximately 70% of the vitreous cavity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOne-month follow-up (8 December 2025)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBCVA improved to 20/80 (logMAR 0.60), a 2-line Snellen gain. IOP was 16.4 mmHg. SD-OCT demonstrated complete Type 1 anatomical closure with restored foveal contour (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). No residual subretinal fluid, intraretinal cysts, or epiretinal membrane was observed. The dAM hydrogel was no longer visible on OCT, consistent with ECM biodegradation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFour-month follow-up (16 March 2026; day 121)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patient reported no metamorphopsia. BCVA further improved to 20/40\u0026thinsp;+\u0026thinsp;1 (logMAR 0.30), a cumulative 3-line Snellen gain. IOP was 12.7 mmHg. Anterior segment examination showed clear cornea, no anterior chamber cells, and an in-place IOL. SD-OCT (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) confirmed sustained Type 1 closure with progressive EZ reconstitution and normal foveal contour. Ultra-wide-field fundus photography (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) confirmed no peripheral complications. Clinical and functional outcomes across all time points are summarized in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eMultifocal ERG\u003c/h3\u003e\n\u003cp\u003emfERG (RETI-scan, 103 segments, Roland Consult, Germany; 28\u0026deg; view angle, DTL electrode, pupil 7 mm dilated) was performed at 4 months (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). P1 amplitudes were reduced across all six rings relative to normative values, expected following a large chronic FTMH. Critically, the Ring 1 (foveal) N1 implicit time (40.2 ms) was within the normal range (36.8\u0026ndash;46.4 ms), confirming preserved foveal photoreceptor signal conduction at the former macular hole site. Measurable responses were present across all rings with no electrophysiological evidence of intraocular hydrogel toxicity.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eSummary of Clinical and Functional Outcomes\u003c/h2\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummary of clinical and functional outcomes.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003ePreoperative\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1 Month\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4 Months\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eBCVA (Snellen / logMAR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e20/200 / 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e20/80 / 0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e20/40\u0026thinsp;+\u0026thinsp;1 / 0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eIOP OD (mmHg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e9.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e16.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e12.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eMacular hole status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eOpen (MD 572 \u0026micro;m, Gass 4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eType 1 closure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eType 1 closure (maintained)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eEllipsoid zone (OCT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eDisrupted\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eImproved\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eProgressive recovery\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eFoveal contour (OCT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eRestored\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eMetamorphopsia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eAC cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003edAM hydrogel on OCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eNot visible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eNot visible\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003emfERG R1 P1 (nV/deg\u0026sup2;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e34.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003emfERG R1 N1 implicit time (ms)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e40.2 (normal 36.8\u0026ndash;46.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eAdverse events\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo our knowledge, this is the first reported use of an injectable dAM hydrogel as a surgical scaffold for large idiopathic FTMH closure, with prospective follow-up to four months. The technique achieved sustained complete Type 1 anatomical closure at both one and four months, with progressive BCVA improvement from 20/200 to 20/40\u0026thinsp;+\u0026thinsp;1 (a cumulative 3-line Snellen gain) and objective electrophysiological confirmation of photoreceptor viability by mfERG, without any intraocular adverse events.\u003c/p\u003e \u003cp\u003eThe biological rationale is grounded in the established properties of the amniotic ECM. Decellularization preserves structural proteins (collagen I, III, IV; laminin; fibronectin) and bioactive molecules\u0026mdash;including anti-inflammatory⁸ and anti-fibrotic⁹ factors\u0026mdash;that promote cell migration, proliferation, and tissue regeneration.⁷ The hydrogel form confers critical intraocular advantages: injectability through 25-gauge cannulas, precise in-hole delivery, uniform gap-filling regardless of hole geometry, and thermosensitive in-situ gelation at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe four-month mfERG findings provide objective functional corroboration absent from prior FTMH biologic scaffold reports. The foveal N1 implicit time of 40.2 ms\u0026mdash;within the normal range of 36.8\u0026ndash;46.4 ms\u0026mdash;indicates that surviving foveal photoreceptors retain adequate response kinetics. The absence of electrophysiological deterioration provides direct in vivo evidence against dAM hydrogel-related intraocular toxicity, corroborating the pre-clinical CCK-8 biocompatibility data.\u003c/p\u003e \u003cp\u003eCompared with the inverted ILM flap, dAM hydrogel injection avoids meticulous flap manipulation and iatrogenic foveal trauma risk. Unlike the solid hAM patch, the hydrogel is delivered through the standard surgical workflow without additional instruments. The biodegradation of the hydrogel\u0026mdash;confirmed by its absence on OCT at one month\u0026mdash;is a desirable property for any intraocular biomaterial.\u003c/p\u003e \u003cp\u003eThis report has inherent limitations as a single-case observation without a preoperative mfERG baseline. The absence of baseline electrophysiology limits direct functional comparison; however, the postoperative mfERG findings are interpretable in the context of expected deficits from a large chronic FTMH. Whether dAM hydrogel offers advantages over the inverted ILM flap is the subject of an ongoing prospective RCT (MACROHOLE trial; ClinicalTrials.gov: NCT06433284; Walailak University IRB No. WUEC-24-331-01) comparing both techniques in idiopathic FTMH with MD 400\u0026ndash;1500 \u0026micro;m.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eInjectable dAM hydrogel achieves stable Type 1 FTMH closure with progressive visual improvement to 20/40\u0026thinsp;+\u0026thinsp;1 and electrophysiological recovery over four months. mfERG evidence of preserved foveal photoreceptor function without intraocular adverse events supports this approach as a feasible and safe biologic scaffold for large FTMH repair and justifies prospective comparative evaluation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eThis study was conducted in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Board of Walailak University (IRB No. WUEC-24-331-01). Written informed consent was obtained prior to all study procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eWritten informed consent for publication of clinical data and all accompanying images was obtained from the patient. A copy of the signed consent is available to the Editor-in-Chief upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets supporting the conclusions of this article are included within the article. Deidentified clinical data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing financial interests. A utility model patent application relating to the dAM hydrogel preparation method has been filed with the Thai Department of Intellectual Property.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eSupported by the Medical Council of Thailand Research Grant (2567). The funding body had no role in study design, data collection, analysis, interpretation, or manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u0026nbsp;\u003c/strong\u003eJJ: conceptualization, surgical procedure, clinical data collection, manuscript drafting. HI: dAM hydrogel preparation, pre-clinical biocompatibility testing, manuscript revision. MS: clinical data collection, figures. AM: project supervision, hydrogel development, critical manuscript revision. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe authors thank the staff of the Ophthalmology Unit and Research Facility at Walailak University Hospital for technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial registration:\u0026nbsp;\u003c/strong\u003eClinicalTrials.gov: NCT06433284 (MACROHOLE trial). Registered April 2024.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBikbova G, Oshitari T, Baba T, Yamamoto S, Mori K. Pathogenesis and management of macular hole: review of current advances. J Ophthalmol. 2019;2019:3467381.\u003c/li\u003e\n\u003cli\u003eRossi T, Gelso A, Costagliola C, Trillo C, Ferrari A, Corbo G, et al. Macular hole closure patterns associated with different internal limiting membrane flap techniques. Graefes Arch Clin Exp Ophthalmol. 2017;255(6):1073\u0026ndash;1078.\u003c/li\u003e\n\u003cli\u003eCaporossi T, De Angelis L, Barca F, Rizzo S. Long-term outcomes of large macular holes treated with inverted internal limiting membrane flap technique. Retina. 2020;40(7):1295\u0026ndash;1302.\u003c/li\u003e\n\u003cli\u003eMichalewska Z, Michalewski J, Adelman RA, Nawrocki J. Inverted internal limiting membrane flap technique for large macular holes. Ophthalmology. 2010;117(10):2018\u0026ndash;2025.\u003c/li\u003e\n\u003cli\u003eRizzo S, Caporossi T, Tartaro R, Finocchio L, Franco F, Barca F, et al. A human amniotic membrane plug to promote retinal breaks repair and recurrent macular hole closure. Retina. 2019;39(Suppl 1):S95\u0026ndash;S103.\u003c/li\u003e\n\u003cli\u003eZhang H, Li Y, Chen G, Han F, Jiang W. Human amniotic membrane graft for refractory macular hole: a single-arm meta-analysis and systematic review. J Fr Ophtalmol. 2023;46(3):276\u0026ndash;286.\u003c/li\u003e\n\u003cli\u003eKafili G, Niknejad H, Tamjid E, Simchi A. Amnion-derived hydrogels as a versatile platform for regenerative therapy: from lab to market. Front Bioeng Biotechnol. 2024;12:1358977.\u003c/li\u003e\n\u003cli\u003eFitriani N, Wilar G, Narsa AC, Mohammed AFA, Wathoni N. Application of amniotic membrane in skin regeneration. Pharmaceutics. 2023;15(3):748.\u003c/li\u003e\n\u003cli\u003eMao Y, Hoffman T, Wu A, Fealk A, Bhowmick S, Vasile D, et al. Endogenous viable cells in lyopreserved amnion retain differentiation potential and anti-fibrotic activity in vitro. Acta Biomater. 2019;94:330\u0026ndash;339.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-retina-and-vitreous","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"IJRV","sideBox":"Learn more about [International Journal of Retina and Vitreous](https://jneurodevdisorders.biomedcentral.com/)","snPcode":"40942","submissionUrl":"https://submission.nature.com/new-submission/40942/3","title":"International Journal of Retina and Vitreous","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"macular hole, amniotic membrane hydrogel, decellularized ECM, vitrectomy, ILM peeling, mfERG, intraocular scaffold, first-in-human","lastPublishedDoi":"10.21203/rs.3.rs-9176578/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9176578/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eLarge idiopathic full-thickness macular holes (FTMH) with minimum diameter \u0026gt;400 μm carry surgical success rates of approximately 56% with standard vitrectomy and gas tamponade. Novel biologic scaffolds have improved closure rates but are limited by intraoperative handling challenges. We report the first clinical application of thermosensitive injectable decellularized human amniotic membrane (dAM) hydrogel as an intraocular scaffold for large FTMH, with four-month anatomical, functional, and electrophysiological outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA 62-year-old man with a Gass stage 4 idiopathic FTMH (minimum diameter 572 μm) and preoperative BCVA of 20/200 underwent combined phacoemulsification and 25-gauge pars plana vitrectomy (PPV) with ILM peeling. Following fluid-air exchange, dAM hydrogel (10 mg/mL, thermosensitive, prepared by detergent decellularization and pepsin solubilization) was injected via a 25-gauge soft-tip cannula, followed by SF6 gas tamponade. Pre-clinical biocompatibility was confirmed by CCK-8 assay and transwell invasion assay prior to clinical use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAt one month, SD-OCT confirmed complete Type 1 anatomical closure and BCVA improved to 20/80. At four months (day 121), BCVA further improved to 20/40+1 (logMAR 0.30), with no metamorphopsia, IOP 12.7 mmHg, and sustained Type 1 closure with progressive ellipsoid zone (EZ) reconstitution on OCT. Multifocal ERG (103 segments) confirmed measurable foveal P1 responses (Ring 1: 34.19 nV/deg²) with normal central N1 implicit time (40.2 ms; normal range 36.8–46.4 ms). No intraocular adverse events occurred at any time point.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eInjectable dAM hydrogel achieves stable Type 1 FTMH closure with progressive visual and electrophysiological recovery over four months. mfERG evidence of preserved foveal photoreceptor function supports this approach as a feasible and safe biologic scaffold for large FTMH repair. Prospective comparative evaluation is ongoing (ClinicalTrials.gov: NCT06433284).\u003c/p\u003e","manuscriptTitle":"First-in-Human Application of Decellularized Human Amniotic Membrane Hydrogel as an Injectable Intraocular Scaffold for Large Idiopathic Full- Thickness Macular Hole Closure: Four-Month Clinical Outcomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-27 13:08:55","doi":"10.21203/rs.3.rs-9176578/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-29T12:26:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99129726966072136425007451250310705090","date":"2026-03-29T11:42:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46335986309246829050094813165298799230","date":"2026-03-27T15:00:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81930499660296314745286431034049688946","date":"2026-03-27T10:56:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-25T10:26:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-25T10:17:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-24T10:39:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Retina and Vitreous","date":"2026-03-20T08:04:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-retina-and-vitreous","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"IJRV","sideBox":"Learn more about [International Journal of Retina and Vitreous](https://jneurodevdisorders.biomedcentral.com/)","snPcode":"40942","submissionUrl":"https://submission.nature.com/new-submission/40942/3","title":"International Journal of Retina and Vitreous","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"47cae6d4-52fc-4034-a4e8-3f420d67e272","owner":[],"postedDate":"March 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-27T13:08:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-27 13:08:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9176578","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9176578","identity":"rs-9176578","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}