Feasibility Study of Decellularized Corneal Lenticule in a Porcine Model for Macular Hole Closure

Abstract

Introduction

To develop an innovative preclinical full-thickness macular hole (FTMH) model using porcine eyes and to evaluate the feasibility of decellularized human corneal lenticule (hCL) as a novel bio-graft for macular hole closure.

Methods

Twenty-five eyes (n = 25) were obtained from pigs euthanized for food consumption and transported to the laboratory within 3 h. Surgical procedures were performed by the same experienced vitreoretinal surgeon using a Leica Proveo microscope (Proveo 8, Leica Microsystems Inc.) with integrated intraoperative optical coherence tomography (iOCT) technology. After core vitrectomy and posterior vitreous detachment, FTMH was induced using diathermy and a vitrectomy probe. hCLs (thickness 100–120 µm), collected from refractive surgery patients, were decellularized and introduced into the vitreous cavity. With the aid of a viscoelastic agent for stability, hCLs were positioned into the FTMH using microsurgical forceps. Placement and integration were evaluated by iOCT and immunofluorescence (IF) analysis.

Results

OCT images and IF analysis confirmed the successful creation of FTMHs ex vivo in porcine eyes, demonstrating the feasibility of hole induction in an ex vivo model. Preliminary OCT and IF analyses showed the successful implantation of decellularized hCLs at the FTMH site, which were correctly positioned in the subretinal space between the choroid and the retinal pigment epithelium.

Conclusions

Our preliminary results suggest that decellularized hCLs can be successfully implanted to close FTMHs in our preclinical ex vivo model of study, paving the way for future research into bioengineered hCLs for therapeutic purposes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40123-026-01333-0.

Key Summary Points

The management of recurrent and relapsing macular holes is still challenging, requiring the development of innovative techniques.

Porcine eyes can be used to develop an innovative ex vivo preclinical full-thickness macular hole (FTMH) model to test potential successful treatments.

Decellularized human corneal lenticules (hCLs) can be successfully implanted into the FTMH, demonstrating macroscopical and microscopical successful positioning.

Our proof-of-concept study supports the feasibility of decellularized hCLs as novel bio-grafts for macular hole closure and future bioengineered therapies.

Digital Features

This article is published with digital features, including a video, to facilitate understanding of the article. To view digital features for this article, go to 10.6084/m9.figshare.31109569.

Introduction

Full-thickness macular holes (FTMH) are a relatively common ophthalmic condition, with a reported estimated prevalence of 3.3/1000 people [1]. First described by Johnson and Gass in 1988, they are characterized by the interruption of all neuroretina layers in the fovea, resulting in reduced visual acuity, scotoma, and metamorphopsia [2, 3]. FTMH pathogenesis can be found in an anomalous posterior vitreous detachment (PVD), resulting in tractional and centripetal forces acting on the macular area as a result of the remaining vitreous cortex, causing the interruption of all retinal layers [4–7]. To date, surgery is the only treatment shown to be successful in obtaining hole closure and re-establishing macular integrity [8]. Despite the high overall success rate of greater than 90%, cases of suboptimal recovery are recorded which lack either functional or anatomical success [9]. Moreover, primary surgery failure or FTMH reopening may occur after the primary surgery (i.e., refractory) or at least 4 weeks after initial successful closure (i.e., recurrent) in up to 10% of cases, leading to challenging surgical scenarios [10].

Therefore, a better understanding of the disease genesis is needed to improve and develop new treatments aimed at ensuring both anatomical and functional success [11].

Recent studies have explored the use of natural scaffolds, such as human amniotic membrane (hAM), for FTMH repair [12, 13]. However, the use of hAM has several limitations, including the limited mechanical strength and the risk of immune responses or rejection, especially if not properly processed [14]. Additionally, as perinatal-derived tissue, hAM may not fully align with the specific biological and structural requirements of ocular tissues, potentially affecting its integration and functionality in retinal applications [15]. To overcome these limits, the human corneal lenticule (hCL) has recently been introduced as a groundbreaking scaffold for ocular regenerative therapy [16]. This natural, collagen-rich tissue is routinely obtained as a discarded tissue of keratorefractive lenticule extraction (KLEx) refractive surgery performed to correct myopia. Its availability, alongside its potential for banking, makes it a cost-effective resource for ocular regenerative therapies. [17]

Human corneal lenticules, as a discarded byproduct of refractive surgery, have been clinically used in the recent years as allogenic therapeutical inlays for the treatment of various corneal diseases, including corneal ulcers and ectasia, providing evidence of the excellent biocompatibility and capacity of intra-tissue integration. [18, 19] Furthermore, as a thin ocular tissue characterized by transparency, mechanical strength, and biocompatibility, the hCL could represent an ideal scaffold for retinal tissue engineering.

While the current literature has already explored and validated the use of existing models in obtaining FTMH closure such as hAM or other natural scaffolds, these do not appear to promote complete functional recovery [9, 10, 12, 13]. Nevertheless, these materials have shown comparable profiles in terms of biocompatibility. As a result, our plan to use the hCL lies in the possibility of bioengineering the hCL with factors potentially promoting regeneration, to use the hCL as a delivery vector. The ex vivo porcine eye is a model system previously used in other ophthalmic studies which has shown high comparability to the human eye and is free of cost and ethics-related issues [20–22]. The possibility of reproducing the FTMH in an ex vivo porcine eye opens the potential for evaluating macro- and microstructural changes occurring in retina layers once the retina is interrupted in the foveal area, to reproduce and develop new potential treatment.

Thus, this study aims to report the development of a new ex vivo porcine eye model for FTMHs and to test the feasibility of hCLs in obtaining structural hole closure through an imaging-histopathological study in our newly established ex vivo model.

Methods

hCL Collection and Preparation

hCLs were obtained from patients undergoing KLEx refractive surgery with the SMILE (small incision lenticule extraction) technique, using the VisuMax (Carl Zeiss Meditec, Jena, Germany) femtosecond laser platform (Visumax^® 800). Specifically, the objectives of our study were to study and test (1) the feasibility of creating an ex vivo porcine FTMH model and (2) the feasibility of surgical placement of the hCLs.

In brief, the laser cut energy index range was 150 nJ, and spot spacing ranged from 2.5 to 4.5 μm; hCLs were obtained after moderate to high myopic treatments (SE ranging from −4.00 to −8.00 D), with optical zone diameter of 6.00–6.80 mm and thickness of 100–120 µm. The study was conducted in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Board and Ethical Committee of the G. d’Annunzio University of Chieti-Pescara (authorization no. 03/07-02-2019).

The extracted hCLs (n = 25) were transferred into 0.9% sodium chloride physiological solution and stored at −80 °C in glycerol until use in experiments. Both non-decellularized and decellularized hCLs were employed. Non-decellularized hCLs were used for initial implantation trials and for macroscopic visualization of their positioning, for which they were stained with eosin prior to implantation. In contrast, decellularized hCLs (keratocyte-free hCLs) were employed for immunofluorescence (IF) experiments, following previously described protocols [23, 24]. Briefly, after thawing, hCLs were washed with phosphate-buffered saline (PBS 1X, Sigma-Aldrich) and then incubated in a 0.1% sodium dodecyl sulfate (SDS) solution for 24 h at room temperature under continuous agitation (300 rpm), according to an established protocol [23]. Subsequently, the hCLs underwent three consecutive washes in PBS 1X, each lasting 24 h. After undergoing a dehydration process at 60 °C for 2 h, hCLs were rehydrated in PBS 1X immediately before the implantation experiments.

Porcine Eye Handling and Surgical Technique

A total of 25 eyes were obtained from 6-month-old pigs, euthanized for food consumption by electrocution at a local abattoir, and transported to the laboratory within 3 h of the animals’ death. Eyes were positioned on a mannequin head support and fixed in order to obtain the necessary stability to proceed for surgery. All surgeries were performed by the same vitreo-retinal experienced surgeon (RM) using the Leica Proveo microscope (Proveo 8, Leica Microsystems Inc.).

With the aid of Vannas scissors, the hCL was surgically prepared for implantation by adjusting its size from the original 7.5 mm to 2–3 mm, according to the macular hole size. After the core vitrectomy, PVD was induced, and the generation of a macular hole was obtained through the use of diathermy and a 25-gauge vitrectomy probe.

Once obtained, the macular hole was identified and scanned through integrated optical coherence tomography (iOCT; Leica Microsystems Proveo microscope). The hCL was then inserted into the vitreous cavity with the help of a microsurgical forceps through a sclerotomy using a micro-vitreo-retinal (MVR) blade and inserted in the FTMH, promoting stability with Viscoelastic Healon GV (Johnson & Johnson Vision). Afterward, eyes were tamponaded with balanced salt solution (BSS). The definition of surgical success was established by positioning the hCL under the retinal pigment epithelium (RPE) through the macular hole by covering its borders at 360°.

Eye Preserving Technique

To identify the most suitable approach for subsequent analyses, the protocols used to preserve porcine eyes after the surgical procedures were as follows:

In the first step, two eyes (n = 2) were analyzed through fixation in formalin for 24 h followed by paraffin embedding. Two eyes (n = 2) were analyzed through fixation in a 15% sucrose solution for 12 h, cryoprotection with Tissue-Tek, and freezing in liquid nitrogen. Due to the ineffectiveness of prior techniques, 21 eyes (n = 21) were fixed as follows: after viscoelastic agent insertion, the preservation procedure involved cryoprotection with Tissue-Tek (Sakura, Umkirch, Germany) followed by freezing in liquid nitrogen. Finally, the preserved eyes were sectioned using a cryostat.

Immunofluorescence and Histological Analyses

Porcine eye cryosections were washed twice with PBS 1X and fixed with 4% paraformaldehyde (PFA), after which 1 h of 5% bovine serum albumin (BSA) blocking was performed. After additional washing, porcine eye slices were stained with anti-collagen I primary antibody (1:300; Abcam, Cat# ab34710) in BSA 5%. Following three washes in PBS 1X (5 min each), slices were incubated for 1 h with an anti-rabbit Cy3 secondary antibody (Jackson ImmunoReasearch Laboratories, Cat# 111-165-144). Finally, slices were counterstained with DAPI (4′6′diamidino-2-phenylindole; Sigma-Aldrich, St. Louis, MO, USA, Cat D9542) to identify cell nuclei and mounted using fluorescent mounting (Dako). Images were captured using a confocal microscope (Zeiss LSM-800; Carl Zeiss Meditec, Jena, Germany).

Data Analysis

A qualitative and image-based analysis was conducted by processing OCT images and videos, along with IF micrographs, to assess the feasibility of reproducing ex vivo hole formation and supporting hCL implantation. Each OCT scan was evaluated by two retina specialists. In case of disagreement, a third expert was consulted. The same protocol was used for IF micrographs analysis. No statistical analyses were performed, as the data consisted principally of imaging results. Interpretation of the findings was based on qualitative evaluation and comparison of the observed patterns.

Results

(1) Establishment of the Preclinical Porcine Full-Thickness Macular Hole Model

Following core vitrectomy and the induction of PVD, an FTMH was successfully created in all eyes (25/25) using a combination of diathermy and a vitrectomy probe. As shown in the video (see Video 1) and in Fig. 1A, OCT analyses provided high-resolution imaging of the retinal layers, confirming the complete formation of the FTMH (Fig. 1B). This result was further validated through immunofluorescence (IF) analyses in 21/25 fixed eyes, which clearly demonstrated the presence of an FTMH, as illustrated in Fig. 2. This approach ensured accurate and reproducible modeling of FTMHs, essential for subsequent experimental investigations.

Fig. 1.

OCT analyses. A Photographs captured from OCT video recordings during the setup of the ex vivo FTMHs model with hCL implantation. B High-resolution OCT magnifications of the induced FTMHs in the porcine eye (B1 and B2) with a focus on hCL implantation (B3)

Fig. 2.

IF analyses. Representative IF image of the FTMHs. Control (CTRL) sample without FTMH (A) and sample with FTMH (B). Nuclei were stained with DAPI (in blue)

Video available for this article.

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Video 1: OCT video recordings during the setup of the ex vivo FTMH model with hCL implantation (MP4 157096 kb)

(2) Feasibility Test for hCL in Inducing FTMH Closure

After the establishment of the preclinical ex vivo model of FTMH, the feasibility of using hCL as a bio-graft to close the hole was investigated. Initially, implantation trials were conducted using non-decellularized hCL pre-stained with eosin to facilitate visualization. As shown in the representative photographs (Fig. 3), the hCL was correctly positioned at the site of the hole, demonstrating successful implantation in all eyes (25/25).

Fig. 3.

hCL implantation. Representative photograph of a bisected eye following the implantation of the hCL

Subsequently, immunofluorescence (IF) analyses were performed on eye sections (21/25 eyes) with induced FTMHs and implanted with decellularized hCL. The decellularization process enabled precise identification of the hCL through staining with a type I collagen antibody (red) and DAPI (blue). Representative images at different magnifications (Fig. 4A, B) clearly show the hCL delineated by the red staining of collagen fibers, while the absence of a blue signal confirms the decellularized nature of the lenticules.

Fig. 4.

Representative IF images at different magnifications of FTMH ex vivo model after the hCL implantation. A Low-magnification view highlighting the overall positioning of the hCL in the subretinal space. B Medium-magnification view providing a closer look at the structural integration of the hCL with the surrounding retinal and choroidal layers

Additionally, using the scale bar, the thickness of the hCL was measured to further validate its successful implantation. During the trials, hCLs with thicknesses ranging from 100 to 120 µm were used. As observed in the representative cross-sectional IF image of the porcine eye, the implanted hCL in the figure measures approximately 100 µm, consistent with the dimensions of the lenticules used in the experiments.

Additionally, as shown in Fig. 5, the hCL was correctly positioned within the subretinal space, precisely located between the choroid and the RPE. Indeed, to better appreciate the positioning of the corneal lenticule, a wider image was reconstructed using confocal microscopy at a lower magnification. This approach allowed us to capture a broader area of the entire eye, ensuring a comprehensive assessment of the hCL placement. This accurate positioning highlights the feasibility of the surgical procedure and ensures that the hCL is situated in an anatomically relevant position to facilitate potential integration with the surrounding retinal tissues.

Fig. 5.

Representative IF images. Low-magnification view clearly showing the precise positioning of the hCL between the RPE and choroidal layers, with red staining for collagen fibers and blue staining for the nuclei (DAPI)

Discussion

The management of recurrent FTMHs still appears to be challenging, with no validated approach able to obtain complete functional and structural surgical success. In this study, we present the first ex vivo model of FTMH, investigating the potential of hCL application to achieve structural anatomical success and facilitate hole closure. In vitro and in vivo models have been widely used in surgical technique development over the years, despite not yielding good outcomes in model reproducibility and comparability with in vivo models. However, in vivo models are often limited by ethical and regulatory concerns associated with their use in experimental research [25–27]. As a result, the development of an ex vivo FTMH model provides a promising platform for advancing surgical techniques, offering high reproducibility while overcoming the limitations of traditional in vitro and in vivo models. To our knowledge, this is the first study to establish an ex vivo model of FTMH using hCLs as a structural scaffold to restore foveal integrity in cases requiring surgery. As a proof of concept, both instrumental and histopathological evidence supported the effectiveness of the hCL in closing the hole, shedding light on future studies investigating possible clinical implications.

Although the porcine eye does not have a macula, we created the retinal hole in a region corresponding to the “'visual streak”—a horizontal band with a high density of ganglion cells and photoreceptors, like the human parafoveal macula [28]. This area, despite lacking a macula and fovea, shares some functional similarities with the human retina and has therefore widely been used as an animal model for diseases affecting the macula [21, 22, 29].

In our study, we used a combination of video imaging, OCT, and IF to assess the feasibility of inducing a retinal hole. Our results demonstrated that we were able to successfully replicate FTMH formation ex vivo in the porcine model, as evidenced by OCT scans showing characteristic retinal disruption and hole formation. Furthermore, IF analysis revealed appropriate changes in retinal architecture, confirming the reproducibility of the FTMH in this model. These findings validate the porcine eye as a suitable ex vivo model for studying retinal hole formation and for evaluating potential surgical interventions aimed at FTMH closure. However, we encountered several challenges in the inclusion and preservation of the porcine eyes. As detailed previously, we tested various preservation methods following the surgical intervention. Unfortunately, we were unable to produce reliable data on cryopreservation using formaldehyde and 15% sucrose solutions. The only effective preservation technique we found was the use of a viscoelastic agent, which not only ensured proper cutting and sectioning with the cryostat but also maintained the stability of the implanted hCL.

Indeed, after validating the feasibility of reproducing an ex vivo model of FTMHs, we assessed the use of the hCL as a bio-graft to close the hole previously created. The idea of using hCLs for the treatment of diseases deriving from discontinuity in ophthalmological structures has recently been appearing in the literature, with interesting evidence for future promising approaches. In a recent article, Zhang et al. described the use of hCL transplantation for the treatment of optic disc maculopathy in a young patient treated with pars plana vitrectomy, hCL sealing, and CF38 tamponade. In their case, the authors achieved significant anatomical improvement, with improvement in visual acuity suggesting hCL transplantation as a promising therapeutic option able to achieve functional and structural success in cases of optic disc maculopathy [30].

In our cases, the IF results demonstrated that the hCL was successfully implanted into the correct anatomical position in the subretinal space, between the choroid and the RPE. Previous studies have demonstrated the ability of stromal lenticules to integrate within tissues, either in intracorneal implantation (i.e., in corneal ectatic or ulcerative disorders) or when transplanted into different ocular tissues such as sclera and conjunctiva [16, 31].

Our results provide compelling evidence that the hCL can maintain its intended position, suggesting its potential as a valuable tool for retinal repair in FTMH surgery. Certainly, the mechanical advantage of an hCL is to be able to plug the hole, creating a physical barrier to promote centripetal adjustment at the FTMH edges. Moreover, the hCL transparency would not interfere with patient vision over time. The advantage of using the hCL not only relies on the possibility of using already available tissues but opens the possibility of using it as a vehicle for substances which have been shown to promote tissue regeneration. In fact, the common substrate on which all available scaffold techniques rely is the ability to induce anatomical closure despite no functional improvements. The possibility of bioengineering the hCL opens the potential for new surgical approaches, pursuing both functional and structural improvement. Previously, Mastropasqua and colleagues demonstrated the feasibility of bioengineering hCLs by incorporating recombinant human nerve growth factor (rhNGF) into polylactic-co-glycolic acid microparticles (PLGA-MPs), thus creating a natural ocular drug delivery system for the controlled release of rhNGF [23]. Building upon this work, Pelusi et al. advanced the bioscaffold by incorporating human amniotic fluid stem cells (hAFSCs) to create a device capable of simultaneously releasing neurotrophic and regenerative factors [24]. In their study, they also examined the effects of hAFSCs and rhNGF delivered by hCLs on a high-glucose-induced ex vivo model, aiming to simulate the molecular mechanisms underlying diabetic retinopathy (DR). Interestingly, the authors found that hAFSCs and rhNGF were able to modulate key molecular mechanisms involved in DR, presenting the bioengineered hCL as a promising ocular delivery system for ophthalmological treatments. Additionally, with hCLs extracted from young, healthy humans, the high amount of collagen fibers must be considered, which may biochemically contribute to the closure process. Moreover, researchers have noted high tissue biological compatibility, which makes hCL a highly maneuverable tissue, as enhanced by the exploration of clinical application potential in human corneal transplantation and correction of hyperopia, presbyopia, and keratoconus [32].

Nevertheless, our study has limitations. First, it must be acknowledged that the use of ex vivo models precludes the assessment of long-term graft stability and functionality. As a result, this study should be regarded as a proof of concept demonstrating the feasibility of hCL integration into retinal tissue. Nevertheless, future studies should evaluate in vivo outcomes, to better characterize the efficacy, stability, and functional contribution of hCL grafts in the treatment of macular holes.

Conclusions

To conclude, our model is the first ex vivo FTMH model showing structural success for the use of hCL as bio-graft in the surgical management of FTMHs. Future studies should focus on testing the hCL in FTMH management by investigating functional outcomes and its potential for future clinical applications. In this light, building upon previous findings regarding the bioengineering of hCLs, the combination with neurotrophic and regenerative factors may aid in the closure of macular holes and in restoring the functionality of the surrounding retinal areas.

Author Contributions

Rodolfo Mastropasqua, Mario Nubile and Domitilla Mandatori: Conceptualization. Maria Ludovica Ruggeri, Letizia Pelusi, Alberto Quarta: Methodology. Alessia Lamolinara, Marcello Allegretti: Software. Samuele Faieta, Nadia Di Pietrantonio, Francesco Del Pizzo: Data curation. Maria Ludovica Ruggeri and Letizia Pelusi: Writing–Original draft preparation. Leonardo Mastropasqua and Assunta Pandolfi: Visualization. Alberto Quarta and Maria Ludovica Ruggeri: Investigation. Rodolfo Mastropasqua: Supervision. Domitilla Mandatori and Rodolfo Mastropasqua: Writing–Review and editing,

Funding

None. The Journal’s Rapid Service Fee will be funded by the University of Chieti-Pescara.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of Interest

Marcello Allegretti: Commercial relationship: Dompé Farmaceutici SpA: Code F (Financial support): Salary. Maria Ludovica Ruggeri, Letizia Pelusi, Mario Nubile, Alberto Quarta, Alessia Lamolinara, Marcello Allegretti, Samuele Faieta, Nadia Di Pietrantonio, Francesco Del Pizzo, Leonardo Mastropasqua, Assunta Pandolfi, Domitilla Mandatori, Rodolfo Mastropasqua declare no conflicting relationship.

Ethical Approval

The study was conducted in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Board and Ethical Committee of the G. d’Annunzio University of Chieti-Pescara (authorization no. 03/07-02-2019).