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A Human Amniotic Membrane Plug to Promote Retinal Breaks Repair and Recurrent Macular Hole Closure

Rizzo, Stanislao MD; Caporossi, Tomaso MD; Tartaro, Ruggero MD; Finocchio, Lucia MD; Franco, Fabrizio MD; Barca, Francesco MD; Giansanti, Fabrizio MD

Section Editor(s): Williams, George A.

doi: 10.1097/IAE.0000000000002320
Surgical Technique

Department of Translational Surgery and Medicine, Ophthalmology, University of Florence, Careggi, Florence, Italy.

Reprint requests: Tomaso Caporossi, MD, Department of Translational Surgery and Medicine, Ophthalmology, University of Florence, Careggi, Largo Brambilla 3, 50134 Firenze, Florence, Italy; e-mail:

None of the authors has any financial/conflicting interests to disclose.

S. Rizzo and T. Caporossi equally contributed to this work.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (

The human amniotic membrane (hAM) is the innermost layer of the fetal membranes. It has a stromal matrix, a thick collagen layer, and an overlying basement membrane with a single layer of epithelium.1 The use of amniotic membrane transplantation to treat ocular surface abnormalities was first reported seven decades ago.2–4

The amniotic membrane transplantation has been successfully used on patients with persistent epithelial lesions,5–8 corneal ulceration,9 symptomatic bullous keratopathy,10 band keratopathy,11 chemical and thermal burns,12–15 conjunctival surface reconstruction, ocular cicatricial pemphigoid, and Steven–Johnson syndrome.16

In these cases, hAM work as an optimal biological support for conjunctival cell growth. Recent publications show that hAM constitute an adequate substrate for in vitro growth and expansion of conjunctival epithelial progenitor cells.17,18

Rosenfeld used a rabbit model to demonstrate that subretinal implantation of hAM seems to be well-tolerated without evidence of inflammation.19

Carmen Capeans20 has demonstrated that human retinal pigmented epithelium (RPE) cells seeded over hAM in the first 24 hours. Moreover, human RPE cells maintain epithelial features and can proliferate over epithelium-free hAM, constituting a tight monolayer with well-defined intercellular and cell–substrate interactions.

Ohno-Matsui21 showed that RPE cells cultured on hAM demonstrated an epithelial phenotype and secreted several growth factors important for maintaining retinal homeostasis. Recently, hAM was implanted in induced RPE and Bruch membrane, which had been damaged mechanically in pigs.22

Human amniotic membrane demonstrated excellent growth support properties on RPE cells in the subretinal space.22

In vivo hAM plug transplantation was recently used for choroidal hole repair in a case of globe rupture.23

We have reported the clinical data of 14 patients with vitreoretinal pathologies, 8 recurrent macular holes and 6 retinal detachments, who underwent subretinal space hAM transplantation to repair retinal breaks or macular holes. Furthermore, we have performed an optical coherence tomography (OCT) analysis for all the patients.

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Materials and Methods

This is a prospective, interventional, consecutive case series conducted at Careggi Hospital affiliated with Florence University School of Medicine. All the patients signed an informed consent. The institutional Review Board approved the study, which is in accordance with the tenets of the Declaration of Helsinki.

We enrolled 14 patients from July 2017 to January 2018. Eight patients suffered from a recurrent macular hole (Table 1), and six had a retinal detachment, one rhegmatogenous retinal detachment (RRD) and five retinal detachment recurrences, with various grades of proliferative vitreoretinopathy (PVR) (Table 2). The patients underwent a standard 3-port, 23-gauge transconjunctival pars plana vitrectomy (Alcon Laboratories, Fort Worth, TX) under retrobulbar anesthesia and hAM plug transplantation in the subretinal space under the retinal tear or the recurrent macular hole.

Table 1

Table 1

Table 2

Table 2

All the surgical procedures in our hospital were performed by the same experienced surgeons (S.R. and T.C.).

Preoperatively, an ophthalmic history and a complete ophthalmic examination including refraction with assessment of best-corrected visual acuity (BCVA, Snellen, and logarithm of the minimal angle of resolution [logMAR]), Goldmann applanation tonometry, standard fundus dilated ophthalmic examination, spectral domain OCT (AngioVue Optovue, Fremont, CA) analysis of the macular hole with an accurate measurement of the internal and external diameter and an ultrawide retinography (Daytona, Optos Inc, Marlborough, MA).

The 23-gauge was chosen to facilitate the insertion of the hAM through the valved trocar.

A complete vitrectomy was performed with an accurate vitreal base shaving (Constellation; Alcon Surgical). In phakic eyes (4 patients in the macular hole group), phacoemulsification and intraocular lens implantation were performed at the beginning of surgery. A chandelier endoilluminator was inserted to facilitate bimanual maneuvers.

A hAM plug was taken from the hAM patch, and the final dimensions were adjusted with vitreoretinal scissors before insertion into the vitreous chamber.

The hAM plug was flipped and rolled inside the vitreoretinal forceps and inserted through the trocar into the vitreous chamber. Inside the vitreous cavity, the hAM was manipulated under fluid or perfluorocarbon (PFCL) (perfluorodecalin, Biofluor, Bucine [AR], Italy) and transplanted through the retinal break or macular hole into the subretinal space; it was then spread to cover the entire portion of the break or the hole. An intraoperative OCT scan was performed to check the correct position of the amniotic membrane plug under the retina (see Video, Supplemental Digital Content 1,

Afterward, perfluorocarbon was injected to a complete overfilling of the break to permit good adhesion of the hAM plug to the underlying RPE and the overlying retina. No laser retinopexy was performed.

Fluid–air exchange and an endotamponade injection was performed at the end of surgery. Standard 1000 cSt silicone oil (Oxane 1300; Bausch + Lomb, Incorporated, Rochester, NY) was chosen as the endotamponade for the retinal detachment group and 20% sulfur hexafluoride (SF6) for the macular hole group.

Silicone oil was removed 3 to 4 months after surgery in all the cases. No additional laser treatment was performed during the silicone oil extraction procedure (see Video, Supplemental Digital Content 2,

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Fourteen patients, 6 men and 8 women were included in the study. The mean age was 66.86 ± 11.45 years (range 42–81 years). Patients were divided into two groups: a retinal detachment group (four women and two men) and a recurrent macular hole group (four men and four women).

In the recurrent macular hole group, all the patients had already undergone PPV with ILM peeling and gas tamponade. In the retinal detachment group, the first patient was affected by RRD with grade B PVR and a superior retinal break. The second patient had a posterior pole retinal detachment with a large recurrent macular hole with grade B PVR. This patient had already undergone PPV with gas endotamponade for a macular hole and a secondary PPV with silicone oil for retinal detachment; patients number 3, 4, and 5 had pathological myopia and were affected by posterior pole recurrent retinal detachment with posterior pole retinal breaks and grade C1 PVR. They had already undergone PPV with silicone oil endotamponade. Patient number 6 had a complete retinal detachment with one posterior pole retinal break and one big midperipheral retinal break with PVR. He had already undergone two PPV procedures with perfluorocarbon as the medium-term endotamponade and standard silicone oil endotamponade after the second operation.

The mean preoperative BCVA was 1.48 ± 0.49 logMAR (20/800) ranging from 2 to 0.7 logMAR (20/2000–20/100) for the macular hole group and 2.33 ± 0.51 logMAR (20/2000) ranging from 3 to 2 logMAR (20/20,000–20/2000) in the retinal detachment group. Four patients in the macular hole group were phakic and underwent a combined phacovitrectomy; in the retinal detachment group, five patients were pseudophakic and one was aphakic (he underwent iris claw intraocular lens implantation during the first operation).

Twenty percent SF6 was used as endotamponade in the macular hole group, and standard 1000 cSt silicone oil in the retinal detachment group.

Silicone oil extraction in the retinal detachment group was performed 3 months after the first procedure; sterile air was injected as a temporary endotamponade in all the cases.

Patients in the macular hole group were examined after 1 week and 1, 3, and 6 months after surgery. An OCT scan was performed at every examination. In all the 8 cases who received a gas injection after the operation, the OCT performed at 1 week already demonstrated the macular hole closure with the neurosensory retina overfilling the hAM plug (Figures 1–4). Best-corrected visual acuity improved from 1.48 ± 0.49 logMAR (20/800) preoperatively to 0.71 ± 0.37 logMAR (20/100) 3 months after the operation, and to 0.48 ± 0.14 logMAR (20/50) 6 months after the operation (Figure 5). No adverse events such as an increase of intraocular pressure (IOP) or endo-ocular inflammation were reported during the follow-up period.

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Fig. 4

Fig. 4

Fig. 5

Fig. 5

In the retinal detachment group, BCVA improved from 2.33 ± 0.51 logMAR (20/2000) preoperatively to 1.2 ± 0.62 logMAR (20/400) after 3 months, and to 0.8 ± 0.47 logMAR (20/125) 2 months after the silicone oil removal (Figure 6).

Fig. 6

Fig. 6

Optical coherence tomography analysis showed a retinal adhesion over the amniotic membrane plug in the retinal break site during the first week after surgery. During the first 2 to 3 months, a retinal ingrowth on the retinal break's margin over the hAM plug was reported in all the cases (Figure 7). During silicone oil extraction, intraoperative OCT was used to check the adhesion of the retina over the hAM plug (Figure 8). Because we obtained a good retinopexy using the hAM plug, we did not perform retinal photocoagulation. No PVR was revealed after a vital-die injection.

Fig. 7

Fig. 7

Fig. 8

Fig. 8

No eyes had postoperative adverse events such as endophthalmitis, hypotony, IOP elevation, or retinal or choroidal reactions; no rejection of the hAM implanted in the subretinal space was reported after more than 1 year of follow-up.

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Human amniotic membrane has been used in superficial ocular pathologies since 1940. Although hAM reduces inflammation and scarring and facilitates epithelialization, there are still uncertainties regarding the integration of the graft and the mechanisms through which it exerts its long-term effects.

Recently, the interest of authors has been directed toward the interactions of the hAM with retinal structures and in particular with the RPE. Amniotic membranes induce RPE cell proliferation and differentiation.

In vivo transplantation of the hAM to repair a choroidal wound has already been performed in one case, and good integration of the plug to the suprachoroidal structures was observed.23

More recently, the Seprafilm Adhesion Barrier (Sanofi, Bridgewater, NJ), a bioreabsorbable translucent membrane composed of sodium hyaluronate and carboxymethylcellulose, was developed to prevent pathological adhesions after abdominal and pelvic surgery. It was used with success to repair retinal breaks in retinal detachment in four cases.24

Haruta et al applied laser retinopexy around the retinal breaks and used the seprafilm sheet to cover the breaks to avoid intravitreal dispersion of proliferating retinal pigment epithelial cells. In our cases, we did not apply laser retinopexy. We implanted the hAM plug in the subretinal space to maximize its interaction with the RPE, to stimulate tissue proliferation and retinal breaks closure. After 3 months, we decided to remove the silicone oil in all the cases of retinal detachment. Preoperative and intraoperative OCT showed a thin retinal substrate over the plug of hAM, which covered the previous retinal break. The repaired retina seemed organized in normal layers as well as the original retina, and it adhered well to the underlying hAM. The results led us to conclude that hAM plugs stimulate retinal proliferation and organization in layers, which can virtually recover the original retinal function.

The treatment of recurrent macular holes is still a challenge for a retinal surgeon. The failure rate of primary surgery in idiopathic macular hole is less than 10%. It may be due to residual epiretinal traction, insufficient gas tamponade, poor compliance by the patient in keeping prone position, or no obvious cause. In recent years, many management options have been proposed, such as laser treatment,25 a second operation with transforming growth factor-beta 2 application,26 or autologous platelet concentrate27 inserted inside the macular hole to achieve its closure.

Some authors have tried alternative endotamponades such as semifluorinated alkane F6H8,28 standard silicone oils,29 or heavy silicone oils30–32 to induce macular hole closure.

Modern approaches to recurrent macular holes are the use of a plug of ILM peeled from the periphery of the posterior pole and inserted into the macular hole,33 a neurosensory retinal free flap,34,35 or capsular lens fragments.36,37

The above mentioned procedures have gained from 60% to 90% of anatomical success and a BCVA improvement of 80% in the case of anatomical success.

In our series, the recurrent macular hole was resolved during the first week after the operation in all the cases. After the gas endotamponade disappeared, the OCT scan demonstrated the macular hole closure without glial process but with a fully stratified retinal layer over the hAM patch. During the follow-up period, the neuroretina over the hAM plug differentiated to form retinal layers, in particular in the outer layer such as the external limiting membrane and ellipsoid zone. This process can probably be associated with the visual acuity improvement found during the follow-up.

Some authors have described retinal fibrosis and pigment epithelium dystrophy in the macular area, after internal limiting membrane autologous transplantation for recurrent macular hole, which can affect final visual recovery.38 The OCT analysis on our patients did not show these pathological findings (Figure 9).

Fig. 9

Fig. 9

In conclusion, we have described a new technique that uses a subretinal placement of a hAM plug in patients with recurrent macular hole and complex retinal detachment with posterior breaks and PVR.

Anatomical success was reached in all cases. The amniotic membrane stimulated a retinal ingrowth in all cases. The new retina showed a time-dependent external layer differentiation that in the macular hole group can be associated with the visual acuity improvement. In the retinal detachment group, we did not observe PVR development even in the case of complex recurrent retinal detachment.

Some authors proposed the possibility of amniotic membrane rejection when implanted in the subretinal space,20 but Rosenfeld et al. did not report any visible signs of rejection or retinal disorganization in a rabbit model.19

Our study has mid-term follow-up, but we do not report any complications such as hAM rejection, retinal detachment, or PVR.

Several studies have demonstrated that hAM is an optimal substrate for conjunctival growth and corneal re-epithelization probably because amniotic stroma can secrete growth factors or express adhesion molecules17,18; recently some studies have demonstrated that hAM is an adequate support to RPE growth and plays an important role in facilitating RPE integration into the subretinal space.19–22

We believe that these hAM characteristics can play a role even in retinal repair. The growth factors that are secreted can induce retinal growth and differentiation with retinal activity increase.

Human amniotic membrane patches are easy to obtain from the tissue bank, and rather than other substrates used to close macular holes, such as ILM plugs, autologous retinal plugs, or capsular lens fragments, they are also easier to manipulate inside the eye. Further studies are necessary to determine the efficacy of this new technique, and more intraoperative and postoperative analyses would be useful to understand the interaction between the amniotic membrane and the retina. Inducing retinal ingrowth and stimulating regeneration of the retinal inner layers, the external limiting membrane, and the ellipsoid zone is a new frontier for vitreoretinal surgery.

We believe that this technique could be a starting point, and we will extend it to other retinal pathologies.

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recurrent macular hole; retinal detachment; proliferative vitreoretinopathy; vitrectomy; human amniotic membrane

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