From the Department of Ophthalmology (AK, KS), Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; and the Ocular Surface Center (WL, SCGT), Miami, Florida.
Dr. Tseng has obtained a patent for the preparation and clinical use of the amniotic membrane.
Supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
Address correspondence to Akira Kobayashi, MD, PhD, Department of Ophthalmology, Kanazawa University Graduate School of Medical Science, 13–1 Takaramachi, Kanazawa, Ishikawa-prefecture 920–8641, Japan.
Recently, use of the amniotic membrane as a graft or a temporary patch has been shown to reduce inflammation successfully and to facilitate wound healing in persistent corneal epithelial defects in several ocular surface diseases,1–3 including acute chemical or thermal burns4,5 and acute stage Stevens–Johnson syndrome.6–8 When the amniotic membrane is used as a graft to restore the corneal stromal and surface integrity, it is anticipated that it is integrated into the host tissue via wound remodeling. Nevertheless, insight into the outcome of transplanted amniotic membranes is limited, due in part to the scarcity of such tissues available for histopathologic studies9,10 and to the lack of an effective in vivo imaging method.
In vivo corneal confocal microscopy allows nonin-vasive real-time spatial sectioning of living corneal tissues at the cellular level.11,12 The clinical feasibility of this method has been documented in studies of both normal and diseased human corneas.11–14 To determine whether in vivo corneal confocal microscopy could be an ideal approach to investigate the outcome of amniotic membrane transplants on corneas, we previously used a dermatological in vivo laser confocal microscope (Vivascope 1000; Lucid Inc., Henrietta, NY) and a cornea-specific white-light confocal microscope (ConfoScan 2; Nidek Technologies, Vigonza, Italy) to identify three layers of the amniotic membrane—an epithelium, a basement membrane, and the amniotic stroma—in cryopreserved amniotic membrane.15 Herein, we used a cornea-specific in vivo laser scanning confocal microscope, the Heidelberg Retina Tomograph II Rostock Cornea Module (HRT II-RCM) (Heidelberg Engineering GmbH, Dossenheim, Germany),16 to disclose more layers in both fresh and cryopreserved amniotic membrane.
Materials and Methods
Preparation of Fresh and Cryopreserved Human Amniotic Membrane
The preparation and clinical use of human amniotic membrane was approved by the Ethical Committee of Kanazawa University Graduate School of Medical Science. The method of preparing cryopre-served amniotic membrane was described previously.4 In brief, after written informed consent was obtained, the human placenta was obtained shortly after elective cesarean delivery from the donor mother, from whom human immunodeficiency virus, hepatitis virus type B, hepatitis virus type C, and syphilis had been serologically excluded. Under a lamellar flow hood, the placenta was cleaned of blood clots with sterile saline containing 50 μg/mL of penicillin, 50 μg/mL of streptomycin, 100 μg/mL of neomycin, and 1.25 μg/mL of amphotericin B. The amnion was separated from the rest of the chorion by blunt dissection through the potential spaces situated between these two tissues and then spread onto a sheet of nitrocellulose paper with the epithelium/basement membrane surface up. The paper, with the adherent amniotic membrane on it, was then cut into small (approximately 5.0 × 5.0 cm) pieces and stored at −80°C in a sterile vial containing Dulbecco’s modified Eagle’s medium and glycerol at the ratio of 1:1 (v/v). Fresh amniotic membrane was subjected directly to laser confocal microscopic analysis.
The amniotic membrane consists of two portions, the placental and the fetal. The former is adjacent to the placenta portion, whereas the latter is mostly enwrapping the fetus. Amniotic membrane prepared from the placenta portion was in general thicker than the fetal portion. For clinical uses, the fetal portion of the amniotic membrane is used most of the time because of its larger area and uniformity in thickness. For this study, the fetal portion of the amniotic membrane was used.
In Vivo Laser Confocal Microscopic Observation
Three fresh amniotic membrane samples from different donors and 10 cryopreserved amniotic membrane samples from different donors were used. Fresh amniotic membranes were kept at room temperature and were examined within 2 hours after harvesting, whereas cryopreserved amniotic membranes within 3 months after harvesting were examined after being thawed at room temperature for at least 1 hour. Three different areas from each sample were examined. A large drop of contact gel (Comfort Gel ophthalmic ointment; Bausch & Lomb, GmbH, Berlin, Germany) was applied on the front surface of the microscope lens, with care taken to avoid the enclosure of air bubbles. The Tomo-cap (Heidelberg Engineering GmbH) was mounted on the holder to cover the microscope lens. Then, the epithelial side of the amniotic membrane samples was attached to the front surface of the Tomo-cap and each amniotic membrane sample was examined layer by layer with the HRT II-RCM, which uses a 60× water-immersion objective lens (Olympus Europa GmbH, Hamburg, Germany), is powered by a 670-nm diode laser as a light source, and covers 400 × 400 μm of the area of observation.17
In Vivo Laser Confocal Microscopy of Fresh Amniotic Membrane
The epithelial cell layer of fresh amniotic membrane was visualized as an irregularly arranged polygonal pattern (approximately 10 to 20 μm in diameter presumably applies to the individual cells) (Fig. 1A). At a deeper plane of approximately 10 to 20 μm beneath the surface, a basement membrane of relatively bright homogeneity was seen (Fig. 1B). Further down, in a posterior aspect of the basement membrane layer (facing the fibroblast layer), some striae could be seen (Fig. 1C). At a slightly deeper plane of approximately 20 to 30 μm beneath the surface, there was a cellular layer containing presumably “live” mesenchymal cells, which gave highly reflective “fibroblastic” shapes (Fig. 1D). Below the cell-rich stromal layer was a less dense connective tissue with a reticular pattern with occasional cross sections of mesenchymal cells (Fig. 1E). This layer was contiguous with the spongy layer, located approximately 100 to 150 μm beneath the surface, where multiple lacunae were outlined by fibrous connective tissues (Fig. 1F). The same results were observed in three different fresh amniotic membrane samples obtained from three separate donors.
Figure 1. Representative Confocal Images of Fresh Amniotic Membrane, Showing (A) an Epithelial Cell Layer of Approximately 10 to 20 μm in Diameter, (B) a Basement Membrane of Approximately 10 to 20 μm Beneath the Surface (the Outline of 10 to 15 Cells Visible in the Far Left Region of the Photomicrograph Depicts Epithelial Cells), (C) a Posterior Aspect of the Basement Membrane Layer with Folds, (D) a Dense Connective Tissue Layer with Fibroblastic Cells Revealing High Reflectivity, (E) a Less Cellular but Dense Connective Tissue, and (F) a Spongy Layer (approximately 100 to 150 μm Beneath the Surface). Bar = 100 μm.
In Vivo Laser Confocal Microscopy of Cryopreserved Amniotic Membrane
Similar to fresh amniotic membrane, on the surface of cryopreserved amniotic membrane, there was a monolayer of polygonal cells arranged in an irregular mosaic pattern, with a diameter of approximately 10 to 20 μm. Each cell was defined by low reflective borders (Fig. 2A). At a deeper plane of approximately 10 to 20 μm beneath the surface, there was a basement membrane, which was of relatively bright homogeneity (Fig. 2B). Further down, in a posterior aspect of the basement membrane layer (facing the fibroblast layer), some striae can be seen but with less microstriae than the fresh amniotic membrane (Fig. 2C). Further below, the dense connective tissue contained few elongated “fibroblastic” mesenchymal cells (Fig. 2D). At a deeper plane, the connective tissue turned into a reticular pattern, in which cells became less distinct (Fig. 2E). At an even deeper plane (approximately 100 to 150 μm beneath the surface), the reticular pattern evolved into a spongy pattern in which cells were not discernable (Fig. 2F). The same results were observed in all 10 samples obtained from 10 separate donors.
Figure 2. Representative Confocal Images of Cryopreserved Amniotic Membrane Showing (A) an Epithelial Cell Layer of Approximately 10 to 20 μm in Diameter, (B) a Basement Membrane with Relatively Bright Homogeneity, (C) a Posterior Aspect of the Basement Membrane Layer with Fewer Folds, (D) a Dense Connective Tissue with Few Cells with Less Reflectivity (approximately 20 μm Beneath the Surface), (E) a Less Dense Connective Layer with a Reticular Pattern, and (F) a Spongy Layer of Loose Connective Tissue. Bar = 100 μm.
With the HRT II-RCM, four distinct layers (a simple epithelium, a basement membrane, a fibro-blastic layer, and a spongy layer) could be consistently identified in both cryopreserved and fresh human amniotic membrane samples. Interestingly, some striae could be seen in a posterior aspect of the basement membrane layer (facing the fibroblast layer). These results were almost comparable to those from histologic sections, which identified (1) a simple epithelium, (2) a basement membrane, (3) a compact stromal layer, (4) a cell-rich loose connective tissue layer, and (5) a cell-poor spongy stromal layer.18 However, we could not identify a compact stromal layer by HRT II-RCM.
The major differences between fresh and cryo-preserved amniotic membrane were found in the posterior side of the basement membrane layer (facing the fibroblast layer) and the cell-rich connective tissue layer. Fresh amniotic membranes had more prominent folds in a posterior side of the basement membrane layer than cryopreserved amniotic membranes (Figs. 1C vs 2C). However, it is not clear whether the degree of folding and other confocal findings are consistent all over the amniotic membrane because we observed only three different areas in each sample. Mesenchymal cells were alive in fresh amniotic membranes; thus, they appeared as highly reflective stellate shapes in the cell-rich superficial stromal layer (Fig. 1D) and also as highly reflective specks in the connective tissue cords extending to the deeper stromal layers (Fig. 1E). In cryopreserved amniotic membranes, they turned into less highly reflective images (Figs. 2D and 2E).
We have previously reported that the human cryo-preserved amniotic membranes harvested in our hospital showed a thickness of approximately 100 to 150 μm.19 However, human amniotic membrane thickness varies between donors and depends on its location in the placenta, and is possibly affected by the technician who prepared the amniotic membrane for ophthalmic use even though the same fetal portion was used. Also, the cryopreservation may have some effects on its thickness that could affect the quality of the confocal images. In this study, however, we did not compare the thickness variation between fresh and cryopreserved amniotic membrane.
The HRT II laser confocal microscope has been used to acquire quantitative three-dimensional images of the retina and optic disc. With the addition of the RCM, the HRT II is converted to a cornea-specific in vivo laser confocal microscope16,17,20 that allows layer-by-layer analysis of the cornea with a longitudinal optical resolution of nearly 4 μm,21 which is much better than that of the conventional white-light confocal microscope (10 μm in longitudinal optical resolution by ConfoScan 2).22 That likely explains why four distinct layers of amniotic membrane tissue could be resolved by the HRT II-RCM, whereas only three layers (an epithelium, a basement membrane, and the amniotic stroma) were visualized when we previously used the white-light confocal microscope (ConfoScan 2) to observe cryopreserved amniotic membrane.15
The dermatological in vivo laser confocal microscope (Vivascope 1000) also has a high resolution (5 μm in longitudinal optical resolution) but we could not disclose four layers in the previous study by Viva-scope 1000.15 This might be due to the instability of the head of the Vivascope 1000 when inappropriately used without “tissue stabilizer.” Also, the use of a different laser beam wavelength in each device (HRT II-RCM: 670-nm diode laser vs Vivascope 1000: 830-nm diode laser) may have some role in the superior resolution of the HRT II-RCM.
Although fresh amniotic membrane (used within 24 hours after harvesting) has been used for ocular surface reconstruction,23 its clinical value has been questioned, especially when the potential risk of disease transmission is considered.24 Now that we have demonstrated how these two membranes might be distinguished by the HRT II-RCM, their baseline characterization can be used for future investigation of the in vivo remodeling of the amniotic membrane after transplantation.
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Figure 3. Light Microscopy Cross Section of Fresh Amnion Tissue (hematoxylin–Eosin; Original Magnification ×200). Amniotic Membrane Includes an Amniotic Epithelium, a Basement Membrane, a Compact Layer, a Fibroblast Layer, and a Sponge Layer as Marked.