Journal of Refractive Surgery

Original Article Supplemental Data

Risk Evaluation of Human Corneal Stromal Lenticules From SMILE for Reuse

Yanfeng Shang, MD; Yu Li, MD, PhD; Zhiqun Wang, MD; Xuguang Sun, MD, PhD; Fengju Zhang, MD, PhD

Abstract

PURPOSE:

To investigate the pathogenicity and immunogenicity of human corneal stromal lenticules from small incision lenticule extraction (SMILE).

METHODS:

Serological testing was completed prior to sample collection to rule out infectious diseases. Pathogens herpes simplex viruses (HSV) type 1 and type 2 were screened for by real-time fluorescent quantitative polymerase chain reaction, and bacteria, fungi, and Acanthamoeba from 128 lenticules of 64 patients were cultured. A total of 132 lenticules from 93 patients were randomly assigned to the fresh group, −78 °C anhydrous glycerol preservation group (glycerol group), and 0.1% sodium dodecyl sulfate decellularization group (SDS group) in pairs and detected by immunohistochemistry, Western blot, transmission electron microscopy, transmittance, and nanoindentation.

RESULTS:

The fresh lenticules were all negative for HSV-1, HSV-2, bacteria, fungi, and Acanthamoeba. HLA-I A/B/C and HLA-II DR antigens were all expressed in fresh lenticules but were clearly reduced after preservation at −78 °C in anhydrous glycerol or decellularization in 0.1% SDS. The collagen fibers of the lenticules in the fresh group were regularly arranged, and the keratocytes were intact. The fibers in the glycerol group were regularly arranged, and the integrity of keratocytes was destroyed. The fibers in the SDS group were disordered and had no cellular structure. The transmittance and Young's modulus were highest in the fresh group, lower in the glycerol group, and lowest in the SDS group.

CONCLUSIONS:

Risk of infection is low, but risk of rejection exists on the reuse of fresh human corneal stromal lenticules from SMILE. Anhydrous glycerol preservation at −78 °C is an ideal method for reducing antigens without damaging the structure and function of lenticules.

[J Refract Surg. 2021;37(1):32–40.]

Abstract

PURPOSE:

To investigate the pathogenicity and immunogenicity of human corneal stromal lenticules from small incision lenticule extraction (SMILE).

METHODS:

Serological testing was completed prior to sample collection to rule out infectious diseases. Pathogens herpes simplex viruses (HSV) type 1 and type 2 were screened for by real-time fluorescent quantitative polymerase chain reaction, and bacteria, fungi, and Acanthamoeba from 128 lenticules of 64 patients were cultured. A total of 132 lenticules from 93 patients were randomly assigned to the fresh group, −78 °C anhydrous glycerol preservation group (glycerol group), and 0.1% sodium dodecyl sulfate decellularization group (SDS group) in pairs and detected by immunohistochemistry, Western blot, transmission electron microscopy, transmittance, and nanoindentation.

RESULTS:

The fresh lenticules were all negative for HSV-1, HSV-2, bacteria, fungi, and Acanthamoeba. HLA-I A/B/C and HLA-II DR antigens were all expressed in fresh lenticules but were clearly reduced after preservation at −78 °C in anhydrous glycerol or decellularization in 0.1% SDS. The collagen fibers of the lenticules in the fresh group were regularly arranged, and the keratocytes were intact. The fibers in the glycerol group were regularly arranged, and the integrity of keratocytes was destroyed. The fibers in the SDS group were disordered and had no cellular structure. The transmittance and Young's modulus were highest in the fresh group, lower in the glycerol group, and lowest in the SDS group.

CONCLUSIONS:

Risk of infection is low, but risk of rejection exists on the reuse of fresh human corneal stromal lenticules from SMILE. Anhydrous glycerol preservation at −78 °C is an ideal method for reducing antigens without damaging the structure and function of lenticules.

[J Refract Surg. 2021;37(1):32–40.]

Since 2011, small incision lenticule extraction (SMILE) has been developed extensively for the correction of myopia and astigmatism.1 As byproducts, a large number of corneal stromal lenticules were extracted intact during the procedure, leading some experts to explore their reuse in recent years. The lenticules have been successfully reimplanted into autologous or especially allogenic corneas of human subjects for correcting hyperopia,2,3 patching corneal perforations,4–7 and other procedures.8–13

However, there are two potential risks in the reuse of the lenticules: infection and rejection. On the one hand, various sources of infections can be transmitted by corneal transplantation.14 Among these sources, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, Treponema pallidum, rabies virus, and Jacob Kreutzfeld prion can be avoided by serological detection and disease history inquiry, whereas pathogenic microorganisms causing infectious keratopathy must be excluded by specific tissue detection. In this study, the main pathogenic microorganisms of infectious keratopathy,15 herpes simplex virus (HSV), bacteria, fungi, and Acanthamoeba in fresh human lenticules from SMILE were screened for to assess their safety for reuse. On the other hand, although the immunogenicity of the corneal stroma is lower than that of the epithelium and endothelium,16 Zhang et al17 found rejection in 3 eyes of 2 patients (5.66%) during a 1-year follow-up study of 29 patients (53 eyes) who underwent allogeneic lenticule implantation for hyperopia. To investigate the antigen expression in lenticules, in this study, the antigen expression, structure, and function of the lenticules from SMILE were observed after different treatments (fresh, −78 °C anhydrous glycerol preservation, and 0.1% sodium dodecyl sulfate solution [SDS] decellularization) to evaluate their immune risks for reuse and explore an ideal method for reducing antigenicity.

Patients and Methods

Ethics Statement

This study was approved by the institutional review board of Tongren Hospital, and written informed consent was obtained from all participants in accordance with the tenets of the Declaration of Helsinki.

Participants

From a total of 157 participants, 260 eyes were randomly selected from 314 consecutive myopic patients who underwent SMILE at Beijing Tongren Eye Center of Beijing Tongren Hospital from April to September 2019 using an online random number generator. All patients met the following inclusion criteria: no systemic diseases, no history of systemic, local hormone, or immunosuppressive medications, no history of HSV systemic or ocular disease, no ocular diseases except for refraction error, no history of prior ocular surgery or trauma, stable refraction with no more than a 0.50-diopter (D) annual change as documented by an optometrist and the surgeon (FZ) for 2 years prior to surgery, and no use of soft spherical contact lenses within 1 week, toric soft contact lenses and hard contact lenses within 3 weeks, or orthokeratology contact lenses within 3 months preoperatively. Routine preoperative examinations were conducted to rule out contraindications for SMILE, such as corneal ectatic conditions (including keratoconus, keratoconus suspect, pellucid marginal degeneration, or irregular astigmatism detected by corneal topography screening using Pentacam HR; Oculus Optikgeräte GmbH), moderate to severe dry eyes (Schirmer I test < 10 mm), severe Meibomian gland disease, and severe contact lens–induced allergy, and serological tests were conducted to rule out human immunodeficiency virus, syphilis, and hepatitis in all patients. The participants' ages ranged from 18 to 45 years (mean: 27.32 years), and 41 participants (26.11%) were men and 116 (73.89%) were women. The preoperative spherical equivalent to be corrected was −1.75 to −10.00 D (mean: −6.47 D).

SMILE Procedure

All SMILE procedures were performed by the same experienced surgeon (FZ) using a VisuMax femtosecond laser system (Carl Zeiss Meditec AG) with a repetition rate of 500 kHz and a pulse energy of 130 nJ. In all cases, the intended central lenticule thickness was 70 to 151 µm (mean: 113.00 µm), the diameter (optical zone) varied from 6 to 6.7 mm (mean: 6.48 mm), and the cap thickness was between 120 and 130 µm (mean: 126.46 µm). All patients received 0.3% ofloxacin eye drops (Tarivid; Santen, Inc) four times a day for 3 days before surgery. The procedure was performed as described in Wang et al,18 with the diameter of the laser spot set at 4.5 µm and line separations at 2 µm, and four tissue planes were created: the posterior surface of the intrastromal lenticule, the lenticular border, the anterior surface of the intrastromal lenticule, and a single small 90-degree angled side cut incision with a circumferential length of 2 mm at the 12-o'clock position. A thin blunt dissector was used to break the remaining tissue bridges and loosen the lenticule, and then the lenticule was grasped by a pair of forceps and extracted through the incision. No incomplete dissection or torn lenticules were noted after extraction, and the lenticules were separately treated for immediate pathogen detection and antigen detection. The subsequent experimental flow is shown in Figure A (available in the online version of this article).

The experimental flow chart. HSV = herpes simplex virus; SDS = sodium dodecyl sulfate

Figure A.

The experimental flow chart. HSV = herpes simplex virus; SDS = sodium dodecyl sulfate

Pathogen Detection

After extraction, the lenticule of one eye was immediately placed into a sterile tube and stored at −80 °C until processing for HSV detection, and the lenticule of the other eye was cut into three pieces with sterilized micro-scissors and placed in 1 mL of 0.9% sterile sodium chloride solution for the culture of bacteria, fungi, and Acanthamoeba in each specimen.

Detection of HSV by Real-time Fluorescent Quantitative Polymerase Chain Reaction (RTFQ-PCR). According to the instructions of the HSV I & HSV II Real Time PCR Kit (SD-0017-02, Liferiver; ZJ Bio-Tech Co, Ltd), PCR was performed by a real-time quantitative PCR detection system (FQD-96A; BIOER, Co, Ltd). The experiments were performed in triplicate, and the data were analyzed by cycle threshold value: a sample with no cycle threshold was negative, and a sample with a cycle threshold value of less than 38 and a clear “S” type amplification curve was positive.

Bacterial Culture. The specimens were transferred into nutrient broth and cultured in a thermostat at 37 °C and 50% humidity for 48 hours. When turbidity occurred, the nutrient broth was mixed and transferred onto a blood-agar plate for 24 hours. If bacterial colony growth was observed, species identification was done by an automatic microorganism analyzer (VITEK 32; BioMerieux, Inc).

Fungal Culture. The specimens were inoculated on potato glucose slant solid medium and cultured in a thermostat at 28 °C and 50% humidity for 10 days. If filamentous fungi grew, aerial mycelium could be observed, and the species were identified according to the morphological characteristics of the colonies and small cultures. If yeast-like fungi grew, cheese-like smooth colonies could be observed, and the species were identified by the automatic microorganism analyzer (VITEK 32).

Acanthamoeba Culture. The specimens were inoculated on Page's nonnutritive agar medium, added to a dense suspension of living Escherichia coli, and incubated in a thermostat at 28 °C and 50% humidity for 15 days. The presence of amoeba trophozoites and cysts was observed under a light microscope every day, and the parts of interest in more than one region were focused on after searching all areas.

Antigen Detection

The specimens were divided into three groups for treatment in pairs according to age, sex, central lenticular thickness, and diameter. For the fresh group, the lenticules were placed into 0.9% sodium chloride solution or stationary solution or tested directly after extraction during the operation. For the −78 °C anhydrous glycerol preservation group (glycerol group), the lenticules were immediately put into anhydrous glycerol (Hebei Wuyi Yanwei Medical Technology Co, Ltd) and stored at −78 °C for 1 to 6 months after being obtained during the operation, then removed, thawed at room temperature, and rehydrated with 0.9% sodium chloride solution for 30 minutes before experimenting. For the 0.1% SDS decellularization group (SDS group), the lenticules were immersed in 0.1% SDS immediately after removal, followed by three more 0.01 M phosphate-buffered saline (PBS) rinses, each for 24 hours under agitation (300 r.p.m.).

Immunohistochemistry. Sections of eyeball stationary liquid (4% paraformaldehyde; Solarbio) fixed, paraffin-embedded tissues were used. Primary antibodies (HLA Class I A/B/C antibody [ab70328]; 1:100; HLA Class II DR antibody [ab20181]; 1:100; Abcam) were applied at 4 °C overnight, and secondary antibodies marked by horseradish peroxidase (Goat Anti-Mouse IgG H & L [ab205719]; 1:5000; Abcam) were applied at room temperature for 50 minutes. Thereafter, sections were treated with 3,3'-diaminobenzidine (Dakocytomation, Inc) for 5 minutes, counterstained with hematoxylin for 3 minutes, and examined by light microscopy. A positive reaction was indicated by a brown color, and the nuclei stained with hematoxylin were blue. Sections for which primary antibodies were omitted were used as negative controls. Sections of fresh thymus tissue served as positive controls.19

Western Blot. The three groups were matched quantitatively according to the diameter (6.5 mm) and central thickness (Table A, available in the online version of this article) of the lenticules. Only a small amount of protein was present in each lenticule (approximately 30 µg); thus, lenticules from six different patients were mixed to create a sample for protein extraction (approximately 190 µg/sample; concentration, 1.21 µg/µL). After protein quantification by the BCA Protein Assay Kit (CW0014S; ComWin Biotech Co, Ltd), equal amounts of proteins (25 µg/sample) were loaded and separated by 10% SDS-PAGE gel and then transferred onto a PVDF membrane. The membrane was incubated with primary antibodies (HLA Class I ABC antibody [ab70328]; 1:500; HLA Class II DR antibody [ab20181]; 1:500; Abcam) at 4 °C overnight and secondary antibody (HRP-labeled goat anti-mouse IgG [A0216]; 1:1000, Beyotime) at room temperature for 1 hour. After exposure and imaging, semiquantitative analysis was performed by measuring band density using ImageJ software (V1.8.0; National Institutes of Health).

Central Thickness of the Lenticules Used for Western Blot

Table A:

Central Thickness of the Lenticules Used for Western Blot

Transmission Electron Microscopy. The lenticules were fixed in 2.5% glutaraldehyde (Sigma) for 2 hours and postfixed in 1% osmium tetroxide for 2 hours. After gradient alcohol dehydration, propylene oxide immersion and epoxy resin embedding, transverse sections were obtained and double-stained with uranyl acetate and lead citrate and examined under a transmission electron microscope (HT7700; Hitachi Ltd).

Transmittance. The transmittance of the central lenticules was determined by an Evolution 300 UV-Vis spectrophotometer (Thermo Fisher Scientific) at a wavelength of 380 to 780 nm. The lenticule was adhered to the inner wall of a colorimetric dish and inserted into the spectrophotometer chamber for measurement. Data were collected at 1-nm wavelength increments. Experiments were performed in triplicate, and the mean transmittance was calculated.

Nanoindentation. The mechanical properties of the lenticules were measured by a cell nanoindenter test system (Chiaro; Optics11). A spherical probe (P190029; Optics11) with 26.5-µm tip radius and 0.50 N/m stiffness was selected, and the loading speed was 2 µm/s. The lenticule was fixed at the bottom of a Petri dish, a 3 × 3 matrix indentation point was made in the central surface of each specimen with a depth of 5 µm and an interval of 100 µm, and the load-displacement curves and Young's modulus were obtained.

Statistical Analysis

SPSS software version 24.0 (SPSS, Inc) was used for all statistical analyses, and measurements are presented as the mean ± standard deviation. Considering the limited sample size, this was an exploratory study. Data distributions were not normal, and comparisons among groups were performed using nonparametric analyses of variance (ANOVA). For each experiment, one primary outcome variable was established. For pairwise comparisons among groups, a Bonferroni test was used for correction, and a P value of less than .05 after correction (equivalent to P < .0167 before correction because three groups were involved in this study; hereafter, we used ‘P’ as ‘corrected P’ unless otherwise explained) was considered statistically significant.

Results

Pathogen Detection

The RTFQ-PCR detection of HSV-1 and HSV-2 and the cultures of bacteria, fungi, and Acanthamoeba were all negative in the fresh corneal stromal lenticules of 128 eyes of 64 patients.

Antigen Detection

Immunohistochemistry. Both HLA-A/B/C and HLA-DR were readily detected on the cell surface of the corneal stromal lenticules in the fresh group, but no obvious positive reactions were observed in the glycerol group or the SDS group (Figure B, available in the online version of this article).

Immunohistochemistry of corneal stromal lenticules. In the fresh group (A and D), positive reactions (brown) for HLA-A/B/C and HLA-DR were observed on the cell surface. In the −78 °C anhydrous glycerol preservation group (B and E) and 0.1% sodium dodecyl sulfate (SDS) group (C and F), no obvious positive reactions were found (bar = 50 µm).

Figure B.

Immunohistochemistry of corneal stromal lenticules. In the fresh group (A and D), positive reactions (brown) for HLA-A/B/C and HLA-DR were observed on the cell surface. In the −78 °C anhydrous glycerol preservation group (B and E) and 0.1% sodium dodecyl sulfate (SDS) group (C and F), no obvious positive reactions were found (bar = 50 µm).

Western Blot. The expression bands of HLA-A/B/C and HLA-DR appeared at molecular weights of 40 and 35 kDa, respectively, and were obvious in the fresh group and very weak in the glycerol group and the SDS group (Figure C, available in the online version of this article). The gray value of HLA-A/B/C in the fresh group was significantly higher than those in the glycerol group and the SDS group (P = .001 and P < .001, respectively), but there was no significant difference between the glycerol group and the SDS group (P = 1.00) (Table 1). Similarly, the gray value of HLA-DR in the fresh group was significantly higher than that in any other group (both P < .001), whereas no significant difference was observed between the glycerol group and the SDS group (P = 1.00) (Table 1). GAPDH, as an internal reference, was not expressed in either the glycerol group or the SDS group because the cell was destroyed (Figure C).

HLA expression of corneal stromal lenticules by Western blot (F: fresh group; G: −78 °C anhydrous glycerol preservation group; S: 0.1% sodium dodecyl sulfate (SDS) decellularization group). The expression bands of HLA-A/B/C and HLA-DR were obvious in the fresh group and very weak in the −78 °C anhydrous glycerol preservation group and SDS group.

Figure C.

HLA expression of corneal stromal lenticules by Western blot (F: fresh group; G: −78 °C anhydrous glycerol preservation group; S: 0.1% sodium dodecyl sulfate (SDS) decellularization group). The expression bands of HLA-A/B/C and HLA-DR were obvious in the fresh group and very weak in the −78 °C anhydrous glycerol preservation group and SDS group.

Gray Band Density Value of HLA Expression in Corneal Stromal Lenticules by Western Blot

Table 1:

Gray Band Density Value of HLA Expression in Corneal Stromal Lenticules by Western Blot

Transmission Electron Microscopy. In the fresh group, the collagen fibers of the lenticules were regularly arranged, and the keratocytes were intact with a large oblong nucleus and little cytoplasm (Figures 1A, 1D, and 1G). In the glycerol group, the fibers were also regularly arranged, but the integrity of the keratocytes was destroyed: the cytomembrane and karyotheca (nuclear membrane, which is located at the junction of the nucleus and cytoplasm) were broken and disintegrated into fragments, and the intracytoplasmic mitochondria, endoplasmic reticulum, and Golgi complex were swollen and disintegrated (Figures 1B, 1E, and 1H). In the SDS group, the fiber arrangement was disordered, and there were many lacustrine open lacunae formed by fiber dissolution and fracture. Cells and cellular debris were rarely observed, and empty original cell spaces were left (Figures 1C, 1F, and 1I).

Ultrastructure of corneal stromal lenticules (A–C, bar = 2 µm; D–I, bar = 500 nm). The collagen fibers were regularly arranged, and the keratocytes with a large oblong nucleus and little cytoplasm were intact in the fresh group (A, D, and G). The fibers were regularly arranged in the −78 °C anhydrous glycerol preservation group, but the integrity of the keratocytes was destroyed (B, E, and H). The fiber arrangement was disordered and dissolved, and no cell structure was observed in the 0.1% sodium dodecyl sulfate (SDS) group (C, F, and I).

Figure 1.

Ultrastructure of corneal stromal lenticules (A–C, bar = 2 µm; D–I, bar = 500 nm). The collagen fibers were regularly arranged, and the keratocytes with a large oblong nucleus and little cytoplasm were intact in the fresh group (A, D, and G). The fibers were regularly arranged in the −78 °C anhydrous glycerol preservation group, but the integrity of the keratocytes was destroyed (B, E, and H). The fiber arrangement was disordered and dissolved, and no cell structure was observed in the 0.1% sodium dodecyl sulfate (SDS) group (C, F, and I).

Transmittance. The transparency of lenticules in the glycerol group (Figure 2B) was close to that in the fresh group (Figure 2A), whereas edema and turbidity were obvious in the SDS group (Figure 2C). In the 380- to 780-nm visible spectrum, the transmittance of central lenticules in the fresh group was the highest, that of the glycerol group was slightly lower, and that of the SDS group was the lowest (Figure 2D). The average transmittance of the three groups was 89.32 ± 2.82%, 87.94 ± 3.11%, and 82.09 ± 5.69%, respectively. Both the fresh group and the glycerol group showed significant differences compared with the SDS group (both P < .001), whereas no significant difference was observed between the former two groups (P = .45).

Transparency and transmittance of corneal stromal lenticules. The lenticules of the (A) fresh and (B) −78 °C anhydrous glycerol preservation group were transparent, and the lenticules in the (C) sodium dodecyl sulfate (SDS) group were turbid and edematous. The transmittance in the 380- to 780-nm spectrum of central lenticules was highest in the fresh group, slightly lower in the glycerol group, and lowest in the (D) 0.1% SDS group.

Figure 2.

Transparency and transmittance of corneal stromal lenticules. The lenticules of the (A) fresh and (B) −78 °C anhydrous glycerol preservation group were transparent, and the lenticules in the (C) sodium dodecyl sulfate (SDS) group were turbid and edematous. The transmittance in the 380- to 780-nm spectrum of central lenticules was highest in the fresh group, slightly lower in the glycerol group, and lowest in the (D) 0.1% SDS group.

Nanoindentation. As shown in Figure 3, the load-displacement curve of the glycerol group is consistent with that of the fresh group, whereas that of the SDS group is low and flat. Young's modulus of the lenticules was 49.21 ± 0.96 kPa in the fresh group, 50.6 ± 2.0 kPa in the glycerol group, and 24.26 ± 8.02 kPa in the SDS group. There was no significant difference between the fresh group and the glycerol group (P = 1.00), but there were significant differences between the SDS group and any other group (P < .001).

Indentation curves of corneal stromal lenticules. With increasing loading force, the indentation depth increased gradually. Under the same force, the 0.1% sodium dodecyl sulfate (SDS) group was pressed into the deepest position, whereas the other two groups were almost the same.

Figure 3.

Indentation curves of corneal stromal lenticules. With increasing loading force, the indentation depth increased gradually. Under the same force, the 0.1% sodium dodecyl sulfate (SDS) group was pressed into the deepest position, whereas the other two groups were almost the same.

Discussion

The reuse of corneal stromal lenticules from SMILE will undoubtedly supplement the shortage of materials for keratoplasty. As a special method of corneal transplantation, the lenticules are sometimes used for healthy corneas,2,3 so the complications of keratoplasty, infection, and rejection should be given more attention.

Although donor-to-host transmission of infectious agents is rare in corneal transplantation, the consequences are catastrophic. A systematic review from January 2000 to November 2016 showed that the positive cultures of microorganisms from donors' corneoscleral rims matched the post-keratoplasty keratitis or endophthalmitis of hosts. Of the 7,870 grafts, 954 had a positive rim culture (12.1%), with 12 patients developing keratitis or endophthalmitis (1.3%). Of the 12 infections, 9 were fungal infections and 3 were bacterial infections.20 Therefore, we studied the main pathogenic microorganisms of infectious keratopathy in the fresh lenticules from SMILE.

In this study, HSV-1 DNA and HSV-2 DNA were not detected in 64 fresh lenticules. Herpes simplex keratitis is the most common type of infectious keratitis, and mostly caused by HSV-1.15 In addition to the trigeminal ganglion, HSV may be latent in corneal stromal cells for a long time without any clinical manifestations after infection. Openshaw et al21 observed HSV-1 DNA in patients without known HSV eye disease in the eye bank by PCR and found HSV-1 in 10 of 24 eye bank corneas, of which 8 were from the 4-mm wide corneal rim and 2 were from the 8-mm diameter central cornea. HSV-1 DNA was detected in 2 donor corneoscleral rims, and HSV-2 DNA was not detected in 273 donor corneoscleral rims by PCR in the report of Remeijer et al.22 The inconsistency between our study and the prior studies may be due to three main reasons. First, the participants in the two studies were from the eye bank, and the history of HSV may not be precise, but the participants of this study were healthy young people and definitely had no HSV history. Second, the samples were harvested from the central 6- to 6.5-mm diameter anterior corneal stroma in our study, whereas they were mainly obtained from the corneoscleral rims in other studies. HSV may be latent in the peripheral cornea.23 Third, due to the pilot nature of this study, a larger sample size would be needed to validate the assessment outcomes.

Simultaneously, no bacteria, fungi, or Acanthamoebae were observed in 64 fresh lenticules of this study. The cornea is located on the ocular surface and vulnerable to contamination by microorganisms in the conjunctival sac. In previous studies, the positive rate of conjunctival sac bacterial culture in a Chinese population with myopia ranged from 21%24 to 29%,25 and fungi culture in a healthy Chinese population ranged from 6.72%26 to 12.92%.27 Few studies have been conducted on the culture of Acanthamoeba in the conjunctival sac of normal humans. The positive rate of bacterial culture of the blades used in laser in situ keratomileusis (LASIK) was 5%, as reported by Khan et al28 and Pang et al.29 Therefore, unlike the bacteria-carrying state of corneal stromal layers of LASIK, the risk of microbial contamination of the conjunctival sac during SMILE is low because of the lack of microkeratome blade, corneal flap, and exposure to the matrix bed.

Immune rejection is the main cause of corneal transplantation failure.30 The human leukocyte antigen system (HLA) is the main antigen that causes the host immune response in human allografts and can be divided into class I (HLA-I) and class II (HLA-II) antigens. Although the distribution of different HLA types in different parts of the cornea is controversial, several studies have confirmed the presence of both HLA-A/B/C (class I) and HLA-DR (class II) antigens in normal human corneal stromal cells,31 rejected allografts,32 and pathological corneas,33 and determined that the frequencies of the HLA alleles in cornea donors were increased in recipients who had developed graft failure.34 Therefore, grafts lacking cell membrane surface antigens are more suitable for lenticule reim-plantation, which does not require cell activity. Thus, we compared two different methods of removing antigens from corneal stromal lenticules in this study: −78 °C anhydrous glycerol cryopreservation and 0.1% SDS decellularization.

Our immunohistochemical and Western blot results showed that both HLA-I and HLA-II antigens were expressed in fresh human lenticules and were clearly decreased after preservation at −78 °C in anhydrous glycerol or decellularization in 0.1% SDS (Figures BC, Table 1). Liang et al35 preserved the lenticules from SMILE at 4 °C using three methods and found that destruction of the cell structure (cell membrane separation and organelle lysis) in the glycerol group occurred earlier and was more severe than that in the silica gel desiccant group and the medium-term corneal preservation solution group. As a dehydrating agent, glycerol is a colorless, odorless, and viscous liquid that can maintain the corneal structure and has antimicrobial and antiprotease properties, so it is often used for the long-term storage of corneas for lamellar keratoplasty.36,37 Li et al19 reported that HLA-A/B/C and HLA-DR were decreased, and keratocytes were all destroyed and collapsed and exhibited loss of intra-cellular organelles and dissolution of cytoplasm in all glycerol-preserved corneas at different temperatures (room temperature, −20 °C and −78 °C) for 3 months, but the −78 °C cornea was the most transparent and pliable, with the collagen fibrils regularly arranged and maintained in parallel, whereas blank regions formed by missing fibrils were found in corneas at room temperature and at −20 °C. Consistently, we observed that the cell membranes, organelles, and nuclei were extensively ruptured (Figure 1) and inferred that the antigens were reduced by destroying the cells in the −78 °C anhydrous glycerol group. In addition, cellular biological incompatibility may be managed by the decellularization of allogenous tissues prior to transplantation.38 The characteristics of decellularized tissue are strongly affected by the decellularization protocol. Yam et al39 combined 1.5 M hypertonic NaCl, 0.1% Triton X-100, 0.1% SDS and enzymatic decellularization into 10 different protocols to decellularize the lenticules from SMILE, and the results showed that 0.1% SDS was the most effective method of decellularization, and the structure and optical properties of the stroma remained good. Similarly, we also observed that 0.1% SDS could remove the cells completely and concluded that the antigen load or antigens were reduced by removing cells in the 0.1% SDS group.

In contrast to previous reports,19,39 as we found that the antigen was reduced equally in the glycerol group and the SDS group (P = 1.00) (Table 1), we further found that other essential characteristics of corneal grafts, the ultrastructure of collagen fibers, and the optical and mechanical properties of the lenticules were all significantly damaged in the SDS group, whereas these characteristics in the glycerol group were consistent with those of the fresh group (Figures 13). First, the collagen fibers were complete and regularly arranged in the glycerol group, but obvious disorder occurred in the SDS group, and many lacustrine open lacunae were formed due to fiber dissolution, which will affect the corneal structure and light transmittance after transplantation. Second, transmittance is a physical quantity indicating the efficiency of light transmission; the transmittance curves from 380 to 780 nm in the visible spectrum were obviously lower, and edema and turbidity could be observed by the naked eyes in the SDS group (Figure 2) but not in the glycerol group, confirming that the lenticules in the glycerol group were more transparent compared to those in the SDS group. Finally, using the nanoindentation technique, load and displacement were continuously monitored during the loading sequence, and the indentation curve and Young's modulus (the ratio of stress to strain of a given substance) were tested. The results showed that the indentation curve and Young's modulus in the SDS group were significantly lower than those in the glycerol group under the same load, proving that the displacement in the SDS group was larger and easier to deform with the same force and thus confirming that the mechanical strength of the lenticules in the glycerol group were better than those in the SDS group.

Thus, we can infer that −78 °C anhydrous glycerol preservation can not only achieve the effect of reducing antigens by 0.1% SDS, but can also avoid its damage to the normal collagen fiber structure and the optical and mechanical properties of the corneal stromal lenticules.

There are rejection risks but low infectious risks in the reuse of fresh human corneal stromal lenticules from SMILE. Anhydrous glycerol preservation at −78 °C is an ideal method for reducing antigen expression in the lenticules and obtaining high-quality grafts.

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Gray Band Density Value of HLA Expression in Corneal Stromal Lenticules by Western Blot

GroupHLA-A/B/CHLA-DR
Fresh31,114.25 ± 8,524.10a37,374.00 ± 9,212.60a
−78 °C anhydrous glycerol preservation7,917.75 ± 3,375.60b6,359.50 ± 3,568.40b
0.1% SDS decellularization5,566.50 ± 3,396.40b3,316.50 ± 1,576.70b
Pc.018.018

Central Thickness of the Lenticules Used for Western Blot

Paired SamplesGroupingCentral Thickness of Single Lenticules (µm)Total
Pair 1Fresh group124139150136105122776
−78 °C anhydrous glycerol preservation group130139144142104117776
0.1% SDS decellularization group150149128126101122776
Pair 2Fresh group13077101149111119687
−78 °C anhydrous glycerol preservation group13688112139100112687
0.1% SDS decellularization group11693116120128114687
Pair 3Fresh group1371261051149198671
−78 °C anhydrous glycerol preservation group1391219911810094671
0.1% SDS decellularization group1361351141059784671
Pair 4Fresh group1231051178914175650
−78 °C anhydrous glycerol preservation group1231041278014076650
0.1% SDS decellularization group149118112999379650
Authors

From Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Laboratory, Beijing, China (YS, YL, ZW, XS, FZ); and Heping Hospital Affiliated to Changzhi Medical College, Changzhi, China (YS).

Supported by the 215 High Level Talent Fund of the Beijing Health Government (No. 2013-2-023) and the Beijing Economic and Technological Development Zone New Innovation Project Leading Talent Training Fund (No. 20180509).

The authors have no financial proprietary interest in the materials presented herein.

The author thank their colleagues from the Refractive Surgery Center, Beijing Tongren Eye Center of Beijing Tongren Hospital for assistance with collecting corneal stromal lenticules during SMILE.

AUTHOR CONTRIBUTIONS

Study concept and design (YS, FZ); data collection (YS, YL, ZW); analysis and interpretation of data (YS, XS, FZ); writing the manuscript (YS); critical revision of the manuscript (YL, ZW, XS, FZ); administrative, technical, or material support (FZ); supervision (FZ)

Correspondence: Fengju Zhang, MD, PhD, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Laboratory, No. 1 Dongjiaomin Lane, Dongcheng District, Beijing 100730, China. Email: zhangfj126@126.com

Received: February 20, 2020
Accepted: October 30, 2020

10.3928/1081597X-20201030-03

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