1、Introduction

        Cornea is the outermost layer of the eye. Given its special location,corneal epithelium is vulnerable to various damages, such as mechanical injuries, chemical burns, thermal injuries and so on (Baradaran-Rafii

et al., 2017; Leong and Tong, 2015). Corneal wound healing is a highly regulated process and cornea can achieve rapid re-epithelialization after an injury. However, disrupted normal corneal healing process can lead to persistent corneal epithelial defects (PED), which may result in irreversible blindness of patients (Jackson et al., 2020; Wirostko et al.,2015). Currently, the standard managements of PED, such as lubrication, bandage soft contact lenses, debridement or tarsorrhaphy, usually aim to cover and protect cornea, thus to permit the proliferation of limbal stem cells and migration of mature corneal epithelial cells to heal defects (Katzman and Jeng, 2014; Liu and Kao, 2015). Nevertheless,when it comes to refractory cases of PED that do not respond to the standard therapies, especially where limbal stem cells are deficient, limbal stem cell transplants and even corneal transplants may be necessary (Vaidyanathan et al., 2019; Wirostko et al., 2015). Allo- or auto-limbal grafts are usually transplanted to repopulate stem cells for the patients with limbal stem cell deficiency (LSCD) (Le et al., 2018; Yin and Jurkunas, 2018). However, autografts may lead to LSCD of the healthy eye, and the allografts require long-term systemic immunosuppression (Ang et al., 2013). Thus, an alternative tissue transplantation is indispensable to deal with this problem.

       Oral mucosa is composed of a stratified nonkeratinized epithelium,making it feasible to reconstruct epithelial tissues within the body(Bauer et al., 2019). The oral mucosa is recognized as an available  source for ocular surface reconstruction owing to its abundance of stems cells within the epithelial base as well as its similar morphological and cytochemical characteristics to corneal epithelium (Ghareeb et al., 2020; Oliva et al., 2020); therefore, it can be considered as an alternative of limbal tissues to treat injured ocular surfaces. Nishida et al. have studied the use of tissue-engineered oral mucosal epithelial-cell sheets fabricated in vitro for the reconstruction of corneal surface, finding that sheets of cultured autologous cells could achieve complete re-epithelialization of the corneal surface without causing any complications (Nishida et al., 2004). Over the next two decades or so, a great deal of researches has been done on the culture conditions of in vitro oral mucosal epithelial-cell sheets to improve the therapeutic effects, indicating oral mucosal epithelial cells as appropriate resources for the reconstruction of corneal surface (Cabral et al., 2020; Utheim et al., 2016; Yazdanpanah et al., 2019). However, the culture of cell sheets in vitro has certain drawbacks such as expensive cost, legal limitations, long culture period and the need of careful continuous monitoring for producing epithelial cell sheets. Most importantly, although the production of cell sheets requires a complex culture system, the in vivo microenvironment still cannot be well simulated in vitro. Therefore, it’s necessary to find a simple and effective way of oral mucosal epithelium (OME) transplantation for the clinical application. Inamochi et al. have reported a simple technique for transplantation of uncultured OME, which is effective to promote corneal epithelialization in a rabbit model of total LSCD (Inamochi et al., 2019). With the effect of OME transplantation on promoting epithelialization preliminary proven, the internal mechanisms through which OME plays a positive role in the repair of injured corneas still needs to be revealed.

        Our study is based on the exploration of the suitable treatment for refractory PED, with the purpose to explore the differentiation potential of oral mucosal stem cells in addition to their key role in corneal re epithelialization after direct OME transplantation, and to further figure out the interactions between stem cells and corneal surface microenvironment. Thus, this study is designed to investigate whether the direct in vivo transplantation of autologous OME can promote the wound healing of alkali burned rat cornea, and further explore the mechanism of re-epithelization on the de-epithelialized cornea by OME in vitro, so as to provide a simple and effective method for the clinical treatment of refractory PED.

2、Materials and methods

2.1 Study design and animals

       Healthy adult New Zealand white rabbits of both genders weighing approximately 2–2.5 kg and eight-week-old female Sprague–Dawley rats weighing 150–200 g each were used in this study. All animal experimental procedures were approved by the Animal Care and Experimental Committee of the Shanghai Ninth People’s Hospital affiliated to Shanghai Jiao Tong University, School of Medicine. Furthermore, all procedures were carried out according to the guidelines of the Chinese Animal Administration and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

2.2 Rat corneal alkali-burn model

       Corneal alkali-burn model was established as previously described (Chen et al., 2016; Liu et al., 2011). Rats were anesthetized with 40 mg/kg pentobarbital intraperitoneally and received topically administration of tetracaine eye drops. A round Whatman III filter paper (3.5 mm in diameter) presoaked with 1.0 N NaOH (Sigma–Aldrich, St. Louis, MO, USA) for 3 s was placed on the central cornea of the right eye for 30 s, followed by flushing the wound surface with 0.9% physiological saline for 1 min. Eighteen rats were randomly assigned into three groups (six rats each) as follows: Normal group, without alkali burn; Alkali Burn  group, alkali burn without treatment; OME Transplantation group, alkali burn with autologous OME transplantation.

2.3 Autologous OME transplantation

       To obtain autologous OME, 3-by-3mm oral mucosa tissue from each rat in the OME Transplantation group was harvested and then digested in 10 mg/ml dispase II (Sigma-Aldrich) at 37 ◦C for 30 min to separate OME. Afterwards, the OME sheet was adhered to the center of cornea with the epithelial surface upward using fibrin sealant (Fig. 1A). Combined steroid and antibiotic eye drops (0.5% moxifloxacin and 0.1% dexamethasone) (Vigadexa, Alcon Laboratories, Inc., Fort Worth, Tex,USA) were applied to rat eyes. And then the eyelids were sutured with two vertical stitches by 6-0 nylon threads (Brown, 2007). As the negative control, rats in Alkali Burn group were also treated with the fibrin glue, eye drops and sutures.

2.4 Clinical examinations

       Clinical examinations were conducted for all three groups of rats on day 4 and day 8 after transplantation. Optical coherence tomography (OCT; Heidelberg Engineering, Germany) was used to measure the central corneal thickness, which could indicate the edema degree of the cornea. Corneal opacity was recorded by the slit-lamp microscope (SLD7, Topcon Inc., Tokyo, Japan) and scored according to a system of scaling from 0 to 4: 0 = no opacity, completely clear cornea; 1 = slightly hazy, iris and pupils visible; 2 = slightly opaque, iris and pupils still detectable; 3 = severely opaque, iris and pupils hardly visible; and 4 = completely opaque, with no view of iris and pupils (Yoeruek et al.,2008). Afterwards, the ocular surface was stained with 2% fluorescein sodium (Sigma-Aldrich), and the corneal epithelial defects were visualized and photographed under cobalt blue light by the slit-lamp microscope. In accordance to the National Eye Institute/Industry (NEI) grading scale, the scores of corneal epithelial defects were evaluated and recorded. The investigators were blinded to group allocation during data collection and analysis.

2.5 Preparation of human oral mucosal biopsies

       This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee for Biomedical Research of the Institutional Review Board of the Ninth People’s Hospital affiliated to Shanghai Jiao Tong University, School of Medicine. Oral mucosal biopsies were collected from the patients with cleft lip and palate who underwent operations at the Department of Plastic and Reconstructive Surgery. All patients provided written informed consents before surgeries.

2.6 Construction of in vitro organ culture model

        The rabbits were euthanized and whole intact rabbit eyes were excised, followed by scrape of the corneal epithelia totally and ringcutting of the cornea at 1 mm inside the limbus of the cornea. The obtained human buccal oral mucosa biopsy specimens were cut into small pieces for around 4 mm2 each and digested with 10 mg/ml Dispase II at 37 ◦C for 60 min. Subsequently, the epithelial sheets were peeled off. Agarose gel made from agarose of low gelling temperature (SigmaAldrich), suitable for cell culture, was used to support the cultured cornea. The procedures were as follows: a 10 mm-diameter iron ring mould was put in the center of cell culture dish. Subsequently, agarose powder dissolved in the distilled deionized water (10 mg/ml) was heated to melt and added into the mould forming gels by natural cooling. After removing the mould, the denuded rabbit cornea was put on the approximately cylindrical agarose gel. And then the human epithelial sheet was sticked to the cornea stroma by fibrin sealant. Afterwards, DMEM/F-12 medium (Invitrogen, Carlsbad, CA, USA) supplemented  with 5% fetal calf serum (Invitrogen), 5 μg/ml of insulin and transferrin (Life Technologies, Carlsbad, CA), 20 nM hydrocortisone (Wako, Osaka, Japan), 10 ng/ml epidermal growth factor (Life Technologies); 100 units/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies) was added into the dish for organ culture (Grant, 2020; Sugiyama et al., 2014). The culture medium was changed every 2 days and the culture period was two weeks.

2.7 Histological examination

       The eyeballs enucleated from the ethically sacrificed rats on day 8, as well as the rabbit corneas organ cultured for one or two weeks, were paraffin-embedded and cut into 6-μm-thick sections. Afterwards, the sections were stained with hematoxylin & eosin (H&E) or Masson’s trichrome (Sigma-Aldrich) according to the manufacturer’s instructions.

       For immunofluorescence staining, the rat eyeballs and the organ cultured corneal tissues were embedded in Tissue-Tek Optimal Cutting Temperature compound (Sakura Seiki, Tokyo, Japan) and then cut into 8-μm sections. The sections were fixed with 4% paraformaldehyde for 20 min, followed by 0.3% Triton X-100 (Sigma-Aldrich) treating for 15 min and 5% bovine serum albumin (Sigma-Aldrich) blocking for 1 h at room temperature. Then the tissue sections were incubated with  primary antibodies of Lamin A + Lamin C (Abcam, San Francisco, CA, USA), Keratin13 (CK13, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Keratin 3 (CK3, Abcam), Keratin 12 (CK12, Abcam), δNp63 (Abcam), E-cadherin (Abcam), ZO-1 (Invitrogen), or α-smooth muscle actin (α-SMA, Abcam) overnight at 4 ◦C, followed by Alexa Fluorconjugated secondary antibodies (1:400, Thermo Fisher Scientific, Waltham, Massachusetts) for 1 h at room temperature. Nuclei were stained with DAPI (Invitrogen). The stained slides were viewed and recorded by a fluorescence microscope (Nikon Eclipse 80i; Nikon Instruments, Tokyo, Japan).

2.8 Statistical analysis

       Data were presented as mean ± standard error of the mean (SEM).The statistical analysis of clinical examination results was performed with two-way analysis of variance test (ANOVA) followed by Bonferroni post hoc test in GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA). And the statistical analysis of corneal epithelial thickness, corneal stromal thickness and immunofluorescence intensity was conducted with one-way ANOVA followed by post hoc analysis Tukey test. A p value of <0.05 indicated statistical significance.

3、Results

3.1 Autologous OME transplantation promoted the recovery of alkaliinjured rat corneas

Fig. 1A revealed the process of autologous OME transplantation after  alkali burn injury for OME Transplantation group. Clinical examinations were performed on day 4 and day 8 after injury. The sodium fluorescein staining results showed that there were larger corneal epithelial defects (represented by the green area) in the rat of Alkali Burn group than those of the OME Transplantation group (Fig. 1B). The silt-lamp microscope examinations revealed that the corneal transparency of the OME Transplantation group was significantly better than that of the Alkali Burn group on both day 4 and day 8 (Fig. 1C). Moreover, consistent with the previous examinations, the corneas of the Alkali Burn group were significantly thicker than those of the OME Transplantation group on both day 4 and day 8 measured by OCT, indicating that OME transplantation alleviated corneal edema after alkali injury (Fig. 1D). Thus, autologous OME transplantation had a good healing effect on corneal alkali burn injury, as it was able to promote the recovery of corneal epithelial integrity, corneal transparency and corneal thickness by improving re-epithelialization and alleviating corneal edema.

3.2 Autologous OME transplantation was beneficial to the recovery of corneal histological morphology and function

       To detect the histological differences of corneal morphology in each group, H&E staining and Masson’s trichrome were performed. As shown in Fig. 2A and Fig. 2B, the cornea of Alkali Burn group had relatively fewer epithelial cell layers comparing to the normal cornea, and the stroma was loose with collagen fibers arranged irregularly. In the OME Transplantation group, the corneal epithelial morphology was much better, with much resemblance to normal corneal epithelium, and the stromal collagen fibers were more compact and orderly arranged.Additionally, the thickness of the corneal epithelium and corneal stroma in each group was measured according to the staining results of histological sections, which statistically verified the corneal injury caused by alkali burn and the admirable healing effect of autologous OME transplantation (Fig. 2C and D).

       Next, immunofluorescence staining was performed in order to further detect the recovery of corneal morphology and function in each group. The staining results of corneal epithelial keratin markers CK3 and CK12 showed that the corneal epithelium in the OME Transplantation group, close to the normal cornea, was intact and well-shaped (Fig. 3A, B, E, F). In contrast, the corneal epithelium in the Alkali Burn group was relatively thin with irregular arrangement of epithelial cells (Fig. 3A, B, E, F). The better expression of E-cadherin in the OME Transplantation group indicated the satisfactory barrier function of the epithelium (Fig. 3C, G). In addition, the degree of corneal stromal fibrosis in the OME Transplantation group was less than that in the Alkali Burn group revealed by α-SMA staining, which further proved that OME transplantation could alleviate the scar formation and was beneficial to the damage repair of corneal alkali burn (Fig. 3D, H).

3.3 The organ cultured cornea achieved re-epithelialization with the transplanted OME

       An organ culture model was established in vitro to further investigate the healing mechanisms of OME transplantation on the epithelial defective cornea (Fig. 4A). H&E staining showed that OME could be completely separate from the connective tissue through digestion by dispase II and the corneal epithelium was thoroughly scraped in order to prevent the proliferation of the residual corneal epithelial cells to affect the culture results (Fig. 4B, a, b). After culturing, the corneal surface could re-form smooth epithelium after one-week culture, although the epithelium was relatively thin with 2 or 3 epithelial layers (Fig. 4c, e).

       When the culture duration reached two weeks, it could be noticeable that the corneal surface had formed stratified and smooth epithelium with cells tightly connected (Fig. 4d, f). The morphology of the epithelium was close to that of normal corneal epithelium, and the magnified image clearly showed the re-formed epithelium was 5–7 layers with regular and compact arrangement (Fig. 4d, f). In addition, the simple deepithelialized corneas without the adhesion of OME were also organ cultured for 2 weeks as the control culture group, and the results showed that no regenerative epithelium formed on the denuded corneal stroma (Supplementary Fig. 1).

       To verify that the newly formed epithelium was derived from transplanted human OME, the section of the organ cultured cornea was stained with anti-Lamin A + C antibody which merely recognized the nuclear envelope protein of human cells. The staining results showed that Lamin A + C was expressed in the epithelium of organ cultured cornea, as in the epithelium of human oral mucosa but not of rabbit cornea, which indicated that the newly formed epithelial cells were derived from human oral mucosa (Fig. 4D). Immunofluorescence staining was further performed to detect the function and phenotype of the epithelium formed by organ culture. Staining results of the common epithelial barrier function markers E-cadherin and ZO-1 indicated that the re-formed epithelium had good intercellular junction function (Fig. 4E and F).

3.4 The epithelium formed by organ culture maintained stemness and transformed to corneal epithelial phenotype

        Stemness was an important characteristic representing proliferation and differentiation potentials, and the isoforms δNp63α and δNp63β of p63 were reported to be specific markers for oral mucosal stem cells (Tao et al., 2009). The immunofluorescence staining showed that the epithelium of the central cornea didn’t express δNp63, but the base of the oral mucosal epithelium existed abundant stem cells, which implied the proliferation potential of the basal cells of the OME. Moreover, on the surface of the organ cultured cornea, we observed a layer of δNp63 positive cells tightly adhered to the corneal stroma, which may be considered as the source of seed cells for epithelial formation and indicated a good proliferative potential of the epithelium (Fig. 5A).

        In addition, we counterstained the commonly used differentiationassociated marker CK13 of non-keratinized oral mucosal epithelial cells and the specific keratin marker CK12 of corneal epithelial cells for different tissue epitheliums. Interestingly, the oral epithelial cells and corneal epithelial cells merely expressed CK13 and CK12 respectively, but the newly-formed epithelium by organ culture showed co-positive of these two markers (Fig. 5B). The oral epithelial marker CK13 was mainly expressed in the upper 2–3 layers of the epithelium, while corneal epithelial marker CK12 was expressed in the whole epithelium. The staining results might indicate the transformation process of oral epithelial cells into corneal epithelial-like cells, which firstly take place in the cells that were in direct contact with the corneal stroma.

4、Discussion

      In clinical application, oral mucosa used for ocular surface reconstruction usually needed to undergo the in vitro single cell culture or explant culture process before performing cultivated oral mucosal epithelial transplantation (COMET). For instance, Sotozono et al. proved that COMET enabled complete epithelialization of PED and stabilization of the ocular surface in patients with severe ocular disease (Sotozono et al., 2014). To avoid being limited by in vitro culture conditions, recently some clinical cases reported the simple oral mucosal epithelial transplantation (SOMET) to promote the corneal epithelization in the patients with complete LSCD, which achieved good postoperative results (Kara and Dogan, 2021; Ngowyutagon et al., 2021). In these ocular surface reconstruction surgeries, the oral mucosal grafts were trimmed manually to remove excess submucosal fat in order to obtain the thinnest possible epithelial tissue. However, no matter how thin the ocular mucosa was cut during the surgery, there would still be residual connective tissue, which may somewhat limit the migration of stem cells to the corneal surface. In our research, we used dispase II to separate the complete epithelium from the submucosal connective tissue, which was conductive to the adhesion of stem cells with corneal stroma and the achievement of rapid re-epithelialization. The dispase II treatment had also been reported by Inamochi et al., which could promote the attachment ability of OME while maintaining the basal layer (Inamochi et al., 2019). Besides, our study focused more on the derivation of seed cells, the process of re-epithelization under the effect of oral mucosal stem cells, the differentiation potential of stem cells as well as the possible interactions between stem cells and corneal stroma.

       In our study, we firstly established the rat corneal alkali burn model to explore the wound healing effect of autologous OME transplantation, as alkali burn was a leading cause of corneal injury and PED (Baradaran-Rafii et al., 2017; Bizrah et al., 2019). The clinical examinations showed that OME could obviously promote the healing rate of the alkali-burn injured corneas (Fig. 1B–D). Although the corneas in the Alkali Burn group also recovered to a large extent on day 8 likely due to their remaining self-repair ability, the histological staining results verified the better recovery of corneal structures and the alleviated scar formation in the corneas of OME Transplantation group (Figs. 2 and 3). The desirable in vivo results encouraged us to further study the effect and mechanisms of OME transplantation on the epithelial defective corneas in vitro, so as to reveal the process of epithelial regeneration and the phenotypic transformation of stem cells. In in vitro experiments, the critical step was the innovatively establishment of the organ culture model to simulate the in vivo OME therapy (Fig. 4A). In this model, the rabbit corneal limbus was cut off and the corneal epithelium was completely scraped away to prevent the proliferation and migration of the residual cells. This procedure could help to simulate the status of persistent epithelial defect failing to heal under PED conditions. The simple de-epithelialized corneas were organ cultured for two weeks with the result of no epithelium could re-form, proving the validity of the model (Supplementary Fig. 1). In addition, oral mucosa from human was used considering that tissues from different species could better distinguish the origin of regenerative epithelial cells. For the organ cultured corneas with OME transplanted, the staining results clarified that the newly formed epithelial cells were indeed derived from the human oral epithelial cells and revealed the formation process of the regenerative epithelium, which presented with stratified and smooth morphology after two weeks of culture (Fig. 4B–F).

       Since the formation of epithelium was generally depended on the migration and differentiation of the stem cells, we also detected the expression of stem cell markers in tissues of each group (Clevers et al., 2014; Gonzalez et al., 2018). Oral mucosa was abundant with stem cells within the epithelial base, and the epithelium of the organ cultured cornea also maintained stemness with a layer of δNp63 positive cells (Fig. 5A). It could be considered that the stem cells derived from OME were the main seed cells for the formation of new corneal epithelium. As adult stem cells presented a promising future for regenerative medicine, oral mucosal epithelial stem cells were essential for the reconstruction of different epithelial tissues (Bauer et al., 2019; Hassan and AbdelAziz, 2018; Papagerakis et al., 2014). The regulation of stem cells mainly relied on the surrounding microenvironment they reside, which not only help to maintain their stemness and self-renewing status, but also control their differentiation precisely for the organogenesis and tissue homeostasis (Birbrair, 2017; Moore and Lemischka, 2006). And a large number of studies had shown that the microenvironment where the oral mucosa tissues were transplanted, played an important role in the remodeling of the grafts (Lu et al., 2010; Martín-Cano et al., 2013; Gaddipati et al., 2014).

      In our study, we transplanted the oral epithelial grafts onto the deepithelialized cornea for organ culture and it could be observed that the epithelium of the organ cultured cornea showed co-positive of the CK12 and CK13 (Fig. 5B), leading us to speculate that the deepithelialized cornea may influence the phenotypes of oral epithelial cells. The corneal stroma, made up of keratocytes and extracellular matrix (ECM), formed structural and proliferative support for corneal epithelium (Espana and Birk, 2020; Sridhar, 2018). Keratocytes were able to interact with epithelial cells through secreting signaling pathway ligands, and ECM was capable to regulate the stem cell differentiation (Smith et al., 2018; Sridhar, 2018; Zhang et al., 2017). In addition, stem cells could mechanosense the stiffness of their microenvironment, and ECM stiffness was transduced into gene expression via adhesion and cytoskeleton proteins that tuned fates (Smith et al., 2018). It could be deduced that the microenvironment of the corneal stroma could promote the oral epithelial cells to transdifferentiate into corneal epithelial like cells. To further explain our speculation, a schematic diagram was presented in Fig. 5C. The newly formed epithelium by organ culture could be derived from the migration and proliferation of oral epithelial cells, especially epithelial stem cells, and the culture microenvironment composed of corneal stroma and corneal culture medium may play a vital role in promoting the gradual trans-differentiation of the oral epithelial cells into corneal epithelial-like cells.

       The phenotypic transformation of regenerative epithelium emphasized the differentiation potential of oral stem cells and the important role of the corneal surface microenvironment. However, the interactions between oral stem cells and ocular surface went beyond what was described above. It was also reported that the transplanted oral stem cells could provide abundant growth factors and chemotactic stimuli, for the rejuvenation and the sustained proliferation of the residual limbal stem cells, leading to the improvement of vision function (Hassan and AbdelAziz, 2018; Sugiyama et al., 2014). Transplantation of oral stem cells likely provided an ideal environment for corneal epithelial cells derived from residual limbal stem cells in the LSCD model by reducing inflammation and neovascularization (Sugiyama et al., 2014). And our in vivo experimental results were somewhat consistent with the above study. The corneas in the OME Transplantation group had a faster healing rate compared to those in the Alkali Burn group, and the reason might be that the transplanted OME provided a suitable environment for the proliferation of limbal stem cells in the alkali-burned corneas. Therefore, we suggested that the application of OME transplantation might not be limited to the LSCD conditions. On the one hand, for patients with stem cell deficiency, OME transplantation could provide plenty of stem cells with differentiation potential under the effect of corneal surface environment. On the other hand, OME could also be an alternative for other patients with refractory PED that did not respond to the standard managements, as OME could provide an appropriate environment for the proliferation of limbal stem cells and corneal epithelial cells, thereby accelerating the healing rate of the injured corneal epithelium. Just as defined clinically, PED resulted from the failure of rapid corneal re-epithelialization within 10–14 days even with standard supportive treatments (Vaidyanathan et al., 2019). No matter there was LSCD or not, it would be significant to promote corneal epithelial healing by OME transplantation thus to alleviate the discomfort and suffering of patients. In the near future, we hope to further explore the underlying mechanisms of the corneal surface reconstructed by OME transplantation, as well as the regulatory factors affecting the epithelial growth and transformation, so as to lay a solid foundation for the clinical treatment of refractory PED.

5、Conclusion

       Here, we demonstrated that the autologous OME transplantation could effectively promote the wound healing of alkali burned rat corneas morphologically and functionally. And then the in vitro organ culture results showed that de-epithelialized corneas could achieve reepithelialization with the transplantation of OME. The re-formed  epithelium maintained stemness and could express the corneal epithelial marker, implying the possibility of the transformation of oral stem cells into corneal epithelial-like cells. Thus, autologous OME transplantation could be a simple, accessible and promising treatment of refractory PED when the standard drugs or managements were not effective.

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