American Journal of Transplantation 2015; 15: 1068–1075 Wiley Periodicals Inc.
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Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13096
Lamellar Keratoplasty Treatment of Fungal Corneal Ulcers With Acellular Porcine Corneal Stroma M.-C. Zhang1,*, X. Liu1, Y. Jin2, D.-L. Jiang1, X.-S. Wei1 and H.-T. Xie1 1
Department of Ophthalmology, Wuhan Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei, China 2 Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi’an, Shaanxi, China Corresponding author: Ming-Chang Zhang, [email protected]
The fundamental problem of corneal transplantation is a severe shortage of donor tissues worldwide, resulting in approximately 1.5 million new cases of blindness annually. To explore an alternative to donor corneas, we conducted a clinical study in which implanted acellular porcine corneal stromas (APCSs) replaced the pathologic anterior corneas in 47 patients who had experienced fungal corneal infections. Subsequently, we demonstrated the safety and efficacy of APCSs in human keratoplasty for a minimum follow-up period of 6 months, during which time no recurrence of infection was observed. All corneal ulcers healed with the return of neovascularization. In addition, our results indicated that epithelialization occurred in all APCS grafts except four grafts; for these four, the grafts dissolved to varying degrees. Furthermore, most porcine grafts (n ¼ 41) gradually became transparent without rejection, and an improvement of more than two lines in best corrected visual acuity (BCVA) was achieved in 34 eyes (72%). Finally, no patients showed any severe adverse reaction or any significant change in postoperative systemic safety indicators. Thus, we concluded that APCS grafts are safe and efficacious during lamellar keratoplasty in treating corneal fungal ulcers and potentially for other clinical diseases. Abbreviations: ANOVA, analysis of variance; APCS, acellular porcine corneal stroma; BCVA, best corrected visual acuity; HHP, high-hydrostatic pressurization; LKP, lamellar keratoplasty; NIFDC, National Institutes for Food and Drug Control; PBS, phosphate-buffered saline; PKP, penetrating keratoplasty Received 07 July 2014, revised 05 October 2014 and accepted for publication 08 November 2014
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Introduction Corneal blindness remains a global challenge in view of supply shortages of donor corneas, particularly in Asian countries, such as China (1). Several attempts have been made in searching for suitable corneal substitutes. Recently, several groups have succeeded in preparing an acellular corneal stroma by using non-ionic detergents and/or enzymes. Hashimoto et al used high-hydrostatic pressurization (HHP) to decellularize porcine corneas, which were then transplanted into rabbit corneas (2). No immune reaction was observed, and the turbid corneas became clear. Although the prospect of acellular porcine corneal stroma (APCS) as an alternative to donor corneas has been demonstrated in several in vitro and animal studies (3–5), their safety and efficacy in human patients has not previously been demonstrated. Fungal keratitis is one of the most common causes of corneal blindness in developing countries such as India and China (6,7), especially in agricultural areas, where early diagnosis and treatment are hampered by a lack of access to medical care (8). Although, various antifungal chemotherapies have been used, effective management of fungal keratitis continues to be problematic, especially when the infection occurs in deeper lesions (9). Overall, one-third of all corneal fungal ulcers require surgical intervention because of treatment failures or corneal perforations (10). A lack of corneal donors for keratoplasty further complicates the treatment of corneal fungal ulcers in China. We have previously reported good histocompatibility and low immunogenicity of APCS in vitro and in rabbit models (11–13). These accomplishments have resulted in the approval of APCS for clinical trials by the National Institutes for Food and Drug Control (NIFDC) of China. In this study, we aimed to investigate the safety and efficacy of APCS used for lamellar keratoplasty (LKP) in patients suffering from fungal corneal ulcers.
Materials and Methods This study was approved by the Ethics Committee of Wuhan Union Hospital and was registered in the Chinese Clinical Trial Registry under identifier ChiCTR-ONC-12002510. All eligible patients were enrolled after obtaining
Acellular Porcine Cornea in Keratoplasty their written informed consent. All of the procedures were performed following the tenets of the Helsinki Declaration, if applicable.
Materials The APCS grafts used in this study were prepared by the AiNear Corneal Engineering Corporation (Shenzhen, China) according to the method previously reported by Jin and coworkers (13). In short, pig eyes were harvested from a quarantined animal facility certified by the Bureau of Animal Quarantine Department of China. The eyes were enucleated immediately after death and thoroughly washed with phosphate-buffered saline (PBS). Whole corneas containing parts of the sclera were cut from the eyes and soaked in ultrapure water to allow swelling for 12 h. The corneal stroma then underwent agitation in 2 M NaCl for 30 min, followed by ultrapure water for 30 min. The process was repeated three times. Next, 0.2% Triton X-100 was used to wash the corneas for 6 h. After a thorough washing in PBS, the APCSs were dehydrated in glycerol to the normal thickness of a native cornea. Finally, sterilization was performed by Co60 irradiation. Furthermore, the prepared APCSs used for this study also passed cytotoxicity and histocompatibility tests, and all procedures were approved by the NIFDC of China.
Participants Patients meeting the inclusion and exclusion criteria (Data S1) were enrolled, all of whom suffered from active corneal fungal ulcers diagnosed by either microscopic examination of a direct smear or confocal microscopy but failed to respond to routine antifungal drug therapies including 5% natamycin (Alcon, Fort Worth, TX) and 0.5% fluconazole (Bausch & Lomb, Rochester, NY). Moreover, all of these ulcers were at risk of perforation, potentially leading to the loss of a functioning globe.
Surgical procedure After retrobulbar anaesthesia by equal volumes of 2% lidocaine and 0.75% bupivacaine, the globe was fixated by traction sutures to the superior and inferior rectus muscles. The ulcer surface was scraped with a blade, and the conjunctival sac was irrigated with 0.5% fluconazole. A trephine of appropriate size was used to mark the area 1 mm larger than the corneal ulcer border. The lamellar dissection was carried out by a blade to create a smooth plane including all ulcer tissues. If required, more than one plane of the lamellar dissection was created to ensure that the remaining stromal bed was clean, before being repeatedly rinsed with 0.5% fluconazole. The tissue removed during the surgery was submitted for microorganism identification through microbiological culturing and histopathology. The APCS was then trephined to the same size and sutured to fill in the stromal defect with 10-0 nylon suture (Alcon, Fort Worth, TX).
Postoperative management All patients included in this study were examined for follow-up at 3 days, 7 days, 1 month, 3 months and 6 months after the operation. They all received topical applications of 0.5% fluconazole and 5% natamycin four times a day. Fluorescent staining was used on the third day to monitor epithelialization. Once completed, the cornea received 0.5% pranoprofen (Senju Pharmaceutical Co., Ltd.) and 1% cyclosporin (North China Pharmaceutical Co., Ltd.) three times a day. At the 1-month follow-up visit, anti-fungal drops were discontinued if there was no suggestive sign of fungal infection or recurrence, while 0.1% tobradex (Alcon, FortWorth, TX) was administered four times a day with weekly tapering. The corneal sutures were removed 1 to 3 months after the surgery. At the 3-month follow-up visit, an extra glucocorticoid was selectively administered according to the neovascularization status and rejection reaction. Topical 1% cyclosporine
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(North China Pharmaceutical Co., Ltd.) was used for 6 months. All medications were discontinued if the cornea was stable. Routine blood and urine tests, as well as hepatic and renal function tests, were performed before the surgery and 6 months after the surgery to assess the safety of the treatment. The symptoms, visual acuity, size and depth of the corneal defect, and neovascularization of the cornea were recorded by slit-lamp examinations at each visit. The APCS’s transparency and the corneal edema were graded from 0 to 3. The corneal neovascularization was graded as 0 if there were no new vessels; it was graded 1, 2, or 3 if new vessels grew into the cornea, to the border of the APCS, or into the APCS, respectively. Corneal photography occurred at each follow-up visit to document the above changes and adverse events, including dissolution of the APCS.
Statistical analysis For purposes of statistical analysis, counting fingers was categorized as an acuity of 0.004, hand motion as 0.002, light perception as 0.001 (14). Final postoperative visual acuity was defined as the visual acuity at the most recent visit. The values were compared with preoperative values using a scatter diagram. Other data were first transformed to rank cases and were then compared by one-way analysis of variance (ANOVA), with follow-up LSD-t or Dunnett T3 tests, using SPSS 18.0, where p < 0.05 was considered to be statistically significant. The staff members performing the statistical analysis for this study were not involved in the clinical observation or data recording.
Results A total of 47 eyes were included in this study from 33 men (70%) and 14 women (30%). The average age was 53.5 9.1 years old, ranging from 18 to 72 years old. Twenty-three patients (nearly 50%) had a definite history of trauma caused by agricultural objects while working in the countryside. The average time period before the corneal ulcer’s being surgically managed was 36 6.3 days, with the longest wait being 8 months. The diameters of corneal ulcers ranged from 2.5 to 8 mm. The depth of the ulcers ranged from one-third of the corneal stromal thickness to the depth of Descemet’s membrane (for details, see Table S1). Forty-three patients were diagnosed by microscopic examination of the direct smear, and 12 were confirmed by preoperative confocal microscopy. Thirty-nine (83%) had tissues removed during the surgery that were also confirmed to have fungal infections via postoperative microbiological culturing or histopathology. Fungal spores and hyphae were found under in vivo confocal microscopy before the operation but not after surgery (Figure 1A and B). Compared to those of normal eyes, the smoothness of the surgical corneal surface curves was retained upon examination by ultrasound biomicroscopy (Figure 1C and D). Compared to the preoperative scores, the irritation symptoms from all patients were significantly reduced after the surgery (p < 0.05, Table 1). Bandage contact lenses were only used in nine patients (19%) who experienced severe eye irritation in the initial postoperative
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Figure 1: In vivo confocal microscopic and ultrasound biomicroscopic (UBM) images of patients. (A) The observation of fungal spores and hyphae under confocal microscopy before surgery. (B) No fungal infection was found in the keratocytes under confocal microscopy from a patient’s cornea 1 year after surgery. The surgical corneal surface curve (C) remained smooth under ultrasound biomicroscopy 1 year after the surgery, as compare to the counterpart eye (D).
Table 1: The evaluation score before and after surgery Irritation score before and after surgery
Corneal neovascularization score before and after surgery
Postoperative graft transparency score
Score
Preoperative1
3 day1
7 day1,2
1 month1,2
3 month1,2
6 month2
0 n (%) 1 n (%) 2 n (%) 3 n (%) Total n (%)
1 (2%) 14 (30%) 30 (64%) 2 (4%) 47 (100%)
0 28 (60%) 19 (40%) 0 47 (100%)
0 39 (83%) 8 (17%) 0 47 (100%)
5 (11%) 38 (80%) 4 (9%) 0 47 (100%)
19 (40%) 28 (60%) 0 0 47 (100%)
41 (87%) 6 (13%) 0 0 47 (100%)
Score
Preoperative3
3 day3
7 day3
1 month3
3 month3
6 month
0 n (%) 1 n (%) 2 n (%) 3 n (%) Total n (%)
16 (34%) 17 (36%) 13 (28%) 1 (2%) 47 (100%)
22 (47%) 24 (51%) 0 1 (2%) 47 (100%)
22 (47%) 24 (51%) 0 1 (2%) 47 (100%)
22 (47%) 20 (42%) 4 (9%) 1 (2%) 47 (100%)
30 (64%) 10 (21%) 5 (11%) 2 (4%) 47 (100%)
39 (83%) 7 (15%) 0 1 (2%) 47 (100%)
Score
3 day4
7 day4
1 month5
3 month5
6 month5
0 n (%) 1 n (%) 2 n (%) 3 n (%) Total n (%)
0 14 (30%) 33 (70%) 0 47 (100%)
0 26 (55%) 21 (45%) 0 47 (100%)
0 30 (64%) 17 (36%) 0 47 (100%)
0 40 (85%) 7 (15%) 0 47 (100%)
1 (2%) 40 (85%) 5 (11%) 1(2%) 47 (100%)
Has significant statistical difference compare with 6 months (p < 0.05). Has significant statistical difference compare with preoperative irritation score (p < 0.05). 3 Dunnett T3 test, has significant statistical difference compare with 6 months score (p < 0.05). 4 Has significant statistical difference compare with 6 months (p < 0.05). 5 Dunnett T3 test, has significant statistical difference compare with 3 days (p < 0.05). 1 2
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Figure 2: Slit-lamp biomicroscopic photographs of pre- and post-operative corneas. The epithelial healing times after surgery ranged from 3 days to 1 week, which was not different from times experienced following human corneal transplantation (A2, B2). The postoperative graft in one fungal infection case, comorbid with leucoma caused by viral keratitis in childhood, had the surgical treatment and healed well (B). The graft remained transparent, even though the pterygium had been growing into the graft of the patient, who had also experienced pterygium preoperatively (D). After 3 years of follow-up (C4, E4), the transparency of the grafts significantly improved compared with observations made at the 6-month follow-up (C3, E3) and was similar to the surrounding normal cornea. A1, B1, C1, D1, and E1 were taken preoperatively; A4, B4, C3, D4, and E3 were taken six months after surgery; C4 and E4 were taken 3 years after surgery.
period. The epithelial healing times ranged from 3 days to 1 week after the surgery and showed no significant difference to healing times following human corneal transplantation (Figure 2A and B). Although, various degrees of neovascularization were noted in 25 patients after the surgery, most neovascularization gradually regressed, and only seven patients (15%) needed the extra topical 0.1% tobradex treatment at the 3-month follow up visit (Table 1, Figure 2). One of the typical patients had a American Journal of Transplantation 2015; 15: 1068–1075
fungal corneal infection lasting for 6 months (Figure 3). With continuous use of anti-fungal drugs, the scope of the ulcer became smaller, but a large number of new vessels grew into the cornea from the superior direction, and ledging could be seen between the ulcer and the surrounding cornea (Figure 3A–C). After the operation, the graft healed well, with no any infection recurrence, and all new vessels regressed during a 3-month follow-up (Figure 3F). Despite the pterygium’s having grown into the grafts in five patients 1071
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Figure 3: Six-month disease course of the fungal infection in a typical patient. (A) The patient was hospitalized in March 2012 with a fungal infection diagnosed via corneal scraping and was given antifungal drug therapy. (B) The scope of the infection was demonstrated after the anti-fungal treatment one and a half months later. (C) After five months, the ulcer was localized in the pupil area, with a large number of new vessels growing into the cornea from the superior direction, and ledging with the normal tissue was observed. (D) By the third postoperative day, the epithelium had healed well. (E) Seven days after the surgery, the corneal graft showed little edema, and a large number of new vessels had also grown into the graft bed. (F) At the 3-month follow up, the new vessels had completely disappeared, and naked vision acuity was 0.4.
who had preoperative pterygia, the grafts remained transparent, and no patient required any additional surgical treatment at the 6-month follow-ups (Figure 2D). For patients with eccentric infections, the corresponding grafts also achieved good results, with no significant postoperative rejection being observed (Figure S1). All of these results collectively suggest that APCS has good histocompatibility and low immunogenicity, which benefits its clinical application, particularly in high-risk transplantation.
roidism, without exophthalmus or hypophasis. Despite of the dissolution of the grafts, all grafts eventually epithelialized, and no perforations or infection relapses occurred. Overall, during minimum follow-ups of 6 months, we observed no infection recurrences. All corneal ulcers
APCS grafts became transparent, and edema disappeared after the 1-month follow-up. The best corrected visual acuities (BCVAs) of 34 eyes, 72% of all patients, showed improvement of more than 2 lines. Of the remaining 13 eyes, only 2 showed a loss of more than one line, whereas the other 11 showed improvements of no more than 2 lines (Figure 4). For example, BCVAs improved to 1.0 in three patients during the 6-month follow-up period. More interestingly, in four patients for whom the follow-up period was prolonged to 3 years, the transparency of the grafts had noticeably improved when compared to only observed for 6 months. These grafts showed no obvious differences from the surrounding normal cornea (Figure 2C3, C4, E3, and E4). These results indicate that APCS grafts can gradually become transparent after an extended period in the absence of a rejection reaction, similar to results found using human donor corneas. During follow-up, various degrees of graft dissolution took place in four patients (Figure 5). Among them, two had infections approaching the endothelial layers pre-operatively. The other two had histories of well-controlled hyperthy1072
Figure 4: Changes in visual acuity after surgery. HM denotes ‘‘hand motion,’’ and CF means ‘‘counting fingers.’’ The diagonal line represents the point at which the values for visual acuity preoperatively and postoperatively were the same.
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Figure 5: The dissolved graft observed with slit-lamp biomicroscopy. (A) Preoperatively, the patient presented with hypopyon and anamnestically reported hyperthyroidism, but these were well controlled, with neither exophthalmus nor hypophasis being observed. One week after the operation, the graft started to dissolve. At the 1-month follow-up, the graft bed had completely epithelialized. There was no sign that the infection had recurred. The ulcer depth of the patient (B) and (C) was unable to be evaluated by the slit lamp preoperatively. However, during the operation, the ulcer infiltration was found to reach the endothelial layer. At 7 days postoperatively, double anterior chambers could be seen in (B2) with the dissolved graft. At one month postoperatively, the double anterior chambers had disappeared, as shown in (B3). All of the graft bed had completely epithelialized, with no recurrent infection noted in the review for 6 months (B, C).
healed, and neovascularization regressed. No patient showed severe adverse reaction, and no significant changes in postoperative systemic safety indicators were observed. Eventually, all of the APCS grafts gradually became transparent over the prolonged follow-up period.
Discussion With the increasing worldwide shortage of donor corneas for keratoplasty, there is a greater need to find a suitable substitute for donor corneas (15). As early as 2003, Amano et al suggested that heterogeneous corneal stromas, especially porcine corneal stromas, could be ideal alternatives to human corneas due to their lower antigenicities, as demonstrated in in vitro corneal tissue engineering, and transparently healing, as shown in animal experiments (16). In addition, studies regarding the successful construction of tissue-engineered hearts, lungs, and livers using acellular biomaterials have also been reported (17–19), which indicates that the acellular stroma is a suitable carrier for tissue engineering. Wang et al analyzed cellular and humoral immunity for three layers of porcine corneas and found that the immunogenicities of the intact endothelia, epithelia and stromas (all of equal thickness), as detected by American Journal of Transplantation 2015; 15: 1068–1075
cellular immunity, were 70.75%, 27.63%, and 1.62%, respectively. Similarly, the immunogenicities, as measured by humoral immunity, were 62.11%, 31.77%, and 6.12%, respectively (20). APCS has a cell affinity and can provide a relative healthy corneal stroma microenvironment as well as a substrate—the basement membrane that is conducive to corneal epithelial cell adhesion and growth. The mesh structure in the matrix layer can provide space for the growth of mesenchymal cells and the exchange of metabolites. In addition, APCSs can also be used as seed carriers (21). Our previous animal study (13), through implantation of APCSs into rabbit corneal pockets and mouse subcutaneous tissue, showed that the APCS is unable to stimulate a host response in the cornea. In subcutaneous tissue, the APCS only triggered an innate immune reaction, which had decreased by 28 days postimplantation. These results demonstrated that complete removal of the cell components of the cornea, which are thought to be the main source of the major histocompatibility complex antigens responsible for allograft/xenograft rejection, can obviously alleviate the immune response to the grafts. In addition, APCS implants could be integrated with body tissues and sufficiently support new tissue regeneration and reconstruction (22). Animal experiments have confirmed that APCS transplants can also be used 1073
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as cosmetic corneal lens implants (23). Although many studies in vitro and in animals have confirmed that APCS has excellent potential for solving the problem created by the serious lack of donor corneas, no large-scale clinical study of ACPS has ever been reported. For the first time, our study has proven that APCS is safe and effective in clinical application. Our clinical studies have shown that, similar to human donor corneas, the transplanted areas generally require three days for epithelialization in which the epithelial regeneration is the most important factor for reducing postoperative complications (Figure 2A and B). Compared with the biosynthetic cornea reported by Dr. May Griffith in 2010 (24), which showed that the highest corrected visual acuity of 0.4 was only achieved in six patients with markedly increased post-surgical astigmatism due to degradation of the biomaterials and delayed full epithelialization (for at least one month), APCS is far superior because it is similar to the natural cornea on the elastic modulus, has good toleration of sutures, provides cutting tension and permits enzymatic degradation in vivo. With a prolonged follow-up period, APCS grafts can gradually become transparent in patients with no rejection reactions, and patients’ vision also improves (Figure 3). Compared with the grafts observed at the 6-month follow-up, the transparency of the grafts at the 3-year follow up was almost equivalent to the surrounding normal corneal tissues (Figure 2C3, C4, E3, and E4). Considering that no obvious rejection reaction was observed, despite our inability to leverage anti-rejection glucocorticoid drugs during the early postoperative period on account of the fungal infections, as well as considering that this was high-risk transplantation in which large numbers of new vessels may have grown into the graft beds preoperatively, APCS appears to offer good histocompatibility and low immunogenicity in clinical applications. The study has also shown that APCS had a unique effect on the treatment of the fungal corneal infection, which has a high rate of morbidity and blindness in China and other developing countries. No fungal infection recurrence occurred in any of the 47 patients after the surgery, even in the cases where the grafts dissolved. This suggests that the porcine cornea, unlike human corneas, might not be susceptible to fungal invasion. However, this hypothesis requires further investigation. Our study suggests that to reduce the recurrence rate, it is necessary to rinse planting beds repeatedly with antifungal drugs during the surgery, and all ulcer lesions should be removed completely. Conservative treatment of fungal keratitis often disappoints because of limited tissue penetration, a narrow antimicrobial spectrum, and toxicity of the currently available antifungal agents (25,26). Furthermore, early diagnosis is difficult. The excessive use of antibiotics and glucocorticoid also aggravates the fungal infection situation. Because of poor responses to drug treatment, many patients eventually have to choose surgery. Penetrating keratoplasty (PKP) 1074
is always the first choice in the traditional surgical treatment (27). However, compared with LKP, traditional PKP causes more intraocular complications, especially in infection cases, and the postoperative endophthalmitis risk also increases significantly (28). Because most fungal keratitis lesions are mainly localized in the stromal layer, LKP not only can provide useful vision with few complications but also can save limited donor cornea resources (29). From experimental and clinical studies, Xie and coworkers proposed that LKP leads to lower endothelial cell disturbance and endophthalmitis incidence compared with PKP, with no statistically significant differences in infection recurrence rates (30). Our study has also proven that APCS is a suitable alternative to donor corneas in the treatment of fungal corneal ulcer using LKP. Because postoperative grafts healed well in one of the fungal infection cases that was comorbid with leucoma caused by viral keratitis in childhood (Figure 2B), we have deduced that, besides fungal corneal ulcer treatment, APCS also has other application potential, such as in the treatment of viral corneal ulcers. Taken together, our approaches may allow the restoration of diseased or damaged corneas that cannot be treated using currently available techniques without human donor corneas. Our study, through 3 years clinical observations, has demonstrated that all patients’ ulcers healed well and that neovascularization gradually regressed. No obvious rejections or other systemic or local side effects were observed. Overall, APCS grafts appear to be safe and efficacious during LKP in treating corneal fungal ulcers and may have other potential clinical applications.
Acknowledgments The APCSs were kindly provided by the Shenzhen AiNear Corneal Engineering Corporation. We thank Chong Tian for providing extensive statistical assistance to us.
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
References 1. Shang X, Zhang M. Body and organ donation in Wuhan, China. Lancet 2010; 376: 1033–1034. 2. Hashimoto Y, Funamoto S, Sasaki S, et al. Preparation and characterization of decellularized cornea using high-hydrostatic pressurization for corneal tissue engineering. Biomaterials 2010; 31: 3941–3948. 3. Pang K, Du L, Wu X. A rabbit anterior cornea replacement derived from acellular porcine cornea matrix, epithelial cells and keratocytes. Biomaterials 2010; 31: 7257–7265.
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Acellular Porcine Cornea in Keratoplasty 4. Li N, Wang X, Wan P, et al. Tectonic lamellar keratoplasty with acellular corneal stroma in high-risk corneal transplantation. Mol Vis 2011; 17: 1909–1917. 5. Shao Y, Yu Y, Pei CG, et al. Evaluation of novel decellularizing corneal stroma for cornea tissue engineering applications. Int J Ophthalmol 2012; 5: 415–418. 6. Xie L, Zhong W, Shi W, Sun S. Spectrum of fungal keratitis in north China. Ophthalmology 2006; 113: 1943–1948. 7. Nath R, Baruah S, Saikia L, Devi B, Borthakur AK, Mahanta J. Mycotic corneal ulcers in upper Assam. Indian J Ophthalmol 2011; 59: 367–371. 8. Gopinathan U, Sharma S, Garg P, Rao GN. Review of epidemiological features, microbiological diagnosis and treatment outcome of microbial keratitis: experience of over a decade. Indian J Ophthalmol 2009; 57: 273–279. 9. Thomas PA, Kaliamurthy J. Mycotic keratitis: Epidemiology, diagnosis and management. Clin Microbiol Infect 2013; 19: 210–220. 10. Jhanji V, Young AL, Mehta JS, Sharma N, Agarwal T, Vajpayee RB. Management of corneal perforation. Surv Ophthalmol 2011; 56: 522–538. 11. Zhang C, Nie X, Hu D, et al. Survival and integration of tissueengineered corneal stroma in a model of corneal ulcer. Cell Tissue Res 2007; 329: 249–257. 12. Lin XC, Hui YN, Wang YS, Meng H, Zhang YJ, Jin Y. Lamellar keratoplasty with a graft of lyophilized acellular porcine corneal stroma in the rabbit. Vet Ophthalmol 2008; 11: 61–66. 13. Luo H, Lu Y, Wu T, Zhang M, Zhang Y, Jin Y. Construction of tissueengineered cornea composed of amniotic epithelial cells and acellular porcine cornea for treating corneal alkali burn. Biomaterials 2013; 34: 6748–6759. 14. Tsubota K, Satake Y, Kaido M, et al. Treatment of severe ocularsurface disorders with corneal epithelial stem-cell transplantation. N Engl J Med 1999; 340: 1697–1703. 15. Lawlor M, Kerridge I, Ankeny R, Dobbins TA, Billson F. Specific unwillingness to donate eyes: the impact of disfigurement, knowledge and procurement on corneal donation. Am J Transplant 2010; 10: 657–663. 16. Amano S, Shimomura N, Kaji Y, Ishii K, Yamagami S, Araie M. Antigenicity of porcine cornea as xenograft. Curr Eye Res 2003; 26: 313–318. 17. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science 2010; 329: 538–541. 18. Shupe T, Williams M, Brown A, Willenberg B, Petersen BE. Method for the decellularization of intact rat liver. Organogenesis 2010; 6: 134–136. 19. Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: Using nature’s platform to engineer a bioartificial heart. Nat Med 2008; 14: 213–221.
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20. Wang Z, Ge J, Xu J, Chen J. Relative quantitative analysis of corneal immunogenicity. Zhonghua Yan Ke Za Zhi 2002; 38: 535– 538. 21. Zhu J, Zhang K, Sun Y, et al. Reconstruction of functional ocular surface by acellular porcine cornea matrix scaffold and limbal stem cells derived from human embryonic stem cells. Tissue Eng Part A 2013; 19: 2412–2425. 22. Du L, Wu X. Development and characterization of a full-thickness acellular porcine cornea matrix for tissue engineering. Artif Organs 2011; 35: 691–705. 23. Liu Z, Zhou Q, Zhu J, et al. Using genipin-crosslinked acellular porcine corneal stroma for cosmetic corneal lens implants. Biomaterials 2012; 33: 7336–7346. 24. Fagerholm P, Lagali NS, Merrett K, et al. A biosynthetic alternative to human donor tissue for inducing corneal regeneration: 24-month follow-up of a phase 1 clinical study. Sci Transl Med 2010; 2: 46ra61. 25. Abdel-Rhaman MS, Soliman W, Habib F, Fathalla D. A new longacting liposomal topical antifungal formula: human clinical study. Cornea 2012; 31: 126–129. 26. FlorCruz NV, Peczon IV, Evans JR. Medical interventions for fungal keratitis. Cochrane Database Syst Rev 2012; 2: CD004241. 27. Sharma N, Sachdev R, Jhanji V, Titiyal JS, Vajpayee RB. Therapeutic keratoplasty for microbial keratitis. Curr Opin Ophthalmol 2010; 21: 293–300. 28. Tan DT, Dart JK, Holland EJ, Kinoshita S. Corneal transplantation. Lancet 2012; 379: 1749–1761. 29. Xie L, Shi W, Liu Z, Li S. Lamellar keratoplasty for the treatment of fungal keratitis. Cornea 2002; 21: 33–37. 30. Shi W, Wang T, Xie L, et al. Risk factors, clinical features, and outcomes of recurrent fungal keratitis after corneal transplantation. Ophthalmology 2010; 117: 890–896.
Supporting Information Additional Supporting Information may be found in the online version of this article. Figure S1: Slit-lamp biomicroscopic photographs of the patients with eccentric infections. Data S1: Inclusion and exclusion criteria. Table S1: The evaluation scores of preoperative ulcer depths and widths.
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Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13096
Lamellar Keratoplasty Treatment of Fungal Corneal Ulcers With Acellular Porcine Corneal Stroma M.-C. Zhang1,*, X. Liu1, Y. Jin2, D.-L. Jiang1, X.-S. Wei1 and H.-T. Xie1 1
Department of Ophthalmology, Wuhan Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei, China 2 Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi’an, Shaanxi, China Corresponding author: Ming-Chang Zhang, [email protected]
The fundamental problem of corneal transplantation is a severe shortage of donor tissues worldwide, resulting in approximately 1.5 million new cases of blindness annually. To explore an alternative to donor corneas, we conducted a clinical study in which implanted acellular porcine corneal stromas (APCSs) replaced the pathologic anterior corneas in 47 patients who had experienced fungal corneal infections. Subsequently, we demonstrated the safety and efficacy of APCSs in human keratoplasty for a minimum follow-up period of 6 months, during which time no recurrence of infection was observed. All corneal ulcers healed with the return of neovascularization. In addition, our results indicated that epithelialization occurred in all APCS grafts except four grafts; for these four, the grafts dissolved to varying degrees. Furthermore, most porcine grafts (n ¼ 41) gradually became transparent without rejection, and an improvement of more than two lines in best corrected visual acuity (BCVA) was achieved in 34 eyes (72%). Finally, no patients showed any severe adverse reaction or any significant change in postoperative systemic safety indicators. Thus, we concluded that APCS grafts are safe and efficacious during lamellar keratoplasty in treating corneal fungal ulcers and potentially for other clinical diseases. Abbreviations: ANOVA, analysis of variance; APCS, acellular porcine corneal stroma; BCVA, best corrected visual acuity; HHP, high-hydrostatic pressurization; LKP, lamellar keratoplasty; NIFDC, National Institutes for Food and Drug Control; PBS, phosphate-buffered saline; PKP, penetrating keratoplasty Received 07 July 2014, revised 05 October 2014 and accepted for publication 08 November 2014
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Introduction Corneal blindness remains a global challenge in view of supply shortages of donor corneas, particularly in Asian countries, such as China (1). Several attempts have been made in searching for suitable corneal substitutes. Recently, several groups have succeeded in preparing an acellular corneal stroma by using non-ionic detergents and/or enzymes. Hashimoto et al used high-hydrostatic pressurization (HHP) to decellularize porcine corneas, which were then transplanted into rabbit corneas (2). No immune reaction was observed, and the turbid corneas became clear. Although the prospect of acellular porcine corneal stroma (APCS) as an alternative to donor corneas has been demonstrated in several in vitro and animal studies (3–5), their safety and efficacy in human patients has not previously been demonstrated. Fungal keratitis is one of the most common causes of corneal blindness in developing countries such as India and China (6,7), especially in agricultural areas, where early diagnosis and treatment are hampered by a lack of access to medical care (8). Although, various antifungal chemotherapies have been used, effective management of fungal keratitis continues to be problematic, especially when the infection occurs in deeper lesions (9). Overall, one-third of all corneal fungal ulcers require surgical intervention because of treatment failures or corneal perforations (10). A lack of corneal donors for keratoplasty further complicates the treatment of corneal fungal ulcers in China. We have previously reported good histocompatibility and low immunogenicity of APCS in vitro and in rabbit models (11–13). These accomplishments have resulted in the approval of APCS for clinical trials by the National Institutes for Food and Drug Control (NIFDC) of China. In this study, we aimed to investigate the safety and efficacy of APCS used for lamellar keratoplasty (LKP) in patients suffering from fungal corneal ulcers.
Materials and Methods This study was approved by the Ethics Committee of Wuhan Union Hospital and was registered in the Chinese Clinical Trial Registry under identifier ChiCTR-ONC-12002510. All eligible patients were enrolled after obtaining
Acellular Porcine Cornea in Keratoplasty their written informed consent. All of the procedures were performed following the tenets of the Helsinki Declaration, if applicable.
Materials The APCS grafts used in this study were prepared by the AiNear Corneal Engineering Corporation (Shenzhen, China) according to the method previously reported by Jin and coworkers (13). In short, pig eyes were harvested from a quarantined animal facility certified by the Bureau of Animal Quarantine Department of China. The eyes were enucleated immediately after death and thoroughly washed with phosphate-buffered saline (PBS). Whole corneas containing parts of the sclera were cut from the eyes and soaked in ultrapure water to allow swelling for 12 h. The corneal stroma then underwent agitation in 2 M NaCl for 30 min, followed by ultrapure water for 30 min. The process was repeated three times. Next, 0.2% Triton X-100 was used to wash the corneas for 6 h. After a thorough washing in PBS, the APCSs were dehydrated in glycerol to the normal thickness of a native cornea. Finally, sterilization was performed by Co60 irradiation. Furthermore, the prepared APCSs used for this study also passed cytotoxicity and histocompatibility tests, and all procedures were approved by the NIFDC of China.
Participants Patients meeting the inclusion and exclusion criteria (Data S1) were enrolled, all of whom suffered from active corneal fungal ulcers diagnosed by either microscopic examination of a direct smear or confocal microscopy but failed to respond to routine antifungal drug therapies including 5% natamycin (Alcon, Fort Worth, TX) and 0.5% fluconazole (Bausch & Lomb, Rochester, NY). Moreover, all of these ulcers were at risk of perforation, potentially leading to the loss of a functioning globe.
Surgical procedure After retrobulbar anaesthesia by equal volumes of 2% lidocaine and 0.75% bupivacaine, the globe was fixated by traction sutures to the superior and inferior rectus muscles. The ulcer surface was scraped with a blade, and the conjunctival sac was irrigated with 0.5% fluconazole. A trephine of appropriate size was used to mark the area 1 mm larger than the corneal ulcer border. The lamellar dissection was carried out by a blade to create a smooth plane including all ulcer tissues. If required, more than one plane of the lamellar dissection was created to ensure that the remaining stromal bed was clean, before being repeatedly rinsed with 0.5% fluconazole. The tissue removed during the surgery was submitted for microorganism identification through microbiological culturing and histopathology. The APCS was then trephined to the same size and sutured to fill in the stromal defect with 10-0 nylon suture (Alcon, Fort Worth, TX).
Postoperative management All patients included in this study were examined for follow-up at 3 days, 7 days, 1 month, 3 months and 6 months after the operation. They all received topical applications of 0.5% fluconazole and 5% natamycin four times a day. Fluorescent staining was used on the third day to monitor epithelialization. Once completed, the cornea received 0.5% pranoprofen (Senju Pharmaceutical Co., Ltd.) and 1% cyclosporin (North China Pharmaceutical Co., Ltd.) three times a day. At the 1-month follow-up visit, anti-fungal drops were discontinued if there was no suggestive sign of fungal infection or recurrence, while 0.1% tobradex (Alcon, FortWorth, TX) was administered four times a day with weekly tapering. The corneal sutures were removed 1 to 3 months after the surgery. At the 3-month follow-up visit, an extra glucocorticoid was selectively administered according to the neovascularization status and rejection reaction. Topical 1% cyclosporine
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(North China Pharmaceutical Co., Ltd.) was used for 6 months. All medications were discontinued if the cornea was stable. Routine blood and urine tests, as well as hepatic and renal function tests, were performed before the surgery and 6 months after the surgery to assess the safety of the treatment. The symptoms, visual acuity, size and depth of the corneal defect, and neovascularization of the cornea were recorded by slit-lamp examinations at each visit. The APCS’s transparency and the corneal edema were graded from 0 to 3. The corneal neovascularization was graded as 0 if there were no new vessels; it was graded 1, 2, or 3 if new vessels grew into the cornea, to the border of the APCS, or into the APCS, respectively. Corneal photography occurred at each follow-up visit to document the above changes and adverse events, including dissolution of the APCS.
Statistical analysis For purposes of statistical analysis, counting fingers was categorized as an acuity of 0.004, hand motion as 0.002, light perception as 0.001 (14). Final postoperative visual acuity was defined as the visual acuity at the most recent visit. The values were compared with preoperative values using a scatter diagram. Other data were first transformed to rank cases and were then compared by one-way analysis of variance (ANOVA), with follow-up LSD-t or Dunnett T3 tests, using SPSS 18.0, where p < 0.05 was considered to be statistically significant. The staff members performing the statistical analysis for this study were not involved in the clinical observation or data recording.
Results A total of 47 eyes were included in this study from 33 men (70%) and 14 women (30%). The average age was 53.5 9.1 years old, ranging from 18 to 72 years old. Twenty-three patients (nearly 50%) had a definite history of trauma caused by agricultural objects while working in the countryside. The average time period before the corneal ulcer’s being surgically managed was 36 6.3 days, with the longest wait being 8 months. The diameters of corneal ulcers ranged from 2.5 to 8 mm. The depth of the ulcers ranged from one-third of the corneal stromal thickness to the depth of Descemet’s membrane (for details, see Table S1). Forty-three patients were diagnosed by microscopic examination of the direct smear, and 12 were confirmed by preoperative confocal microscopy. Thirty-nine (83%) had tissues removed during the surgery that were also confirmed to have fungal infections via postoperative microbiological culturing or histopathology. Fungal spores and hyphae were found under in vivo confocal microscopy before the operation but not after surgery (Figure 1A and B). Compared to those of normal eyes, the smoothness of the surgical corneal surface curves was retained upon examination by ultrasound biomicroscopy (Figure 1C and D). Compared to the preoperative scores, the irritation symptoms from all patients were significantly reduced after the surgery (p < 0.05, Table 1). Bandage contact lenses were only used in nine patients (19%) who experienced severe eye irritation in the initial postoperative
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Figure 1: In vivo confocal microscopic and ultrasound biomicroscopic (UBM) images of patients. (A) The observation of fungal spores and hyphae under confocal microscopy before surgery. (B) No fungal infection was found in the keratocytes under confocal microscopy from a patient’s cornea 1 year after surgery. The surgical corneal surface curve (C) remained smooth under ultrasound biomicroscopy 1 year after the surgery, as compare to the counterpart eye (D).
Table 1: The evaluation score before and after surgery Irritation score before and after surgery
Corneal neovascularization score before and after surgery
Postoperative graft transparency score
Score
Preoperative1
3 day1
7 day1,2
1 month1,2
3 month1,2
6 month2
0 n (%) 1 n (%) 2 n (%) 3 n (%) Total n (%)
1 (2%) 14 (30%) 30 (64%) 2 (4%) 47 (100%)
0 28 (60%) 19 (40%) 0 47 (100%)
0 39 (83%) 8 (17%) 0 47 (100%)
5 (11%) 38 (80%) 4 (9%) 0 47 (100%)
19 (40%) 28 (60%) 0 0 47 (100%)
41 (87%) 6 (13%) 0 0 47 (100%)
Score
Preoperative3
3 day3
7 day3
1 month3
3 month3
6 month
0 n (%) 1 n (%) 2 n (%) 3 n (%) Total n (%)
16 (34%) 17 (36%) 13 (28%) 1 (2%) 47 (100%)
22 (47%) 24 (51%) 0 1 (2%) 47 (100%)
22 (47%) 24 (51%) 0 1 (2%) 47 (100%)
22 (47%) 20 (42%) 4 (9%) 1 (2%) 47 (100%)
30 (64%) 10 (21%) 5 (11%) 2 (4%) 47 (100%)
39 (83%) 7 (15%) 0 1 (2%) 47 (100%)
Score
3 day4
7 day4
1 month5
3 month5
6 month5
0 n (%) 1 n (%) 2 n (%) 3 n (%) Total n (%)
0 14 (30%) 33 (70%) 0 47 (100%)
0 26 (55%) 21 (45%) 0 47 (100%)
0 30 (64%) 17 (36%) 0 47 (100%)
0 40 (85%) 7 (15%) 0 47 (100%)
1 (2%) 40 (85%) 5 (11%) 1(2%) 47 (100%)
Has significant statistical difference compare with 6 months (p < 0.05). Has significant statistical difference compare with preoperative irritation score (p < 0.05). 3 Dunnett T3 test, has significant statistical difference compare with 6 months score (p < 0.05). 4 Has significant statistical difference compare with 6 months (p < 0.05). 5 Dunnett T3 test, has significant statistical difference compare with 3 days (p < 0.05). 1 2
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Figure 2: Slit-lamp biomicroscopic photographs of pre- and post-operative corneas. The epithelial healing times after surgery ranged from 3 days to 1 week, which was not different from times experienced following human corneal transplantation (A2, B2). The postoperative graft in one fungal infection case, comorbid with leucoma caused by viral keratitis in childhood, had the surgical treatment and healed well (B). The graft remained transparent, even though the pterygium had been growing into the graft of the patient, who had also experienced pterygium preoperatively (D). After 3 years of follow-up (C4, E4), the transparency of the grafts significantly improved compared with observations made at the 6-month follow-up (C3, E3) and was similar to the surrounding normal cornea. A1, B1, C1, D1, and E1 were taken preoperatively; A4, B4, C3, D4, and E3 were taken six months after surgery; C4 and E4 were taken 3 years after surgery.
period. The epithelial healing times ranged from 3 days to 1 week after the surgery and showed no significant difference to healing times following human corneal transplantation (Figure 2A and B). Although, various degrees of neovascularization were noted in 25 patients after the surgery, most neovascularization gradually regressed, and only seven patients (15%) needed the extra topical 0.1% tobradex treatment at the 3-month follow up visit (Table 1, Figure 2). One of the typical patients had a American Journal of Transplantation 2015; 15: 1068–1075
fungal corneal infection lasting for 6 months (Figure 3). With continuous use of anti-fungal drugs, the scope of the ulcer became smaller, but a large number of new vessels grew into the cornea from the superior direction, and ledging could be seen between the ulcer and the surrounding cornea (Figure 3A–C). After the operation, the graft healed well, with no any infection recurrence, and all new vessels regressed during a 3-month follow-up (Figure 3F). Despite the pterygium’s having grown into the grafts in five patients 1071
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Figure 3: Six-month disease course of the fungal infection in a typical patient. (A) The patient was hospitalized in March 2012 with a fungal infection diagnosed via corneal scraping and was given antifungal drug therapy. (B) The scope of the infection was demonstrated after the anti-fungal treatment one and a half months later. (C) After five months, the ulcer was localized in the pupil area, with a large number of new vessels growing into the cornea from the superior direction, and ledging with the normal tissue was observed. (D) By the third postoperative day, the epithelium had healed well. (E) Seven days after the surgery, the corneal graft showed little edema, and a large number of new vessels had also grown into the graft bed. (F) At the 3-month follow up, the new vessels had completely disappeared, and naked vision acuity was 0.4.
who had preoperative pterygia, the grafts remained transparent, and no patient required any additional surgical treatment at the 6-month follow-ups (Figure 2D). For patients with eccentric infections, the corresponding grafts also achieved good results, with no significant postoperative rejection being observed (Figure S1). All of these results collectively suggest that APCS has good histocompatibility and low immunogenicity, which benefits its clinical application, particularly in high-risk transplantation.
roidism, without exophthalmus or hypophasis. Despite of the dissolution of the grafts, all grafts eventually epithelialized, and no perforations or infection relapses occurred. Overall, during minimum follow-ups of 6 months, we observed no infection recurrences. All corneal ulcers
APCS grafts became transparent, and edema disappeared after the 1-month follow-up. The best corrected visual acuities (BCVAs) of 34 eyes, 72% of all patients, showed improvement of more than 2 lines. Of the remaining 13 eyes, only 2 showed a loss of more than one line, whereas the other 11 showed improvements of no more than 2 lines (Figure 4). For example, BCVAs improved to 1.0 in three patients during the 6-month follow-up period. More interestingly, in four patients for whom the follow-up period was prolonged to 3 years, the transparency of the grafts had noticeably improved when compared to only observed for 6 months. These grafts showed no obvious differences from the surrounding normal cornea (Figure 2C3, C4, E3, and E4). These results indicate that APCS grafts can gradually become transparent after an extended period in the absence of a rejection reaction, similar to results found using human donor corneas. During follow-up, various degrees of graft dissolution took place in four patients (Figure 5). Among them, two had infections approaching the endothelial layers pre-operatively. The other two had histories of well-controlled hyperthy1072
Figure 4: Changes in visual acuity after surgery. HM denotes ‘‘hand motion,’’ and CF means ‘‘counting fingers.’’ The diagonal line represents the point at which the values for visual acuity preoperatively and postoperatively were the same.
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Figure 5: The dissolved graft observed with slit-lamp biomicroscopy. (A) Preoperatively, the patient presented with hypopyon and anamnestically reported hyperthyroidism, but these were well controlled, with neither exophthalmus nor hypophasis being observed. One week after the operation, the graft started to dissolve. At the 1-month follow-up, the graft bed had completely epithelialized. There was no sign that the infection had recurred. The ulcer depth of the patient (B) and (C) was unable to be evaluated by the slit lamp preoperatively. However, during the operation, the ulcer infiltration was found to reach the endothelial layer. At 7 days postoperatively, double anterior chambers could be seen in (B2) with the dissolved graft. At one month postoperatively, the double anterior chambers had disappeared, as shown in (B3). All of the graft bed had completely epithelialized, with no recurrent infection noted in the review for 6 months (B, C).
healed, and neovascularization regressed. No patient showed severe adverse reaction, and no significant changes in postoperative systemic safety indicators were observed. Eventually, all of the APCS grafts gradually became transparent over the prolonged follow-up period.
Discussion With the increasing worldwide shortage of donor corneas for keratoplasty, there is a greater need to find a suitable substitute for donor corneas (15). As early as 2003, Amano et al suggested that heterogeneous corneal stromas, especially porcine corneal stromas, could be ideal alternatives to human corneas due to their lower antigenicities, as demonstrated in in vitro corneal tissue engineering, and transparently healing, as shown in animal experiments (16). In addition, studies regarding the successful construction of tissue-engineered hearts, lungs, and livers using acellular biomaterials have also been reported (17–19), which indicates that the acellular stroma is a suitable carrier for tissue engineering. Wang et al analyzed cellular and humoral immunity for three layers of porcine corneas and found that the immunogenicities of the intact endothelia, epithelia and stromas (all of equal thickness), as detected by American Journal of Transplantation 2015; 15: 1068–1075
cellular immunity, were 70.75%, 27.63%, and 1.62%, respectively. Similarly, the immunogenicities, as measured by humoral immunity, were 62.11%, 31.77%, and 6.12%, respectively (20). APCS has a cell affinity and can provide a relative healthy corneal stroma microenvironment as well as a substrate—the basement membrane that is conducive to corneal epithelial cell adhesion and growth. The mesh structure in the matrix layer can provide space for the growth of mesenchymal cells and the exchange of metabolites. In addition, APCSs can also be used as seed carriers (21). Our previous animal study (13), through implantation of APCSs into rabbit corneal pockets and mouse subcutaneous tissue, showed that the APCS is unable to stimulate a host response in the cornea. In subcutaneous tissue, the APCS only triggered an innate immune reaction, which had decreased by 28 days postimplantation. These results demonstrated that complete removal of the cell components of the cornea, which are thought to be the main source of the major histocompatibility complex antigens responsible for allograft/xenograft rejection, can obviously alleviate the immune response to the grafts. In addition, APCS implants could be integrated with body tissues and sufficiently support new tissue regeneration and reconstruction (22). Animal experiments have confirmed that APCS transplants can also be used 1073
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as cosmetic corneal lens implants (23). Although many studies in vitro and in animals have confirmed that APCS has excellent potential for solving the problem created by the serious lack of donor corneas, no large-scale clinical study of ACPS has ever been reported. For the first time, our study has proven that APCS is safe and effective in clinical application. Our clinical studies have shown that, similar to human donor corneas, the transplanted areas generally require three days for epithelialization in which the epithelial regeneration is the most important factor for reducing postoperative complications (Figure 2A and B). Compared with the biosynthetic cornea reported by Dr. May Griffith in 2010 (24), which showed that the highest corrected visual acuity of 0.4 was only achieved in six patients with markedly increased post-surgical astigmatism due to degradation of the biomaterials and delayed full epithelialization (for at least one month), APCS is far superior because it is similar to the natural cornea on the elastic modulus, has good toleration of sutures, provides cutting tension and permits enzymatic degradation in vivo. With a prolonged follow-up period, APCS grafts can gradually become transparent in patients with no rejection reactions, and patients’ vision also improves (Figure 3). Compared with the grafts observed at the 6-month follow-up, the transparency of the grafts at the 3-year follow up was almost equivalent to the surrounding normal corneal tissues (Figure 2C3, C4, E3, and E4). Considering that no obvious rejection reaction was observed, despite our inability to leverage anti-rejection glucocorticoid drugs during the early postoperative period on account of the fungal infections, as well as considering that this was high-risk transplantation in which large numbers of new vessels may have grown into the graft beds preoperatively, APCS appears to offer good histocompatibility and low immunogenicity in clinical applications. The study has also shown that APCS had a unique effect on the treatment of the fungal corneal infection, which has a high rate of morbidity and blindness in China and other developing countries. No fungal infection recurrence occurred in any of the 47 patients after the surgery, even in the cases where the grafts dissolved. This suggests that the porcine cornea, unlike human corneas, might not be susceptible to fungal invasion. However, this hypothesis requires further investigation. Our study suggests that to reduce the recurrence rate, it is necessary to rinse planting beds repeatedly with antifungal drugs during the surgery, and all ulcer lesions should be removed completely. Conservative treatment of fungal keratitis often disappoints because of limited tissue penetration, a narrow antimicrobial spectrum, and toxicity of the currently available antifungal agents (25,26). Furthermore, early diagnosis is difficult. The excessive use of antibiotics and glucocorticoid also aggravates the fungal infection situation. Because of poor responses to drug treatment, many patients eventually have to choose surgery. Penetrating keratoplasty (PKP) 1074
is always the first choice in the traditional surgical treatment (27). However, compared with LKP, traditional PKP causes more intraocular complications, especially in infection cases, and the postoperative endophthalmitis risk also increases significantly (28). Because most fungal keratitis lesions are mainly localized in the stromal layer, LKP not only can provide useful vision with few complications but also can save limited donor cornea resources (29). From experimental and clinical studies, Xie and coworkers proposed that LKP leads to lower endothelial cell disturbance and endophthalmitis incidence compared with PKP, with no statistically significant differences in infection recurrence rates (30). Our study has also proven that APCS is a suitable alternative to donor corneas in the treatment of fungal corneal ulcer using LKP. Because postoperative grafts healed well in one of the fungal infection cases that was comorbid with leucoma caused by viral keratitis in childhood (Figure 2B), we have deduced that, besides fungal corneal ulcer treatment, APCS also has other application potential, such as in the treatment of viral corneal ulcers. Taken together, our approaches may allow the restoration of diseased or damaged corneas that cannot be treated using currently available techniques without human donor corneas. Our study, through 3 years clinical observations, has demonstrated that all patients’ ulcers healed well and that neovascularization gradually regressed. No obvious rejections or other systemic or local side effects were observed. Overall, APCS grafts appear to be safe and efficacious during LKP in treating corneal fungal ulcers and may have other potential clinical applications.
Acknowledgments The APCSs were kindly provided by the Shenzhen AiNear Corneal Engineering Corporation. We thank Chong Tian for providing extensive statistical assistance to us.
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
References 1. Shang X, Zhang M. Body and organ donation in Wuhan, China. Lancet 2010; 376: 1033–1034. 2. Hashimoto Y, Funamoto S, Sasaki S, et al. Preparation and characterization of decellularized cornea using high-hydrostatic pressurization for corneal tissue engineering. Biomaterials 2010; 31: 3941–3948. 3. Pang K, Du L, Wu X. A rabbit anterior cornea replacement derived from acellular porcine cornea matrix, epithelial cells and keratocytes. Biomaterials 2010; 31: 7257–7265.
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Acellular Porcine Cornea in Keratoplasty 4. Li N, Wang X, Wan P, et al. Tectonic lamellar keratoplasty with acellular corneal stroma in high-risk corneal transplantation. Mol Vis 2011; 17: 1909–1917. 5. Shao Y, Yu Y, Pei CG, et al. Evaluation of novel decellularizing corneal stroma for cornea tissue engineering applications. Int J Ophthalmol 2012; 5: 415–418. 6. Xie L, Zhong W, Shi W, Sun S. Spectrum of fungal keratitis in north China. Ophthalmology 2006; 113: 1943–1948. 7. Nath R, Baruah S, Saikia L, Devi B, Borthakur AK, Mahanta J. Mycotic corneal ulcers in upper Assam. Indian J Ophthalmol 2011; 59: 367–371. 8. Gopinathan U, Sharma S, Garg P, Rao GN. Review of epidemiological features, microbiological diagnosis and treatment outcome of microbial keratitis: experience of over a decade. Indian J Ophthalmol 2009; 57: 273–279. 9. Thomas PA, Kaliamurthy J. Mycotic keratitis: Epidemiology, diagnosis and management. Clin Microbiol Infect 2013; 19: 210–220. 10. Jhanji V, Young AL, Mehta JS, Sharma N, Agarwal T, Vajpayee RB. Management of corneal perforation. Surv Ophthalmol 2011; 56: 522–538. 11. Zhang C, Nie X, Hu D, et al. Survival and integration of tissueengineered corneal stroma in a model of corneal ulcer. Cell Tissue Res 2007; 329: 249–257. 12. Lin XC, Hui YN, Wang YS, Meng H, Zhang YJ, Jin Y. Lamellar keratoplasty with a graft of lyophilized acellular porcine corneal stroma in the rabbit. Vet Ophthalmol 2008; 11: 61–66. 13. Luo H, Lu Y, Wu T, Zhang M, Zhang Y, Jin Y. Construction of tissueengineered cornea composed of amniotic epithelial cells and acellular porcine cornea for treating corneal alkali burn. Biomaterials 2013; 34: 6748–6759. 14. Tsubota K, Satake Y, Kaido M, et al. Treatment of severe ocularsurface disorders with corneal epithelial stem-cell transplantation. N Engl J Med 1999; 340: 1697–1703. 15. Lawlor M, Kerridge I, Ankeny R, Dobbins TA, Billson F. Specific unwillingness to donate eyes: the impact of disfigurement, knowledge and procurement on corneal donation. Am J Transplant 2010; 10: 657–663. 16. Amano S, Shimomura N, Kaji Y, Ishii K, Yamagami S, Araie M. Antigenicity of porcine cornea as xenograft. Curr Eye Res 2003; 26: 313–318. 17. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science 2010; 329: 538–541. 18. Shupe T, Williams M, Brown A, Willenberg B, Petersen BE. Method for the decellularization of intact rat liver. Organogenesis 2010; 6: 134–136. 19. Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: Using nature’s platform to engineer a bioartificial heart. Nat Med 2008; 14: 213–221.
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20. Wang Z, Ge J, Xu J, Chen J. Relative quantitative analysis of corneal immunogenicity. Zhonghua Yan Ke Za Zhi 2002; 38: 535– 538. 21. Zhu J, Zhang K, Sun Y, et al. Reconstruction of functional ocular surface by acellular porcine cornea matrix scaffold and limbal stem cells derived from human embryonic stem cells. Tissue Eng Part A 2013; 19: 2412–2425. 22. Du L, Wu X. Development and characterization of a full-thickness acellular porcine cornea matrix for tissue engineering. Artif Organs 2011; 35: 691–705. 23. Liu Z, Zhou Q, Zhu J, et al. Using genipin-crosslinked acellular porcine corneal stroma for cosmetic corneal lens implants. Biomaterials 2012; 33: 7336–7346. 24. Fagerholm P, Lagali NS, Merrett K, et al. A biosynthetic alternative to human donor tissue for inducing corneal regeneration: 24-month follow-up of a phase 1 clinical study. Sci Transl Med 2010; 2: 46ra61. 25. Abdel-Rhaman MS, Soliman W, Habib F, Fathalla D. A new longacting liposomal topical antifungal formula: human clinical study. Cornea 2012; 31: 126–129. 26. FlorCruz NV, Peczon IV, Evans JR. Medical interventions for fungal keratitis. Cochrane Database Syst Rev 2012; 2: CD004241. 27. Sharma N, Sachdev R, Jhanji V, Titiyal JS, Vajpayee RB. Therapeutic keratoplasty for microbial keratitis. Curr Opin Ophthalmol 2010; 21: 293–300. 28. Tan DT, Dart JK, Holland EJ, Kinoshita S. Corneal transplantation. Lancet 2012; 379: 1749–1761. 29. Xie L, Shi W, Liu Z, Li S. Lamellar keratoplasty for the treatment of fungal keratitis. Cornea 2002; 21: 33–37. 30. Shi W, Wang T, Xie L, et al. Risk factors, clinical features, and outcomes of recurrent fungal keratitis after corneal transplantation. Ophthalmology 2010; 117: 890–896.
Supporting Information Additional Supporting Information may be found in the online version of this article. Figure S1: Slit-lamp biomicroscopic photographs of the patients with eccentric infections. Data S1: Inclusion and exclusion criteria. Table S1: The evaluation scores of preoperative ulcer depths and widths.
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