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Ocular Surface Reconstruction With Autologous Nasal Mucosa in Cicatricial Ocular Surface Disease
Ocular Surface Reconstruction With Autologous Nasal Mucosa in Cicatricial Ocular Surface Disease JAE HOON KIM, YEOUN SOOK CHUN, SEOK HYUN LEE, SEOG KYUN MUN, HAENG SUN JUNG, SU HYON LEE, YOUNGSOOK SON, AND JAE CHAN KIM ● PURPOSE: To investigate the possibility of replacing the
metaplastic ocular surface with nasal mucosa, and to evaluate the results of autologous nasal and oral mucosal transplantation in cicatricial ocular surface diseases. ● DESIGN: Retrospective interventional case series. ● METHODS: We studied 6 eyes in 6 patients with chemical burns, which were characterized by a cicatricial ocular surface. After removal of cicatricial tissues and symblepharolysis, autologous nasal mucosa was transplanted in all patients. In 3 patients with extensive damage, oral mucosal autografting was performed concurrently. The nasal and oral mucosa was evaluated using immunohistochemical analysis for p63, K3, MUC5AC, and CD34. Clinical outcomes were assessed based on visual acuity, ocular manifestations, and liquid-based cytology. ● RESULTS: Immunohistochemical analysis revealed a plentitude of p63 and K3 in nasal mucosal epithelium. Goblet cells and MUC5AC expression were only observed in nasal mucosal epithelium, not in oral mucosal epithelium. Well-developed parallel vasculature was demonstrated in the nasal mucosa. In contrast, perpendicular vasculature was demonstrated in the oral mucosa. This vascular feature remained after transplantations. In all patients, ocular surface stability recovered with no major complications and increased goblet cells were observed on ocular surface. However, delayed epithelialization and ischemic thinning were seen at oral mucosal graft sites. ● CONCLUSIONS: Nasal mucosa, which has the advantage of well-developed parallel vasculature, enriched goblet cells, and plenty of stem cells, may be an ideal substitute for a cicatricial ocular surface. Transplantation of autologous nasal mucosa is a very effective method for achieving ocular surface reconstruction in cicatricial ocular surface diseases. (Am J Ophthalmol Accepted for publication Jul 22, 2009. From the Departments of Ophthalmology (J.H.K., Y.S.C., Se.H.L., J.C.K.) and Otorhinolaryngology–Head and Neck Surgery (S.K.M.), College of Medicine, Chung-Ang University, Yongsan Hospital, Seoul, Republic of Korea; Research and Development Institute, Modern Cell and Tissue Technologies Incorporated, Seoul National University of Technology, Seoul, Republic of Korea (H.S.J., Su.H.L.); and the Department of Genetic Engineering, College of Life Science, Kyung Hee University, Gyeonggi-do, Republic of Korea (Y.S.). Inquiries to Jae Chan Kim, Department of Ophthalmology, ChungAng University, Yongsan Hospital, 65-207, Hangangro-3Ga, YongsanGu, Seoul 140-757, Republic of Korea; e-mail: [email protected] 0002-9394/10/$36.00 doi:10.1016/j.ajo.2009.07.030
©
2010 BY
2010;149:45–53. © 2010 by Elsevier Inc. All rights reserved.)
C
ICATRICIAL OCULAR SURFACE DISEASES SUCH AS
Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and chemical/thermal burns are very difficult and challenging disorders for ophthalmologists. These chronic and excessive inflammatory diseases induce destruction of corneal epithelial stem cells located in the limbus, loss of conjunctival goblet cells producing mucins, and ultimately squamous metaplastic epithelium-like skin.1–3 These changes cause severe visual loss, ocular pain, limitations in eyeball movement, and cosmetic problems. Regarding the treatment of these diseases, ocular surface reconstruction may be required to replace the cicatricial tissues with stem cells or a stem cell niche and conjunctival goblet cells. Normal, healthy ocular surface tissues of the fellow eye, such as conjunctiva or limbus,4 may be the best source of ocular surface reconstruction materials. However, in bilateral disease, this approach is not an option. Furthermore, harvesting of the fellow eye is restrictive and often provides an insufficient quantity of tissue. Therefore, amniotic membrane (AM)5–7 and various autologous mucosal tissues, such as oral mucosa,8 hard palate mucosa,9 maxillary sinus mucosa,10 or nasal mucosa have been used for ocular surface reconstruction.11,12 With the recent development of tissue engineering techniques, methods using autologous cultivation of corneal epithelial cells,13 limbal cells,14 or oral mucosal epithelial cells have come into the spotlight.15–17 However, tissue engineering techniques are possible in only some restricted hospitals, because these techniques are fastidious and require much effort and expense. Hence, nasal mucosa and oral mucosa, which can be easily accessed and obtained, may be practical tissue sources for clinicians. Especially, nasal mucosa has enriched goblet cells in the epithelium and submucosa, which secrete the mucin required for tear film stabilization. In 1990, Naumann and associates11 introduced autologous nasal mucosa transplantation in severe conjunctival mucus deficiency syndrome. After that, there were a few reports in which the nasal mucosa was used in eyelid reconstructive surgeries.18,19 However, nasal mucosal transplantation has not been used broadly in ocular surface reconstruction as of yet because most ophthalmologists hesitate to harvest the nasal mucosa. There have been no studies assessing the
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FIGURE 1. Histology and immunohistochemistry of the nasal mucosa and oral mucosa (magnification ⴛ400). (Top left) Periodic acid–Schiff (PAS) staining demonstrated goblet cells only in the nasal mucosal epithelium, (Top right) not in the oral mucosal epithelium. (Bottom left) MUC5AC immunostaining showed goblet cells containing mucin only in the nasal mucosal epithelium, (Bottom right) not in the oral mucosal epithelium.
immunohistochemical characteristics of the nasal mucosa and comparing them to those of the ocular surface. Therefore, we investigated the possibility of replacing the metaplastic ocular surface with autologous nasal mucosa by analyzing the histologic and immunohistochemical characteristics of the nasal mucosa. We also evaluated and compared the results of ocular surface reconstruction with autologous nasal mucosa and oral mucosa in cicatricial ocular surface diseases, especially ocular chemical burns.
to determine the state of the nasal and oral mucosa. After preparation of a 5% povidone-iodine solution for antiseptic purposes, nasal mucosal tissues, each measuring 2 cm2, were stripped in situ from the one-third portion to the two-thirds portion of the inferior nasal turbinate using a blade and scissors in all patients. After harvest, bleeding was controlled by bipolar cauterization under an endoscope. In 3 patients who exhibited entire cicatricial ocular surface change, and for whom ocular surface reconstruction using only AM and nasal mucosa was expected to be insufficient, oral mucosal tissues, each measuring 1 cm2, were taken from the lower inner lip mucosa. Nasal and oral submucosal connective tissues were removed and trimmed into thin, flat mucosal tissues 2 mm in thickness with scissors and a blade.
METHODS ● PATIENTS: We obtained informed consent from 6 patients (6 eyes) with chemical burns. They had all undergone prior ocular surgery with AMs more than twice. Nevertheless, they all had devastating cicatricial squamous metaplasia, recurrent corneal ulcers, total limbal deficiency, conjunctivalization, fibrovascular proliferation, and symblepharon. Hence, we performed ocular surface reconstruction using AM transplantation, nasal mucosal autografting, oral mucosal autografting, and conjunctival limbal autografting on these 6 damaged eyes.
● SURGICAL PROCEDURES:
All surgical procedures were performed by one surgeon (J.C.K.) on a case-by-case basis, with each patient under retrobulbar anesthesia. All patients underwent symblepharolysis and excision of cicatricial and fibrovascular tissue. In 3 patients who had no vision, but who had cosmetic problems, corneal tattooing was performed using India ink (True Black Eye Liner; Biotouch Products, Walnut, California, USA). Thereafter, permanent AM grafting was performed on bare sclera and whole cornea in all patients using cryopreserved AM (AmniSite-Cornea; Bioland Ltd, Cheonan, Korea) with
● PREPARATION OF AUTOLOGOUS ORAL MUCOSA AND NASAL MUCOSA: All patients were examined carefully
before the operation by an otorhinolaryngologist in order 46
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FIGURE 2. K3 and P63 immunohistochemistry of the nasal mucosa (magnification ⴛ400). (Left) Keratin 3 was expressed in the nasal mucosal epithelium, similar to cornea and conjunctiva. (Right) P63, a stem cell marker, was highly expressed (arrow) in the basal layer of the nasal mucosal epithelium.
FIGURE 3. Comparison of the nasal mucosal and oral mucosal vasculature. (Top left) Flat-mount immunohistochemical staining of CD34, which was expressed in vascular endothelial cells, showed a parallel vascular direction in the nasal mucosa. (Top right) In contrast, CD34 staining showed mainly a round dot pattern, which indicated a perpendicular vascular direction in the oral mucosa (magnification ⴛ100). These vascular features remained after transplantation of nasal and oral mucosa as well. (Bottom left) One year after surgery, the vascular plexus at the nasal mucosal graft site (inside white loop) in Case 1 showed a parallel direction, (Bottom right) while that at the oral mucosal graft site in Case 2 showed a perpendicular vascular direction.
interrupted 10-0 nylon sutures. Nasal mucosal autografting (in 6 eyes) and oral mucosal autografting (in 3 eyes; Cases 1 to 3) were performed on sclera over the permanent AM grafting with interrupted 10-0 nylon sutures. Conjunctival limbal autografting was performed in the perilimbal area over the permanent AM grafting in 2 patients (Cases 2 and 5) using the method described by Kenyon and Tseng.4 In the 4 remaining patients, it was impossible to harvest limbal tissue from the fellow eye, because of previous harvesting of conjunctivolimbal tissue or because of VOL. 149, NO. 1
chemical burn–induced damage to the ocular surface. Lastly, temporary AM patches were placed in all patients. After surgery, 20% autologous serum eye drops were instilled every 2 hours, 0.5% levofloxacin (Levaquin; Ortho-McNeil Pharmaceutical, Titusville, New Jersey, USA) was used topically 4 times a day, and topical steroid ointment (dexamethasone [Decadron; Merck & Co Inc, Whitehouse Station, New Jersey, USA] polymyxin B sulfate, and neomycin sulfate) was administered 3 times daily until epithelialization was complete. During the first
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FIGURE 4. Clinical appearance before and after ocular surface reconstruction using autologous nasal mucosa in Cases 2 (Top), 3 (Middle), and 4 (Bottom). (Top, Middle, and Bottom left) Preoperatively, there was total conjunctivalization with severe scarring, symblepharon, and ulceration of the ocular surface. One year after surgery, (Top, Middle, and Bottom center and Bottom right) we noted stabilized ocular surfaces and (Top and Middle right) abundant mucin secretion (arrow).
week after surgery, prednisolone (30 mg per day) and third generation cephalosporins (cefditoren pivoxil; [Spectracef; Purdue Pharmaceutical Products, Stamford, Connecticut, USA] 300 mg per day) were administered orally. Five days after surgery, temporary AM patches were removed and preservative-free artificial tears were used.
and MUC5AC were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). The mucosal tissues were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) at 4 C for 10 minutes and then permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 minutes. The endogenous peroxidase was quenched with 0.3% H2O2 in 0.5% horse serum in PBS and incubated with 5% horse serum to block the nonspecific sites. Monoclonal antibodies against p63, K3, CD34, or MUC5AC were applied and incubated for 1 hour at room temperature, followed by incubation with biotinylated second antibodies, anti-mouse, or anti-rabbit IgG, using a Vectastain Elite ABC Kit (PK6101; Vector Laboratories, Burlingame, California, USA), according to the manufacturer’s protocol. Samples were finally incubated with diaminobenzidine (DAB) peroxidase substrate to give a brown stain and were then counterstained with hematoxylin. After washing with PBS, the samples were mounted and analyzed.
● CLINICAL EVALUATION: Preoperative and postoperative best-corrected visual acuity (BCVA) and intraocular pressure were measured. Ocular surface manifestations, the presence of corneal erosion, corneal neovascularization, the recurrence of symblepharon, epithelial integrity, and survival of the mucosal graft were evaluated at the first day, third day, first week, second week, fourth week, and every month postoperatively with a slit-lamp microscope (SL 130; Carlzeiss Meditec Inc., Dublin, California, USA) and fluorescein staining. Clinical success was defined as intact ocular surface epithelium without epithelial erosion, maintained for greater than 4 weeks.
● LIQUID-BASED CYTOLOGY: Liquid-based cytology was performed to evaluate for the presence of goblet cells, before surgery and 1 year after surgery, in Cases 4 and 5. Each patient turned his head to the operative eye in the supine position. After that, 1 ml of balanced salt solution (BSS; Alcon Laboratories, Fort Worth, Texas, USA) was dropped from the medial bulbar conjunctiva to the lateral canthus and collected in an Eppendorf tube. A commer-
● HISTOLOGIC AND IMMUNOHISTOCHEMICAL ANALYSIS:
Hematoxylin-eosin staining, periodic acid–Schiff (PAS) staining, and immunohistochemical staining for p63, keratin 3 (K3), CD34, and MUC5AC were performed to evaluate the epithelial characteristics in the remnant nasal mucosa and oral mucosa of Case 1, after the mucosa was trimmed. Monoclonal antibodies against K3, p63, CD34, 48
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FIGURE 5. Liquid-based cytologic findings before and after ocular surface reconstruction using autologous nasal mucosa in Cases 4 (Top) and 5 (Bottom). Before surgery, (Top left and Bottom left) only squamous cells and inflammatory cells without any goblet cells were observed. However, 1-year after surgery, (Top right and Bottom right) several goblet cells (arrow) were easily found.
FIGURE 6. Illustrations of ocular surface reconstructive procedures and postoperative slit-lamp photographs in Cases 1 (Top) and 2 (Bottom). Ten months after surgery, (Top center) well-developed horizontal vascularization (black arrow) was present at the nasal mucosal autograft site, (Top right) whereas ischemic thinning (white arrow) was present at the oral mucosal autograft site. Two weeks after surgery, (Bottom center) abundant mucus (white arrowhead) and (Bottom right) complete epithelialization (white asterisk) were observed at the nasal mucosal autograft site, but the entire epithelial defect (black asterisk) was still present at the oral mucosal autograft site.
cially manufactured fixative (CytoRich Red; TriPath Imaging Inc, Burlington, North Carolina, USA) was added to the collected specimen and vortex-mixed for 15 minutes. After the specimen sat for 30 minutes, it was centrifuged at 600 g for 10 minutes; the supernatant was then decanted. As for washing, 10 ml of water was added to the specimen, which was then vortexed. The specimen was then centrifuged at 600 g for 5 minutes, and the supernatant was decanted again. After the specimen was loaded onto a PrepStain system (TriPath Imaging Inc) for processing, a VOL. 149, NO. 1
uniform thin layer of cells was produced in a 13-mm-diameter circle. Interpretations were made by applying fundamental cytologic guidelines practiced by pathologists.20
RESULTS ● HISTOLOGIC AND IMMUNOHISTOCHEMICAL ANALYSIS: PAS staining and MUC5AC immunostaining dem-
onstrated goblet cells containing mucin only in the nasal
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TABLE. Characteristics and Clinical Outcomes of Patients Receiving a Nasal Mucosal Autograft in Cicatricial Ocular Surface Disease BCVA Case
Gender/Age
Disease
Previous Operative
Combined Operative
1
M/58
Alkali burn
FR, AMT(⫻2)
FR, AMT, OMAU
2
M/60
Acid burn
FR, AMT(⫻2)
FR, AMT, CLAU, OMAU
3
M/48
Alkali burn
FR(⫻2), AMT(⫻4)
4
M/50
Flame burn
5
M/42
6
M/53
Follow-up (months)
Preoperative
Last
Postoperative Status
LP(⫹)
HM(⫹)
20/1000
20/200
FR, AMT, CT, OMAU
LP(⫺)
LP(⫺)
AMT(⫻2)
FR, AMT, CT
LP(⫺)
LP(⫺)
Alkali burn
AMT(⫻2)
FR, AMT, CLAU
20/1000
20/320
Thermal burn
AMT(⫻2)
FR, AMT, CT
LP(⫺)
LP(⫺)
Ischemic thinning on OMAU Delayed epithelialization on OMAU Stabilized ocular surface Stabilized ocular surface Stabilized ocular surface Stabilized ocular surface
16 16 18 14 12 12
AMT ⫽ amniotic membrane transplantation; BCVA ⫽ best-corrected visual acuity; CLAU ⫽ conjunctiva limbal autograft; CT ⫽ corneal tattooing; FR ⫽ fornix reconstruction; HM ⫽ hand motion; LP ⫽ light perception; OMAU ⫽ oral mucosal autograft.
mucosal epithelium, but not in the oral mucosal epithelium (Figure 1). K3 was strongly expressed in the nasal mucosal epithelium, similar to cornea and conjunctiva. Furthermore, P63, a stem cell marker, was expressed throughout the whole basal layer of the nasal mucosal epithelium (Figure 2). Flat-mount immunostaining for CD34, which was expressed in vascular endothelial cells, showed a longish pattern representing the parallel vascular network to the epithelium in the nasal mucosa. However, the oral mucosa stained with CD34 showed a mainly round-dot vascular pattern (though a partially longish pattern was found) (Figure 3). This implies that the oral mucosa has a perpendicular vascular network to the epithelium, as contrasted with the parallel vasculature of the nasal mucosa. These vascular features remained at 1-year after transplantation of nasal or oral mucosa in all patients (Figure 3).
were observed on liquid-based cytology. However, 1- year after surgery, several goblet cells were easily found (Figure 5). All transplanted conjunctival limbal tissues around the nasal mucosal graft were well healed and living. However, poor, delayed epithelialization and pale ischemic thinning were found at the oral mucosal graft sites (Figure 6). Preoperative BCVAs were no light perception (3 eyes), light perception (1 eye), and 20/1000 (2 eyes). Postoperative BCVAs had improved in 3 eyes (excluding the 3 eyes that underwent corneal tattooing) at the last follow-up examination (Table). ● CASE 1:
A 58-year-old Korean man presented with fibrovascular tissue covering the entire ocular surface, symblepharon, and limited ocular motility following two AM transplantations and 1 fornix reconstruction surgery at another hospital. He had incurred an alkali burn in July 2005. After removal of the scarred fibrovascular tissue, symblepharolysis, and permanent AM grafting on the cornea and conjunctiva, we performed nasal mucosal autografting on the superior nasal bulbar conjunctiva and oral mucosal autografting on the superior temporal bulbar conjunctiva (Figure 6). Finally, temporary AM patch was performed on the whole ocular surface. The patient’s BCVA improved from light perception to hand motion. Ocular motility improved, and the ocular surface stabilized with abundant mucin secretion at the last follow-up examination (16 months after the operation). However, ischemic thinning was observed at the oral mucosal graft site, starting 3 months after the operation. Well-developed horizontal vascularization was noted at the nasal mucosal graft site (Figure 6).
● CLINICAL OUTCOME:
The mean follow-up period in our study was 14.7 months (range, 12 to 18 months). Nasal and oral mucosal tissues were safely excised without any complications, and their mucosal harvest sites were completely healed in 2 weeks. In all patients, the ocular surface was stabilized, and there were no recurrences of ulceration, conjunctivalization, fibrovascular tissue, or symblepharon (Figure 4). Clinical success was achieved without any major postoperative complications. Eyeball movement was improved in all patients. All patients noted improvement in ocular comfort, and 3 patients with corneal tattooing were very satisfied with their cosmetic results. We noted rapid epithelial healing, well-developed horizontal vascularization, and abundant mucin in all nasal mucosal graft sites. The existence of mucin and goblet cells was confirmed by liquid-based cytology. Before surgery, only squamous cells and inflammatory cells without any goblet cells
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● CASE 2:
A 60-year-old Korean man presented with conjunctivalization, recurrent ulceration, symblepharon,
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and limited ocular motility in spite of two ocular surface reconstructions using AMs in another hospital, secondary to alkali burns. After removal of the conjunctivalized fibrovascular tissue, symblepharolysis, permanent AM grafting on the cornea, and conjunctival limbal autografting in the fellow eye, nasal mucosal autografting and oral mucosal autografting were performed on the superior nasal bulbar conjunctiva and inferior nasal bulbar conjunctiva (Figure 6). Temporary AM patch was performed on the whole ocular surface as well. Two weeks after surgery, the nasal mucosal autografting had completely healed and was secreting mucin, but the whole epithelial defect was still present at the oral mucosal autografting site (Figure 6). Four weeks after surgery, the ocular surface was stabilized, and the epithelial defect at the oral mucosal graft site had healed. BCVA improved significantly from 20/1000 to 20/200, and ocular motility improved as well.
DISCUSSION THIS STUDY DEMONSTRATES THE POSSIBILITY OF REPLAC-
ing cicatricial ocular surface tissues with nasal mucosa by identifying the characteristics of nasal mucosa and documenting successful clinical autologous nasal mucosal transplantation. The expression of K3 in nasal mucosal epithelium, like normal cornea and conjunctiva, suggests that nasal mucosa may be one of the appropriate tissues to replace the cicatricial ocular surface.21 The expression of p63, a stem cell marker, in the whole basal layer of the nasal mucosal epithelium indicates that nasal mucosa may restore the cicatricial ocular surface as a source of stem cells/stem cell niche. K3 and p63 are also expressed in the oral mucosal epithelium, according to previous reports.22–24 However, a point of difference is that goblet cells and MUC5AC expression are only present in nasal mucosal epithelium, not in the oral mucosal epithelium. In the normal ocular surface, cornea and conjunctival nonkeratinizing squamous epithelial cells produce the membrane-associated mucins MUC1, MUC4, and MUC16.25–27 The gel-forming mucin MUC5AC is secreted solely by conjunctival goblet cells, and the secretory mucins MUC2 and MUC7 are produced in corneal and conjunctival epithelium.28 –30 The MUC genes of the nasal mucosa are very similar to those of the conjunctiva. MUC1, MUC2, MUC4, MUC5AC, and MUC5B are expressed in nasal mucosal epithelium, and MUC7 is expressed in nasal mucosal glands.31 Hence, nasal mucosal transplantation can replenish these mucins and stabilize the tear film and ocular surface naturally. As expected on the basis of these nasal mucosal characteristics, using liquid-based cytology, we observed a stabilized and recovered ocular surface with abundant mucin secretion and increased goblet cells on the ocular surface more than 1-year after nasal mucosal VOL. 149, NO. 1
transplantation. Although Weinberg and associates reported a loss of goblet cells in nasal mucosal autografting on the posterior eyelid surface,19 goblet cell loss was observed only on the grafts placed in the palpebral conjunctiva, not in the bulbar conjunctiva. One report noted that functional goblet cells persisted in autologous nasal mucosa for up to 10 years after transplantation on the ocular surface.12 This difference in the survival of goblet cells in nasal mucosal grafts may be associated with environmental conditions, especially air flow. Because the nasal mucosa is in contact with air flow normally, we believe that nasal mucosa transplanted on the bulbar conjunctiva, which is in contact with air flow, may maintain its goblet cells. There are other potential mucosal donor sites, such as rectal mucosa and vaginal mucosa, both of which have a significant number of goblet cells. However, when considering additional time to harvest, risks of donor site complications, and donor site pain, ophthalmologists and patients may hesitate to use these donor sites. In this study, it took a short time (about 10 minutes) to harvest nasal mucosal tissues. After harvesting, there were no instances of severe pain and no complications at the nasal mucosal donor sites. Hence, we believe nasal mucosal harvesting is not difficult or dangerous to perform. Another remarkable histologic and immunohistochemical finding is the vascular direction in nasal mucosa and oral mucosa. Flat-mount histology and immunostaining for CD34, a vascular endothelial cell marker, showed that the nasal mucosa had a horizontal vascular direction. In contrast, on histologic analysis and immunostaining for CD34, we found that the oral mucosa had a vertical vascular direction. Clinically, well-developed horizontal vasculature, rapid healing, and graft survival were seen at all nasal mucosal autografting sites. Meanwhile, delayed, poor epithelialization and ischemic thinning were observed at the oral mucosal autografting sites. Even if these findings are observed in only 3 cases, these suggest that the differing vascular patterns in nasal mucosa and oral mucosa may be correlated with the differing clinical results obtained with each graft. Although these vascular features have not yet been fully explained, they may have relevance to the fact that nasal mucosa has an extensive system of arteriovenous anastomoses and a profuse subepithelial capillary network that connects to a plexus of deeper vessels in the submucosa to exchange heat with flowing air.32,33 Pinkish, well-developed vascularizations of nasal mucosa may induce cosmetic problems, though they are advantageous for restoring the ocular surface. Hence, transplantation of nasal mucosa would be preferable at conjunctival sites eclipsed by the upper eyelids. Also, we observed that all conjunctival limbal autografting around nasal mucosal autografting healed well without any complications. We believe that nasal mucosal autografting may support the conjunctival limbal autograft-
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ing with its well-developed vasculature, enriched goblet cells and mucins, and increased p63 expression, though further studies about outcomes of conjunctival limbal autografting in the presence of nasal mucosal autografting are required. Cultivated oral mucosal epithelial sheets, which have been recently developed and introduced, also have the proper characteristics for ocular surface reconstruction, such as expression of p63, K3, and membrane-associated mucins MUC1, MUC4, and MUC16.17,34 Many clinical studies have demonstrated sufficient effects and good clinical outcomes with cultivated oral mucosal epithelial transplantation in severe ocular surface disorders.16,17,35,36 However, there are no goblet cells that express the secretory mucin MUC5AC in cultivated oral mucosal epithelial cells. Hence, nasal mucosa may be a more appropriate substrate for the conjunctiva, though cultivated oral mucosal sheets may be the ideal substrate for cornea. Furthermore, the tissue-engineering technique is fastidious and requires much effort and expense, and it is still possible in only some restricted hospitals.
Only patients with severe ocular burn were included in this study. However, successful nasal mucosal autografting results have been reported in other cicatricial ocular surface diseases, such as Stevens-Johnson syndrome and ocular pemphigoid.12 If the nasal mucosal donor site is not involved in these diseases on examination before harvesting, nasal mucosal autografting can be performed and may be effective. In summary, we noted that ocular surface stability recovered after surgery in all patients, leading to cosmetic and functional improvement with no major postoperative complications. Moreover, we verified the suitability of the nasal mucosa for the ocular surface based on histologic and immunohistochemical analysis. This study suggests that nasal mucosa, which has a welldeveloped horizontal vasculature, enriched goblet cells, and plenty of stem cells, may be an ideal substitute tissue for a cicatricial ocular surface, especially conjunctiva. We reaffirm that transplantation of autologous nasal mucosa is a very effective and beneficial method for achieving ocular surface reconstruction in cicatricial ocular surface diseases.
THIS STUDY WAS SUPPORTED BY A GRANT OF THE KOREA HEALTHCARE TECHNOLOGY R&D PROJECT, MINISTRY FOR HEALTH, Welfare & Family Affairs, Republic of Korea (A084721). The authors indicate no financial conflict of interest. Involved in design of study (J.C.K.); conduct of study (J.H.K., Se.H.L., S.K.M., J.C.K.); data collection (Se.H.L.); management, analysis, and interpretation of data (J.H.K.); histologic and immunohistochemical analysis (H.S.J., Su.H.L., Y.S.); and preparation (J.H.K.), review (Y.S.C.), and approval of manuscript (J.C.K.). This study was approved by the Institutional Review Board for Human Studies of Chung-Ang University Yongsan Hospital.
10. Fry TL, Wood CI. Readily available full-thickness mucous membrane graft. Arch Otolaryngol Head Neck Surg 1987; 113:770 –771. 11. Naumann GO, Lang GK, Rummelt V, Wigand ME. Autologous nasal mucosa transplantation in severe bilateral conjunctival mucus deficiency syndrome. Ophthalmology 1990; 97:1011–1017. 12. Wenkel H, Rummelt V, Naumann GO. Long-term results after autologous nasal mucosal transplantation in severe mucus deficiency syndromes. Br J Ophthalmol 2000;84:279 – 284. 13. Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, de Luca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 1997;349:990 –993. 14. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000;343:86 –93. 15. Nakamura T, Kinoshita S. Ocular surface reconstruction using cultivated mucosal epithelial stem cells. Cornea 2003; 22:S75–S80. 16. Nakamura T, Inatomi T, Sotozono C, Amemiya T, Kanamura N, Kinoshita S. Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders. Br J Ophthalmol 2004;88:1280 – 1284. 17. Nishida K, Yamato M, Hayashida Y, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 2004;351: 1187–1196.
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29.
30.
31.
32.
33.
34.
35.
36.
human conjunctival epithelia. Invest Ophthalmol Vis Sci 1996;37:1684 –1692. Argueso P, Gipson IK. Epithelial mucins of the ocular surface: structure, biosynthesis, and function. Exp Eye Res 2001;73:281–289. Jumblatt MM, McKenzie RW, Steele PS, Emberts CG, Jumblatt JE. MUC7 expression in the human lacrimal gland and conjunctiva. Cornea 2003;22:41– 45. McKenzie RW, Janblatt JE, Janblatt MM. Quantification of MUC2 and MUC5AC transcripts in human conjunctiva. Invest Ophthalmol Vis Sci 2000;41:703–708. Groneberg DA, Peiser C, Dinh QT, et al. Distribution of respiratory mucin proteins in human nasal mucosa. Laryngoscope 2003;113:520 –524. Cauna N. Blood and nerve supply of the nasal lining. In: Proctor DF, Anderson IB, editors. The Nose: Upper Airway Physiology and the Atmospheric Environment. Amsterdam: Elsevier Biomedical Press, 1982:45–70. Baraniuk JN, Lundgren JD, Okayama M, et al. Substance P and neurokinin A in human nasal mucosa. Am J Respir Cell Mol Biol 1991;4:228 –236. Hori Y, Sugiyama H, Soma T, Nishida K. Expression of membrane-associated mucins in cultivated human oral mucosal epithelial cells. Cornea 2007;26:S65–S69. Inatomi T, Nakamura T, Kojyo M, Koizumi N, Sotozono C, Kinoshita S. Ocular surface reconstruction with combination of cultivated autologous oral mucosal epithelial transplantation and penetrating keratoplasty. Am J Ophthalmol 2006; 142:757–764. Inatomi T, Nakamura T, Koizumi N, Sotozono C, Yokoi N, Kinoshita S. Mid-term results on ocular surface reconstruction using cultivated autologous oral mucosal epithelial transplantation. Am J Ophthalmol 2006;141:267–275.
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Biosketch Jae Hoon Kim, MD, is currently an Ophthalmology resident at Chung-Ang University Yongsan Hospital, Seoul, Korea. He graduated and received his medical degree from the Chung-Ang University in 2002. His research interests include ocular surface and ophthalmic plastic and reconstruction surgery.
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Biosketch Jae Chan Kim, MD, PhD, is a Professor and Chief instructor of the Ophthalmology and a Director of the Department of Ophthalmology at Chung-Ang University Yongsan Hospital, Seoul, Korea. He graduated and received medical degree and PhD from the College of Medicine in Chung-Ang University, and completed his residency training at the Department of Ophthalmology in Chung-Ang University Hospital. His research interests include amniotic membrane, stem cells, and ocular surface reconstruction using tissue engineering.
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metaplastic ocular surface with nasal mucosa, and to evaluate the results of autologous nasal and oral mucosal transplantation in cicatricial ocular surface diseases. ● DESIGN: Retrospective interventional case series. ● METHODS: We studied 6 eyes in 6 patients with chemical burns, which were characterized by a cicatricial ocular surface. After removal of cicatricial tissues and symblepharolysis, autologous nasal mucosa was transplanted in all patients. In 3 patients with extensive damage, oral mucosal autografting was performed concurrently. The nasal and oral mucosa was evaluated using immunohistochemical analysis for p63, K3, MUC5AC, and CD34. Clinical outcomes were assessed based on visual acuity, ocular manifestations, and liquid-based cytology. ● RESULTS: Immunohistochemical analysis revealed a plentitude of p63 and K3 in nasal mucosal epithelium. Goblet cells and MUC5AC expression were only observed in nasal mucosal epithelium, not in oral mucosal epithelium. Well-developed parallel vasculature was demonstrated in the nasal mucosa. In contrast, perpendicular vasculature was demonstrated in the oral mucosa. This vascular feature remained after transplantations. In all patients, ocular surface stability recovered with no major complications and increased goblet cells were observed on ocular surface. However, delayed epithelialization and ischemic thinning were seen at oral mucosal graft sites. ● CONCLUSIONS: Nasal mucosa, which has the advantage of well-developed parallel vasculature, enriched goblet cells, and plenty of stem cells, may be an ideal substitute for a cicatricial ocular surface. Transplantation of autologous nasal mucosa is a very effective method for achieving ocular surface reconstruction in cicatricial ocular surface diseases. (Am J Ophthalmol Accepted for publication Jul 22, 2009. From the Departments of Ophthalmology (J.H.K., Y.S.C., Se.H.L., J.C.K.) and Otorhinolaryngology–Head and Neck Surgery (S.K.M.), College of Medicine, Chung-Ang University, Yongsan Hospital, Seoul, Republic of Korea; Research and Development Institute, Modern Cell and Tissue Technologies Incorporated, Seoul National University of Technology, Seoul, Republic of Korea (H.S.J., Su.H.L.); and the Department of Genetic Engineering, College of Life Science, Kyung Hee University, Gyeonggi-do, Republic of Korea (Y.S.). Inquiries to Jae Chan Kim, Department of Ophthalmology, ChungAng University, Yongsan Hospital, 65-207, Hangangro-3Ga, YongsanGu, Seoul 140-757, Republic of Korea; e-mail: [email protected] 0002-9394/10/$36.00 doi:10.1016/j.ajo.2009.07.030
©
2010 BY
2010;149:45–53. © 2010 by Elsevier Inc. All rights reserved.)
C
ICATRICIAL OCULAR SURFACE DISEASES SUCH AS
Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and chemical/thermal burns are very difficult and challenging disorders for ophthalmologists. These chronic and excessive inflammatory diseases induce destruction of corneal epithelial stem cells located in the limbus, loss of conjunctival goblet cells producing mucins, and ultimately squamous metaplastic epithelium-like skin.1–3 These changes cause severe visual loss, ocular pain, limitations in eyeball movement, and cosmetic problems. Regarding the treatment of these diseases, ocular surface reconstruction may be required to replace the cicatricial tissues with stem cells or a stem cell niche and conjunctival goblet cells. Normal, healthy ocular surface tissues of the fellow eye, such as conjunctiva or limbus,4 may be the best source of ocular surface reconstruction materials. However, in bilateral disease, this approach is not an option. Furthermore, harvesting of the fellow eye is restrictive and often provides an insufficient quantity of tissue. Therefore, amniotic membrane (AM)5–7 and various autologous mucosal tissues, such as oral mucosa,8 hard palate mucosa,9 maxillary sinus mucosa,10 or nasal mucosa have been used for ocular surface reconstruction.11,12 With the recent development of tissue engineering techniques, methods using autologous cultivation of corneal epithelial cells,13 limbal cells,14 or oral mucosal epithelial cells have come into the spotlight.15–17 However, tissue engineering techniques are possible in only some restricted hospitals, because these techniques are fastidious and require much effort and expense. Hence, nasal mucosa and oral mucosa, which can be easily accessed and obtained, may be practical tissue sources for clinicians. Especially, nasal mucosa has enriched goblet cells in the epithelium and submucosa, which secrete the mucin required for tear film stabilization. In 1990, Naumann and associates11 introduced autologous nasal mucosa transplantation in severe conjunctival mucus deficiency syndrome. After that, there were a few reports in which the nasal mucosa was used in eyelid reconstructive surgeries.18,19 However, nasal mucosal transplantation has not been used broadly in ocular surface reconstruction as of yet because most ophthalmologists hesitate to harvest the nasal mucosa. There have been no studies assessing the
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FIGURE 1. Histology and immunohistochemistry of the nasal mucosa and oral mucosa (magnification ⴛ400). (Top left) Periodic acid–Schiff (PAS) staining demonstrated goblet cells only in the nasal mucosal epithelium, (Top right) not in the oral mucosal epithelium. (Bottom left) MUC5AC immunostaining showed goblet cells containing mucin only in the nasal mucosal epithelium, (Bottom right) not in the oral mucosal epithelium.
immunohistochemical characteristics of the nasal mucosa and comparing them to those of the ocular surface. Therefore, we investigated the possibility of replacing the metaplastic ocular surface with autologous nasal mucosa by analyzing the histologic and immunohistochemical characteristics of the nasal mucosa. We also evaluated and compared the results of ocular surface reconstruction with autologous nasal mucosa and oral mucosa in cicatricial ocular surface diseases, especially ocular chemical burns.
to determine the state of the nasal and oral mucosa. After preparation of a 5% povidone-iodine solution for antiseptic purposes, nasal mucosal tissues, each measuring 2 cm2, were stripped in situ from the one-third portion to the two-thirds portion of the inferior nasal turbinate using a blade and scissors in all patients. After harvest, bleeding was controlled by bipolar cauterization under an endoscope. In 3 patients who exhibited entire cicatricial ocular surface change, and for whom ocular surface reconstruction using only AM and nasal mucosa was expected to be insufficient, oral mucosal tissues, each measuring 1 cm2, were taken from the lower inner lip mucosa. Nasal and oral submucosal connective tissues were removed and trimmed into thin, flat mucosal tissues 2 mm in thickness with scissors and a blade.
METHODS ● PATIENTS: We obtained informed consent from 6 patients (6 eyes) with chemical burns. They had all undergone prior ocular surgery with AMs more than twice. Nevertheless, they all had devastating cicatricial squamous metaplasia, recurrent corneal ulcers, total limbal deficiency, conjunctivalization, fibrovascular proliferation, and symblepharon. Hence, we performed ocular surface reconstruction using AM transplantation, nasal mucosal autografting, oral mucosal autografting, and conjunctival limbal autografting on these 6 damaged eyes.
● SURGICAL PROCEDURES:
All surgical procedures were performed by one surgeon (J.C.K.) on a case-by-case basis, with each patient under retrobulbar anesthesia. All patients underwent symblepharolysis and excision of cicatricial and fibrovascular tissue. In 3 patients who had no vision, but who had cosmetic problems, corneal tattooing was performed using India ink (True Black Eye Liner; Biotouch Products, Walnut, California, USA). Thereafter, permanent AM grafting was performed on bare sclera and whole cornea in all patients using cryopreserved AM (AmniSite-Cornea; Bioland Ltd, Cheonan, Korea) with
● PREPARATION OF AUTOLOGOUS ORAL MUCOSA AND NASAL MUCOSA: All patients were examined carefully
before the operation by an otorhinolaryngologist in order 46
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FIGURE 2. K3 and P63 immunohistochemistry of the nasal mucosa (magnification ⴛ400). (Left) Keratin 3 was expressed in the nasal mucosal epithelium, similar to cornea and conjunctiva. (Right) P63, a stem cell marker, was highly expressed (arrow) in the basal layer of the nasal mucosal epithelium.
FIGURE 3. Comparison of the nasal mucosal and oral mucosal vasculature. (Top left) Flat-mount immunohistochemical staining of CD34, which was expressed in vascular endothelial cells, showed a parallel vascular direction in the nasal mucosa. (Top right) In contrast, CD34 staining showed mainly a round dot pattern, which indicated a perpendicular vascular direction in the oral mucosa (magnification ⴛ100). These vascular features remained after transplantation of nasal and oral mucosa as well. (Bottom left) One year after surgery, the vascular plexus at the nasal mucosal graft site (inside white loop) in Case 1 showed a parallel direction, (Bottom right) while that at the oral mucosal graft site in Case 2 showed a perpendicular vascular direction.
interrupted 10-0 nylon sutures. Nasal mucosal autografting (in 6 eyes) and oral mucosal autografting (in 3 eyes; Cases 1 to 3) were performed on sclera over the permanent AM grafting with interrupted 10-0 nylon sutures. Conjunctival limbal autografting was performed in the perilimbal area over the permanent AM grafting in 2 patients (Cases 2 and 5) using the method described by Kenyon and Tseng.4 In the 4 remaining patients, it was impossible to harvest limbal tissue from the fellow eye, because of previous harvesting of conjunctivolimbal tissue or because of VOL. 149, NO. 1
chemical burn–induced damage to the ocular surface. Lastly, temporary AM patches were placed in all patients. After surgery, 20% autologous serum eye drops were instilled every 2 hours, 0.5% levofloxacin (Levaquin; Ortho-McNeil Pharmaceutical, Titusville, New Jersey, USA) was used topically 4 times a day, and topical steroid ointment (dexamethasone [Decadron; Merck & Co Inc, Whitehouse Station, New Jersey, USA] polymyxin B sulfate, and neomycin sulfate) was administered 3 times daily until epithelialization was complete. During the first
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FIGURE 4. Clinical appearance before and after ocular surface reconstruction using autologous nasal mucosa in Cases 2 (Top), 3 (Middle), and 4 (Bottom). (Top, Middle, and Bottom left) Preoperatively, there was total conjunctivalization with severe scarring, symblepharon, and ulceration of the ocular surface. One year after surgery, (Top, Middle, and Bottom center and Bottom right) we noted stabilized ocular surfaces and (Top and Middle right) abundant mucin secretion (arrow).
week after surgery, prednisolone (30 mg per day) and third generation cephalosporins (cefditoren pivoxil; [Spectracef; Purdue Pharmaceutical Products, Stamford, Connecticut, USA] 300 mg per day) were administered orally. Five days after surgery, temporary AM patches were removed and preservative-free artificial tears were used.
and MUC5AC were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). The mucosal tissues were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) at 4 C for 10 minutes and then permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 minutes. The endogenous peroxidase was quenched with 0.3% H2O2 in 0.5% horse serum in PBS and incubated with 5% horse serum to block the nonspecific sites. Monoclonal antibodies against p63, K3, CD34, or MUC5AC were applied and incubated for 1 hour at room temperature, followed by incubation with biotinylated second antibodies, anti-mouse, or anti-rabbit IgG, using a Vectastain Elite ABC Kit (PK6101; Vector Laboratories, Burlingame, California, USA), according to the manufacturer’s protocol. Samples were finally incubated with diaminobenzidine (DAB) peroxidase substrate to give a brown stain and were then counterstained with hematoxylin. After washing with PBS, the samples were mounted and analyzed.
● CLINICAL EVALUATION: Preoperative and postoperative best-corrected visual acuity (BCVA) and intraocular pressure were measured. Ocular surface manifestations, the presence of corneal erosion, corneal neovascularization, the recurrence of symblepharon, epithelial integrity, and survival of the mucosal graft were evaluated at the first day, third day, first week, second week, fourth week, and every month postoperatively with a slit-lamp microscope (SL 130; Carlzeiss Meditec Inc., Dublin, California, USA) and fluorescein staining. Clinical success was defined as intact ocular surface epithelium without epithelial erosion, maintained for greater than 4 weeks.
● LIQUID-BASED CYTOLOGY: Liquid-based cytology was performed to evaluate for the presence of goblet cells, before surgery and 1 year after surgery, in Cases 4 and 5. Each patient turned his head to the operative eye in the supine position. After that, 1 ml of balanced salt solution (BSS; Alcon Laboratories, Fort Worth, Texas, USA) was dropped from the medial bulbar conjunctiva to the lateral canthus and collected in an Eppendorf tube. A commer-
● HISTOLOGIC AND IMMUNOHISTOCHEMICAL ANALYSIS:
Hematoxylin-eosin staining, periodic acid–Schiff (PAS) staining, and immunohistochemical staining for p63, keratin 3 (K3), CD34, and MUC5AC were performed to evaluate the epithelial characteristics in the remnant nasal mucosa and oral mucosa of Case 1, after the mucosa was trimmed. Monoclonal antibodies against K3, p63, CD34, 48
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FIGURE 5. Liquid-based cytologic findings before and after ocular surface reconstruction using autologous nasal mucosa in Cases 4 (Top) and 5 (Bottom). Before surgery, (Top left and Bottom left) only squamous cells and inflammatory cells without any goblet cells were observed. However, 1-year after surgery, (Top right and Bottom right) several goblet cells (arrow) were easily found.
FIGURE 6. Illustrations of ocular surface reconstructive procedures and postoperative slit-lamp photographs in Cases 1 (Top) and 2 (Bottom). Ten months after surgery, (Top center) well-developed horizontal vascularization (black arrow) was present at the nasal mucosal autograft site, (Top right) whereas ischemic thinning (white arrow) was present at the oral mucosal autograft site. Two weeks after surgery, (Bottom center) abundant mucus (white arrowhead) and (Bottom right) complete epithelialization (white asterisk) were observed at the nasal mucosal autograft site, but the entire epithelial defect (black asterisk) was still present at the oral mucosal autograft site.
cially manufactured fixative (CytoRich Red; TriPath Imaging Inc, Burlington, North Carolina, USA) was added to the collected specimen and vortex-mixed for 15 minutes. After the specimen sat for 30 minutes, it was centrifuged at 600 g for 10 minutes; the supernatant was then decanted. As for washing, 10 ml of water was added to the specimen, which was then vortexed. The specimen was then centrifuged at 600 g for 5 minutes, and the supernatant was decanted again. After the specimen was loaded onto a PrepStain system (TriPath Imaging Inc) for processing, a VOL. 149, NO. 1
uniform thin layer of cells was produced in a 13-mm-diameter circle. Interpretations were made by applying fundamental cytologic guidelines practiced by pathologists.20
RESULTS ● HISTOLOGIC AND IMMUNOHISTOCHEMICAL ANALYSIS: PAS staining and MUC5AC immunostaining dem-
onstrated goblet cells containing mucin only in the nasal
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TABLE. Characteristics and Clinical Outcomes of Patients Receiving a Nasal Mucosal Autograft in Cicatricial Ocular Surface Disease BCVA Case
Gender/Age
Disease
Previous Operative
Combined Operative
1
M/58
Alkali burn
FR, AMT(⫻2)
FR, AMT, OMAU
2
M/60
Acid burn
FR, AMT(⫻2)
FR, AMT, CLAU, OMAU
3
M/48
Alkali burn
FR(⫻2), AMT(⫻4)
4
M/50
Flame burn
5
M/42
6
M/53
Follow-up (months)
Preoperative
Last
Postoperative Status
LP(⫹)
HM(⫹)
20/1000
20/200
FR, AMT, CT, OMAU
LP(⫺)
LP(⫺)
AMT(⫻2)
FR, AMT, CT
LP(⫺)
LP(⫺)
Alkali burn
AMT(⫻2)
FR, AMT, CLAU
20/1000
20/320
Thermal burn
AMT(⫻2)
FR, AMT, CT
LP(⫺)
LP(⫺)
Ischemic thinning on OMAU Delayed epithelialization on OMAU Stabilized ocular surface Stabilized ocular surface Stabilized ocular surface Stabilized ocular surface
16 16 18 14 12 12
AMT ⫽ amniotic membrane transplantation; BCVA ⫽ best-corrected visual acuity; CLAU ⫽ conjunctiva limbal autograft; CT ⫽ corneal tattooing; FR ⫽ fornix reconstruction; HM ⫽ hand motion; LP ⫽ light perception; OMAU ⫽ oral mucosal autograft.
mucosal epithelium, but not in the oral mucosal epithelium (Figure 1). K3 was strongly expressed in the nasal mucosal epithelium, similar to cornea and conjunctiva. Furthermore, P63, a stem cell marker, was expressed throughout the whole basal layer of the nasal mucosal epithelium (Figure 2). Flat-mount immunostaining for CD34, which was expressed in vascular endothelial cells, showed a longish pattern representing the parallel vascular network to the epithelium in the nasal mucosa. However, the oral mucosa stained with CD34 showed a mainly round-dot vascular pattern (though a partially longish pattern was found) (Figure 3). This implies that the oral mucosa has a perpendicular vascular network to the epithelium, as contrasted with the parallel vasculature of the nasal mucosa. These vascular features remained at 1-year after transplantation of nasal or oral mucosa in all patients (Figure 3).
were observed on liquid-based cytology. However, 1- year after surgery, several goblet cells were easily found (Figure 5). All transplanted conjunctival limbal tissues around the nasal mucosal graft were well healed and living. However, poor, delayed epithelialization and pale ischemic thinning were found at the oral mucosal graft sites (Figure 6). Preoperative BCVAs were no light perception (3 eyes), light perception (1 eye), and 20/1000 (2 eyes). Postoperative BCVAs had improved in 3 eyes (excluding the 3 eyes that underwent corneal tattooing) at the last follow-up examination (Table). ● CASE 1:
A 58-year-old Korean man presented with fibrovascular tissue covering the entire ocular surface, symblepharon, and limited ocular motility following two AM transplantations and 1 fornix reconstruction surgery at another hospital. He had incurred an alkali burn in July 2005. After removal of the scarred fibrovascular tissue, symblepharolysis, and permanent AM grafting on the cornea and conjunctiva, we performed nasal mucosal autografting on the superior nasal bulbar conjunctiva and oral mucosal autografting on the superior temporal bulbar conjunctiva (Figure 6). Finally, temporary AM patch was performed on the whole ocular surface. The patient’s BCVA improved from light perception to hand motion. Ocular motility improved, and the ocular surface stabilized with abundant mucin secretion at the last follow-up examination (16 months after the operation). However, ischemic thinning was observed at the oral mucosal graft site, starting 3 months after the operation. Well-developed horizontal vascularization was noted at the nasal mucosal graft site (Figure 6).
● CLINICAL OUTCOME:
The mean follow-up period in our study was 14.7 months (range, 12 to 18 months). Nasal and oral mucosal tissues were safely excised without any complications, and their mucosal harvest sites were completely healed in 2 weeks. In all patients, the ocular surface was stabilized, and there were no recurrences of ulceration, conjunctivalization, fibrovascular tissue, or symblepharon (Figure 4). Clinical success was achieved without any major postoperative complications. Eyeball movement was improved in all patients. All patients noted improvement in ocular comfort, and 3 patients with corneal tattooing were very satisfied with their cosmetic results. We noted rapid epithelial healing, well-developed horizontal vascularization, and abundant mucin in all nasal mucosal graft sites. The existence of mucin and goblet cells was confirmed by liquid-based cytology. Before surgery, only squamous cells and inflammatory cells without any goblet cells
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A 60-year-old Korean man presented with conjunctivalization, recurrent ulceration, symblepharon,
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and limited ocular motility in spite of two ocular surface reconstructions using AMs in another hospital, secondary to alkali burns. After removal of the conjunctivalized fibrovascular tissue, symblepharolysis, permanent AM grafting on the cornea, and conjunctival limbal autografting in the fellow eye, nasal mucosal autografting and oral mucosal autografting were performed on the superior nasal bulbar conjunctiva and inferior nasal bulbar conjunctiva (Figure 6). Temporary AM patch was performed on the whole ocular surface as well. Two weeks after surgery, the nasal mucosal autografting had completely healed and was secreting mucin, but the whole epithelial defect was still present at the oral mucosal autografting site (Figure 6). Four weeks after surgery, the ocular surface was stabilized, and the epithelial defect at the oral mucosal graft site had healed. BCVA improved significantly from 20/1000 to 20/200, and ocular motility improved as well.
DISCUSSION THIS STUDY DEMONSTRATES THE POSSIBILITY OF REPLAC-
ing cicatricial ocular surface tissues with nasal mucosa by identifying the characteristics of nasal mucosa and documenting successful clinical autologous nasal mucosal transplantation. The expression of K3 in nasal mucosal epithelium, like normal cornea and conjunctiva, suggests that nasal mucosa may be one of the appropriate tissues to replace the cicatricial ocular surface.21 The expression of p63, a stem cell marker, in the whole basal layer of the nasal mucosal epithelium indicates that nasal mucosa may restore the cicatricial ocular surface as a source of stem cells/stem cell niche. K3 and p63 are also expressed in the oral mucosal epithelium, according to previous reports.22–24 However, a point of difference is that goblet cells and MUC5AC expression are only present in nasal mucosal epithelium, not in the oral mucosal epithelium. In the normal ocular surface, cornea and conjunctival nonkeratinizing squamous epithelial cells produce the membrane-associated mucins MUC1, MUC4, and MUC16.25–27 The gel-forming mucin MUC5AC is secreted solely by conjunctival goblet cells, and the secretory mucins MUC2 and MUC7 are produced in corneal and conjunctival epithelium.28 –30 The MUC genes of the nasal mucosa are very similar to those of the conjunctiva. MUC1, MUC2, MUC4, MUC5AC, and MUC5B are expressed in nasal mucosal epithelium, and MUC7 is expressed in nasal mucosal glands.31 Hence, nasal mucosal transplantation can replenish these mucins and stabilize the tear film and ocular surface naturally. As expected on the basis of these nasal mucosal characteristics, using liquid-based cytology, we observed a stabilized and recovered ocular surface with abundant mucin secretion and increased goblet cells on the ocular surface more than 1-year after nasal mucosal VOL. 149, NO. 1
transplantation. Although Weinberg and associates reported a loss of goblet cells in nasal mucosal autografting on the posterior eyelid surface,19 goblet cell loss was observed only on the grafts placed in the palpebral conjunctiva, not in the bulbar conjunctiva. One report noted that functional goblet cells persisted in autologous nasal mucosa for up to 10 years after transplantation on the ocular surface.12 This difference in the survival of goblet cells in nasal mucosal grafts may be associated with environmental conditions, especially air flow. Because the nasal mucosa is in contact with air flow normally, we believe that nasal mucosa transplanted on the bulbar conjunctiva, which is in contact with air flow, may maintain its goblet cells. There are other potential mucosal donor sites, such as rectal mucosa and vaginal mucosa, both of which have a significant number of goblet cells. However, when considering additional time to harvest, risks of donor site complications, and donor site pain, ophthalmologists and patients may hesitate to use these donor sites. In this study, it took a short time (about 10 minutes) to harvest nasal mucosal tissues. After harvesting, there were no instances of severe pain and no complications at the nasal mucosal donor sites. Hence, we believe nasal mucosal harvesting is not difficult or dangerous to perform. Another remarkable histologic and immunohistochemical finding is the vascular direction in nasal mucosa and oral mucosa. Flat-mount histology and immunostaining for CD34, a vascular endothelial cell marker, showed that the nasal mucosa had a horizontal vascular direction. In contrast, on histologic analysis and immunostaining for CD34, we found that the oral mucosa had a vertical vascular direction. Clinically, well-developed horizontal vasculature, rapid healing, and graft survival were seen at all nasal mucosal autografting sites. Meanwhile, delayed, poor epithelialization and ischemic thinning were observed at the oral mucosal autografting sites. Even if these findings are observed in only 3 cases, these suggest that the differing vascular patterns in nasal mucosa and oral mucosa may be correlated with the differing clinical results obtained with each graft. Although these vascular features have not yet been fully explained, they may have relevance to the fact that nasal mucosa has an extensive system of arteriovenous anastomoses and a profuse subepithelial capillary network that connects to a plexus of deeper vessels in the submucosa to exchange heat with flowing air.32,33 Pinkish, well-developed vascularizations of nasal mucosa may induce cosmetic problems, though they are advantageous for restoring the ocular surface. Hence, transplantation of nasal mucosa would be preferable at conjunctival sites eclipsed by the upper eyelids. Also, we observed that all conjunctival limbal autografting around nasal mucosal autografting healed well without any complications. We believe that nasal mucosal autografting may support the conjunctival limbal autograft-
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ing with its well-developed vasculature, enriched goblet cells and mucins, and increased p63 expression, though further studies about outcomes of conjunctival limbal autografting in the presence of nasal mucosal autografting are required. Cultivated oral mucosal epithelial sheets, which have been recently developed and introduced, also have the proper characteristics for ocular surface reconstruction, such as expression of p63, K3, and membrane-associated mucins MUC1, MUC4, and MUC16.17,34 Many clinical studies have demonstrated sufficient effects and good clinical outcomes with cultivated oral mucosal epithelial transplantation in severe ocular surface disorders.16,17,35,36 However, there are no goblet cells that express the secretory mucin MUC5AC in cultivated oral mucosal epithelial cells. Hence, nasal mucosa may be a more appropriate substrate for the conjunctiva, though cultivated oral mucosal sheets may be the ideal substrate for cornea. Furthermore, the tissue-engineering technique is fastidious and requires much effort and expense, and it is still possible in only some restricted hospitals.
Only patients with severe ocular burn were included in this study. However, successful nasal mucosal autografting results have been reported in other cicatricial ocular surface diseases, such as Stevens-Johnson syndrome and ocular pemphigoid.12 If the nasal mucosal donor site is not involved in these diseases on examination before harvesting, nasal mucosal autografting can be performed and may be effective. In summary, we noted that ocular surface stability recovered after surgery in all patients, leading to cosmetic and functional improvement with no major postoperative complications. Moreover, we verified the suitability of the nasal mucosa for the ocular surface based on histologic and immunohistochemical analysis. This study suggests that nasal mucosa, which has a welldeveloped horizontal vasculature, enriched goblet cells, and plenty of stem cells, may be an ideal substitute tissue for a cicatricial ocular surface, especially conjunctiva. We reaffirm that transplantation of autologous nasal mucosa is a very effective and beneficial method for achieving ocular surface reconstruction in cicatricial ocular surface diseases.
THIS STUDY WAS SUPPORTED BY A GRANT OF THE KOREA HEALTHCARE TECHNOLOGY R&D PROJECT, MINISTRY FOR HEALTH, Welfare & Family Affairs, Republic of Korea (A084721). The authors indicate no financial conflict of interest. Involved in design of study (J.C.K.); conduct of study (J.H.K., Se.H.L., S.K.M., J.C.K.); data collection (Se.H.L.); management, analysis, and interpretation of data (J.H.K.); histologic and immunohistochemical analysis (H.S.J., Su.H.L., Y.S.); and preparation (J.H.K.), review (Y.S.C.), and approval of manuscript (J.C.K.). This study was approved by the Institutional Review Board for Human Studies of Chung-Ang University Yongsan Hospital.
10. Fry TL, Wood CI. Readily available full-thickness mucous membrane graft. Arch Otolaryngol Head Neck Surg 1987; 113:770 –771. 11. Naumann GO, Lang GK, Rummelt V, Wigand ME. Autologous nasal mucosa transplantation in severe bilateral conjunctival mucus deficiency syndrome. Ophthalmology 1990; 97:1011–1017. 12. Wenkel H, Rummelt V, Naumann GO. Long-term results after autologous nasal mucosal transplantation in severe mucus deficiency syndromes. Br J Ophthalmol 2000;84:279 – 284. 13. Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, de Luca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 1997;349:990 –993. 14. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000;343:86 –93. 15. Nakamura T, Kinoshita S. Ocular surface reconstruction using cultivated mucosal epithelial stem cells. Cornea 2003; 22:S75–S80. 16. Nakamura T, Inatomi T, Sotozono C, Amemiya T, Kanamura N, Kinoshita S. Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders. Br J Ophthalmol 2004;88:1280 – 1284. 17. Nishida K, Yamato M, Hayashida Y, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 2004;351: 1187–1196.
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Biosketch Jae Hoon Kim, MD, is currently an Ophthalmology resident at Chung-Ang University Yongsan Hospital, Seoul, Korea. He graduated and received his medical degree from the Chung-Ang University in 2002. His research interests include ocular surface and ophthalmic plastic and reconstruction surgery.
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Biosketch Jae Chan Kim, MD, PhD, is a Professor and Chief instructor of the Ophthalmology and a Director of the Department of Ophthalmology at Chung-Ang University Yongsan Hospital, Seoul, Korea. He graduated and received medical degree and PhD from the College of Medicine in Chung-Ang University, and completed his residency training at the Department of Ophthalmology in Chung-Ang University Hospital. His research interests include amniotic membrane, stem cells, and ocular surface reconstruction using tissue engineering.
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