Original Article Clinical outcome of autologous ...

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January 2006

Original Article Clinical outcome of autologous cultivated limbal epithelium

transplantation

Virender S. Sangwan, MS; Himanshu P. Matalia, MS; Geeta K. Vemuganti, MD; Anees Fatima, MSc; Ghazala Ifthekar, M.Sc; Shashi Singh, PhD; Rishita Nutheti, MSc;Gullapalli N. Rao, MD Purpose: To report the clinical outcome of autologous cultivated limbal epithelial transplantation. Methods: Eighty-six patients’ records and their clinical photographs were reviewed for demographics, primary etiology, type of limbal transplantation, ocular surface stability, visual acuity, final outcome, and possible factors affecting outcome and complications. Results: Eighty-eight eyes of 86 patients with limbal stem cell deficiency (LSCD) underwent autologous cultivated limbal epithelium transplantation between March 2001 and May 2003, with a mean follow-up of 18.3 months. The etiology of LSCD was alkali burns in 64% patients. Sixty-one eyes had total LSCD. Thirty two of the 88 eyes had undergone amniotic membrane transplantation and 10 eyes had previously undergone limbal transplantation with unfavorable outcome. Nineteen eyes underwent penetrating keratoplasty, of which 11 grafts survived at the final follow-up. Finally, 57 eyes (73.1%, 95% CI: 63.3–82.9) had a successful outcome with a stable ocular surface without conjunctivalization, 21 eyes (26.9%, 95%CI: 17.1–36.7) were considered failures, and 10 patients were lost to follow-up. Conclusion: LSCD can be successfully treated by autologous cultivated limbal epithelium transplantation in majority of the cases. Key Words: Cultivated autologous limbal epithelium transplantation, limbal stem cell deficiency Indian J Ophthalmol 2006;54:29-34

The physiology as well as molecular and cellular biology of the ocular surface and its components has not only increased our understanding but has also opened a new chapter in regenerative medicine with the use of tissue engineering.1–3 Various scientific discoveries in the past two decades have led to the identification of the limbal location of corneal epithelial stem cells and the role it plays in regenerating the corneal epithelium.1,4–7 Simultaneously, various surgical techniques of limbal transplantation using cadaveric2 or live-related donor tissues have evolved.8,9 However, these methods have met with limited success and require indefinite immunosuppression to avoid limbal allograft rejection.10,11 Long-term systemic immunosuppression involves the risk of serious eye and systemic complications apart from being a significant economic burden.

Novel techniques of ex vivo epithelium cell culture have allowed us to treat Limbal Stem Cell Deficiency (LSCD) with a better surgical approach, which requires a very small donor tissue, obviating the need for immunosuppression, minimizing the risk to the donor site, and increasing the possibility of obtaining an autologous donor tissue from a small, uninvolved area of the limbus. 12–14 This method is called autologous cultivated limbal epithelium transplantation. A few such methods using different culture techniques and carriers have been reported in the literature, 12,13,15,16 but being a relatively new technique there is little information regarding its clinical outcome.

Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory (VSS, HPM, GKV, AF, GI), Cornea and Anterior Segment Service (VSS, HPM, GNR), Ophthalmic Pathology Laboratory (GKV), International Center for Advancement of Rural Eye Care (RN), L. V. Prasad Eye Institute, Banjara Hills, Hyderabad, India and Center for Cellular and Molecular Biology (SS), Hyderabad, India.

Materials and Methods

Correspondence to Virender S. Sangwan, L.V. Prasad Eye Institute, L. V. Prasad Marg, Banjara Hills, Hyderabad 500034, India. E-mail: Manuscript received: 29.7.05; Revision accepted: 31.12.05

We report the results of our cases of ocular surface reconstruction using autologous cultivated limbal epithelium transplantation. To the best of our knowledge, ours is the largest series of any form of limbal transplantation ever reported.

During the study period (March 2001–May 2003), 88 autologous cultivated limbal epithelium transplantation procedures were performed at the L. V. Prasad Eye Institute, Hyderabad, with a diagnosis of LSCD. The medical records and clinical photographs of all consecutive patients following cultivated limbal epithelium transplantation were retrospectively reviewed for the demographics, primary etiology, previous surgeries, and preoperative and

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postoperative best corrected visual acuity, the type of cultivated limbal epithelium transplantation, complications, and final outcome. All the grafted eyes were clinically diagnosed as having LSCD with conjunctivalization, absence of limbal palisades of Vogt (radial pigmented folds at the limbus and a biological marker of the location of corneal epithelial stem cells), chronic inflammation, persistent corneal epithelial defect, and/or recurrent corneal epithelial defect.1 Cases with follow-up of less than 6 weeks were not considered for analysis. Our surgical techniques for cultivated limbal epithelium transplantation have been reported.17 Informed consent was obtained from the patients or guardians. Limbal biopsy was performed on the healthy contralateral eye or a healthy area of the ipsilateral eye. The procedure included careful dissection of a 1 x 2 mm2 piece of limbal epithelium with 0.5 mm into clear corneal stromal tissue at the limbus under strict aseptic conditions. The limbal tissue that contained limbal epithelial cells at the pigmented line (palisades of Vogt) and a part of the corneal stroma was excised. The tissue was transported in human corneal epithelium (HCE) medium to the tissue culture laboratory, where, under strict aseptic conditions, the donor limbal tissue was shredded into small pieces. Human amniotic membrane (HAM) prepared and preserved by our eye bank was used as a carrier. HAM, measuring 3 x 4 cm2 was de epithelialized using 0.25% trypsin and EDTA solution for 15 min. Limbal tissue procured by limbal biopsy, shredded into small pieces, was explanted over the center of the de epithelialized HAM with the basement membrane side-up [Figure 1(A)]. A similar parallel culture was prepared as a backup. Ours is a submerged explant culture system without the use of any feeder cell layer. We use HCE medium with 10% fetal bovine serum to nurture the culture. Since October 2002 we have been using autologous serum instead of fetal bovine serum for our culture. The HCE medium was prepared using 9.7 g/l modified Eagle medium with the addition of 16.2 g/l Ham F12 serum, 0.01 mg/l epidermal growth factor, 0.25 mg/l insulin, 0.1 mg/l cholera toxin, and hydrocortisone. The medium was filtered with 0.22 mm membrane filters using a vacuum pump. The sterile medium was supplemented with

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10% fetal calf serum or autologous serum at the time of use. The culture was incubated at 37°C with 5% CO2 and 95% air. The growth was monitored daily under phase-contrast microscope and the medium was changed every alternate day [Figure 1(B)]. The culture was terminated when a monolayer of the cells growing from the explants became confluent, in 10–14 days [Figure 1 (C)]. At the time of limbal transplantation, the fibrovascular pannus covering the ocular surface was excised from the cornea and sent for histopathological examination. A drop of epinephrine (1: 1000) was instilled in the conjunctival cul-de sac prior to pannus excision to achieve hemostasis. After release of the symblephara and adequate hemostasis with cautery, the HAM with the monolayer of cultivated limbal epithelial cells with the epithelial side up was spread over the defect. The graft was then secured to the limbal side by interrupted, circumferential 10-0 nylon sutures as well as to the surrounding conjunctival edge by interrupted 8-0 polyglactin sutures. A bandage contact lens was applied. We used to apply bandage contact lens postoperatively in all our earlier cases to prevent any damage from lid action, but we discontinued the use of the same as we failed to make out any beneficial effect. Following the cultivated limbal epithelium transplantation, all the patients were treated with 1% prednisolone acetate eye drops eight times a day tapered to once a day in 5–6 weeks and 0.3% ciprofloxacin hydrochloride eye drops four times a day for 1 week. 0.3% Ciprofloxacin hydrochloride eye drops were continued if an epithelial defect was present or as long as the bandage contact lens was used. The patients were seen on postoperative day 1, week 1, week 2, week 5, and monthly thereafter. Each examination included a complete history, noting down of new ocular or systemic symptoms, a complete evaluation of recipient as well as donor sites, and any sign of neovascularization or surface instability. Sutures were removed when indicated (loose or vascularized). Epithelialized sutures were left in place indefinitely. The success of the procedure was defined by stable ocular surface and subjective improvement in the symptoms of the

C

Figure 1: Ex vivo cultivation of limbal epithelium cells. (A) Diagrammatic representation of our culture technique showing human amniotic membrane as a carrier (gray color) with small bits of tissue explanted over it (white color), submerged in human corneal epithelium medium (blue color). (B) Photomicrograph of cultivated limbal epithelium (ex vivo) under phase contrast microscope, showing confluent and compact monolayer of the growing cells (original magnification x200). (C) Photomicrograph of whole mount of cultivated limbal epithelium, harvested over human amniotic membrane showing a confluent growth of cells from the central tissue explants (original magnification x100, H & E stain).

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Table 1: Effect of factors affecting outcome of cultivated autologous limbal epithelium transplantation Factor

Numbers

Failure (%)

HR (95% CI)

Contralateral eye

61

14 (23.0)

1.00

Ipsilateral eye

16

6 (37.5)

1.71 (0.66–4.46)

No

58

12 (20.7)

1.00

Yes

19

8 (42.1)

2.14 (0.87–5.24)

P value

Autograft

0.272

0.096

AMT before cultured limbal transplantation 29

6 (20.7)

1.00

No

48

14 (29.2)

1.63 (0.63–4.26)

0.315

Number of surgeries before limbal transplantation 0

42

11 (26.2)

1.00

1

19

5 (26.3)

0.95 (0.33–2.75)

0.930

>1

16

4 (25.0)

0.90 (0.29–2.83)

0.855

Etiology Acid injury

13

2 (15.4)

1.00

Alkali injury

47

12 (25.5)

1.77 (0.39–7.90)

0.457

Other chemicals

7

1 (14.3)

0.78 (0.07–8.63)

0.841

10

5 (50.0)

3.94 (0.76–20.41)

0.102

Others

effect of previously described possible risk factors on survival of autologous cultivated limbal epithelial transplantation was studied by univariate survival analysis and Cox proportional hazards survival regression. SPSS version 12.0 (SPSS Inc., Chicago, IL) was used for data analysis.

Results

Coculture

Yes

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HR, hazard ratio; AMT, amniotic membrane transplantation; CI, confidence interval

patient with a minimum follow-up of 6 weeks. The cut-off duration of 6 weeks was decided based on our previous study, in which we found that the ocular surface stabilized in a mean of 6 weeks.17 The stability of the ocular surface was assessed clinically, based on absence of recurrent breakdown of the corneal epithelium, increased permeability to fluorescein, or conjunctivalization. Failure was defined by conjunctivalization, recurrent corneal epithelium breakdown, or persistent corneal epithelial defect. We also studied the complications during the study period, related or unrelated to the procedure. Corneal graft failure was defined as the primary graft failure, in which nonresolving corneal graft edema following penetrating keratoplasty (PKP) persisted more than 2 weeks and secondary graft failure was defined as nonresolving graft edema 3 months following graft rejection. The factors that could potentially affect the outcome were demographics of the patients, etiology of the LSCD, previous surgical procedures performed, and laterality of the donor tissue (ipsilateral or contralateral). Best corrected visual acuity (BCVA) was measured at each visit on a standard Snellen visual acuity chart. The ambulatory visual acuity was defined as the BCVA of more than or equal to 20/200 and nonambulatory visual acuity of less than 20/ 200. Statistical analysis The data were analyzed with the help of the Biostatistics Department at the L. V. Prasad Eye Institute. Kaplan-Meier survival analysis was performed to evaluate the survival of autologous cultivated limbal epithelial transplantation. The

Eighty-eight eyes of 86 patients underwent autologous cultivated limbal epithelium transplantation between March 2001 and May 2003. The patients ranged in age from 3 to 47 years (mean ± SD, 21.1 ± 12.5 years); there were 12 females and 74 males. The etiology of LSCD was chemical burns in 78 (88.6%) eyes (Table 1); alkali burn was the most common (n=56). Sixty-one (69.3%) of the 88 eyes had total LSCD with loss of limbal palisades of Vogt and conjunctivalization in 360°, in which the donor tissue was taken from the contralateral eye. Seventeen (19.3%) eyes had partial LSCD with partial loss of limbal palisades of Vogt and focal conjunctivalization in the same area (range 90°–330°) where limbal biopsy was taken from the clinically normal area of the same eye. On clinical examination, 19 (21.6%) eyes showed severe conjunctival damage, including symblephara in 16 eyes. Success was achieved in 57 of the 78 eyes (73.1%, 95% CI: 63.3–82.9) and 21 eyes (26.9%, 95% CI: 17.1–36.7) had failure with a mean follow-up of 18.3 ± 11.2 months (range 3–40.5 months) following cultivated limbal epithelium transplantation (Figures 2 and 3). Ten of the first 86 patients (88 eyes) were lost to follow up. Considering the best-case scenario, if we presume all those cases lost to follow up had a successful outcome, the mean success rate would be 76.1% (95% CI: 67.2– 85.0). Similarly, for the worst-case scenario, in which we considered all the 10 cases lost to follow up as failures, the mean success rate would be 64.8% (95% CI: 54.8–74.8). A Kaplan-Meier analysis of probability of survival of the cultivated limbal epithelium transplantation showed that most of the failures occurred in the first six months. The latter part of the follow-up showed fewer failures (Figure 4). Nineteen eyes had undergone PKP following cultivated limbal epithelium transplantation for optical purposes. Eleven of these grafts were successful with a mean follow-up of 17.4 months. BCVA on Snellen chart improved from 17/78 (21.8%) eyes to 41/78 (52.6%) eyes having functional ambulatory vision (> 20/200) postoperatively; 11 of these eyes had undergone PKP. Thirty-three of the 41 eyes with postoperative ambulatory visual acuity had visual acuity of more than or equal to 20/80 (Figures 5 and 6); 9 of these eyes had undergone PKP. The outcome of PKP following cultivated limbal stem cell transplantation has already been reported.17 Univariate analysis of factors affecting final outcome of the transplantation did not demonstrate any statistically significant effect of the type of transplantation, duration of symptoms, and etiology of LSCD or previous surgical intervention on the outcome of cultivated limbal epithelium transplantation (Table 1). Two patients developed phthisis bulbi. One of these cases had corneal perforation and developed panophthalmitis. Two cases with corneal grafts had microbial keratitis and two had uncontrolled glaucoma, which did not respond to the

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treatment and developed glaucomatous optic atrophy.

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Discussion Reconstruction of the ocular surface in cases of LSCD is one of the most challenging problems in ophthalmology. The therapeutic measure to treat the condition aims at replenishing the deficient limbal epithelium with the stem cells for corneal epithelium, which would maintain the stable ocular surface indefinitely.1,3 Various techniques have been reported with the aim of restoring limbal stem cell functions by using allogenic tissue of cadaveric origin (cadaveric keratolimbal allograft) or from live-related donors (conjunctival limbal allograft) with limited success.8,10,11 Apart from the limited growth potential of the cadaveric tissue, 18 long-term immunosuppression to avoid limbal allograft rejection may have significant systemic adverse effects and place an economic burden on the recipient. The use of autologous tissue to reconstruct the ocular surface was reported by Kenyon and Tseng with conjunctival limbal autograft.19 However, cases with total LSCD were seldom treated by this method owing to concerns about donor site deficiency. Pellegrini et al. in 1997 reported successful reconstruction of the ocular surface using autologous limbal epithelium cultivated ex vivo on amniotic membrane as substrate. 12 Use of this tissue engineering technology for ocular surface reconstruction is considered the best of current methods. Our group has developed the technique of ex vivo cultivated limbal epithelium. Our technique, reported earlier, is ingenious and unique in several aspects.17,20 Ours is a submerged explant culture technique in which we use de-epithelialized amniotic membrane as a substrate and human corneal epithelium medium for ex vivo cultivation. We do not use the complicated system of 3T3 fibroblast feeder layer or air-lifting contrary to previous reports that have stressed their need. We do not wait for the ex vivo stratification or the epithelium tight junction to form. Instead, we transplant a confluent monolayer of the cultivated cell. We hypothesized and successfully proved that

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Figure 2: Slit-lamp photographs of cultivated limbal epithelium transplantation for a case of total limbal stem cell deficiency. (A) Preoperative condition showing total conjunctivalization with symblephara formation superiorly. (B) Postoperatively, showing stable ocular surface and a clear visual axis with minimal residual scarring in superior corneal periphery (20 months postoperatively).

B

Figure 3: Slit-lamp photograph of penetrating keratoplasty following cultivated limbal epithelium transplantation for the optical purpose. (A) Preoperative condition showing total limbal stem cell deficiency with extensive symblephara all around. (B) Clear and compact corneal graft with a stable ocular surface after penetrating keratoplasty following successful autologous cultivated limbal epithelium transplantation (30 months postoperatively).

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the inherent nature of this epithelium would lead to in vivo stratification.17 The 2-week culture that we use is shorter than that previously reported. 12–15 Lately, we have successfully started using autologous serum instead of bovine serum for our culture medium. We report here the outcome of our cases with autologous cultivated limbal epithelium transplantation, which is to the best of our knowledge the largest series of any kind of limbal transplantation procedure reported till date.

1.0

Cumulative Survival Probability

.9 .8 .7 .6

Survival probability SE 6 months = 77.9 % 5.1 1 year = 72.5 % 5.6 2 years = 67.5 % 6.2 3 years = 67.5% 6.2

.5 .4 .3 .2 .1 0.0

n = 49

36

26

15

7

6

6

12

18

24

30

36

0

42

48

Follow up duration (in months)

Figure 4: Kaplan-Meier survival curve showing cumulative of probability of survival of cultivated limbal epithelium transplantation.

Preop Vs Postop BCVA

Preoperative BCVA

20/20

20/30

20/50

20/80

20/125

20/200 CF

PL+PR acc

PL+PR acc

CF

20/200

20/125

20/80

20/50

20/30

20/20

Postoperative BCVA

Figure 5: Comparison of preoperative and postoperative visual acuity at the last follow-up. BCVA, Best corrected visual acuity

90 80 70 60 %

50 40 30 20 10 0 Preop BCVA

Postop BCVA

Visual Acuity 20/200

Figure 6: Visual outcome following cultivated limbal epithelium transplantation

In this study, we report the outcome as defined by stable ocular surface following cultivated autologous limbal epithelium transplantation with a minimum follow-up of six weeks. The six week cut-off period was based on our previous studies, which showed that the ocular surface took a mean of six weeks to stabilize.17 The analysis of probability of survival of our cases interestingly showed the first six months to be a critical period when most of the failures occurred, whereas there were fewer failures as time progressed. This observation not only suggests that a closer follow-up is needed in the first six months of the postoperative period, but that this may also help in prognostication of the cases. The literature about the clinical outcomes of cultivated limbal epithelium transplantation is not only sparse but most studies have a smaller sample size and/or inadequate follow-up.12–14,21 Our study had a very encouraging overall initial success of 73.1% and we presume it to remain good in the long run owing to the autologous nature of our grafts. The fact that the long term success rate declines with time, as reported earlier, owing to clinical or subclinical limbal allograft rejection, may therefore not hold true in our cases. However, it does not decrease the need for the long-term follow-up. The population of our cultivated limbal epithelium cells is presumed to be limbal stem cells, are responsible for the indefinite production of progeny and maintenance of a stable ocular surface. Although the aim of cultivated limbal epithelium transplantation is not to improve vision but to stabilize the ocular surface epithelium, the functional ambulatory visual acuity (