Journal of Refractive Surgery

Therapeutic Refractive Surgery Supplemental Data

Femtosecond Laser–Assisted Stromal Lenticule Addition Keratoplasty for the Treatment of Advanced Keratoconus: A Preliminary Study

Leonardo Mastropasqua, MD; Mario Nubile, MD, PhD; Niccolò Salgari, MD; Rodolfo Mastropasqua, MD

Abstract

PURPOSE:

To investigate the in vivo effect of a novel femtosecond laser–assisted procedure termed stromal lenticule addition keratoplasty for advanced keratoconus.

METHODS:

Ten patients with stage III and IV stable keratoconus were included. Negative meniscus-shaped stromal lenticules were produced from corneoscleral eye bank buttons with a refractive lenticule extraction procedure with a 500-kHz VisuMax femtosecond laser (Carl Zeiss Meditec, Jena, Germany). Recipient corneas underwent a modified femtosecond laser flap-cut procedure to produce an intrastromal pocket and lenticules were implanted. Patients were followed up for 6 months after surgery with determination of uncorrected (UDVA) and corrected (CDVA) distance visual acuity, subjective refraction and topographic corneal curvature changes, anterior segment optical coherence tomography (AS-OCT), and in vivo confocal microscopy.

RESULTS:

Comparison of preoperative and 6-month postoperative UDVA and CDVA showed statistically significant improvements (P = .024 and .007, respectively) from 1.58 ± 0.36 to 1.22 ± 0.37 and from 1.07 ± 0.17 to 0.70 ± 0.23 logMAR. Eight of 10 eyes showed an improvement in UDVA (P < .001) that ranged between one and three lines, whereas all but one eye presented improved CDVA. Corneal topography documented a decrease between preoperative and 6-month postoperative anterior mean curvature (AVG-K at 3 mm) and anterior Q values (P = .005). AS-OCT showed a significant increase in thickness of the central and mid-peripheral cornea produced by the lenticule implantation (P = .005).

CONCLUSIONS:

The stromal lenticule addition keratoplasty procedure was clinically efficient in improving the corneal shape and vision in patients with keratoconus. Negative meniscus-shaped lenticule addition induced a flattening of the cone while increasing corneal thickness.

[J Refract Surg. 2018;34(1):36–44.]

Abstract

PURPOSE:

To investigate the in vivo effect of a novel femtosecond laser–assisted procedure termed stromal lenticule addition keratoplasty for advanced keratoconus.

METHODS:

Ten patients with stage III and IV stable keratoconus were included. Negative meniscus-shaped stromal lenticules were produced from corneoscleral eye bank buttons with a refractive lenticule extraction procedure with a 500-kHz VisuMax femtosecond laser (Carl Zeiss Meditec, Jena, Germany). Recipient corneas underwent a modified femtosecond laser flap-cut procedure to produce an intrastromal pocket and lenticules were implanted. Patients were followed up for 6 months after surgery with determination of uncorrected (UDVA) and corrected (CDVA) distance visual acuity, subjective refraction and topographic corneal curvature changes, anterior segment optical coherence tomography (AS-OCT), and in vivo confocal microscopy.

RESULTS:

Comparison of preoperative and 6-month postoperative UDVA and CDVA showed statistically significant improvements (P = .024 and .007, respectively) from 1.58 ± 0.36 to 1.22 ± 0.37 and from 1.07 ± 0.17 to 0.70 ± 0.23 logMAR. Eight of 10 eyes showed an improvement in UDVA (P < .001) that ranged between one and three lines, whereas all but one eye presented improved CDVA. Corneal topography documented a decrease between preoperative and 6-month postoperative anterior mean curvature (AVG-K at 3 mm) and anterior Q values (P = .005). AS-OCT showed a significant increase in thickness of the central and mid-peripheral cornea produced by the lenticule implantation (P = .005).

CONCLUSIONS:

The stromal lenticule addition keratoplasty procedure was clinically efficient in improving the corneal shape and vision in patients with keratoconus. Negative meniscus-shaped lenticule addition induced a flattening of the cone while increasing corneal thickness.

[J Refract Surg. 2018;34(1):36–44.]

Despite recent advancements in keratoconus management with the introduction of corneal cross-linking (CXL) procedures, the surgical approach remains the only solution able to improve visual acuity when spectacles or contact lenses do not permit patients to achieve a useful visual function.1–4 It has been reported that 15% to 28% of patients with keratoconus may require surgical intervention that includes penetrating or deep anterior lamellar keratoplasty.5,6

Intracorneal ring segment implantation in selected cases has been gaining acceptance as an alternative to corneal transplantation, inducing favorable changes to the geometry and refractive power of an ectatic cornea.7,8 Another option proposed to obtain corneal regularization is the combination of CXL with photorefractive/phototherapeutic keratectomy.9–11 Despite the promising refractive outcomes,10,12 it is worth noting that tissue subtractive techniques may further affect corneal biomechanical stability, increasing the risk of ectasia progression.

The introduction of a new all-femtosecond laser technique called small incision lenticule extraction (SMILE) represented an important innovation, as an alternative to excimer laser ablation, in corneal refractive surgery.13 Lenticule extraction can be performed either with a hinged stromal flap similarly to LASIK (femtosecond lenticule extraction [FLEx])13,14 or flapless through a small superficial incision (SMILE).14–16 With these techniques, it is possible to cut and extract a viable lenticule of corneal stromal tissue, consistent with the intended refractive correction.15

Thanks to the minimal tissue damage induced by the femtosecond laser and the structural integrity of the extracted stromal lenticule, it has been recently proposed that SMILE lenticules can then be preserved and reimplanted to reverse the effect of the refractive treatment or to treat different types of ametropia.17–19 Lenticules produced with a myopic correction algorithm are thicker in the center and gradually become thinner toward the periphery, with the shape of a positive meniscus lens.14 The implantation of such shaped lenticules has already been used to correct hyperopia in humans,20–22 inducing an increase in anterior central corneal curvature as an alternative to tissue-subtractive ablation procedures. The refractive correction of hyperopia through lenticule removal (extraction), using a hyperopic SMILE algorithm that produces a negative meniscus lenticule is currently under investigation.23–25 This kind of lenticule presents an inverted thickness profile that gradually becomes thicker from the center toward the periphery of the optical zone. Conversely, stromal addition procedures (implantation) with hyperopic lenticules can cause central corneal flattening.

In a previous ex vivo study on human corneas, we showed that the implantation of negative meniscus-shaped intracorneal stromal lenticules was a feasible and reproducible procedure for inducing central corneal flattening while increasing the stromal thickness.26

In this preliminary study, we aimed to investigate the in vivo effect of this novel femtosecond laser–assisted procedure called stromal lenticule addition keratoplasty for advanced keratoconus.

Patients and Methods

Patients

This non-comparative interventional case series study was approved by the institutional review board of the Department of Medicine and Ageing Sciences (University of Chieti, Italy). After a detailed explanation of the procedure, informed consent was obtained from all patients in accordance with the tenets of the Declaration of Helsinki. All patients underwent a complete baseline clinical examination that included evaluation of uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), slit-lamp biomicroscopy, dilated fundus examination, corneal topography (Sirius; CSO Costruzioni Oftalmiche, Firenze, Italy), anterior segment optical coherence tomography (AS-OCT) (RTVue; Optovue Inc., Fremont, CA), and in vivo confocal microscopy (Heidelberg Engineering GmbH, Heidelberg, Germany) examination.

Inclusion criteria were patients with stage III to IV stable keratoconus who were already candidates for keratoplasty27 without documented evidence of progression of keratoconus,28 CDVA worse than 20/200, intolerance to any type of contact lenses for keratoconus, myopic refraction, central keratoconus on topography with the apex of the cone within a 1.5-mm zone, steep keratometry (range: 53.00 to 70.00 D), transparent cornea, minimum thinnest pachymetry of 300 μm, and normal endothelial cell density for age range. Patients with apical scarring, eccentric cone distribution on topography, previous history of laser ablative surgery (LASIK/photorefractive keratectomy) or CXL, or concurrent ocular surface diseases such as dry eye and active atopic keratoconjunctivitis were excluded from the study.

Surgical Technique

Lenticules for implantation obtained from human eye bank donor cornea with scleral rims that were suitable for transplantation were obtained for this study from the Rome Eye Bank (Azienda Ospedaliera San Giovanni Addolorata, Rome, Italy). The mean donor age was 60.1 years (range: 49 to 66 years). Negative serologic test results were obtained from all donors. The average death-to-enucleation time was 8 hours (range: 5 hours and 30 minutes to 10 hours) and the mean storage time (between eye bank procedures and femtosecond laser cut) was 28.8 hours (range: 22 to 48 hours). To avoid the effect of storage-induced stromal edema, tissue was harvested for 6 hours in dextran-enriched storage medium (Thin-C; Alchimia, Padova, Italy). Central corneal thickness (CCT) was measured by ultrasound pachymetry and was less than 600 μm in all cases.

The surgical procedure for lenticule preparation was previously described.26 Briefly, the corneoscleral buttons were rinsed with balanced salt solution and mounted on an artificial anterior chamber (Network Medical Products Ltd., Coronet House, United Kingdom) with standardized pressure. Corneal epithelium was gently removed using a blunt spatula under a surgical microscope prior to performing the femtosecond laser stromal cuts. The FLEx procedure with a hyperopic profile was performed with a 500-kHz VisuMax femtosecond laser (Carl Zeiss Meditec, Jena, Germany) to create the stromal lenticules (Figure 1A).

Schematic representation of the principles and phases of stromal lenticule addition keratoplasty surgical technique. (A) Stromal lenticule profile (negative meniscus) to be dissected with the femtosecond laser in the donor cornea. (B) Intrastromal pocket dissection in the recipient keratoconic cornea, site of the lenticule implantation. (C) Dissection and separation of the stromal lenticules after femtosecond laser cut in the donor cornea using an artificial anterior chamber. (D) The plane of femtosecond laser dissection forming the pocket in the recipient cornea is dissected and opened by using a blunt small incision lenticule extraction spatula. (E and F) The stromal lenticule is grasped at this edge and inserted into the recipient stromal pocket using dedicated forceps through the small superior incision. (G) The lenticule is distended inside the stromal pocket thanks to the cross-like opening of the forceps. (H) Fine centration and complete distension of the lenticule edges are ensured by using a small stromal spatula.

Figure 1.

Schematic representation of the principles and phases of stromal lenticule addition keratoplasty surgical technique. (A) Stromal lenticule profile (negative meniscus) to be dissected with the femtosecond laser in the donor cornea. (B) Intrastromal pocket dissection in the recipient keratoconic cornea, site of the lenticule implantation. (C) Dissection and separation of the stromal lenticules after femtosecond laser cut in the donor cornea using an artificial anterior chamber. (D) The plane of femtosecond laser dissection forming the pocket in the recipient cornea is dissected and opened by using a blunt small incision lenticule extraction spatula. (E and F) The stromal lenticule is grasped at this edge and inserted into the recipient stromal pocket using dedicated forceps through the small superior incision. (G) The lenticule is distended inside the stromal pocket thanks to the cross-like opening of the forceps. (H) Fine centration and complete distension of the lenticule edges are ensured by using a small stromal spatula.

The flap thickness was set to 110 μm and a diameter of 8 mm. Lenticules were programmed for 8.00 D of hyperopic correction with a 6-mm optical zone in the donor corneas. The maximum lenticule thickness was 148 μm with a central lenticule minimal thickness of 30 μm. The overall transition zone in the periphery of the lenticules was 0.70 mm in diameter (Figure 1A). After completion of the femtosecond laser dissection, the flap was lifted as in the LASIK procedures. With the aid of a thin blunt stromal dissector, the underlying lenticule was separated and isolated. All of the lenticules were intact without signs of rupture or tearing, as observed under the surgical microscope with high magnification. This tissue was soaked in Thin-C storage medium while maintaining its orientation and subsequently used for implantation.

The femtosecond laser parameters were set as follows: repetition rate of 500 kHz and spot separation between 2 and 2.5 μm for the lamellar cuts and 3 μm for the side cuts. The spot energy was between 135 and 150 nJ.

Lenticule Implantation Technique

The recipient corneas underwent a modified femtosecond laser flap-cut procedure with the femtosecond laser system (Figure 1B) to produce an intrastromal pocket capable of hosting the donor lenticule.26 Intraoperative details of the surgical techniques are shown in Figures 1C–1H. All surgical procedures were performed under topical anesthesia. The flap hinge length was set to 21.7 mm to obtain a single small superior incision of 4 mm. The incision side cut was angled at 100° from the surface plane. The stromal pocket diameter was set to 8.2 mm (1.5 mm larger than the total diameter of the donor lenticule), whereas the thickness was set to 120 μm from the corneal surface. Laser settings included an energy cut index of 35 nJ and a spot separation of 5 μm.

Intrastromal pockets were centered on the patient's cornea, using the corneal fixation-based centration previously described by Reinstein et al.29 for SMILE procedures and the correct position and depth of the pocket dissections were assessed using AS-OCT before proceeding with implantation. Patients were then moved under a surgical microscope and the incision was opened with a Seibel spatula. The plane of the pocket was dissected using a blunt instrument (SMILE spatula) to remove eventual residual tissue bridges. Correct lenticule orientation was guaranteed by distending the lenticule onto a keratoplasty spoon-shaped glide during transfer on the patient's eye. Subsequently, the lenticule was placed on the patient's cornea with the center marked with gentian violet dye.

Lenticule implantation was performed with specifically designed forceps (Janach, Como, Italy) with a cross-action opening to drag the lenticule inside the pocket and distend the tissue while maintaining the grip in a single maneuver. While grasping the edge of the lenticule with the forceps, it was gently inserted into the pocket through the superior incision. During implantation, the original orientation with the anterior surface directed upward was respected and the lenticule was spread out from the surface using a blunt spatula. The lenticule's position was carefully centered onto the apex of the cone. Incomplete unfolding at the lenticule edges, lenticule striae, or microdistortions were avoided by gentle centrifugal-oriented massage on the corneal surface after insertion.

At the end of the procedure, distension and centration were assessed by the operator using an intraoperative microscope, AS-OCT, and topography. In case a mild offset of the center of the lenticule with respect to the apex of the cone was detected, the lenticule was dragged to the target position with the use of a blunt spatula and spread out again. Once centration was ensured, the incision was mopped with a sterile sponge to soak residual fluid from the interface and a bandage contact lens was applied to the surface of the eye.

Postoperatively, all patients received topical ofloxacin 0.3% six times daily and dexamethasone 0.15% four times daily. After 2 weeks, topical antibiotics were discontinued and the topical steroid was tapered over an 8-week period.

Postoperative Examinations and Outcome Measures

Determination of UDVA, CDVA using logMAR visual acuity charts, manifest refraction, and topographic analysis were performed on all eyes before surgery and at 7 days and 1, 3, and 6 months postoperatively. Corneal topography was performed by corneal Scheimpflug-based topography (Sirius). Curvature changes were assessed using the average keratometric values in the central 3-mm zone (AvgK) for both the anterior and posterior corneal surface. Anterior corneal asphericity (Q value at 8 mm) was also calculated.

At the same time points, a clinical slit-lamp examination and morphologic analyses using AS-OCT (RTVue, Optovue Inc., Fremont, CA) and laser scanning in vivo confocal microscopy (HRT II Rostock Cornea Module, Heidelberg Engineering GmbH, Germany) were also performed.

An AS-OCT pachymetry map was acquired to assess changes in central cone apex and 6-mm thickness. Corneal tomography was acquired using AS-OCT with single 8-mm scan length mode on the horizontal and vertical meridians. Lenticule dimensions were measured with the caliper tool on horizontal and vertical scans. Central lenticule thickness, maximum lenticule thickness, and lenticule diameter were measured by averaging the four readings of the scans and differences between the preoperative and postoperative values in recipient corneas were calculated. In vivo confocal microscopy examination was performed according to a previously described technique.30 The technical configuration of the confocal laser scanning device has been previously described.31 In vivo confocal microscopy was performed under topical anesthesia with 0.4% oxybuprocaine. Proper alignment and positioning of the head were maintained with the help of a dedicated movable target (red fixation light) for the contralateral eye. A drop of 0.2% polyacrylic gel served as the coupling medium between the polymethylmethacrylate contact cap of the objective lens and cornea. All corneas were examined centrally by performing three complete z-axis scans from epithelium to endothelium. Confocal micrographs were analyzed with the Heidelberg Eye Explorer software (version 1.5.1; Heidelberg Engineering GmbH).

Statistical Analysis

Given the number of patients enrolled in this pilot study, only non-parametric (Wilcoxon signed-rank and Fisher's exact tests) statistical tests were used. All statistical analyses were performed using SPSS software (version 24.0; IBM Corporation, Armonk, NY) and evaluated at an alpha level of 0.05.

Results

Safety, Visual Acuity, and Refraction

The follow-up period extended to 6 months after surgery. We did not observe any intraoperative or relevant postoperative complications. Two patients required repositioning of the lenticule to achieve target centration during surgery. Total operation times ranged from 15 to 35 minutes. All patients tolerated the procedure well and experienced only mild symptoms within the first week after surgery, which included foreign body sensation and mild blurred vision. Slit-lamp examination on day 1 showed mild interface edema and few lenticular tissue folds, which subsequently cleared by day 7. Slit-lamp photographs showing the postoperative appearance after surgery are presented in Figure 2. There were no cases of infection, epithelial defects, stromal infiltration, or immunologic rejection during the entire follow-up period. The preoperative data of recipient patients are reported in Table 1.

Slit-lamp photographs showing the (A) preoperative and (B and C) postoperative features of the cornea after stromal lenticule addition surgery. (B) The cornea appears clear with a transparent interface at 1 month after surgery and the intrastromal lenticule is barely visible. (C) Transillumination slit-lamp photograph highlights the lenticule profile.

Figure 2.

Slit-lamp photographs showing the (A) preoperative and (B and C) postoperative features of the cornea after stromal lenticule addition surgery. (B) The cornea appears clear with a transparent interface at 1 month after surgery and the intrastromal lenticule is barely visible. (C) Transillumination slit-lamp photograph highlights the lenticule profile.

Patient Demographics and Refractive Data of Patients Treated With SLAK

Table 1:

Patient Demographics and Refractive Data of Patients Treated With SLAK

At 6 months postoperatively, UDVA significantly improved from 1.58 ± 0.36 to 1.22 ± 0.37 logMAR (P = .024; Wilcoxon signed-rank test), whereas CDVA improved from 1.07 ± 0.17 to 0.70 ± 0.23 logMAR (P = .007).

Eight of 10 eyes showed an improvement in UDVA (P < .001; Fisher's exact test), whereas all but one eye had improved CDVA. Particularly, 1 eye gained three lines, 3 eyes gained two lines, 5 eyes gained one line and 1 eye had no change in lines of CDVA (Table 1 and Figure 3). Cumulative Snellen CDVA values before and after surgery are shown in Figure 4. Changes of spherical equivalent over time after surgery and vector analysis of refractive astigmatism are presented in Figure 5 and Figure A (available in the online version of this article), respectively.

Change in lines of corrected distance visual acuity (CDVA) after lenticule implantation at 6 months.

Figure 3.

Change in lines of corrected distance visual acuity (CDVA) after lenticule implantation at 6 months.

Corrected distance visual acuity (CDVA) at 6 months postoperatively (postop) compared with preoperative CDVA (preop). CF = counting fingers

Figure 4.

Corrected distance visual acuity (CDVA) at 6 months postoperatively (postop) compared with preoperative CDVA (preop). CF = counting fingers

Stability of spherical equivalent over the 6-month follow-up period. SD = standard deviation; SEQ = spherical equivalent; D = diopters

Figure 5.

Stability of spherical equivalent over the 6-month follow-up period. SD = standard deviation; SEQ = spherical equivalent; D = diopters

Refractive astigmatism vector analysis represented with double-angle cylinder plot. Centroid (green dot) and standard deviation (green ellipse) of (A) preoperative and (B) 6-month postoperative astigmatism. D = diopters

Figure A.

Refractive astigmatism vector analysis represented with double-angle cylinder plot. Centroid (green dot) and standard deviation (green ellipse) of (A) preoperative and (B) 6-month postoperative astigmatism. D = diopters

Topographic Curvature Changes

Changes in curvature and asphericity values detected by corneal topography are reported in Table A (available in the online version of this article).

Changes in Topographic Curvature and Corneal Asphericity

Table A:

Changes in Topographic Curvature and Corneal Asphericity

In all cases, corneal topography showed a generalized flattening of the cone as expressed by the mean anterior keratometry value (mean K). The mean anterior keratometry value decreased from 58.69 ± 3.59 to 53.59 ± 3.50 at 6 months after surgery. Corresponding posterior corneal mean K values showed negligible variation (Table A). Figure B (available in the online version of this article) shows the topographic changes of a representative case treated with the lenticule implantation procedure and the differential tangential curvature maps. In all treated eyes, a reduction of the anterior corneal asphericity (Q value) was detected at 6 months (Table A), indicating a more regular prolate corneal profile.

Anterior and posterior corneal topographical changes after stromal lenticule addition keratoplasty. Preoperative (A) anterior and (D) posterior tangential maps showing a stage 4 central keratoconus. Postoperative topographies showing a significant central cone flattening, surrounded by a mid-peripheral annular increase in curvature in the anterior tangential maps (B) 1 month and (C) 6 months after surgery. Postoperative posterior surface topographies at (E) 1 month and (F) 6 months do not show evident changes compared to the preoperative cone in the central cornea. Differential tangential (G) anterior curvature map shows a central flattening of the cone, with respect to preoperative appearance, whereas minimal changes are visible in the (H) posterior surface differential map.

Figure B.

Anterior and posterior corneal topographical changes after stromal lenticule addition keratoplasty. Preoperative (A) anterior and (D) posterior tangential maps showing a stage 4 central keratoconus. Postoperative topographies showing a significant central cone flattening, surrounded by a mid-peripheral annular increase in curvature in the anterior tangential maps (B) 1 month and (C) 6 months after surgery. Postoperative posterior surface topographies at (E) 1 month and (F) 6 months do not show evident changes compared to the preoperative cone in the central cornea. Differential tangential (G) anterior curvature map shows a central flattening of the cone, with respect to preoperative appearance, whereas minimal changes are visible in the (H) posterior surface differential map.

The decrease between preoperative and 6-month postoperative anterior corneal curvature (AVG-K) and anterior Q values was statistically significant (P < .005; Wilcoxon signed-rank test) (Table A).

Pachymetry and Corneal Profile Changes on AS-OCT

Corneal cross-sectional visualization using AS-OCT allowed identification of the intrastromal lenticule profiles in contrast to the surrounding recipient corneal stroma, as shown in Figure 6. Mild irregularities of the lenticule and recipient stroma, attributable to tissue edema, were detected during the first week after surgery and receded thereafter. OCT imaging confirmed the precise lenticular profile inside the intrastromal pocket across the whole diameter, which corresponded to the lenticule shape before extraction (Figure 6B). Lenticule morphometric values at 6 months are reported in Table B (available in the online version of this article). Particularly, central lenticule thickness and maximum lenticule thickness measurements were consistent with the expected values as programmed in donor lenticules dissected by femtosecond laser, although the measurements of central lenticule thickness were slightly higher than programmed thickness in all cases.

High-resolution anterior segment optical coherence tomography (OCT) scans of the recipient cornea (A) before and (B) 6 months after surgery and (C and D) corresponding OCT pachymetry maps. The intrastromal lenticule appears well centered and integrated with its profile identifiable within the recipient stroma. Lenticule interfaces show moderate reflectivity in the central zone, whereas the stromal component of the lenticule presents a lower reflectivity compared to the surrounding recipient stroma. Pachymetry maps show a corneal thickness increase in both central corneal cone apex and mid-peripheral cornea after surgery, consistent with the lenticule morphology.

Figure 6.

High-resolution anterior segment optical coherence tomography (OCT) scans of the recipient cornea (A) before and (B) 6 months after surgery and (C and D) corresponding OCT pachymetry maps. The intrastromal lenticule appears well centered and integrated with its profile identifiable within the recipient stroma. Lenticule interfaces show moderate reflectivity in the central zone, whereas the stromal component of the lenticule presents a lower reflectivity compared to the surrounding recipient stroma. Pachymetry maps show a corneal thickness increase in both central corneal cone apex and mid-peripheral cornea after surgery, consistent with the lenticule morphology.

AS-OCT Changes in Pachymetry and Lenticule Parameters Analysis

Table B:

AS-OCT Changes in Pachymetry and Lenticule Parameters Analysis

Postoperative CCT pachymetry values increased in all cases, as reported in Table B. The mean CCT values increased from 406 ± 43 to 453 ± 39 μm at the end of the follow-up (P < .005; Wilcoxon signed-rank test). An increase in peripheral corneal thickness at 6 mm was observed in all cases and was consistent with the local thickness of the implanted lenticule (Table B), as shown in Figures 6C–6D.

Statistically significant linear correlations between lenticule thickness and the induced modification were not observed.

In Vivo Confocal Microscopy

The lenticule–host interfaces could be identified in all cases. Both anterior and posterior lenticule interfaces were characterized by acellular structure and dishomogeneous hyperreflectivity (Figures CA and CE, available in the online version of this article), with some bright spots interspersed as shown in Figure C. Reflectivity values of the confocal micrograph at the interface decreased over time in all eyes. Activated and lower reflective elongated keratocytes within the lenticule lamellae were observed with disordered extracellular collagen arrangement by week 1 (Figure CC). Inflammatory cell infiltration or diffuse lamellar keratitis were not observed in any of the cases during follow-up. At 6 months, keratocyte morphology appeared close to normal, with most of the cells within the lenticule visible as bright keratocytes with more regular shape and regular reflectivity, whereas the extracellular tissue presented normal transparency (Figures CB, CD, and CF).

In vivo confocal microscopy micrographs of the recipient stroma after surgery. At 1 week after surgery, the (A) anterior and (E) posterior lenticule interfaces are characterized by acellularity and high reflectivity with bright dots. (C) Disordered collagen arrangement and activated keratocytes are visible within the lenticule lamellae mild tissue edema. (B and F) After 6 months, the stromal lenticule interfaces showed reduction of reflectivity and some visible keratocyte nuclei. (D) Distinct viable keratocyte cells, with elongated morphology, were seen in the lamellae of the lenticule.

Figure C.

In vivo confocal microscopy micrographs of the recipient stroma after surgery. At 1 week after surgery, the (A) anterior and (E) posterior lenticule interfaces are characterized by acellularity and high reflectivity with bright dots. (C) Disordered collagen arrangement and activated keratocytes are visible within the lenticule lamellae mild tissue edema. (B and F) After 6 months, the stromal lenticule interfaces showed reduction of reflectivity and some visible keratocyte nuclei. (D) Distinct viable keratocyte cells, with elongated morphology, were seen in the lamellae of the lenticule.

Discussion

The possibility of transplanting refractive lenticules was already validated in previous studies conducted with animal models.17,18,32 These studies reported that intrastromal lenticule implantation can be performed with integration and survival of the tissue within the host cornea.17,18,32

Stromal transplantation of refractive lenticules derived from myopic SMILE has been proposed as a possible tissue-addition approach to steepen the cornea for treating hyperopia. The approach is different than subtractive procedures such as LASIK that induce an increase in central corneal curvature by ablating the mid-peripheral tissue. The first case of allogenic lenticule implantation for the treatment of high hyperopia in an aphakic patient was described by Pradan et al.20 Ganesh et al.22 studied the use of cryopreserved refractive lenticules extracted after myopic SMILE as allogeneic human subject transplantation. The initial results obtained with this technique, called femtosecond laser intrastromal lenticular implantation, yielded promising outcomes. However, the accuracy and predictability of the refractive outcome, especially for higher hyperopic treatments, requires further improvement.21,22

Based on these studies, Ganesh and Brar33 later proposed the application of intrastromal implantation of donut-shaped stromal tissue, obtained by a 3-mm central punch of cryopreserved myopic lenticules. The addition of this tissue in the corneal mid-periphery and around the cone technique induced a relative flattening in the center and modification in the corneal shape to a less hyperprolate morphology.33 The possible mechanism of action of this approach is likely to be partially similar to the implantation of intracorneal ring segments because both techniques involve addition of material and local elevation in the mid-periphery.7,8

In this pilot study, we investigated the in vivo morphological and refractive effects of negative meniscus-shaped stromal lenticule transplantation, obtained from eye bank donor corneas, for the treatment of advanced cases of non-progressive keratoconus. The principle of the stromal lenticule addition keratoplasty technique is to reshape the anterior corneal profile by stromal tissue addition that increases the thickness and improves corneal curvature.

After surgery, a statistically significant improvement in UDVA, CDVA, and spherical equivalent was observed. After surgery, 9 of 10 eyes showed an increase in CDVA that ranged from one to three Snellen lines. All treated eyes had a concomitant reduction of myopic refractive error and manifest astigmatism, although the degree of improvement was neither homogeneous nor predictable among the patients.

Topographical analysis showed that all eyes had a detectable reduction of central anterior corneal curvature, indicating a significant relative flattening of the cone, with negligible effects on the posterior corneal curvature. Correspondingly, a significant improvement of corneal asphericity (Q) values was noted, indicating a less hyperprolate shape of the central keratoconic corneas, after surgery. Notable variations of corneal curvature and asphericity were observed in the patients treated regardless of the preoperative curvature values.

AS-OCT showed that lenticule morphometry after implantation corresponded to the values of the lenticule design programmed by the laser, although minimal differences in the central lenticule thickness were observed; on average the central lenticule thickness was measured to be 47 μm compared to a programmed thickness of 30 μm. This difference could be due to postoperative factors such as interface spacing effects or differences in tissue imbibition in vivo with respect to the condition of the donor corneas in storage conditions. The lenticule peripheral thickness values showed a greater variability that might be related to similar factors and to the compressive effects of the peripheral recipient lamellae on the lenticule. AS-OCT measurements also revealed a corresponding significant increase in both central and mid-peripheral corneal pachymetry that was consistent with the shape and thickness profile of the lenticule implanted. The mid-peripheral thickening corresponded to the thickest part of the lenticule, whereas the central thinner part of the lenticule, placed at the apex of the cone, was responsible for the mild thickness increase observed in the central cone area. It is known that the epithelium remodels following a change in stromal surface curvature, which therefore likely influenced the total corneal thickness.34

In their series, Ganesh and Brar33 reported an increase in CCT after implantation of donut-shaped myopic lenticules, although there was no addition of stroma in the central area in this procedure. They proposed that this might be related to a mild lifting of the anterior corneal layers and creation of a potential space in the center after peripheral tissue addition. They also observed a trend toward a decrease in CCT at 6 months after surgery, suggesting that the space created may collapse over time.33 This phenomenon should not occur in our technique because of the presence of the central part of the lenticule tissue addition in the cone apex. However, stromal wound healing processes and epithelial remodeling may influence corneal thickness changes during the postoperative course.

To our knowledge, this is the first study that investigated the in vivo stromal wound healing after lenticular implantation in keratoconic corneas in humans. Interestingly, in vivo confocal microscopy showed a mild stromal interface wound healing phenomena in the early postoperative phase, characterized by minimal tissue edema that subsided after 1 week and activated keratocytes. Corneal and interface transparency were not affected during the follow-up and resident quiescent keratocytes were visible within the lenticular lamellae 6 months after surgery. Confocal microscopic observations documented that the stromal lenticular transplantation was easily integrated within the recipient corneas, similarly to what was reported for lenticule implantation in animal models.17,18 However, further specific studies are needed to better characterize in vivo the integration processes, on a cellular basis, of stromal addition keratoplasty procedures.

The improvement of corneal curvature and keratoconus stromal thickness induced by the procedure tested in our study might be further consolidated by CXL as already proposed.33 Although we only evaluated patients who had a stable corneal ectasia, we believe that the lenticule addition technique combined with CXL may also be beneficial in the treatment of progressive keratoconus.4 The addition of stromal tissue should favor a biomechanical strengthening of the cornea, whereas the CXL effect may further help in stabilizing the keratoconus progression, similar to what was suggested for intracorneal ring segment implantation.4,8 It is believed that the lamellar cut performed for intrastromal pocket creation should not significantly alter the biomechanical properties of the cornea. It has been shown that femtosecond laser creation of vertical side cuts through corneal lamellae rather than horizontal delamination contributes to the loss of structural integrity during LASIK flap creation.35 Ganesh and Brar33 found some improvements in corneal biomechanical parameters after tissue addition combined with CXL in keratoconus, but specifically designed clinical studies are needed to clarify this point.

Another advantage related to the principle of this technique may be that corneas with thin stroma, having thinnest pachymetry below the recommended values for CXL, may indeed be treated thanks to the stromal expansion induced by the lenticule implantation.

The major limitations of our study include the small sample size and the fact that we selected only patients with central keratoconus to facilitate the procedure of lenticule implantation and centration. Specific studies investigating whether the lenticule profile used in our investigation might also be useful in patients with more eccentric keratoconus, which are the most common cases. In addition, variation of lenticule shapes, profiles, diameter, optical power, and depth of insertion should be analyzed in a prospective randomized fashion.

Although the visual improvement in our series did not exceed three Snellen lines, it has to be underlined that all patients had advanced keratoconus and were intolerant to contact lenses. In further investigations, we will evaluate whether the corneal curvature improvement induced by the procedure in cases with less severe keratoconus may favor contact lens tolerance, with the possibility of having a greater impact on vision.

Our preliminary study demonstrated that stromal lenticule addition keratoplasty was a feasible and effective technique for stromal remodeling that improved vision and corneal regularity in advanced central keratoconus.

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Patient Demographics and Refractive Data of Patients Treated With SLAK

No.Age / GenderEyeStagePreoperativeFollow-up (6 Mo)Change in Lines of VA


Manifest RefractionUDVACDVAManifest RefractionUDVACDVA
153 / MODIV−4.00 / −5.50 × 801.31.0−2.00 / −3.00 × 901.30.7+1
240 / MOSIV−3.00 / −4.00 × 1001.31.0−1.25 / −2.50 × 750.60.4+3
364 / MODIV−2.00 / −3.50 × 901.31.00.00 / −2.00 × 851.30.7+1
427 / MODIV−10.00 / −5.00 × 952.01.3−6.00 / −3.00 × 802.01.0+1
535 / FODIV−5.00 / −3.00 × 851.31.0−2.00 / −1.50 × 951.00.5+2
645 / MOSIV−7.00 / −4.00 × 1052.01.3−4.00 / −2.25 × 1001.31.0+1
737 / FOSIV−6.00 / −2.75 × 851.31.0−2.00 / −1.50 × 900.80.5+2
842 / FOSIV−6.50 / −3.50 × 952.01.0−3.50 / −2.75 × 851.31.00
939 / MODIII−4.00 / −4.00 × 901.30.80.00 / −2.50 × 901.30.5+1
1048 / MOSIV−8.00 / −3.00 × 1102.01.3−4.00 / −1.75 × 1001.30.7+2

Changes in Topographic Curvature and Corneal Asphericity

No.Anterior AVG-K (3 mm)Posterior AVG-K (3 mm)Anterior Q Value



Preop6 MonthsDiff.DPPreop6 MonthsDiff.DPPreop6 MonthsDP
155.249.5−5.7−10.33−10.44−10.65−0.212.01−2.46−1.96−20.33
256.8252.86−3.96−6.97−8.87−9.69−0.829.24−1.83−0.11−93.99
356.1850.55−5.63−10.02−9.07−9.17−0.11.10−1.64−0.15−90.85
466.5360.49−6.04−9.08−10.79−11.54−0.756.95−1.85−0.65−64.86
557.351.4−5.9−10.30−8.02−8.78−0.769.48−1.75−0.20−88.57
663.0558.3−4.75−7.53−8.5−8.75−0.252.94−1.82−0.66−63.74
759.453.7−5.7−9.60−9.04−9.53−0.495.42−1.93−0.13−93.26
857.0353.9−3.13−5.49−8.86−9.24−0.384.29−1.97−0.27−86.29
955.950.08−5.82−10.41−9.29−9.52−0.232.48−2.17−0.28−87.10
1059.555.1−4.4−7.39−10.22−10.78−0.565.48−1.99−0.91−54.27
Mean58.6953.59−5.10−8.71−9.31−9.77−0.464.94−1.94−0.53−74.33
SD3.593.590.991.740.890.930.262.930.230.5723.68

AS-OCT Changes in Pachymetry and Lenticule Parameters Analysis

No.Central Corneal Thickness6-mm ThicknessLenticule Parameters



Preop6 MonthsDiff.DPPreop6 MonthsDiff.DPCLTMLTLD
1453494419.0558768810117.21431486.8
2460505459.7859373714424.28511566.71
3443481388.585856567112.14441407.03
43203725216.2542458315937.50341586.14
53704235314.3254569515027.52631436.35
63894364712.0845357512226.93551156.45
73954404511.3946159313228.63531216.57
83834415815.1449764915230.58631456.91
9427467409.3751361510219.88471156.76
104234765312.5347259512326.06361166.97
Mean4064534711.8551363812525.07511356.67
SD433962.716155277.199170.29
Authors

From National Centre of High Technology in Ophthalmology of the University “G d'Annunzio,” Chieti-Pescara, Italy (LM, MN, NS); and the Eye Treatment Center, Whipps Cross University Hospital, London, United Kingdom (RM).

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

Drs. Mastropasqua and Nubile contributed equally to this work and should be considered as equal first authors.

AUTHOR CONTRIBUTIONS

Study concept and design (LM, MN, NS, RM); data collection (MN, NS); analysis and interpretation of data (LM, MN, NS, RM); writing the manuscript (MN, NS); critical revision of the manuscript (LM, MN, NS, RM); statistical expertise (MN); administrative, technical, or material support (LM, MN, NS, RM); supervision (LM, MN, RM)

Correspondence: Mario Nubile, MD, PhD, Ophthalmology Clinic, National Centre of High Technology in Ophthalmology of the University “G d'Annunzio,” Clinical Hospital Colle dell'Ara, Chieti-Pescara, Italy. E-mail: m.nubile@unich.it

Received: June 23, 2017
Accepted: September 21, 2017

10.3928/1081597X-20171004-04

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