The advances of corneal preparation – what is to come?

Abstract

Significant advances have been made over the last decade in the field of corneal transplantation techniques, which have given rise to new challenges for eye banks. As the new developments in corneal transplantation surgeries allow selective replacement of the diseased corneal layer and the group of patients that can be successfully treated expanded, the demand for available corneal tissue is increasing steadily. Thus, eye banks are challenged to develop more efficient, validated and standardized corneal lamellar graft preparation (split corneal grafts) and preservation methods. Furthermore, precut and preloaded lamellar corneal grafts that are customized to patient’s needs and easier to transplant are developed. Such developments are not only strengthening the cooperation among eye banks, collaborating surgeons and patients, but also increase the need to interact in a constructive way with different national and international regulatory bodies, in order to update and/or establish legal, safety and quality standards for future corneal tissue transplantation.

Keywords:

• corneal lamellar graft preparation
• corneal transplantation
• eye banks
• precut corneal grafts
• preloaded corneal grafts

At the end of previous century, the notable achievements in eye banking corneal tissue storage methods enabled prolongation of corneal preservation time and the advent of scheduled elective transplantation surgeries. Since then, a remarkable evolution in the field of corneal transplantation procedures has been achieved. Thus, entering an era of selective lamellar keratoplasty (LK) procedures has given rise to new challenges for eye banks.

LK procedures enable the replacement of only the diseased corneal layers and can be divided into anterior or posterior (endothelial) LK [1]. In anterior LK procedures, only the diseased anterior corneal layers are transplanted, leaving the healthy deep posterior corneal layers of the recipient intact [2]. Due to its distinct advantages over full-thickness penetrating keratoplasty (PKP), deep anterior LK (DALK) has become the gold standard approach for treating patients with ectatic and anterior corneal layer disorders [2]. However, suboptimal visual outcomes due to interface haze are still an issue after DALK [1]. As smoothness of the stromal interface relates to better visual acuity results, more precise anterior lamellar grafts preparation methods as well as improvements in surgical techniques for the posterior recipient stroma dissection (e.g., pre-Descemetic DALK) have been sought. Currently, femtosecond laser (FSL)-assisted creation of shaped wound configurations in DALK are reported to enable better mechanical stability and wound-healing properties, which were previously demonstrated in stepped corneal wounds in PKP (e.g., top hat, mushroom, zig-zag, Christmas tree) [3]. The use of FSL shaped wound incisions (e.g., zig-zag incision) not only facilitates big bubble formation with better baring of Descemet’s membrane (DM), it also creates better donor–recipient fit with better surface area contact, resulting in faster wound healing [3]. Thus, the introduction of FSL enabled more precise shaped and technically easier donor and recipient corneal preparation in DALK [3,4].

Since 1998, different endothelial keratoplasty (EK) procedures have evolved to treat patients with isolated corneal endothelial dysfunction [5]. EK include surgical variations, such as deep lamellar endothelial keratoplasty, Descemet stripping (automated) endothelial keratoplasty (DSEK/DSAEK) and Descemet membrane endothelial keratoplasty (DMEK) [5], which differ in posterior lamellar graft thicknesses due to variations in residual stromal content [1]. As DMEK replaces only the diseased endothelium with DM, the DM endothelial graft (DEG) should not involve layers of corneal donor stroma [6]. Based on the clinical trial results, stroma-free posterior lamellar grafts (as in DEG) provide the best and the fastest visual rehabilitation [7], thus preferring DMEK for corneal endothelia dysfunction. However, due to the difficulties in DEG preparation and to perform the surgical procedure, some surgeons hesitate in starting with DMEK [8]. Therefore, due to easier graft handling and a still rapid visual outcome, some specialized ophthalmic centers prefer the DSAEK and ultrathin-DSAEK techniques to DMEK [6,9]. However, although many factors can contribute to optimal visual outcomes following EK, a high-quality corneal graft is highly desirable, be it prepared by the surgeons immediately before the keratoplasty or delivered as a precut donor lamellar corneal graft by eye banks. Moreover, the introduction of new advanced instruments and preparation tools (e.g., microkeratomes, excimer lasers, FSL, artificial chambers) has further enabled the development of more accurate and safer lamellar corneal graft preparation methods with better cellular viability [1]. Thus, a lot of surgeons and eye bankers still debate different techniques to prepare optimal donor lamellar grafts.

Moving forward from full-thickness PKP to selective lamellar corneal transplantation procedures, eye banks started to develop and prepare precut corneal tissues to support corneal surgeons. The precut and prevalidated lamellar corneal grafts can overcome the technical and logistical difficulties of cutting the donor cornea in the operating room, which can prolong the operation time and increase the wastage of precious donor tissues [10]. Furthermore, comparable endothelial cell loss, visual outcomes and detachment rates were reported between eye bank precut and surgeon prepared corneal lamellar tissue, with no higher clinical infections rates being noticed [6]. However, some surgeons still have concerns regarding the possible varying quality and safety of the tissue preparation between different eye banks, preventing them to exclusively use precut tissue [11]. Thus, standardization and validation of precut and preloaded corneal graft tissue is very much needed, and is on the way.

Currently, DSAEK is more standardized, efficient and safe for generating corneal grafts [1] in comparison to DMEK grafts. In DSAEK, the posterior lamellar grafts are commonly prepared with a microkeratome [6]. However, if graft thickness asymmetry and irregularity are present, a known postoperative hyperopic refractive shift is noticed [6]. Preparation of thinner and more precise grafts is therefore desired [9,12]. Double-pass microkeratome ‘ultrathin’ (100 μm or less) yielded DSAEK grafts are being under active research [9,12]. To facilitate the supply of graft tissues, we presented that DSAEK grafts could be precut, trephined and then preserved as a ready-to-use graft in organ culture (OC) for up to 2 weeks without causing apparent endothelial damage or tissue thickening [9]. Recently, we designed and validated the efficacy of a three-dimensional (3D) printed smart storage glide which is capable of preserving and delivering posterior corneal grafts for DSAEK allowing transportation of quality-controlled precut tissues [12]. The designed smart storage glide ensured safe delivery (no negative influences on preservation, transportation and implantation) of the graft tissues and allowed the surgeon to perform graft insertion easier. We additionally showed that 3D printing could be used for the development of surgical tools [12]. DSAEK precut grafts could also be successfully shipped worldwide in hypothermic conditions and clinically used after several days with minimal endothelial loss [13].

On the other hand, the standardization of DMEK precut grafts and the development of ready-to-transplant preloaded DEG tissues are even more challenging for eye banks and surgeons [1,5,14–16]. Since a perfect DEG tissue has a thickness of less than 30 μm, it is extremely vulnerable to damage [1]. Without the stromal layer, the DEGs are likely to roll, which can additionally decrease the endothelial cell viability after graft preparation [1]. Therefore, endothelial cell mortality analysis has been desired to standardize a mortality threshold. Currently, various methods for DEG dissections are used including manual mechanical peeling methods (forceps dissection) [5], pneumatic dissection and hydro-dissection methods [1,16–18]. With the relatively high mortality rate of endothelial cells (>8%) and the high donor tissue wastage (>16%), preparation of the donor DEG tissues is a challenge [16]. With the forceps dissection method, using either the 1-point nontoothed forceps technique or the 2-point nontoothed forceps technique to grasp the DM, strong, localized radial lines of tension can develop with inadvertent peripheral rents, which predispose to tissue wastage [19]. The introduction of a curvilinear forceps by Yoeruek and colleagues enabled equal distribution of the tensile strength over the DM, reporting decreased tissue wastage and preparation time compared to the 1-point forceps technique [19]. Moreover, due to the time investment and difficulties in balancing the tensile forces in nontoothed forceps dissection, the air dissection method to harvest DEG was supposed to be faster and easier to prepare [20]. In the first comparative study evaluating air and forceps dissection of DEG, which were further OC for 1 week, both techniques showed equal endothelial cell loss, apoptosis and ultrastructural findings [20]. However, although air injection for DEG separation is a routinely performed method in DALK, high manual manipulation skills are required (controlling injected air), the success rates of creating the big bubble being variable [17]. In contrast, the submerged hydro-separation method that we have recently described allows a higher success rate and decreases the time required for preparing the DEG tissue [1,16,17]. Since Dua et al. suggested the controversial acellular pre-Descemetic layer called Dua’s layer to be responsible for different types of bubble formation, we believe that with liquid bubble formation the liquid separates the DM-stroma phase or DM-Dua’s layer phase, the tissue obtained being without stromal residues as was further confirmed with histological examination of the grafts [16] in comparison to ‘residual stroma’ contacting DEG tissues after pneumodissection [21]. We compared the DEG preparation using exclusively air or liquid bubble. By using a liquid bubble, we generated a greater yield, larger diameters and higher maintenance of endothelial cell density and integrity [17]. Moreover, as the liquid helps to keep the tissue intact in the artificial chamber, further cut/trephination is enabled in comparison to an air bubble separation method that sticks DEG to the stroma as soon as the anterior cornea is cut/trephined [16,17]. Thus, the procedure enables standardized, prevalidated (quality assured), precut, no-touch, ready-to-use DEG tissue, allowing preservation for up to 7 days in a deturgescent medium [16]. Additionally, the pre-trephined tissues could be either rolled or on a contact lens delivered using a surgical glide, to ease the handling of such a delicate tissue, thus supporting the surgeons in transplantation procedures [16].

Due to donor corneal shortage, the development of split corneal transplantation grafts is also being evaluated. With the appropriate graft preparation technique, a single cornea can be used for two different lamellar procedures, minimizing donor tissue wastage. Melles et al. [5] reported a standardized ‘no-touch’ donor tissue preparation for DALK and DMEK. In this technique DM was stripped with a peripheral rim of trabecular meshwork enabling easier manipulation of the DEG, which was transferred to a soft contact lens to be subsequently trephined. Due to the elastic properties a ‘Descemet roll’ with the endothelium on the outside was formed, the rolls could be further stored in OC medium at 31°C until transplantation. Since a soft contact lens was used as a matrix during trephination, the anterior corneal tissue was left intact for further use [5]. Similarly, Chu et al. [22] reported the storage of anterior corneal grafts that were separated from donor DSAEK tissue. The remaining anterior corneal grafts were successfully stored in Optisol GS hypothermic storage for up to 4 weeks before being used in patch grafting of cornea and sclera [22]. Additionally, as recently reported by Kanavi et al. [23] and Javadi et al. [24], cryopreserved corneas were successfully used for DALK in eye banks that do not use OC media as the long-term preservation method, with no observed differences in clinical outcomes and postoperative complications after DALK comparing cryopreserved and fresh corneal grafts [24]. Thus, freezing of surplus eye globes could enable an enlarged and a reliable source of donor corneas for DALK [23], especially in the case of shortage of fresh donor corneas.

Furthermore, to increase the availability of donor tissues, Melles and colleagues [7] evaluated the technical feasibility of using semicircular, large-diameter hemi-DMEK grafts, with reported good clinical outcomes in three patients [7]. Taking EK a step further, with a better understanding of endothelial wound healing processes, a technically simplified procedure of DM endothelial transfer was recently introduced, where no need to unfold the Descemet roll or to completely attach the endothelial graft exists [8].

To conclude, over the recent years, new achievements in corneal surgical techniques and lamellar tissue graft preparation methods evolved, as we strived to optimize clinical outcomes of transplant patients. These new surgical and storage procedures have prompted new studies not only in the field of endothelial cell physiology and stem cell investigations, but also new research reinvestigating and reconsidering the ultra-structural corneal anatomy [21]. This knowledge may further influence the future approach to the processing of corneal tissues and surgical techniques. Thus, as we speculate, eye banks will play an important role also in the future in the development of new surgical technical procedures in a good collaboration with the clinicians.

Financial & competing interests disclosure

Z Lužnik is the recipient of a Boehringer-Ingelheim travel grant. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.