Ординатура / Офтальмология / Английские материалы / Corneal Endothelial Transplant (DSAEK, DMEK & DLEK)_John_2010

Histology

The cross-sections of control samples and HCEC sheets were stained for DAPI to examine the histological structure of endothelium. The endothelial cells from the human donor corneas were organized on the Descemet’s membrane as a monolayer (Figure 38-9A). The detached cell sheets also showed a monolayered architecture of cells that mimicked native endothelium (Figure 38-9B).

A

to correspond to the cell polarity as in vivo (Figures 38-1D and E). After cell separation from thermoresponsive culture substrates at 20°C, a bioadhesive gelatin disk was placed on the apical surface of the harvested HCEC sheets, and the gelatin-HCEC sheet constructs were spontaneously formed by a 5-min incubation at room temperature (Figure 38-10).

B

Figures 38-9A and B: Histological examination of control samples (A) and HCEC sheets (B) with DAPI labeling show a monolayer of endothelial cells (arrows) was located on corneal stroma (asterisk) and cell carrier membrane (double asterisk), respectively. Scale bars, 20 µm.

Gelatin Carriers for HCEC Sheet Transplantation

Given that HCECs in vivo possess polarity and pump water from corneal stroma into the anterior chamber, a correct orientation of the transplanted HCECs must be maintained with the apical side facing the aqueous humor in anterior chamber. Accordingly, the detached HCEC sheets were delivered using the 7 mm gelatin disks (700-800 µm thick, MW = 100,000, IEP = 5) with the HCECs apical side down

Figure 38-10: Gelatin carriers for intraocular delivery of thermally detached HCEC sheets. After cell release from PNIPAAm-grafted culture surfaces at 20°C, the gelatin-HCEC sheet constructs were made by using the 7-mm diameter gelatin disks (arrow) as a transparent and bioadhesive supporter for HCEC sheets with a size of around 0.75 cm2. Scale bar, 5 mm.

Postoperative Evaluations

For in vivo transplantation studies, the central 7 mm of corneal endothelium was removed with a silicone-tipped cannula (Figure 38-11A). In HCEC sheet groups, after surgery, slit-lamp biomicroscopy revealed that the anterior chamber was filled up with the gelatin-HCEC sheet constructs (Figure 38-11B). Moreover, an intact, roundshaped layer of HCECs was positioned onto the denuded corneal posterior surface. The following day, severe corneal swelling was noted, and persisted until completion of the experiment in wound (Figure 38-11C) and gelatin groups. At postoperative 2 weeks, the gelatin disks largely dissolved and HCEC sheets were attached onto the denuded surface of Descemet’s membrane in the HCEC sheet groups. The swollen cornea returned to clarity and a nearly normal corneal thickness after implantation of HCEC sheets 4 weeks postoperatively (Figure 38-11D). The corneal thickness of traumatized corneas with transplanted HCEC sheet improved more significantly than that of the control groups during the first postoperative 2 weeks (Figure 38-12). All corneas in the control groups did not return to

normal during the follow-up. Figures 38-13A to C illustrate the images of flat-mounts and cross-sections of the grafted corneal samples in the HCEC sheet groups. Under fluorescence microscopy, the HCEC sheet grafts were shown on the recipient corneas and maintained intact tight junctions at 2 weeks postoperatively (Figure 38-13A). Histological examination under light and fluorescent microscopy revealed that, after surgery for 2 weeks, the implanted HCECs labeled with PKH26 red fluorescent dye remained attached (Figures 38-13B and C).

Discussion

HCECs in vivo demonstrate an age-related decrease in cell density and cannot be compensated due to their limited regenerative capacity.2 When the cell density is less than a critical level of 1000 cells/mm2, the endothelium no longer functions, causing corneal edema and loss of visual acuity. In these cases, HCEC transplantation aims to restore vision with the hope of reconstituting a structural and functional endothelial monolayer. Central to the tissue reconstruction

is the cellular arrangement and organization of grafts (i.e. the well-organized cell sheets or isolated cell suspensions). Therefore, the ability to obtain and deliver an intact monolayer of HCECs would be beneficial for developing an effective therapeutic strategy to rescue damaged endothelium. In this chapter, we presented a novel method for harvesting cultured human corneal endothelium for tissue repair. In contrast with traditional enzymatic digestion (i.e. trypsinization), a cell culture system using thermoresponsive supports has been designed to allow the bioengineered endothelial equivalents to retain cellular activity, organization, function, and ECM integrity.

Cultivation of adult HCECs from older donors has been proven to be difficult.30 To overcome this, a growth factorenriched medium was used to succeed in mass culturing untransformed adult HCECs. In this study, when cultivated on the nanostructured PNIPAAm-grafted surfaces, the HCECs derived from older donor tissue keep their phenotypic characteristics and proliferative capacity as grown on the commercial tissue culture plates. For strengthening of the harvested HCEC monolayers, the confluent cultures could be stimulated to become thicker by a further 2-week incubation to enhance cell production of ECM. This unique phenomenon of ECM formation in cultured HCECs possibly indicated the same property of increasing thickness of Descemet’s membrane with aging in the human cornea.31 In addition, the cell density of confluent HCEC monolayers is comparable to those as in vivo. It suggests that these bioengineered tissue equivalents have sufficient cell numbers to support their barrier and ionic pump functions.

After 45 min of incubation at 20°C, the area of harvested HCEC sheets decreased approximately from 9.6 to 0.75 cm2. These results support the report by Shimizu et al demonstrating that spread cells are compacted after the temperature-modulated detachment of cultured cardiomyocyte sheets.15 One possible explanation for the shrinkage of HCEC sheets is that the reorganization of cytoskeleton is induced by the low-temperature treatment. When autologous transplantation is considered, the HCEC sheet grafts should be designed to fit the size of the initial biopsy in the recipient. Despite the contraction observed in these bioengineered tissue grafts, the size of harvested HCEC sheets can be arbitrarily adjusted by controlling the surface size of thermoresponsive culture supports.

It has been reported that the hypothermic preservation (4°C) of cells would result in decreased activity of the Na+ pump32 or induce apoptosis.33 Since the cell sheet detachment is performed at 20°C, which is lower than its normal culture temperature (37°C), the effects of lowtemperature treatment on the viability of harvested tissue

equivalents should be investigated. Our data indicate that the detached HCEC monolayers are tolerant of 45 min incubation at 20°C. In this study, we performed viability testing in cell sheets after immediate separation from thermoresponsive supports. Although the HCEC monolayers were detached and preserved in serum-free OPTI-MEM, the storage time (time between detachment and begin of implantation) is not a concern for our study. Using cell-adhesive and transparent gelatin hydrogel carriers, the detached HCEC sheets with good viability can be immediately transplanted through a 7.5 mm sclerocorneal incision to recipient corneas denuded of endothelium.10

In addition to retain their deposited ECM, the harvested HCEC sheets with cellular interconnections are consisted of closely packed, small, polygon-shaped cells when studied morphologically. These findings are consistent with previous publications regarding morphological observations of cultured corneal endothelial cells on carrier substrates5,7-9 or corneas denuded of endothelium.1,34-40 Immunohistochemistry studies demonstrated the proper location of ZO-1 and Na+, K+-ATPase proteins implying the HCEC sheets are capable of maintaining intact barrier functions as well as ionic pump functions. By histological examination, the monolayered architecture of detached cell sheets is also confirmed. It is important to note that these characteristics of cultured HCEC monolayers are similar to those observed on the native endothelium of eye bank donor corneas.

In an effort to develop alternative therapy techniques, we have evaluated the feasibility of using HCEC sheets for corneal endothelial reconstruction.10 As a graft source, cell sheets have an intact cellular arrangement and a cellular organization, which are important factors for successful graft-host integration and tissue repair. Our previous report on transplantation of intact retinal sheets has demonstrated that these grafts positioned with correct polarity could grow into ordered and viable laminated retinas.24 However, the dissociated retinal cell suspensions or microaggregates simply develop the rudimentarily differentiated rosettes after being grafted into the subretinal space.41

Despite having a tissue-like architecture, the thermally detached cell sheets were easily wrinkled and folded during removal of the thermoresponsive culture substrates.42 In the field of cell sheet transfer, Okano et al have introduced poly(vinylidene difluoride) (PVDF) membranes as a supporter, which renders for the three-dimensional manipulation of cardiomyocyte sheets into layered constructs.15 Moreover, using a doughnut-shaped PVDF supporter, recent attempts have been made to reconstruct ocular surface by transplantation of cultured epithelial cell sheets originating from autologous corneas17 or oral

mucosae.18 The results of these works have been encouraging since the visual acuity of patients who received bioengineered cell sheets was significantly improved. However, intraocular grafting is different from the trials in corneal epithelial cell therapy because the unique physiological environments (i.e. anterior chamber and subretinal space) are perfused with large amounts of tissue fluid, which may cause unstable attachment of implanted cell sheets to lesion sites. In these cases, to deliver and retain the cells at the site of injury is important in the design of related cell therapy techniques. Therefore, it is necessary to provide a temporary support structure for enhancing the graft-host integration during tissue reconstruction. Compared with other reports on corneal endothelial cell transplantation studies using different carriers,4-9 we have developed a novel method to deliver the cultivated HCEC monolayers by utilizing a biodegradable and cell-adhesive gelatin disk without permanent residence of carrier materials in vivo. We have shown that the gamma-sterilized hydrogel disks made from raw gelatins (IEP = 5.0, MW=100 kDa) with appropriate dissolution degree and acceptable cytocompatibility are capable of providing stable mechanical support, making these carriers promising candidates for intraocular delivery of cultivated HCEC sheets.11

Results from a short-term study have suggested that the transplanted HCEC sheets could be integrated into rabbit corneas denuded of endothelium.10 Additionally, the corneas have returned to a nearly normal thickness indicating the function of bioengineered HCEC sheets. For intraocular delivery of the cultured cell sheets, the use of gelatin carriers is very attractive because of the highly transparent and deformable nature of this hydrogel material. It is known that surgery with smaller incisions may provide faster rehabilitation and reduce postoperative morbidity. Currently, PK is the most common way to treat corneas that are opacified due to endothelial dysfunction. When compared with PK, the cell therapy techniques proposed in the present study enable intraocular grafting of HCEC sheets to be performed to occur with minimal surgical incisions. Since the foreign supporting materials are substantially completely absorbed in vivo, we believe that this novel approach will have a high success rate in treating corneal endothelial cell loss. Although these data are encouraging, long-term efficacy and safety data need further investigation.

In summary, this study described a novel cell therapeutic method for HCEC loss, by mass cultivating HCECs from adult human corneal donors, harvesting HCECs as a cell sheet after detaching from a thermoresponsive PNIPAAmgrafted surface and delivering HCECs with a negatively

charged, high molecular weighted gelatin disk. Based on the current methodology, the adult HCEC monolayers having normal morphology and viability can be obtained without the need for cell carriers during cultivation. After transplantation, the functional HCEC sheets were integrated into the denuded corneas, with the returned corneal clarity. Results of this study demonstrated the feasibility of transplanting HCEC sheet for corneal endothelial cell loss and as a possible alternative to PK.

References

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12.Lai JY, Chen KH, Hsu WM, Hsiue GH, Lee YH. Bioengineered human corneal endothelium for transplantation. Arch Ophthalmol 2006;124:1441-8.

13.Yamato M, Okano T. Cell sheet engineering. Mater Today 2004;7:42-47.

14.Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H, Fukai F, Okano T. Cell sheet engineering: Recreating tissues without biodegradable scaffolds. Biomaterials 2005;26:6415-22.

15.Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, Kikuchi A, Umezu M, Okano T. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res 2002;90:e40-e48.

16.Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med 2006;12:459-65.

17.Nishida K, Yamato M, Hayashida Y, Watanabe K, Maeda N, Watanabe H, et al. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Transplantation 2004;77:379-85.

18.Nishida K, Yamato M, Hayashida Y, Watanabe K, Yamamoto K, Adachi E, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium.

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Introduction

Descemet stripping automated endothelial keratoplasty (DSAEK)1-11 is a new form of corneal transplant surgery where the donor corneal disk comprising of deep corneal stroma, Descemet’s membrane (DM) and healthy endothelium is added onto the inner corneal surface of the patient’s cornea. An air-bubble facilitates the initial intraoperative adhesion of the donor disk to the patient’s cornea, followed by the donor corneal endothelium helping in the continued adherence of the donor disk to the patient’s cornea. Further, over time, this adhesion becomes even stronger as there is continued tissue remodeling and integration of the stromal donor-recipient tissues at the level of the interface.

From the Present to the Future

In DSAEK surgery, stroma, endothelium and DM are transplanted. However, only the healthy endothelium is necessary to clear a cloudy host cornea. Hence, the elimination of the donor corneal stroma will be beneficial and also will decrease the overall increased corneal thickness of the transplanted host cornea. The donor stromal tissue acts much like a carrier layer for the healthy donor endothelium in this form of transplantation surgery. This progression of transplantation technique without the donor corneal stroma has already been performed12,13

[See also Chapter 36, True Endothelial Cell (TEnCell) Transplantation, and Chapter 37, Descemet Membrane Endothelial Keratoplasty (DMEK)]. In 2006, Melles et al used organ-cultured DM with the endothelium in one patient and in 10 cadaveric eyes and called the procedure Descemet membrane endothelial keratoplasty (DMEK).12 In 2007, Tappin reported using donor endothelial cells with only a Descemet’s carrier in 3 patients with endothelial cell failure, and called it true endothelial cell (TEnCell) transplant.13 However, this technique of transplanting only the healthy donor endothelium with the attached Descemet’s membrane (DM) is called by the author (TJ) “Descemet’s membrane endothelial transplant (DECT)” [See also Chapter 36, True Endothelial Cell (TEnCell) Transplantation, and Chapter 37, Descemet Membrane Endothelial Keratoplasty (DMEK) and Chapter 13, Definition, Terminology and Classification of Lamellar Corneal Surgery] has not been perfected for mass duplication by corneal surgeons at the present time (at the time of writing this chapter). The terms DMEK and TEnCell transplant are synonymous with DECT. However, DMEK is the more commonly known name for this procedure. The future directions in DSAEK surgery may be in way of further improvements and refinements in

this surgical technique for easier duplication. This may also include the designing and manufacturing of new surgical instruments to facilitate this goal. In this type of procedure, some of the hurdles include handling a thin, flexible layer of tissue and attaching such a layer to the recipient cornea without any folds or wrinkles in the transplanted thin circular disk.

Another surgical direction in the future would be the possible transplantation of healthy donor endothelial cells without any carrier tissue, onto the inner surface of the patient’s cornea (See also Chapter 38, Corneal Endothelial Reconstruction with a Bioengineered Cell Sheet). This type of surgery is called by the author (TJ) endothelial cell transplantation (ECT) (See also Chapter 13, Definition, Terminology and Classification of Lamellar Corneal Surgery).

In this case the healthy donor endothelial cells have to adhere to the host cornea, continue to function and clear the host cornea. Some of the difficulties in ECT would include the preparation of healthy donor endothelial cells, introduction and transfer of these cells into the recipient anterior chamber (AC), and having these endothelial cells adhere to the inner corneal surface of the recipient cornea without being detached and washed away by the aqueous humor. Also, if endothelial cells are detached and dislocated to another region in the AC such as the iris, lens surface or the anterior chamber angle this may have some potential deleterious effects. Additionally, in ECT, the transplanted cells have to be healthy and continue to function in the recipient environment to clear the host cloudy cornea. This type of surgery will eliminate some of the current surgical steps used in DSAEK surgery, including the donor disk preparation using the artificial anterior chamber, the tacofolding of the donor corneal disk, and the use of an airbubble to attach the disk to the host cornea. However, at the present time (time of writing this chapter), ECT is not a reality.

The ultimate step in the future directions of DSAEK surgery would be to eliminate the use of all donor corneal tissues and only work with the patient’s own corneal endothelial cell layer. This may mean, some form of technique(s) to activate the decompensated corneal endothelium to function again to clear the patient’s cloudy cornea. Such a type of procedure may be called endothelial cell activation (ECA) (See also Chapter 13, Definition, Terminology and Classification of Lamellar Corneal Surgery).

ECA may be limited to those cases of early endothelial cell dysfunction, prior to complete endothelial decompensation and irreversible cellular death. This type of surgery is not possible at the time of writing this chapter.

The current advancements in Selective Tisuue Corneal Transplantation (STCT), a term that the author (TJ)

introduced,1-3 is highly beneficial in the field of corneal transplantation, since it has eliminated a full-thickness corneal wound and the use of any corneal sutures. The future of DSAEK surgery looks even more promising, and such surgical techniques as DECT, ECT, and ECA may become an everyday reality with improved qualitative and quantitative visual outcome for the patient.

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