WO2011040888A1

WO2011040888A1 - A biodegradable composition or combination and uses thereof

Info

Publication number: WO2011040888A1
Application number: PCT/SG2010/000373
Authority: WO
Inventors: Donald Tan; Jod Mehta
Original assignee: Singapore Health Services Pte Ltd
Priority date: 2009-10-01
Filing date: 2010-09-30
Publication date: 2011-04-07

Abstract

The present invention provides a composition or combination comprising at least one biodegradable material for use in protecting and/or preserving cells. For example, the composition or combination of the invention may be for use in corneal transplant, against injury, damage and/or trauma. The invention also provides a method of corneal transplant as well as in protecting cells against damage and/or trauma using a composition or combination. In particular, the composition or combination comprises fibrinogen and thrombin.

Description

A biodegradable composition or combination and uses thereof

Field of the invention

The present invention relates to a biodegradable material in a composition or combination for protecting and/or preserving cells. The biodegradable material may be biological or artificially synthesised. For example, fibrin sealant or fibrin glue in the form of a composition or combination comprising fibrinogen and thrombin, may be used for protecting and/or preserving cells. In particular, the composition or combination of the invention may be for use in corneal transplant, against injury, damage and/or trauma. The invention also relates to a method of corneal transplant by using the composition or combination of the invention. There is also provided a method of protecting cells against damage and/or trauma using a composition or combination of the invention. The composition or combination may be for protecting and/or preserving at least one layer of cells, in particular, corneal endothelial cells and/or Descemet's Membrane.

Background of the invention

Corneal transplantion (penetrating keratoplasty (PK)) was first performed by Eduard Zirm over 104 years ago and there have been few changes compared to the original surgery. However, over the last decade, there have been developments in reducing the complexity of the surgical techniques used. The cornea comprises of essentially three layers, the epithelium, the stroma and the Descemet's Membrane (DM)/endothelial cell complex. Conventional (full- thickness) penetrating keratoplasty generally relates to the transplantation of all of these layers. However, there can be many complications related to conventional penetrating keratoplasty, for example, poor ocular healing, neutrophic cornea, suture related complications, graft rejection, tectonically weak eyes and high graft astigmatism. In order to circumvent some of these problems, surgeons have looked at more selective tissue transplantation techniques. Selective tissue transplantation procedures have taken the form of either anterior lamellar keratoplasty (ALK) in which the epithelium and stroma are replaced or endothelial keratoplasty (EK) in which the DM/endothelial cell complex is replaced. Endothelial dysfunction is the leading cause of corneal transplantation in Singapore and USA. In patients with purely corneal dysfunction, endothelial keratoplasty has emerged as a viable alternative to conventional full-thickness penetrating keratoplasty.

The procedure of endothelial keratoplasty involves manual dissection of both the donor and recipient cornea and transplantation of essentially the donor posterior stroma with DM and endothelium, a procedure also known as Deep Lamellar Endothelial keratoplasty (DLEK). This surgery obviated the need for surface sutures required in PK and results were equivalent to PK. However, this surgery often took many hours, required special instrumentation and haze often developed at the interface between the donor and recipient stroma leading to a reduction in best spectacle corrected visual acuity.

To combat the problem of the interface and reduce manipulation of the recipient cornea, an improved technique known as Descemet Stripping Endothelial Keratoplasty (DSEK) has been described, involving stripping off the recipient endothelium. DSEK removed the need for dissection of the recipient cornea, thus reducing surgical time. The transplanted layers comprise a 50-250 micron thick lenticule comprising posterior stroma DM/endothelium from the donor. Since there was minimal dissection of the recipient cornea, the donor lenticule protruded from the posterior edge of the cornea into the anterior chamber. DSEK was further improved by using an automated microkeratome to cut the donor lenicule, providing a smoother stromal interface and a more consistent donor lenticule thickness. This surgery was coined Descemet Stripping Automated Endothelial Keratoplasty (DSAEK). DSAEK has become synonymous with endothelial transplantation and the outcomes have been shown to be equivalent to and in some centres superior to PK. It is essentially sutureless, small incision corneal transplantation surgery since there are no sutures on the ocular surface and the posterior cornea is accessed by a small scleral incision of approximately 5 mm. Even though visual acuity results and outcomes have been encouraging with DSAEK, the procedure is not an exact anatomical tissue replacement. The donor tissue is made up of a lenticule of stroma and DM/endothelial layers which is not an exact anatomical replacement of the diseased DM/endothelium which has been stripped off the recipient cornea. This may compromise the recipient due to the extra thickness in eyes with a shallow anterior chamber or with an anterior chamber intra-ocular lens. It also adds an interface between the donor stroma and the recipient. Furthermore, DSAEK techniques to insert the donor tissue through a small 4-5mm incision currently has limitations due to endothelial cell damage during the various folding or coiling stages of insertion, using a variety of devices. Endothelial damage in these situations relates to endothelium to endothelium touch during the folding or coiling process, or damage due to forceps manipulation of donor tissue whilst inserting the donor into the eye and positioning the donor in the appropriate anatomical plane adjacent to the posterior corneal surface of the recipient eye by the surgeon. Subsequently, another technique known as Descemet's Membrane Endothelial Keratoplasty (DMEK) was developed where the DM/endothelium alone was harvested from a donor and transplanted into a recipient cornea with the DM/endothelium stripped off. DMEK potentially offers major advantages over the earlier three techniques. Firstly, visual rehabilitation is much faster. Secondly, there is a near perfect anatomical restoration of the recipient cornea in DMEK which may provide better optical quality of the recipient cornea. Thirdly, the donor DM/endothelial may be peeled off directly in DMEK and does not require additional expensive surgical instrument for stromal lamellar dissection, particularly when compared with DSAEK, where an automated microkeratome is required.

However, the major challenge of DMEK is the harvesting of the DM/endothelial complex without excessive endothelial cell damage or tearing/disintegration of DM, and the difficulty in handling the thin layers of tissue of approximately 10 micron in thickness. A second major challenge in DMEK is placement of the donor into the eye and unscroliing the donor DM/endothelial complex in the correct orientation (endothelial surface down), and attaching the unscrolled donor DM/endothelial complex onto the posterior corneal surface of the donor eye in the correct anatomical position, usually with the help of Balanced Salt Solution (BSS) or air, all to be performed without excessive damage or loss to the endothelial cells of the donor tissue.

One further challenge in corneal transplantation today involves donor harvesting, processing, storage and transportation in a series of eye banking procedures. Throughout all these stages, endothelial damage may occur by inadvertent physical touching of the endothelial layer, or by excessive donor tissue distortion or manipulation.

Accordingly, there is a need to improve on protecting and/or preserving the corneal endothelial and/or Descemet's Membrane for all the above procedures, Summary of the invention

The invention provides a composition or combination comprising at least one biodegradable material for use in protecting and/or preserving cells. In particular, the composition or combination according to the invention may be for use in corneal transplant, in protecting and/or preserving against injury, damage and/or trauma. For example, the composition or combination according to the invention may be for protecting and/or preserving at least one layer of cells, in particular, corneal endothelial cells and/or Descemet's Membrane.

According to another aspect, the present invention relates to a composition or combination comprising at least one biodegradable material for use in corneal transplant, and/or in protecting and/or preserving at least one layer of corneal endothelial cells and/or Descemet's Membrane.

According to another aspect, the present invention relates to a method of corneal transplant comprising applying and/or administering a composition or combination comprising at least one biodegradable material. In particular, the method comprises administering and/or applying the composition or combination of the invention to the patient's or donor's eye or part thereof.

There is also provided a method of protecting and/or preserving at least one layer of corneal endothelial cells and/or Descemet's Membrane comprising applying and/or administering a composition or combination comprising at least one biodegradable material to at least one layer of cells.

The composition or combination comprising at least one biodegradable material may be for preserving the integrity of the layer of cells. The composition or combination may further improve or maintain pliability to the layer(s) of cells. The combination and/or combination may also reduce and/or prevent injury, damage and/or trauma to the layer(s) of cells. In particular, the composition or combination may be used for protecting the corneal endothelial cells and/or Descemet's Membrane for donor preparation, tissue harvesting, storage, transportation and/or corneal transplantation.

There is also provided the use of at least one biodegradable material in the preparation of a medicament for use in corneal transplantation. There is also provided the use of at least one biodegradable material in the preparation of a medicament for protecting and/or preserving at least one layer of corneal endothelial cells and/or Descemet's Membrane. The invention also comprises applying and/or administering the composition or combination to at least one layer of cells. In particular, the medicament may be for preserving the integrity of the layer of cells and/or improve or maintain pliability to the layer(s) of cells. The composition or combination may also reduce and/or prevent injury, damage and/or trauma to the layer(s) of cells, for example, for protecting the corneal endothelial cells and/or Descemet's Membrane for donor preparation, tissue harvesting, storage, transportation and/or corneal transplantation. The medicament according to the invention may be in the form of a composition or combination.

Any biodegradable material with suitable properties for protecting and/or preserving cells may be used for the invention. For example, biological or synthetic polymers, gels, glycans and the like may be used for the invention. Also, the biodegradable material for the invention may be biological and/or artificially synthesised. In particular, the biodegradable material may be a composition or combination comprising and/or consisting essentially of fibrinogen and thrombin. Accordingly, the invention also provides fibrin sealant or fibrin glue in the form of a composition or combination comprising fibrinogen and thrombin for use in protecting and/or preserving cells. In particular, the fibrin sealant or fibrin glue in the form of composition or combination according to the invention may be for use in corneal transplant, in protecting and/or preserving against injury, damage and/or trauma. For example, the composition or combination according to the invention may be for protecting and/or preserving at least one layer of cells, in particular, corneal endothelial cells and/or Descemet's Membrane. According to one aspect, the present invention relates to a composition or combination comprising fibrinogen and thrombin for use in corneal transplant, and/or in protecting and/or preserving at least one layer of corneal endothelial cells and/or Descemet's Membrane.

According to another aspect, the present invention relates to a method of corneal transplant comprising applying and/or administering a composition or combination comprising fibrinogen and thrombin. In particular, the method comprises administering and/or applying the composition of the invention to the patient's or donor's eye or part thereof.

There is also provided a method of protecting and/or preserving at least one layer of corneal endothelial cells and/or Descemet's Membrane comprising applying and/or administering a composition or combination comprising fibrinogen and thrombin to at least one layer of cells.

The composition or combination comprising fibrinogen and thrombin may be for preserving the integrity of the layer of cells. The composition or combination may further improve or maintain pliability to the layer(s) of cells. The combination and/or combination may also reduce and/or prevent injury, damage and/or trauma to the layer(s) of cells.

In particular, the composition or combination may be used for protecting the corneal endothelial cells and/or Descemet's Membrane for donor preparation, tissue harvesting, storage, transportation and/or corneal transplantation.

There is also provided the use of fibrinogen and thrombin in the preparation of a medicament for use in corneal transplantation. There is also provided the use of fibrinogen and thrombin in the preparation of a medicament for protecting and/or preserving at least one layer of corneal endothelial cells and/or Descemet's Membrane comprising applying and/or administering a composition or combination comprising fibrinogen and thrombin to at least one layer of cells. In particular, the medicament may be for preserving the integrity of the layer of cells and/or improve or maintain pliability to the layer(s) of cells. The combination and/or combination may also reduce and/or prevent injury, damage and/or trauma to the layer(s) of cells, for example, for protecting the corneal endothelial cells and/or Descemet's Membrane for donor preparation, tissue harvesting, storage, transportation and/or corneal transplantation. The medicament according to the invention may be in the form of composition or combination.

Brief description of the figures

Figure 1 shows a graph plotting the mean intraocular pressure (IOP) of the rabbits in the three different groups: rabbits intracamerally injected with TISSEEL fibrin sealant with normal IOP, rabbits intracamerally injected with TISSEEL fibrin sealant with raised IOP and rabbits intracamerally injected with control (BSS) against Post-Operative Day (POD).

Figure 2 shows a graph plotting the mean central pachymetry of the rabbits in the three different groups: rabbits intracamerally injected with TISSEEL fibrin sealant with normal IOP, rabbits intracamerally injected with TISSEEL fibrin sealant with raised IOP and rabbits intracamerally injected with control (BSS) against Post-Operative Day (POD).

Figure 3 illustrates an example of a test rabbit eye from the pre-op stage and up to Post-Operative Day (POD) 21 after intracameral injection with TISSEEL fibrin. This rabbit showed normal IOP after the intracameral injection. Figure 4 illustrates an anterior segment optical coherence tomography showing the disappearance of fibrin sealant up to Post-operative Day (POD) 21.

Figure 5 shows a graph of the endothelial staining for live/dead cell assay of the rabbits in the three different groups: rabbits intracamerally injected with TISSEEL fibrin sealant with normal lOP, rabbits intracamerally injected with TISSEEL fibrin sealant with raised lOP and rabbits intracamerally injected with control (BSS).

Figure 6 shows a graph of the TUNEL assay indicating the percentage of TUNEL positive cells in the different layers of the cornea. Figure 7 shows fibrin sealant spots obtained with testing the EasySpray system at different pressures and distances on paper. The left panel indicates the different pressure for the row and the top panel indicates the distance from the paper for the column.

Figure 8 shows a graph of the average surface diameter obtained with testing the Easy Spray at different pressures and distances on paper.

Figure 9 illustrates cryostat sections of paper sprayed with the EasySpray system at a distance of 5.0 cm at (a) 10 psi (b) 15 psi and (c) 20 psi. A scale bar corresponding to 50 μΐη is indicated,

Figure 10 illustrates the fibrin sealant layer on H&E stained pig cornea sections after spraying with the EasySpray system at 20 psi and a distance of 5 cm. A scale bar corresponding to 50 μΐη is indicated,

Figure 1 shows the eye of patient #1 (a) 1 day post-operative and (b) 1 week post-operative.

Figure 12 shows the eye of patient #2 1 day post-operative.

Figure 13 shows the eye of patient #3 1 day post-operative. Definitions

"Biodegradable" refers to any material being able to be degraded or broken down by natural or biological processes. Biodegradable also encompasses bioabsorbable. An example of a biodegradable material is a composition or combination comprising and/or consisting of fibrinogen and thrombin.

"Bioabsorbable" refers to being able to be absorbed by tissues or organs of an organism.

A "combination" of components, for the purpose of the present invention, refers to components, which are in separate form. In particular, the components are for administration in combination such as when two or more components are administered simultaneously or independently such that the components may act at the same time. Components administered independently can, for example, be administered separately (in time) or concurrently. The period of time elapsing between the administration of the components of the combination of the invention can be determined by a worker of skill in the art, and will be dependent upon, for example, the age, health, and weight of the recipient, nature of the combination treatment, side effects associated with the administration of other component(s) of the combination, frequency of administration(s), and the nature of the effect desired. The components may also be mixed together just prior to administration. Combination also includes mixtures, blends and the like. The components may be packaged together in a kit.

Similarly, combination spray refers to two or more components sprayed simultaneously or independently such that they act at the same time. The components may be sprayed independently or separately (in time) so long as they act at the same time. For example, the fibrinogen may be administered first, followed shortly by the thrombin or vice versa, so long as the fibrinogen and thrombin mixes together and thickens. Alternatively, the components may be administered by a combination spray device wherein on activating the combination spray device, separated components are sprayed concurrently.

Accordingly, the "combination" according to the present invention refers to fibrinogen and thrombin in any association, like in the form of a package, a kit, a paste, a device, for example a syringe, and the like comprising fibrinogen and thrombin. For example, a "combination" relates or encompasses the fibrin sealant or fibrin glue available in the market comprising fibrinogen and thrombin as separate components. Examples, of such combination are given in the paragraph below under definition of "fibrin sealant or fibrin glue". "Composition" for the purpose of the present invention refers to a product of mixing or combining two or more components. For example, fibrin sealant or fibrin glue obtained by mixing or combining fibrinogen and thrombin is within the scope of the definition of composition.

"Comprising" is herein defined as "including principally, but not necessarily solely". Furthermore, the term "comprising" will be automatically read by the person skilled in the art as including "consisting of. The variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

"Donor preparation" refers to the action or process of making the donor tissue ready for extraction or harvesting.

"Fibrin sealant or fibrin glue" refers to a paste comprising two main components, fibrinogen and thrombin. Fibrin sealant or fibrin glue is usually used as a clinical tissue adhesive and is usually packaged for sale with the fibrinogen and thrombin as separate components. For example, the two components may be in separate syringes packed in the kit or device. Both components may contain other components, including pharmaceutically acceptable excipients. The fibrinogen component may include aprotonin and/or Factor XIII. The thrombin component may include thrombin and a calcium chloride solution. The fibrinogen and thrombin are usually mixed together prior to use. For example, the two components may be loaded into two syringes, the tips of the syringes may then be joined together and pressure may be applied to the plunges of the two syringes to mix the two components. Alternatively, the fibrinogen or thrombin may be separated in a double-barrelled syringe with a common plunger which mixes the two components from the two separate barrels for administration on activatingss the plunger. On mixing, the thrombin acts as an enzyme, converting fibrinogen into fibrin and the composition thickens. The presence of Factor XIII causes the fibrin to crosslink, which may give the composition additional resilience. Any suitable proportion of fibrinogen and thrombin may be mixed to prepare the thickened composition, as long as the thrombin used is sufficient to catalyse the polymerisation of fibrinogen to fibrin. An example of commercially available fibrin sealant is known by the trade name of TISSEEL. Another example of commercially available fibrin sealant is Evicel.

"Harvest or harvesting", in a biological and/or clinical context, refers to extracting from a culture or a living or recently deceased body, especially for transplantation.

"Pliable or pliability" refers to the capability of being bent or folded without breaking or falling apart.

"Protect" refers to keeping safe from injury, harm or damage. Preserve also refers to keeping safe from injury, harm or damage but also refers to keeping alive or intact or keeping free from decay.

"Preserving the integrity" refers to preserving an intact or unimpaired condition.

"Trauma" refers to an injury to living tissue caused by an extrinsic agent and/or an agent, force or mechanism causing such an injury. Detailed description of the invention

The present invention relates to a biodegradable material in a composition or combination for protecting and/or preserving cells

According to one aspect, the invention relates to fibrin sealant or fibrin glue in the form of a composition or combination comprising fibrinogen and thrombin for use in medicine. In particular, the composition or combination according to the invention is for use in corneal transplant. There is also provided the composition or combination according to the invention for use in protecting and/or preserving cells against injury, damage and/or trauma. For example, the composition or combination may be for protecting and/or preserving at least one layer of cells, in particular, corneal endothelial cells and/or Descemet's Membrane.

According to another aspect, the present invention relates to a composition or combination comprising fibrinogen and thrombin for protecting and/or preserving at least one layer of corneal endothelial cells and/or Descemet's Membrane.

The composition or combination comprising fibrinogen and thrombin may be for preserving the integrity of the layer(s) of cells. The composition or combination comprising fibrinogen and thrombin may preserve the integrity of the layer(s) of cells by providing a physical barrier with the formation of a polymerized fibrin layer over the cells, preventing direct contact with the underlying cell layer, and also by providing some degree of thickness to the endothelial/DN complex, thereby reducing the risk of wrinkling or distortion of the DM layer which may further lead to endothelial damage. The composition or combination may also maintain or improve pliability to the layer(s) of cells. The combination and/or combination may also reduce and/or prevent injury, damage and/or trauma to the layer(s) of cells. In particular, the composition or combination may be for protecting the cells in a variety of situations, including, but not limited to:

(i) corneal donor preparation procedures

(ii) donor cornea storage, storage in eye banks

(iii) donor transportation

(iv) surgical transplantation procedures, including penetrating keratoplasty (PK), and all forms of endothelial keratoplasty (EK), including Deep Lamellar Endothelial Keratoplasty (DLEK), Descemets Stripping Automated Endothelial Keratoplasty (DSAEK), Descemet's Membrane Endothelial Keratoplasty (DMEK) or femtosecond laser-assisted forms of DLEK, DSAEK or DMEK.

The composition or combination comprising fibrinogen and thrombin may be applied and/or administered to at least one layer of cells in vitro and/or in vivo. For example, the composition or combination may be applied to at least one layer of cells harvested from a donor or in vitro cultured cells.

Accordingly, the corneal endothelial and/or Descemet's Membrane may comprise isolated corneal endothelial and/or Descemet's Membrane.

Alternatively, the composition or combination may be applied and/or administered intracamerally for protecting corneal Descemet's Membrane/endothelial cells. In particular, corneas may first be harvested from donors. Fibrinogen and thrombin may be combined together and then injected into intracamerally into the harvested corneas.

A further method of application may be by a combination spray of fibrinogen and thrombin. For example, a combination spray device may be used to deliver the combination spray of fibrinogen and thrombin for providing a uniform and thin coating onto the corneal endothelial surface. Fibrinogen and thrombin on combining forms a thickened composition. This composition can be applied immediately after cornea harvesting as a protectant, so that the donor endothelium is well-protected from the time the cornea is removed from the donor, during storage in the eye bank, during transportation, up to the time of transplantation.

The composition may be used as a protectant for full thickness penetrating keratoplasty. The composition may also be used as a protectant for endothelial keratoplasty. In particular, the composition may be used as a protectant for either Descemets Stripping Automated Endothelial Keratoplasty (DSAEK), or Descemet's Membrane Endothelial Keratoplasty (DMEK). The composition thickens the DM/endothelial cell complex and assist in preserving the integrity of the DM/endothelial layer(s), particularly for DMEK. The composition may further provide mechanical protection, reducing and/or preventing trauma and/or damage during harvesting of the DM/endothelial complex and subsequent manipulation during transplantation when the tissue is inserted intra-ocularly. The protected layer(s) of cells may be substantially rigid and damage to the cells through excessive wrinkling, folding and other deformation may thus be reduced. However, the protected layer(s) of cells may maintain or have improved pliability, thus reducing the likelihood of disintegration during manipulation.

The composition or combination of fibrinogen and thrombin may also be used on in vitro expanded corneal endothelial cells. Following expansion of human corneal endothelial cells on a collagen carrier, the composition or combination may be added to protect the cells prior to transplantation. Fibrinogen and thrombin may be kept or stored as separate components (a combination), for example in a kit and mixed together prior to use. The fibrinogen component may include aprotonin and/or Factor XIII. The thrombin component may include thrombin and a calcium chloride solution. Both components may contain other components, including pharmaceutically acceptable carriers, excipients and/or diluent. When combined or mixed, thrombin acts as an enzyme converting fibrinogen to fibrin and the mixture or composition thickens and may then be applied and/or administered to the layer(s) of cells.

Any suitable proportion of fibrinogen and thrombin may be mixed together to form the thickened composition. For example, the thickened composition is applied or administered to the corneal endothelial and/or Descemet's Membrane to form a layer which may be 100 to 150 μιη thick. Fibrinogen and thrombin preparations are available commercially in a kit for preparing fibrin sealant or fibrin glue according to the manufacturer's instructions. An example of a commercially available product comprising fibrinogen and thrombin is TISSEEL. Other fibrinogen and thrombin kits may also be used. Alternatively, fibrinogen or thrombin may be prepared from blood samples and combined as previously described (Thompson et a/., 1998). The fibrinogen and thrombin may be from an animal (e.g. bovine) source or may be from human. The fibrinogen and thrombin may be obtained from the blood of a single subject or from pooled from multiple subjects. Although donors of blood for fibrinogen and thrombin are usually screened, the fibrinogen and thrombin may be obtained from the patient, for example the recipient and this may reduce or prevent the incidence of transmitted infections.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention. EXAMPLES

Example 1 Intracameral injection of fibrinogen and thrombin into rabbit corneas

Example 1 shows that the morphology and functional status of the corneal endothelium remains unaffected by the coating of fibrinogen and thrombin in a rabbit model. Safety of the polymerized fibrinogen-thrombin combination was shown in a series of rabbit eye experiments as fibrin formation directly over the corneal endothelium did not have deleterious effects on the morphology or functional capabilities (such as maintaining the cornea in a relatively dehydrated state through the Na-ATPASe driven endothelial pump) of the corneal endothelial layer.

Materials and Methods

The animal study adhered to the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic Vision and Research, and was approved by the institutional review board and ethics committees of the Singapore National Eye Centre and Singapore Eye Research Institute, Singapore.

Ten female New Zealand white rabbits weighing between 2 and 2.5 kg were used for this study. Twenty eyes of the 10 rabbits were divided equally into 2 groups. One eye of each rabbit received an intracameral injection of fibrin sealant with thrombin 500 IU and fibrinogen concentration of 72 - 110 mg per ml as stated by the manufacturer (TISSEEL, Baxter, USA), and the fellow eye received an intracameral injection of balanced salt solution (BSS, Santen, Osaka, Japan) as a control. Surgical Procedures

The rabbits were anesthetized with an intramuscular injection of ketamine (40mg/kg), xylazine (4mg/kg) and topical xylocaine before the surgery. A wire lid speculum was placed to separate the eyelids. An operating microscope was positioned over the eye undergoing surgery. The anterior chamber was entered in the superotemporal quadrant through a long corneal tunnel using a 30-G needle bent approximated at 60 degrees. After entry, 0.03 ml of aqueous humor was withdrawn from the eye and 0.3 ml of air was injected into the anterior chamber to achieve a complete air fill to push back the iris and the lens. TISSEEL fibrin sealant components were constituted according to the standard manufacturer's instructions prior to surgery, and pre-warmed to 37 degrees Celsius. The dual syringe system with a common plunger (Duploject) that ensured the feeding of equal volumes of the two components into a common joining piece, mixed just before contact, was assembled. Sealer Protein Concentrate was used with Thrombin 500 IU for a rapid setting composition. A 25-G needle was attached to the end of the Duploject and was used to inject between 0.03 to 0.05 ml of fibrin sealant intracamerally to coat the endothelium. The anterior chamber was then irrigated with 0.3 ml of BSS. A 25-G needle was used as a smaller gauge needle was unable to inject the fibrin sealant through. The procedure was repeated in the fellow eye, and the fibrin sealant was replaced with an injection of 0.03 ml of BSS as a control.

Clinical Evaluation

Detailed clinical examinations were performed preoperatively and daily postoperatively on anesthetized rabbits. The examinations included slit lamp examination (FS-3V Zoom Photo Slit Lamp, Nikon, Japan) to check the degree of external inflammation, cornea clarity, and the degree of resolution of intracameral fibrin sealant, serial slit lamp photography (Nikon D 00, Japan), high resolution anterior segment optical coherence tomography (OCT) scans of the cornea and anterior segment (Visante OCT, Carl Zeiss Meditec, Inc, Dublin, California, USA) and intraocular pressures (IOP) measurements (Tono-pen XL, Medtronic Solan, Jacksonville, Florida). Corneal thickness measurements were obtained using the measurement calipers provided by the Visante software. The anterior segment quadratic scans were analyzed using a graphic program (Image J, NIH) and the mean area of intracameral fibrin sealant was calculated daily. All rabbits received the following topical medications in both eyes during the entire duration of follow-up: neomycin, polymyxin B and dexamethasone (Maxitrol, Alcon Laboratories, Fort Worth, Texas, USA) 6 hourly, Predforte 6 hourly, atropine 1 % 12 hourly and vidisic gel once a day. The rabbits were followed up until complete dissolution of the fibrin sealant (duration of follow-up ranged from 14 days to 30 days). One rabbit died during the follow-up period and was excluded from the study. The rabbits were sacrificed with an intravenous injection of sodium pentobarbitol (100mg/kg). The rabbit eyes were enucleated, and their corneas and a 2 mm rim of sclera were removed with a blade and scissors and the irises were peeled from the corneas. The corneoscleral buttons and irises were stored in Optisol-GS solution until further testing.

Corneal Specimens

Endothelial Staining for Live/Dead Cell Assay A viability/cytotoxicity assay using calcein-AM and ethidium hompdimer-1 (EthD-1 ) (Molecular Probes, Eugene, OR, USA) was used to assess corneal endothelial cell viability. The components of the viability/cytotoxicity assay were first allowed to thaw to room temperature in a dark room. Calcein-AM is cleaved by cellular esterases present within viable cells to form a fluorescent green product which is membrane impermeable. Ethidium homodimer- is a fluorescent red marker which binds to nucleic acids and only passes through the compromised membrane of non-viable cells. Phosphate buffered saline (PBS) (10 ml) was added to calcein AM (15 pL) to obtain a solution of 6μΜ of calcein AM, and PBS (10 ml) was added to EthD-1 (30 μ1_) to obtain a solution of 6 μΜ of EthD-1. The corneo-scleral buttons were initially stored in Optisol GS at 4° C after harvesting from the rabbits (for less than 30 minutes). In preparation for the staining, each corneal button was gently submersed in PBS to remove any residual growth medium and the centre 8.5 mm of the cornea was trephined using a Coronet corneal punch (Network Medical Products, North Yorkshire, UK). Each trephined control and corneal button was then placed into an individual well of a 12-well plate. 50 pL of each of the reagent was then gently placed on the endothelial surface (to minimize staining of the epithelium). The plate was covered in foil and allowed to incubate at room temperature in darkness for 45 minutes.

At the end of the incubation, the tissue was gently washed with PBS and placed endothelial side up onto a glass slide. Slides were then subsequently examined with a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Oberkochen, Germany) using sequential illumination at wavelengths of 500 nm and 625 nm. This will illuminate the calcein-AM positive (live) and EthD-1 positive (dead) cells respectively. The photographs were analyzed using a graphic program (Image J, NIH) and the percentage of dead cells per slide was calculated_:

Electron Microscopy (EM) - Scanning EM and Transmission EM The rabbit corneas were first fixed in cold 2.0% glutaraldehyde, 2% paraformaldehyde and 0.1 M sodium cocodylate, pH 7.4 (Electron Microscopy Sciences, Washington, USA) overnight at 4°C. The tissue was then washed in sodium cacodylate buffer and rinsed with distilled water. The tissues were secondarily fixed in 1 % osmium tetra-oxide and then dehydrated, dried and mounted on SEM stubs. They were then sputter coated and examined with a SEM (JSM-5600; JEOL, Tokyo, Japan) at 15W. Low power (x18) and high power x500 SEM images were taken. Thirteen micrographs were taken per corneal button, one in the centre and in one in each clock hour around the button. For TEM the tissue was trimmed into smaller pieces. These samples were then post-fixed in 1 % osmium tetra-oxide (Electron Microscopy Sciences, Washington, USA). After extensive rinsing with distilled water, tissues were dehydrated in a graded series of ethanol, and embedded in Araldite (Electron Microscopy Sciences, USA). All semi-thin sections of 0.5 - 1 pm thickness were cut with Reichert-Jung Ultracut E Ultramicrotome (C. Reichert Optische Werke AG, Vienna, Austria), counter-stained with toluidine blue/basic fuchsin stain and examined using an Axioplan, Zeiss Light Microscope (Carl Zeiss, Germany). All ultra-thin sections of 60-80nm were collected on copper grids, doubled-stained with uranyl acetate and lead citrate for 8 minutes each, then viewed and photographed on a JEM 1220 electron microscope. (JEOL, Tokyo, Japan) at 100kv.

Histopathology

The rabbit corneal specimens were embedded in optimal cutting temperature freezing compound (OCT). Six micron thick sections were cut and positioned on poly-lysine coated glass slides and then air dried for 20 minutes. They were then subjected to hematoxylin and eosin staining by standard techniques or evaluated for apoptotic cell death by terminal deoxynucleotidyl transferase assay dUTP nick end labeling (TUNEL), using a terminal deoxynucleotidyl transferase assay kit (In Situ Cell Detection Kit, TMR red; Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer's instructions. Nulcei were counterstained with 4',6-diamidino-2-phenlindole (DAPI; Molecular Probes, Eugene, OR, USA). Slides were then mounted and subsequently examined with a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Oberkochen, Germany) using sequential illumination at wavelengths of 540 nm and 580 nm. This will illuminate the TUNEL positive cells red and rest of the DAPI stained cells blue. The photographs were analyzed using a graphic program (Image J, NIH) and the percentage of apoptotic positive cells per slide was calculated. Trabecular Meshwork Specimens

Election Microscopy (EM) - Transmission EM

The corneal limbi from the rabbits underwent transmission EM as described above.

Histopathology

The corneal limbi from the rabbits underwent H&E staining and were evaluated for apoptotic cell death by TUNEL assay as described above.

Iris specimens

Election Microscopy (EM) - Transmission EM The rabbit irises underwent transmission EM as described above.

Statistical Analysis

The eyes were divided into 3 different groups (eyes with intracameral TISSEEL with normal lOP, eyes with intracameral TISSEEL with raised lOP and control eyes). Variables of the 3 groups were compared by analysis of variance using a statistical software (SPSS 9.0). Pairwise comparisons were made by unpaired Student's t test. All tests were two-tailed. The level of significance was a P: value of 0.05. Data was reported as mean +/- standard error of mean (SEM).

Results

Surgical procedures The corneal endothelium was coated with the intracameral injection of fibrin sealant in all the rabbits. In all cases, fibrin sealant inadvertently coated the iris and this was left in-situ. The fibrin sealant took between 30 seconds to 1 minute to set. Clinical Evaluation

All eyes showed mild hyperaemia soon after surgery, which resolved within the first few days postoperatively. In the eyes with the fibrin sealant, two eyes had normal IOP and pachymetry post-operatively, while the other seven eyes had IOP spikes and raised pachymetry. The eyes with normal IOP had corneas that remained thin and clear, and the anterior chamber showed mild inflammation which settled on 6 hourly topical corticosteroids (Figures 3 and 4).

Among the eyes with raised IOP, all but one showed corneal oedema, two eyes had irido-corneal contact with angle closure post-procedure, which resolved spontaneously with the dissolution of the fibrin sealant. The eyes with raised IOP received the following topical anti-glaucoma treatment: timol 0.5% (MSD, Hertfordshire, UK) twice a day, travopost 0.004% (Travatan, Alcon laboratories, Fort Worth, Texas, USA) once a day and brimonidine tartrate 0.1 % (Alphagan, Allergen, Irvine, California) three times a day. The eyes that had an intraocular pressure of greater than 25 mmHG despite intensive topical anti-glaucoma medication underwent therapeutic anterior chamber paracentesis, which managed to relieve the pressure.

In all cases, partial dissolution of injected fibrin sealant within the anterior chamber was noted to occur within 3 days after injection, with complete dissolution occurring between POD 15 to 30. The IOP and pachymetry were noted to be increased after intracameral injection of fibrin sealant. Figures 1 and 2 show the mean IOP and central pachymetry of the rabbits in the 3 different groups. The amount of intracameral fibrin sealant was shown to correlate significantly with IOP on POD 1. There was no correlation between the amount of intracameral fibrin sealant and IOP over other time points, with P value adjusting for type I error. The amount of intracameral fibrin sealant was shown to correlate significantly with pachymetry on POD 7 and POD 10, with the IOP taken into consideration. There was moderate univariate correlation between increased lOP and increased pachymetry over time, and the correlation was significant. Figure 2 shows the univariate and multivariate analysis correlating amount of fibrin sealant, lOP and pachymetry. Hence the raised lOP in the initial period was caused by the over-excessive amount of fibrin sealant injected into the anterior chamber. This was most likely due to blockage of the trabecular meshwork by the sealant.

Corneal specimens

Endothelial staining for live/dead cell assay

There was no significant difference between the percentage of dead corneal endothelial cells evaluated using live/dead cell assay between the three groups with p > 0.05 (Figure 5).

Electron microscopy (EM)- Scanning EM and Transmission EM.

Scanning EM and Transmission EM showed that corneal endothelial cells remained hexagon-shaped and presented well-defined borders in all three groups.

Histopathology

Histopathology of the cornea was normal in all three groups. There was no significant difference between the number of apoptotic cells in the epithelium (F=0.739, p > 0.05), stroma (F=2.367, p > 0.05) and endothelium (F=4.161 , post hoc Games-Howell and Hochberg GTS p > 0.05) using TUNEL assay between the three groups (Figure 6). Trabecular Meshwork Specimens

Electron Microscopy (EM) - Transmission EM Transmission EM showed normal trabecular meshwork in all the three groups. Histopathology Histology of the trabecular meshwork was normal in all the three groups. There was no significant difference between the percentage of apoptotic trabecular meshwork cells using TUNEL assay between the three groups (p > 0.05).

Iris Specimens

Electron Microscopy (EM) - Transmission EM Transmission EM showed normal iris structure in all the three groups.

The above initial animal studies indicated that fibrin sealant can be safely injected into the anterior chamber of the eye without detrimental damage to the corneal endothelial cells, trabecular meshwork or the iris.

Example 2 Optimisation of spray delivery Materials and Methods

Preparation of Tisseel fibrin tissue sealant

Fibrin sealant was reconstituted according to the manufacturer's instructions (Tisseel VH Fibrin Sealant, Baxter Healthcare, Singapore), with some modifications. The setting time of fibrin sealant depends on the concentration of the thrombin component. This Tisseel kit contains the Sealer protein concentrate (fibrinogen) and thrombin at the concentration of 500 lU/ml for a rapid setting mixture (with 60 seconds). Aprotinin (fibronolysis inhibitor) was added to sealer protein concentrate and calcium chloride (CaC^) was added to the thrombin. 20 μΙ of 0.5 % tryphan blue was added to 2 ml of thrombin-CaCb for detecting the spread of fibrin glue after spraying. The reconstitution procedure was carried out in a fibrinotherm-heating device which maintains an optimal physiological temperature of 37°C with constant stirring. The two final components were drawn in their respective syringes. Extreme care was taken to avoid formation of air bubbles. The two syringes were then connected to a Duploject syringe holder, which ensured the feeding of equal quantities of the two components.

Applicator systems EasySpray and DuploSray are two different applicator systems tested for delivery of the fibrin sealant. The pressure or flow rate of the gas may be adjusted for both systems to control the spray of fibrin sealant.

The EasySpray system was used according to the manufacturer's instructions. With the EasySpray sytstem, the two syringes, each containing a component of Tisseel sealant were attached to a Duploject syringe holder. The Duploject plunger was connected to the EasySpray system via set filters. The EasySpray system was operated using CO2 gas maintained at a controlled pressure range. The spray of fibrin sealant is activated by occluding a clip centre using the thumb. The DuploSpray system was also used according to the manufacturer's instructions. The arrangement of the DuploSpray applicator system is similar to EasySpray but the applicator is different. The DuploSpray system uses a long shaft with dual lumen tubing each carrying the separate components of tisseel sealant, attached to the spray head of the Duploject syringe holder. The flow rate of the gas can be varied between 1 L/min to 2 L/min. Optimisation of fibrin sealant spray on paper

The optimisation experiment involved changing the pressure or flow rate and varying the distance of application of fibrin sealant in the spray system: The distances of 2.5 cm. 5 cm, 7.5 cm and 10 cm were tested for both the applicator systems. Pressures of 10, 15 and 20 psi were tested for the EasySpray system and flow rate of 1 Umin and 2L/min were tested for the DuploSpray system.

Fibrin sealant was sprayed with these combinations of distances and pressures/flow rate on a sheet of paper of thickness 0.183 ± 0.002 mm. The sprayed glue was in the form of spots with sizes and thickness depending on the combination tested (Figure 7). The sprayed spots on the paper were cut into half (-3-4 cm in length) and embedded in Optimal Cutting Temperature mixture. 40 μιη thick sections were then cut using a cryostat and the sections were mounted on slides and examined using bright field mode.

The EasySpray system was observed to produce a spot of consistent diameter at a distance of 5 cm from the paper (Figures 7 and 8).

In general, the EasySpray system produced a more consistently smooth layer of fibrin coating (Figures 7 and 9) than the Duplojet system. A pressure of 20 psi at a distance of 5 cm with the EasySpray system produced a consistent layer of fibrin glue on the paper. (Figures 7 and 9(c)). Optimisation of fibrin sealant on pig corneas

Based on the preliminary results, pig corneas were sprayed with fibrin sealant using EasySpray applicator at distances of 5 and 7.5 cm at a pressure of 20 psi. The whole corneas were then embedded in Optimal Cutting Temperature freezing mixture. 8 μητι thick sections were then cut using a cryostat and the sections were positioned on glass slides and stained with Haematoxyiin and Eosin (H&E) staining as described below. Haematoxylin and Eosin Staining

Any conventional H&E staining method may be used. For example the following method may be used. The glass slides with the 8 μΐη thick sections of pig corneas were air dried and redydrated with 95% ethanol for 5 minutes. The sldies were then washed and stained with haemotoxylin for 1 minute, followed by treating with Scott's tap water for 5 minutes for intensifying the nucleus staining. The slides were then counter stained with Eosin for 2 minutes and followed with washing in tap water. A series of dehydration with 95% and 100% ethanol were carried out for 5 minutes each. The slides were mounted after two changes of xylene for 2 momutes each and examined under bright field mode.

The results similarly show that using the EasySpray at 20 psi and 5 cm produced a consistent layer of fibrin glue on the pig corneas (Figure 10).

Example 3 Clinical patients undergoing DSAEK

DSAEK DSAEK is a form of selective lamellar corneal transplant surgery that allows replacement of diseased endothelium while maintaining the patient's own corneal stroma. However, significant loss in endothelial cell density (ECD) occurs during DSAEK, due partly to the surgical trauma sustained by the graft during insertion into the anterior chamber, especially if the technique: used involves folding of the graft and insertion with forceps, and also particularly in the case of Asian eyes, which have shallow anterior chambers and high vitreous pressures. In extreme cases, extensive endothelial cell loss may result in 'iatrogenic' primary graft failure (IPGF), or contribute to graft dislocation. A commercial insertion device EndoGlide (AngioTech, Reading PA. USA/Network Medical Products, North Yorkshire, UK) had been developed to minimize donor endothelial cell injury during DSAEK and is an FDA Class 1 medical device approved for use in the United States for DSAEK surgery, and obtained CE mark status in June 2009.

In this example, fibrin sealant was used to provide extra endothelial protection together with the insertion device EndoGlide. Patients

Consecutive patients with visually-significant corneal oedema from endothelial dysfunction and who were suitable for DSAEK were enrolled into the study with full informed consent. Prospective data was collected before surgery, and included demographic details and prior ophthalmic history. Ethnicity was classified into the following four categories as defined by the Singapore Department of Statistics (singstat.gov.sg/statsres/glossary/population.html#E): Chinese, Malay, Indian, and Others. Intra-operative and post-operative complications, including IPGF, graft dislocation and pupil-block glaucoma, were documented. Outcome measures monitored were best corrected visual acuity (BCVA), refractive error, and central ECD before surgery, at one month, three months, six months, and twelve months after surgery. Pre-operative donor ECD was based on specular microscopy performed by the Singapore Eye Bank with the Konan Keratoanalyzer EKA-98 (Konan Medical Corp, Japan), Post-operative ECD was performed using the Noncon Robo Specular Microscope NSPr9900 (Konan Medical Corp, Japan) in the SNEC by independent and experienced ophthalmic technicians. The ECD was considered acceptable if least 100 endothelial cells were counted and marked on a high-quality image of the central corneal endothelium; otherwise the ECD was excluded from the results. Post-operative endothelial cell loss was calculated as a percentage of the preoperative donor ECD. Other measures monitored include pre- and post-operative anterior segment imaging with the Visante OCT (Carl Zeiss Meditec, Dublin, CA, USA ), corneal topography with the Pentacam (Oculus, Lynnwood, WA, USA), and contrast sensitivity with the Functional Acuity Contrast Sensitivity (Stereo Optical Co., Chicago, IL, USA).

Statistical Analysis

Descriptive statistics for normally distributed variables are reported as mean + standard deviation, otherwise median and range are reported. The 95% confidence intervals (95% CI) are reported along with the mean 3-month, 6- month and 12-month percentage ECD loss. All data analysis was carried out with SPSS Statistics 17.0 (SPSS Inc, Chicago, III)

Donor Cornea Preparation and Placement

Lamellar dissection of the donor cornea was first performed with an automated lamellar therapeutic keratoplasty system (Moria, Antony, France). Following ALTK dissection, the endothelial surface of the donor cornea was left to dry slightly to remove excess balanced salt solution, and the donor endothelial layer was then sprayed with fibrin sealant using the EasySpray system at 5cm 20psi pressure. The donor tissue was then transferred to the Hanna trephine block for trephination and trephined to the desired diameter which usually ranges from 8.0mm to 9.5mm. The edges of the anterior cap and posterior donor lenticule were first separated by gentle irrigation of balanced salt solution (BSS) with a cannula and then then transferred together onto the donor well of the EndoGlide Preparation Base, with endothelial surface facing up.

Pre-operative donor ECD averaged 2957 ± 242 cells/mm², median graft diameter was 8.75 mm (range 8.25-9.5 mm), and average graft thickness was 187 ± 32 microns. . Surgical Technique in recipient

A temporal scleral tunnel incision measuring 4.5 mm wide by 1.5 mm to 2.0 mm deep was used for graft insertion with the EndoGlide. Alternatively, a similar clear corneal approach may also be used. An anterior chamber (AC) maintainer was pre-placed to prevent AC collapse during insertion. Descemet membrane was stripped in the conventional manner. A clear cornea paracentesis was also made at the nasal limbus directly across from the temporal for entry of the curved EndoGlide Placement forceps to pull the graft from the EndoGlide into the AC. Through a limbal stab incision, an inferior peripheral iridectomy iwas also performed to prevent pupil block glaucoma. An air bubble was left in the AC at the end of surgery.

All phakic patients had phacoemulsification and intraocular lens implantation performed prior to graft insertion (phaco-DSAEK to maximize the anterior chamber depth. Graft Coiling Procedure

Under the operating microscope, the internal lumen of the Glide Capsule was lubricated with BSS. The straight Loading EndoGlide forceps (AngioTech / Network Medical Products) was then introduced through the anterior opening of the Glide Capsule and used to grasp the leading stromal edge of the posterior donor lenticule. As the graft was slowly drawn into the Glide Capsule, the sides will begin to coil upwards to adopt a double coil configuration when the edges of the graft encounter the central internal ridge. The double coiling may be facilitated by gentle upwards strokes of the edges with a BSS cannula or Sinskey hook. The graft, with the endothelial surface protected by the layer of fibrin glue, should be drawn forward, fully coiled, until the leading edge just reached the anterior opening of the Glide Capsule. Placement of the Glide Introducer

The Glide Introducer was then inserted into the posterior opening of the Glide Capsule and locked into place to seal the Glide Capsule from behind. This posterior seal prevented egress of BSS or aqueous from the EndoGlide during graft insertion, and enabled a deep AC while minimizing aqueous flow and turbulence even while the AC maintainer was open. The entire EndoGlide complex was removed from the Preparation Base and inverted right-side up for insertion into the eye.

EndoGlide Insertion The Introducer of the EndoGlide was grasped with thumb and forefinger, and the anterior glide surface of the EndoGlide was smoothly inserted into the temporal wound until the anterior opening was seen through the cornea to be fully within the AC. Moderate flow from the AC maintainer and the tight wound seal around the EndoGlide during the insertion process helped maintain the AC depth.

Graft Pull-Through and Positioning

While holding the EndoGlide in position, the contralateral hand was used to insert the Placement forceps through the nasal paracentesis into the AC and over the glide surface of the EndoGlide. The leading stromal edge of the graft was then grasped and pulled out of the Glide Capsule and into the AC where it will automatically begin to uncoil in the correct anatomical position, i.e. endothelial surface down. Full uncoiling may be achieved by gentle sideways or to-and-fro movements of the graft with the Placement forceps, or by increasing the flow of the AC maintainer to further deepen the AC. Whilst still holding onto the graft with the forceps, the EndoGlide was removed, and a small air bubble (e.g. 2-3 mm in size) was initially injected beneath the graft in order to float it against the recipient stromal surface. The graft may now be released by the forceps, as the air bubble prevents descent of the graft. The surgery was then completed by suturing the main scleral wound and AC maintainer paracentesis site, centering the graft and injecting more air for a full air tamponade of at least 6 minutes to facilitate graft adhesion to the posterior corneal surface. At the conclusion of surgery, an air bubble, measuring slightly less than the graft diameter, was left in place.

Results

Surgery was performed on 4 eyes of 4 Asian patients, based on the procedure described above. All 4 patients exhibited the most common indications for DSAEK, pseudophakic bullous keratopahty (PBK), followed by Fuchs Endothelial Dystrophy, and were therefore suitable for DSAEK.

Postoperative Results.

After completion of the surgery, a minimum of 2 weeks follow-up was completed with all patients. In all 4 cases, recipient corneas and donor transplants were noted to be crystal clear from postoperative day 1 , with no stromal straie or edema, suggesting healthy and fully functional endothelial status (Figures 11(a), 12 and 13).Patient #1 exhibited 20/80 vision and Patient #3 exhibited 20/60 vision on postoperative day 1. In the immediate post-operative period, no patient had graft dislocation of IPGF. For Patient #1 , the donor endothelial cell count was 2857 cells mm^"2. At one week postoperatively, endothelial cell count was measured at 2653 cells/mm^"2, representing only a 7.15% loss or endothelial cells. The eye of patient #1 showed a slit-lamp appearance at 1 week but on the whole, the endothelium should be functioning normally since the rest of the eye was clear. Overall, the fibrinogen and thrombin was observed not to cause any averse effect or damage to the endothelium and was suitable for use with the DSAEK procedure. Reference

Thompson, D. F., Letassy, N. A. and Thompson, G. D. Fibrin glue: a review ofits preparation, efficacy, and adverse effects as a topical haemostat. Drug Intelligence & Clinical Pharmacy 22(12):946-952.

Claims

A composition or combination comprising at least one biodegradable material for use in protecting and/or preserving at least one layer of corneal endothelial cells and/or Descemet's Membrane.

The composition or combination according to claim 1 , for use in preserving the integrity of the layer of cells.

The composition or combination according to claim 1 or 2, for use in maintaining and/or improving pliability of the layer of cells.

The composition or combination according to any one of the preceding claims, for use in reducing and/or preventing injury, damage and/or trauma to the layer of cells.

The composition or combination according to any one of the preceding claims, for use in protecting and/or preserving the layer(s) of cells for donor preparation, tissue harvesting, storage, transportation and/or corneal transplantation.

The composition or combination according to any one of the preceding claims, wherein the composition or combination is for in vitro and/or in vivo administration and/or application.

The composition or combination according to claim 6, wherein administration and/or application comprises intracameral injection and/or aerosol spraying.

The composition or combination according to claim 6 or 7, wherein administration comprises intracameral injection into donor corneas.

The composition or combination according to any one of the preceding claims, wherein the corneal endothelial cells and/or Descemet's Membrane comprises isolated corneal endothelial and/or Descemet's Membrane.

The composition or combination according to any one of the preceding claims, comprising fibrinogen and thrombin.

A method of protecting and/or preserving at least one layer of cells comprising administering and/or applying a composition or combination comprising at least one biodegradable material to at least one layer of corneal endothelial cells and/or Descemet's Membrane.

12. The method according to claim 11 , for protecting and/or preserving the integrity of the layer(s) of cells.

13. The method according to claim 1 1 or 12, for maintaining and/or improving pliability of the layer(s) of cells.

14. The method according to any one of claims 11 to 13, for reducing and/or preventing injury, damage and/or trauma to the layer(s) of cells.

15. The method according to any one of claims 11 to 14 wherein the method is for protecting and/or preserving the layer(s) of cells for donor preparation, tissue harvesting, storage, transportation and/or corneal transplantation.

16. The method according to any one of claims 1 1 to 15, wherein administration of the composition or combination is performed in vitro and/or in vivo.

17. The method according to claim 16, wherein administration of the composition or combination comprises intracameral injection and/or by aerosol spraying.

18. The method according to claim 16 or 17, wherein administration of the composition or combination comprises intracameral injection into donor corneas.

19. The method according to any one of claims 1 to 18 wherein the corneal endothelial and/or Descemet's Membrane comprises isolated corneal endothelial and/or Descemet's Membrane.

20. The method according to any one of claims 1 to 19, wherein the composition or combination comprises fibrinogen and thrombin.

21 . Use of at least one biodegradable material in the preparation of a composition or combination for use in protecting and/or preserving at least one layer of corneal endothelial cells and/or Descemet's Membrane.

Use according to claim 21 , wherein the biodegradable material preserving the integrity of the layers(s) of cells

23. Use according to claim 21 or 22, wherein the biodegradable material is for maintaining and/or improving pliability of the layer(s) of cells.

24. Use according to any one of claims 20 to 23, wherein biodegreadable material is for reducing and/or preventing damage and/or trauma to the layer(s) of cells.

25. Use according to any one of claims 21 to 24, for protecting and/or preserving the layer(s) of cells for donor preparation, tissue harvesting, storage, transportation and/or corneal transplantation.

26. Use according to any one of claims 21 to 25, wherein the composition is for in vitro and/or in vivo administration and/or application to the layer(s) of cells.

27. Use according to any one of claims 21 to 26, wherein the composition is for administration by intracameral injection and/or aerosol spraying.

28. Use according to any one of claims 21 to 26, wherein the composition is for administration by intracameral injection into donor corneas.

29. Use according to any one of claims 21 to 28, wherein the corneal endothelial cells and/or Descemet's Membrane comprises isolated corneal endothelial and/or Descemet's membrane.

30. A composition or combination comprising at least one biodegradable material for use in corneal transplant.

31. Use of at least one biodegradable material in the preparation of a composition or combination for use in corneal transplant.

32. A method of corneal transplant comprising applying and/or administering a composition or combination comprising at least one biodegradable material.