OPHTHALMOLOGICAL DEVICE FOR THE TREATMENT OF LSCD AND SUBSTRATE FOR USE IN SAME

Abstract

The invention pertains to an ophthalmological device (100, 200) for the treatment of Limbal Stem Cell Deficiency, the device (100, 200) comprising: a stem cell carrier substrate; and a culture of limbal epithelial stem cells cultivated on said stem cell carrier substrate; wherein said stem cell carrier substrate comprises a hydrogel containing collagen or collagen-mimicking peptides; and wherein a ring-shaped area on a surface of said stem cell carrier substrate is provided with a pattern of niches (110, 210). The invention also pertains to a method for producing the ophthalmological device.

Claims

1. An ophthalmological device for treatment of Limbal Stem Cell Deficiency, the ophthalmological device comprising: a stem cell carrier substrate; and a culture of limbal epithelial stem cells cultivated on said stem cell carrier substrate; wherein said stem cell carrier substrate comprises a hydrogel containing collagen or collagen-mimicking peptides; and wherein a ring-shaped area on a surface of said stem cell carrier substrate is provided with a pattern of niches, positioned so as to coincide, after transplantation, with a limbus.

2. The ophthalmological device according to claim 1, wherein said stem cell carrier substrate has a shape of a dome, wherein a rim portion of said dome presents said niches and wherein a central portion of said dome is devoid of said niches.

3. The ophthalmological device according to claim 1, wherein said stem cell carrier substrate has a shape of a ring.

4. The ophthalmological device according to claim 1, wherein said stem cell carrier substrate comprises a 12% Collagen-Like Peptide (CLP-12) hydrogel.

5. The ophthalmological device according to claim 4, wherein said CLP-12 hydrogel is crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC).

6. The ophthalmological device according to claim 4, wherein said CLP-12 hydrogel is crosslinked with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

7. The ophthalmological device according to claim 1, wherein said stem cell carrier substrate comprises a 18% Collagen-Like Peptide (CLP-18) hydrogel, cross-linked with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

8. The ophthalmological device according to claim 1, wherein said stem cell carrier substrate comprises a RHC Type I based hydrogel.

9. The ophthalmological device according to claim 1, wherein said pattern consists of regularly spaced grooves.

10. The ophthalmological device according to claim 9, wherein said regularly spaced grooves have a width in a range of 15-150 μm.

11. The ophthalmological device according to claim 1, wherein said stem cell carrier substrate is loaded with one or more pharmaceutical components.

12. (canceled)

13. A method for producing the ophthalmological device according to claim 1, the method comprising: chemically crosslinking the collagen or collagen-mimicking peptides to produce said hydrogel forming said stem cell carrier substrate; pressing a stamp onto said hydrogel, said stamp having a template of said ring-shaped area provided with said pattern of niches; and after pressing the stamp onto said hydrogel, applying at least some of the limbal epithelial stem cells to said hydrogel.

14. A method of using the ophthalmological device according to claim 1 as an implant for the treatment of Limbal Stem Cell Deficiency.

15. The ophthalmological device according to claim 2, wherein said stem cell carrier substrate comprises a 12% Collagen-Like Peptide (CLP-12) hydrogel.

16. The ophthalmological device according to claim 15, wherein said CLP-12 hydrogel is crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC).

17. The ophthalmological device according to claim 15, wherein said CLP-12 hydrogel is crosslinked with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

18. The ophthalmological device according to claim 3, wherein said stem cell carrier substrate comprises a 12% Collagen-Like Peptide (CLP-12) hydrogel.

19. The ophthalmological device according to claim 18, wherein said CLP-12 hydrogel is crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC).

20. The ophthalmological device according to claim 18, wherein said CLP-12 hydrogel is crosslinked with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

21. The ophthalmological device according to claim 9, wherein said regularly spaced grooves have a width in a range of 50-90 μm.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0057] These and other features and advantages of embodiments of the present invention will now be describe in more detail with reference to the accompanying drawings, in which:

[0058] FIG. 1 is a schematic representation of a first embodiment of the device according to the present invention;

[0059] FIG. 2 is a schematic representation of a second embodiment of the device according to the present invention; and

[0060] FIG. 3 presents a flow chart of an embodiment of the process according to the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

[0061] With reference to FIG. 1, a device 100 according to a first embodiment of the present invention has a dome-shape, with a curvature resembling the native cornea. FIG. 1a shows a schematic top view of the device. FIG. 1b shows a schematic elevation of the device. The width of the niches or grooves 110 has been exaggerated for illustrative purposes—consequently, the number and spacing of the illustrated niches or grooves 110 is not meant to represent realistic values. The curvature of the dome has also been exaggerated for illustrative purposes.

[0062] The diameter d of the disk is preferably in the range of 10-14 mm, more preferably in the range of 11-12 mm. The hydrogel must have sufficient mechanical strength and thickness for stem cell transfer. The thickness t of the hydrogel can be adjusted according to the patient's need, normally ranging from 50 to 500 μm. The outer skirt or rim of the device has a 3D groove pattern with a width w of the grooves 110 and ridges ranging from 50 to 90 μm, preferably around 70 μm. In the illustrated case, the grooves are located on the superficial side of the hydrogel (the convex side). Alternatively or additionally, niches can be located at the concave side of the outer skirt, facing the ocular tissue that has been freed of the fibro-vascular tissue, depending on the degree of primary destruction or absence of the limbal niches.

[0063] The niches or grooves 110 are positioned in a ring-shaped area at the periphery of the disk so as to coincide, after transplantation, with the limbus. A central portion 120 of the device 100 is free from niches and forms a clear window. After transplantation, this central window portion 120 covers the cornea and therefore any interference with the optical function of the eye should be avoided or at least minimized. Optionally, this central window portion 120 is made of a different material than the ring-shaped area bearing the grooves 110. Preferably, the central window portion 120 is made of a biodegradable or resorbable material, which presents the advantage that the area over the cornea opens up to expose the cornea to air after the time required for the degradation or resorption of the material.

[0064] With reference to FIG. 2, a device 200 according to a second embodiment of the present invention has a ring-shape, with a curvature resembling the native cornea. FIG. 2a shows a schematic top view of the device. FIG. 2b shows a schematic elevation of the device. The width of the niches or grooves 220 has been exaggerated for illustrative purposes—consequently, the number and spacing of the illustrated niches or grooves 200 is not meant to represent realistic values. The curvature of the ring has also been exaggerated for illustrative purposes.

[0065] The outer diameter d of the ring is preferably in the range of 10-14 mm, more preferably in the range of 11-12 mm. The outer diameter d of the ring may have any other value, based on measurements of the implantation site in the target patient. Other preferred parameters of the device are as discussed above in the context of FIG. 1. The niches or grooves 210 are positioned on the ring-shaped device 200 so as to coincide, after transplantation, with the limbus. As the device 200 is ring-shaped, the central portion 220 is a void. After transplantation, this central portion 220 leaves the cornea uncovered.

[0066] Several collagen hydrogels may be used in the present invention. These include a 12% Collagen-Like Peptide (CLP-12) hydrogel, which may be crosslinked with, for example, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM); a 18% Collagen-Like Peptide (CLP-18) hydrogel, which may be crosslinked with, for example, DMTMM.

[0067] It is also possible to use hydrogels based on plant-based collagens, such as RHC Type I. The inventors have found that anisotropic RHC I hydrogels are less favorable for CLET than isotropic hydrogels (see also FIG. 2, below).

[0068] The choice of the type of collagen to be used is constrained by the requirement that the resulting hydrogel be sufficiently solid to allow patterning. The inventors have found that patterning is generally successful in yeast-derived and synthetic collagens, but less so in collagens derived from Nicotiana tabacum plants.

[0069] While various specific collagen hydrogels have been discussed hereinabove, the skilled person will appreciate that the invention can also be practiced with various other chemical formulations of collagen (and peptide) hydrogels.

[0070] With reference to FIG. 3, the invention also pertains to a method for producing the ophthalmological device as described above.

[0071] In a first step 310, collagen is chemically crosslinked to produce the hydrogel forming the stem cell carrier substrate. Chemical crosslinking of collagen to produce a hydrogel is known in the art. For example, international patent application publication no. WO 2015/032985 A1 discloses a method of preparing a hydrogel comprising: [0072] providing a solution of a first polymer comprising a natural polymer comprising methacrylate and/or acrylate functional groups; [0073] providing a second polymer comprising a synthetic and/or a natural polymer having at least two functional groups selected from thiol, acrylate and/or methacrylate, or synthetic and/or natural monomers having thiol acrylate and/or methacrylate functional groups; [0074] mixing the first and the second polymer, or monomers, in water to a total polymer concentration of at least 2 weight %; and [0075] letting the functional groups of the first and the second polymer chains cross-link, optionally applying UV radiation to the mixture when the second polymer has acrylate and/or methacrylate functional groups.

[0076] The reader will find further information about this method in the cited publication. While this method of preparing the hydrogel is a preferred embodiment of the present invention, the invention is not limited thereto.

[0077] The hydrogel is formed into the desired dome shape or ring shape during or after the crosslinking step.

[0078] In a second step 320, the pattern is applied by microcontact printing. A stamp presenting a template of the ring-shaped area provided with the desired pattern of niches is pressed onto the hydrogel (or vice versa). The stamp may be made of polydimethylsiloxane (PDMS) or any other suitable material. Preferably, the stamp is incubated, prior to contact with the hydrogel, with a suitable “ink” such as a 0.1 mg/mL human fibronectin dilution in phosphate-buffered saline.

[0079] In a third step 330, the substrate is primed for cell cultivation by applying the initial (previously harvested or cultured) limbal epithelial stem cells to the molded (patterned) hydrogel. The device can be loaded with cultivated stem cells derived from the contralateral eye of the patient or from a highly compatible donor eye or other cells derived from stem cells. It shall be noted that the implant may be reloaded at a later stage (not illustrated) in a secondary procedure with cell cultures in case of early or late failure of the primary transplantation due to stem cell death.

[0080] The inventors' experiments have shown that surface patterning does not impact cell phenotype or genotype.

[0081] The present invention also pertains to the use of the ophthalmological device as described above as an implant for the treatment of Limbal Stem Cell Deficiency. The ophthalmological device may be so used in humans or in animals other than humans.

[0082] The present invention also pertains to a stem cell carrier substrate for use in the ophthalmological device as described above, the stem cell carrier substrate comprising a hydrogel containing collagen or collagen-mimicking peptides. A ring-shaped area on a surface of the stem cell carrier substrate is provided with a pattern of niches. All other optional features and technical effects disclosed above for the substrates that form part of embodiments of the ophthalmological device according to the invention, apply equally to the substrate as a separate inventive aspect. The substrate may produced according to steps 310 and 320 as described above in the context of FIG. 3. Such a substrate can be produced and stored for a period of time before being used. Prior to implantation, it is primed for cell cultivation by applying the initial stem cells to the patterned area, as described in step 330 of FIG. 3 above. The substrate may additionally be loaded with diverse pharmaceutical components that may be deemed therapeutically useful.

[0083] In a more general way, the present invention also pertains to a pre-formed substrate comprising a hydrogel containing collagen or collagen-mimicking peptides, the substrate being provided with a pattern of niches. Such a patterned substrate, when loaded with the appropriate payload (in particular, stem cells and/or pharmaceutical components) can be used as an implant in various other medical applications.

[0084] While the invention has been described hereinabove with reference to specific embodiments, this has been done to clarify and not to limit the invention, the scope of which is determined by the accompanying claims.