Stabilization treatment of collagen scaffolds

Abstract

To disclose shape-stabilized collagen scaffolds and methods of obtaining such stabilized scaffolds.SOLUTION: Stroma can be harvested, for example, from human or porcine corneal stroma. The stroma can be shaped during excision or in a separate step after excision. Following shaping (and preferably decellularization), the excised stroma portion is subject to pressure, force or vacuum to reduce fluid content and then irradiated or otherwise treated to induce crosslinking of collagen chains or fibrils 16. Various sources of energy can be employed to induce peptide bond crosslinking of collagen including, for example, ultraviolet (UV) radiation. The scaffolds can also be selectively densified or patterned. The invention is particularly useful in forming stable lenticules 10A for intracorneal implantation in additive ocular surgery.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] The present invention relates to the stabilization treatment of a collagen scaffold. (Related Application) This application claims the priority of U.S. Provisional Patent Application No. 62 / 693,192, filed on Jul. 2, 2018, which is hereby incorporated by reference in its entirety.

Background Art

[0002]

[0001] Abnormal refractive states, such as visual impairments caused by refractive anomalies, can be a serious problem for patients of all ages and are often, but not always, treatable by subtractive laser procedures. In recent years, an additive technique involving the implantation of a lenticule into a patient's cornea after cutting and folding back a flap to expose an intrastromal region of the cornea has been developed. The shape of the lenticule modifies the refractive power of the patient's cornea by changing the curvature of the cornea. The flap can then be replaced over the lenticule. In other situations, such as keratoconus, the implanted lenticule can mechanically stabilize or normalize the abnormal stroma and delay the progression of the disease. In cases of keratoconus, stromal pockets are more frequently formed rather than flaps.

[0003]

[0002] However, several problems limit the wider acceptance of additive (lenticule) techniques. First, the availability of donor human corneas for forming lenticules is severely limited. In addition, when lenticules are obtained from non-human sources, they need to be decellularized to minimize immune responses. Furthermore, due to the nature of lenticules (structured layers of collagen), they are fragile and require special handling. In general, decellularization further weakens the structure and / or modifies the permeability properties, such that decellularized lenticules are prone to postoperative swelling, and as a result, the proper refractive correction during surgery may not be maintained. Therefore, a better method for stabilizing implantable lenticules and other collagen-based scaffolds is needed.

[0004]

[0003] Collagen is widely present in the human body. For example, collagen is found in the intestines, veins, joints, skin, intima, ventricular valves, and cornea.

[0005]

[0004] However, corneal stroma collagen is unique in many respects. Apart from water, collagen constitutes the main component of the corneal stroma. Other components of the stroma include glycoaminoglycans (GAGs) and proteoglycans. Living cells make up only about 1-4 percent of the corneal stroma. The human stroma consists of approximately 100-150 lamellae, each containing parallel collagen fibrils. Because it is transparent, the layers of collagen fibrils exhibit in-plane conformation, with the spacing between layers or lamellae being almost ideally distributed. No other organ contains collagen fibrils configured in this way.

[0006]

[0005] While solid collagen exhibits high tensile strength in the in-plane direction, it is considerably more brittle in the direction perpendicular to the plane. Decellularized solid collagen is even more brittle. When using such decellularized solid collagen as a scaffold, a better method is needed to increase its mechanical strength. [Overview of the Initiative] [Means for solving the problem]

[0007]

[0006] A method for stabilizing a collagen scaffold has been disclosed, and by the method of the present invention, a collagen scaffold can be formed using water extraction and / or compression, as well as cross-linking, to mechanically strengthen the collagen scaffold and prevent the collagen scaffold from swelling in an aqueous environment. The method of the present invention may be particularly useful for preparing a collagen scaffold as a lenticle for intrastromal or intracorneal implantation as part of additive refractive surgery.

[0008]

[0007] Scaffolds formed from collagenous tissue can provide mechanical advantages to the cornea. However, since scaffolds are usually mechanically brittle due to decellularization, they need to be strengthened and oriented before being placed in the stroma. One aspect of the present invention discloses a method for forming and strengthening a scaffold from donor collagenous tissue, comprising the steps of: excising a portion of the tissue from the central region of a donor collagenous tissue source (e.g., donor corneal stroma); shaping the tissue portion to form a scaffold of a first desired shape; decellularizing the scaffold; compacting the scaffold (e.g., generally perpendicular to the lamellar structure of the scaffold) to remove excess fluid present in the scaffold and increase the collagen density; and crosslinking at least a portion of the scaffold to mechanically strengthen it and, if the scaffold is exposed to an aqueous environment, preventing subsequent swelling. Thus, the method of the present invention can enhance the mechanical strength and chemical stability of the scaffold. If the scaffold is intended for use as a lenticle that can be implanted in the cornea, cross-linking can be used to restore and / or ensure the optical transparency (e.g., clarity) of the scaffold.

[0009]

[0008] In certain embodiments, the excision step and the molding step can be performed simultaneously. The decellularization step of the scaffold may further include lysing cells, removing cellular debris from the lenticles with a detergent or surfactant, and optionally further including enzymatically removing at least one immunogenic epitope from the lenticles. A useful solution for decellularizing the scaffold may include water, ethyl alcohol, and glycerol. Orientation of the scaffold can also be manipulated. The steps of the method described herein may be performed in any order, if feasible.

[0010]

[0009] Although not bound by any particular operating theory, the mechanical strengthening of the collagen scaffold is not only a result of crosslinking, but also a result of the compaction of collagen fibrils into tighter, more rigid bundles. Crosslinking of the densified fibril bundles can result in material strength that exceeds the improvements achievable by crosslinking alone.

[0011]

[0010] In the method of the present invention, the crosslinking step may further include exposing at least a portion of the compressed scaffold to a crosslinking agent or energy carrier, or exposing at least a portion of the compressed scaffold to radiation to induce crosslinking by peptide bond formation between collagen fibrils, with or without the assistance of an energy carrier. A combination of chemical crosslinking and photocrosslinking is also disclosed, in which the chemical agent can induce different chemical bonds and physically form mechanical bridges between collagen fibrils.

[0012]

[0011] The crosslinking step can be carried out by direct exposure, exposure to such radiation at a line-of-sight angle, or by exposing at least a portion of the compressed (or dehydrated) scaffold to radiation via an evanescent waveguide connected to the surface of the scaffold. The crosslinking step may further include exposing the surface portion of the compressed scaffold to radiation, resulting in the surface portion exhibiting a greater degree of crosslinking and a higher collagen density than the bulk region of the scaffold.

[0013]

[0012] Preferably, the compression and crosslinking steps result in at least a portion of the scaffold having a higher collagen density than the initial decellularized scaffold segment. For example, at least a portion of the compressed and crosslinked scaffold may have a composition of at least 20 percent collagen, preferably more than 35 percent collagen.

[0014]

[0013] The method of the present invention can be put into practice such that the scaffold is configured for use as an implantable intracorneal lenticle having a lenticle body, an oriented front and rear surface, and the method of the present invention further includes treating at least a portion of the rear surface of the lenticle with a crosslinking agent or by selective application of patterning radiation to promote adhesion of the lenticle to the parenchymal bed. In certain embodiments, the method of the present invention may include the steps of forming a lenticle from a donor parenchyma; removing a portion of the tissue from a central region of the donor parenchyma by lenticle extraction; shaping the removed tissue portion into a lenticle of a first desired shape, wherein the lenticle has a lenticle body, an front and rear surface; removing cellular material from the lenticle; removing excess fluid present in the lenticle (e.g., water originally present in the parenchyma and any other fluid that may have been introduced into the lenticle during decellularization); and crosslinking at least a portion of the lenticle to define the final desired shape and prevent subsequent swelling if the lenticle is exposed to an aqueous environment. Lenticular formation can be performed by excision or extraction of the lenticular using a corneal incision knife, femtosecond laser, excimer laser, or water jet.

[0015]

[0014] Lenticle crosslinking can be performed by exposing at least a portion of the compressed scaffold to a crosslinking agent or energy carrier, or by exposing at least a portion of the compressed scaffold to radiation, with or without the assistance of an energy carrier, to induce crosslinking by peptide bond formation between collagen fibrils. For example, lenticle crosslinking can be performed by exposing at least a portion of the compressed scaffold to radiation by direct exposure, exposure at a line-of-sight angle, or via an evanescent waveguide connected to the surface of the scaffold. In certain applications, ultraviolet radiation is a preferred energy source.

[0016]

[0015] Chemical methods have been reported for the crosslinking of collagen. These chemical methods may include the use of chemical agents (e.g., genipin) or photochemical agents. For example, U.S. Patent Application Publication 2017 / 0119928 discloses a method for forming an artificial heart valve by decellularizing pericardial tissue, crosslinking it with a glutaraldehyde-containing solution, and obtaining a collagenous matrix material that can be molded into a desired structure for implantation.

[0017]

[0016] Radiation crosslinking may be more advantageous than chemical crosslinking because the resulting lenticles are often more transparent, resulting in better visual acuity after implantation into the recipient's cornea.

[0018]

[0017] Radiation bridging of a compressed scaffold can be performed with substantially less energy than the energy required for an uncompressible (fluid-rich) scaffold. For example, in the case of a typical lenticle after compression, bridging can be performed with approximately 15 joules / cm². 2 Less than, or in some cases approximately 2500 joules / cm³ 2 This can be done with ultraviolet radiation at a fluence of less than 15 joules / cm³. More generally, the desired fluence is about 15 joules / cm³. 2 ~Approximately 600 joules / cm² 2 The range is as follows. One particular range of wavelengths useful for crosslinking collagenous scaffolds generally corresponds to a portion of the "UV-C" wavelength band, for example, from about 185 nm to about 280 nm. Other UV wavelength bands (the UV-B band from about 280 nm to about 315 nm (or 320 nm), or the UV-A band from about 315 nm (or 320 nm) to about 400 nm) can also be used in some cases, similar to X-rays, gamma radiation, or electron beams, to induce at least partial crosslinking and / or sterilization of the scaffold.

[0019]

[0018] Decellularized collagen scaffolds exhibit a certain degree of "shape memory" or "stress hysteresis," so it is possible to compress the decellularized collagen scaffold separately and then crosslink it, but the decellularized collagen scaffold is no longer constrained by the compression mold. Such separation is an (continuous) alternative manufacturing process. However, the preferred method is, in most cases, to induce crosslinking while the scaffold is compressed in the mold. In either case, crosslinking of the compacted collagen bundles results in greater strength than crosslinking of the dispersed collagen fibrils.

[0020]

[0019] In another aspect of the method of the present invention, the crosslinking step may further include exposing the surface portion of the compressed scaffold to radiation such that the surface portion exhibits a greater degree of crosslinking and higher collagen density than the bulk region of the scaffold. Furthermore, the crosslinking step may also include applying radiation sufficient to inactivate any microbial factors and sterilize the lenticles. In certain embodiments, the decellularization step of the scaffold may include removing cellular debris from the lenticles using a detergent or surfactant, and optionally further including enzymatically removing at least one immunogenic epitope from the lenticles.

[0021]

[0020] Furthermore, the compression and crosslinking steps can result in at least a portion of the scaffold having a greater collagen density than the initial collagenous tissue segment. The crosslinking step may further include selectively applying radiation to the front surface so that the front surface region exhibits a greater degree of crosslinking or a greater collagen density than the bulk region of the lenticle body.

[0022]

[0021] In yet another aspect of the present invention, a decellularized collagen lenticle is disclosed having a lenticle body derived from donor tissue, having an anterior and posterior surface formed to provide the lenticle with a desired shape and orientation. For example, the lenticle may have convex and concave surfaces, which often, though not always, coincide with the anterior and posterior surfaces of the lenticle. The lenticle body may include layers of decellularized and compressed collagen to obtain a composition of more than 15 or 25 percent collagen, and may further be at least partially crosslinked to prevent axial swelling. In the case of bulk crosslinked lenticles, the composition may be more than 30 percent collagen. Furthermore, if a locally high-density layer is desired (for example, to approximate a corneal structure such as Bowman's membrane), the local collagen concentration may be even higher (for example, more than 35 percent, more than 40 percent, or even more than 60 percent collagen).

[0023]

[0022] In certain embodiments, lenticles can be characterized by layers of collagen that are crosslinked by the application of radiation, and lenticles can also be further characterized by induced peptide bonds between collagen fibrils. Lenticles may have 90 to 100 percent, or preferably 95 to 99.99 percent, of their cellular material removed, and following decellularization, compression, and crosslinking, the lenticles can be implanted in a patient's eye to alter the refractive power of the cornea and to replenish or strengthen areas of stromal damage or lesions.

[0024]

[0023] The lenticles according to the present invention typically have a curved disc shape and a diameter ranging from about 0.5 mm to about 10 mm. The lenticles also typically have a maximum thickness which can range from about 600 to about 50 micrometers, more preferably from about 400 to about 100 micrometers. The lenticles are not typically of uniform thickness, and the minimum thickness can be less than about 50 micrometers, more preferably less than about 30 micrometers or less than about 15 micrometers.

[0025]

[0024] The lenticle needs to exhibit low immunoreactivity due to the degradation of immunogenic epitopes. In certain embodiments, at least one surface of the lenticle further includes a pattern of variable crosslinking to facilitate attachment of the lenticle to the parenchyma bed when substantially implanted into the patient's parenchyma bed. The lenticle can also have a front face with a front region having a collagen density greater than that of the bulk region of the lenticle body. For example, the collagen density of the front region can be at least about 35 percent or 40 percent or 60 percent collagen.

[0026]

[0025] Thus, the present invention discloses a method for stabilizing the shape of a collagen lenticle and a scaffold. The collagenous tissue can be harvested from any collagenous source, human or animal-derived. In certain preferred embodiments, the tissue can be harvested from human or porcine parenchyma. The source tissue can be shaped in a separate step during or after excision. The tissue can be shaped, decellularized, and stabilized by a variety of techniques, some of which are described in more detail below. Following shaping and decellularization, the excised collagenous tissue segment is subjected to compression to reduce fluid content and irradiated to induce crosslinking of collagen chains or collagen fibrils. Crosslinking can be induced with or without a chemical mediator, such as a crosslinking agent. In certain preferred embodiments, crosslinking is obtained by formation of peptide bonds between collagen fibrils by exposure to sufficiently high energy radiation. Since the creation of peptide bonds between collagen chains is usually an exothermic reaction, this energy radiation is desirable. A variety of energy sources can be used to induce crosslinking of peptide bonds. In certain preferred embodiments, the energy is delivered by ultraviolet (UV) radiation.

[0027]

[0026] The present invention is generally applicable particularly to the spatial stabilization treatment of a layered collagenous tissue, particularly a collagen scaffold taken from the corneal stroma. The layered collagen composition of the source tissue may have evolved in nature for optical transparency, but when exposed to an aqueous environment, it may result in mechanical fragility and / or a tendency to swell and be permeable. Therefore, one object of the present invention is to prevent such swelling and / or provide greater axial mechanical strength with the final scaffold. The present invention can also be applied to non-layered (or disordered) excised collagenous tissue segments.

[0028]

[0027] In another aspect of the present invention, when the scaffold is a lenticule for corneal transplantation, a method for modifying the collagen density or smoothness of at least one surface of the scaffold, for example, the front surface, is disclosed. Such surface modification can facilitate the manipulation of the scaffold after transplantation, for example, when it is necessary to re-incise the overlying flap to access the implant. The surface modification can be performed by irradiation and / or application of a chemical agent.

[0029]

[0028] A decellularized and shaped corneal tissue lenticule from a donor of an allograft and / or xenograft, and a method for obtaining such a lenticule are disclosed. The lenticule is particularly useful as an intrastromal or intracorneal lenticule implant in corneal transplantation, in which case a hinged flap is formed within the patient's cornea and folded back along this hinge to expose the corneal stromal bed. Then, the shaped lenticule is applied to the stromal bed and the flap is returned to its original position, resulting in a new curvature being imparted to the cornea and the desired refractive correction being achieved. Fine adjustment of the new refractive power can be performed by laser ablation either simultaneously with the transplantation or at a later time if regression or tension changes occur.

[0030]

[0029] In one aspect of the present invention, decellularized corneal lenticles and methods for decellularizing corneal tissue are disclosed to reduce the patient's potential immunogenic response to transplanted lenticles. Only about 1–4 percent of a normal cornea is composed of cells. The remaining 96–99 percent is almost entirely extracellular matrix (ECM), i.e., mainly collagen, glycoaminoglycans (GAGs) and proteoglycans, as well as water. In a preferred embodiment, the cellular components of the lenticles are removed, for example, by treatment with a surfactant such as sodium tetradecyl sulfate (STS), or by enzymatic solubilization. If desired, especially if the source is a non-human (xenogeneic) donor, further steps can be taken to further reduce the immunogenicity of the lenticles. For example, two non-human epitopes that can be present in xenogeneic tissue are neu5GC and alpha-Gal. These undesirable epitopes can be located not only within but also on the surface of parenchymal cells; some epitopes may be embedded within GAGs, also known as mucopolysaccharides, surrounding ECM collagen fibrils. In such cases, such epitopes can be selectively removed, at least partially, by kinase treatment and further washing.

[0031]

[0030] The decellularized lenticles of the present invention typically have 95% to 100% of their cellular material removed. Preferably, the lenticles have 95% to 99.99% of their cells removed. It is obvious that all possible partial ranges between 95% and 100% are intended and considered as part of the present invention, without detailing any such partial ranges. For example, the lenticles may have 95% to 97%, 97% to 99%, or 98% to 99.9% of their cellular material removed.

[0032]

[0031] In another aspect of the present invention, a disc-shaped lenticle according to the present invention is obtained by cutting a disc-shaped tissue segment from a donor cornea. The tissue segment can be sliced ​​and / or further shaped or cut during the slicing procedure to obtain a desired shape. Cutting can be performed mechanically, for example, by laser processing using a microcorneal cutting knife, for example, by light cutting using a femtosecond laser or by an excimer laser or by a water jet. To reduce the possibility of asymmetry, it is preferable that the tissue segment is taken from the central portion of the donor cornea, for example, with the optical axis or geometric axis of the donor cornea secured at the center of the lenticle. The shape of the tissue segment will be determined by the change in dioptric power required to correct the patient's refractive error. For example, in the case of correcting hyperopia (hypermetropia) and / or presbyopia, the goal is usually to increase the curvature of the cornea, and the desired lenticle shape will be slightly convex on at least one side. Typically, the maximum thickness of a lenticle is less than 400 micrometers, or often less than 200 micrometers, or less than 100 micrometers, or less than 50 micrometers. For almost all applications, the maximum thickness is less than 600 micrometers. In some situations, visual improvement in patients with macular degeneration can be achieved when the disc-shaped lenticle is formed as a prism that redirects light to different parts of the retina.

[0033]

[0032] Treatment of keratoconus may include not only the reduction of visual aberrations but also the mechanical strengthening of the pathological parenchyma. For this purpose, a coplanar disk with limited refractive effects, having a transitional slope (wedge-shaped) band at the periphery of the disk and a thickness between 50 and 300 micrometers, may be preferred. Alternatively, a lenticle for treating keratoconus may take advantage of the natural curvature inherited from the donor cornea, or a further degree of curvature may be introduced during compression and / or bridging by the use of a curved mold. In some cases, it may be advantageous to implant the lenticle "inverted," for example, so that the curvature of the lenticle is opposite to that of the recipient cornea, when treating keratoconus.

[0034]

[0033] In some cases, a curved mold is preferable, but the compression and bridging steps can also be performed in a flat mold. (Flat molding may be advantageous for storage and transport or manufacturing efficiency.) In another aspect of the present invention, a sealed mold can be used for both compression and storage.

[0035]

[0034] The curvature of the surface can also be altered and / or the collagen fibrils can be newly aligned (or maintained) by applying pressure to one surface of the scaffold. For example, the scaffold can be fixed to the opening of a chamber, and the chamber can then be filled with pressurized fluid to apply pressure to one side of the scaffold, thereby imparting a horizontal / tangential force to the scaffold. The pressure of the fluid will cause the scaffold to expand (like a balloon). A compressible plate can optionally be applied to the opposite surface. For example, a curved (quasi-concave) compression plate can be used to limit the extent to which the scaffold can be extended or reformed. Once the desired curvature is achieved, bridging can be used to maintain the desired shape and / or prevent the tendency of the thus manipulated scaffold to swell during intrastromal or intracorneal implantation.

[0036]

[0035] In some embodiments, it may be advantageous to ensure the upper stromal surface, i.e., the so-called "Bowman's membrane," such that the anterior surface of the lenticle exhibits a different texture from the other (posterior) surfaces, because the natural anterior segment of the corneal upper stromal surface is denser and smoother due to the natural compression of the outermost layer of stromal tissue. Alternatively, an excised segment can be taken from the central region of the stroma, and the anterior surface can be densified following shaping and excision by selective cross-linking, as described in more detail below. The posterior surface (opposite the anterior or Bowman's membrane) is rougher due to the lower density of the stromal tissue and the fact that it is formed by mechanical or laser cutting of the tissue. This difference in roughness may be particularly advantageous when the lenticle is used for intrastromal or intracorneal implantation, because it is highly desirable that the lenticle adheres firmly to the stromal bed. If suboptimal refraction results are observed after the procedure, it may be necessary to fold the flap again to allow for further corneal transplantation (renticle resculpting) using laser ablation or other methods. Any movement of the lenticle from its original position on the parenchymal bed could impair the effectiveness of such corneal transplantation. Furthermore, the smoothness of the anterior surface of the lenticle also reduces the likelihood that re-incising the flap would cause the lenticle to dislodge.

[0037]

[0036] In yet another aspect of the present invention, the posterior surface may be treated after excision, shaping, and decellularization to allow the posterior surface to adhere to the parenchyma. For example, a crosslinking agent may be applied before sterilization and packaging. Alternatively, an adhesion enhancer may be applied by a clinician during pre-transplant treatment. The front surface may be treated to make it less likely to adhere to the flap. [Brief explanation of the drawing]

[0038] [Figure 1A] This is a schematic cross-sectional view of the extracted parenchymal tissue segment. [Figure 1B]This figure shows a schematic cross-section of an excised parenchymal tissue segment after decellularization, illustrating the spacing resulting from tissue swelling and collagen fibrils. [Figure 1C] This is a schematic cross-sectional view of an excised parenchymal tissue segment after decellularization, compression, and cross-linking according to the present invention. [Figure 2] This is a schematic cross-sectional view of an excised parenchymal tissue segment after decellularization, compression, and selective cross-linking of the anterior region. [Figure 3] This is a schematic cross-sectional view of an excised parenchymal tissue segment after decellularization, compression, and selective patterning of the posterior region. [Figure 4A] This figure illustrates a lenticle according to the present disclosure in a flat shape, which is a typical shape during manufacturing and / or transport. [Figure 4B] This figure illustrates a lenticle in its final curvature state, prepared for intracorneal implantation. [Figure 5A] This diagram illustrates a lenticle designed for intracorneal implantation to correct hyperopia. [Figure 5B] This diagram illustrates a lenticle designed for intracorneal implantation to correct myopia. [Figure 5C] This diagram illustrates a lenticle designed for intracorneal implantation to correct presbyopia. [Figure 5D] This diagram illustrates a lenticle designed for intracorneal implantation to correct a condition known as keratoconus. [Figure 6A] This figure illustrates another embodiment of a lenticle manufactured with localized spots of strong crosslinking. [Figure 6B] This figure illustrates yet another embodiment of a lenticle, which includes a central optically active zone with moderate crosslinking and an outer or peripheral zone with strong crosslinking. [Figure 7A] This diagram illustrates the use of a lenticle for deep lamellar corneal transplantation (DALK). [Figure 7B]This figure illustrates the use of a lenticle configured to be placed on the Bowman membrane of the patient's intact tissue. [Figure 7C] This figure illustrates yet another embodiment of a lenticle for full-thickness corneal transplantation (PK). [Figure 8A] Figures 8A and 8B illustrate two alternative designs for the periphery of a thick lenticle. Figure 8A shows a lenticle with a simple periphery, for example, a cylindrical or conical shape. [Figure 8B] Figures 8A and 8B illustrate two alternative designs for the periphery of a thick lenticle. Figure 8B shows a lenticle with a zigzag or stepped edge around its periphery. [Figure 8C] This diagram shows a lenticle with a "key" edge around its periphery. [Figure 9] This is a schematic perspective view of a press apparatus for use in the steps of compressing and crosslinking a collagen scaffold according to the present invention. [Figure 10A] Figures 10A and 10B illustrate a two-part sealed compression and storage mold according to the present invention. Figure 10A shows the mold before compression of the scaffolding. [Figure 10B] Figures 10A and 10B illustrate a two-part sealing, compression, and storage mold according to the present invention. Figure 10B illustrates the mold after compression. [Figure 11] This figure illustrates a further apparatus according to the present invention for extending scaffolding. [Figure 12] This figure shows yet another alternative apparatus, similar to the apparatus in Figure 11, but with the addition of a compressive plate element for simultaneous extension and compression of the scaffolding, and to facilitate exposure to bridging radiation. [Figure 13] This figure illustrates an apparatus 130 for measuring the transparency of a lenticle prepared in accordance with this disclosure. [Figure 14] This is a graph of the luminance distribution curve obtained using the apparatus shown in Figure 13, for quantifying optical transparency. [Modes for carrying out the invention]

[0039]

[0065] Because processes, compositions, or methodologies can vary, the present invention is not limited to any particular process, composition, or methodology described herein. The terms used herein are solely for the purpose of describing a particular version or embodiment of the present invention and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Any publications described herein are incorporated in their entirety by reference. Nothing herein should be construed as admitting that the present invention does not have prior rights to such disclosure on the grounds of prior art.

[0040]

[0066] The term “dissection” encompasses any known method relating to the dissection, ablation, or removal of biological materials, for example, by the action of a mechanical blade, ultraviolet (UV) laser, femtosecond laser, or water jet.

[0041]

[0067] The term "compression" includes compaction by the application of pressure or by other techniques such as vacuum, or moisture extraction driven by centrifugal force.

[0042]

[0068] The term "radiation" includes infrared radiation, visible light radiation, ultraviolet radiation (for example, from about 400 nm down to about 193 nm or below), X-rays, gamma rays, and electron beams.

[0043]

[0069] The term "biological sample" refers to tissue, cells, cell extracts, homogenized tissue extracts, or mixtures of one or more cell products. Biological samples may be used or present in a suitable physiologically acceptable carrier.

[0044]

[0070] As used herein and in the appended claims, unless otherwise explicitly stated in the context, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, a reference to “cell” refers to one or more cells and their equivalents known to those skilled in the art.

[0045]

[0071] As used herein, the term "approximately" means plus or minus 10% of the numerical value used with it. Therefore, approximately 100 μm means a range of 90 μm to 110 μm.

[0046]

[0072] As used herein, the terms “animal,” “patient,” or “subject” include, but are not limited to, humans and non-human vertebrates such as wild animals, domesticated animals, and farmed animals. The terms “animal,” “patient,” or “subject” also refer to the recipient of a corneal lenticle transplant. The term “xenograft” refers to tissue collected from donor animals, including pigs (porcine), cattle, apes, monkeys, baboons, other primates, and any other non-human animals. “Allograft” refers to tissue taken from a donor of the same species as the recipient.

[0047]

[0073] Generally, the term "tissue" refers to any collection of cells that are similarly specialized and integrated in the performance of a particular function. The corneal stroma, although mostly noncellular (about 1-5% cellular), is an example of "tissue."

[0048]

[0074] The term "lenticular" refers to decellularized and processed donor corneal tissue ready for transplantation into or onto the cornea of ​​a recipient. Unless otherwise specified, the terms "lenticular" and "scaffold" are used synonymously herein. The terms "collagen concentration" and "collagen percentage" are used synonymously herein and refer to the amount of collagen present in the lenticular or scaffold. This concentration or percentage can be measured as a fraction of the weight of fully dried lenticular, for example, vacuum-dried lenticular, relative to the weight of lenticular before drying (in perfect equilibrium with moisture). In some cases, drying can be improved using ethanol.

[0049]

[0075] In relation to lenticle transplantation, the term “intracorneal” refers to any procedure in which the lenticle is placed inside or on the cornea. One type of intracorneal transplantation is “intrastromal” transplantation, in which the lenticle is placed within the stroma of the eye without excising the anterior Bowman’s membrane or epithelium, for example, by folding a flap of anterior tissue or by direct insertion via a lateral approach. Other types of intracorneal use of lenticles include deep lamellar keratopathy (DALK) and full-thickness keratopathy (PK), in which the lenticle completely replaces the anterior segment of the eye, as will be discussed in more detail below. Yet another applicable “intracorneal” procedure is the so-called “superficial keratopathy,” which will also be discussed in more detail below.

[0050]

[0076] The term "axial direction" refers to the orientation of the lenticle or scaffold relative to its orientation. Typically, a shaped lenticle is curved in the shape of a spheroid or ellipsoidal disc, and the axial direction, or "axis," is generally perpendicular to the center of the disc. Unless otherwise specified, the "axial direction" is also generally nearly parallel or coaxial with the visual axis, or optical axis, of the eye from which the tissue segment is extracted or the recipient eye into which the lenticle is designed to be implanted. (In a natural eye, the optical axis usually passes almost through the center of the cornea, though not precisely.)

[0051]

[0077] Planetary physics (spherical geometry) can provide convenient terminology to describe the spatial configuration of collagen fibrils in a substance. The majority of collagen fibrils are in high- to mid-latitude orbits, with only a small fraction existing in polar orbits. Therefore, the substance is usually thinnest in the central region / location. The axial direction of compression can be called polar cap compression or radial compression. The central region of the donor substance exhibits strong rotational symmetry in fibril matching. If the implant is specified to function not only under compression but also under tensile stress, it is preferable that the lenticles extracted from the central substance location impart rotationally symmetrical tensile properties to the implant.

[0052]

[0078] This disclosure also relates to decellularized corneal lenticles from allograft and / or xenograft sources, and methods for forming lenticles from donor stroma. The decellularized corneal lenticles of this disclosure can be used to correct abnormal refractive conditions such as myopia, hyperopia, presbyopia, and astigmatism, as well as other ophthalmic conditions.

[0053]

[0079] The cornea can generally be considered to consist of five layers from anterior to posterior: the corneal epithelium, the thin but dense upper stromal layer (usually called Bowman's membrane in the human eye), the corneal stroma, Descemet's membrane, and the corneal endothelium. The corneal epithelium consists of about six layers of non-keratinized stratified squamous epithelial cells, which are rapidly growing and readily regenerating. The anterior stromal layer (e.g., Bowman's membrane) is a robust layer composed mostly of randomly organized and tightly interwoven collagen type I fibrils. The corneal stroma is a thick, transparent layer composed of collagen type I fibrils arranged in parallel layers. Descemet's membrane is a thin, noncellular layer that functions as the basement membrane of the corneal endothelium and is composed of low-rigidity collagen type IV fibrils. Finally, the corneal endothelial cells consist of a single flattened or low-cubic monolayer of mitochondrial-rich cells.

[0054]

[0080] As used herein, the term “Bowman’s membrane” is used to describe any anterior stromal region of the cornea, whether from a human or donor animal. While the densifying layer of the human cornea may be more pronounced (hence known as Bowman’s membrane in the human cornea), all corneas exhibit, to some extent, a somewhat higher anterior densification and smoothness (relative to the stromal bed tissue), depending on the animal species and age. Thus, “Bowman’s membrane” is a term used throughout this application to describe such anterior segments.

[0055]

[0081] Therefore, the harvesting and processing of donor corneas, as well as the fabrication of lenticles, are crucial elements in correcting refractive errors of visual acuity. Lenticles are particularly useful as lenticle implants in corneal transplantation, in which a hinged flap is formed within the patient's cornea and folded back along this hinge to expose the corneal stroma. The shaped lenticle is then applied to the stroma, and the flap is returned to its original position, resulting in a new curvature in the cornea and the desired refractive correction. Fine-tuning of the new refractive power can be performed by laser ablation either simultaneously with the transplant or at a later time if regression or tension changes occur.

[0056]

[0082] In certain embodiments of the present invention, a decellularized corneal lenticle may include a lenticle body derived from a donor parenchyma, having a front surface including at least a portion of the uppermost layer from the donor parenchyma and a rear surface formed to provide a lenticle having a desired shape; where the donor parenchyma is decellularized. In other embodiments, the lenticle is formed without regard to the preservation of Bowman's membrane and any upper portion of the donor parenchyma. For example, the lenticle may be formed by intrastromal excision of a parenchymal tissue segment, for example, by excision with a femtosecond laser pulse.

[0057]

[0083] In certain embodiments, donor parenchyma is harvested and decellularized to produce a lenticle that has a reduction in any potential immunogenic response on the patient's side. Only about 1–4 percent of a normal cornea is composed of cells. The remaining 96–99 percent is almost entirely extracellular matrix (ECM), primarily collagen, as well as water, glycoaminoglycans, and proteoglycans. As described above, the decellularized lenticle of the present invention typically has 95–100% of its cellular material removed. Preferably, the lenticle has 95–99.99% of its cells removed. It is evident that all possible sub-ranges between 95% and 100% are intended and considered as part of the present invention, without detailing them. For example, the lenticle may have 95–97%, 97–99%, or 98–99.9% of its cellular material removed. The amount of cellular material remaining in the lenticle can be assessed, for example, by the content of residual DNA or RNA. Preferably, the DNA or RNA content is less than 1 weight percent, less than 0.1 weight percent, or less than 0.01 weight percent of the initial DNA or RNA content.

[0058]

[0084] Decellularization, i.e., removal of cellular material from donor stroma, can be performed using a variety of techniques. In one preferred embodiment, corneal cellular material is removed by chemical treatment. Chemicals used to dissolve and remove cells from the cornea include surfactants such as sodium tetradecyl sulfate (STS), acids, alkaline treatments, ionic detergents such as sodium dodecyl sulfate (SDS), nonionic detergents such as Triton X-100, and amphoteric detergents. In some embodiments, corneal cellular material is removed using enzymatic treatment. Lipases, thermolysins, galactosidases, nucleases, trypsins, endonucleases, and exonucleases are used to remove cellular material from the cornea. In some embodiments, corneal cellular material is removed using physical techniques. These physical techniques include methods used to dissolve, kill, and remove cells from the tissue matrix through the use of temperature, force, and pressure, as well as electrical disruption. Thermal methods are often used in rapid freeze-thaw mechanisms. Thermal methods preserve the physical structure of the ECM scaffold. Pressure decellularization involves the controlled use of hydrostatic pressure at high temperatures to avoid unsupervised ice crystal formation that could damage the scaffold. Electrical disruption of the plasma membrane is another option for dissolving corneal cellular material.

[0059]

[0085] In the embodiments described herein, lenticles can exhibit even lower immunoreactivity due to the degradation of immunogenic epitopes. This can be an important step when using heterologous offerings. For example, two non-human epitopes that may be present in heterologous tissues are N-glycolylneuraminic acid (Neu5GC) and galactose-alpha-1,3-galactose (alpha-Gal). These undesirable epitopes can be present not only inside but also on the surface of parenchymal cells; a small portion of epitopes may be embedded in glycoaminoglycans (GAGs), also known as mucopolysaccharides, surrounding ECM collagen fibrils. In certain embodiments, epitopes can be selectively removed by enzymatic treatment, such as galactosidase treatment, and further washing. Alternatively, corneal tissue can be taken from a knockout transgenic animal (e.g., a transgenic pig) that lacks any immunogenic epitopes, and thus non-immunogenic lenticles can be produced without the need for an epitope degradation step.

[0060]

[0086] In the embodiments described herein, the decellularized lenticles can be further sterilized in conjunction with packaging and sealing. Sterilization can be carried out using a wet agent, radiation, or an electron beam. In one preferred embodiment, sterilization of the decellularized lenticles is performed using UV radiation because there is a low probability of damage to the collagen scaffold. The use of UV radiation may be advantageous in improving the optical transparency of the lenticles.

[0061]

[0087] In another aspect of the present invention, the shape and orientation of the lenticles are designed for best results. In some embodiments, the diameter of the lenticles is from about 0.5 mm to about 10 mm, or from about 3 mm to about 9 mm, or from about 4 mm to about 8 mm, or from about 5 mm to about 7 mm. Again, it is clear that all possible partial ranges between 0.5 mm and 10 mm are intended and considered as part of the present invention, without detailing all such partial ranges.

[0062]

[0088] The donor corneal stroma can be sliced ​​and / or further shaped to obtain the desired form. Dissection can be performed mechanically, for example, using a microcorneal dissection knife or by laser processing, such as photoablation with an excimer laser or photocutting with a femtosecond laser. To reduce the possibility of asymmetry, the corneal tissue segment is preferably taken from the central portion of the donor cornea, for example, with the optical or geometric axis of the donor cornea centered at the lenticle. The shape of the corneal tissue segment will be determined by the change in diopter power required to correct the patient's refractive error. For example, in the case of correcting hyperopia (farsightedness) and / or presbyopia, the goal is usually to increase the curvature of the cornea, and the desired lenticle shape will be slightly convex on at least one side. In some embodiments, the maximum thickness of the lenticle is less than 600 micrometers, less than 400 micrometers, less than 200 micrometers, less than 100 micrometers, or less than 50 micrometers. The smaller the diameter and the thinner the lenticle, the faster the lenticle will integrate with the patient's actual body.

[0063]

[0089] The parenchymal collagen fibrils are long polymer (polypeptide) strings. These fibrils are triple-twisted proteins. The length of a single collagen fibril is nearly macroscopic, and therefore each fibril can individually be a strong scatterer of light. The fact that the parenchyma is transparent in the axial direction is the result of the negative sum of all these strong scattering contributions. That is, despite their individual scattering, the collagen fibrils collectively contribute to a collective, nearly zero scattering overall. This collective transparency is achieved when the fibrils are arranged parallel to a single plane. In the corneal parenchyma, the arrangement plane is perpendicular to the optical axis. This unique arrangement is present in the cornea but not in other organs. In other organs such as the intestines, myocardium, and ventricular valves, the fibrils are not precisely aligned, and therefore light is scattered. The same is true for the limbal collagen of the cornea.

[0064]

[0090] In addition to transparency, natural selection has similarly optimized the structure of the cornea in terms of strength. The length and alignment of collagen fibrils substantially contribute to the in-plane (tangential) tensile strength of the stroma. The natural shape of the eye is maintained by hydrostatic intraocular pressure, which applies tangential tensile stress to the stroma. The in-plane orientation of collagen fibrils contributes to corneal transparency and results in significant mechanical tensile strength. However, this strength is almost entirely limited to forces applied in-plane. In comparison, the strength of the corneal stroma in the optical axis direction is substantially brittle. One manifestation of this axial strength deficiency is the swelling of the scaffold when immersed in water, for example, buffered saline solution (BSS). The swelling is almost without exception unidirectional in the optical axis direction. In-plane swelling is negligible.

[0065]

[0091] Since the shape conformity of the optically active lenticles is crucial for successful additive refractive surgery, this fragility of the scaffold is a source of concern. Axial scaffold swelling may induce refractive errors.

[0066]

[0092] The amount of swelling often correlates with the immersion solution. Adding surfactants and / or detergents to the water results in the highest degree of swelling, approximately 250–400% of the scaffold's nominal thickness. BSS typically induces swelling of approximately 150–250% of the nominal thickness. Alcohol (both light and heavy) usually causes less swelling. (Nominal thickness can be defined, for example, as the axial thickness of the initial excised parenchymal segment (lamellae) before the decellularization step, e.g., less than about 60 seconds, or therefore after excision, if the tissue specimen is very fresh, or it may be longer if the specimen retains epithelial and / or endothelial cells).

[0067]

[0093] In cases of thick lenticles, swelling can make the lenticle so thick that it becomes difficult to reposition the flap onto the parenchymal bed. The flap may also be too short to cover the added material (increasing the risk of epithelial hypoplasia), and may require further mechanical extension of the flap onto the parenchymal bed and / or suturing.

[0068]

[0094] Along with changes in permeability and / or water affinity of the scaffold (compared to the pre-decellularized substance), the unidirectional mechanical brittleness of the collagen scaffold may cause significant unidirectional swelling in the direction of the scaffold's thickness (i.e., axial direction).

[0069]

[0095] Free water is present within collagenous tissue. In intact natural corneas, the water content of the stroma is controlled by the overall structure of the cornea, such as the epithelial and endothelial membranes that form the corneal boundary. However, when exogenous stroma tissue is transplanted during additive refractive surgery, this balance is often disrupted, and the water content of the transplanted lenticle tends to increase, leading to postoperative swelling beyond its nominal thickness.

[0070]

[0096] According to the present invention, by placing a flat (cylindrical) collagenous tissue specimen between two plates to which steady pressure is applied, the fluid content can be reduced (or, in some cases, the collagen concentration can be increased). The same applies to decellularized shaped lenticles. The press must be equipped with drainage so that excess free fluid is drawn laterally from the tissue. In the case of shaped lenticles, at least one of the plates of the press must be curved to accommodate the radial change in the thickness of the lenticle. Preferably, the pressure is applied gently for a desired time. Depending on the desired degree of compression, the pressure can be applied to the collagen scaffold for a predetermined period ranging from a few seconds to several hours, for example, from 30 seconds to 1 hour, or in some cases from 5 minutes to 30 minutes.

[0071]

[0097] The distance moved by the press plates allows for the approximate calculation of collagen concentration. For example, if the nominal thickness of a flat excised tissue segment is 100 micrometers, and the segment swells to 200 micrometers after the decellularization process, the collagen content of the composition can be approximately 15%. If the segment is then recompressed to a thickness of 100 micrometers, the nominal collagen concentration will recover to a level of approximately 30%. Further compression of the scaffold to a thickness of 50 micrometers will result in a collagen concentration of approximately 60%. Further compression of the scaffold to a thickness of 40 micrometers will result in a collagen concentration of approximately 75%. (The collagen content percentage can be measured as the weight fraction of vacuum-dried lenticle relative to the weight of lenticle before drying, assuming the lenticle is in perfect equilibrium with water.) This value is only obtained after prolonged pressure and is close to the upper limit of a reasonably achievable collagen concentration. At this point, residual water is firmly bound to the protein, for example, by van der Waals forces, and further pressure may impair the integrity of the collagen fibrils.

[0072]

[0098] The process of compressing collagenous tissue is nearly reversible when the scaffold is removed from the press and immersed again in the fluid. The scaffold reswells to approximate its thickness before compression. However, in another aspect of the present invention, most notably, a method is disclosed to prevent reswelling by strengthening the collagen scaffold in the axial strain direction through cross-linking of the collagen scaffold during pressing. Compression of the scaffold and / or removal of excess fluid can also be carried out by exposing the scaffold to acceleration, for example, by centrifugal separation. For example, 10G~100G (981m / s²). 2 Accelerations up to or beyond ) can be used. Excess fluid removal can also be done or assisted by exposing the scaffold to a vacuum or reduced pressure.

[0073]

[0099] In certain embodiments, crosslinking can be carried out by chemical agents. External chemical molecules can be added (primarily in aqueous solutions or other fluids) at a sufficient concentration, duration, and temperature. The molecules can be constructed to bond with one collagen fibril at one end and another collagen fibril at the other end. The type of bond can be agent-specific or does not need to be peptide-type bond. The chemical molecules create physical bridges with chemical bonds (covalent bonds) as strong adhesion. A sufficiently dense collection of such bridges imparts new strength and / or rigidity to the axial collagen scaffold. The collagen molecules do not need to be in contact with each other, but they need to be located at approximately the molecular size of the agent. This mild operating requirement facilitates the crosslinking process. Examples of chemical crosslinking agents are glutaraldehyde, genipine, and monosaccharides.

[0074]

[0100] Collagen fibrils are themselves powerful light scatterers, but the orientation and statistical structure of collagen fibrils collectively eliminate scattering. A potential drawback of crosslinking agents and energy mediating molecules is that when collagen scaffolds are used as implantable lenticles in refractive surgery, these molecules introduce exogenous substances into the collagen scaffold that act as light scatterers and may adversely affect the optical transparency of the collagen scaffold.

[0075]

[0101] In other embodiments, collagen fibrils can also be strengthened by stable chemical bonds directly established between them. This occurs when the fibrils are in contact with each other, but does not occur spontaneously. So-called peptide bonds are endothermic and require the localized and timely delivery of external energy to the site of contact between the fibrils. In some embodiments, specialized mediating molecules that receive energy from photons can be used. The mediating molecule can then provide binding energy or catalyze the crosslinking process without itself participating in the link structure. Thus, light is an indirect energy source for building stable bonds. An example of a mediating molecule is riboflavin when exposed to light from, for example, a light-emitting diode (LED).

[0076]

[0102] In yet another variation, the collagen fibrils of the scaffold can be directly bonded to each other by radiation that is locally absorbed at the points where the fibrils are in contact with one another. Exogenous mediating molecules do not need to capture the energy quantum (although endogenous molecules such as glycoaminoglycans (GAGs) present in collagenous tissues may provide a similar function). The quantum is absorbed directly and in a timely manner at the junction of the contact event. This method can utilize various forms of direct irradiation, e.g., visible light, blue light, or UV radiation, gamma rays, or even electron beams. A preferred energy source is UV light having an energy density of at least 100 joules per square centimeter, or at least 200 joules per square centimeter, or at least 300 joules per square centimeter. For example, the desired energy density of the active radiation can be in the range of approximately 100 to approximately 5000 joules per square centimeter, or between approximately 200 to approximately 1000 joules per square centimeter, or between approximately 300 to approximately 600 joules per square centimeter.

[0077]

[0103] The density (compression) of the collagen scaffold can play a role in the rate of crosslink formation. When the collagen concentration is higher (i.e., when the collagen scaffold is more compressed), the crosslink formation process can proceed more quickly.

[0078]

[0104] The critical (threshold) dose required for pure radiation-induced crosslinking may correlate with the decellularization process on its own (without any mediator such as riboflavin). The threshold dose for non-decellularized parenchyma can be 3 to 300 times greater than the threshold dose for decellularized collagen scaffolds. (This ratio may also correlate with the wavelength of the radiation applied.) In another aspect of the invention, it has been found that the threshold for collagen crosslinking decreases when decellularization removes more of the extracellular material (such as GAGs) present in the parenchyma. The difference in thresholds can vary by an order of magnitude depending on the decellularization protocol applied and the wavelength of the radiation. This decrease in the crosslinking threshold appears to correlate with the intensity of the decellularization protocol.

[0079]

[0105] Crosslinking can be performed simultaneously with or in a subsequent step of compression, via a dedicated UV radiation source (or by ambient UV radiation, such as sunlight).

[0080]

[0106] When using light energy to induce crosslinking, light absorption can be determined by Beil's law; that is, more light will be absorbed in the surface layer of the material upon which the light strikes, and less will be absorbed in deeper layers of the material. The amount of light available to induce crosslinking decreases exponentially, essentially attenuating. If there are differences in the amount of light-scattering molecules within the scaffold, this can also affect the energy distribution because the scattering agent reduces the amount of light that can pass into the region beneath the irradiated material. In this invention, such effects can be effectively used to produce crosslinking of varying degrees and / or to impart various properties to the surface region of the scaffold exposed to active radiation.

[0081]

[0107] Therefore, the innate absorption profile of light energy alone, or in conjunction with the introduction of a light scattering agent (e.g., as a surface coating), provides an option for selective surface crosslinking and / or a lesser degree of crosslinking beneath the surface of the collagen scaffold. Selective crosslinking of the lenticle surface may have advantages in conferring different adhesion, permeability, or smoothness to the surface, or in facilitating greater or lesser penetration by recipient cells after transplantation. For example, if a scaffold is compressed to a collagen density of about 60% and selectively crosslinked at the surface to a depth of about 5-10 micrometers, the surface will retain its high density, while the rest of the scaffold will remain unchanged from its expanded state after the decellularization process. Such a high-densification surface can form a pseudo-Bowman membrane.

[0082]

[0108] Furthermore, by using surface crosslinking to selectively treat a portion of the surface with a light scatterer, or by exposing a scaffold through a patterned mask, a patterned effect can be imparted to the front, back, or both sides. Surface patterning can selectively modify the friction or adhesion of a portion of the surface.

[0083]

[0109] Three methods exist for selectively crosslinking the surface of a scaffold. Firstly, the penetration depth can be limited by using short-wavelength UV radiation that has strong absorption in the scaffold. In this approach, the preferred wavelength range is from about 230 to about 150 nanometers, and more preferably from about 215 to about 193 nanometers (e.g., 193 nm). The impact angle of the radiation is usually the normal to the surface, but may vary between 0 and 60 degrees relative to the surface normal.

[0084]

[0110] Alternatively, longer wavelength UV radiation can be used, for example, resulting in wavelengths up to about 400 nanometers and penetrating the scaffold at minute incidence angles greater than 60 degrees, preferably greater than or greater than 75 degrees, for example, in the range of about 80 to about 89.9 degrees. One advantage of this method is that reliable and powerful UV radiation sources with wavelengths of about 280 nanometers or more are readily available, as such radiation sources are for industrial applications. The use of laser radiation as a light source can also be preferable because the spatial coherence of the laser light allows for the demarcation of minute incidence angles with good precision.

[0085]

[0111] Another alternative method involves using evanescent wave slab waveguides. These waveguides allow for very shallow bridging depths (approximately the wavelength of the incident light). For example, when using 380 nanometer UV radiation, bridging can be limited to a surface layer of less than a micrometer.

[0086]

[0112] Importantly, by increasing the density of the anterior surface of the lenticle (or more strongly and selectively cross-linking it), the anterior surface of the lenticle will exhibit a different texture from the posterior surface, because Bowman's membrane is denser and smoother due to tightly interwoven collagen type I fibrils. The posterior surface (opposite the anterior surface) will be rougher due to the lower density of the composition of this part of the lenticle (and the fact that it is formed by mechanical or laser cutting of the tissue). This difference in roughness can be particularly advantageous when the lenticle is used for intrastromal or intracorneal implantation, because it is highly desirable for the lenticle to adhere firmly to the stromal bed. If suboptimal refractive results are observed after the procedure, it may be necessary to fold the flap again to allow for further corneal implantation (re-cutting of the lenticle) by laser ablation or other means. Any movement of the lenticle from its initial position in the stromal bed may impair the effectiveness of such corneal implantation. Furthermore, the smoothness of the anterior surface of the lenticle also reduces the likelihood that re-incising the flap would cause the lenticle to detach.

[0087]

[0113] In yet another aspect of the present invention, the posterior surface may be treated after excision, shaping, and decellularization to allow it to adhere to a substantial bed. For example, a crosslinking agent or adhesion enhancer may be applied before sterilization and packaging. Alternatively, the crosslinking agent or adhesion enhancer may be applied by a clinician during pre-transplant treatment.

[0088]

[0114] In some embodiments, the harvested, shaped, and decellularized lenticles are marked in a way that allows the clinician to maintain proper orientation of the lenticles during flap re-incision and laser re-cutting adjustment. The markings can be made in various ways, but in all cases they are invisible to the patient once the lenticles are placed on the substantial floor. In some embodiments, the markings can be microscopic notches on the upper anterior or bottom anterior portion of the lenticle. In some embodiments, the markings can be lines or dots made with dye placed on the upper anterior or bottom anterior portion of the lenticle.

[0089]

[0115] Methods for forming a lenticle from a donor stroma are also disclosed herein. In some embodiments, the method for forming a lenticle from a donor stroma includes the steps of removing a portion of the stroma from the central region of the donor cornea, and shaping the posterior surface of the donor stroma to form a lenticle body of a desired shape. To reduce the possibility of asymmetry, the tissue segment is preferably taken from the central portion of the donor cornea, for example, so that the optical or geometric axis of the donor cornea is centered on the lenticle. The shape of the tissue segment will be determined by the change in diopter power required to correct the patient's refractive error. For example, in the case of correcting hyperopia (farsightedness) and / or presbyopia, the goal is usually to increase the curvature of the cornea, and the desired lenticle shape will be slightly convex on at least one side. The lenticle may also include one or more asymmetrical markers (such as the letter "L") on the periphery of the lenticle to identify the anterior and posterior surfaces of the lenticle.

[0090]

[0116] The scaffolds of the present invention can further offer advantages as refractive (e.g., additive) lenticles in that they can be designed to have a higher refractive index than natural parenchymal tissue. Currently, the use of implantable lenticles to modify corneal curvature is usually limited to total diopter powers (e.g., the sum of the diopter values ​​of the natural cornea and the additive lenticle) of less than about 50 D (measured near the apex) before epithelial instability or unacceptable epithelial erosion occurs. Implantable scaffolds with higher refractive indices offer the potential for greater hyperopia correction. The standard refractive index of parenchymal tissue is typically about 1.376. The compression techniques of the present invention can provide scaffolds with refractive indices greater than 1.377, greater than 1.378, greater than 1.379, greater than 1.38, or higher. The refractive index gradient of the lenticle can also be obtained by controlled bridging formation. (Such gradients can also be used for the correction of higher-order refractive errors, the term which can be described by higher-order Zernike polynomials relating to the human eye).

[0091]

[0117] In the embodiments described herein, the method of the present invention produces lenticles having a shape and density designed for best results. Lenticles are obtained by first slicing a disc-shaped tissue segment from the donor parenchyma so as to ensure that Bowman's membrane is the front surface. In some embodiments, the diameter of the lenticles is about 0.5 mm to about 10 mm, or about 3 mm to about 9 mm, or about 4 mm to about 8 mm and about 5 mm to about 7 mm. The tissue segment can be sliced ​​and / or further shaped or sliced ​​to obtain the desired shape during the slicing procedure. Slicing can be performed mechanically, for example, by laser processing using a microcorneal dissection knife, or by optical cutting using a femtosecond laser. Slicing can be performed using equipment such as that disclosed in International Patent Application PCT / IB2016 / 054793, entitled "Surgical Apparatus and Blade Elements for slicing Lamellar Segments From Biological Tissue," which is incorporated herein in its entirety by reference.

[0092]

[0118] Lenticles can also be obtained by femtosecond laser ablation, excimer laser ablation, or by water jet dissection. If preservation of the anterior segment is not required, lenticles can also be obtained by the Small incision Lenticule Extraction (SMILE) technique disclosed in U.S. Patent No. 6,110,166, entitled "Method For Corneal Laser Surgery," which is also incorporated herein in its entirety by reference.

[0093]

[0119] In some embodiments described herein, the maximum thickness of the lenticle is less than 600 micrometers, less than 400 micrometers, less than 200 micrometers, less than 100 micrometers, or less than 50 micrometers. The smaller the diameter and the thinner the lenticle, the faster it can integrate with the patient's physical bed.

[0094]

[0120] In certain embodiments, donor parenchyma is decellularized to produce lenticles in a state where the likelihood of adverse patient reactions to cell-derived immunogens is reduced. Decellularized lenticles produced using the methods of the present invention have 90 percent to 100 percent, or preferably 95 percent to 99.99 percent, or 98 percent to 99.9 percent of cells and / or cellular residue removed. Only about 2 percent of a typical cornea consists of cells. The remaining 98 percent is almost entirely extracellular matrix (ECM), mainly collagen, water, GAGs, and proteoglycans. Preferred, primarily practiced methods for characterizing the amount of cellular residue after the decellularization process are based on the detection of DNA residues or, alternatively, RNA residues. These methods are highly sensitive and specific. These measurements are typically standardized to the amount of DNA / RNA present in the lenticles before the decellularization step. Without detailing any possible sub-range between 90% and 99.99%, it is evident that all such sub-ranges are intended and considered as part of the present invention. For example, the prepared lenticles have 90%, 95%, 99%-99.7%, 99.7%-99.9%, or better, of the natural cellular material removed (evaluated by residual DNA / RNA content). In other words, the amount of cellular material remaining in the lenticles can be less than 1% by weight, less than 0.1% by weight, or less than 0.01% by weight of the original DNA or RNA content, as evaluated by the residual DNA or RNA content. (Thanks to the lenticle extraction itself, a considerable amount of decellularization can be performed. As much as 95% of the total corneal cell content is present in the epithelium and endothelium, which can be mechanically disposed of, leaving only the corneal stroma for further decellularization.)

[0095]

[0121] The removal of cellular material from donor stroma (decellularization) can be carried out using various techniques. In one preferred embodiment, corneal cellular material is removed by chemical treatment. Chemicals used to dissolve and remove cells from the cornea include acids, bases, surfactants (e.g., sodium tetradecyl sulfate (STS)), ionic detergents (e.g., sodium dodecyl sulfate (SDS)), nonionic detergents (e.g., Triton X-100), and amphoteric detergents.

[0096]

[0122] Alternatively, or in addition, corneal cellular material is removed using enzymatic treatment. Lipases, thermolysins, galactosidases, nucleases, trypsins, endonucleases, and exonucleases can be used to remove cellular material from the cornea. In some embodiments, corneal cellular material is removed using physical techniques. These physical techniques include methods used to dissolve, kill, and remove cells from the tissue matrix through the use of temperature, pressure, and / or electrical disruption. Temperature-based decellularization methods may include rapid freezing protocols. Such temperature-based methods preserve the physical structure of the ECM scaffold. Pressure decellularization involves the controlled use of hydrostatic pressure at high temperatures to avoid unsupervised ice crystal formation that could damage the scaffold. Electrical disruption of the plasma membrane is another option for dissolving corneal cellular material.

[0097]

[0123] In some embodiments, lenticles can be further processed to exhibit even lower immunoreactivity due to the degradation of immunogenic epitopes. This is an important step when using heterologous offerings. For example, two non-human epitopes that may be present in heterologous tissues are N-glycolylneuraminic acid (Neu5GC) and galactose-alpha-1,3-galactose (alpha-Gal). These undesirable epitopes are present not only inside but also on the surface of parenchymal cells; a small portion of epitopes may be embedded in glycoaminoglycans (GAGs), also known as mucopolysaccharides, surrounding ECM collagen fibrils. In certain embodiments, such epitopes can be removed or their conformation modified (to neutralize immunogens) by enzymatic treatment such as kinase or galactosidase treatment and further washing. Alternatively, corneal tissue can be harvested from knockout transgenic pigs lacking epitopes, thus allowing for the production of non-immunogenic lenticles without the need for a degradation step. In some cases, it may be preferable to remove the epithelial cell layer and / or endothelial cell layer or residue from the lenticle before epitope neutralization. This can be done, for example, by curettage with a surgical scalpel, or by rubbing with an abrasive of appropriate coarseness.

[0098]

[0124] In embodiments described herein, the method for producing decellularized lenticles may further include a sterilization step, which may be combined with packaging and sealing. Sterilization can be carried out using a wet agent, gamma radiation, or an electron beam. In one embodiment, sterilization of the decellularized lenticles is performed using an electron beam because there is a low probability of damage to the collagen scaffold. Alternatively, radiation used to induce crosslinking can also provide sufficient energy for sterilizing the lenticles.

[0099]

[0125] In some embodiments, the preparation of harvested, shaped, and decellularized lenticles may include a step of marking the lenticles in such a way that the clinician can maintain the proper orientation of the lenticles during flap re-incision and laser adjustment. The markings can be made in various ways, but in all cases they will be invisible to the patient once the lenticles are in place. In some embodiments, the markings may be microscopic notches on the upper anterior or bottom anterior portion of the lenticles. In some embodiments, the markings may be lines or dots made with dye placed on the upper anterior or bottom anterior portion of the lenticles. These methods may also include a step of imprinting one or more asymmetrical markers (such as the letter "L") on the periphery of the lenticles to identify the front and rear surfaces of the lenticles.

[0100]

[0126] The present invention and embodiments illustrating the methods and materials used will be further understood by referring to the following exemplary protocol. Corneal tissue can be harvested from a pig donor. Lenticles can be taken from a region within the donor stroma so as to maintain high-density collagen type I fibrils or Bowman's membrane on the anterior and less dense posterior surfaces of the lenticles. Alternatively, corneal tissue can be taken from a stroma region below a naturally densified surface.

[0101]

[0127] As shown in Figure 1A, the target region of the donor cornea can be dissected into a disc-shaped lenticle 10A having an anterior surface 12, a posterior surface 14, and organized layers of collagen fibrils 16. At this stage, the lenticle will typically have a diameter of approximately 0.5 to 10 millimeters and a thickness of less than 250 micrometers.

[0102]

[0128] Figure 1B shows a lenticle after decellularization, for example by chemical treatment, enzymatic treatment, or physical technique, to produce a lenticle from which 95–99.99% of cellular material has been removed. If desired, the decellularized, shaped lenticle can be further processed to degrade immunogenic epitopes. The lenticle can be further washed and sterilized, and if desired, a crosslinking agent can be applied to the front and / or back surfaces of the lenticle. The decellularized lenticle (typically swollen by the application of a washing agent, surfactant, and / or washing solution) exhibits greater spacing of the fibrillary layers 16. In some embodiments, the upper front surface of the lenticle can also be marked with notches to assist in lenticle orientation on the patient's parenchyma.

[0103]

[0129] In Figure 1C, the decellularized (and potentially swollen) lenticles are then subjected to compression to expel excess fluid from the lenticle body and increase collagen density. The collagen fibril layer is compressed together and then, at least partially, crosslinked to prevent further swelling.

[0104]

[0130] Figure 2 shows a lenticle 20 having a front region 22 in which the collagen fibrillary layer is compressed and crosslinked. The bulk region 24 of the lenticle 20 can also be crosslinked to prevent swelling, but not to the same extent as the front region 22.

[0105]

[0131] Figure 3 shows a lenticle 30 in which one surface (e.g., the posterior surface 14) is formed with a pattern 32 to promote postoperative integration. The pattern 32 can be formed by selective application of a drug, selective irradiation, or a combination of these techniques. (It is clear that the pattern can be applied to the anterior surface 12, the posterior surface 14, or both surfaces.)

[0106]

[0132] Figure 4A illustrates a lenticle 40A according to the present disclosure in a flat shape, which is often a typical shape during manufacturing and / or transport.

[0107]

[0133] Figure 4B illustrates a lenticle 40B in an exemplary final curvature for intracorneal implantation. Lenticles can take various shapes to correct a variety of refractive errors or ocular conditions (further discussed below). While lenticles are commonly exemplified as spherical or ellipsoidal shapes, it is clear that they may also be ellipsoidal or any desired shape (e.g., partially ring-shaped). Such shapes may be useful for correcting astigmatism or higher-order aberrations in the patient's visual acuity. Such aspherical shapes may be useful in matching the shape of the patient's cornea or limbus.

[0108]

[0134] Figure 5A illustrates lenticle 50A, designed for intracorneal implantation to correct hyperopia. Figure 5B illustrates lenticle 50B, designed for intracorneal implantation to correct myopia. Figure 5C illustrates lenticle 50C, designed for intracorneal implantation to correct presbyopia. Figure 5D illustrates lenticle 50D, designed for intracorneal implantation to correct a condition known as keratoconus, in which the cornea's natural collagenous structure is fragile due to injury, genetics, or other eye conditions, such as an imbalance in enzyme or signaling activity within the cornea. Lenticles 50A, 50B, 50C, and 50D are typically designed for intrastromal (interlaminar) implantation within the cornea. By placing the lenticle within the stromal tissue 2, epithelial regions 4 and endothelial regions 6 are secured.

[0109]

[0135] Figure 6A illustrates another embodiment of the lenticle, in which case the lenticle 60A is manufactured with localized spots 61 of strong crosslinking to form areas of further mechanical strength for the attachment of surgical sutures, for example, during full-thickness corneal transplantation (PK) or deep lamellar corneal transplantation (DALK), as will be discussed in more detail below.

[0110]

[0136] Figure 6B illustrates yet another embodiment of the lenticle. In this embodiment, the lenticle 60B includes a central optically active zone 65 with moderate bridging (e.g., having a main diameter dimension of about 3 to 6.5 millimeters) and an outer or peripheral zone 63 with strong bridging, which again, form an area of ​​further mechanical strength, for example, for the attachment of surgical sutures or in supporting corneally fragile parenchyma outside the visual zone. It should be noted that zones 63 and 65 do not need to be concentric, and it may be desirable to offset the optically active zone from the center of the lenticle, for example, in the treatment of keratoconus.

[0111]

[0137] Figure 7A illustrates the use of the lenticle 70A according to this disclosure for a different type of intracorneal transplant, namely deep lamellar corneal transplantation (DALK). In this procedure, the anterior segment of the eye is first removed, and the lenticle 70 is placed in the eye to replace a portion of the natural Bowman's membrane 3 and stroma 2. The lenticle 70A can be initially fixed in place by sutures 7. Optionally, the lenticle 70A may be formed from natural donor tissue having intact Bowman's membrane, which is secured through the decellularization and compression steps of lenticle formation. Alternatively, the anterior surface of the lenticle may be selectively treated, for example, by shallow radiocrosslinking to form a Bowman's membrane-like structure. In either case, following intracorneal transplantation, the patient's peripheral epithelium may grow over the anterior surface of the transplanted lenticle.

[0112]

[0138] Figure 7B illustrates the use of the lenticle 70B, which is similar to the DALK lenticle shown in Figure 7A, but is typically thinner (e.g., less than 200 micrometers) and is configured for placement on the patient's intact Bowman membrane 3 but beneath the epithelium 4. This procedure is sometimes called a "superficial corneal transplant," in which the lenticle 70B can also be fixed in place with sutures 7.

[0113]

[0139] Figure 7C illustrates yet another embodiment of the lenticle 70C according to this disclosure, in which case it is designed for full-thickness corneal transplantation (PK). The lenticle 70C is similar to the DALK lenticle shown in Figure 7A, but is configured to completely replace the entire depth of the central corneal tissue, i.e., the posterior corneal endothelium 6, stroma 2, and Bowman's membrane 3. The lenticle 70C can also be fixed in place by sutures 7. Following intracorneal transplantation of the lenticle 70C, the patient's peripheral epithelium can grow across the anterior surface of the transplanted lenticle.

[0114]

[0140] Figures 8A, 8B, and 8C illustrate two alternative designs for the periphery of a thick lenticle, such as those useful in DALK and PK procedures. Figure 8A shows a lenticle 80A with a simple, for example, cylindrical or conical periphery. Figure 8B shows a lenticle 80B with a zigzag or stepped rim on the periphery of the lenticle. The shaped rim can be designed to align with complementary structures formed in the natural cornea to further assist in the bonding of the lenticle 80B to the remaining natural corneal tissue. Stepped variations (e.g., reverse zigzag) can also be used. As shown in Figure 8C, more advanced rim shapes can be used to enable mechanical latch-in or lock-in to the natural cornea. The lock-in function can, in some cases, reduce or eliminate the need for surgical suturing along the latch-in edge. The periphery of such a lenticle can be bridged more strongly than the central portion.

[0115]

[0141] In some cases, the thickness of these thick lenticles can vary from the center to the edge. For example, the thickness of the lenticles can vary from about 400 micrometers in the center to 550 micrometers at the periphery. This is consistent with the natural thickness variation seen in most corneas, in which case a typical intact cornea will have a central corneal thickness of about 500 micrometers, while the peripheral segment of the cornea can have a thickness of about 550 to 650 micrometers.

[0116]

[0142] Figure 9 schematically illustrates a press 90 for use in compressing a collagen scaffold according to the present invention. The press 90 may include a frame 92 and a movable stage 94. The frame holds an upper press element 96, and the stage holds a lower press element 98. At least one of these elements is non-planar and molded to conform to the desired final shape of the scaffold. Elements 96 and 98 can be brought to compression by the application of a thrust force ( schematically illustrated by a worm screw 91). In certain embodiments, at least one of the press elements 96, 98 can be made transparent so that the scaffold held in compression between the press elements 96 and 98 can be irradiated, for example, by a UV radiation source 93 at the same time the scaffold is being molded.

[0117]

[0143] Figures 10A and 10B illustrate a two-part sealed compression and storage container or mold 101 according to the present invention. The container / mold 101 may comprise a mold base 102 and a mold upper 104, with a chamber 105 defined between the mold base 102 and the mold upper 104. The purpose of the mold 101 is to compress a scaffold 100A, for example, a decellularized collagenous (extracellular matrix) scaffold, and at the same time remove fluid from the scaffold. Figure 10A illustrates the mold 101 in the state before the scaffold is compressed. A seal 106, for example, an O-ring or flat gasket seal, initially separates the mold upper 104 from the mold bottom 102. In some cases, it may be desirable to fill the remaining part of the chamber 105 not occupied by the scaffold 100A with fluid 107 to avoid gas entrapment in the scaffold during compression. The seal 106 is configured to contain the fluid expelled from the scaffold, as well as any fluid initially surrounding the scaffold of the chamber 101. Figure 5A shows the mold before compression, at the initial moment when the seal 106 creates isolation from the surrounding environment.

[0118]

[0144] The upper portion 104 of the mold can be at least partially transparent or semi-transparent to allow active radiation to pass through the scaffold in order to bridge the scaffold. Alternatively, or in addition, the bottom portion 102 of the mold can be transparent to active radiation. The transparent portion of the mold can be made of plastic, glass, ceramic, or metal, or any combination thereof, as long as the mold is sufficiently transparent or semi-transparent to allow radiation transmission.

[0119]

[0145] Figure 10B illustrates the mold 101 after compression of the scaffold 100B. A seal, such as an O-ring 106, can move within the groove 103, which in turn contains the expelled fluid. (Various other drainage mechanisms can also be used.) In this state, the mold 101 can be used for crosslinking and sterilization. Such sterilization can be performed simultaneously with crosslinking if the appropriate wavelength and fluence of radiation are selected. (In many cases, the amount of radiation required for viral inactivation is far less than the dose required for crosslinking.) The compressed and sterilized mold can then be used for further transport or long-term storage. In the case of active crosslinking radiation, such as UV radiation, the radiation needs to reach all surfaces of the lenticle during this process in order to also be effective for sterilization purposes. Therefore, it may be desirable to use a seal (e.g., an O-ring or flat gasket that is permeable to the selected active radiation). For example, when UV light is used to both crosslink and sterilize the lenticles during molding, the seal is a composition of fluoropolymers such as polytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP), ethylene tetrafluoroethylene (ETFE), or perfluoroalkoxy (PFA), and can be made from UV-permeable or semi-permeable materials.

[0120]

[0146] The matching shape of the scaffolding or lenticle before compression with the mold shape typically ensures that the density of the lenticle remains uniform after compression. For example, compression can halve the vertical dimensions and uniformly double the density.

[0121]

[0147] Figure 11 illustrates a further apparatus 110 according to the present invention for extending a scaffold 161, which may be a natural decellularized parenchyma excision or a scaffold that has been decellularized and subjected to an initial compression treatment. In this embodiment, the scaffold 161 is attached to the opening of the chamber 163 by a sealing mechanism 167, for example, by crimping the periphery of the scaffold 161 between two flat flanges. (Instead of, or in addition to, crimping, various other sealing mechanisms can be used to form a liquid-tight connection between the scaffold and the opening of the chamber.) A fluid 166 is then introduced into the chamber 163 via a controlled pressure conduit 165 to fill the inside of the chamber and apply pressure to the scaffold 161. For example, horizontal and / or tangential extension can be achieved by maintaining a sufficient hydrostatic pressure in the chamber, for example, up to about 30 to about 500 mbar, preferably up to about 50 to about 200 mbar (or up to about 22.5 to about 375 Torr, preferably up to about 37.5 to about 150 Torr).

[0122]

[0148] Even when the fluid applying mechanical stress to the scaffolding is pure water or an equilibrium salt solution (BSS), the application of this fluid pressure can also cause the scaffolding to deswell (release free water). The fluid can flow out of the scaffolding in any direction (e.g., by movement into the pressurized fluid chamber, by seepage from the outer surface of the scaffolding, or both). This effect can also be enhanced or limited by using a hypertonic or hypotonic liquid in chamber 163.

[0123]

[0149] By exposing the ECM scaffold to this type of stretching, the collagen fibrils can be aligned to construct a curved shape. Once the desired curve is achieved, the shape can be secured (or fixed) by crosslinking, for example by active radiation 168, as shown.

[0124]

[0150] Figure 12 shows yet another alternative apparatus 120, similar to the apparatus in Figure 6, but with the addition of a compressive plate element 171 for simultaneous extension and compression of the scaffold, and for facilitating exposure to bridging radiation. Horizontal and / or tangential extension is also achieved here by maintaining a sufficient hydrostatic pressure of about 30 to about 500 mbar, preferably about 50 to about 200 mbar, within the chamber. Vertical / radial compression can be applied simultaneously by the upper plate 171. Preferably, the upper plate 171 is at least partially transparent, e.g., clear or semi-transparent, to allow active radiation to pass through the upper plate to the scaffold in order to bridge it.

[0125]

[0151] Although the upper plate 171 is exemplified as a flat plate, the topography of this compressible element can take any desired shape and / or curvature to ensure that the collagen fibrils are properly fixed and that lenticles of the desired shape are constructed. Such any desired shape and / or curvature can also be secured or fixed here by radiation-induced crosslinking.

[0126]

[0152] Figure 13 illustrates an apparatus 130 for measuring the transparency of a lenticle fabricated according to this disclosure. The apparatus 130 includes a light source 132, a cuvette 134 (on which a lenticle 135 can be placed), a lens 136, and a detector 138. The light source, for example, a light-emitting diode (LED) with a waveguide / fiber and a beam collimator (not shown), preferably generates a beam of collimated light 133. The beam needs to be spatially coherent with a flat (non-Gaussian) intensity profile over its beam width D (e.g., about 5-7 mm). For example, the light source 132 can generate green light having a mid wavelength of about 500 nanometers and a bandwidth of about 5-10 nanometers.

[0127]

[0153] Light from light source 132 is directed to a cuvette 134 in which a lenticle 135 is suspended in a refractive index matching fluid 137 (for example, a silicone oil or other fluid that is miscible with water and chemically inert, with a refractive index n of approximately 1.376). The value n = 1.376 represents the nominal or average refractive index of natural corneal tissue. In some cases, it may be advantageous to have lenticles with different refractive indices. In such cases, the fluid in the cuvette would be selected to match a specific n value of the lenticle under test. The light passing through the cuvette 134 (and lenticle 135) is then directed to a lens 136, which is a high-quality lens without spherical aberration at a selected beam wavelength. The lens 136 has a focal length f and directs the focused light towards a detector 138 located at the focal plane of the lens 136. If the lenticle were perfectly transparent (assuming ideal optical transparency for the optical elements through which light from light source 132 passes to detector 138), the size of the beam colliding between the detector (located at the focal plane) would be a diffraction-limited spot of uniform intensity. In reality, the lenticle is never perfectly transparent, and the beam intensity image at the detector will exhibit some degree of haze due to forward scattering of light within the scaffold. (The focal length of the lens, along with the beam diameter, must be chosen to impose a desirable resolution of the diffraction-limited angle.)

[0128]

[0154] Therefore, the transparency of the lenticle can be evaluated by measuring the intensity profile of the detected beam. Suitable photodetectors for measuring the intensity or brightness profile include, for example, photographic plates, CCD arrays, or scanning pinhole detectors.

[0129]

[0155] Figure 14 illustrates an example of a method for quantifying transparency by measuring the scattering angle of a lenticle (referred to herein as "Θ" or "theta"). Figure 14 illustrates a luminance distribution curve measured at the focal plane of the lens. This Gaussian distribution represents the sum of all contributions from the scattering wavefront. The more scattering at the lenticle, the wider the detected beam. One measure of scattering (decrease in optical transparency) is the full width at vertical half maximum (FWHV), as shown in Figure 14. It should be understood that the Gaussian curve in Figure 14 is idealized, and actual luminance curves may be distorted by noise or other spurious signals. Multiple measurements and averaging (or other known noise reduction signal processing techniques) can be used to obtain the best data representation of scattering by the lenticle. The FWHV value divided by the focal length f gives a measurement of the beam's angular divergence (in radians). Θ = FWHV / f [rad]

[0130]

[0156] Theta is dependent only on the amount of scattering and can also be expressed in arc minutes using the conversion formula: 1 arcmin = 291 microrads. Lenticles produced by the methods disclosed herein exhibit satisfactory transparency when the angle Θ is less than 4 arc minutes, preferably less than 3 or 2 arc minutes, and more preferably less than 1 arc minute in some cases (e.g., in the case of intracorneal implantation).

[0131]

[0157] All patent documents or publications cited herein are incorporated in their entirety by reference. While the applicant's teachings are described in conjunction with various embodiments, they are not intended to be limited to such embodiments. Rather, as will be understood by those skilled in the art, the applicant's teachings encompass a variety of alternatives, modifications, and equivalents. Any element or feature shown in relation to one embodiment can be used interchangeably with or in addition to any element or feature shown in any other embodiment, and all such substitutions of elements and features should be understood as being encompassed by this disclosure. Therefore, it will be understood that the present invention should not be limited to the embodiments disclosed herein, but should be understood from the following claims, which should be interpreted as broadly as permitted by law.

Claims

1. A method for forming a scaffold from a donor corneal stroma extracted from the central region of a donor corneal source, The step of decellularizing the donor corneal stroma to obtain a scaffold; The steps of at least partially removing the fluid present on the scaffolding; and A step of bridging the aforementioned scaffolding to prevent subsequent swelling. Includes, The scaffold is configured for use as a portable lenticle for refraction correction, the lenticle having a lenticle body and front and rear surfaces that provide the lenticle with a desired shape, A method wherein the step of removing fluid from the scaffold further includes compressing the scaffold, so that at least a portion of the compressed and crosslinked scaffold exhibits a collagen concentration of at least 20 percent.

2. The method according to claim 1, wherein the front region exhibits a greater degree of cross-linking or a higher collagen concentration than the bulk region of the lenticle body.

3. The method according to claim 1 or 2, further comprising the step of forming the scaffold, wherein the forming step is performed simultaneously with or in succession to the extraction of the donor corneal stroma.

4. The method according to any one of claims 1 to 3, wherein the step of removing the fluid further comprises applying pressure, acceleration (e.g., centrifugal force) or vacuum to the scaffold.

5. The method according to claim 4, wherein the step of removing the fluid further comprises exposing the scaffold to an acceleration of 10G to 100G.

6. The method according to any one of claims 1 to 5, wherein the decellularization step further comprises treating the scaffold with a chemical decellularizing agent, and the decellularization step is performed before or after the step of removing fluid, or before or after the step of crosslinking.

7. The method according to any one of claims 1 to 6, wherein the decellularization step further comprises removing cellular debris from the scaffold using a detergent or surfactant.

8. The method according to any one of claims 1 to 7, further comprising enzymatically removing at least one immunogenic epitope of the scaffold or modifying its conformation.

9. The method according to any one of claims 1 to 8, wherein the decellularized scaffold has 95% to 100% of its original cellular material removed.

10. The method according to any one of claims 1 to 9, wherein the decellularized scaffold exhibits a residual amount of cellular material that is less than 1 weight percent of the original DNA or RNA content, as evaluated by the content of residual DNA or RNA.

11. The method according to any one of claims 1 to 10, wherein at least a portion of the compressed and crosslinked scaffold exhibits a collagen concentration of more than 45%.

12. The method according to any one of claims 1 to 11, wherein the crosslinking step further comprises exposing the compressed scaffold to active radiation.

13. The method according to any one of claims 1 to 12, further comprising the step of crosslinking by exposing the compressed scaffold to radiation to induce crosslinking by the formation of peptide bonds between collagen fibrils.

14. The method according to claim 12, wherein the compressed scaffold is exposed to radiation without the use of a crosslinking accelerator or other energy carrier.

15. The method according to any one of claims 1 to 14, wherein the crosslinking step further comprises irradiating the scaffold with ultraviolet radiation, X-rays, gamma radiation, or an electron beam.

16. The step of irradiating the scaffolding with approximately 15 joules / cm² of light is performed on the scaffolding. ２ ~Approximately 2500 joules / cm² ２ The method according to claim 15, further comprising irradiating with UV radiation having a fluence in the range up to to induce crosslinking.

17. The step of irradiating the scaffolding with approximately 15 joules / cm² of light is performed on the scaffolding. ２ ~Approximately 600 joules / cm² ２ The method according to claim 15 or 16, further comprising irradiating with UV-C radiation (approximately 100 nm to approximately 280 nm) having a fluence in the range of to induce crosslinking.

18. The method according to any one of claims 15 to 17, wherein the bridging step further comprises exposing the compressed scaffold to ultraviolet radiation by direct exposure, exposure at a minute incidence angle, or via an evanescent waveguide connected to the surface of the scaffold.

19. The method according to any one of claims 1 to 18, further comprising the step of crosslinking by selectively exposing the front region of the compressed scaffold to radiation such that the front region exhibits greater crosslinking and a higher collagen concentration than the bulk region of the lenticle body.

20. The method according to any one of claims 1 to 19, wherein the crosslinking step further comprises applying radiation sufficient to inactivate any microbial factors and / or sterilize the scaffold.

21. The method according to any one of claims 1 to 20, wherein the crosslinking step further comprises treating at least a portion of the rear surface of the lenticle with a crosslinking agent or by selective application of patterning radiation to promote adhesion of the lenticle to the patient's parenchyma when implanted in the parenchyma.

22. The method according to any one of claims 1 to 21, further comprising the step of crosslinking the scaffolding to enhance its optical transparency.

23. The method according to any one of claims 1 to 22, wherein the scaffold exhibits a scattering angle theta of less than 4 arcminutes.

24. The step of compressing the scaffolding is, The method further includes fixing the scaffold across the opening of the fluid chamber and applying fluid pressure to one surface of the scaffold to form a scaffold of a first desired shape. The method according to any one of claims 1 to 23.

25. The method according to any one of claims 1 to 24, wherein the donor corneal source is of human or non-human origin.

26. The method according to any one of claims 1 to 25, wherein the donor corneal source is derived from an allograft or a xenograft.

27. A transplantable, collagenous, decellularized lenticle for refractive correction, A lenticle body derived from a donor corneal source, having a front and rear surface that provides the lenticle with a desired shape; The lenticle body further comprises collagen that is at least partially compressed and crosslinked to prevent axial swelling. A lenticle comprising a lenticle exhibiting a collagen concentration of at least 20 percent.

28. The lenticle according to claim 27, wherein the front region has cross-linking or a higher collagen concentration than the bulk region of the lenticle body.

29. The lenticle according to claim 27 or 28, wherein the collagen layer is crosslinked by the application of radiation, and the collagenous lenticle is further characterized by chemical bonds induced between collagen fibrils.

30. The lenticle according to claim 29, wherein the collagen layer is crosslinked by the application of UV-C radiation.

31. A lenticle according to any one of claims 27 to 30, which does not contain a crosslinking accelerator or other energy carrier.

32. A lenticle according to any one of claims 27 to 31, which does not contain riboflavin.

33. The lenticle according to any one of claims 27 to 32, wherein the amount of cellular material remaining in the collagenous lenticle is evaluated by the amount of residual DNA or RNA, and is less than 1 weight percent of the initial DNA or RNA content.

34. A lenticle according to any one of claims 27 to 33, which is formed in a desired shape so that it can be implanted in the stroma region of a patient's eye to alter the refractive power of the cornea.

35. A wrench tool according to any one of claims 27 to 34, having a disc-like shape and a diameter of approximately 0.5 mm to approximately 10 mm.

36. A lenticle according to any one of claims 27 to 35, having a maximum thickness of less than approximately 600 micrometers.

37. A lenticle according to any one of claims 27 to 36, which exhibits low immunoreactivity due to the removal or degradation of immunogenic epitopes.

38. The lenticular according to any one of claims 27 to 37, wherein the rear surface of the collagenous lenticular further includes a crosslinking agent or a pattern of variable crosslinking to promote adhesion of the collagenous lenticular to the parenchymal bed when implanted in a patient parenchymal bed.

39. A lenticle according to any one of claims 27 to 38, having a curvature fixed / stabilized by crosslinking.

40. A lenticle according to any one of claims 27 to 39, having sufficient optical transparency for use as an intracorneal implant.

41. A lenticle according to any one of claims 27 to 40, exhibiting a scattering angle of less than 4 arcminutes and theta.

42. A lenticle according to any one of claims 27 to 41, exhibiting a collagen concentration of more than 45%.

43. A lenticle according to any one of claims 27 to 42, which is sterilized by radiation.

44. A lenticular according to any one of claims 27 to 43, exhibiting a refractive index greater than 1.

377.

45. A lenticular according to any one of claims 27 to 44, which exhibits a gradient in refractive index.

46. The lenticle according to any one of claims 27 to 45, wherein the donor corneal source is of human or non-human origin.

47. The lenticle according to any one of claims 27 to 46, wherein the donor corneal source is derived from an allograft or a xenograft.

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