HK1113755A - Ophthalmic devices and related methods and compositions - Google Patents

Description

Ophthalmic devices and related methods and compositions

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application 60/601,270 filed on 8/13/2004, the entire contents of which are incorporated herein by reference.

Background

1. Field of the invention

The present invention relates to devices, methods, and compositions for improving vision or treating a traumatic eye (or ophthalmic) disease or disorder in an individual. In particular, the present invention relates to corneal onlay, and corneal implants made of materials that provide one or more benefits to an individual.

2. Description of the related Art

U.S. patent 5,713,957 discloses a corneal onlay comprising a non-biodegradable, non-hydrogel ocular biocompatible material and having a porosity (porosity) sufficient to allow passage therethrough of interstitial fluid components having a molecular fluid molecular weight (molecular weight) greater than 10,000 daltons.

U.S. Pat. No. 5,716,633 discloses collagen/PHEMA hydrogels for promoting epithelial cell growth and regeneration of the matrix. This collagen hydrogel can be provided as an optical lens (optical lens) for attachment to the anterior corneal boundary layer (Bowman's membrane), which can be effective in promoting and supporting the growth of epithelial cells or the attachment of the corneal epithelium to the anterior surface of the lens. The collagen hydrogel is a hydrogel polymer formed by radical polymerization of a hydrophilic monomer solution that gels and crosslinks in the presence of a stored aqueous solution of collagen to form a three-dimensional polymer network for anchoring the collagen. The final concentration of collagen in the implant is from about 0.3% to about 0.5% (wt/wt).

Us patent 5,836,313 discloses a method of forming implantable composite corneal prostheses. The method provides a corneal prosthesis designed to provide a suitable substrate for corneal epithelial cell growth. The corneal prosthesis is formed by: placing corneal tissue in a mold having the shape of a corneal implant, crosslinking the polymer solution, and chemically bonding a biocompatible hydrogel having a thickness of about 50-100 microns to the corneal tissue to form the corneal prosthesis. Alternatively, the polymer solution is placed between the corneal tissue and the preformed hydrogel and then polymerized, thereby bonding the polymer solution to both the hydrogel and the corneal tissue.

U.S. Pat. No. 6,454,800 discloses a corneal onlay or corneal implant comprising a plurality of surface indentations that support the attachment and growth of tissue cells.

U.S. patent 6,689,165 discloses a synthetic device for strengthening and replacing the cornea with tethered corneal enhancers to increase the adhesion and migration of corneal epithelial cells.

Some of the problems associated with existing collagen-based materials are that collagen-based materials are not optically clear, which may be due to formation or transformation into a fiber-based material that can result in unwanted light scattering.

Accordingly, there is a need for biocompatible, ophthalmically acceptable materials suitable for placement in the eye to enhance vision in an individual.

Summary of The Invention

Ophthalmic devices comprise a body comprising a composition effective to promote nerve growth through or on the body when the device is placed in an eye of an individual. In certain embodiments, the device is an ophthalmic device that enhances vision. In an alternative embodiment, the device is a therapeutic ophthalmic device. The vision-improving device of the present invention may be understood as a device configured to correct one or more refractive errors. In other words, the device of the invention may be understood as a device for correcting ametropia. The entity of the inventive device can be formed with an optical power.

The compositions of the present invention are optically clear and may contain collagen in an amount of about 1% (w/v or w/w) to about 50% (w/v or w/w). In certain embodiments, the amount of collagen is greater than 2.5% (w/w or w/v). As used herein, the amount of collagen and/or other components of the compositions and devices may be understood as a w/w percentage or a w/v percentage without departing from the spirit of the present invention. In other embodiments, the amount of collagen is greater than about 5.0%. For example, the material may comprise collagen in an amount of about 10% to about 30%. In certain embodiments, this material comprises crosslinked collagen in an amount of about 1% to about 50%, wherein the collagen is crosslinked with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC; CAS #1892-57-5) and N-hydroxysuccinimide. In a further embodiment, the amount of crosslinked collagen is from 2.5% to about 50%. The material may comprise a first collagen polymer cross-linked with a second collagen polymer. In certain embodiments, the ophthalmic devices disclosed herein do not require glutaraldehyde for preparation. For example, glutaraldehyde is not used as a crosslinking agent in the preparation of ophthalmic devices. The use of glutaraldehyde as a crosslinking agent may not be desirable or preferred for handling and safety requirements of glutaraldehyde and/or the compositions and devices of the invention. In certain embodiments, the ophthalmic devices are prepared without a cytotoxic component, or in other words, with a less cytotoxic component.

The aforementioned device may be a corneal onlay, or a full-thickness corneal implant, such as a device configured to replace an individual's natural cornea. The devices of the present invention are transparent and may be prepared with a composition that is transparent prior to the composition being formed into the device.

The material of the aforementioned devices may also comprise one or more cell growth enhancing agents or one or more other biopolymers.

In accordance with the present disclosure, a method of making an ophthalmic device, such as a device for correcting ametropia, includes crosslinking a collagen polymer with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC and NHS). Crosslinking occurs at an acidic pH, such as a pH of about 5.0 to about 5.5. The method may also include one or more steps of adding a cell growth enhancer to the crosslinked composition. The method comprises placing the composition in a mold and allowing the composition to cure to form the ophthalmic device.

Any feature or combination of features described herein is included within the scope of the present invention provided that the features in any such combination are not mutually inconsistent as determined by the context, this specification, and the general knowledge of one of ordinary skill in the art. In addition, any feature or combination of features may be specifically excluded from any embodiment of the present invention.

Other advantages and aspects of the invention will be apparent from the following detailed description, drawings, embodiments and claims.

Brief Description of Drawings

FIG. 1 is a schematic representation of a cross-sectional view of a T-pipe joint (adapter) of a system for making the compositions and devices of the present invention.

Fig. 2 is a schematic representation of a cross-sectional view of a female Luer adapter (femaleLuer adapter) of a system for preparing the compositions and devices of the present invention.

Fig. 3 is a plan view of the T-junction of fig. 1 connected to one septum (septum) and two syringes to prepare the compositions and devices of the invention.

FIG. 4 is a graph of cell count as a function of time for the human recombinant hydrogel material designated F1.

FIG. 5 is a graph of cell count as a function of time for the human recombinant hydrogel material designated F3.

FIG. 6 is a graph of cell count as a function of time for the human recombinant hydrogel material designated F6.

FIG. 7 is a photograph of a human recombinant hydrogel material designated F3 in rat localization.

Figure 8 is a schematic view of an embodiment of an ophthalmic device for correcting refractive error according to the present invention.

Fig. 8A is a schematic view of a lens edge configuration of one embodiment of an implant of the present invention.

FIG. 9 is swell ratio (swell ratio) with EDC and NH₂Graph of the change in molar ratio of (a).

FIG. 10 shows tensile Strength (tensile Strength) with EDC and NH₂Graph of the change in molar ratio of (a).

Fig. 11 provides a graph of tensile strength as a function of collagen concentration (left graph) and a graph of expansion ratio as a function of collagen concentration (right graph).

FIG. 12 provides information for different EDC and NH₂The composition of (3), the heat flow as a function of temperature (left panel), and for a composition of (3)Graph of heat flow versus temperature for compositions with different CSC concentrations (right panel).

Figure 13 is a graph of neurite length as a function of the ratio of chondroitin sulfate in the dry weight of collagen.

Detailed Description

Typically the human eye has a lens and an iris. The posterior chamber is located behind the iris and the anterior chamber is located in front of the iris. As discussed herein, the eye has a cornea that consists of five layers. One of these layers, the corneal epithelium, is disposed along the anterior outer surface of the cornea. The corneal epithelium is a stratified squamous epithelium extending laterally to the limbus (limbus).

The five layers of the cornea include the corneal epithelium, anterior lamina limica, stroma, posterior lamina limica (Descemet' smembrane), and endothelium. The corneal epithelium is typically about 5-6 cells thick (about 50 microns thick) and typically regenerates when the cornea is injured. The corneal epithelium provides a relatively smooth diopter and helps prevent infection of the eye. Corneal stroma is a layered collagen structure that contains cells such as fibroblasts and corneal cells dispersed therein. The stroma constitutes about 90% of the corneal thickness. The anterior portion of the stroma, which is located under the epithelium, is acellular and is referred to as the anterior boundary layer. The anterior boundary layer, which is located between the epithelium and the stroma, is believed to protect the cornea from damage. The corneal endothelium is typically a monolayer of short cubic or squamous cells that dehydrate the cornea by removing water from the cornea. The cornea of an adult is typically about 500 μm (0.5mm) thick and is generally free of blood vessels.

Ophthalmic devices have been invented that provide one or more benefits to individuals, such as humans, who wish to improve or improve their vision or who require treatment for diseases, disorders or trauma to the eye. The devices described herein may be provided as corneal onlays, corneal inlays, or full-thickness corneal implants. The device of the present invention can improve vision in individuals with reduced vision or provide vision to individuals without vision. The devices described herein do not specifically include intraocular lenses (intraocular lenses).

As used herein, "optically clear" refers to a white light transmittance of at least 85%. In certain embodiments, "optically clear" refers to an optical clarity comparable to a healthy cornea, e.g., having a white light transmission of more than 90% and a scattering of less than 3%.

As used herein, a "corneal implant" is an ophthalmic implant or device that is configured (e.g., sized and shaped) to be placed between the epithelium or epithelial cell layer and the anterior boundary layer of an eye of an individual, such as the eye of a human or animal. In contrast, contact lenses are configured to be placed on the epithelium of the eye. Thus, the corneal onlay may be disposed entirely above the anterior layer, or it may include one or more portions that extend into the anterior layer. These portions constitute a minor portion of the device, e.g. less than 50% of the area or volume of the device.

As used herein, a "corneal implant" is a device or implant that is configured to be placed in the stroma of the eye. The corneal implant may be placed within the stroma by forming a flap or pocket within the stroma. The corneal insert is placed under the anterior border layer of the eye.

As used herein, a "full thickness corneal implant" refers to a device configured to replace all or a portion of an unhealthy cornea of an eye, which is located in front of the aqueous humor of the eye.

The ophthalmic devices of the present invention have reduced cytotoxicity or are non-cytotoxic and provide one or more benefits to an individual in whom the device is placed. For example, the apparatus provides one or more of: (i) a desired refractive index (reactive index), (ii) a desired optical clarity (for visible light, light transmission and light scattering equal to or better than that of a material of a healthy human cornea with comparable thickness), (iii) a desired light intensity, such as a light intensity that enhances vision, (iv) improves comfort, (v) promotes corneal and epithelial health, and (vi) a therapeutic benefit, such as in the treatment of diseases, disorders or trauma to the eye. The ophthalmic devices of the present invention are transparent or made of transparent materials. Some examples of such devices include optically clear devices.

The above benefits, as well as others, may be obtained by forming a device from a material that is (i) formable, e.g., moldable, to form a matrix having an acceptable light intensity, (ii) optically clear or visually transparent, and (iii) effective to promote nerve penetration and/or growth on the device. When the device is a corneal implant, the device is effective to promote re-epithelialization on the anterior surface of the device.

The device is made of a material having mechanical or structural characteristics sufficient to withstand manipulation, implantation, which may include suturing and post-installation abrasion. The device provides or allows sufficient nutrient and gas exchange to promote ocular health. The device, such as a corneal onlay, prepared in-mold is made of a material that can be molded into the appropriate size and shape, including the edge gradient (edge gradient) and vision correction curvature discussed herein.

In one embodiment of the invention, an ophthalmic device for improving vision comprises a body comprising a material effective to promote nerve growth through the body when the device is placed in an eye of an individual. By promoting nerve growth through this entity, the corneas of individuals receiving the one or more devices maintain their contact sensitivity. The entity is formed to have a light intensity. Thus, the entity may be understood as a lens entity. As discussed herein, the device may be configured (e.g., sized and shaped) as a corneal onlay, or a full-thickness corneal implant. In certain embodiments, the device for correcting refractive error of the present invention may not have a light intensity. For example, a device for correcting refractive error in accordance with the present disclosure may be understood as a blank (blanks) that may be placed between the corneal epithelium and the anterior border layer of a patient, or within the corneal stroma of a patient.

For corneal implants, the materials from which the implant is made provide or allow for the exchange of gases and nutrients such as glucose between the anterior lamina and the epithelium to maintain a viable, fully functional epithelium. Other nutrients include factors or agents that promote or enhance survival, growth and differentiation of cells, such as epithelial cells. The exchange should be comparable or superior to that of a healthy human cornea. The permeability of the material to nutrients and/or drugs can be monitored using conventional techniques. In addition, the movement of nutrients and/or drugs through the material should not alter the optical properties of the material. The implant or lenticule is completely biocompatible, allows the epithelium to rapidly adhere to the implant, and allows innervation and restoration of sensitivity, such as contact sensitivity.

The ophthalmic devices of the present invention may comprise an extracellular matrix (ECM) component. In certain devices, the material of the body comprises, consists essentially of, or consists of collagen. In the preparation of the device, the collagen may be crosslinked, for example, by using EDC/NHS. The amount of collagen provided in the hydrogel device of the invention is higher than that currently used in other ophthalmic devices. For example, the amount of collagen provided in the device of the invention is typically greater than 1% (w/w) or (w/v), as discussed herein. In certain embodiments, the amount of collagen is greater than 2.5%. For example, the amount of collagen may be about 5.0% or higher. In certain embodiments of the devices of the present invention, the amount of collagen is from about 1% (w/w) to about 50% (w/w), such as from 2.5% to about 50%. For example, the amount of collagen is greater than about 6% (w/w). Alternatively, the material may comprise collagen in an amount of about 10% (w/w) to about 30% (w/w). As understood by those of ordinary skill in The art, about 15% by weight of hydrated (hydrated) human Cornea is collagen (Maurice D M: The Cornea and Sclera, pp489-600.The Eye, Vol I, Second ed., Ed. H Davson. academic Press, New York, 1969). Thus, the devices of the present invention include collagen in higher amounts than existing ophthalmic devices and in amounts more similar to the amount of collagen present in a human cornea. In addition, the amount and type of collagen provided in the devices of the present invention is effective to provide the desired refractive index, the desired optical clarity, moldability, allow manipulation of the device in the eye, implantation and suturing, and post-installation abrasion.

The remainder of the ophthalmic device, e.g., the non-collagen based portion, may be a liquid, such as water or saline, or may also include one or more other polymers, such as biopolymers and the like. For example, an ophthalmic device comprising about 24% (w/w) collagen, as disclosed herein, can comprise about 76% (w/w) of a liquid, such as water or saline. In other words, in the hydrated state, the ophthalmic device can have a collagen component that is 24% by weight of the hydrated ophthalmic device. As another example, an ophthalmic device may comprise a collagen component that comprises 24% by weight of the hydration device, and a second polymeric component that comprises 6% by weight of the hydration device, with 70% by weight being liquid.

As will be appreciated by those of ordinary skill in the art, the amount of collagen in the device may be higher in the non-hydrated state than in the hydrated state by a percentage.

Collagen comprises three polypeptide chains and is helical in structure. The term "collagen polymer" as used herein shall refer to a triple helical collagen molecule. Collagen is a rod-like molecule with a length and diameter of about 300nm and about 1.5nm, respectively. Collagen molecules have amino acid sequences at their N-and C-termini called "telopeptides" which comprise most of the antigenicity of collagen. Atelocollagen (Atelocollagen) was obtained by pepsin digestion [ dellustroet et al, J Biomed Mater res.1986 jan; 20(1): 109-20], and a non-terminal peptide, indicating low immunogenicity [ Stenzel et al, Annu Rev biophysis bioeng.1974; 3(0): 231-53].

The collagen used in the devices described above may be obtained or derived from any suitable source of collagen, including animal, yeast and bacterial sources. For example, the collagen may be human collagen, bovine collagen, porcine collagen, avian collagen, murine collagen, equine collagen, and the like, or the collagen may be recombinant collagen. The recombinant collagen in the device of the invention may include one or more structural or physical features not present in collagen obtained from normal animal sources, as the recombinant collagen is obtained from bacteria, yeast, plants, or transgenic animals. For example, recombinant human collagen may contain different glycosylated components that may not be present in animal-derived and processed collagen. In addition, recombinant collagen may have a different degree of cross-linking relative to collagen derived from an animal, which may have a variable composition. Variations in the degree of cross-linking in animal-derived collagen can lead to inconsistencies in the collagen and variability in chemical and physical properties, which may not be desirable. Recombinant human collagen not only has tightly controlled purity, but is not associated with viral and/or prion contamination, but animal-derived collagen may be associated with viral and/or prion contamination. Collagen for use in the device of the invention is publicly available or may be synthesized using conventional techniques. For example, recombinant collagen can be obtained from Fibrogen (from multigenic yeast bioreactor cultures or pharming (Netherlands) from milk from transgenic cows or rabbits), or recombinant collagen can be prepared and obtained using the methods disclosed in PCT publication No. WO93/07889 or WO 94/16570. in certain devices, the collagen can be type I collagen. the devices can also be formed from atelocollagen (e.g., collagen without telopeptides). in certain embodiments, the collagen is a non-denatured type of collagen. the atelocollagen can be obtained from companies such as Koken Japan (supplier A, as used herein), where bovine collagen is present at 3.5% (w/v) in a neutral composition, 3.0% (w/v) in an acidic composition, 10% (w/v) in an acidic composition, and wherein the porcine collagen present in the acidic composition is 3.0% (w/v), or the porcine collagen is present as an acidic lyophilized porcine collagen powder. Acidic lyophilized porcine collagen powder is also available from Nippon Ham (Japan) (supplier B, as used herein). Becton Dickinson (supplier C, as used herein) provided 0.3% acidic collagen composition, and 10% acidic collagen composition.

Among the various collagen types, the atelocollagen I is easily solubilized, manipulated, and provides clarity to the final device. This collagen (bovine, porcine or recombinant, in neutral or acidic solution, or as an acidic lyophilized powder) can be obtained from several of the companies described herein above. The lyophilized acidic porcine collagen was readily dissolved in cold water at a concentration of up to 33% (w/v) by stirring at 4 ℃ to give a homogeneous (non-milky) aqueous solution. These clarified collagen compositions (e.g., solutions) have a pH of about 3 (supplier B), or about 5 (supplier a). Commercially available acidic collagen compositions, as low as 0.3% (w/v), can be concentrated by vacuum evaporation and stirring at 0 ℃ -4 ℃ to give a final clear solution with a collagen concentration of up to about 10% (w/v), which can then be used for the preparation of the device of the present invention.

Relatively tough or firm ophthalmic devices can be obtained with type I collagen that is not denatured (i.e., loses all or a substantial portion of its triple helical conformation to become gelatin) during isolation and purification.

Differential Scanning Calorimetry (DSC) is a useful tool to determine the quality of a collagen solution from a supplier based on the content of the triple helix in the collagen (table 1). DSC enthalpy of denaturation (Δ H) for near-perfect triple helix content_{Denaturation of the material}) In the range of 65-70J/g (based on collagen dry weight). By DSC data,. DELTA.H_{Denaturation of the material}The results show that the solutions obtained from the commercial acidic lyophilized porcine collagen, and some of the commercial bovine collagen solutions, are in a complete triple helix morphology.

Collagen solution with low triple helix content (Δ H)_{Denaturation of the material}< 5J/g, supplier C, Table 1), has a relatively low viscosity and produces a poor gel compared to a composition or solution of collagen of the same concentration having a triple helix content close to 100%. It has been found that Δ H_{Denaturation of the material}A collagen composition (solution) of > about 60J/g to produce an acceptable ophthalmic device.

TABLE 1 denaturation enthalpy of collagen solution

Commercially available collagen samples	Composition of	ΔHDenaturation of the material(J/g of Dry collagen)
			Koken (Japan) supplier A	10% solution of cattle	65.3
Koken (Japan) supplier A	10% bovine collagen solution, concentrated from 3% acidic solution	67.5
			Koken (Japan) supplier A	5% bovine collagen solution, concentrated from 3.0% acidic solution	66.4
Koken (Japan) supplier A	3.5% neutral solution of cattle	68.1
			Koken (Japan) supplier A	3.5% bovine neutral solution after heat denaturation	24.4
Koken (Japan) supplier A	3.0% porcine collagen solution	72.0
			Koken (Japan) supplier A	5% solution of collagen (acidic) from lyophilized pig	68.1
Nippon Ham, supplier B	10% solution of collagen from lyophilized pig (which is very acidic)	63.4
			Becton Dickinson, supplier C	From a 0.3% concentrated solution of 5% bovine	59.4
Becton Dickinson, supplier C	Solution of 10% cattle	4.8
			FibroGen recombinant human collagen	10% solution, concentrated from 0.3 wt/wt%	67.7

In certain embodiments, including those described herein above, the solid material may comprise a cross-linked collagen polymer. Alternatively, or in other words, the material of the entity may comprise two or more cross-linked collagen polymers. For example, the solid material can comprise a first collagen polymer, a second collagen polymer, and a third collagen polymer. Other materials may comprise more than three collagen polymers. The crosslinked collagen polymer may be understood as the collagen component of the ophthalmic device.

Thus, the vision improving ophthalmic device according to the present invention may comprise a collagen component in an amount of about 1% (w/w) to about 50% (w/w) collagen and formed to have light intensity. As discussed herein, in certain embodiments, the amount of collagen is greater than 2.5%, such as at least about 5.0%. For example, in certain embodiments, the collagen is greater than about 6% (w/w). For example, the collagen is present in an amount of about 10% (w/w) to about 30% (w/w). For example, the collagen may be present in an amount of about 10% (w/w) to about 24% (w/w). In some devices, the collagen is the only water-swellable (e.g., hydrogel) polymer of the device. In other devices, the collagen may be the only device or lens-forming polymer. For example, the device may comprise 100% collagen in the dry state. As discussed herein, in certain devices, the collagen may be crosslinked, or at least partially crosslinked, for example using EDC/NHS.

The collagen polymers used in the preparation of the compositions and devices of the invention may be derived from the same collagen source, or from different collagen sources. Or, in other words, a single type of collagen, such as type I endless peptide collagen (which comprises multiple collagen polymer chains), is processed in a manner effective to allow the collagen polymers to crosslink with one another. In one embodiment, the collagen polymer is recombinant collagen. In other embodiments, the collagen polymers are all derived from the same animal source. The individual collagen polymers in a single composition may have different molecular weights.

It is understood that the device for correcting ametropia of the present invention comprises cross-linked recombinant collagen. The amount of collagen present in this device is greater than that found in other collagen-based devices previously disclosed for correcting ametropia. The device may be formed to have light intensity.

The devices disclosed herein are transparent. For example, the device should be optically clear. For example, the device should provide minimal light scattering (comparable or better than healthy human corneal tissue) when placed in an eye of an individual. In addition, the devices disclosed herein have a refractive index. In certain embodiments, the refractive index is from about 1.34 to about 1.37. For example, the refractive index may be in the range of 1.341 to 1.349. When the device is configured as a corneal onlay or a corneal onlay, the device is configured to be placed within a healthy eye of an individual; whereas for an eye with a damaged or diseased cornea, a full-thickness corneal implant may be required. In certain embodiments of the devices of the present invention, the devices are not yellowish or yellow in color. For example, the device may be designed to reduce or eliminate the yellowish or yellow color that may be associated with some collagen-containing compositions.

The device of the present invention has a front surface and a back surface. Thus, the solid or collagen component of the device may have an anterior surface and a posterior surface. The front and rear faces are generally opposed surfaces. The anterior surface of the device refers to the surface that faces away from the retina when the device is placed in the eye, and the posterior surface of the device refers to the surface that faces toward the retina when the device is placed in the eye. When the device is a corneal onlay, its posterior surface will be adjacent to, and may contact, the anterior layer, while its anterior surface will be adjacent to, and may contact, the corneal epithelium. When the device is a corneal implant, the anterior surface is adjacent to or facing the anterior border layer and the posterior surface is in the stroma and facing the retina of the eye. When the device is a full thickness corneal implant, the anterior surface will face the corneal epithelium and the posterior surface will be adjacent to and may contact the corneal endothelium.

The device of the invention may comprise no additional surface modification or the device may comprise surface modifications that affect cell growth and/or differentiation on the anterior surface or/and the posterior surface. For example, the corneal onlays may not comprise surface modifications that affect cell growth on the anterior or posterior surfaces. As used herein, "cell growth" refers to the expansion of a cell or group of cells. Thus, cell growth refers to the physical growth of an individual cell, such as an increase in surface area, volume, etc., the proliferation of one or more cells, such as cell division, and cell migration, sometimes forming a layered multilayer found on a healthy human cornea. Cell growth refers to the growth of nerve cells, such as one or more neuronal processes extending onto, under, or through the device, and the growth or migration or proliferation of epithelial or endothelial cells on the surface of the device. As used herein, "cell differentiation" refers to the morphological, biochemical and physiological changes undergone by an individual cell or population of totipotent, pluripotent or immature precursor cells (including stem cells) to obtain its final phenotype. In certain embodiments of the devices of the present invention, epithelial cells are grown on and intimately associated with (coupled) the corneal onlays, e.g., directly attached to the onlays, in particular the anterior surface of the onlays.

In certain corneal onlays, the entity or collagen component includes a modification to the posterior surface that is effective to reduce epithelial cell growth under the onlay when the onlay is placed in an eye of an individual. Additionally, or alternatively, the corneal onlay may comprise a solid or collagen component that includes a modification to the anterior surface effective to promote epithelial cell growth, including migration, on the anterior surface of the onlay when the onlay is placed in the eye of an individual. Relatedly, the full thickness corneal implant may include a solid or collagen component that includes a modification of the posterior surface that is effective to reduce endothelial cell growth on the posterior surface of the full thickness corneal implant when the implant is placed in the eye. A full-thickness corneal implant may not include a modification to the anterior surface.

Examples of surface modifications that can reduce cell growth include fluorinated monolayers providing plasma multimerization, such as CF on one or both of the anterior and posterior surfaces₄Or C₃F₈Providing low surface free energy on one or both of said surfaces, and/or by rendering one or both surfaces hydrophilic. The surface may be rendered hydrophilic by providing one or more surfaces with an alginate (alginate) coating.

The device may comprise one or more cell growth enhancing agents that promote cell growth on or through the device. In certain embodiments, the cell growth enhancer agent comprises a peptide. For example, the cell growth enhancer may be a peptide having an amino acid sequence comprising RGD, YIGSR, or IKVAV. Type I collagen itself is a rich source of the RGD sequence. In certain embodiments, the cell growth enhancer agent is a neurotrophic factor, or a biologically active or neurotrophic portion of the molecule. For example, the neurotrophic factor may be Nerve Growth Factor (NGF), epidermal growth factor (EGF or HB-EGF), or basic fibroblast growth factor (bFGF or FGF-2). The cell growth enhancer agent may be integrally formed with the collagen component or entity of the device, or in other words, the cell growth enhancer agent may be provided substantially throughout the device. In contrast, some ophthalmic devices include peptides provided on only one surface of the device.

In certain embodiments, a collagen-based ophthalmic device comprises a collagen component that is processed at an acidic pH in the preparation of the device. An acidic pH is particularly useful when the collagen component comprises a first collagen polymer cross-linked to a second collagen polymer. The acidic pH used in the preparation of the device is typically below about 6.0, for example, the pH may be from about 5.0 to about 5.5. By maintaining an acidic pH and preventing or reducing fluctuations in pH during pH adjustment, collagen fibrillation is reduced. In addition, by maintaining a pH above about 5.0, the collagen does not degrade as quickly as a pH below 5.0.

The collagen polymer may be crosslinked with any small or polymeric collagen-reactive agent or molecule. The crosslinking chemistry may use conventional methods that are conventional to those skilled in the art, or use novel reagents. By crosslinking the collagen polymers, the devices retain their optical clarity and are able to withstand biodegradation.

In certain embodiments, the collagen polymer is crosslinked by using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC; CAS #1892-57-5) and N-hydroxysuccinimide (NHS). In other words, the crosslinking reagent used in the preparation of the present device is EDC/NHS. The collagen polymer and the EDC/NHS crosslinker are mixed at an acidic pH while preventing fluctuations in pH. After sufficient mixing, portions of the mixed composition are placed in a mold and allowed to cure in the mold to form the ophthalmic device. One benefit of using water soluble EDC/NHS chemistry to crosslink collagen and CSCs is that it produces zero-length (amide) bonds. This reduces the possibility of the grafted toxic substance leaching into the tissue. In addition, unreacted reagents and by-products of the EDC/NHS reaction are water soluble and therefore can be easily removed after gel formation.

In certain embodiments, the collagen polymers are crosslinked by using a crosslinking agent or a crosslinking reagent that has reduced cytotoxicity. These cross-linking agents preferably do not cause irritation or produce a negative response when the ophthalmic device is placed in an eye of an individual. In some embodiments, the crosslinking agent is a crosslinking agent other than glutaraldehyde. While glutaraldehyde may be a useful crosslinking agent in certain embodiments, glutaraldehyde may not be preferred due to handling and safety requirements.

In further embodiments, the method may further comprise using at least one of the following components: poly (N-isopropylacrylamide-co-acrylic acid)), chondroitin sulfate, keratan sulfate, dermatan sulfate, elastin, chitosan, N, O-carboxymethyl chitosan, hyaluronic acid aldehyde (hyaluronic acid aldehyde), and alginate, which may be mixed with the collagen composition. Thus, the ophthalmic devices of the present invention can comprise a collagen component, such as a matrix of crosslinked collagen polymers, and one or more non-collagen polymers, including biopolymers. The non-collagen polymers may be crosslinked together and/or with collagen polymers to form a network or matrix of crosslinked polymers.

In certain embodiments, the compositions are mixed together through relatively narrow passageways or channels to induce high shear between the different compositions. In one embodiment, the composition is mixed with a syringe-based system. The mixing relies on a syringe to induce high shear between the viscous collagen solution and the agent by aspiration through a narrow channel. The diameter of the channel is chosen to accommodate the viscosity and burst force (burst strength) of the syringe or other similar device. For high viscosity (e.g., 20-30% (w/v) collagen solution), a small volume syringe with a small diameter syringe plunger is used because higher pressures can be achieved by hand. Mixing is carried out at an acidic pH, e.g., from about 5.0 to about 5.5, and mixing is carried out at a reduced temperature, e.g., from about 0 ℃ to about 5 ℃.

In contrast to other ophthalmic devices, the devices of the present invention are prepared without the use of living cells. Thus, the present inventors have invented a novel method for preparing compositions and ophthalmic devices using relatively high, near-physiological concentrations of collagen, without the use of viable corneal cells. In addition, the devices of the present invention are substantially or completely free of synthetic dendritic (dendrimer) components that have been used to enhance collagen cross-reactivity in other devices.

Other neuro-compatible (nerve-friendly) materials may be used in the manufacture of the devices of the invention. These materials can be made using the methods disclosed herein and tested for neural compatibility, such as neural growth, using conventional methods common to those of ordinary skill in the art, such as cell culture systems and the like. For example, the material can be tested and characterized using the method disclosed in WO 2004/015090 filed on 8/11/2003.

The devices disclosed herein are configured (e.g., sized and shaped) to be placed in an eye, near the corneal region of the eye. When the device is a corneal onlay, the diameter of the onlay is about 4mm to about 12mm, for example about 6 mm. The peripheral thickness of the insert may be less than about 30 μm, for example from about 10 μm to about 30 μm. The outer body may have a central thickness of about 70 μm.

The overmold, in the shape of an overmold, may be made from polypropylene and may have a diameter of 4mm, 6mm, 8mm, or 12 mm. The mold should be relatively stiff (e.g., not bend during closing) and transparent to allow visual filling. The mold is set to have a well tapered (taper) (e.g., about 10 μm) peripheral edge, or a more tapered (tip) (e.g., about 30 μm) peripheral edge.

The corneal implant mold (full or partial thickness) may have a diameter of about 12 mm. The implant mold is shaped to have the desired corneal curvature and thickness. If necessary, the ophthalmic device (e.g., hydrogel) may be trepanned out as needed for the implantation procedure.

An example of a device for correcting refractive error in accordance with the present invention is shown in figures 8 and 8A.

The corneal onlays disclosed herein may also be configured to correct one or more wavefront aberrations (aberrations) of an individual's eye. Us patent 6,086,204(Magnate) and WO 2004/028356 (almmann) provide descriptions of wavefront techniques and wavefront aberration measurements. The corneal onlay may be shaped to correct wavefront aberrations by shaping the mold to a configuration required to allow the onlay to achieve a corrective shape. Methods of using wavefront aberration measurements in corneal implants are disclosed in us application 60/573,657 filed 5/20, 2004. The inclusions may also be ablated to correct wavefront aberrations. For example, the outer body may be cut using a laser or laser-like device, a lathe, and other suitable lens forming devices.

The corneal onlays disclosed herein may also comprise a plurality of distinct zones. For example, the corneal onlay may comprise an optic zone and a peripheral zone. Generally, the optic zone is surrounded by a peripheral zone, or in other words, the optic zone is generally centrally disposed about the visual axis of the onlay (e.g., central visual axis), with the peripheral zone being located between the edge of the optic zone and the peripheral edge of the corneal onlay. The implant may provide additional zones and implant configurations depending on the particular visual deficit of the patient.

In addition, the corneal onlays of the present invention may have junctional zones (junctional zones), such as two or more zones that have no visually or optically detectable junctional zone. The zones of the implant may be smooth and continuous, and the implant may be optically optimized to correct not only ametropia, but also other optical aberrations of the eye and/or optical device independently or in combination with correcting ametropia. As understood by those skilled in the art, corneal onlays may be constructed to correct visual defects including, but not limited to, myopia, hyperopia, astigmatism, and presbyopia. The implant may enhance or ameliorate visual deficits by optical means (means) or physical means or a combination thereof applied to the ocular substrate. Thus, the corneal onlay may be a monofocal lens or a multifocal lens, including but not limited to bifocal lenses.

Additionally, or alternatively, the corneal onlay may be a toric lens (toric lenses). For example, the implant may include a toric (toric) zone effective to correct or reduce the effects of astigmatism when the implant is placed on an astigmatic eye. The enclosure may include a toric zone on a rear surface of the enclosure or the enclosure may include a toric zone on a front surface. Advantageously, toric onlays can be used without the need for ballast (ballast) to maintain proper positioning of the onlay on the eye, as the onlay can be held in a relatively fixed position by the epithelium of the appliance (appliance). However, ballast may be provided if desired. In certain embodiments, the outer object may comprise a ballast, such as a prism; or it may comprise one or more thin regions, such as one or more thin regions below and/or above. In an implant configured to correct presbyopia, the implant may include one or more designs, such as concentric, aspheric (with positive and/or negative spherical aberration), diffractive, and/or multi-zone refractive (multi-zone reactive).

The invention also encompasses compositions, such as synthetic or non-naturally occurring compositions. The composition may be wholly or partially synthetic. For example, the present invention relates to optically clear compositions. Such compositions are useful in the preparation of one or more ophthalmic devices as disclosed herein. Alternatively, the composition may be used in a non-ophthalmic device as a non-ophthalmic composition, or may be used in an ophthalmic device and does not provide correction of refractive error. In another embodiment, a composition according to the present disclosure comprises collagen in an amount greater than about 1% (w/w) in a hydrated state, and is optically clear. As discussed herein, the amount of collagen may be greater than 2.5%, such as at least about 5.0%. For example, the composition may comprise collagen in the hydrated state in an amount of from about 1% (w/w), or 2.5%, or about 5.0%, to about 30% (w/w). In certain embodiments, the composition may comprise about 6% (w/w) collagen. In other embodiments, the composition may comprise collagen in an amount of about 10% (w/w) to about 24% (w/w). The composition may comprise crosslinked collagen in a hydrated state in an amount greater than about 1% (w/w), wherein the collagen is crosslinked with EDC/NHS.

The compositions of the present invention may comprise two or more collagen polymers. In certain embodiments, the composition comprises a first collagen polymer cross-linked with a second collagen polymer, as discussed above. The composition may be substantially or completely free of cytotoxic agents, such as glutaraldehyde.

The ophthalmic devices disclosed herein can be placed in the eye using any suitable method or technique.

For example, corneal onlays may be placed on the anterior layer of the eye by removing or separating portions of the epithelium from the anterior layer. In some cases, an amount of an alcohol, such as ethanol, may be applied to the corneal epithelium to delaminate the epithelium from the eye (delaminanate). The concentration of the alcohol may be about 10% to about 60%, for example, about 20% or about 50%. Warming the ethanol to about 37 ℃ (e.g., body temperature) is effective in enhancing epithelial removal. This de-epithelialization technique is similar to the currently used LASEK technique.

In other cases, the corneal onlay may be placed on the anterior border layer by placing the onlay under an epithelial flap or in an epithelial pocket. Such flaps and pockets may be prepared with cutting instruments, blunt dissection tools, and the like. Examples of methods for placing corneal onlays in the eye are disclosed in U.S. application 10/661,400 filed on 12.9.2003 and U.S. application 60/573,657 filed on 20.5.2004.

The corneal implant may be placed in the eye by forming an intrastromal pocket or flap and placing the implant in the pocket or under the flap.

The full-thickness corneal implant can be placed in the eye by removing a damaged or diseased portion of the cornea and placing the corneal implant in or adjacent to the area of the removed portion of the cornea.

The ophthalmic devices disclosed herein may be placed in the eye with forceps, or any other suitable insert, such as those described in U.S. application 10/661,400 filed on 12/9 2003 and U.S. application 60/573,657 filed on 20/5 2004.

To facilitate placement of the ophthalmic device in the eye, the device may include a visualization (visualization) component. The visualization component may be any suitable feature that allows the device to be easily seen when inserted or placed in the eye. For example, the visualization component may include one or more markers (marking) that may aid in rotational positioning of the device, or the visualization component may include a dye, such as a biocompatible or non-cytotoxic dye, or a staining reagent.

Specific details relating to the ophthalmic devices of the present invention and related methods of making and using the devices are provided in the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

Examples

Example 1

Preparation of collagen-based corneal onlays.

Typically, 0.5mL to 2.0mL of the collagen solution in aqueous buffer is mixed with 0.01mL to 0.50mL of the crosslinker formulation in aqueous buffer at about 0 ℃ without bubble entrapment. In some compositions, a second biopolymer other than collagen is added to the composition.

To mix the composition, a syringe containing the composition was connected to ethylene-tetrafluoroethylene copolymer T-tubes (Tee-pieces) (upstight finishes) to form a micro-manifold (micro-manifold) that allowed for thorough mixing and/or controllable neutralization of the viscous collagen solution without pH fluctuation. pH fluctuations often lead to irreversible fibrillation of collagen, resulting in an opaque matrix.

More specifically, the first Luer fitting is used to retain a septum that is cut to size to closely fit the bottom of a needle hole of a T-shaped tube. "Ice Blue" 17mm custom 22397 membranes from Restek corporation were cut to size. A first syringe containing a buffer, such as MES (2- [ N-morpholino ] ethanesulfonic acid) buffer, is immobilized (lock) into the second Luer adapter and any air bubbles are forced out with the buffer. The collagen solution was placed in a second syringe and then connected to a third Luer fitting of an ethylene tetrafluoroethylene copolymer T-tubing (shown in figure 1) equipped with three Luer fittings (figure 2). The complete assembly is shown in fig. 3.

The collagen solution was thoroughly mixed with MES buffer solution by repeated aspiration through the T-tube between the first and second syringes, thereby vigorously shearing the liquid via flow in the narrow inner diameter channel of the T-tube (e.g., about 0.5mm to about 0.25 mm). The pH is adjusted to 5.0-5.5. The collagen/buffer mixture was then mixed with EDC and NHS solutions (EDC: NHS in a 1: 1 molar equivalent ratio) at 0 deg.C-4 deg.C by directing the composition through a manifold using another syringe.

Dispensing aliquots of each substantially homogeneous solution immediately into a housing mold and first curing at room temperature for 5-24 hours, such as 15 hours; then at 37 deg.C for 15-24 hours, and at both temperatures in a 100% humidity environment.

After 2 hours immersion in Phosphate Buffered Saline (PBS), each final insert sample was carefully separated from its mold.

In some instances, these gels are immersed in an aqueous solution of a second reactive biopolymer to further crosslink and add new biological agents.

Finally, the crosslinked hydrogel of the implant was immersed in PBS solution (0.5% in PBS, 1% chloroform) at 20 ℃ to terminate any reactive residues and extract reaction by-products. These sterile, equilibrated hydrated explants were rinsed thoroughly with PBS prior to all testing.

For gels prepared with some collagen/EDC-NHS chemistries, which have higher collagen concentrations (10% and above), the gel is first soaked in buffer pH 9.1 to stop any residual reactivity and the reaction product is extracted extensively before storage in chloroform-saturated PBS. This alkaline extraction eliminates the epithelial toxicity problem of these samples. For many stoichiometries, soaking in chloroform-saturated PBS followed by removal of the chloroform residue can result in sterile, non-cytotoxic gels.

Example 2

Ophthalmic device with cell growth enhancing agent

Cell growth enhancers, such as pentapeptides (YIGSR, the active unit in the laminin macromolecule) themselves, or in combination with synergistic peptides (synergicpeptides), such as synergistic peptides including IKVAV, synergistic IGFs, and substance P peptides that promote epithelial health, EGF, NGF, FGF, or portions of these molecules, may be incorporated into any collagen/EDC-NHS cross-linked device, including those with a second EDC-NHS reactive biopolymer. For YIGSR, the attachment of the cell growth enhancer may be achieved by the reactivity of the free amine end group of the tyrosine residue on the reagent. Extensive extraction after gelation can be used to remove any unbound cell growth enhancer.

Details of the specific formation of ophthalmic devices are provided in examples 3-13 below, as well as in table 2.

Example 3

Ophthalmic devices were prepared as described in example 1: 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) + collagen were heated to 21 ℃ for 15 hours and then to 37 ℃ for 15 hours in MES buffer at 0 ℃ to 4 ℃ and pH 5.5. EDC: NHS is 1: 1 molar equivalent ratio.

Example 4

Ophthalmic devices were prepared as described in example 1: at 0 deg.C-4 deg.C, pH 5.5 in MES buffer, using COP + EDC-NHS + collagen, warm to 21 deg.C for 15 hours, then at 37 deg.C for 15 hours. EDC: NHS is 1: 1 molar equivalent ratio. [ COP, copolymer, poly (N-isopropylacrylamide-co-acrylic acid) was prepared by free radical polymerization of NiPAAm and AAc in 1, 4-dioxane in liquid nitrogen at-70 ℃ with 2, 2 '-azobis-isobutyronitrile (2, 2' -azo bis-isobutronitril) as the initiator (initiator) ].

Example 5

Ophthalmic devices were prepared as described in example 1: EDC-NHS + chondroitin sulfate C (ChS) + collagen was warmed to 21 ℃ for 15 hours and then 37 ℃ for 15 hours in MES buffer at 0 ℃ to 4 ℃ and pH 5.5. EDC: NHS is 1: 1 molar equivalent ratio.

Example 6

Ophthalmic devices were prepared as described in example 1: collagen + EDC-NHS + N, O-carboxymethyl chitin (CMC) was used in MES buffer at 0 deg.C-4 deg.C, pH 5.5, and warmed to 21 deg.C for 15 hours, then 37 deg.C for 15 hours. EDC: NHS is 1: 1 molar equivalent ratio.

Example 7

Ophthalmic devices were prepared as described in example 1: the temperature was raised to 21 ℃ for 2 hours at 0 ℃ to 4 ℃ in MES buffer at pH 5.5 using collagen + EDC-NHS + N, O-carboxymethyl chitosan (CMC), and then a second cross-linking agent was added when the gel was immersed in chitosan in PBS (1% aqueous solution, 5000Da) for 4 hours. Finally, the temperature was raised to 37 ℃ for 15 hours. EDC: NHS is 1: 1 molar equivalent ratio.

Example 8

Ophthalmic devices were prepared as described in example 1: the temperature was raised to 21 ℃ for 15 hours and then 37 ℃ for 15 hours at 0 ℃ to 4 ℃ in MES buffer using collagen + EDC-NHS + Hyaluronic Acid (HA) at pH 5.5. EDC: NHS is 1: 1 molar equivalent ratio.

Example 9

Ophthalmic devices were prepared as described in example 1: the temperature was raised to 21 ℃ for 15 hours and then to 37 ℃ for 15 hours in MES buffer at 0 ℃ to 4 ℃ with collagen + EDC-NHS + chondroitin sulfate (ChS) + Hyaluronic Acid (HA). EDC: NHS is 1: 1 molar equivalent ratio.

Example 10

Ophthalmic devices were prepared as described in example 1: heating collagen + hyaluronic acid aldehyde (HA-CHO) + sodium cyanoborohydride in PBS at pH 7-8 at 0-4 deg.C to 21 deg.C for 15 hr, and then 37 deg.C for 15 hr. HA-CHO was prepared from HA (0.1g) by oxidative cleavage with sodium periodate (0.05g) at 21 ℃ for 2 hours. The aqueous solution was dialyzed against water for 2 days.

Example 11

Ophthalmic devices were prepared as described in example 1: the temperature was raised to 21 ℃ for 15 hours and then 37 ℃ for 15 hours in MES buffer at 0 ℃ to 4 ℃ with collagen + EDC-NHS + alginate at pH 5.5. EDC: NHS is 1: 1 molar equivalent ratio.

Example 12

Ophthalmic devices were prepared as described in example 1: collagen was heated to 21 ℃ for 2 hours at 0 ℃ to 4 ℃ in MES buffer using glutaraldehyde ("Glut", diluted to 1% in water) + collagen, and then + a second crosslinker when the gel was immersed in chitosan in PBS (1% aqueous, 5000Da) for 4 hours at pH 5.5. The gel in the mold was warmed to 37 ℃ for 15 hours and then removed in PBS.

Example 13

Ophthalmic devices were prepared as described in example 1: the temperature was raised to 21 ℃ for 15 hours and then 37 ℃ for 15 hours in MES buffer at pH 5.5 with collagen + EDC-NHS + chitosan at 0 ℃ to 4 ℃. EDC: NHS is 1: 1 molar equivalent ratio.

Example 14

Ophthalmic devices were prepared as described in example 1: at pH 7-8 in PBS buffer, at 0 deg.C-4 deg.C, the temperature is raised to 21 deg.C for 15 hours with EDC-NHS + chondroitin sulfate C (ChS) + collagen, and then 37 deg.C for 15 hours. EDC: NHS is 1: 1 molar equivalent ratio.

All of the devices of examples 3-14 produced strong, clear and elastic gels using all of the commercially available collagen and reactant ratios shown in table 2 below.

DSC for some hydrogels for implant applicationsOptical clarity and refractive index, measurements, tensile properties (stiffness), maximum tensile strength, elongation at break (elongation), table 2) and in vivo performance. After the reactions of all examples, from the results of DSC measurement of the gel, an increase in denaturation temperature and Δ H were found_{Denaturation of the material}This is consistent with cross-linking of collagen. The refractive index of all formulations in table 2 ranged from 1.341 to 1.349.

Example 15

External storage performance of body (watch 2)

Pnas 100: 15346-15351(2003) evaluates how epithelial cells (human immortalized corneal epithelial cells, HCEC) grow to confluence on hydrogels (days to confluence), to evaluate how HCEC cells stratify on hydrogels (stritify), and to evaluate the growth of dorsal root ganglion nerves of chickens on and into hydrogels (growth into hydrogels is reported in microns/day growth when data is available).

Human corneas will re-establish their epithelium within 3-5 days after complete removal.

In vitro experiments often last about 6-8 days, but better formulations may allow re-epithelialization to confluence in 3-5 days or less. For the thicker gels (> 5% collagen), extensive above (over) nerve growth (300 micron extension) was found in vitro experiments for many formulations. When the gel hardness increased, the nerve ingrowth slowed rapidly, but was still observed by deep-depth microscopy.

TABLE 2 hydrogels⁺Composition and Properties of

Examples	Collagen supplier (Table 1) (initial concentration wt/vol%)	collagen/XL equivalence ratio or (wt/wt)	Final collagen concentration in gel (w/v%)	Maximum stress, gram force*	Strain at break (strain) mm*	Hardness g/mm*	Days to confluency of epithelial cells in vitro	Nerve growth in vitro within 6 days
									2	B, AFDP: (dissolved at 10%)	Col-NH2∶EDC＝5∶1Col∶YIGSR＝5∶0.0001	7.2				3-5	Over: fast In: 27 μm/d
3	A, (10% of cattle)	Col-NH2∶EDC＝5∶1	7.3	8.0	2.6	4.0
									3	B, AFDP: (dissolved at 10%)	Col-NH2∶EDC＝5∶1	7.3	9.7	4.0	4.4	2-3	Over: fast In: 40 μm/d
3	B, AFDP: (at 15% dissolution)	Col-NH2∶EDC＝5∶1	108	13.08	4.6	3.0	2-3
									3	B, AFDP: gel (dissolved at 20%) 350 μm thickness	Col-NH2∶EDC＝10∶1	143	11.95	4.3	3.0	2-3
3	B, AFDP: (at 32% dissolution)	Col-NH2∶EDC＝1∶1	18.0	14			2-3	Over: fast In: 30 μm/d
									3	A, (3.5% neutral bovine)	Col-NH2∶EDC＝1∶1	2.7	3.1	2.0	1.7	3-5	Over: fast-acting toy
5	A, (3.5% neutral bovine)	Col-NH2∶EDC＝2∶1Col∶ChS＝(9∶1)	2.7	2.5	1.8	1.4	3	Over: fast In: 41 μm/d
									5	A, (35% neutral bovine)	Col-NH2∶EDC＝2∶1Col∶ChS＝(4∶1)	2.7	2.7	1.8	1.5	3	Over: fast In: 73 μm/d
5	A, (35% neutral bovine)	Col-NH2∶EDC＝2∶1Col∶ChS＝(3∶1)	27	3.1	1.5	1.5	3	OverIn：＝70μm/d
									6	A, (3.5% neutral bovine)	Col-NH2∶EDC＝1∶1Col∶CMC＝(1∶0.5)		3.6	1.6	2.0
6	B, (AFDP: 32% dissolution)	Col-NH2∶EDC＝1∶13Col∶CMC＝(15∶1)	14.5	6.0			3
									7	A, (3.5% neutral bovine)	Col-NH2EDC 1: 1Col CMC 2: 1 + soluble chitosan		2.9	1.6	1.9
8	A, (3.5% neutral bovine)	Col-NH2∶EDC＝2∶1Col∶HA＝(9∶1)	2.2	2.5	2.2	1.08	3-5
									8	A, (5% neutral bovine)	Col-NH2∶EDC＝2∶1Col∶HA＝(4∶1)	2.2	2.4	2.2	2.07	3-5
8	A, (10% neutral)	Col-NH2∶EDC＝2∶1	2.2	2.0	1.8	1.13	3-5

	Of cattle)	Col∶HA＝(3∶1)
									9	A, (3.5% neutral bovine)	Col-NH2∶EDC＝0.5∶1.0Col∶HA∶ChS＝9∶1∶1	2.3	3.0	1.7	1.7	3-5	Overgrowth and ingrowth
10	A gel of 350 μm thickness (3.5% neutral bovine)	Col-NH2∶HA-CHO＝1∶1	3.2	1.0		0.7
									11	A, (3.5% neutral bovine)	Col-NH2∶EDC＝2∶1Col∶Alg＝4∶1	2.7	3.0	2.0	1.6	3-5	Over: fast In: 41 μm/d
11	A, (3.5% neutral bovine)	Col-NH2∶EDC＝2∶1Col∶Alg＝2∶1	2.7	3.4	2.5	1.5	3-5	Over: fast In: 13 μm/d
									12	A, (35% neutral bovine)	Col: Glut? Is there a + soluble chitosan		3.1	2.1	1.5
13	B, (11% AFDP), 900 μm thick gel	Col-NH2EDC 033: 1.0 Col: chitosan (15: 1)	5.8	8.32	3.29	2.57	4
									13	B, (11% AFDP), gel 500 μm thick	Col-NH2EDC 0.66: 1.0Col chitosan 15: 1	5.8	4.25	4.02	1.32
13	B (11% AFDP)900 μm thick gel	Col-NH2EDC 066: 1.0Col chitosan (15: 1)	5.8	8.46	5.23	2.17

⁺Abbreviations: col collagen protein; glut glutaraldehyde; HA hyaluronic acid; ChS chondroitin sulfate C; Col-NH₂Free amine components of collagen; AFDP acidic lyophilized porcine; epithelial; ND is not determined.

^*A 500 μm thick, 12mm diameter implant, unless otherwise specified. Stress, strain (strain) and hardness values are derived from the following sources such as Li et al pnas 100: 15346-15351 (2003).

^**over: neurites from dorsal root ganglia overgrow on hydrogels. In: neurite outgrowth into the hydrogel reached the indicated length within 6 days.

Example 16

In vivo performance of the implant

The external preparation was prepared as described in example 1. The first set of (set) explants was prepared from 10% (w/v) porcine collagen with EDC/NHS. A second set of explants was prepared from 3.5% (w/v) bovine collagen using Chondroitin Sulfate (CSC) and EDC/NHS. The outer diameter of the insert was about 6mm, the center thickness was about 70 μm, and the beveled edge (sloped edge) was 30 μm.

To implant the implant, the porcine epithelium was treated with 45% ethanol for 30-45 seconds. A butterfly incision is made and a pocket is formed in the epithelium. With blue non-cytotoxic dyes (Gel-Code)^TM) Staining the outsources for visualization. Inserting the pre-dyed external object into the pocket. The protective contact lens is sutured to the eye.

Visual inspection was performed to assess inflammation, redness, and/or vascular invasion of the cornea. Corneal clarity was assessed by slit lamp examination. Intraocular pressure was measured with an intraocular pressure measuring pen (tonopenn). The contact sensitivity of the cornea was measured using a Cochet-Bonnet aesthesiometer. Contact sensitivity may be useful for assessing the presence of functional nerves that can be confirmed with in vivo confocal imaging and immunohistochemistry of harvested cornea with implant. Corneal topography was examined immediately prior to implantation, and three weeks post-operatively using the PAR Corneal Topography System (CTS).

Corneal topography was performed by placing the anesthetized pig eyes in line with the CTS. A diluted solution of fluorescein and artificial tears is applied to the eye to coat the corneal surface so that the target grid can be seen. The focal plane of the instrument is adjusted to bring the target grating into focus on the anterior corneal surface. The digital image of the grating is captured. The digital image is analyzed to provide a measure of the shape of the anterior corneal surface. The digital images of the implant before and after implantation are compared for evaluation of corneal shape changes due to the implant.

In vivo confocal microscopy can capture images of different depths of the cornea in live pigs, and thus can monitor the eye's response to the ophthalmic device. For example, confocal microscopy can be used to monitor the presence of nerves in the device. In vivo confocal microscopy was performed by examining anesthetized pigs with a Nidek Confoscan 3 in vivo confocal microscope before implantation of the ophthalmic device and 3 weeks post-surgery. An artificial tear was dropped on the eye to be examined. Two drops of local anesthetic are applied to the eye to reduce eye movement. Confocal lenses (immersion gels) were brought into contact with the cornea, with a layer of gel on the anterior lens surface, to match refractive indices. The focal plane of the instrument is adjusted to focus the corneal endothelium and then an image of the cornea is taken as the focal plane of the lens is scanned through a depth equal to the thickness of the cornea.

In addition to histopathological examination of hematoxylin-eosin (H & E) stained tissue sections, immunohistochemistry was used to determine whether corneal epithelium was restored in the implants, and early indications of corneal epithelium adhesion and interaction with underlying implants. Immunohistochemistry is also used to determine the presence or absence of nerves and any infiltration of immune and inflammatory cells. Anti-neurofilament staining was performed by conventional techniques on the half of the cornea with and without implanted implant, respectively, after detergent penetration. Bound antibodies were visualized using immunofluorescence.

The cornea that received the corneal onlays as described above healed well and remained optically clear with minimal or no redness or inflammation. There was no evidence of vascular infiltration. Normal intraocular pressure was observed. The cornea after surgery showed contact sensitivity. Topographic measurements show that the implanted implant can alter the corneal topography. The implant causes a change in corneal thickness of about 50 μm at the central corneal height (elevation). The epithelium adheres well to the external agents. In vivo corneal microscopy showed good general corneal structure with both the superior subcutaneous and stromal (stromal) nerves, and cells from the epithelium to the endothelium. H & E stained cryosections showed the incorporation of the exosomes into the host cornea. Immunohistochemistry using staining for E-cadherin showed little, if any, change in cell adhesion in the cornea implanted with the implant compared to the untreated cornea. Staining for keratin-3 and E-cadherin was compared to controls. Collagen VII staining against anchored fibers with basement membrane complex (anchoring fiber) showed less pronounced staining compared to control. Staining for α 6 integrin showed localization in the basal epithelial cells of both the operated and untreated controls. Staining with anti-neurofilament 200 antibody showed the presence of nerves in the implantation site of the cornea with the implant. Staining with anti-CD 45 antibody showed no inflammatory or immune response.

Example 17

Corneal onlay ablation

collagen/EDC and collagen/chitosan explants were cut with a VISX Star S4 excimer laser (table 3). Surface topography measurements of the implant are obtained before and after treatment with the PAR Corneal Topography System (CTS). For treatment, the outer body is removed from the stock solution and placed on a spherical surface made of PMMA.

Phototherapeutic keratectomy (PTK) procedure delivers a uniform number of laser pulses (or energy) throughout the ablation area. Photorefractive keratectomy (PRK) surgery varies the pulse density over the ablation area to achieve the desired change in curvature. AZD refers to the cutting zone diameter. Depth is the expected treatment depth on a human cornea as reported by the laser manufacturer.

Table 3: cutting parameters

Surgery	Type (B)	AZD(mm)	Ball (D)	Cyl(D)	Depth (mum)
						1234567	PTKPTKPRKPRKPRKPRKPRK	5566666	--------2+2-4+4-4	--------0000+2	10202619513830

The preoperative and postoperative topography of collagen/EDC external placement was used to generate a differential map (differencemap) to show the effect of ablation.

The PTK cut is expected to produce a fairly constant central blue region (for the difference plot) of diameter 5 mm. A small gradient in the amount of tissue removed is expected because the implant is a curved surface. The spherical (sphere) PRK cut for myopia is expected to remove the greatest depth of tissue from the center. The depth of tissue removed is expected to gradually drop to 0 at the edge of the cut. The correction of the hyperopic sphere is not intended to contact the 1mm diameter of the center and to remove tissue to the greatest extent at the edges of the treatment zone, and is also intended to create a transition zone extending 9mm from the center to the periphery. The disparity map after hyperopia correction is expected to show a blue ring around the central green zone. The disparity map from myopic astigmatic correction is expected to show similarities to the map for myopic spherical correction, except that the blue image of the former is expected to be elliptical.

The difference map from the excised artifact shows the expected tissue removal image in all of the difference maps described above. The maximum depth of tissue removed indicates that it is deeper than the expected depth of a human cornea. For example, the corneal onlay material removal rate is about 1.7 to about 2 times the corneal removal rate. The difference in ablation rates between the implant material and the cornea is not consistent from sample to sample. This difference in velocity may be due to measurements of treatment depth, material density and surface roughness, water content of the material, and others.

It was observed that the collagen/chitosan inclusion cut faster than the collagen/EDC inclusion. This difference may be due to hydration problems. For example, the post-operative collagen/chitosan implant may have a lower water content than the collagen/EDC implant.

The ophthalmic devices may also include a strength-enhancing component, such as a urethane.

Example 18

Human recombinant collagen ophthalmic devices

The crosslinked collagen hydrogel was prepared by mixing 0.3ml of 13.7 wt% human recombinant type I collagen from fibrigen (San Francisco, CA) and 0.3ml of 0.625M morpholinoethanesulfonic acid (MES) using a syringe-based system as described above. Mixing was performed at reduced temperature by mixing in an ice-water bath.

After obtaining a homogeneous solution, 57 μ l of EDC/NHS was added to react with collagen free amine groups (Coll-NH)₂) The molar equivalent ratio was 3: 1. To adjust the pH of the solution to about 5, NaOH (2N) was added to the mixture.

The mixture is poured into a glass or plastic mold and left at room temperature and 100% humidity for 16 hours. The mold was then transferred to a 37 ℃ incubator for 5 hours for post-curing (post-curing).

Other hydrogels with type I human recombinant collagen were also prepared using this method, having EDC/NHS to collagen Coll-NH2 group ratios of 1: 1 and 6: 1.

The Refractive Index (RI) was measured on a VEE GEE refractometer. Light transmission was measured at white light wavelength, 450nm, 500nm, 550nm, 600nm and 650 nm. Direct tensile property measurements such as stress, strain at break (break strain) and modulus (modulius) were measured on an Instron electromechanical tester (model 3340). The size of the sample was 5mm by 0.5 mm. The water content of the hydrogel was calculated according to the following formula:

(W-W₀)/W％，

wherein W₀And W represent the dry and swollen sample weights, respectively.

EDC/NHS/Coll-_NH2The yield of the human recombinant collagen hydrogel (designated as F1) at a ratio of 1/1/1 (molar equivalents) was 1.3457 ± 0.0013. EDC/NHS/Coll-_NH2The yield of the human recombinant collagen hydrogel (designated as F3) at a ratio of 3/3/1 (molar equivalents) was 1.3451 ± 0.0002. EDC/NHS/Coll-_NH2The refractive index of the human recombinant collagen hydrogel (designated as F6) with a ratio of 6/6/1 (molar equivalents) was 1.3465. + -. 0.0001。

Table 4 summarizes the light transmission of the different hydrogels.

TABLE 4 light transmission

Wavelength (nm)	White light	450	500	550	600	650
							Average transmission (%)
F1F3F6	86.7±0.990.7±2.575.5±1.5	69.7±1.285.8±3.548.7±0.4	76.0±1.386.4±2.957.7±1.1	79.2±1.486.7±2.662.7±1.1	82.4±1.388.0±2.467.6±1.4	84.9±1.489.6±2.671.6±1.7

The hydrogel material designated F3 showed the most acceptable optical properties. Visually or macroscopically, F3 showed the highest transparency compared to other human reconstituted hydrogels.

Table 5 provides the mechanical properties of the human recombinant hydrogels of the present invention.

TABLE 5 mechanical Properties

Sample(s)	F1	F3	F6
				Average maximum stress (KPa) average breaking strain (%) average modulus (MPa)	62.6±9.967.1±21.062.60±6.820.281±0.032	117.2±36.9110.5±49.750.20±7.550.525±0.124	149.9±57.799.5±60.823.51±9.031.949±0.939

Hydrogel F3 was shown to have a relatively low modulus (modulius), but acceptable other mechanical properties.

Table 6 provides the water content values of the hydrogel materials

TABLE 6 equilibrium Water content

Sample(s)	F1	F3	F6
				Water content (%)	92.82±0.68	92.63±0.61	91.40±0.38

It is clear that the hydrogel is highly hydrated.

In this example, a pH indicator was added to MES buffer to aid in monitoring pH changes. A specific indicator used in this example was Alizarin Red S (Sigma Aldrich).

Fig. 4 is a graph of growth of human corneal epithelial cells on the human recombinant collagen sample F1 over the course of 7 days. Fig. 5 is a graph of growth of human corneal epithelial cells on the human recombinant collagen sample F3 over the course of 7 days. Fig. 6 is a graph of growth of human corneal epithelial cells on the human recombinant collagen sample F6 over the course of 7 days.

More cell growth was observed on the recombinant hydrogel material than was observed in the control experiment.

Fig. 7 is a photograph showing that the hydrogel material F3 remained in vivo for at least 30 days.

Example 19

Collagen-poly (NIPAAm-co-AAC) compositions

Compositions (example 4) were prepared using the EDC/NHS crosslinking procedure described herein. The initial collagen concentration was 15%. The final collagen concentration was 11%. The final poly (NIPAAm-co-Aac) concentration was 3%. The total solids concentration in the gel was 14%.

The material has a yield of 1.3542, a tensile strength of 11 g-force, an elongation of 3.3mm, and a modulus of 3.8 g-force/mm, as measured by a method such as Li et al, PNAS 100: 15346-15351 (2003).

The denaturation temperature is increased from 40 ℃ before crosslinking to 50 ℃ after crosslinking. The material has higher light transmission and lower back scattering than either human or rabbit corneas (back scatter. e.g., for white light, the percent transmission of the hydrogel material is about 102%, for human corneas about 93%, and for rabbit corneas 78%. the percent transmission of the hydrogel is about 90%, 96%, 100%, 101%, and 103% for light at wavelengths of 450nm, 500nm, 550nm, 600nm, and 650nm, respectively, the human and rabbit corneas exhibit a percent transmission of less than 100% at all wavelengths tested and consistently lower percent transmission at each wavelength than the hydrogel material.

The hydrogel material showed confluency of corneal epithelial cells 7 days after seeding.

Example 20

Collagen/chondroitin sulfate composition

A biosynthetic matrix with high optical clarity and tensile strength was developed with collagen I, with Chondroitin Sulfate (CSC) as the proteoglycan equivalent. Hydrogels containing CSCs up to 30% (wt/wt) of the collagen dry weight were prepared under controlled conditions, but without aggregation or collagen fibril formation that would result in a loss of optical clarity. The hydrogels are characterized by physics and biochemistry. In vitro experiments showed that Human Corneal Epithelial Cells (HCECs) grew well on the gel surface and layered successfully. The stroma supports good nerve ingrowth. Similar results were obtained in vivo.

The composition was prepared analogously to example 14 as described above. CSCs were covalently bound to collagen using EDC and NHS chemistry.

As disclosed herein, by using EDC/NHS (1: 1 molar equivalents) crosslinking technology, different dry weight ratios of CSC to collagen and different EDC to collagen-NH were prepared₂Collagen (3.5 w/v%) and CSC gel at molar equivalent ratio. All gels were visually clear. As shown in table 7, the composition has higher light transmission and low light scattering (about 87% transmission and 3% backscattering) compared to human cornea.

TABLE 7 Transmission and light Scattering

Weight ratio of CSC to collagen (%)	0	5	10	20	30
						Transmission (%)	89.9	95.5	93.0	90.5	97.3
Back scattering (%)	0.30	0.19	0.19	0.17	0.19
						EDC and NH2Ratio of (A) to (B)	0.25	0.5	1.0	2.0
Transmission (%)	96.7	100	99.9	88.2
						Back scattering (%)	0.24	0.28	0.16	0.20

The refractive index is 1.34-1.35, which is similar to the refractive index of human cornea (1.376).

The swelling ratio of gels with different EDCs and collagen-NH was measured and calculated using the following formula₂Preparing the following components in molar ratio:

expansion ratio of (W)_w-W_d)/W_d

Wherein W_wIs the weight of the hydrated gel, and W_dIs the weight of the xerogel.

Crosslinking of collagen-CSCs with EDC/NHS will form crosslinks between carboxylic acid and amine groups. The results (FIG. 9) suggest that EDC and collagen-NH were increased₂The ratio of (a) will reduce the swelling ratio of the collagen-CSC gel because it introduces a more dense network structure.

Pnas 100: 15346-15351(2003) measures the mechanical properties of the implant. The implant was fully hydrated in PBS and pulled out at a rate of 10 mm/min. The tensile strength was monitored at the time of breakage of the implant (thickness 500 μm and diameter 12 mm). By increasing EDC and NH₂The molar ratio of (a) to (b) enhances the tensile strength of the gel (fig. 10). However, the material becomes brittle when the amount of EDC is increased significantly, so that EDC becomes brittle when it is mixed with collagen-NH₂At a molar ratio of 2 or more, the tensile strength is slightly decreased.

The tensile strength of the gel was also enhanced by increasing the concentration of collagen, as shown in figure 11 (compare using 10% collagen instead of 3.5%). The tensile strength increased from 2.65 to 10.02 gram-force and the expansion ratio decreased from 21.5 to 12.1.

The crosslinking efficiency was evaluated by Differential Scanning Calorimetry (DSC). Heating the collagen or crosslinked collagen hydrogel induces a structural transformation of the native triple helical structure at a temperature that depends on the nature and extent of crosslinking. Complete cross-linking of collagen solution in sealing discs (commercial sealed pan)The hydrated collagen hydrogel was characterized and the temperature of the sample was increased at a constant rate of 2 ℃/min. The temperature at the highest peak was recorded as the denaturation temperature. When EDC and NH₂When the ratio of (a) was increased, the denaturation temperature was increased from 42.4 ℃ to 56.6 ℃ (fig. 12A), suggesting that the introduction of covalent cross-linking would increase the stability of the triple helix, thereby increasing the denaturation temperature. The denaturation temperature of the collagen-CSC gel was higher than that of the collagen-only gel (fig. 12B). However, changing the molar ratio of CSC to collagen in the collagen-CSC gel did not affect the denaturation temperature.

Human corneal epithelial cells from established cell lines were observed for in vitro growth. In vitro nerve growth was performed using dorsal root ganglia implanted in a gel. Neurites were grown for 7 days, gels were stained for neurofilaments, and neurite extension was measured. Neurites grew well in all collagen-CSC gels (fig. 13).

Increasing the CSC concentration from 5% to 20% significantly increased the length of neurite extension within the gel. Other benefits were not apparent in the 30% CSC containing gel (fig. 13). Excellent epithelial coverage and integration of the implant was observed.

Example 21

Collagen type III composition

Materials: human recombinant type III collagen (5.1% w/w fibrigen Inc), 0.625M morpholinoethanesulfonic acid [ MES, Aalizarin Red S containing pH indicator (6.5mg/100ml water) ], 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hcl (edc), N-hydroxy-succinimide (NHS).

The hydrogel was prepared with 18.3% (w/w) collagen type III solution. In two bubble-free syringes connected by a plastic T-tube, 0.3ml of 18.2 wt% human recombinant type III collagen (concentrated from 5.1% w/w human recombinant type III collagen (FibroGen Inc)) and 0.3ml of MES (0.625M) were mixed under ice-water bath conditions. After forming a homogeneous solution, 33.5mg EDC and 20.1mg NHS were dissolved in 0.125ml MES, 57. mu.l of which was taken and mixed with EDC: NHS: collagen-NH₂The molar ratio was 3: 1 and injected into the syringe. No NaOH solution was added as the mixture was pink indicating a pH of about 5. The mixture was thoroughly mixed and poured into a glass mold (thickness 434 μm) and left at room temperature and 100% humidity for 16 hours. The mold was then transferred into an incubator and post-cured at 37 ℃ for 5 hours. The resulting flat hydrogel was removed and soaked in 10mM PBS, with fresh buffer changed every 8 hours. The obtained hydrogel was immersed in 10mM PBS containing 1% chloroform and stored in a refrigerator at 4 ℃.

EDC, NHS, collagen-NH was also prepared by the above method₂Other collagen type III hydrogels in the ratio of 2: 1 and 1: 1. All the gels obtained were transparent.

A5.1% (w/w) collagen type III solution was also used to prepare the hydrogel. 0.3ml of 5.1 wt% human recombinant type III collagen and 50. mu.l MES (0.625M) were mixed in two bubble-free syringes connected by a plastic T-tube under ice-water bath conditions. After forming a homogeneous solution, 9.3mg of EDC and 5.6mg of NHS were dissolved in 0.125ml of MES, 57. mu.l of which were taken and added as EDC: NHS: collagen-NH₂The molar ratio was 3: 1 and injected into the syringe. No NaOH solution was added, as the mixture was pink, indicating a pH of about 5. The mixture was thoroughly mixed and poured into a glass mold (thickness 434 μm) and left at room temperature and 100% humidity for 16 hours. The mold was then transferred into an incubator and post-cured at 37 ℃ for 5 hours. The resulting flat hydrogel was removed and soaked in 10mM PBS with fresh buffer changes every 8 hours. Finally, the obtained hydrogel was immersed in 10mM PBS containing 1% chloroform and stored in a refrigerator at 4 ℃. The resulting gel was optically clear.

For an initial collagen concentration of 18.3% w/w, the final collagen content was 8.36% (w/v) (calculated based on the dilution factor after addition of each component) or about 10% (w/v) (measured).

For a collagen starting concentration of 5.1% w/w, the final collagen content was 3.76% (w/v) (calculated based on the dilution factor after addition of each component), or approximately 4% (w/v) (measured).

While the invention has been described with respect to various specific examples and embodiments, it will be understood that the invention is not limited thereto and that other embodiments are also within the scope of the invention.

Some documents, patents and patent applications have been cited above. Each of the cited documents, patents, and patent applications is incorporated by reference in its entirety.

Claims (29)

1. An optically clear biosynthetic composition comprising cross-linked collagen, wherein the composition comprises an amount of collagen from about 5% to about 50% by weight or volume.

2. The composition of claim 1, wherein the amount of collagen is at least 6% by weight or volume.

3. The composition of claim 2, wherein the amount of collagen is at least 10% by weight or volume.

4. The composition of claim 3, wherein the amount of collagen is from about 10% to about 30% by weight or volume.

5. The composition of claim 4, wherein the amount of collagen is from about 10% to about 24% by weight or volume.

6. The composition of claim 1, wherein the cross-linked collagen comprises one type of collagen.

7. The composition of claim 1, wherein the cross-linked collagen comprises two or more types of collagen.

8. The composition of claim 1, further comprising a cell growth enhancer.

9. The composition of claim 8, wherein the cell growth enhancer agent is a peptide.

10. The composition of claim 9, wherein the peptide has an amino acid sequence of RGD, YIGSR, or IKVAV.

11. The composition of claim 8, wherein the cell growth enhancer agent is selected from the group consisting of: neurotrophic factors, nerve growth factors, and epidermal growth factors.

12. The composition of claim 11, wherein the cell growth enhancer agent is distributed substantially throughout the composition.

13. The composition of claim 1, wherein said collagen is the only water-swellable polymer in said device.

14. The composition of claim 1, wherein the collagen is crosslinked at an acidic pH.

15. The apparatus of claim 14, wherein the acidic pH is between about 5.0 and about 5.5.

16. The composition of claim 1, wherein the crosslinked collagen comprises atelocollagen, type I collagen, type III collagen, or a combination thereof.

17. The composition of claim 1, wherein the crosslinked collagen comprises recombinant collagen.

18. The composition of claim 1, wherein the crosslinked collagen comprises collagen isolated from an animal.

19. The composition of any one of claims 1-18, wherein the crosslinked collagen is prepared by a process of crosslinking a collagen polymer using a crosslinking agent other than glutaraldehyde.

20. The composition of claim 19, wherein the crosslinked collagen is prepared by a process of crosslinking a collagen polymer using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

21. The composition of claim 1, further comprising poly (N-isopropylacrylamide-co-acrylic acid), chondroitin sulfate, N, O-carboxymethyl chitosan, hyaluronic acid aldehyde, or alginate.

22. The composition of any one of claims 1-21, which is effective to promote nerve growth on and/or into the composition.

23. A method of preparing the composition of any one of claims 1-22, comprising:

combining a collagen polymer with a cross-linker formulation at an acidic pH; and

solidifying the resulting combination to form a composition comprising cross-linked collagen.

24. The method of claim 23, wherein the combining step comprises mixing the collagen polymer and the cross-linker formulation in a system configured to generate high shear forces to the resulting combination.

25. The method of claim 23, wherein the combining occurs at a temperature of about 0 ℃ to about 5 ℃.

26. The method of claim 27, further comprising adding a cell growth enhancer to the combination.

27. A method of treating an ophthalmic disease, disorder or injury, comprising:

contacting an eye of a subject suffering from said ophthalmic disease, disorder or injury with an ophthalmic device, wherein the ophthalmic device is manufactured from the composition of any one of claims 1-23.

28. Use of the composition of any one of claims 1-23 for the manufacture of an ophthalmic device for treating an ophthalmic disease, disorder or injury in a subject in need thereof.

29. Use of the composition of any one of claims 1-23 for treating an ophthalmic disease, disorder or injury in a subject in need thereof.