Injectable and biocompatible composite matrices for the synthesis of biosynthetic corneas and methods for their preparation

Abstract

The present patent of invention relates to injectable, biocompatible collagenous composite matrices for the synthesis of biosynthetic corneas, composed of decellularized tissues incorporated with nanoparticulate materials, specifically developed for the medical-ophthalmological field. These matrices are derived from animal tissues rich in collagen, particularly porcine tissues, and are biomechanically enhanced through the addition of nanoparticles, especially metallic ones (e.g., gold, silver, and copper), using an innovative method of preparation, comminution, lyophilization, solubilization, and hydrolysis of collagen fibers. The proposed new methodology not only enables the efficient integration of nanoparticles into the tissue, but also enhances the mechanical and biofunctional properties of the resultant matrices, providing a promising framework for corneal tissue regeneration. Furthermore, these matrices have the potential to democratize access to the treatment of conditions affecting the cornea, overcoming geographic and economic barriers that limit access to corneal transplants. This advance represents a significant step toward equality in ocular health, enabling individuals in remote regions or underserved communities to have the same access to treatment as those in urban centers with greater resources.

Description

INJECTABLE AND BIOCOMPATIBLE COMPOSITE MATRICES FOR THE SYNTHESIS OF BIOSYNTHETIC CORNEAS AND THEIR METHODS OF OBTAINING THEM

[0001] This invention patent relates to injectable, biocompatible collagenous composite matrices for the synthesis of biosynthetic corneas, consisting of decellularized tissues incorporated with nanoparticulate materials, specifically developed for the medical-ophthalmological field. These matrices originate from collagen-rich animal tissues, particularly porcine, and are biomechanically enhanced by the addition of nanoparticles, particularly metallic ones (e.g., gold, silver, and copper), using an innovative method of preparation, comminution, lyophilization, solubilization, and hydrolysis of collagen fibers.

[0002] The proposed new methodology not only promotes efficient integration of nanoparticles into the tissue, but also enhances the mechanical and biofunctional properties of the resulting matrices, offering a promising scaffold for corneal tissue regeneration with direct applicability in transplants and ophthalmic treatments. Description of the state of the art:

[0003] The cornea is an avascular, transparent tissue located at the front of the eyeball. It is the interface between the body and the environment, serving as the eye's primary structural barrier against external agents and mechanical impacts. The central thickness of a human cornea is approximately 500 pm, and the peripheral thickness is 700 pm. It also acts as the primary lens of the visual system, refracting two-thirds to 75% of incident light and scattering less than 1% of it.

[0004] In addition to transmitting and focusing light rays, the cornea also helps maintain the structural integrity of the visual system while supporting intraocular pressure from external forces exerted by the pressurized, fluid-filled globe. From a macroscopic perspective, the native cornea is composed of hundreds of layers of collagen fibrils, each with a distinct angular direction.

[0005] The cornea's transparency is intrinsically related to its structure and extracellular components. The absence of blood and lymphatic vessels is also essential for maintaining the optical properties of this tissue. Furthermore, the spacing and diameter of type I collagen fibrils, the cornea's largest constituent by weight, allow for non-destructive interaction with visible light wavelengths precisely toward the pupil and retina, where image formation occurs.

[0006] The human cornea is usually divided into five distinct regions: epithelium, Bowman's membrane, stroma, Descemet's membrane, and endothelium. The stroma is the thickest region and, consequently, most relevant to the tissue's mechanical and optical properties. Bowman's membrane separates the endothelium from the stroma, while Descemet's membrane separates the stroma from the endothelium.

[0007] The human corneal epithelium consists of five to seven layers of cohesive cells, representing approximately 10% of the total tissue thickness, usually ranging from 50 to 60 pm, with the greatest thickness at its periphery. Stratified, squamous, and non-keratinized, the surface epithelium provides the primary protection against foreign bodies and retains the internal fluid content of the visual system.

[0008] From the outermost to the innermost cell layer of the epithelium, there are two or three layers of elongated, squamous superficial cells, two or three layers of polygonal winged cells, and a layer of prismatic basal cells, which exhibit a higher rate of cell reproduction. As these cells reproduce through mitosis, they begin to occupy the space of older, more superficial cells, which in turn begin to slough off. It has a high capacity for regeneration and healing, being fully renewed in approximately seven days.

[0009] Bowman's layer lies just below the basement membrane. Unlike the epithelium, it is a dense, acellular structure measuring 6 to 15 pm thick, composed of a disorganized and compact aggregate of primarily type I, III, and IV collagen fibrils. These components are essential for maintaining the cornea's structural and physiological functions. Bowman's membrane does not regenerate in the event of injury and can form scars.

[0010] The stroma is a highly organized structure, representing approximately 80% of the total thickness of the cornea and the main region responsible for the transparency and mechanical strength of this tissue. Its main composition comprises an extracellular matrix with highly organized collagen fibrils. Type I collagen is predominantly present, with other types of collagen, such as type V and type VI, also being observed. It also contains metalloproteinases and glycosaminoglycans (GAGs), of which keratan sulfate is the most abundant GAG, accounting for approximately 65% ​​of the total amount.

[0011] The stroma, in contrast to other collagen structures, is transparent. This is due to the delicate organization of collagen fibers and extracellular matrix components. These fibers are arranged in parallel bundles called fibrils, and these fibrils are packaged in layers or lamellae. The stroma of a human cornea typically contains 200 to 250 of these lamellae (DELMONTE; KIM, 2011; MIROTSOU; ABE; LANZA, 2020).

[0012] The average fiber diameter typically ranges from 22.5nm to 35nm, depending on the collagen type, and the spacing between them is approximately 41.5nm. Within these fibers are cells called keratocytes, which are responsible for producing the components of the extracellular matrix. Keratocytes are differentiated cells of mesenchymal origin and, despite being dispersed throughout the stroma, are still capable of making contact with one another. These cells do not naturally proliferate and constitute only 3% to 5% of the stroma's volume. However, after injury, keratocytes can increase their metabolic activity and secrete extracellular matrix components, such as collagen molecules and glycosaminoglycans, to promote tissue repair.

[0013] The fourth layer, Descemet's membrane, is an amorphous, acellular structure composed of collagen fibrils, primarily type IV, and glycoproteins that are continuously synthesized and secreted by endothelial cells. This layer slowly increases in thickness with age. It is one of the thickest basement membranes in the human body, reaching up to 10 pm in thickness. In addition to type IV collagen fibers, this membrane is also formed by filaments of type III fibers. Type III collagen fibers also confer porosity to this structure, thus playing an important role in tissue strength and hydration.

[0014] The endothelium consists of only one layer of flat, hexagonal cells, approximately 4 pm thick, which have little replication capacity. Therefore, this thickness tends to decrease with age. the individual's age. The endothelium allows the passage of nutrients and the maintenance of tissue hydration.

[0015] At birth, an individual has an average endothelial cell density of 3,500 cells / mm 2 The rate of decline is approximately 0.6% per year. Trauma, injury, and other pathological processes can accelerate this process. However, the remaining cells have the ability to adapt, modifying their morphology to occupy the space of the degenerated endothelial cells.

[0016] The biomechanical properties of the cornea play a crucial role in its functionality, with the arrangement of collagen fibrils in the stroma being a determining factor. This arrangement is influenced by the concentration, diameter, and orientation of the collagen fibrils. These fibrils are organized to provide rigidity and strength to the cornea. Physiological processes seek to maintain the ultrastructure and properties of this tissue, and a cornea that is too rigid or too flexible can cause serious vision problems.

[0017] The cornea's stiffness is also important for its mechanical function, as it protects the eye from damage. An important parameter for measuring this stiffness is the modulus of elasticity, which, for tensile and compressive stresses, is called Young's modulus and can be defined as the ratio of the stress applied to the material to the resulting deformation. In general, the modulus of elasticity is highest in the central region of the cornea, where the stroma is thickest, and tends to decrease with age due to the degradation of collagen fibrils.

[0018] It is well known that, once outside its natural biological environment, the cornea exhibits a widely variable Young's modulus. This complexity emphasizes the need for rigorous evaluation of experimental conditions and the methodology used when investigating biomechanical properties. This is essential to ensure reliable results applicable to clinical settings and tissue engineering. In this context, the stroma emerges as the most relevant layer for characterizing these properties. Thinner layers, such as the epithelium and Descemet's and Bowman's membranes, have proven irrelevant in the biomechanical evaluation of this tissue.

[0019] Furthermore, corneas exhibit notable mechanical anisotropy, primarily due to the different orientations of collagen fibrils in specific directions within the cornea. This non-uniformity is also one of the factors that explains the discrepancies in the values ​​found for the modulus of elasticity, which can range from 0.05 to 27.5 MPa depending on the methodology employed. The direction corresponding to the highest Young's modulus is due to the fact that it contains a greater number of collagen fibrils oriented in that particular direction. Conversely, the direction associated with the lowest Young's modulus has fewer collagen fibrils oriented in that same direction. Other parameters, such as the degree of fibril compaction and corneal swelling, also alter the mechanical properties of this tissue.

[0020] Any change in corneal tissue that causes damage to the visual system can be considered a corneal dysfunction. Diseases such as bullous keratopathy, leukoma, trachoma, Fuchs' dystrophy, and infectious keratitis are some examples that can damage the structure of this tissue and lead to partial or complete loss of visual acuity. It is estimated that corneal diseases are the fourth leading cause of blindness worldwide, preceded only by cataracts, glaucoma, and macular degeneration.

[0021] A meta-analysis, conducted by Flaxman et al. (2017), found that in 2015 there were approximately 216.6 million people considered Of the visually impaired worldwide, 4.5 million of this group had visual impairment due to corneal diseases. In addition to pathological manifestations, it is important to emphasize that various physical injuries, such as ulcerations, burns, and ocular perforations, have the potential to trigger vascularization in the ocular tissue, which, in turn, can result in impaired visual acuity. Similarly, it is worth noting that severe corneal ectasia conditions, such as keratoconus and myopia, are also associated with this scenario.

[0022] Vascularization of corneal tissue is a common pathology observed in ocular surface diseases and unsuccessful procedures, and is the main cause of corneal transplant failure. Because corneal tissue is transparent and lacks blood vessels, it is crucial that the corneal structure not become vascularized, as this could intensify connections between the immune system and transplant antigens, triggering a strong immune response that can even lead to vision loss.

[0023] Angiogenesis is a specific subset of vascularization and consists of the formation of new blood capillaries from preexisting vessels. Pro-angiogenic and anti-angiogenic factors interact in the corneal ultrastructure in a highly regulated process that can be divided into the following phases: activation, progression, migration, differentiation, and cell maturation.

[0024] Although several factors mitigate angiogenesis in the cornea, in situations of hypoxia, inflammation, or vascular injury, angiogenic signals stimulate quiescent endothelial cells to activate. This improves vascular permeability through the action of several mediators, one of the main ones being VEGF (Vascular Endothelial Growth Factor).

[0025] Studies that elucidated the mechanisms and stages of corneal vascularization have led to the development of medications to suppress the formation of new blood vessels. In this context, bevacizumab (AVASTIN®), a monoclonal antibody that inhibits local permeability induced by VEGF, stands out.

[0026] Recent research has shown that gold nanoparticles (AuNPs) can also trigger antiangiogenic effects by interacting with VEGF, reducing its activity or inhibiting its binding to receptors, thus preventing the signaling that triggers the formation of new blood vessels. This vascular endothelial growth factor is recognized as the main stimulator of new blood vessel growth in the corneal ultrastructure. Deactivating this signaling protein through the addition of molecular elements has the potential to effectively inhibit the generation of new blood vessels.

[0027] Studies on corneal dysfunction have played a fundamental role in the development of medications and methods aimed at preserving the integrity and function of this tissue, even when affected by trauma and / or disease. In situations where ectasia occurs due to genetic factors, treatments such as glasses, contact lenses, and even the insertion of intrastromal polymer rings remain simple and effective options. However, when corneal damage reaches a more advanced stage, it is often necessary to completely or partially replace the damaged tissue with healthy tissue. In this context, keratoplasty, or corneal transplant, has emerged as the most widely accepted and frequently used treatment to restore patients' visual acuity.

[0028] corneal transplant is a surgical procedure in which degenerated corneal tissue is removed and replaced with a healthy cornea. With technological advancements, notably in the field of ophthalmology and the use of new techniques and equipment, such as the femtosecond laser, it is possible to replace the entire cornea, a process known as Penetrating Keratoplasty, or just specific regions of it, described as Lamellar Keratoplasty.

[0029] Corneal transplantation is considered the most widely accepted treatment worldwide and is commonly used to restore visual acuity in patients with severe corneal dysfunction. One of the factors behind this procedure's high success rate is the cornea's unique immunological condition. This characteristic results from the cornea's relative isolation from the immune system and the absence of blood and lymphatic vessels in their normal state.

[0030] In fact, it is estimated that approximately 80% to 90% of patients experience no post-surgical complications within one year of transplantation. Of the possible complications, transplant rejection is described as the most common cause of tissue failure, with approximately 20% of these cases classified as irreversible. The most significant risk factor is neovascularization of the transplanted cornea. Anterior synechiae, intraocular inflammation, glaucoma, and herpes can also lead to rejection.

[0031] Neovascularization results from an immune response against the "foreign body." This mechanism aims to improve the connection between graft antigens and the recipient's immune system. Although it is a natural mechanism, this process can lead to corneal opacity and vision loss. Therefore, it must be suppressed as much as possible.

[0032] Inhibiting neovascularization is not the only concern for increasing the success of corneal transplants. By analyzing the behavior Regarding the long-term survival of transplanted material, statistics reveal a situation that requires even greater attention. Ten years after the surgical procedure, the graft survival rate can range from 35% to 89%. In cases of retransplantation, the success rate is even lower, around 53% at five years, and drops to 41% ten years after surgery. In other words, a significant number of patients, especially those with inflammation and vascularization of the cornea, require a new transplant within five years.

[0033] As if these limitations weren't enough, studies and indicators indicate that the demand for corneas is on an upward trend and is not being met, primarily due to the shortage of human donors. A study by Gain et al. (2016) revealed that approximately 12.7 million people worldwide are waiting for a cornea transplant. Comparing this with the number of transplants performed, the alarming statistic is that only one in seventy individuals in need receives a transplant.

[0034] In this context, it is becoming increasingly difficult for eye banks to meet the growing demand for transplantable tissue. Furthermore, the procedure of corneal transplantation from human donors, despite its high success rate, also presents other limitations that must be addressed. These include, for example, the difficulty in quality control; the costs involved in surgical procedures; and possible delays in the availability of tissue for transplantation. It is crucial to track the donor's history and determine whether they have any potentially transmissible diseases.

[0035] Furthermore, tissue quality can be affected by the donor's age, health, cause of death, and other parameters specific to graft preparation, screening, and preservation. Therefore, alternatives to human donor corneas are necessary to meet global demand. Strategies in the field of Tissue Engineering have been widely adopted, such as the production of artificial corneas and the use of xenografts for the synthesis of decellularized matrices. Corneal Tissue Engineering as a Medical Alternative

[0036] The challenge of regenerating, repairing, and even improving the function of organs and tissues is such that it requires the integration of several fields of knowledge. In Tissue Engineering, the interdisciplinary scope integrates natural sciences and engineering. Reproducing and mimicking biological structures, to the point of mitigating the effects of rejection in the recipient body, is crucial to the success of materials developed within this technological field.

[0037] The scarcity of viable corneas and the growing demand for them have been a driving force in tissue engineering for the development of new materials and systems. When considering these, it is expected that the alternatives developed will achieve equivalence or superiority in application when compared to native tissues. To achieve this, many requirements must be met. In the case of the cornea, the new material must be biocompatible, have a similar geometry and structure, notably preserving the tissue's transparency, and possess adequate mechanical strength to withstand the stresses to which it will be subjected.

[0038] Similarly, biological functions such as cell anchoring and solute and solvent permeability for oxygen and nutrient transfer within the structure, for example, must be mimicked. All of these characteristics must be coupled with a manufacturing process that is reproducible and high-quality, ideally with high production speed and affordable cost.

[0039] Nevertheless, artificial corneas, such as keratoprostheses, have been developed and applied clinically. They can be understood as laboratory-made structures, with or without the help of biological material, designed primarily to replace the function of the native human cornea. Although these materials possess adequate levels of mechanical strength, transparency, and biocompatibility, they have not established themselves as a viable alternative to meet the growing demand for corneal tissue, especially in underdeveloped countries. In fact, their use has been directed at patients identified as having a high risk of rejection of human cadaveric tissue.

[0040] Only two types of artificial corneas, so far, have proven successful in clinical applications. The first, known as Boston KPro, is made from a polymethyl methacrylate (PMMA) matrix with a titanium backplate. The second, called Osteo-Odonto-Keratoprosthesis (OOKP), is fabricated from a piece of tooth that is used to support a PMMA matrix.

[0041] The main limitation of implementing Boston KPro on a larger scale is that it still requires a donor cornea. Furthermore, it is indicated for patients with an intact ocular surface. In turn, OOKP is indicated in cases of bilateral corneal blindness, trachoma, and chemical and thermal injuries, for example. Its application requires the extraction of the patient's tooth to accommodate a PMMA optical cylinder in the dentin. This material is then embedded in the patient's cheek for several months, for later implantation in the eye. Clearly, the cost of clinical surgical practices is one of the major limitations to the widespread use of OOKP. Furthermore, the materials used for these artificial corneas, notably PMMA, are not are conducive to the adhesion and proliferation of keratocytes, as a result of which the need to improve these systems was perceived.

[0042] New versions of keratoprostheses have been developed to improve their functionality, such as replacing PMMA with less rigid, hydrophilic polymers, thus resembling the characteristics of native corneal tissue. However, they still pose a high risk of serious complications in patients, such as calcification, glaucoma, retroprosthetic membrane formation, retinal detachment, corneal melting, and extrusion of the prosthesis itself.

[0043] From this perspective, new approaches began to be analyzed. The use of 3D printing has enabled the construction of biosynthetic corneas, seeking to replicate the functions, geometry, and biomechanics of native tissue for in vivo applications (HOLLAND et al., 2021; ULAG et al., 2020). These corneas were engineered from various polymeric biomaterials for scaffold fabrication, whether synthetic such as poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA), or naturally occurring (biopolymers) such as collagen, chitosan, gelatin, alginate, and silk fibroin.

[0044] All of these materials can induce the secretion of extracellular matrix proteins by keratocytes, enabling the formation of adhesion complexes that can recreate basement membranes—that is, they have the ability to regenerate sublayers such as the corneal epithelium, stroma, and endothelium.

[0045] The use of biopolymers, however, has demonstrated better results for the adhesion, migration, and proliferation of fibroblasts, keratocytes, and limbal epithelial stem cells when compared to synthetic polymers. The use of corneal scaffolds containing collagen as constituents, for example, is especially indicated because of their sequence. of amino acids known as RGD. This tripeptide is directly related to the process of cell adhesion and anchoring.

[0046] In turn, Wu et al. (2014), using silk fibroin with the tripeptide RGD conjugated to its surface, together with growth factors such as TGF-3, were able to mimic an in vivo microenvironment, inducing the secretion of extracellular matrix components by human corneal stromal stem cells, which differentiated into keratocytes. Furthermore, silk fibroin was shown to support the growth of corneal epithelial and endothelial cells, in addition to enhancing the attachment and differentiation of mesenchymal stem cells.

[0047] The aforementioned studies revealed that the 3D printing technique was partially capable of reproducing the complex geometry of the cornea, allowing the manipulation of collagen fibril spacing and pore size to stimulate cell adhesion and proliferation. This technique is enabling the fabrication of scaffolds tailored to the characteristics of a patient's cornea and has the advantage of being suitable for other groups, those with low to medium risk for corneal transplantation, in contrast to keratoprostheses.

[0048] However, clinical applications and studies, especially those examining the long-term behavior of these materials in vivo, have identified that keratocyte adhesion is still limited. The normal cell density of healthy tissue has not been achieved. Although 3D printing and in vitro cell culture techniques were able to mimic much of the cornea's structure, its ultrastructure, with the presence of different types of collagen, proteoglycans, glycoproteins, and other extracellular matrix components, makes the process of fully replicating the cornea unfeasible to meet current demand.

[0049] Compared to the aforementioned alternatives, a new possibility has emerged: the use of xenografts to produce corneal matrices. Compared to keratoprostheses and biosynthetic corneas, xenografts provide a more conducive microenvironment for cell growth, migration, and differentiation due to the presence of growth factors and other extracellular matrix components, such as GAGs.

[0050] The fact that the cornea is considered an immunologically privileged tissue, relatively distant from blood and lymphatic vessels, is a key factor in the use of xenografts. Another factor that drives their use is the high material availability, along with their easy acquisition and histological structure similar to that of humans, especially when dealing with tissues from porcine corneas.

[0051] However, it is still necessary to minimize the possibility of rejection of the material in the host body. To achieve this, a technique is used to remove the animal's native biological material while preserving the remaining extracellular components—a process known as decellularization.

[0052] Decellularization is a process by which cells from organs and / or tissues are removed to form an acellular structure with preserved structural framework. According to Gonzalez-Andrades et al. (2011), the use of decellularization techniques provides advantageous pathways for the development of viable corneas for transplantation. Several studies have been conducted to determine the best protocols for removing cells from these tissues. However, there is still no consensus on which technique is most effective.

[0053] Decellularization procedures can be categorized into three distinct approaches or combinations of them: chemical, physical, and biological. Chemical decellularization involves, for example, the use of detergents, including sodium dodecyl sulfate (SDS), Triton X-100, formic acid, ammonium hydroxide, and sodium chloride. Physical decellularization, on the other hand, encompasses techniques such as agitation, freeze-thaw cycles, electrophoresis, application of high hydrostatic pressure, osmotic pressure, supercritical CO2, ultrasound, glycerol, and lyophilization. Finally, biological methods include the use of enzymes such as pepsin, trypsin, dispases, phospholipase A2, human serum, and nucleases.

[0054] SDS is a powerful ionic detergent and, due to its remarkable effectiveness in removing cells and solubilizing cell membranes, is widely used in corneal decellularization, either alone or in combination with other agents. However, it is important to note that the effectiveness of SDS may vary depending on the methodology and concentration used, as high concentrations and exposure times can lead to protein denaturation, which in turn affects the structure of the extracellular matrix (ECM). Most protocols include a subsequent washing step to remove any remaining residue.

[0055] The ideal SDS concentration for corneal decellularization typically ranges from 0.1% to 1% (w / v), as this range allows for effective cell removal without compromising corneal ultrastructure or reducing GAG and collagen levels. However, it is important to note that some conflicting studies report a reduction in total GAG content, damage to the basement membrane, and loosening of collagen fibers, even at lower concentrations. These discrepancies may be attributed to species variations, contact time with the detergent, or the presence of inhibitors. of proteases, which can protect the structure and components of the extracellular matrix. Therefore, the need for standardization of procedures and characterization techniques to ensure the effectiveness of the decellularization process using SDS is evident.

[0056] Furthermore, it is important to highlight that some methodologies have achieved good results in corneal decellularization without the need for detergents. In a study conducted by Gonzalez-Andrades et al. (2011), it was demonstrated that the use of a 1.5 M sodium chloride (NaCl) solution for the decellularization of porcine corneas resulted in a scaffold with adequate histological and optical properties. This approach provided a favorable microenvironment for the penetration of human keratocytes and the maintenance of their differentiation within the scaffold.

[0057] In turn, Wu et al. (2009) demonstrated that the use of phospholipase A2 to remove biological material from porcine corneal stromas preserved collagen fibers and a sufficient amount of proteoglycans and GAGs, maintaining the ultrastructure of the region. The developed material presented adequate biocompatibility and biomechanics, undetectable immunogenicity, and high transparency and stability.

[0058] It is clear that decellularization processes, even the most efficient, cause, to some degree, damage to the extracellular matrix. The choice of methodology for corneal decellularization should be based on the best balance between maintaining structural integrity and eliminating native cells. It is worth noting, however, that continuous advances in the study of decellularization techniques are enabling the use of xenografts to provide replacement and reconstruction matrices for corneal tissue. As long as the developed material is stored properly, it is possible to offer greater flexibility in patient and surgical intervention planning. This advantage can significantly optimize procedures, potentially increasing the number of transplants performed.

[0059] When choosing which animal species can provide the best matrix for the production of decellularized corneal scaffolds, some considerations are worth highlighting. Although primates are phylogenetically closer to humans, which would a priori lead to a lower transplant rejection rate, they have smaller ocular structures than humans and are more susceptible to zoonotic transmission.

[0060] Another important factor is the growing ethical concern surrounding the procurement of organs and tissues from primates. Pigs, in turn, are slaughtered annually in greater numbers than the demand for corneas. They also have a size and visual system similar to that of humans, and the costs of care and feeding are relatively low.

[0061] Furthermore, experiments involving the genetic modification of pigs are enabling their tissues to become resistant to damage caused by the immune response of primates, notably humans. Genetic engineering can make pig corneas comparable to, or even superior to, human corneas in their ability to resist rejection, allowing these matrices to be produced under pathogen-free conditions.

[0062] Nevertheless, in vivo clinical studies indicate the need for further research to improve matrices from decellularized porcine corneas, largely because these tissues experience long-term loss of optical and mechanical properties, leading to corneal opacity. Another limitation of using these matrices for transplantation is related to the expense and risks of clinical practices, highlighting the invasive nature of the surgical procedure. The use of an injectable material that provides the components and structures necessary to restore corneal defects could be an alternative to circumvent these restrictions. In this context, the possibility of forming hydrogels from decellularized corneal matrices arises.

[0063] Hydrogels can be understood as polymeric materials with interconnected three-dimensional networks, capable of retaining large amounts of water or biological fluids within their structure, while also allowing the diffusion of oxygen, water, and glucose through their networks. The ability to absorb and retain fluids, such as water, is related to the presence of hydrophilic functional groups in their structure. Some commonly observed examples are amino, carboxyl, and hydroxyl groups. In this context, corneal tissue can be conceptualized as collagen hydrogels composed of collagen fibrils that maintain their transparency at visible light wavelengths.

[0064] Hydrogels can be produced from synthetic or natural polymers, known as biopolymers. In ophthalmology, chitosan, collagen, hyaluronic acid, alginate, and silk fibroin are commonly used biopolymers. Synthetic polymers are also used to manufacture hydrogels, including polyethylene glycol and cyanoacrylates. However, decellularized extracellular matrices are emerging as the best option for producing injectable hydrogels for corneal tissue restoration, as they can also be used to fill and restore both regular and irregular regions.

[0065] The stabilization of the structure of hydrogels derived from extracellular matrices tends to be achieved through the cross-linking process, following hydrolysis of the collagen fibers, in which the peptides in the polymer chains establish chemical interactions with each other. The cross-linking of these chains can lead to a process called sol-gel transition, in which a colloidal solution, a sol system, acquires aspects of a gel system, that is, acquiring a structure with greater rigidity and viscosity. The physicochemical conditions of the environment, such as temperature and pH, directly influence the aspects of this transition.

[0066] Several agents can be used to promote the crosslinking of hydrogels from decellularized corneas, and to different degrees, either using chemical crosslinkers, such as riboflavin, genipin and proanthocyanidin, or physical crosslinkers, such as ultraviolet radiation;

[0067] When considering the application of an injectable hydrogel for corneal tissue restoration, whose sol-gel transition occurs inside the eye, i.e., in an in vivo environment, it is crucial to regulate the physicochemical parameters of this reaction to avoid damaging the patient's ocular biological structure. Therefore, it is necessary to choose cross-linking agents carefully. Therefore, Wang et al. (2020) analyzed the combination of two such agents: l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) and N-hydroxysuccinimide (NHS). The choice of these molecules is intrinsically related to the fact that the collagen cross-linking reaction occurs at room temperature and neutral pH, preserving the components of the extracellular matrix, GAGs, and collagen itself, which also contributes to the gelation of the hydrogel.This behavior is clinically important, as the biomaterial still in solution can be used in surgical procedures and quickly reach a gel state, protecting the. tissue integrity and allowing the patient's native cells to anchor and proliferate.

[0068] The results of experiments by Wang et al. (2020) with in vitro cells indicated that the hydrogel cross-linked with CMC and NHS did not exhibit significant cytotoxicity, promoting the synthesis of extracellular matrix components through keratocyte adhesion. Furthermore, the hydrogel accelerated the healing of corneal epithelial and stromal wounds induced in animals. Another highly relevant finding was that no toxic byproducts were released into the tissue during or after the cross-linking reaction.

[0069] In this context, other studies have also been conducted using injectable hydrogels derived from decellularized porcine corneas, such as that conducted by Zhou et al. (2021), which also obtained satisfactory results in the recovery of animal corneal tissue, demonstrating their effectiveness in promoting tissue repair, not only during in vitro cell culture but also when injected into corneal defects in vivo. It should be noted, however, that both studies still observed some limitations regarding the mechanical properties of the hydrogels, which were inferior to those of native tissue. Therefore, further improvement of these decellularized matrices is necessary. The addition of nanocomponents presents a possible alternative for this purpose. The present patent aims to apply functionalized nanoparticles, especially AuNPs, as agents that induce collagen cross-linking.

[0070] Delving deeper into the analysis of the cytotoxicity of AuNPs on the tissues present in the eyeball, Masse et al. (2019) concluded that most studies reported low or no toxicity due to the inert nature of gold and the biocompatibility of the chosen ligands. It is noteworthy, however, that these Characteristics are also associated with the dimensions and shapes of the nanoparticles, as well as the amount applied. For example, Azharuddin et al. (2014) introduced 20nm to 30nm AuNPs stabilized with citrate, administered topically, in the eyes of mice. This material remained present in different regions of the cornea for thirty days without inducing any morphological changes in the tissue (MASSE et al., 2019).

[0071] In concluding their review, Masse et al. (2019) demonstrated the undeniable potential of AuNPs in the field of ophthalmic therapeutics. This is largely due to the great tunability of these particles' properties through modifications to their morphological aspects, such as size and shape. The AuNPs studied were applied through various routes, including topical application, intravenous administration, perfusion, and even incubation. Furthermore, the non-cytotoxicity coupled with the antiangiogenic and anti-inflammatory effects of AuNPs make them excellent candidates for the treatment of ocular diseases and dysfunctions, including those of the cornea.

[0072] Indeed, during the search for materials to prevent corneal neovascularization, Cho et al. (2015) demonstrated that topical administration of AuNPs, with an average diameter of 20 nm, significantly mitigated this process in mice. The use of this material led to a 39.8% decrease in vascularization without causing significant side effects. One of the reasons suggested for this effect is that AuNPs inhibited VEGF expression.

[0073] Thus, in vivo and ex vivo models, such as the chicken embryo chorioallantoic membrane (CAM), have corroborated this by demonstrating the antiangiogenic activity of AuNPs. Research conducted by Darweesh (2019) highlighted that gold nanoparticles demonstrated the ability to decrease vascular density and permeability while preserving blood vessel integrity. As a result, Most of the investigated biomedical applications revealed that AuNPs are effective as angiogenic inhibitors, as they were able to significantly reduce the total complexity of vascular tubules, the overall vessel length, and the number of vascular junctions.

[0074] Regarding the synthesis of AuNPs, several production routes can be employed. However, when considering biomedical applications, it is advisable to use non-toxic reagents or, otherwise, to ensure they are completely consumed. Based on this assumption, the Turkevish method, which uses sodium citrate as a gold ion reducing agent, and some of its variants, which use citric acid, stand out.

[0075] In typical syntheses of AuNPs for biomedical applications using the Turkevish method, chloroauric acid (HAuCl4) is used as a precursor for gold ions (Au+3) and citrate as a reducing agent, triggering nucleation and subsequently stabilizing the gold nanoparticles through adsorption. Increasing the amount of citrate used tends to stabilize the AuNPs at smaller sizes.

[0076] The larger surface area attributed to AuNPs, with polar molecules adsorbed on their surface, allows for a greater degree of interaction with collagen fibers. Thus, the use of nanoparticles tends to better stabilize these fibers than the use of conventional crosslinkers. Furthermore, these commonly used crosslinkers, such as glutaraldehyde, are incompatible in biological applications and may lead to adverse effects in the long term.

[0077] Since the entire decellularization process degrades, to some degree, the structure of the cornea's collagen fibers, the use of AuNPs appears to be an excellent alternative to improve the properties biomechanics of scaffolds derived from this tissue. Furthermore, the aforementioned characteristics of low cytotoxicity and antiangiogenic effect support its use in this field.

[0078] Based on all of the above, the new composite collagen matrices proposed here are justified. These matrices are developed from a novel method of preparing animal tissue with the insertion of nanocomponents, especially AuNPs. From all the studies presented here, it was possible to identify the characteristics that demonstrate the novelty and inventive step of the present invention. This premise is further corroborated by the extensive research conducted on prior art patents, the following being the lists that most closely approximate the technology required herein:

[0079] W02011109712 describes a method for creating human corneal stromal tissue from the recellularization of scaffolds. The primary objective is to offer a viable alternative to traditional corneal transplants, which are limited by donor scarcity and the risk of rejection. The method involves harvesting corneal stromal cells from a donor and expanding them in culture. The cells are then carefully seeded onto a support that mimics the natural structure of the cornea. Through cultivation under controlled conditions, the cells develop and differentiate into mature stromal cells, creating the tissue. Although this approach also seeks to solve significant ophthalmological problems, it fundamentally differs from the technology discussed here in the type of base tissue used (human vs. animal) and, most importantly, in the absence of any mention of the use of metal nanoparticles to enhance the properties of the biological tissue.

[0080] WO2021152322 describes methods for creating novel transparent and curved corneal stromal tissues, as well as their corresponding decellularized versions. These tissues are intended for use in ophthalmology, specifically for corneal transplantation and other applications. Again, this application does not mention the insertion and use of metal nanoparticles to enhance the properties of the biological tissue. A biodegradable scaffold for cultivating corneal stromal cells in a three-dimensional environment is cited as a disruptive innovation. The scaffold provides the cells with a structure to grow and differentiate, resulting in a tissue with enhanced optical properties. The technique allows the curvature of the corneal tissue to be controlled during the production process, which is crucial for ensuring precise adaptation to the individual anatomy of the patient.

[0081] W02020008258 proposes a method for stabilizing collagen scaffolds, aiming to improve their strength and durability for biomedical applications. The invention focuses on two main aspects: the addition of specific cross-linking agents to strengthen the bonds between collagen molecules and the addition of biocompatible polymers.

[0082] By reviewing and detailing the state of the art, it becomes clear that, despite the proposal of numerous techniques and approaches to improve tissue matrices in order to create viable corneas for transplantation, these efforts have not yet yielded concrete successes. Such initiatives have not been able to effectively address the challenge posed by the limited availability of corneas, maintaining the discrepancy between supply and the high demand of patients awaiting corneal tissue transplants. It is possible to infer, therefore, that only experimentation with the addition of innovative biocompatible polymers, the implementation of advanced decellularization techniques, and the application of more effective cross-linking processes will still lead to are not enough, which demonstrated the need for more disruptive approaches. Brief description of objectives

[0083] To overcome the aforementioned challenges and limitations, this patent was proposed. To this end, the invention aimed to incorporate nanocomponents, particularly gold nanoparticles (AuNPs), with controlled dimensions and morphologies, to optimize decellularized collagen matrices derived from animal sources, with a particular emphasis on porcine tissues. This approach resulted in a significant improvement in both the biomechanical properties and biocompatibility of the tissues produced, demonstrating the potential of this innovation for advanced biomedical applications. The developed matrices thus become viable substitutes for damaged human corneas. The details and functionalities of the present invention can be better understood by the following detailed description, in conjunction with the attached figures, where: Figure 1 shows images obtained by transmission electron microscopy of biosynthetic corneas, at different scales, in the following sequence: (A) and (A') corneal matrix without nanoparticles, (B) and (B') corneal matrix with gold nanoparticles at a concentration of 25 ppm, (C) and (C') corneal matrix with gold nanoparticles at a concentration of 50 ppm and (D) and (D') corneal matrix with gold nanoparticles at a concentration of 75 ppm. Figure 2 shows the proportion of light transmitted by the corneal matrices - HEDS (corneal matrix without nanoparticles), HEDS25 (corneal matrix with gold nanoparticles at a concentration of 25 ppm), HEDS50 (corneal matrix with gold nanoparticles at a concentration of 50 ppm) and HEDS75 (corneal matrix with gold nanoparticles at a concentration of 75ppm) - in light wavelengths ranging from 300nm to 800nm Figure 3 shows FTIR spectra of native porcine cornea, decellularized porcine cornea with 0.1% SDS, and corneal matrices without AuNPs (HEDS) and with AuNPs (HEDS-Au). Figure 4 shows the value of the Elasticity Modulus (KPa) for different frequencies obtained by dynamic-mechanical analysis (DMA), comparing the values ​​of Native Corneas, Decellularized Corneas and the developed matrices (HEDS, HEDS25, HEDS50 and HEDS75). Figure 5 shows the tan(õ) value for different frequencies obtained by dynamic-mechanical analysis (DMA), comparing the values ​​of Native Corneas, Decellularized Corneas and the developed matrices (HEDS, HEDS25, HEDS50 and HEDS75). Figure 6 shows the result of the Cell Viability assay with human fibroblast cells after 24 hours of contact with the samples. *Indicates statistical difference in relation to the Control group (***p<0.001). Figure 7 shows the graph with the comparative values ​​between the percentage occupied by blood vessels in the photos obtained in a CAM (Chorio-Allantoic Membrane) assay of the seven groups studied (Negative control - PBS; HEDS; HEDS25, HEDS50, HEDS75, in addition to the groups with only AuNPs and the positive control - Bevacizumab). Figure 8 shows the graph with the comparative values ​​between the number of blood vessel junctions in the photos obtained in a CAM (Chorio-Allantoic Membrane) assay of the seven groups studied (Negative control - PBS; HEDS; HEDS25, HEDS50, HEDS75, in addition to the groups with only AuNPs and the positive control - Bevacizumab). Figure 9, in turn, presents the graph with the comparative values ​​of the lacunarity of the blood vessels in the photos obtained in a CAM (Chorio-Allantoic Membrane) test of the seven groups studied (Negative control - PBS; HEDS; HEDS25, HEDS50, HEDS75, in addition to the groups with only AuNPs and the positive control - Bevacizumab). Detailed description of the invention:

[0084] BIOCOMPATIBLE COMPOSITE MATRICES FOR BIOSYNTHETIC CORNEAL SYNTHESIS, as explained, were produced with an intricate and meticulous combination of components and proportions to result in materials with mechanical, optical, and biological properties similar to human corneas. The goal was to create materials that not only replicate but also seamlessly integrate with human ocular tissue, providing a long-lasting and effective solution for restoring corneal structure and, by extension, visual acuity. In this context, the matrices synthesized here are engineered as composite hydrogels.

[0085] The relevance of this development lies in its ability to offer a significantly improved quality of life for those who face limitations with available conventional treatments. Many patients, for various reasons—from donor scarcity to medical contraindications for human corneal transplantation—find themselves without viable treatment options.

[0086] This was made possible by extensive testing and trials to determine the best proportions of nanoparticle materials that could be incorporated into the hydrogel structure. Furthermore, it was necessary to define the best size, morphology, concentration, and stabilizing agents for the nanoparticles to achieve optimal biological and mechanical properties.

[0087] Furthermore, all processing steps for the synthesis of corneal matrices were carefully selected and studied to obtain a material with only biocompatible and contamination-free components. All of this was verified and endorsed by the physicochemical characterizations presented in Figures 1 to 9.

[0088] After all the studies, innovative formulations for these matrices can be achieved, which include the following components: • Decellularized and processed animal tissue; • Integrated metallic nanoparticles, especially AuNPs, with an average diameter ranging from 10nm to 40nm; • Biocompatible Crosslinking Agents.

[0089] Among the various sources of animal tissue, cornea and skin stand out due to the quantity and proportion of collagen, a fundamental component of both human and animal corneas. The use of these tissues, however, must be guided by strict ethical standards, ensuring that obtaining these materials does not imply additional slaughter beyond that for food consumption. This minimizes the ethical impact associated with their acquisition, aligning the practice with animal welfare and sustainability guidelines.

[0090] In this context, pigs are particularly prominent as a substantial source of these tissues, mainly due to their physiological compatibility with humans and the frequency of their slaughter for consumption. In 2022, for example, Brazil recorded the slaughter of more than 50 million pigs, according to data from the Brazilian Institute of Geography and Statistics (IBGE). Notably, parts such as the eyeball, which includes the cornea, are often overlooked in the production chain, representing a significant opportunity for regenerative medicine.

[0091] The premise of this patent also lies in the valorization of currently underutilized organic materials, proposing their reuse within an innovative and sustainable context. The focus is the development of biocompatible matrices, made from this wasted organic matter, designed to promote the regeneration or replacement of living tissues and structures, with particular attention to human corneas. This process involves the application of advanced bioengineering techniques to transform these materials into scaffolds that mimic the natural extracellular matrix, thus providing a favorable environment for cell regeneration and tissue integration.

[0092] Once a xenogeneic tissue has been selected, it must undergo the decellularization process. Extensive studies and tests have demonstrated that the resulting matrices possess optimal properties using the following methodology: • Extraction and sterilization of animal tissue immediately after slaughter and preservation in balanced saline solution. • Decellularization using chemical, physical or biological agents, especially the ionic detergent SDS in proportions of up to 1% (w / v).

[0093] The animal tissue is then kept under agitation for 24 hours at room temperature in the decellularizing solution. After this period, the tissues are rinsed with PBS for up to 120 minutes, with constant agitation, at a temperature of 4°C. During the rinsing process, the PBS must be replaced every 30 minutes. After the washing cycles are completed, the tissues are again left in PBS under mechanical agitation for a period of 72 hours, with PBS changes every 24 hours.

[0094] After obtaining the decellularized extracellular matrices, they are dispersed and comminuted, with the aim of fragmenting them. structures of collagen lamellae, fibers, and fibrils. This step is followed by freeze-drying of the samples, which is carried out for a period of up to 48 hours.

[0095] After freeze-drying and comminution, the material should have an average particle size <40 pm. This allows the collagen fibers to be effectively hydrolyzed in an acidic solution using digestive enzymes, particularly pepsin. This process produces a homogeneous solution of dissolved collagen. The resulting solution is neutralized, and pepsin activity is inhibited by the addition of 1 M NaOH, adjusting the pH of the solution to 7.4.

[0096] Metal nanoparticles are added to the previously prepared solution, maintaining a maximum concentration of 75 ppm to ensure the system's biocompatibility. It is crucial that these nanoparticles are stabilized exclusively with biocompatible agents, such as citrate, thus ensuring the system's integrity and safety for biomedical applications. To complete the process, the system is crosslinked by adding chemical agents, particularly carbodiimides (EDC and / or CMC) in a 10xPBS solution.

[0097] Before cross-linking, all formulations undergo homogenization. The homogenized systems are then transferred to molds of the desired shape and incubated for 20 minutes, allowing the cross-linking reaction to complete. Since the cross-linking reaction takes a few minutes to complete, the material can be injected directly into the patient's ocular structure, which must be adequately prepared during this intermediate period. Notably, the reaction does not generate heat, allowing for in situ completion. After the reaction is complete, the sol-gel transition is observed within a few minutes, resulting in a hydrogel with properties analogous to those of the human cornea.

[0098] The optical, mechanical, and biological tests conducted and shown in Figures 1 to 9 demonstrated that the developed matrices have excellent optical transmittance (Figure 2), with values ​​above 70% for visible light wavelengths, even with the addition of nanoparticles. The evaluation of the mechanical properties (Figures 4 and 5), conducted through the DMA test, revealed a clear correlation between the increase in AuNP concentration and the increase in the elastic modulus, for the samples subjected to an oscillating compressive stress in PBS at 37 °C. 5 C, until reaching a ratio of 75 ppm. These results suggest that the incorporation of these nanoparticles has the potential to improve the strength of hydrogels. Specifically, the addition of 75 ppm AuNPs resulted in an approximately 75% increase in the elastic modulus of biosynthetic corneas.

[0099] The cell viability assay (Figure 6), conducted using the resazurin method, revealed no statistically significant differences between the control group and the various samples, indicating that the developed matrices are not cytotoxic to the cell line used (human dermal fibroblasts). The CAM assay (Figures 7 to 9) clearly demonstrated an inhibitory effect on angiogenesis, reflected in the reduction in the percentage of vascular area and the decrease in the number of vascular junctions, while there was an increase in lacunarity as the AuNP concentration increased, reaching a maximum proportion of 75 ppm. Application Example - 1

[0100] Based on all the studies employed, below is an example of the components and proportions used to develop the injectable composite matrices: • 40mg of decellularized porcine stroma with 0.1% SDS (m / v), comminuted and lyophilized, following the aforementioned methodology. • 4mg of pepsin, • 1ml of 0.01M HCI.

[0101] This mixture remains under constant stirring for 24 hours at room temperature, promoting the hydrolysis of the collagen chains and homogenization of the system. Subsequently, the pepsin is deactivated by adding 1M NaOH until the pH of the solution reaches 7.4. To incorporate gold nanoparticles, they are added to the medium at a specific concentration of 75 parts per million (ppm), followed by a homogenization process to ensure uniform distribution of the particles in the system. Subsequently, a mixture containing 26 mg of l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) and 7 mg of N-hydroxysuccinimide (NHS) in 200 pL of a tenfold (10x) concentrated phosphate buffered saline (BS) solution is incorporated to facilitate the crosslinking of the collagen peptides. This procedure aims to improve the structure and functionality of the composite, allowing an effective interaction between the biomimetic components and the decellularized and lyophilized porcine stroma.It is important to highlight that, when calculating the concentration of gold nanoparticles, only the mass fraction of gold present in the solution was taken into consideration, in relation to the total mass of the treated porcine stroma, to ensure the accuracy and effectiveness of the dosage used. Application Example - 2

[0102] From all the studies employed, here is another example of the components and proportions used to develop injectable composite matrices: • 50mg of decellularized pig skin with 0.5% SDS (m / v), comminuted and lyophilized, following the aforementioned methodology. • 5mg of pepsin, • 1ml of 0.01M HCI.

[0103] This mixture is subjected to constant stirring for 24 hours at room temperature, which promotes efficient hydrolysis of the collagen chains and homogenization of the system. Pepsin activity is neutralized with the gradual addition of 1M NaOH until the pH of the medium reaches 7.4, ensuring ideal conditions for the next step of the process.

[0104] To integrate silver nanoparticles into the composite, they are added at a new specific ratio of 60 parts per million (ppm). This concentration adjustment aims to optimize the distribution and functionality of the nanoparticles within the matrix. The mixture is homogenized again to ensure uniform dispersion of the silver nanoparticles. Next, a solution containing 24 mg of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide hydrochloride (EDC) and 8 mg of N-hydroxysuccinimide (NHS) in 250 pL of tenfold (10x) concentrated phosphate-buffered saline (PBS) is added to the system. Application Example - 3

[0105] From all the studies employed, here is another example of the components and proportions used to develop injectable composite matrices: • 50mg of decellularized porcine horny skin with 1.0% Triton-X (m / v), comminuted and lyophilized, following the aforementioned methodology. • 3.5mg of pepsin, • lml of 0.01M HCI.

[0106] This preparation is kept under continuous stirring for 20 hours at room temperature, facilitating the hydrolysis of the collagen chains and ensuring complete homogenization of the system. Pepsin is inactivated by the careful addition of 1M NaOH, adjusting the pH of the solution to 7.3, an optimal value for the subsequent phases.

[0107] The incorporation of copper nanoparticles into the system is accomplished by adding a meticulously calculated amount of these, fixed at 50 ppm. This concentration was chosen to promote synergistic interaction with the other components, optimizing the biofunctional properties of the matrix. A rigorous homogenization step follows to ensure uniform distribution of the copper nanoparticles. Subsequently, a solution containing 28 mg of l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide hydrochloride (EDC) and 6 mg of N-hydroxysuccinimide (NHS) in 220 pL of tenfold (10x) concentrated phosphate-buffered saline (PBS) is introduced, aiming at the effective crosslinking of the collagen peptides and the formation of a stable and functional composite matrix. Final Considerations

[0108] The innovation of injectable biosynthetic composite corneas represents a revolutionary milestone in the field of ophthalmology and regenerative medicine, promising to transform the treatment of eye diseases and injuries that affect vision. This advancement is not just a scientific triumph; it is a spark of hope that lights the way for countless individuals around the world facing the prospect of blindness or severely impaired vision.

[0109] The benefits of biosynthetic corneas extend across several dimensions, both medical and social, offering solutions to long-standing challenges. frequently faced by patients and healthcare professionals. First, they promise a viable and effective alternative to traditional corneal transplants, which are limited by the scarcity of eye tissue donations. This is crucial, considering the discrepancy between the demand for corneal transplants and the availability of suitable donations, a problem that affects millions of people waiting for a chance to regain their sight.

[0110] Beyond the issue of availability, biosynthetic corneas offer a significant reduction in the risk of rejection by the patient's immune system. Traditionally, even after a successful corneal transplant, patients face the risk of rejection, which can compromise the procedure's efficacy and longevity. Biosynthetic corneas, designed to be biocompatible, minimize this risk, providing a more durable and stable solution for vision restoration.

[0111] Another notable benefit is the customizable nature of biosynthetic corneas. Adaptable to each patient's specific needs, they can be manufactured to match the unique characteristics of the recipient eye, including shape, size, and curvature, thus optimizing visual outcomes after the procedure. This customization ensures a more natural and efficient integration of the biosynthetic cornea with the patient's ocular tissue, enhancing vision recovery.

[0112] From a medical-social perspective, the implementation of biosynthetic corneas has the potential to democratize access to treatment for corneal conditions, overcoming geographic and economic barriers that currently limit access to corneal transplants. This advancement represents a significant step toward equality in eye health care, enabling individuals in remote regions or communities less favored have the same opportunity for treatment as those in urban centers with more abundant resources.

[0113] In summary, biosynthetic composite corneas open new frontiers in regenerative medicine, offering practical solutions and renewed hope for millions of people affected by corneal diseases and injuries. This advancement not only significantly improves the prospects for vision recovery for many, but also reflects a broader commitment to innovation and inclusion in healthcare, marking a new chapter in the search for more effective, accessible, and personalized treatments for vision loss.

[0114] Finally, it is reiterated that there are possibilities for modifications and adaptations in the proportions and components used, and the descriptive memory of this document should not be considered as limiting, but rather as illustrative, and there may be constructive variations, for those skilled in the art, which are equivalent without, however, departing from the scope of protection of the invention.

Claims

CLAIMS 1. INJECTABLE AND BIOCOMPATIBLE COMPOSITE MATRICES FOR THE SYNTHESIS OF BIOSYNTHETIC CORNEAS, with mechanical, optical and biological properties that mimic human corneas, offering an environment conducive to cell regeneration and tissue integration, characterized by encompassing: • Decellularized extracellular matrix - preferably of xenogeneic origin - comminuted and lyophilized with an average size <40 pm to allow effective hydrolysis of collagen molecules, followed by chemical crosslinking using, preferably, carbodiimides; • Metallic nanoparticles integrated into the matrices, preferably gold nanoparticles (AuNPs) stabilized with citrate molecules adsorbed on their surface, with an average diameter that can vary from 10nm to 40nm and a concentration of up to 75 parts per million (m / m); • Biocompatible crosslinking agents whose sol-gel transition phase is >60 seconds in contact with the collagen hydrogel, especially carbodiimides such as EDC and l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMC); Therefore, the reticulated matrices have light transmittance - in the visible light wavelength range - greater than 70%.

2. INJECTABLE AND BIOCOMPATIBLE COMPOSITE MATRICES FOR SYNTHESIS OF BIOSYNTHETIC CORNEAS, according to claim 1, characterized in that the decellularization is carried out with chemical, physical and / or biological agents, preferably SDS (Sodium Dodecyl Sulfate) and Triton X-100, in concentrations that do not denature the collagen fibers of the extracellular matrix.

3. INJECTABLE AND BIOCOMPATIBLE COMPOSITE MATRICES FOR THE SYNTHESIS OF BIOSYNTHETIC CORNEAS, according to claims 1 and 2, characterized by the presence of nanoparticles - preferably AuNPs, suppressing the formation of new blood vessels in angiogenic processes in vivo.

4. METHODS FOR OBTAINING INJECTABLE AND BIOCOMPATIBLE COMPOSITE MATRICES FOR THE SYNTHESIS OF BIOSYNTHETIC CORNEAS, characterized by encompassing the following steps: • Extraction and sterilization of animal tissue immediately after slaughter and preservation in balanced saline solution; • Decellularization using chemical, physical and / or biological agents, especially the ionic detergent SDS in proportions of up to 1% (m / v), with the animal tissue being kept under agitation for at least 24 hours at room temperature and then being subjected to rinsing with PBS solution for up to 120 minutes, with constant agitation, at a temperature of 4°C, with the PBS being replaced every 30 minutes; and after the completion of the washing cycles, the tissues are left again in PBS solution under mechanical agitation for a period of 72 hours, with PBS changes every 24 hours; • Dispersion and comminution, wet or dry; • Freeze-drying for up to 48 hours; • Acid hydrolysis, using HCI with pH ranging from 1 to 4, with or without the presence of digestive enzymes; • Deactivation of enzymes and neutralization of pH with the addition of NaOH; • Addition of metallic nanoparticles in proportions of up to 75ppm, preferably AuNPs; • Homogenization of the system; • Chemical crosslinking, preferably by carbodiimides, such as CMC and / or EDC.

5. METHODS FOR OBTAINING INJECTABLE AND BIOCOMPATIBLE COMPOSITE MATRICES FOR THE SYNTHESIS OF BIOSYNTHETIC CORNEAS, according to claim 4, characterized in that the animal tissue preferably originates from porcine collagenous structures, preferably the cornea itself and / or the skin.

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