ES2906715T3 - Biomaterial devices for guided tissue regeneration - Google Patents

Abstract

A guided tissue regeneration (GTR) device, comprising: mutable collagenous tissue (MCT) and chitosan, wherein the chitosan is present in a mass percentage greater than zero and less than or equal to 90, wherein the chitosan is attached electrostatically to MCT forming a matrix, and the matrix of chitosan and MCT is in the form of an MCT-chitosan composite; and an applicator for administering the MCT or matrix to an area to be treated.

Description

DESCRIPTION

Biomaterial devices for guided tissue regeneration

field of invention

The present invention relates to biomaterial devices for guided tissue regeneration (GTR). More specifically, aspects of the invention relate to tissue sponges, wound dressings, cosmeceutical compositions, and other devices and topical compositions comprising mutable collagenous tissue (MCT) for effecting GTR. Still more specifically, aspects of the invention relate to such devices comprising MCT and chitosan (CHT) composites for effecting GTR. Aspects of the invention also relate to the treatment of burns, wounds, ulcers, and other related skin injuries and disorders, by applying MCT, alone or as a composite, in biomaterial devices to promote GTR. Aspects of the invention also relate to the treatment of skin abnormalities, such as scarring and skin discolorations, including chloasma discolorations, by applying MCT and/or MCT-chitosan composites in cosmeceutical formulations.

Background of the invention

Natural polymers have been used in many pharmaceutical applications and medical device technologies. A natural polymer, chitosan (sometimes referred to herein as CHT), has been used for the preparation of nanoparticles, microspheres, hydrogels, films, fibers, and tablets. Chitosan has been used to prepare potential drug delivery systems, such as oral, nasal, parenteral, transdermal, and ophthalmic formulations. Chitosan has also been used to prepare wound dressings and tissue sponges (Kumar et al., Chem. Rev. 2004, 104, 6017-6084). However, chitosan formulations and materials suffer from numerous drawbacks including limited stability, biodegradability, and tensile strength. Materials such as modified chitosan and synthetic composites have been tried for many of the same uses for which chitosan has been evaluated, but many of these materials have similar drawbacks, including insufficient biocompatibility.

Consequently, there is a need for new materials that are biocompatible and biodegradable, and that have adequate stability and mechanical properties and performance for use in human and other mammalian treatments and therapies. These new materials and compositions would preferably have advantages over chitosan alone, such as additional and/or improved biocompatibility, high stability, and improved physical and biological properties. The ability to use these materials as tissue sponges, wound dressings, cosmeceuticals, and/or therapeutic agent delivery systems would further assist researchers in the areas of biomedical engineering, biomaterials, and tissue engineering. C. Ferrario et al., Marine Environmental Research 128, 46 57 (2017) discloses collagen membranes derived from echinoderms (sea urchin, starfish, sea cucumber) for guided tissue regeneration.

Summary

The present invention relates to the subject matter defined in the claims.

Embodiments of the invention provide biodegradable and biocompatible mutable collagenous tissue (MCT) and MCT-chitosan composites. These composite materials can be formed from various materials, such as hydrogels, biofilms, three-dimensional sponges, and nanofibers. MCT-chitosan composites are stronger and have better mechanical properties than known chitosan materials. The MCT component of the composites adds improved biocompatibility, cell adhesion, physical and chemical stability, and mechanical properties relative to the antimicrobial and hemostatic properties of the chitosan component, thereby significantly increasing the efficacy of the composite in therapeutic applications.

Accordingly, in one aspect, the invention provides a composition comprising a matrix of MCTs and chitosan (CHT), in which the MCT is isolated from marine invertebrates. In the matrix, CHT can bind to MCT through electrostatic interactions, such as hydrogen bonding and dipole-dipole interaction, to form the MCT-CHT composite. In one aspect, the MCT-chitosan composite comprises a polyelectrolyte crosslinked structure between the GAG and collagen in the MCT, and their interaction with N-glucosamine units in chitosan. The mutable collagenous tissue may comprise collagen and glycosaminoglycan (GAG).

In one aspect, the MCT can comprise collagen and chondroitin sulfate. The compositions, or the MCT used to form the composition, may be substantially composed of type I collagen. Chitosan may have a degree of deacetylation of from about 60% to about 99%. The average molecular weight of chitosan can be from about 20 kDa to about 400 kDa. In some embodiments, the mass ratio of the MCT in the composite may be greater than 0 to 10:90 of the mass ratio of chitosan in the composite. In some embodiments, the mass ratio of the MCT is from about 100:0 to 50:50 of the mass ratio of chitosan.

By selecting appropriate marine invertebrate sources (eg, echinoderms) and isolation procedures, the amount and proportion of MCT obtained with increased yields of collagen and glycosaminoglycans can be controlled. For example, MCTs higher in type I collagen, a fibrillar type of collagen that is a key structural composition of various connective tissues, can be isolated and used in the compositions described herein. Additionally, isolated MCTs may be biased or controlled for the nature of fibrillar collagen type (I, II, III, V, XI) and glycosaminoglycan (chondroitin sulfate, hyaluronic acid), as well as for their structural heterogeneity.

In one embodiment, the MCT can be isolated from marine invertebrates, such as echinoderms. In a more particular embodiment, the MCT will be isolated from echinoderms of marine invertebrates, such as sea urchins and sea cucumbers. In an even more particular embodiment, the MCT will be isolated from sea cucumbers.

In another embodiment, the MCT can be composed of collagen. In a more particular embodiment, the collagen can be fibrillar collagen type I, II, III, V or XI. In an even more particular embodiment, the fibrillar collagen is type I. By means of the isolation processes described herein, fibrillar type I collagen can be selectively isolated.

In another embodiment, the MCT may comprise collagen and glycosaminoglycan. In some embodiments, the glycosaminoglycan can include chondroitin sulfate, hyaluronic acid, heparin, keratan sulfate, heparan sulfate, and/or dermatan sulfate, or a mixture of both components. In another embodiment, the glycosaminoglycan in the MCT will comprise chondroitin sulfate and/or hyaluronic acid or a mixture of both components.

The MCT-chitosan composition can be a hydrogel, a biofilm, a 3D sponge, or a nanofiber. The nanoparticles can be formulated into various therapeutic agent delivery systems, such as oral solutions, IV solutions, or aerosols. Biofilms can form on wound healing, surface coatings, or packaging materials. 3D sponges can be used as sponges, such as for tissue engineering and wound dressing supports. The nanofibers can be formulated as wound dressing carriers or surface coating agents.

In some embodiments, the MCT and the MCT-CHT composite can be cross-linked, through physical and/or chemical processes. Physical crosslinking can be achieved by radiation treatment (UV, gamma) and/or heat treatment. Chemical crosslinking can be achieved by adding a crosslinking agent to the MCT or MCT-chitosan composite material, the amounts of crosslinking agent used in crosslinked biomaterials, relative to the MCT content, can be from about 0.1 to about 1.0 %. Crosslinking agents that can be used include glutaraldehyde, ethyl-dimethyl-carbodiimide (EDC)-N-hydroxysuccinimide (NHS), riboflavin, genipin, and the like.

The MCT-chitosan composite can also be formulated as a 3D sponge biofilm with improved water absorption, thermal stability, vapor permeation, and cell adhesion. In such embodiments, the biofilms and/or 3D sponges will be suitable as tissue engineering sponges and wound dressing supports for surgical and medical applications.

Also disclosed is a method of administering a bioactive agent to a mammal, the method comprising administering to a mammal the MCT-chitosan composite described herein. MCT-chitosan composites can form a nanoparticle, nanofiber, hydrogel, biofilm, or 3D sponge that encapsulates the bioactive agent, eg, a drug or nutrient. Examples of drugs, vitamins, and nutrients that can be incorporated into formulations include lipids such as fatty acids, including omega-3 and omega-6 fatty acids, fat-soluble vitamins (eg, vitamin A, D, E, and/or K), antibiotics (eg, amoxicillin, ampicillin, clindamycin, doxycycline, erythromycin, metronidazole, penicillin, tetracycline, vancomycin, and the like), probiotics (eg, lactic acid bacteria, bifidobacteria, and the like), dermal active compounds (eg , retinoic acid, tranexamic acid, hydrogen peroxide, hydroquinone, cysteamine, azelaic acid, tyrosinase inhibitors and the like), micronutrients such as p-carotene and/or ascorbic acid, proteins, and peptides. In some embodiments, the MCTs and other nutritional supplements may also be included in an MCT-chitosan composite matrix, or in a composition that includes an MCT-chitosan composite matrix.

The MCT and MCT-chitosan composites can be tailored to degrade at a range of rates under various conditions by varying the amounts of the components and the methods for preparing the MCT and composites. Therefore, a method for preparing MCT and MCT-chitosan composite material is also disclosed. The composition described herein can be used for the manufacture of biomedical devices and medicaments useful for the treatment of conditions such as bacterial infection and/or fungal infection, burns, diabetic foot and inflammatory conditions in a mammal, such as a human. .

In still other aspects, the MCT-chitosan composite biomaterials can be provided as therapeutic cosmetics, or cosmeceuticals, to promote collagen, scar healing, wound healing, melisma and chloasma reduction and other skin-related benefits, using MCT-chitosan composites in conjunction with a pharmaceutically acceptable topical carrier, including, but not limited to, a solution, suspension, liquid, gel, ointment, a lotion or a cream.

Brief description of the drawings

The following drawings form a part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention may be better understood by reference to the accompanying drawings in conjunction with the detailed description presented herein. The accompanying description and drawings may highlight a certain specific example, or a certain aspect of the invention. However, those skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

In the drawings:

Figures 1A and 1B represent the comparative structure between the general structure of collagen and the general structure of MCT.

Figure 2 represents the general structure of glycosaminoglycans.

Figures 3A and 3B depict the comparative morphology of collagen fibril structure between bovine collagen and MCT.

Figure 4 represents the general structure of chitosan.

Figures 5A and 5B are schematic illustrations of the manufacture of MCT-chitosan composites according to one embodiment.

Figure 6 is a schematic illustration of the fabrication of MCT-chitosan composite biofilms according to one embodiment.

Figure 7 is a schematic illustration of the fabrication of 3D MCT-chitosan composite sponges according to one embodiment.

Figure 8 is a schematic illustration of the fabrication of MCT-chitosan composite hydrogels according to one embodiment.

Figure 9A is a schematic illustration of the fabrication of electrospun MCT-chitosan nanofibers, and Figure 9B is a photograph of the resulting nanofibers according to one embodiment.

Figure 10A represents the effect of a cross-linking agent (0.1% v/v glutaraldehyde) on the mechanical properties of biofilms composed of MCT-CHT, and Figure 10B represents the swelling behavior of biofilms composed of MCT-CHT. CHT according to embodiments.

Figures 11A and 11B represent an illustrative representation of MCT-chitosan biofilms and their potential applications as wound dressing supports, and Figures 11C and 11D represent a morphological characterization of electrospun nanofibers composed of MCT-chitosan by scanning electron microscopy ( SEM).

Figure 12 is a schematic illustration of different MCT-chitosan composites, as used in GTR applications.

Figures 13A-13C depict the physical appearance of 3D sponges composed of MCT-chitosan (MCT/CHT) made in different mass ratios of MCT/CHT according to the embodiments.

Figure 14 depicts TGA thermograms for 3D sponges composed of MCT-chitosan formulated at different MCT/CHT mass ratios.

Figure 15 represents the water absorption capacity of different 3D sponges composed of MCT-chitosan formulated in different mass ratios of MCT/CHT.

Figures 16A (no cells added) and 16B (cells added) are SEM micrographs showing adsorption of ADSC cells on 3D MCT-CHT sponges.

Figure 17 represents a proliferation profile of ADSC cells cultured on 3D sponges composed of MCT-chitosan during an incubation period of 15 days.

Figure 18 depicts ATR-FTIR spectra of composite electrospun nanofibers (ESNF) made at different mass ratios of MCT-chitosan composites.

Figure 19 depicts a TGA thermal analysis of chitosan, MCT and MCT-chitosan compounds.

Figures 20A-20C are SEM micrographs of chitosan, and Figures 20D-20F are SEM micrographs of ESNF of MCT-chitosan, scale bar 10 pm (Figures 20A and 20D), scale bar 2 pm (Figures 20B and 20E), and a 200 nm scale bar (Figures 20C and 20F), in which the circles indicate the presence of droplets associated with a poor electrospinning process in chitosan ESNF, showing the improvement of the MCT-chitosan compounds to obtain electrospun nanofibers.

Figure 21 depicts the proliferation of L929 fibroblast cells co-cultured with ESNF chitosan, MCT and MCT-chitosan composite.

Figures 22A-22C are SEM micrographs showing cell adhesion on ESNF chitosan (Figure 22A), MCT-chitosan composite (Figure 22B), and MCT (Figure 22C), after 7 days of incubation. Figure 23 depicts gel electrophoresis (SDS-PAGE) analysis showing major protein bands for MCTs compared to collagen samples extracted from beef and chicken.

Figure 24 depicts gel electrophoresis (SDS-PAGE) analysis showing the efficiency of the sea cucumber MCT isolation process, as shown by the consistency in MCT protein bands from batch to batch.

Figure 25A represents the FTIR infrared spectra for MCT collagen compared to beef collagen, showing the chemical structural differences between both samples, and Figure 25B represents the comparative FTIR spectra showing the efficiency of the isolation process of MCT collagen. MCTs from sea cucumber, as shown by the consistency in the FTIR chemical profile of MCTs from batch to batch.

Figure 26 represents thermogravimetric analysis (TGA) of collagen samples showing differences in thermal behavior of MCT and calf collagen samples.

Figure 27 shows a distribution of collagen structural amino acid composition observed in mutable collagen tissue extracted from sea cucumber and compared to bovine collagen isolated from calf skin. Figures 28A and 28B are scanning electron microscopy (SEM) images showing the morphology and porosity of the MCT-chitosan dressing support (3D sponge) fabricated by a solvent casting technique.

Figures 29A and 29B are scanning electron microscopy (SEM) images showing the surface morphology and structure of the MCT-chitosan nanofiber dressing support fabricated by an electrospinning technique.

Figs. 30A and 30B are before and after images showing the effects of administration of scar cream according to one embodiment.

Figures 31A and 31B are additional before and after images showing the effects of scar cream administration according to one embodiment.

Figures 32A-32C are yet more before and after images showing the effects of scar cream administration according to one embodiment.

Figures 33A and 33B are still additional before and after images showing the effects of scar cream administration according to one embodiment.

Figures 34A-34D are yet more before and after images showing the effects of scar cream administration according to one embodiment.

Figures 35A-35C depict the structure of the MCT-CHT array.

Fig. 36 depicts a wound sponge and its characteristics and features according to one embodiment. Fig. 37 depicts the function of a 3D sponge according to one embodiment.

Fig. 38 depicts the function of a device according to one embodiment.

Detailed description

According to aspects of the invention, the present application describes new biocompatible and biodegradable composite materials comprising a combination of mutable collagenous tissue (MCT) and chitosan. MCT-chitosan composite materials are extremely versatile and can be formulated into various types of biomaterials, such as dermal patches, three-dimensional sponges, biodegradable sutures, and sponges for cell proliferation in tissue engineering, as well as hydrogels and biofilms for tissue engineering. tissue regeneration.

Additionally, the MCT and MCT-chitosan composite can also be formulated as a 3D sponge biofilm with improved water absorption, thermal stability, vapor permeation, and cell adhesion. In such embodiments, the biofilms and/or 3D sponges will be suitable as sponges for guided tissue regeneration in tissue engineering and wound dressing supports for surgical and medical applications.

Definitions: As used herein, certain terms have the following meanings. All other terms and expressions used in this specification have their conventional and usual meanings that would be understood by one skilled in the art. Such conventional and customary meanings may be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

The term "and/or" means any one of the elements, any combination of the elements, or all of the elements to which this term is associated.

The singular forms "un", "uno/una", and "el" or "la", include a plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, such that a compound X includes a plurality of compounds X.

The term "approximately" can refer to a variation of ±5%, ±10%, ±20%, or ±25% from the specified value. For example, "about 50" percent may in some embodiments mean a variation of 45 to 55 percent. For whole number ranges, the term "approximately" may include one or two whole numbers greater than and/or less than a recited whole number. Unless otherwise indicated herein, the term "about" is intended to include values, eg, weight percent, close to the recited range that are equivalent in terms of functionality of the individual ingredient, composition, or embodiment. In addition, a recited range (eg, weight percent, carbon groups, and the like) includes each specific value, integer, decimal, or identity within the range. Specific values listed herein and the like are for illustrative purposes only; they do not exclude other defined values or other values within defined ranges.

The term "one or more" is readily understood by those skilled in the art, particularly when read in the context of its use. For example, "one or more substituents on a phenyl ring" refers to one to five, or one to four, for example, if the phenyl ring is disubstituted.

The term "bringing into contact" refers to the act of touching, making contact with, or coming into close or immediate proximity, including at the molecular level, for example, to cause a chemical reaction or physical change, for example, in a solution or another reaction mixture.

An "effective amount" generally means an amount that provides the desired effect. Thus, an effective amount means a dosage sufficient to enhance the efficacy of treatment for a disease state or condition being treated. Therefore, the effective amount may vary depending on the patient, the disease and the affected treatment.

The term "patient" or "subject" refers to any animal, such as a mammal, including mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans.

With reference to the MCT, the term "substantially comprising collagen" means that the MCT comprises at least fibrillar collagen. For example, an MCT fibrillar collagen may comprise type I, II, III, V and/or XI collagen. In one aspect, Other compounds that may be specifically included or excluded from the composites described herein include fibrillar collagen type II, III, V and/or XI or combinations thereof. The term "glycosaminoglycan" refers to molecules comprising long unbranched polysaccharides containing a repeating disaccharide unit, including, for example, chondroitin sulfate, hyaluronic acid, heparin, keratan sulfate, heparan sulfate, and/or dermatan sulfate.

Mutable Collagen Tissue (MCT): Echinoderms are marine invertebrates widespread throughout the oceans and used as a food source for decades (eg sea cucumbers and sea urchins). They are also well known for their unique connective tissues, called mutable collagenous tissue (MCT), which are capable of rapidly changing their passive mechanical properties (stiffness and viscosity), under the control of the nervous system. MCTs are a unique feature of echinoderms and have been described in all five extant classes (IC Wilkie, "Mutable collagenous tissue: overview and perspectives", V. Matranga (Ed.), Echinodermata. Progress in Molecular and Subcellular Biology. Marine Molecular Biotechnology, Vol 5, Springer, Berlin (2005), pp 221-250 Mutable collagen structures consist of discontinuous collagen fibrils organized into bundles (fibrils) by an elastomeric network of fibrillin microfibrils and interconnected by a matrix transfer tissue consisting of glycosaminoglycan that binds to and aggregates the fibril.This type of tissue has recently been proposed as a potential source of inspiration for "smart dynamic biomaterials" for tissue engineering and regenerative medicine applications (A. Barbaglio, S. Tricarico, C. Di Benedetto, D. Fassini, AP Lima, AR Ribeiro, CC. Ribeiro, M. Sugni, F. Bonasoro, IC Wilkie, M. Barbosa, MDCandia Carnevali, "The smart connective tissue of echinoderms: a materializing promise for biotech applications", Cah. Biol. Mar., 54 (2013), p. 713-720; C. Di Benedetto, A. Barbaglio, T. Martinello, V. Alongi, D. Fassini, E. Cullora, M. Patruno, F. Bonasoro, MA Barbosa, MD Candia Carnevali, M. Sugni, "Production, characterization and biocompatibility of marine collagen matrices from an alternative and sustainable source: the sea urchin Paracentrotuslividus", Mar. Drugs, 12 (2014), pp. 4912-4933. In particular, sea cucumber membrane (a well-known MCT) can provide a sustainable and biocompatible source of native fibrillar collagen to produce thin membranes for regenerative medicine applications. Figure 1 shows the general structure of collagen, in this case bovine collagen, on the left, and the general structure of MCT on the right. In the case of the MCT, the right side of figure 1 shows a detailed view of a part of the structure, with a proteoglycan-CAG crosslink between interfibrillar matrices and between collagen fibrils.

Among the "blue biomaterials", collagen from marine invertebrates appears as a valid substitute for the most widely used mammalian-derived collagen (for example, bovine collagen, represented on the left side of figure 1). Mammalian-derived collagen is commonly used in a wide range of human applications, from large-scale uses, such as in the food, pharmaceutical/nutraceutical, and cosmetic industries, to more specific fields, such as cell culture and biomedical/clinical applications. However, due to allergy concerns, religious and social/lifestyle restrictions, reasons related to disease transmission (e.g., bovine spongiform encephalopathy), and the high costs of recombinant technologies, alternative collagen sources are constantly being investigated. to mammals (T.H. Silva, J. Moreira-Silva, A.L.P. Marques, A. Domingues, Y. Bayon, R.L. Reis, "Marine origin collagens and its potential applications", Mar. Drugs, 12 (2014), pp. 5881- 5901.

Glycosaminoglycans, the general structure of which is shown in Figure 2 below, are long unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit consists of an amino sugar (N-acetylglucosamine or N-acetylgalactosamine) together with a uronic sugar (glucuronic acid or iduronic acid) or galactose. Glycosaminoglycans are highly polar and attract water. Therefore, they are useful to the body as a lubricant or as a shock absorber, mainly located on the surface of cells or in the extracellular matrix (ECM).

Another advantage of echinoderm MCTs is the relative ease of obtaining a large number of native collagen fibrils, which maintain their original structure (Di Benedetto et al., 2014, cited above). In fact, most of the mammalian collagen is usually used in its hydrolyzed form (solubilized in acid), a characteristic that considerably reduces the mechanical performance of the membrane/sponge produced and that can be a limit in those biomedical applications where it is required. very resistant materials, with a three-dimensional organization of fibrils, (for example, regeneration of tendons/ligaments or reconstruction of the dermis). Echinoderm MCTs may be useful for easily and rapidly producing fibrillar collagen membranes with high similarity in terms of ultrastructural and mechanical characteristics to the physiological setting of connective tissue. Figures 3A and 3B show the comparative morphology of the collagen fibril structure of bovine collagen (figure 3a) and MCT (figure 3B). MCT fibrils are internally cross-linked by GAG (glycosaminoglycan), which provides more stability to the macromolecular structure of collagen and reduces its biodegradability, an important aspect for wound healing and an attribute that bovine collagen lacks. Figures 35A-35C depict fibril crosslinking using GAG. Figure 35A shows the macroscopic arrangement of collagen fibrils and illustrates that the aligned fibrillar structures are held together by intrafibrillar crosslinking networks determined by interactions between glycosaminoglycan (GAG) and the collagen core protein. Figure 35B shows the intrafibrillar crosslink networks in more detail. Figure 35C depicts the multidirectional stability of a tightly organized mesh, resulting in improved mechanical performance and biodegradation properties in vivo.

A specific field of regenerative medicine where MCT fibrillar collagen is suitable for commercial applications is guided tissue regeneration (GTR). One of the objectives of GTR is to reduce post-surgical tissue adhesions, a frequent and only partially resolved complication, which prevents correct tissue regeneration. Adhesions are abnormal junctions or mixtures of cells that form between tissues or organs after surgery or due to local inflammation. Only recently have researchers tried to produce effective and successful tools to overcome them. In fact, barrier membranes comprising several different biomaterials (for example, chitosan and hyaluronic acid) have been tested in GTR, but none of them showed all the necessary functional properties, the most important of which is to prevent cell penetration. in the underlying anatomic compartment (S. Tang, W. Yang, X. Mao, "Agarose/collagen composite scaffold as an anti-adhesive sheet", Biomed. Mater., 2 (2007), pp. S129-S134. Membranes based on echinoderm MCT have a porosity and a three-dimensional structure that can be modified as desired.

Chitosan: Chitin is a biopolymer composed of poly N-acetyl glucosamine. Chitin is the second most abundant biopolymer on earth, after cellulose. It is commonly found in the exoskeleton or cuticles of many invertebrates, such as the shells of marine arthropods, and in the cell wall of most fungi and some algae. Chitin is generally insoluble in water, but can be deacetylated by treatment with a caustic, such as sodium hydroxide, to form the soluble cationic polysaccharide chitosan. The chemical name for chitosan is poly(p-(1^4)-2-amino-2-deoxy-D-glucopyranose). Figure 4 shows the general chemical structure of chitosan.

Chitosan-based surgical dressings and bandages produced by HemCon Medical Technologies were recently approved by the US FDA for use as hemostatic dressings with proven antibacterial properties against a wide range of harmful organisms, including MRSA and Acinetobacter baumannii. Bandages and dressings can be used to quickly stop bleeding, including extensive arterial bleeding. Both the blood coagulation and antibacterial properties of the materials can be attributed to chitosan (see US Patent No. 7,482,503 (Gregory et al.). The MCT-chitosan composite can be used instead of chitosan in the compositions described therein, while the useful characteristics of chitosan, such as mucoadhesiveness, biocompatibility and biodegradability, are still maintained.

Chitosan is marketed by many chemical suppliers, such as Sigma Aldrich Co., St. Louis, MO. Chitosan is offered in different qualities, average molecular weights and degrees of deacetylation.

In some embodiments, the chitosan can be a "high molecular weight" chitosan. High molecular weight chitosan refers to chitosan having a number average molecular weight of at least about 100 kDa, and typically from about 170 kDa to about 400 kDa. In some embodiments, the high molecular weight chitosan can have a molecular weight of at least about 100 kDa, at least about 110 kDa, at least about 150 kDa, or at least about 200 kDa. In other embodiments, the high molecular weight chitosan can have a molecular weight of at least about 100 kDa to about 400 kDa, from about 120 kDa to about 400 kDa, from about 150 kDa to about 400 kDa, from about 170 kDa to about 400 kDa, 100 kDa to about 300 kDa, about 120 kDa to about 300 kDa, about 150 kDa to about 300 kDa, about 170 kDa to about 300 kDa. The "DA" value in Figure 4 can be any number or range that results in approximately the values for the N-acetyl-D-glucosamine content of chitosan described herein. As one skilled in the art would readily recognize, chitosan, as illustrated in Figure 4, can also be partially acetylated.

Other embodiments may include low molecular weight chitosan. Low molecular weight chitosan refers to chitosan molecules with less than 100 monomeric units (less than about 18 kDa or less than about 20 kDa). Molecular weights of chitosan can be determined, for example, by gel permeation chromatography and capillary viscosity.

The chitosan may have a degree of deacetylation which is typically at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%. Alternatively, the chitosan can be completely deacetylated.

MCT-Chitosan Composite Materials: Chitosan binds to the structural components in the MCT through electrostatic interactions, determined by its positively charged amino groups, allowing for the formation of strong hydrogen bonds and dipole-dipole interactions, due to the high molecular weights and charge density of both MCT and chitosan. These interactions allow the development of stable biomaterials, such as hydrogels, biofilms, 3D sponges and nanofibers. MCT-chitosan composites can be used in GTR to prepare dermal patches, cosmeceuticals, and dressings for wound healing applications. MCT-chitosan composites can also be used as hydrogels or sponge-like or scaffold-like materials for skin and cartilage tissue cultures, as matrices for tissue engineering, and as biocompatible coatings for biomedical devices.

MCT and MCT-chitosan composites provide greater biocompatibility, improved mechanical performance, and superior biodegradability compared to known synthetic and animal collagen-based devices. In addition to the direct biological effects of MCT-chitosan composites (antimicrobial, antifungal, and wound healing properties), the biomaterials can be used for controlled or targeted release systems to encapsulate therapeutic agents for oral, dermal, or respiratory administration.

Nanoparticles and biofilms can be prepared from MCT and chitosan, as described herein. MCT comprising collagen is particularly useful for preparing nanoparticles. Since MCT mainly comprises fibrillar collagen, MCT is well suited for preparing hydrogels, biofilms, 3D sponges, and nanofibers from MCT-chitosan composite material, providing composite biomaterials with superior mechanical performance compared to animal collagen-based biomaterials. acquaintances.

MCT and MCT-chitosan composite biomaterials can be crosslinked during their preparation by chemical treatment by reaction with biologically compatible crosslinking agents (glutaraldehyde, ECC/NHS) or by heat treatment under vacuum pressure. Swelling of a composite polymeric matrix can lead to faster degradation and reduced mechanical properties. Crosslinking reduces or prevents spontaneous swelling of composite biomaterials, which increases mechanical performance and handling.

In some embodiments, the MCT and MCT-chitosan composite may include or exclude polymers other than chitosan or MCT. For example, some embodiments include dextran, alginate, and/or cellulose-derived materials such as hydroxyethylcellulose; other embodiments exclude some or all of these. Some embodiments include synthetic polymers such as polyvinyl alcohol, polycaprolactone, or polyethylene oxides, while other embodiments exclude some or all of them.

Analysis of MCT, Chitosan, and Combination Products: Various methods can be used to analyze and evaluate MCT, chitosan, and their compound products. These techniques include mass spectrometry, mechanical properties (tensile strength) and swelling properties, scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize surface morphology, differential scanning calorimetry (DSC) for the thermal characterization of the composite biomaterial. The tensile strength and swelling properties of the composite biomaterials were characterized in ASTM measurements.

Analysis of the MCT and MCT-chitosan composites described herein indicate that the composites have improved stability, higher drug loading capacity, better drug release properties, better cellular uptake, higher porosity, better tensile strength and thermal stability compared to compositions including only chitosan. The materials are also non-cytotoxic in vitro.

Wound healing: MCT and MCT-chitosan composite biomaterials also have valuable properties for wound healing applications because they exhibit improved bacteriostatic activity relative to pure chitosan, higher biocompatibility, and improved mechanical properties. MCT-chitosan composites show an increase in the antimicrobial activity of chitosan. The composite materials bind to the negatively charged bacterial surface to disrupt the cell membrane. These properties can be applied for use in GTR applications by formulating injectable hydrogels, dermal patches and wound dressing supports, for example to promote the healing of ulcers and burns. MCT-chitosan composites can also be used as hemostatic agents in wound and surgical dressings.

MCT biomaterials and MCT-chitosan composites can be used in various biomedical applications. As a result of biocompatible properties such as good blood compatibility, enhanced mechanical performance and cell growth efficiency, MCT-chitosan composites can be used in surgical applications and regenerative medicine. The permeability of MCT-chitosan composite membranes can be controlled by plasma treatment. Consequently, such composite membranes can be used in dialysis.

United States Patent No. 7,482,503 (Gregory et al.) describes methods for the preparation of wound dressings. Wound dressings can be prepared according to such methods using the MCT materials and MCT-chitosan composites described herein instead of the chitosan biomaterial that Gregory et al. et al. describes. Additionally, MCT-chitosan composites can be used as coatings for medical devices, such as stents, catheters, and prostheses, to prevent harmful biofilm formation or bacteremia in patients and also to promote biomimicry and osseointegration. The formation of complexes of MCTs with chitosan promotes a surface modification of collagen fibrils that increases the porosity of their 3D sponge scaffolds, as shown in SEM micrographs. MCT has also been found to improve the physical properties of chitosan scaffolds, such as tensile strength, degree of swelling, and thermal stability, as shown by mechanical analysis and DSC calorimetry.

Tissue engineering: Tissue engineering (TE) research is based on the seeding of cells in porous biodegradable polymer matrices. A fundamental factor for successful seeding is the availability of good biomaterials that serve as a temporary matrix or scaffold for cells to proliferate and differentiate. Recently, chitosan and its derivatives have been described as attractive candidates for foam materials. because they degrade as new tissues are formed, eventually without inflammatory reactions or toxic degradation. In ET applications, the cationic nature of chitosan is primarily responsible for electrostatic interactions with anionic glycosaminoglycans, proteoglycans, and other negatively charged molecules.

The formation of complexes of MCTs with chitosan promotes a surface modification of chitosan films that increases the porosity of their 3D sponges, as shown in SEM micrographs. MCT has also been found to improve the physical properties of chitosan biofilms, such as tensile strength, degree of swelling, and thermal stability, as shown by mechanical analysis and DSC calorimetry. MCT biomaterials and MCT-chitosan composites can be used to control cell morphology and function and thus can be used as a tissue engineering scaffold or matrix in wound healing applications for GTR. MCT biomaterials and MCT-chitosan composites can also be chemically modified for ET applications. For example, the composites can be modified by grafting particular sugars onto the MCT backbone. Certain cells can clearly recognize specific sugars, thus providing specific recognition to antigen-presenting cells, such as B lymphocytes, dendritic cells, and macrophages.

The devices of the present invention are useful in therapeutic methods for treating various GTR-associated conditions in a mammal, which involves administering to a mammal having such a condition an effective amount of an MCT-chitosan composite of one or more embodiments of invention. A mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, pig, goat, and the like.

The following examples are intended to illustrate aspects of the present invention and are not to be construed as limiting the scope of the invention. One skilled in the art will readily recognize that the examples suggest other ways in which the invention could be practiced. It is to be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

examples

Example 1. MCT-Chitosan Composite Materials: Preparation, Data and Applications

Pharmaceutical grade chitosan (92% deacetylation grade calculated by 1 H NMR; average molecular weight 185 kDa calculated by specific viscometry) was purchased from Sigma Aldrich (St. Louis, MO, USA). The degree of deacetylation and average molecular weight distribution can be controlled in the production of MCT-chitosan composites to provide chitosan with higher or lower degrees of deacetylation and/or higher or lower average molecular weights.

Mutable collagenous tissue (MCT) was isolated from marine invertebrate echinoderms. Adult specimens of sea urchins, starfish and sea cucumbers were collected by divers in China, Tahiti and Japan, among other places, and immediately dissected. Peristomial samples of sea urchin, aboral walls of starfish arms and whole body walls of sea cucumber were collected and stored at -20 °C for the subsequent collagen extraction protocol described by Ferrario C., Leggio L. ., Leone R., Di Benedetto C., Guidetti L., Cocce V., Ascagni M., Bonasoro F., La Porta CAM, Candia Carnevali MD, Sugni M, "Marine-derived collagen biomaterials from echinoderm connective tissues", Sea Environ Res. Volume 128, pages 46-57. Animal collection and experimental handling were performed in accordance with the laws and regulations of each country. Sea urchins (peristomial membranes) and starfish (aboral arm walls) were dissected into small pieces, rinsed with artificial seawater and left in hypotonic buffer (10 mM Tris, 0.1% EDTA) for 12 h at room temperature (RT) and then in a decellularizing solution (10 mM Tris, 0.1% sodium dodecyl sulfate) for 12 h at room temperature. After several washes in phosphate buffered saline (PBS), the samples were placed in a deaggregation solution (0.5 M NaCl, 0.1 M Tris-HCl, pH 8.0, 0.1 M p-mercaptoethanol). , 0.05 M Na-EDTA). The obtained MCT suspension was filtered and dialyzed against 0.5M Na-EDTA solution (pH 8.0) for 3 hours at RT and against dH2O overnight at RT. Starfish samples underwent an additional step in 1 mM citric acid (pH 3-4) between decellularization and disaggregation solutions to remove calcium carbonate ossicles present in fresh tissue as much as possible. All steps were carried out under stirring conditions. Sea cucumber MCT was extracted from the whole body wall following a different protocol. Briefly, the starting tissue was cut into small pieces, placed in PBS and gentamicin (40 pg/ml) and left under shaking conditions at RT for at least 5 days to obtain an MCT suspension which was subsequently filtered. The suspensions obtained from the three experimental models were stored at -80 °C until use.

Preparation of MCT-chitosan composites: Mutable collagenous tissues (MCTs) were dissolved in 0.5% v/v acetic acid at room temperature overnight and degassed prior to preparation of chitosan biomaterials and composites. A chitosan solution was prepared by dissolving chitosan powder in an aqueous solution of acetic acid (0.5% v/v) at room temperature (RT). Once the chitosan powder was completely dissolved, the solution was filtered and degassed by vacuum filtration. Figure 5A schematically illustrates the dissolution and degassing of the chitosan solution, according to one embodiment. The chitosan solution (0.1-0.5 % w/v) was then mixed with MCT isolated from echinoderm (2.0-10.0 % v/v), in different molar ratios of MCT-CHT (100: 0, 80:20, 60:40, 50:50, 40:60, 8:80 and 10:90). The solution was allowed to react, with stirring, for 1 hour at RT. Figure 5B further illustrates the preparation of MCT-chitosan composites, according to one embodiment. The concentration of chitosan and MCT was controlled in the compounding process by adding different proportions of the respective components to provide the desired composition.

Preparation of MCT-chitosan composite biofilms:: Solutions of MCT-chitosan composites were poured into glass or silicone molds and slowly spread to form uniform liquid films. The liquid films were then evaporated at 80 °C for 24 hours or overnight at 40 °C, to provide 2D-cast composite biofilms. Figure 6 illustrates the pouring of MCT-chitosan biofilms, according to one embodiment.

Preparation of MCT-chitosan composite 3D sponges:: MCT-chitosan 3D sponges are prepared by a cross-linking method, determined by heat treatment under vacuum pressure. Composite sponges (diameter = 12 mm, thickness = 6 mm) were prepared by pouring/lyophilization techniques (Step 3c). One gram of a 2% w/w chitosan solution in water or 0.5% v/v in acetic acid was mixed with aqueous solutions of MCT (0.5 - 2.5% v/v), as illustrated in figure 7. The resulting mixture was poured into a suitable sized glass or silicone mold, frozen at -20°C and lyophilized to remove solvent to provide the porous 3D MCT-chitosan sponge. MCT-chitosan composite 3D sponges are physically similar to known chitosan sponges. However, composite 3D sponges have important additional properties, such as increased water retention (swelling), improved mechanical properties, and superior biocompatibility. MCT-chitosan composite 3D sponges can be used, for example, to provide improved hemostatic (blood coagulation) and wound dressings because the hemostatic effect of chitosan is increased due to the immunostatic properties of the MCT component. The addition of MCT also improves its mechanical performance, adhesion and cell growth.

Preparation of MCT-chitosan composite hydrogels:: The MCT (2% v/v), chitosan (1% w/v) composite solution was frozen at -20°C and lyophilized to remove solvent, leaving a powdery material. Two grams of the lyophilized MCT-chitosan composite were dissolved in 100 mL of deionized water and vigorously stirred while increasing the pH dropwise with a concentrated 6N NaOH solution, as illustrated in Figure 8. Once the solution reached a suitable pH value (~7.2), a composite hydrogel was spontaneously formed and the viscosity of the dispersion increased significantly. In another embodiment, MCT-chitosan hydrogels can also be made by regulating the final concentration of high molecular weight (HMW) chitosan between 2 and 10% w/v or by mixing the final MCT-chitosan composite material with a synthesis-enhancing additive. viscosity, such as hydroxyethylcellulose, glycerol or polyethylene glycol, among others.

Preparation of Electrospun Nanofibers of MCT-Chitosan Composite Material:: MCT-chitosan nonwoven nanofiber mats were prepared by an electrospinning technique, as illustrated in Figures 9A and 9B. The MCT-chitosan composite was dissolved in deionized water at a chitosan concentration ranging from 0.5 to 2.0 (% w/v). The concentration of the MCT-chitosan composite was adjusted so that its viscosity and conductivity were suitable for electrospinning. The dispersion was left overnight in a refrigerator (4°C) for complete hydration before electrospinning experiments. The maximum shear rate was according to the power law of materials in tubular geometry, with volumetric flow (Q) of 2.78 * 10.1° m3/s and an internal radius of the tube of 1 mm (R = 0.5 x 10 '3m). To improve the properties of the MCT-chitosan composite material for electrospinning, the samples were mixed with polyvinyl alcohol (PVA) as an electrospinning aid. PVA (10% w/v) was dissolved in water at 80 °C with vigorous stirring for 4 h. The MCT-chitosan and PVA composite dispersions were mixed in mass ratios of 100:00, 60:40, 50:50, 40:60 and 0:100. The mixed MCT-chitosan/PVA samples (5 ml) were subjected to electrospinning using an electrospinning device and a 30 kV power supply (Gamma High Voltage Research, Ormond Beach, FL , USA). The distance between the needle tip and the collector was set at 20 cm, the voltage was 20 kV, and the solutions were pumped at 1 ml/h. Nanofibers were collected on aluminum foil and stored in a desiccator for further characterization, as Figure 9A also illustrates.

Characterization of MCT-chitosan biofilms: The mechanical properties of biofilms composed of MCT-chitosan were evaluated by comparing tensile strength and swelling behavior. Swelling is the first stage in the physical degradation of biofilms. Rapid swelling promotes rapid and uncontrolled release of active compounds (eg, drugs and/or pesticides) from a biofilm matrix. Glutaraldehyde is commonly added as a crosslinking agent in the production of chitosan biofilms to decrease the rate of swelling. A disadvantage of using glutaraldehyde in a hydrogel formulation is the reduction in biofilm tensile strength. MCT-chitosan composite biofilms were cast according to the methodologies described above. In addition, cross-linked biofilms were formulated by first immersing pre-poured chitosan or MCT-chitosan composite biofilms in a glutaraldehyde solution (0.10% v/v) for 30 minutes, followed by extensive washing with deionized water, followed by culture at 80 °C for 2 hours.

Evaluation of the mechanical properties: The tensile strength measurements were made in a universal mechanical testing machine (model TEST 108 from GT Test, France, equipped with the Test Winner 920 software), with a crosshead speed of 10 mm/ minute and a 2 kN static load cell. Biofilms were cut into standard tensile specimens with a dumbbell-shaped knife (type H3) with a dimension of 17 mm * 4 mm * 0.08 mm (length * width * thickness). At least five samples of each type of biofilm were analyzed after an adequate storage period (3 and 20 weeks) at 50 ± 3% RH and 23 ± 2 °C in a humidity chamber (CIAt, France). The maximum tensile stress (TS) was calculated by dividing the maximum load to break the film by the cross-sectional area. MCT-chitosan composite biofilms showed higher tensile strength than chitosan biofilm alone. Figures 10A and 10B illustrate the mechanical behavior of different MCT-chitosan composite biofilms (at a mass ratio of 50:50 and 100:0, respectively) compared to chitosan (0:100) alone. Data are presented as [mean ± SD; n = 5]. The addition of a crosslinking agent (glutaraldehyde) reduced the tensile strength of all the biofilms compared to the non-crosslinked ones.

Evaluation of swelling behavior: The degree of swelling of chitosan biofilms and MCT-chitosan composite material was evaluated by gravimetric methods. Each dry biofilm was first weighed on an analytical balance (Wd). After weighing, the biofilms were immersed in distilled water for 60 min at room temperature. Biofilms were then removed from the water and weighed (Ws) at 5, 10, 20, 30, 40, 50, and 60 minutes. Prior to weighing on a high-precision balance, each biofilm sample was quickly removed from the water bath and blotted dry with tissue paper to remove excess water. After weighing, the biofilms were returned to the water. Next, the degree of swelling (%) of each biofilm sample was calculated according to the following equation:

Degree of swelling ( %) = [ ( Ws - Wd)/Ws] x100

The results indicate that the MCT-chitosan composite biofilms exhibited lower swelling rates and lower total degrees of swelling compared to the chitosan-alone biofilms. Figure 10B illustrates the degree of swelling of the MCT-chitosan hydrogels as a function of time.

These results indicate that the MCT-chitosan composites act as a strong cross-linking agent or its equivalent, which reduces the swelling of the composites while improving the tensile strength of the biofilm. The properties provided by the MCT were superior to those of a glutaraldehyde crosslinking agent in chitosan biofilms. Therefore, MCTs are suitable, reliable, and biocompatible "green alternatives" to glutaraldehyde for formulating biofilms for packaging, patches, and surgical biomaterials.

Characterization of MCT-chitosan composite wound dressing carriers (biofilms, 3D sponges, and electrospun nanofibers): MCT-chitosan composite wound dressing carriers (biofilms, 3D sponges, and electrospun nanofibers) were characterized according to their chemical profile using attenuated total reflectance with Fourier transform infrared spectroscopy (Nicolet 4700 ATR FT-IR, Thermo Scientific, Grand Island, NY, USA) and thermal properties using thermogravimetric analysis (TGA, Q100, TA Instruments, Lindon, UT, USA). Thermal analyzes (DSc and TGA) were performed at 5 °C/min over a scanning temperature range of 20 to 400 °C in a nitrogen atmosphere (20 mL/min). Figures 11A and 11B illustrate MCT-chitosan biofilms and their potential applications as wound dressing supports. The morphology of the nanofibers was examined by SEM (Leo 1530-FE, Zeiss, Cambridge, UK). The average fiber diameter was determined by analyzing at least 20 fibers in SEM images using ImageJ software. Figures 11C and 11D illustrate the morphology of the surface.

Advantages of MCT-chitosan composite biomaterials compared to chitosan biomaterials: MCT-chitosan composite biomaterials (Figure 12) provide significantly improved properties for various applications, compared to chitosan biomaterials. MCT-chitosan composite biomaterials can be prepared as nanoparticles, hydrogels, biofilms, 3D sponges, or electrospun nanofibers. Each of these forms of biomaterials can be used for a number of specific applications, and each of the composite biomaterials has significant advantages over chitosan biomaterials, as summarized in Table 1 below.

Table 1. Improvements of the MCT-chitosan composite compared to chitosan and collagen biomaterials for GTR applications.

Example 2. MCT-chitosan composite 3D sponges for GTR, wound healing and tissue engineering applications.

Chitosan (CHT) is described as biocompatible and bioabsorbable. In particular, CHT is considered a good wound healing accelerator. On the other hand, collagen (MCT) is one of the most widely used matrix biomaterials in tissue engineering. Highly porous MCT-only 3D sponges have been used to promote in vitro growth of many tissue types. Hybrid 3D sponge biomaterials were fabricated by mixing CHT and MCT, isolated from sea cucumber specimens, in different mass ratios, applying previously developed methodologies involving solvent pouring and lyophilization. Hybrid 3D MCT/CHT sponges were characterized according to their water absorption capacity, mechanical properties, thermal behavior (TGA) and morphology (SEM). Hybrid 3D sponges showed improved stability, higher porosity, higher thermal stability and mechanical properties, as well as higher biodegradation compared to simple 3D sponges. Incubation of cell cultures with adipose-derived stem cells (ADSCs) and SEM images showed that hybrid MCT/CHT 3D sponges allowed ADSCs to adhere, propagate, and grow in vitro.

Fabrication of MCT-chitosan hybrid 3D sponges:: Chitosan (CHT, 2.0% w/v) was dissolved in acetic acid (0.1% v/v) and slowly mixed with an aqueous solution of collagen (MCT , 5% w/v) to produce hybrid solutions in molar ratios of MCT-CHT 100:0, 80:20, 60:40 and 50:50, respectively. Hybrid MCT-chitosan 3D sponges were made by pouring each solution into glass molds, pouring solvent and lyophilizing for 48 hours. The sponges were cut into small fragments (12 mm in diameter and 3 mm thick) for later characterization and cell proliferation studies and storage in a desiccator at controlled relative humidity.

Physical and chemical characterization of MCT-chitosan hybrid 3D sponges: Light microscopy images of the fabricated hybrid sponges were captured on an inverted microscope (LIB-305, USA) at 4x magnification. The morphology of the MCT-chitosan hybrid 3D sponges was examined under a scanning electron microscope (SEM, JSM-5200, JEOL, USA) at 20kx magnification. The tilt angle of each sample was 30 degrees. Thermogravimetric analysis (TGA) was performed on a TGA-7 instrument (Perkin Elmer, USA). Sponge samples (5-10 mg) were poured onto an aluminum stand and analyzed under nitrogen (10 mL/min) at a heating rate of 10 °C/min according to a temperature program set between 50 and 600 °C. Uniaxial mechanical compression tests of hybrid 3D MCT/CHT sponges (n = 5 per condition) were performed under ambient conditions (20 °C and 50 % relative humidity, RH) using a load cell of a compression machine. universal tensile test (TensilonRTG, Japan) with a maximum force of 250 N. Compression (mm) and load (N) were collected at a crosshead speed of 5 mm/min. Compressive elastic moduli were calculated as the tangent slope of the stress-strain curves within the initial linear region of the compression curve. Compressive strength was calculated at 15% strain (within the region where the stress-strain curves were linear for all samples). The dried 3D sponge samples had a cylindrical shape with a diameter of 12 mm and a thickness of 3 mm measured with an electron micrometer (DMH Series 293, Mitotoyo, Japan).

Cell adhesion and proliferation studies: Adipose-derived stem cells (ADSC) were isolated from live horses. ADSC cells (~105 cells/cm2) were placed on top of each MCT-chitosan hybrid 3D sponge. A tissue culture plate (polystyrene) was used as a control. Cultures were placed in the incubator for 1 day and, upon removal, washed with phosphate buffered saline (PBS) and trypsinized. Aliquots of the resulting dissociated cell suspensions were counted in a Coulter multi-dimensional counter (Model 0646, Coulter Electronics, Hialeah, FL, USA). Only counts between 8 and 32 pm in diameter were used. Cell proliferation was also determined by cell counts as described above after 1, 3, 7 and 10 days of culture. In this experiment, six replicate samples were examined. Adhered and/or proliferated ADSC cells were fixed with glutaraldehyde (2.5% v/v) in 0.1 M PBS (pH 7.4) for 30 min and then rinsed with 0.1 M PBS. Fixed cells were lyophilized and plated with gold for morphological analysis by scanning electron microscopy (SEM, Hitachi Model S-2460N, Hitachi Ltd., Tokyo, Japan).

Data and statistical analysis: All data were expressed as mean ± standard deviation of at least three replicates. Statistical analysis was performed using JMP Pro (Version 10.0.0; SAS Institute Inc., Cary, NC, USA), setting p=0.05. The results were analyzed with bidirectional ANOVA models with interaction between the independent variables "sample" and "concentration" to evaluate significant differences.

Results and analysis

Physical and chemical characterization of hybrid MCT-chitosan 3D sponges: Chitosan physically binds to collagen through hydrogen bonding interactions, determined by its available amino and hydroxyl groups. This interaction allows the development of stable biomaterials, such as nanoparticles, biofilms, biofoams and tissue sponges (Madrigal-Carballo et al., Polymerliposome nanoparticles obtained by the electrostatic bio-adsorption of natural polymers onto soybean lecithin liposomes, Intl. J. Nanoparticles 5 (3 ) (2012) 196-209; Madrigal-Carballo et al., Protein-loaded chitosan nanoparticles modulate uptake and antigen presentation of hen egg-white lysozyme by murine peritoneal macrophages, Intl. J. Nanoparticles 3 (2) (2010) 179- 191;Ma et al., "A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a sponge of human neofetal dermal fibroblasts", Biomaterials, 22(4) (2001), pp. 331- 336). Figure 13A shows the fabricated MCT-chitosan hybrid 3D sponges. As seen in Figure 13A, the illustrated hybrid 3D sponges are made in different MCT/CHT mass ratios (50:50, 60:40, 80:20 and 100:0). Figure 13B shows light microscopy images of the sponges, and Figure 13C are sEm micrographs for each 3D sponge fabricated. In Figure 13C, the scale bars are 500 pm.

SEM micrographs in Figure 13C show a change in the surface morphology of simple chitosan sponges when combined with MCT. This change is shown by the decrease in apparent pore size with the addition of MCT in the mCt/CHT hybrid 3D sponge matrix. Therefore, the interaction of MCT with chitosan seems to provide a higher crosslink density, possibly determined by the higher number of possible hydrogen bonding interactions available between both macromolecules, which increases molecular alignment and compactness.

Figure 14 is a graph of thermal analysis of the hybrid 3D sponges of MCT-chitosan by thermogravimetry (TG), which shows that the thermal behavior for the hybrid 3D sponge of m Ct /CHT (50:50) was intermediate between both sponges. of MCT/CHT (100:0) and MCT/CHT (0:100) composite material. The hybrid 3D sponge incorporating MCT showed better thermal stability than the simple chitosan sponge, with an average decomposition temperature of 300 °C, corresponding to a 15-fold increase in thermal stability compared to the simple chitosan sponge. CHT.

Biological 3D sponges need sufficient mechanical properties to maintain their integrity after implantation. Consequently, a compression test was performed on 3D hybrid MCT-chitosan sponges to obtain the mechanical stress-strain curves and to calculate both the elastic modulus and the compressive stress (at 15% strain), respectively, as shown. shown in Table 2. The results show a positive effect on mechanical properties with the addition of MCT to the chitosan 3D sponge matrix. The MCT/CHT (50:50) hybrid 3D sponge showed an increase in Young's modulus of compression of approximately 85-fold, compared to a MCT/CHT (100:0) sponge. Likewise, it was observed that the compressive strength (at a strain of 15%) increased approximately 78 times for the system of MCT/CHT hybrid 3D sponge (60:40). The observed improvement in mechanical resistance can be associated with the formation of internal polymeric networks determined by hydrogen bonds, between collagen and chitosan that promote the mechanical stabilization of the matrix and, therefore, a suitable 3D sponge for possible purposes. implantation.

Table 2. Young's mechanical modulus for different MCT-chitosan hybrid 3D sponges. The results are ________________________________ expressed as the mean ± SD (= 5).________________________________

Sample Compressive Modulus (MPa) Compressive ^{Strength (MPa, at} _{15% strain)} MCT/CHT (100:0) 0.18 ± 0.07 0.12 ± 0.01

MCT/CHT (80-20) 0.34 ± 0.10 0.44 ± 0.02

MCT/CHT (60-40) 0.78 ± 0.55 0.51 ± 0.02

MCT/CHT (50-50) 1.22 ± 0.61 0.55 ± 0.03

Swelling properties are important in a sponge to promote hydration and cell growth. The 3D sponges of MCT-chitosan showed an intermediate behavior between the two simple sponges of MCT and chitosan, being the one with the highest proportion of chitosan the one that showed the highest water absorption capacity. These results can be explained by the presence of more hydrogen bonding points available in the chitosan biomolecule than in the collagen biomolecule, due to the reduced rotation and mobility of the functional groups determined by the quaternary structure of collagen.

Figure 15 shows the water absorption behavior obtained for different hybrid 3D sponges of MCT-chitosan. In Figure 15, data is plotted as mean ± SD, n = 3, and (*) = p < 0.05, compared to a plain 3D sponge of MCT/CHT (100:0) at the same point. of time. Figure 15 also includes an inset image, for illustrative purposes, to show the swelling behavior of the MCT/CHT (50:50) hybrid 3D sponge. The graph in Figure 15 shows the similarity below 70% relative humidity for all the different hybrid 3D sponges. Whereas, after reaching 85% relative humidity, significant differences between systems were observed as the proportion of MCTs in the hybrid 3D sponge matrix increased, showing differences in water absorption ranging 250-fold, showing the hybrid 3D MCT/CHT sponge (50:50) the highest water absorption capacity, almost 300% and the simple 3D MCT/CHT sponge (100:0) showing the lowest water absorption capacity, with values close to 50%. MCT/CHT (80:20) and MCT/CHT (60:40) show a water absorption capacity between MCT/CHT (100:0) and MCT/CHT (50:50).

The ability of a composite sponge to retain water is an important aspect in evaluating its properties and its suitability for skin tissue engineering. The water-binding capacity of the MCT-chitosan sponge could be attributed both to its hydrophilicity and to the maintenance of the three-dimensional structure. Chitosan and MCT have abundant hydrophilic groups, such as hydroxyl, amino, and carboxyl groups, which can retain water in their microstructure. MCT appears to promote an increase in hydrophilicity at higher relative humidity, leading to increased water absorption capacity. The water absorption values obtained for the MCT-chitosan sponges were in agreement with those of similar experiments published previously (Ma et al., "Chitosan porous sponges with improved biostability for skin tissue engineering", Biomaterials. Elsevier, 24(26) ( 2003), pp. 4833-4841, Chhabra et al., "Optimization, characterization, and efficacy evaluation of 2 % chitosan sponge for tissue engineering and wound healing", Journal of pharmacy & bioallied sciences, Medknow Publications, 8(4) (2016 ), p.300).

Growth of Adipose-Derived Stem Cells (ADSCs) on MCT-Chitosan Hybrid 3D Sponges: To study the interactions between ADSCs and MCT-chitosan hybrid 3D sponges, porous structures approximately 12 mm in diameter and 3 mm in diameter were used. of thickness. After 72 hours of culture, the ADSCs reached greater than 90% confluency in the sponges. Cross-sectional SEM images of the MCT/CHT (100:0) 3D sponge (Figures 16A and 16B) showed that after 72 hours of cell seeding, ADSCs adhered and spread over the surface of the sponge. 3D MCT-chitosan sponge (100:0) and completely fused with each other such that intercellular connections were not visible (FIG. 16B), compared to the sponge system without attached cells (FIG. 16A). In Figures 16A and 16B, the scale bar indicates 10 pm. The surface of the porous sponges was filled with cells and a pellicle that could be secreted by the precipitation of ECM from the cells (Lin, Li and Su, "Three-dimensional chitosan sponges influence the extra cellular matrix expression in Schwann cells", Materials Science and Engineering C, 42 (2014), pp. 474-478 Ji et al., "Biocompatibility study of a silk fibroin-chitosan sponge with adipose tissue-derived stem cells in vitro", Experimental and Therapeutic Medicine, 6(2 ) (2013), pp. 513-518).

Figure 17 demonstrates the level of proliferation in a 3D MCT-Chitosan (50:50) composite sponge after an incubation period of 15 days, where the legend (□) indicates a 3D MCT/CHT (100:50) sponge. 0), the legend (+) indicates a simple 3D sponge of MCT/CHT (0:100), and the legend (A) indicates a 3D composite sponge of MCT/CHT (50:50), and in which data are presented as mean ± SD, n = 5. (*) = p < 0.05, compared to a plain 3D MCT/CHT sponge (0:100) at the same time point. The three graphs represent a 3D sponge of MCT/CHT (0:100), MCT/CHT (100:0), and a 3D composite sponge of MCT/CHT (50:50). The MCT/CHT (50:50) composite 3D sponge showed a significant increase in the cell adhesion and proliferation, after three days of incubation with ADSC, in comparison with the 3D sponges of MCT/CHT (0:100) and MCT/CHT (100:0).

Polystyrene plate surfaces are known to have good cell adhesion and show rapid cell confluence during incubation (Jeong Park et al., "Platelet derived growth factor releasing chitosan sponge for periodontal bone regeneration," Biomaterials, 21(2 ) (2000), pp. 153-159). The degree of cell attachment and proliferation implies that the MCT-chitosan hybrid 3D sponges have good cell adaptability. Examination of cell proliferation in the sponge revealed that there was a statistically significant difference between the experimental and control groups after 3 days of incubation with ADSC. This may have resulted in an adaptation process that followed ADSC incorporation into the sponge. The fact that the difference between the samples started to become significant after 3 days of incubation may be because the ADSCs demonstrated an initial slow proliferation rate in the sponge, followed by a return to a more normal proliferation rate three days later. . Likewise, the 3D sponge composed of MCT/CHT (50:50) showed significantly higher cell adhesion and proliferation. High degrees of cell adhesion and proliferation of approximately 50-fold were observed in the MCT/CHT (50:50) composite 3D sponge compared to the MCT/CHT (0:100) and MCT/CHT (100:0) 3D sponges. ), during an incubation period of 15 days. MCT is known to stimulate the proliferation, chemotaxis, and collagen protein synthesis of osteoblast cells and ligamentous fibroblasts (Zhang et al., "Novel chitosan/collagen sponge containing transforming growth factor-p1 DNA for periodontal tissue engineering", Biochemical and Biophysical Research Communications, 344(1) (2006), pp. 362-369). Likewise, MCT has been reported to enhance stem cell proliferation (Costa-Pinto et al., "Adhesion, proliferation, and osteogenic differentiation of a mouse mesenchymal stem cell line (BMC9) seeded on novel melt-based chitosan/polyester 3D porous sponges", Tissue engineering. Part A, 14(6) (2008), pp. 1049-1057). The combination of MCT and chitosan can be very beneficial in increasing the cell proliferation response.

Conclusions: Unlike the rapid degradation of collagen, chitosan biodegrades slowly in vitro. Modification of 3D collagen sponges with chitosan resulted in improved mechanical strength, thermal stability, biocompatibility, and biodegradability. The MCT-chitosan hybrid 3D sponges provide a multidimensional structure for ADSCs both on the surface and in the interior, with the spatial features for cell adhesion, migration and proliferation and facilitating cell growth. After 72 hours of incubation, the ADSCs were observed to fuse and form a complete cell layer on the surface of the sponges, so that the surface was almost covered and only a few pores were visible, and some cells migrated inside the sponges. pores. Hybrid 3D MCT-chitosan sponges promoted ADSC adhesion, proliferation, and differentiation. SEM images showed that the large surface area of the porous sponges allowed ADSCs to attach, spread, and grow on the sponges. The flat morphology and excellent distribution in and around the interconnected porous structure indicated strong cell adhesion and cell growth. Consequently, MCT-chitosan hybrid 3D sponges exhibit biocompatibility for ADSC adhesion and are therefore good candidates for potential tissue engineering applications.

Example 3. Electrospun MCT-Chitosan Composite Nanofibers as Biocompatible Sponges for Cell Proliferation in Wound Healing and Tissue Engineering Applications

Electrospun nanofibers (ESNF) were prepared from MCT-chitosan composites. Polyvinyl alcohol (PVA) was used as a building agent. Mixed MCT-chitosan/PVA solutions of different volume ratios (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) were prepared and adjusted to be similar in viscosity and electrical conductivity and suitable for electrospinning. The morphology of ESNFs was examined by scanning electron microscopy (SEM), Fourier transform infrared spectrometry (FTIR), and differential scanning calorimetry (DSC). Studies were used to characterize the chemical composition and thermal characteristics of the nanofibers (NF). The ability of NFs to promote fibroblast cell proliferation in vitro was investigated using the optimized MCT-chitosan/PVA solutions. The results show that MCT-chitosan based ESNFs are very suitable for the growth of fibroblast cells, significantly better than PVA ESNFs. The results also show that MCT-chitosan is better than chitosan alone in promoting cell proliferation.

Preparation of MCT-chitosan composites for electrospinning: MCT-chitosan powders were swollen in deionized water with vigorous stirring until a homogeneous dispersion (1% v/v) was obtained. Dispersions were left overnight in a refrigerator (4°C) for complete hydration before characterization experiments. MCT-chitosan composite dispersions were characterized by measuring their viscosity by stress sweep test in a rheometer (C-VOR, Bohlin Instruments, Malvern, UK) with cone and plate geometry and electrical conductivity at 25 °C. with a conductivity meter (Orion Star A215, ThermoFisher, Waltham MA, USA) with an electrode conductivity constant of 0.7265 cm-1.

Electrospinning of MCT-chitosan composites: The MCT-chitosan composites were mixed with aqueous acetone (30% v/v) with vigorous stirring until a homogeneous dispersion was obtained. The concentrations of the composite solutions were adjusted so that their viscosities and conductivities were similar and suitable for electrospinning. The dispersion was left overnight in a refrigerator (4°C) to complete hydration before electrospinning experiments. A maximum shear rate was calculated for each sample of MCT-CHT composite material, according to the power law of materials in tubular geometry, with volumetric flow (Q) of 2.78 x 10'1° m3/s and a internal tube radius of 1 mm (R = 0.5 x 10-3 m).

To improve the properties of the electrospinning composite, the samples were mixed with PVA as an electrospinning aid. PVA (10% w/v) was dissolved in water at 80 °C with vigorous stirring for 4 h. The MCT-chitosan and PVA composite dispersions were mixed in volume ratios of 100:00, 60:40, 50:50, 40:60 and 0:100. The mixed MCT-chitosan/PVA samples (5 mL) were subjected to electrospinning using an electrospinning device and a 30 kV power supply (Gamma High Voltage Research, Ormond Beach, FL, USA) (see Figs. 9A and 9B). The distance between the tip of the needle and the collector was set at 20 cm and the solution was pumped at 1 ml/h. The nanofibers were collected on aluminum foil and stored in a desiccator for further characterization.

ESNF characterization of MCT-chitosan: ESNFs were characterized according to their chemical profile using attenuated total reflectance with Fourier transform infrared spectroscopy (Nicolet 4700 ATR FT-IR, Thermo Scientific, Grand Island, NY, USA). ) and thermal properties by thermogravimetric analysis (TGA, Q100, TA Instruments, Lindon, UT, USA). TGA analyzes were performed at 5 °C/min over a scanning temperature range of 100 to 400 °C under a nitrogen atmosphere (20 mL/min). The morphology of the nanofibers was examined by SEM (Leo 1530-FE, Zeiss, Cambridge, UK). The average fiber diameter was determined by analyzing at least 20 fibers in SEM images using ImageJ software.

Cell proliferation assay: Cell proliferation was determined by an MTT cell proliferation assay for the number of viable cells. Briefly, ESNFs previously collected under sterile conditions were placed in different wells of a sterile cell culture plate with media, and 3 ml of fibroblast cell suspension (L929, 1.5*105) was added to each treatment well. The cell culture plate was placed in a 37°C incubator for 3, 7 and 14 days, respectively. After incubation, the medium was removed and MTT solution was added to each treatment well at a 1:10 dilution with fresh medium. Plates were incubated at 37 °C for 4 h and absorbance at 560 nm was measured using a microplate reader (SpectraMax Plus, Molecular Devices, Sunnyvale, CA, United States). Following cell growth experiments (day 7), ESFNs were harvested and washed with medium, fixed with glutaraldehyde (2.5% v/v) at 4 °C for 2 h, and plated with gold before imaging by . SEM.

Statistical analysis: Statistical analysis was performed using AssistatVR software (Statistics, Arlington, TX). Experimental data were presented as mean ± SD. To compare control group and experimental groups, data were analyzed using a generalized linear model followed by least squares means (SAS; Cary, NC). Differences were considered statistically significant at P < 0.05.

Figure 18 shows plots of ESNF ATR-FTIR spectra of MCT/CHT (0:100) (indicated as "+") and MCT/CHT (100:0) (indicated as "o") separately, and also for different MCT/CHT composites (60:40, indicated as "*" and 40:60, indicated as "x"). The arrows in the figure indicate changes in the FTIR spectra of nanofibers associated with the addition of MCT to chitosan. In particular, increased absorption trends can be observed at both 1650 cm-1 and 1000 cm-1. These correspond respectively to the stretching frequencies of carbonyl (continuous arrow) and carbon-oxygen (dashed arrow), associated with the polysaccharide nature of chitosan. In general, the differences between the CHT and the MCT are evident in the figure. In the two composites, there is a deviation in certain places on the graph, but in many places, there is substantial overlap, which is not surprising in view of the relatively similar ratios of CHT and MCT.

Figure 19 shows the TGA thermal analysis of the MCT/CHT composites (100:0, 60:40, 40:60 and 0:100), indicating a positive effect on the thermal stability of chitosan as a result of the addition of MCTs. The MCT-chitosan (60:40) composite shows improved thermal stability compared to chitosan alone. The MCT-chitosan (60:40) composite shows a mean degradation temperature of approximately 320°C, while chitosan alone shows a mean degradation temperature of approximately 280°C.

Figures 20A-20F show SEM micrographs of electrospun chitosan nanofibers and electrospun MCT-chitosan nanofibers. In Figures 20A and 20D, the scale bar is 10 pm; in Figures 21B and 20E, the scale bar is 2 pm and in Figures 20C and 20F, the scale bar is 200 nm. The dotted circles indicate the presence of droplets associated with a poor electrospinning process in the chitosan ESNFs and show the improvement in ESNFs of the MCT-chitosan composites.

Figure 21 shows the proliferation of L929 fibroblast cells co-cultured with ESNF chitosan, MCT and MCT-chitosan composite (50:50). The figures show data from an MTT cell proliferation assay, using PVA ESNF as a control. The mean percentage of viable cells ± standard deviations correspond to experiments performed at three different times. Figs. 22A-22C are SEM micrographs showing cell adhesion on chitosan, MCT-chitosan composite, and MCT, respectively.

Conclusions: MCT-chitosan composite nanofibers were successfully fabricated by electrospinning, using PVA (10% w/v) as auxiliary agent. Electrospinning parameters for the fabrication of MCT-chitosan composite nanofibers were set and the mass ratio between MCT and chitosan was optimized to 50:50. ATR-FTIR analysis shows the presence of MCT-chitosan components in ESNFs. The thermal stability of MCT-chitosan composite ESNFs was compared with that of chitosan alone, suggesting that the addition of MCTs to chitosan improves the thermal stability of ESNFs. Fibroblast cell proliferation results indicate that MCT-chitosan ESNFs are suitable for cell growth and are significantly better than chitosan ESNFs or MCT alone, after 7 days of incubation. SEM images showed that the large surface area of the MCT-chitosan ESNFs allowed good adhesion, spreading, and growth of L929 fibroblast cells. The results indicate that MCT-chitosan ESNFs improve biocompatibility and activation for adhesion of fibroblast cells and are therefore useful in the development of dressing supports for applications in tissue engineering, regenerative medicine and as dressings for wound healing for the treatment of tissue burns.

The following description of Figs. 36-38 provides a summary of the structure and application of the GTR devices according to the embodiments, as well as the advantages of those devices.

Fig. 36 shows a 3D sponge according to one embodiment. In the figure, the sponge comprises MCTs composed of collagen and glycosaminoglycan, which are major components of the neodermis. The neodermis is new tissue that forms during wound healing. The collagen-GAG structure of the MCT promotes integrin binding during the healing process. As noted above, MCT is derived from a marine source, including, in one aspect, marine invertebrates, in a more particular aspect, echinoderms, and, in an even more particular aspect, sea urchins and/or sea cucumbers. sea. The ultrastructure of this MCT bears some resemblance to human connective tissue, pointing to salutary effects of GTR, such as neodermis formation.

The Figure 36 sponge also provides moisture management to promote wound closure and healing. In one aspect, the sponge has a gelling effect, promoting patient comfort through a cooling and soothing effect during healing. The crosslinking treatment of the MCT allows the sponge to be effective for 30 days, since the resulting structure has low physical degradation and resistance to proteolytic enzymes. In particular, the resulting structure targets and deactivates excess enzymes, such as matrix metalloproteinases (MMPs), that can degrade proteins. Such specificity and deactivation promote and improve wound closure and healing.

Figure 37 depicts a 3D sponge according to some embodiments. The sponge may be composed of MCT, or an MCT-CHT matrix, depending on the embodiment. This material in the sponge promotes the ingrowth of tissue and blood vessels. Other aspects of the sponge structure include an absorbent gel-forming composition that maintains a moist environment and controls exudate, promoting tissue healing and regeneration.

Figure 38 depicts a GTR device, which may be composed of MCT or an MCT-CHT matrix, sutured into an opening in the skin as part of treatment. The figure shows the sutures around the device, as well as the neodermis that forms under the device as part of the healing process. A portion of Figure 38 shows a detailed view of the neodermis, showing the MCT, alone or in a CHT composite matrix, with integrin binding sites.

Example 4. Cosmeceutical dosage forms (not according to the invention)

The following formulations illustrate representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of the MCT and MCT-chitosan (MCT-CHT) composition described herein, employing a dermatologically acceptable topical carrier. and/or pharmaceutical, including, but not limited to, a solution, suspension, liquid, gel, ointment, lotion, or cream. In the following examples, a gel or cream is provided: (i)

(i) Gel for GTR % by weight

Part A

MCT or 2.0 Composite Material

MCT-CHT

Polyethylene oxide 0.4

Carbopol 0.4 Water___________________________________ 90.9

Part B

Glycerin ethoxylate 0.5

Polyethylene glycol oleyl ether 1.0

Diethanolamine oleth-3 phosphate 0.8

Crodamol PMP 0.5

(continuation)

(i) Gel for GTR % by weight

Glucose ethoxylated methyl ester___________ 07

Part C

Triethanolamine 0.4

Fragrance________________________________ 0_1_

This formulation is prepared in separate steps as follows: Part A of the mixture was prepared by dispersing the Carbopol in water and then stirring the other components. All components of Part B were mixed and heated to 70°C. Parts A and B were then combined and the triethanolamine and fragrance (Part C) added. The resulting cream was stable and smooth and had good moisturizing qualities and excellent skin feel.

(ii) Cream for GTR 1% by weight

MCT or 2.5 Composite Material

MCT-CHT

Polyethylene oxide 0.3

Glycerin 5.0

Ethyl alcohol 15.0

Oleth-5 2.0

Glucose ethoxylated methyl ester 0.7

Preservative 0.3

Fragrance 0.15

Water up to 100.0

The formulation was prepared by mixing all the components together.

(iii) Cream for GTR 2% by weight

Part A

MCT or 2.5 Composite Material

MCT-CHT

Polyethylene oxide 1.5

Carbopol 0.5

Water___________________________________ 83.5

Part B

Vaseline 5.0

Polyethylene glycol 2.5 stearate

Diethanolamine oleth-3 phosphate 2.0

Silicone copolymer 1.5

Preservative______________________________ 0.3

Part C

Triethanolamine 0.5

Fragrance________________________________ 0_1_

This formulation is prepared in separate steps as follows: Part A of the mixture was prepared by dispersing the Carbopol in water and then stirring the other components. All components of Part B were mixed and heated to 70°C. Parts A and B were then combined and the triethanolamine and fragrance (Part C) added. The resulting cream was rich in excellent moisturizing qualities and did not leave the skin feeling greasy.

(iv) Scar cream composition

Ingredient Weight (mg) Composition (%)

Astaxanthin 50 0.05

Vitamin E 150 0 15

Royal jelly 100 0 10

Moringa extract/Vitamin C 200 0.20

MCT or composite material

_MCT-CHT 1000 1.00

Cream base q.s. 95.00

Total volume 100ml

Preparation procedure:

1- Weigh 150 mg of Vitamin E and mix it with the cream base dispersion with gentle agitation.

2- Weigh 1000 mg of MCT-CHT lyophilized powder (formulated in MCT:CHT mass ratios of 100:0 to 70:30) and dissolve it in 5.00 ml of acetic acid (0.5 M) prepared in distilled water (pH 3.2).

3- Weigh 200 mg of Moringa/Vitamin C extract and dissolve them in the aqueous solution of MCT-CHT.

4- Weigh 50 mg of astaxanthin and dissolve them in the aqueous solution containing MCT-CHT and Moringa/Vitamin C extract.

5- Weigh 100 mg of royal jelly and mix with the aqueous solution containing MCT-CHT, Moringa extract/Vitamin C and Astaxanthin.

6- Mix the aqueous solution containing MCT-CHT, Moringa extract/Vitamin C and Astaxanthin with the cream base containing Vitamin E, with continuous and gentle stirring, until obtaining a homogeneous dispersion. 7- Pour the homogenized scar cream formulation into a suitable glass container and store at room temperature.

The formulations described above can be prepared by conventional procedures well known in the art of pharmacy. It will be appreciated that the above cosmeceutical compositions may be varied in accordance with well known pharmaceutical techniques to accommodate different amounts and types of the MCT-CHT composite as active ingredient.

The scar cream composition just described was applied to several patients. Figures 30A and 30B show before and after results for one patient. Figure 30A is an image of a five year old cesarean section scar, prior to the application of the aforementioned scar cream. Figure 30B is a picture of the same scar after daily application of scar cream for two weeks. As is obvious from Figure 30B, the scar after treatment is shorter and has a different color, closer to the color of the surrounding skin.

Figures 31A and 31B show before and after results for another patient. Figure 31A is an image of a fifteen year old scar of a left knee joint, prior to the application of the aforementioned scar cream. Figure 31B is a picture of the same scar after application of the scar cream. As is obvious from Figure 31B, the scar has a different color, closer to the color of the surrounding skin.

Figures 32A to 32C show before and after results for another patient. Figure 32A is an image of a 25 year old appendectomy scar, prior to the application of the aforementioned scar cream. Figure 32B is an image of the same scar after daily application, and Figure 32C is an image of the same scar after eight days of daily application of a scar cream according to one embodiment. As is obvious from Figures 32B and 32C, the scar after treatment is shorter and has a different color, closer to the color of the surrounding skin.

Figures 33A and 33B show before and after results for another patient. Figure 33A is an image of a six-month-old knee surgery scar, and Figure 33B is an image of the same scar after seven days of daily application of the aforementioned scar cream. As is obvious from Figures 33A and 33B, the scar after treatment is much less prominent than before, and has a different color, closer to the color of the surrounding skin. The patient did not experience any adverse effects or hyperplasia.

Figures 34A-D show progressive results for yet another patient in the treatment of a scar. Figure 34A is an image of a burn scar, Figures 34B and 34C show images of the scar during daily application of the aforementioned scar cream, and Figure 34D shows an image of the scar after seven days of application. treatment. As is obvious from these figures, the scar after treatment is substantially scarred, with a color closer to the color of the surrounding skin compared to the appearance of the scar before treatment.

In addition to the treatment examples above, scar formation can occur in various areas of the body, including in and around the eyelids, for example as a result of injury, surgery, plastic surgery, or other repair and/or healing procedures. . The scar cream described above has also been used in this type of treatment, with good results.

As part of the above work as described, and the examples produced, work was done to determine and show structural differences between MCT extracted from sea cucumbers and bovine collagen isolated from calf skin tissue. Amino acid composition analysis of collagen samples was determined as described in (Cui F., Li Z., Zhang Y., Dong P., Fu X., Gao X., "Characterization and Subunit Composition of Collagen from the Body Wall of Sea Cucumber", Stichopus Japonicus", Food Chem. 100(3) (2007): 1120-5.). Briefly, collagen samples were hydrolyzed with 6 M HCl at 110 °C for 24 h, at The major amino acid composition of the hydrolyzate was then analyzed using a SYKAM S433D amino acid analyzer (SYKAM, Munich, Germany).

Table 3 below shows the amino acid composition for different samples of MCTs isolated from sea cucumber and bovine collagen (calf skin) isolated from calf skin. The analysis of calfskin collagen samples 1-4 in the following table was carried out in investigations that can be found in the following Articles: X. Cheng, Z. Shao, C. Li, L. Yu, MA Raja, C. Liu, "Isolation, Characterization and Evaluation of Collagen from Jellyfish RhopilemaesculentumKishinouye for Use in Hemostatic Applications", PLOS One, 2017, 0169731; Y. Han, JR. Ahh, JW. Wow, CK. Jung, SM. Cho, YB. Lee, S. B. Kim, "Processing Optimization and Physicochemical Characteristics of Collagen from Scales of Yellowfin Tuna (Thunnusalbacares)", Fisheries and Aquatic Sciences, Volume 13, Issue 2, 2010, pp.102-111; H. Li, BL Liu, LZ Gao, HL Chen, "Studies on bullfrog skin collagen", Food Chemistry, Volume 84, Issue 1, January 2004, pp. 65-69; P. Kit-tiphattanabawon, S. Nalinanon, S. Benjakul, and H. Kishimura. "Characteristics of Pepsin-Solubilized Collagen from the Skin of Splendid Squid (Loligoformosana)", Journal of Chemistry, Volume 2015, Article ID 482354, 8 pages.

Table 3. Amino acid compositions.

Composition (%i in p/p)

Amino Acid MCT Code Collagen from Skin Collagen Skin Collagen Skin Collagen from 1 Calf Skin Calf 2 Calf 3 Calf 4

one

0.00 0.00 0.00 0.00 0.00 0.00

7.33 7.46 NA 9.06 8.60 9.40

9.23 8.42 4.93 5.11 4.50 4.50

4.37 3.38 2.35 1.85 1.80 1.80

3.47 4.10 4.09 2.85 3.70 3.90

13.46 14.01 7.84 10.69 7.10 7.50

12.21 11.99 11.69 10.88 13.50 12.10

19.13 19.64 32.56 16.28 32.50 33.00

9.22 9.99 12.91 8.12 11.20 11.90

2.53 2.60 3.97 2.27 2.20 2.10

0.65 0.61 0.86 NA 0.60 0.60

1.72 1.64 2.35 1.37 1.10 1.10

2.73 2.83 3.82 2.86 2.50 2.30

1.46 1.24 0.91 0.48 0.30 0.30

1.42 1.19 1.94 1.84 1.30 0.30

0.33 0.24 0.06 1.96 0.50 0.50

0.65 0.50 4.27 0.27 2.70 2.60

8,54 8,65 5,36 4,19 5,10 5,10

8.54 8.65 5.36 4.19 5.10 5.10

Table 3 shows the amino acid compositions of mutable collagen tissue (MCT) and bovine collagen (BC). The major amino acids of the MCT were glycine (19.0%), glutamic acid (14.0%), proline (12.0%), alanine (9.0%), aspartic acid (9.0%), arginine ( 8.0%) and hydroxyproline (6.7%), which were similar to those found in bovine collagen shown in Table 4 (see references).

The primary structure of type I collagen is characterized by containing domains with continuous repetition of the Gly-X-Y sequence (where X is mainly proline and Y is mainly hydroxyproline), and very short N- and C-terminal regions called telopeptides (15 to 26 residues). of amino acids). The repetitive sequence Gly-X-Y in the a1 chain plays an important role in the formation of the triple helix of the secondary structure. See Gelse K, Poschl E, Aigner T.

2003. Collagens-structure, function, and biosynthesis. AdvDrugDeliverRev 55(12): 1531-46; Gomez-Guillen M, Gimenez B, Lopez-Caballero M, Montero M. 2011. Functional and bioactive properties of collagen and gelatin from alternative sources: a review. Food Hydrocolloid 25(8): 1813-27. As an amino acid with the lowest molecular weight, glycine residues arranged in the center of the triple helix can help the helix structure to fold compactly [3]. See Fraser R, MacRae T, Suzuki E. 1979. Chain conformation in the collagen molecule. J MolBiol 129(3):463-81. Therefore, glycine is the main amino acid in bovine collagen. According to previous references, the glycine content in bovine collagen ranges from 14 to 33%, approximately a quarter of the total amino acids, which is consistent with the glycine content of MCT (19%).

Figures 23 and 24 show the results of collagen matrix purity tests for marine collagen compared to beef and chicken collagen and show that marine collagen is as safe as beef and chicken collagen and therefore therefore, it is safe enough to be used in the applications just described. These figures show a triple helix structure, where the broad blue band at the bottom is an alpha helix, with a beta or gamma helix at the top. MCT 1 and MCT 2 in these figures indicate different batches of MCT, showing the reproducibility of the results.

In Figure 25A, the peaks indicated by "+" on the right side of the figure show the hydrolysis of calf collagen to solubilize it. In the comparison chart, MCT is indicated as "o". Figure 25B shows comparable FTIR spectra, indicating reproducibility of results with respect to the efficiency of the sea cucumber MCT isolation process, with MCT1 denoted as "*" and MCT2 as "x". Figure 25B shows a high degree of consistency in the MCT chemical profile from batch to batch.

Figure 26 shows the results of thermogravimetric analysis (TGA) of collagen samples and shows the differences in thermal behavior of the MCT samples. In figure 26, a reduced stability is shown as the lines head toward the bottom of the graph. The line for beef collagen shows increasing instability at 280°C, while the lines for MCT1 and MCT2 show stability even at 400°C. The graph shows better thermal stability for MCT than beef collagen, indicating an improved ability to store MCT-based products.

The imino acids (proline and hydroxyproline) are important amino acids comprising the Gly-X-Y repeat sequences in the a chain, due to their ability to maintain the stability of the collagen triple helix with its pyrrolidine rings. See Wong, DW. 1989. Mechanism and theory in food chemistry. New York: Van Nostrand Reinhold. The proline and hydroxyproline contents in the MCT were 12.0 and 7.0%, respectively, for a total imino acid content of 20.0%, values slightly lower than those observed for bovine collagen, as shown in figure 27.

It is observed that the contents of proline and hydroxyproline are related to the ambient temperature. See Zhong M, Chen T, Hu C, Ren C. 2015. Isolation and Characterization of Collagen from the Body Wall of Sea Cucumber Stichopus monotuberculatus. Food Chem 80(4):C671-C679. However, compared to bovine collagen, the ratio of Gly to imino acid content (Hyp/Pro) is lower, suggesting more efficient stabilization of the Gly-based triple helix by imino acids in the MCT.

Collagen stability can be affected by amino acid composition, especially imino acids. Proline (PRO) and hydroxyproline (HYP) can maintain the spatial structure of collagen with pyrrolidine rings, while the hydroxyl groups of hydroxyproline form hydrogen bonds with the adjacent chain to enhance triple helix stability. Analysis of the amino acid composition of MCT indicates a high presence of glycine and imino acids, suitable for the formation of a stable collagen triple helix that shows a lower degradation rate than bovine collagen.

Figure 27 is a bar graph of GLY, HYP, PRO and imino acid (sum of HYP and PRO) for MCT1 and MCT2 samples, compared to various calf collagen samples. One of the very favorable aspects of the MCT is the almost 1:1 ratio, consistently reproducible, between GLY and imino acids. By comparison, even the most favorable beef collagen sample, in the middle of the figure, does not show as close to a 1:1 ratio as samples MCT1 and MCT2. A high content of GLY compared to imino acids indicates less stability. While a high GLY content is favorable, a 1:1 ratio of GLY to imino acids indicates greater stability.

Figures 28A and 28B and Figures 29A and 29B are scanning electron microscopy (SEM) images of the MCT formed in two different ways. Figures 28A and 28B show the results of using a solvent casting technique to form the MCT-chitosan composite. Figure 28A shows the morphology of the structure of an MCT-chitosan dressing support (eg, a 3D sponge). Figure 28B indicates a high degree of porosity of the structure. Figures 29A and 29B show the results of using an electrospinning technique to form the MCT-chitosan dressing backing. As in Figures 28A and 28B, respectively, Figure 29A shows the morphology of the structure and Figure 29B indicates a high degree of porosity of the structure. Porosity is an important attribute in guided tissue regeneration, to promote cell adhesion and growth.

Claims (14)

1. A guided tissue regeneration (GTR) device, comprising: mutable collagenous tissue (MCT) and chitosan, wherein chitosan is present at greater than zero mass percent and less than or equal to 90, wherein chitosan is electrostatically bound to MCT forming a matrix, and the matrix of chitosan and MCT it is in the form of an MCT-chitosan composite material; and an applicator for administering the MCT or matrix to an area to be treated. 2. The device of claim 1, wherein the MCT is isolated from invertebrate marine organisms. 3. The device of claim 2, wherein the invertebrate marine organisms are selected from echinoderms. 4. The device of claim 3, wherein the MCT is isolated from echinoderms, selected from the group consisting of sea urchins and sea cucumbers. 5. The device of claim 4, wherein the MCT is isolated from sea cucumbers. 6. The device of claim 1, wherein the MCT comprises collagen and glycosaminoglycan (GAG). 7. The device of claim 1, wherein the MCT consists of fibrillar collagen selected from the group consisting of type I, type II, type III, type V or type XI, or a mixture of two or more of type I, type II, type III, type V and type XI. 8. The device of claim 7, wherein the MCT consists of fibrillar collagen type I. 9. The device of claim 6, wherein the glycosaminoglycan is selected from the group consisting of chondroitin sulfate, hyaluronic acid, heparin, keratan sulfate, heparan sulfate, or dermatan sulfate, or a mixture of two or more of chondroitin sulfate, hyaluronic acid , heparin, keratan sulfate, heparan sulfate, and dermatan sulfate. 10. The device of claim 9, wherein the glycosaminoglycan is selected from the group consisting of chondroitin sulfate and hyaluronic acid, or a mixture of chondroitin sulfate and hyaluronic acid. 11. The device of claim 1, wherein a mass ratio between MCT and chitosan is selected from the group consisting of 10:90, 20:80, 40:60, 50:50, 60:40, 80:20 or 90:10. 12. The device of claim 1, wherein the MCT-chitosan composite material is a hydrogel, a biofilm, a 3D sponge, an electrospun nanofiber, a nanoparticle, or a coating on the surface of a wound dressing support. 13. The device of claim 1, wherein the MCT-chitosan composite comprises a polyelectrolyte crosslinked structure between the GAG and the collagen in the MCT, which interacts with the N-glucosamine units of chitosan. 14. The device of any one of claims 1 to 13 for use in a method of effecting guided tissue regeneration (GTR), the method comprising applying the MCT-chitosan composite material to an area to be treated.