KR102232371B1 - Biomaterial devices and topical compositions for treatment of skin abnormalities - Google Patents

Abstract

Devices for induced tissue regeneration (GTR) include a matrix of chitosan and variable collagen tissue (MCT), wherein the chitosan is electrostatically bound to the MCT to form an MCT-chitosan complex material. The MCT can be isolated from echinoderms. The MCT-chitosan composite material may be formulated as a biofilm, 3D-cavernous body, hydrogel, or electrospun nanofiber, or the MCT-chitosan composite material may coat the surface of the biomaterial. The devices may comprise a wound dressing and tissue cavernous bodies including 3D cavernous bodies. Fields of application include tissue engineering and wound healing, and the wound healing includes burns and others related to induced tissue regeneration applications. The MCT and MCT-chitosan complex materials contained in pharmaceutically acceptable topical carriers have cosmetic uses for treating scars as well as various pigmentation problems including skin discoloration and black spots/stains.

Description

Biomaterial device and topical composition for treatment of skin abnormalities {BIOMATERIAL DEVICES AND TOPICAL COMPOSITIONS FOR TREATMENT OF SKIN ABNORMALITIES}

The present invention relates to biomaterial devices for guided tissue regeneration (GTR).

More specifically, the present invention relates to a topical composition comprising tissue sponges, wound dressings, cosmetic compositions and other devices and variable collagen tissue (MCT) to effect GTR. More specifically, the present invention relates to devices including composites of MCT and chitosan biopolymer (CHT) to exert the effect of GTR. The present invention relates to the treatment of burns, wounds, ulcers, and other lesions and related skin diseases by applying MCT alone or in combination in biomaterial devices to promote GTR. The present invention also relates to the treatment of skin abnormalities such as scars and chloasma by applying MCT and/or MCT-chitosan composites to cosmetic formulations, the discoloration including browning.

Natural polymers have been used in many pharmaceutical applications and medical device technologies. One natural polymer, chitosan (sometimes referred to herein as CHT), has been used in the manufacture 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 been used to prepare wound dressings and tissue cavernous bodies (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 composite materials have been tested for many of the same applications for chitosan to be evaluated, but many of these materials suffer from similar disadvantages including insufficient biocompatibility.

Embodiments of the present invention provide biodegradable and biocompatible variable collagen tissue (MCT) and MCT-chitosan composite materials. These composites can be formed from a variety of materials such as hydrogels, biofilms, three-dimensional sponges and nanofibers. The MCT-chitosan composite materials are stronger and have superior mechanical properties than known chitosan materials. The MCT component of the composites adds bio-stability, cell adhesion, physical and chemical stability, and improved mechanical properties to the antimicrobial and hemostatic properties of the chitosan component, significantly increasing the effect of the composite in therapeutic applications. .

Thus, in one aspect the present invention provides a composition comprising an MCT or a matrix of MCT and chitosan (CHT), wherein the MCT is isolated from echinoderms. In the matrix, the CHT may be connected to the MCT by electrostatic interactions such as hydrogen bonding and dipole-dipole interaction to form an MCT-CHT composite material. In one aspect, the MCT-chitosan composite includes a structure in which a polyelectrolyte is crosslinked between GAG (glycosaminoglycan) and collagen in MCT, and an interaction with N-glucosamine units of chitosan. The variable collagen tissue may be composed of collagen and glycosaminoglycan (GAG).

In one aspect, the MCT may include collagen and chondroitin sulfate. The compositions, or the MCT, were used to form the composition, and it may consist primarily of type 1 collagen. The chitosan may have a deacetylation degree of about 60% to about 99%. The chitosan may have an average molecular weight of about 20 kDa to about 400 kDa. In some embodiments, the mass ratio of the MCT in the composite materials may be 100:0 to 10:90 of the mass ratio of chitosan in the composite material. In some embodiments, the mass ratio of the MCT is about 100:0 to 50:50 of the chitosan mass ratio.

Through the procedure of selecting and isolating appropriate echinoderms, the amount and ratio of MCTs obtained with high yields of collagen and glycosaminoglycans can be controlled. For example, an MCT having a higher fibrillar collagen content, which is a major structural composition of several connective tissues, of type 1 collagen can be isolated and used in the compositions described herein. Additionally, the isolated MCTs are not only in terms of structural heterogeneity, but also in the essential aspect of the types of fibrillar collagen (I, II, III, V, XI) and glycosaminoglycans (chondroitin sulfate, hyaluronic acid). , Can be biased or controlled.

In one embodiment, the MCT may be separated from echinoderms. In a more detailed embodiment, the MCT may be separated from echinoderm animals such as sea urchins and sea cucumbers. In a more detailed embodiment, the MCT may be separated from sea cucumbers.

In another embodiment, the MCT may be composed of collagen. In a more detailed embodiment, the collagen may be type 1, type 2, type 3, type 5, or type 11 fibrillar collagen. In one more detailed embodiment, the fibrillar collagen is type 1. By the separation process described herein, type 1 fibrillar collagen may be selectively separated.

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

The MCT and MCT-chitosan composition may be a hydrogel, a biofilm, a 3D-cavernous body, or a nanofiber. Nanoparticles can be formulated into various therapeutic delivery systems such as oral solutions, fourth solutions, or aerosols. Biofilms can be formed as wound healing, surface coating, and packaging. 3D-cavernous bodies can be used as cavernous bodies such as tissue engineering and wound dressing templates. Nanofibers can be formulated as wound dressing templates or surface coatings.

In some embodiments, the MCT and MCT-CHT composite material may be crosslinked by 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, and the amount of the crosslinking agent used in the crosslinked biomaterials with respect to the above content of MCT may be about 0.1 to about 1.0%. Crosslinking agents that can be used include glutaraldehyde, ethyl-dimethyl-carbodiimide (EDC)-M-hydroxysuccinimide (NHS), riboflavin, genipin, etc. do. The MCT and the MCT-chitosan composite material can also produce a 3D cavernous biofilm with improved moisture absorption, thermal stability, vapor permeation and cell adhesion. In these embodiments, the biofilms and/or 3D-cavernous bodies may be suitable as cavernous bodies for tissue engineering, and as a wound dressing template for surgical and medical applications.

In the present specification, the method of the present invention also provides a method of delivering a bioactive agent to a mammal, comprising administering a mammalian MCT or an MCT-chitosan complex material. The MCT and MCT-chitosan composite materials may form nanoparticles, nanofibers, hydrogels, biofilms, or 3D-cavernous bodies encapsulating bioactive agents such as drugs or nutrients. Drugs, vitamins, and nutrients include fat. For example, fatty acids including omega-3 and omega-6, fat-soluble vitamins (e.g. vitamins A, D, E and/or E), antibiotics (e.g., amoxicillin, ampicillin, clindamycin, doxycycline) , Erythromycin, metronidazole, penicillin, tetracycline, vancomycin, etc.), probiotics (e.g. lactic acid bacteria, bifidobacteria, etc.), active skin compounds (e.g. retinoic acid, tranexamic acid, hydrogen peroxide, hydro Quinone, cysteamine, azelaic acid, tyrosinase inhibitors, etc.), trace elements such as β-carotene and/or ascorbic acid, proteins, and peptides. In some embodiments, MCTs and other nutritional supplements may be included in a composition comprising an MCT-chitosan complex matrix, or an MCT-chitosan complex matrix.

The MCT and MCT-chitosan composite materials can be customized to decompose at various rates under various conditions by varying the number of components and methods for preparing the MCT and the composite materials. Accordingly, the present invention also provides a method of manufacturing an MCT and MCT-chitosan composite material. The present invention further relates to the use of the composition for the manufacture of biomedical devices and medicaments useful for the treatment of conditions such as bacterial infections and/or fungal infections, burns, diabetic feet and inflammatory conditions in mammals such as humans as described herein. to provide.

In another aspect, the MCT and MCT-chitosan complex biomaterials are therapeutic cosmetics to promote collagenization, scar healing, wound healing, reduction of melasma and chloasma, and other skin-related benefits, or Medicinal cosmetics may be provided, and MCT or MCT-chitosan composites are used with a pharmaceutically acceptable topical carrier including, but not limited to, solutions, suspensions, liquids, gels, ointments, lotions, or creams.

Accordingly, there is a need for new materials that are biocompatible and biodegradable and have stability and mechanical properties and performance suitable for use in the treatment and therapy of humans and other mammals. These new substances and compositions may preferably have advantages over chitosan alone, for example additional and/or improved biocompatibility, high stability and improved physical and biological properties. Spongy tissues for delivery of therapeutic agents. , Wound dressings, cosmetics and/or systems, the availability of these materials will further benefit researchers in the fields of biomedical engineering, biomaterials and tissue engineering.

The following drawings are included to form a part of this specification and to further illustrate specific embodiments or various aspects of the invention. In any case, embodiments of the invention may be best understood by reference to the accompanying drawings in conjunction with the above detailed description presented herein. The above description and the accompanying drawings may highlight specific examples or specific aspects of the invention. However, those skilled in the art will understand that some of the above examples or aspects may be used in combination with aspects or other examples according to embodiments of the present invention.
In the drawing:
1A and 1B show a comparative structure between a typical collagen structure and a typical MCT structure.
Figure 2 shows the general structures of glycosaminoglycans.
3A and 3B show a comparative form of fibrillar collagen structure between bovine collagen and MCT.
4 shows the general structure of chitosan.
5A and 5B are schematic diagrams of the preparation of MCT-chitosan composite materials according to an embodiment.
6 is a schematic diagram of the production of MCT-chitosan composite biofilms according to an embodiment.
7 is a schematic diagram of the preparation of MCT-chitosan complex 3D-cavernous bodies according to an embodiment.
8 is a schematic diagram of the preparation of MCT-chitosan complex hydrogels according to an embodiment.
9A is a schematic diagram of the preparation of MCT-chitosan electrospun nanofibers, and FIG. 9B is a photograph of the nanofibers according to the embodiment.
10A shows the effect of a crosslinking agent (glutaraldehyde 0.1% v/v) on the mechanical properties of MCT-CHT composite biofilms, and FIG. 10B shows the expansion behavior of MCT-CHT composite biofilms according to an embodiment. Shows.
11A and 11B show representative examples applicable to MCT-chitosan biofilms and their wound dressing templates, and FIGS. 11C and 11D are MCT-chitosan composite electrospun nanofibers by scanning electron microscopy (SEM). Shows morphological characteristics.
12 is a schematic diagram of various MCT-chitosan composite materials used for GTR application.
13A-13C show the physical appearance of MCT-chitosan (MCT/CHT) composite 3D-cavernous bodies prepared with different MCT/CHT mass ratios according to embodiments.
FIG. 14 shows TGA thermograms for MCT-chitosan complex 3D-cavernous bodies formulated with different MCT/CHT mass ratios.
FIG. 15 shows the absorption capacity for different MCT-chitosan complex 3D-cavernous bodies formulated with different MCT/CHT mass ratios.
Figures 16A (no cells added) and 16B (cells added) show SEM microscopy showing the uptake of ADSC cells into MCT-CHT-3D-cavernous bodies.
Figure 17 shows the proliferation profile of ADSC cells cultured in MCT-chitosan complex 3D-cavernous bodies during a 15-day culture period.
18 shows the ATR-FTIR spectrum of composite electrospun nanofibers (ESNF) made of MCT-chitosan composites of different mass ratios.
19 shows thermal analysis of TGA for chitosan, MCT and MCT-chitosan composites.
Figures 20A-20C are SEM micrographs of chitosan, Figures 20D-20F are SEM micrographs of MCT-chitosan ESNFs, scale bars are 10 mm (Figures 20A and 20D), scale bars are 2 mm (Figures 20B and 20E). , And the scale bar 200 mm (Figs. 20C and 20F), where the circle indicates the presence of droplets associated with the erroneous electrospinning process in chitosan ESNF, and the degree of improvement of the MCT-chitosan composites to obtain electrospun nanofibers.
Figure 21 shows the proliferation of L929 fibroblasts cultured with chitosan, MCT and MCT-chitosan complex ESNFs.
22A-22C are SEM micrographs showing cell adhesion to chitosan (FIG. 22A), MCT-chitosan composite (FIG. 22B) and MCT (FIG. 22C) ESNFs after 7 days of culture.
FIG. 23 is a gel electrophoresis analysis (SDS-PAGE) showing major protein bindings to MCT compared to collagen samples extracted from calves and chickens.
Figure 24 shows a gel electrophoresis analysis (SDS-PAGE) showing the effect of the separation process of the MCT from sea cucumber, and the consistency of protein bindings to MCT from batch to batch is shown.
Figure 25A relates to the FTIR-infrared spectrum for MCT collagen compared to calf collagen, showing the chemical structure differences between the two samples, Figure 25B is a comparative FTIR spectrum showing the effect of the MCT separation process from sea cucumber As shown, the consistency of the FTIR chemical profile for the batch-to-batch MCT is shown.
26 shows a thermal capacity analysis (TGA) of collagen samples showing differences in thermal behavior for calf collagen samples.
Figure 27 shows the distribution of the collagen structure amino acid composition found in variable collagen tissue compared to bovine collagen extracted from sea cucumber and isolated from calf skin.
28A and 28B are scanning electron microscopy (SEM) images showing the morphology and porosity of an MCT-chitosan dressing template (3D-cavernous body) prepared by a solvent casting technique.
29A and 29B are electro-scanning microscopy (SEM) images showing the surface morphology and structure of the MCT-chitosan nanofiber dressing template prepared by electrospinning technology.
Figures 30A and 30B show before and after photographs showing the effect of administration of a scar cream according to an embodiment.
31A and 31B additionally show before and after photographs showing the effect of administration of a scar cream according to an embodiment.
32A-32C further show before and after pictures showing the effect of administration of a scar cream according to an embodiment.
33A and 33B further show before and after pictures showing the effect of administration of a scar cream according to an embodiment.
34A-34D show before and after photographs showing the effect of administration of a scar cream according to an embodiment.
35A-35C show the MCT-DHT composite structure.
36 is a view showing a wound cavernous body and its characteristics and characteristics according to an embodiment.
37 illustrates the function of a 3D cavernous body according to an embodiment.
38 is a diagram illustrating a function of a device according to an embodiment.

This application claims the benefit of U.S. Regular Application No. 16/123,986, filed September 6, 2018, which is incorporated by reference in its entirety in this application.

According to aspects of the present invention, this application describes novel biodegradable, biocompatible composite materials comprising a combination of variable collagen tissue (MCT) and chitosan. The MCT and MCT-chitosan composites are very versatile, e.g. skin patches, three-dimensional cavernous bodies, biodegradable sutures, and cavernous bodies for cell proliferation in tissue engineering, as well as hydrogels and biofilms for tissue regeneration. It can be composed of various types of biomaterials, such as.

Additionally, the MCT and MCT-chitosan composite material may also produce a biofilm such as a 3D-cavernous biofilm with improved water absorption, heat stability, vapor permeation and cell adhesion. In some embodiments, the biofilms and/or 3D-cavernous bodies would be suitable as cavernous bodies for guided tissue regeneration in tissue engineering, and wound dressing templates for surgical and medical applications.

Definition : Certain terms in this specification have the following meanings. All other terms and phrases used herein have a plain and general meaning that can be understood by a person of ordinary skill in the art. This clear and general meaning can be obtained by referring to the technical dictionaries of Hawley's Condensed Chemical Dictionary 14 ^th Edition, by RJ Lewis, John Wiley & Sons, New York, NY, 2001.

In the present specification, "one embodiment", "an embodiment", "exemplary embodiment", etc. may include specific aspects, features, structures, parts, or characteristics, but all embodiments necessarily It indicates that it does not contain structures, parts, or properties. In addition, these phrases may refer to the same embodiment mentioned in other parts of this specification, but are not necessarily the case. In addition, when a particular aspect, feature, structure, part, or feature is described in connection with an embodiment, the feature related to that aspect, feature, structure, part, or other embodiments is conventional in order to be described to a person skilled in the art. See if it is explicitly stated if it is within the knowledge of the technician.

The term "and/or" means one of the items, a combination of items, or all items related to the term.

The singular expressions of "a", "an", and "the" include plural expressions unless the context clearly interprets otherwise. In addition, for example, reference to “a compound” includes a number of such compounds, so compound X includes a number of compounds X.

The term “about” may refer to a change of ± 5%, ± 10%, ± 20%, or ± 25% of the specified value. For example, in some embodiments "about 50" percent may have a variation of 45 to 55 percent. In the case of an integer range, the term “about” may include integers greater than and/or less than the recited integer. Unless otherwise indicated herein, the term “about” includes values close to the stated range that are of equivalent value in terms of the functional aspects of the individual components, compositions, or examples, for example, by weight. Is intended to be. Additionally, stated ranges (eg, weight percentages, carbon groups, etc.) include specific values, integers, decimals, or ranges of identity. Certain values listed herein are for illustrative purposes only and do not exclude other defining values or other values within a defined range.

The phrase “one or more” is readily understood by the skilled person, especially when read in the context of its use. For example, when the phenyl ring is disubstituted, one or more substituents on the phenyl ring may refer to, for example, 1 to 5, or 1 to up to 4

The term "contact" refers to an act of touching, contacting, or bringing immediately or near, including at the molecular level, for example a physical change in a reaction mixture or solution or a physical change in another reaction mixture.

“Effective amount” generally means an amount that provides the desired effect. Therefore, an effective amount means an amount sufficient to improve the therapeutic efficacy for the condition or disease condition being treated. Thus, the effective amount may vary depending on the patient, the disease, and the treatment affected.

The terms such as "patient" or "subject" can be used as any of the mammals, including voles, mice, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, primates, and humans. Refers to an animal of.

In the context of MCT, the phrase “substantially comprising collagen” means comprising at least fibrillar collagen. For example, the MCT fibrillar type collagen may include type 1, 2, 3, 5, and/or type 11 collagen. In one aspect, the MCT fibrillar collagen will be characterized as type 1. Other compounds that may be explicitly excluded or included from the composites described herein include type 2, 3, 5 and/or type 11 fibrillar collagen or combinations thereof. The term "glucose aminoglycan" refers to long non-branched disaccharide units such as, for example, chondroitin sulfate, hyaluronic acid, heparin, keratan sulfate, heparan sulfate and/or dermatan sulfate. It refers to a molecule containing polysaccharides.

Variable Collagen Tissue (MCT): Echinoderms are marine invertebrates that are widespread in all seas and have been used as food for decades (eg sea cucumbers and sea urchins). They are also well known for their unique connective tissue called variable collagen tissue (MCT), which can rapidly change passive mechanical properties (stiffness and viscosity) under nervous system control. MCT is a unique feature of echinoderms and has been described in all five existing classes (IC　Wilkie, "Variable Collagen Tissue: An Overview and Perspective," V.　Matranga　(Ed.), 　Echinodermata. Progress in Molecular and Subcellular Biology. Marine Biology. Marine Molecular Biotechnology, 　 vol. 5, 　 Springer, 　 Berlin 　 (2005), pp. 　221-250.). The variable collagen structures are composed of discontinuous collagen fibers organized into bundles by an elastomer network of fibrillin microfibrils, and glycosamino acids that bind and aggregate into the fibers. They are interconnected by a stress-transfer matrix composed of glycans. This type of tissue has recently been presented as a 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, "Smart Connective Tissues in Echinoderms: Viability for Biotech Applications," Cah. ,　54　(2013), pp.　713-720; C.　Di Benedetto, A.　Barbaglio, T.　Martinello, V.Alongi, D.　Fassini, E. Cullora, M.　Patruno,　F.Bonasoro,　Cansa,MA Carnevali, 　M. Sugni, "Production and characterization and biocompatibility of marine collagen matrices from alternative and sustainable sources: sea urchin Paracentrotus lividus," Mar. Drugs, 　12　 (2014), pp. 　4912-4933. ). In particular, the sea cucumber membrane (well known MCT) can provide a sustainable and biocompatible natural fibrous collagen source to create thin films for regenerative medicine applications. 1 shows a general collagen structure. The left is the case of bovine collagen, and the right is the general structure of MCT. In the case of MCT, the right side of FIG. 1 shows an exploded view of a part of the structure having proteoglycan-CAG cross-links between matrices between fibrils and collagen fibers.

Among the "blue biomaterials", marine invertebrate collagen presents the most commonly used mammalian-derived collagen (eg, bovine collagen, shown on the left in Fig. 1) as an effective substitute. Mammalian-derived collagen is routinely used in human applications, from large-scale applications such as food, pharmaceutical/functional food industries and cosmetics to more targeted fields such as cell culture and biomedical/clinical applications. However, due to allergy issues, religious issues and social/lifestyle constraints, disease transmission-related reasons (e.g. bovine spongiform encephalopathy), and the high cost of recombinant technology, sources of collagen for mammals continue to be studied (TH　Silva,　J). .　Moreira-Silva, 　ALP　Marques, 　A. Domingues, 　Y.　Bayon, 　RL　Reis, "Collagen of marine origin and its potential applications", Mar. Drugs, 　12　 (2014), pp.

Glycose aminoglycans showing the general structure in FIG. 2 at the bottom are long unbranched polysaccharides composed of repeating disaccharide units. The repeating unit of amino sugar (N-acetylglucosamine or N-acetylgalactosamine) is composed of uronic acid (glucuronic acid or iduronic acid) or galactose. Glycose aminoglycans are highly polar and attract water. Therefore, they are mainly useful for the body as lubricants or shock absorbers located on the cell surface or in the extracellular matrix (ECM).

Another advantage of echinoderm MCTs is that they can easily obtain large amounts of natural collagen fibrils that retain their original structure (cited in Di Benedetto et al., 2014,). In fact, most mammalian collagens are generally used in a hydrolyzed (solubilized acid) form, which strongly inhibits the mechanical performance of the resulting membrane/cavernous body, and requires a fibrin three-dimensional structure (e.g., Tendon/Ligament Regeneration or Dermal Regeneration) may be limited in biomedical applications. Echinoderm MCTs can be useful for quickly and easily producing fibrillar collagen membranes having high similarity in terms of superstructure and mechanical properties to the physiological condition of connective tissue. Figures 3A and 3B show a comparative form of collagen fibrous structure for bovine collagen (Figure 3A) and MCT (Figure 3B). MCT fibrils are crosslinked internally by glucoseaminoglycans (GAG), imparting more stability to the macromolecular structure of collagen and reducing biodegradability, an important aspect of wound healing, and bovine collagen deficiency properties. Figures 35A-35C show crosslinking of fibrils using GAG. Figure 35A shows the macroscopic arrangement of the collagen fibrils, and the ordered fibril structures are driven by the interaction between glycosaminoglycan (GAG) and the core protein of collagen by a cross-linking network between fibrils. Show that they stay together. 35B shows the crosslinking network between fibrils in more detail. Figure 35C shows the multidirectional stability of a strongly structured mesh, resulting in improved mechanical performance and biodegradability in bio.

A specific field of regenerative medicine suitable for commercial use of MCT fibrillar collagen is induced tissue regeneration (GTR). One of the above goals of GTR is to reduce post-operative tissue adhesion, a common and partially resolved problem, interfering with proper tissue regeneration. Adhesion is a mixture of cells or abnormal adhesions formed between tissues or organs after surgery or due to local inflammation. Only recently have researchers tried to produce effective and satisfactory tools to overcome them. Indeed, barrier-membrane containing several different biomaterials (e.g. chitosan and hyaluronic acid) were tested for GTR, but did not show all of the required functional properties, most importantly, the underlying anatomical compartment. (S.　Tang,　W.　Yang,　X.　Mao, "Agarose/collagen composite skeleton as an anti-adhesion sheet", Biomed. Mater., 　2　 (2007), pp.　S129-S134). Echinoderm MCT-based membranes have a porous and 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 generally found in the exoskeletons or epithelium of many invertebrates, such as the shells of marine arthropods, and in the cell walls of most fungi and some algae. Chitin is generally insoluble in water, but can be deacetylated by treatment with an oxidizing agent such as sodium hydroxide to form water-soluble cationic polysaccharides. The chemical name of chitosan is poly(β-(1→4)-2-amino-2-deoxy-D-glucopyranose). Figure 4 shows the general chemical structure of chitosan.

Chitosan-based bandages and surgical dressings produced by HemCon Medical Technologies have recently been approved by the United States. FDA for use as a hemostatic bandage has proven antibacterial properties against a wide range of harmful organisms, including MRSA and acinetobacter baumannii. The bandages and dressings can be used for rapid hemostasis, including extensive arterial bleeding. The blood coagulation and the antimicrobial properties of these substances can be attributed to chitosan (US Pat. No. 7,482,503 (Gregory et al.) is incorporated herein by reference). The MCT-chitosan composite material may be used instead of chitosan in the composites described herein, but useful characteristics of chitosan such as mucosal adhesion, biocompatibility, and biodegradability can still be maintained.

Chitosan is Sigma Aldrich Co., St. It is commercially available from many chemical suppliers such as Louis, MO. Chitosan comes in a variety of grades, average molecular weights, and degrees of deacetylation.

In some embodiments, the chitosan may 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, typically about 170 kDa to about 400 kDa. In some embodiments, the high molecular weight chitosan may 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 molecular weight of the high molecular weight chitosan is about 100 kDa to about 400 kDa, about 120 kDa to about 400 kDa, about 150 kDa to about 400 kDa, about 170 kDa to about 400 kDa, about 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 value of "DA" in FIG. 4 may be any number or range, and it may be approximately the values of the N-acetyl-D-glucosamine content of chitosan described herein. As can be quickly recognized by one of ordinary skill in the art, the chitosan shown in FIG. 4 can also be partially acetylated.

In other embodiments, low molecular weight chitosan may be included. Low molecular weight chitosan refers to a chitosan molecule having 100 or less monomer units (about 18 kDa or less or about 20 kDa or less). The molecular weight of chitosan can be determined, for example, by gel permeation chromatography and capillary viscosity.

The chitosan may generally have a degree of deacetylation of 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 material : Chitosan binds to MCT structural components by electrostatic interactions driven by positively charged amino groups, and strong hydrogen bonding and dipoles due to both MCT and chitosan high molecular weight and packing density -Can form dipole interactions. These interactions enable the development of stable biomaterials such as hydrogels, biofilms, 3D-caverns, and nanofibers. MCT-chitosan complex materials can be used in GTR to prepare skin patches, cosmetics, and dressings for a wound healing effect. MCT-chitosan composite materials can be used as tissue engineering matrices for culturing skin and cartilage tissue, such as hydrogels or cavernous-like materials or skeletons, and as biocompatible coatings for biomedical devices.

MCT and MCT-chitosan composite materials provide high biocompatibility, improved mechanical performance, and superior biodegradability compared to known synthetic and animal collagen-based devices. In addition to direct biological effects in the MCT-chitosan composites (antibacterial, antifungal and wound healing properties), the biomaterial can be used in a targeted or controlled output system to encapsulate therapeutics for oral, skin, or respiratory delivery. have.

As described herein, nanoparticles and biofilms can be prepared from MCT and chitosan. MCTs containing collagen are particularly useful for producing nanoparticles. Since MCT mainly contains fibrillar collagen, MCT is suitable for manufacturing MCT-chitosan complex hydrogels, biofilms, 3D-cavernous bodies and nanofibers, and has excellent mechanical performance compared to known animal collagen-based biomaterials. It provides a composite biomaterial with

The MCT and MCT-chitosan composite biomaterials can be crosslinked during preparation by chemical treatment, either by reaction with biologically compatible crosslinking agents (glutaraldehyde, ECC/NHS) or by heat treatment under vacuum. The swelling of the composite polymer matrix can lead to rapid decomposition and reduction of mechanical properties. Crosslinking prevents or reduces spontaneous expansion of composite biomaterials, and increases mechanical performance and handling.

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

Analysis of MCT, Chitosan and Combination Products : A variety of methods can be used to evaluate and analyze MCT, chitosan, and combinations thereof. These techniques include mass spectrometry, mechanical properties (tensile strength) and expansion properties, scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize surface morphology, and differential scanning calories for thermal characterization of composite biomaterials. A method of measurement (DSC) is included. The tensile strength and expansion properties of the composite biomaterials were characterized in ASTM measurements.

The analysis of the MCT and MCT-chitosan composites described herein shows that the composite materials have improved stability, higher drug loading capacity, improved drug release properties, improved cell uptake, when compared to compositions containing only chitosan. It is shown to have greater porosity, improved tensile strength, and thermal stability. These substances are also non-cytotoxic in vitro.

Wound Healing : MCT and MCT-chitosan composite biomaterials also have useful properties of wound healing effect, as they exhibit improved bacteriostatic activity, improved biocompatibility and improved mechanical properties with respect to pure chitosan. The MCT-chitosan composites were found to have increased chitosan antibacterial activity. The composites bind to the negatively charged bacterial surface and disturb the cell membrane. These properties can be applied to GTR use, for example, by formulation of injectable hydrogels, skin patches, and wound dressing templates used to promote ulcer and burn healing. The MCT-chitosan composites can also be used as hemostatic agents in wounds and surgical dressings.

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

U.S. Patent No. 7,482,503, referenced above (Gregory et al.), describes such methods of making wound dressings. Wound dressings can be prepared according to the method of using the MCT and MCT-chitosan composite materials described herein, instead of the chitosan biomaterial described in Gregory et al. In addition, the MCT-chitosan composites can be used as coatings for medical devices such as stents, catheters, and artificial implants to prevent harmful biofilm formation or bacteremia in patients, and to promote biomimetic and skeletal synthesis. . MCT complexation with chitosan promotes the surface modification of collagen fibrils, which increases the porosity of the 3D-cavernous skeleton, as shown by SEM microscopy. MCT has also been found to improve the above physical properties of chitosan skeletons such as tensile strength, expandability, and thermal stability, as shown by mechanical analysis and DSC calorimetry.

Tissue Engineering : Tissue engineering (TE) research is based on sparging cells into porous biodegradable polymer matrices. A major factor for successful application is the availability of superior biomaterials that serve as temporary matrices or scaffolds for cells to proliferate and differentiate. Recently, chitosan and its derivatives have been reported as attractive candidates for cavernous-like substances because they degrade as the new tissues are formed, and eventually there is no inflammatory reaction or toxic decomposition. In TE applications, the cationic nature of chitosan is primarily responsible for electrostatic interactions with anionic glycosaminoglycans, proteoglycans, and other negatively charged molecules.

MCT complexation with chitosan promotes the surface modification of chitosan films, which increases the porosity of 3D-cavernous bodies, as shown by SEM microscopy. MCT was also found to improve the above physical properties of chitosan biofilms, such as tensile strength, expandability, and thermal stability, as indicated by mechanical analysis and DSC calorimetry. The MCT and MCT-chitosan composite biomaterials can be used to control the function and morphology of cells, and thus can be used as a tissue engineering skeleton or matrix in the wound healing process for GTR. The MCT and MCT-chitosan composite biomaterials can also be chemically modified for TE use. For example, these composites can be modified by implanting specific sugars into the MCT spine. Certain cells can specifically recognize these specific sugars and thus provide specific recognition for antigen presenting cells such as B-cells, dendritic cells, and macrophages.

Cosmetic Formulations : The present invention also provides formulations comprising the MCT and MCT-chitosan composites described herein for use as therapeutic cosmetics (cosmeceuticals). MCT and MCT-chitosan composites in powder or solution form can be added to basic cosmetic formulations to form pharmaceutical and/or functional cosmetic products. These cosmetic compositions may be formulated with dermatological and/or pharmaceutically acceptable topical carriers, but are not limited to solutions, suspensions, liquids, gels, ointments, lotions, or creams. The compositions release MCT and CHT into the tissue over a long period of time and promote collagen synthesis, scar healing, wound healing, reduction of melasma/sparse or other skin discoloration, and other benefits to the skin.

Cosmetic compositions can be formulated by general techniques known to those of ordinary skill in the art, such as the techniques described in US Pat. No. 9,980,894 and US Pat. No. 9,962,464 (Herrmann et al.), both of which are described herein. Incorporated by reference.

Examples of drugs, vitamins and nutrients are fatty acids including omega-3 and omega-6 fatty acids, fat-soluble vitamins (e.g. vitamins A, D, E and/or K), water-soluble vitamins (e.g. For example, vitamin C, thiamine, riboflavin, niacin, pantothenic acid, vitamin B6, folic acid, vitamin B12), antibiotics (e.g., amoxicillin, ampicillin, clindamycin, doxycycline, erythromycin, metronidazole, penicillin, tetracycline, vancomycin, etc.), Probiotics (e.g., lactic acid bacteria, bifidobacteria, etc.), active skin compounds (e.g., retinoic acid, tranexamic acid, hydrogen peroxide, hydroquinone, cysteamine, azelaic acid, tyrosinase inhibitors, etc.) and It can be incorporated into formulations containing the same liquid phase. The formulation consists of micronutrients such as beta-carotene and/or ascorbic acid, proteins, and peptides.

These compositions and formulations generally contain at least 0.1% MCT or MCT-chitosan composite materials. The percentages of the compositions and formulations can of course vary and can conveniently be between about 2% and about 60% of the weight of a given unit dosage form. For therapeutically useful compositions, the amount of the MCT and MCT-chitosan complex substances is such that an effective dosage level can be obtained.

Cosmetic compositions and the like may also contain: a binder such as xanthan gum, acacia, corn starch or gelatin; Excipients such as dicalcium phosphate; Disintegrants such as corn starch, potato starch, alginic acid, and the like; And/or lubricants such as magnesium stearate; Some specific cosmetic compositions may contain such types of materials, vegetable oils, or liquid carriers such as polyethylene glycol. The liquid carrier or carrier is, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof, It can be a solution or a liquid dispersion. The proper fluidity can be maintained, for example, by maintenance of the particle size in the case of dispersions or the use of surfactants. Prevention of the action of certain microorganisms can be caused by various additional antimicrobial and antifungal agents, such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thiomeric acid, and the like. In many cases it will be desirable to include, for example, sugars, buffers or isotonic agents such as sodium chloride. For example, the use of an absorption delaying agent such as aluminum monostearate and gelatin in the composition can lead to delayed absorption of injectables. Of course, any substance used to prepare any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amount used.

For topical administration, the MCT and MCT-chitosan composite materials can be applied in pure form. However, it will generally be preferred to be solid or liquid for administering them to the skin as compositions or formulations. Examples of such compositions or formulations are those in combination with a dermatological and/or pharmaceutically acceptable topical carrier.

Useful solid carriers include subdivided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, dimethylsulfoxide (DMSO), alcohols or glycols or water-alcohol/glycol mixtures, and the MCT or MCT-chitosan composite materials can be dissolved or dispersed to an effective level, and non-toxic interface It can be selectively dissolved or dispersed with the aid of the active agents. Adjuvants such as flavorings and additional antimicrobial agents may be added to optimize the properties for a given application. The resulting liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pumped or aerosol sprayers.

To act directly on the skin of the user by using thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, denatured celluloses or denatured mineral substances with liquid carriers, it is diffusible. Pastes, gels, ointments, soaps, etc. can be used directly.

Examples of useful dermatological compositions that can be used to deliver MCT or MCT-chitosan complex substances to the skin are known in the art; For example, Jacquet et al. (US Pat. No. 4,608,392), Geria (US Pat. No. 4,992,478), Smith et al. (US Pat. No. 4,559,157), and Wortzman (US Pat. No. 4,820,508) are all incorporated herein by reference. Included by reference.

Aspects of the present invention provide methods for treating various conditions associated with GTR in mammals, including administration to a mammal under conditions having an effective amount of MCT or MCT-chitosan composite according to one or more embodiments of the present invention. . Mammals include primates, humans, rodents, canines, felines, cattle, sheep, horses, pigs, goats, and the like.

The following examples are for explaining aspects of the present invention and should not be construed narrowly to the scope of the present invention. One of ordinary skill in the art will readily appreciate that the above examples suggest other ways in which the present invention may be practiced. It should be understood that numerous variations and modifications may occur within the above scope of the invention.

[Experimental Example]

Experimental Example 1. MCT-chitosan composites: manufacturing, data and applications

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

Variable collagen tissue (MCT) was isolated from echinoderm. Adult specimens of the sea urchin, starfish and sea cucumber were collected at different locations by Chinese, Tahiti, and Japanese scuba divers and immediately dissected. 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 Mar Environ Res. Volume 128, pp. Samples of sea urchin moss membranes, aboral arm walls and body walls of sea cucumbers, described in 46-57, were collected and stored for collagen extraction protocol at -20 °C. I did. Animal collection and experimental manipulation were carried out in accordance with national laws and regulations. Sea urchin (lichen membranes) and starfish (parenteral arm walls) were dissected into small pieces, rinsed with artificial seawater, left in a low concentration buffer (10 mM Tris, 0.1% EDTA) at room temperature for 12 hours, then dehydrated. -The cell solution (10 mM Tris, 0.1% sodium dodecyl sulfate) was allowed to stand at room temperature for 12 hours. After washing several times in phosphate buffered saline (PBS), the samples were placed in a digestion solution (0.5 M NaCl, 0.1 M Tris-HCl pH 8.0, 0.1 M beta-mercaptoethanol, 0.05 M EDTA-Na). The obtained MCT suspension was filtered and dialyzed against _{dH 2} O with 0.5M EDTA-sodium solution (pH 8.0) for 3 hours at room temperature. Starfish samples were subjected to 1 mM citric acid (pH 3-4) addition step between decellularization and digestion solutions to remove as much of the calcium carbonate vesicles present in fresh cells as possible. All steps were carried out under stirring conditions. Sea cucumber MCT was extracted from the whole body wall according to different protocols. Briefly, the starting tissue was cut into small pieces, placed in PBS and gentamicin (40 μg/mL), filtered, and then placed under stirring conditions at room temperature for at least 5 days to obtain an MCT suspension. The suspensions obtained from the three experimental models were stored at -80 °C until use.

Preparation of MCT-chitosan composites : Variable collagen tissues (MCT) were dissolved in acetic acid (0.5% v/v) overnight at room temperature, and biomaterials and composites using chitosan were degassed prior to fabrication. Chitosan powder was dissolved in an aqueous acetic acid solution (0.5% v/v) at room temperature to prepare a chitosan solution. After the chitosan powder was completely dissolved, the solution was filtered and degassed by vacuum filtration. 5A schematically shows the dissolution and degassing of the chitosan solution according to an embodiment. The chitosan solution (0.1-0.5% w/v) is echinoderm at different MCT-CHT molar ratios (100:0, 80:20, 60:40, 50:50, 40:60, 20:80 and 10:90) It was mixed with the MCTs (2.0-10.0% v/v) isolated from. The solution was stirred and reacted at room temperature for 1 hour. 5B further shows the manufacture of the MCT-chitosan composites according to an embodiment. The concentrations of chitosan and MCT were controlled in the process of forming composite materials by adding different proportions of each of the components to provide the desired composition.

Preparation of MCT-chitosan composite biofilms : The MCT-chitosan composite solutions were injected into glass or silicone molds and slowly diffused to form liquid films. The liquid films were evaporated at 80ºC for 24 hours or overnight at 40ºC to provide a 2D casting of composite biofilms. 6 shows a process of injecting the MCT-chitosan biofilms according to an embodiment.

Preparation of MCT-chitosan complex 3D-cavernous bodies: MCT-chitosan 3D-cavernous bodies are prepared by a crosslinking method driven by heat treatment under vacuum. Composite sponges (diameter = 12 mm, thickness = 6 mm) were prepared by casting/freeze drying techniques (step 3c). As shown in Figure 7, 2% w/w aqueous chitosan solution or 1 gram of 0.5% v/v acetic acid is mixed with aqueous MCT solution (0.5-2.5% v/v). The resulting mixture was poured into a glass or silicone mold, frozen at -20 °C, and freeze-dried to remove the solvent, to give the MCT-chitosan porous 3D-cavernous body. The MCT-chitosan complex 3D-cavernous bodies are physically similar to known chitosan spongy bodies. However, the composite 3D-cavernous bodies have significant additional properties such as high moisture retention (expansion), improved mechanical properties, and good biocompatibility. The MCT-chitosan complex 3D-cavernous bodies can be used, for example, to provide an improved wound and hemostatic (blood clotting) dressing, where the hemostatic effect of chitosan is increased by the immunodynamic properties of the MCT component. Because it becomes. The addition of MCT also improves mechanical performance, cell adhesion and growth.

Preparation of MCT-chitosan complex hydrogels : MCT (2% v/v), chitosan (1% w/v) complex solution was frozen at -20 °C and freeze-dried to remove the solvent, leaving a powdery substance . As shown in FIG. 8, 2 grams of the freeze-dried MCT-chitosan composite was dissolved in 100 mL deionized water, and the pH was increased by applying it as a concentrated solution of NaOH 6N with strong stirring. When the solution reached an appropriate pH value (~7.2), a composite hydrogel spontaneously formed, and the viscosity of the dispersion increased significantly. In another embodiment, MCT-chitosan hydrogels may also be used to control the final concentration of 2 to 10% w/v high molecular weight (HMW) chitosan or the final MCT-chitosan composite and hydroxyethylcellulose, glycerol, or polyethylene. It can be prepared by mixing a viscosity enhancing agent such as glycol, and the like.

Preparation of MCT-chitosan composite electrospun nanofibers : As shown in Figs. 9A and 9B, non-woven MCT-chitosan nanofiber mats were manufactured by electrospinning technology. 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 the viscosity and conductivity were suitable for electrospinning. For complete hydration before electrospinning experiments, the dispersion was placed in a refrigerator (4°C) and left overnight. The maximum shear rate is a tubular volume flow rate (Q) of 2.78 x ^{10 -10} m ³ /s and a radius of 1 mm (R = 0.5 x 10 ^-3 m) inside the tube according to the power method data. To improve the MCT-chitosan composite properties for electrospinning, as an electrospinning aid, samples were mixed with polyvinyl alcohol (PVA). PVA (10% w/v) was dissolved in water with vigorous stirring at 80°C for 4 hours. The mixed dispersions of the MCT-chitosan composite and PVA were mixed in a mass ratio of 100:00, 60:40, 50:50, 40:60 and 0:100. MCT-chitosan/PVA mixed samples (5 mL) were spun 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 to 20 cm, the voltage was set to 20 kV, and the solution was pumped at 1 mL/h. As shown in Figure 9A, the nanofibers were collected in aluminum foil and stored in a dryer for further characterization.

Characteristics of MCT-chitosan biofilms : The mechanical properties of the MCT-chitosan composite biofilms were evaluated by comparing tensile strength and expansion behavior. Expansion is the first step in the physical decomposition of biofilms. Rapid expansion promotes rapid and uncontrolled release of active compounds (eg drugs and/or pesticides) from the biofilm matrix. Glutaraldehyde is generally added as a crosslinking agent in the production of chitosan biofilms to slow the swelling rate. The disadvantage of using glutaraldehyde in hydrogel formulations is the reduction in tensile strength of the biofilm. MCT-chitosan composite biofilms were injected according to the methodologies previously described. In addition, before injection of chitosan or MCT-chitosan composite biofilms, the first immersion was performed in a glutaraldehyde solution (0.10% v/v) for 30 minutes, and then thoroughly washed with deionized water, and then at 80°C for 2 hours. By drying, crosslinked biofilms were prepared.

Evaluation of mechanical properties : Tensile strength measurements were performed with a universal mechanical test machine (TEST 108 model of GT Test in France equipped with TEST Winner 920 software) using a cross head speed of 10 mm/min and a 2 kN fixed load cell. The biofilms were cut into standard tensile samples from a dumbbell-shaped knife (H3 type) having a size of 17 mm X 4 mm X 0.08 mm (length X width X thickness). At least 5 biofilm samples of each type were tested at 50±3% RH and 23±2°C in a humidity chamber (CIAT, France) after an appropriate storage period (3 and 20 weeks). The maximum tensile stress (TS) was calculated by dividing the maximum load to break the membrane by the cross-sectional area. MCT-chitosan composite biofilms showed higher tensile strength than chitosan biofilm alone. 10A and 10B show the mechanical behavior of different MCT-chitosan composite biofilms (50:50 and 100:0 mass ratios, respectively) compared to chitosan alone (0:100). Data are [mean ± SD; n = 5]. This addition of a crosslinking agent (glutaraldehyde) reduced the tensile strength of all biofilms compared to the uncrosslinked ones.

Evaluation of expansion behavior : The degree of expansion of chitosan and MCT-chitosan composite biofilms was evaluated by gravimetric methods. Each dry biofilm was first measured with an _{analytical balance (W d ).} After weighing, the biofilms were immersed in distilled water for 50 minutes at room temperature. _{The biofilms were removed from the water and the weight (W s} ) was measured at 5, 10, 20, 30, 40, 50, and 60 minutes. Before weighing with a high precision balance, each biofilm sample was quickly removed from the water bath and wiped with tissue paper to remove excess water. After weighing, the biofilms were returned to water. The degree of expansion (%) of each biofilm sample was calculated according to the following equation:

Expansion (%) = [(Ws-Wd)/Ws] x 100

The above results show that the MCT-chitosan composite biofilms show a lower degree of expansion and a lower total degree of expansion compared to the chitosan biofilms. 10B shows the degree of expansion of the MCT-chitosan hydrogels as a function of time.

These results indicate that MCT-chitosan composite materials act as strong crosslinking agents or equivalents, thereby reducing the expansion of the composite materials and increasing the tensile strength of the biofilm. The properties provided by MCT were superior to those of the glutaraldehyde crosslinker in chitosan biofilms. Therefore, MCT is a suitable, reliable and biocompatible'green alternatives' to glutaraldehyde to formulate packaging, patches, and surgical biomaterials.

Characteristics of MCT-chitosan composite wound dressing templates (biofilms, 3D-cavernous bodies, and electrospun nanofibers): The MCT-chitosan composite wound dressing templates (biofilms, 3D-cavernous bodies and electrospun nanofibers) Fibers) were subjected to weakened total reflectance Fourier-modified infrared spectroscopy (Nicolet 4700 ATR FT-IR, Thermo Scientific, Grand Island, NY, USA) and thermogravimetric analysis (TGA, Q100, TA Instruments, Lindon, UT, USA). Was characterized according to the chemical profile using. Thermal analysis (DSc and TGA) was performed at 5°C/min over a temperature scan range of 20 to 400°C in a nitrogen atmosphere (20 mL/min). Eh 11A and 11B show potential applications as MCT-chitosan biofilms and wound dressing templates. The morphology of the nanofibers was examined using SEM (Leo 1530-FE, Zeiss, Cambridge, UK). The average fiber diameter was determined by analyzing at least 20 fibers in an SEM image using ImageJ software. 11C and 11D show the shape of the surface.

Advantages of MCT-chitosan composite biomaterials compared to chitosan biomaterials : The MCT-chitosan composite biomaterials (FIG. 12) provide significantly improved characteristics in various fields of application compared to chitosan biomaterials. The MCT-chitosan composite biomaterials may be made of nanoparticles, hydrogels, biofilms, 3D-spongy bodies, or electrospun nanofibers. Each of these types of biomaterials can be used for various target applications, and each of the composite biomaterials has significant advantages over chitosan biomaterials, as summarized in Table 1 below.

Table 1 is a table showing the improvement of the MCT-chitosan complex compared with chitosan and collagen biomaterials for GTR application.

Biomaterial Applicable to Improvement using MCT-chitosan composites Bio films Controlled drug release
Mechanical properties



Antibacterial

Biodegradable
Wound healing MCT crosslinking effect increases controlled release; This can replace glutaraldehyde MCT-chitosan composite biofilms, showing that the tensile strength is increased compared to chitosan; Therefore, these composites are suitable for dressing, patching, and packaging applications.
MCT reduces the biodegradation rate of the biofilms, an important property that promotes sustained delivery.
MCT immunostatic properties can improve the performance of biofilms as an accelerator of wound healing. Wound dressing templates (3D-cavernous bodies and electrospun nanofibers) Antimicrobial biocompatibility
Wound healing and coagulant


Cell growth and tissue engineering
MCT can be made more active and fit like skin patches or bandages by increasing the mechanical properties in MCT-chitosan composites compared to chitosan alone.
MCT immunostatic properties can increase the performance of wound dressing templates as wound healing, cell proliferation and coagulation promoters.
The biocompatibility, antimicrobial properties and porosity of the MCT-chitosan composite wound dressing template make these composites an advantageous cavernous material for tissue engineering and surgical dressing applications. Hydrogels
Biocompatible
Additional biomaterials

MCT-chitosan hydrogels are readily available as additional biomaterials in a variety of formulations (e.g. food, adhesives, pharmaceuticals, biomedical applications) due to their improved mucosal adhesion and viscosity, as well as anti-microbial and immunostatic properties. Can be used

Experimental Example 2. MCT-chitosan complex 3D-cavernous body for application in wound healing and tissue engineering GTR.

Chitosan (CHT) has been reported to be biocompatible and bioabsorbable. In particular, CHT is considered a good wound healing promoter. Meanwhile, collagen (MCT) is one of the most widely used matrix biomaterials in tissue engineering. Porous MCT single 3D-cavernous bodies have been used to support the in vitro growth of many types of tissues. Hybrid 3D-cavernous biomaterials were prepared by applying previously developed methodologies, including solvent casting and freeze-drying, of CHT and MCT isolated from sea cucumber specimens, and mixing them in different mass ratios. MCT/CHT hybrid 3D-cavernous bodies were characterized according to their water absorption capacity, mechanical properties, thermal behavior (TGA), and morphology (SEM). Hybrid 3D-cavernous bodies exhibited improved stability, greater porosity, increased thermal stability, and mechanical properties, as well as higher biodegradability compared to single 3D-cavernous bodies. Cell culture and SEM imaging of adipocyte-derived stem cells (ADSCs) of MCT/CHT hybrid 3D-cavernous bodies show the adhering, proliferation and in vitro growth of ADSCs allowed.

Preparation of MCT-chitosan hybrid 3D-cavernous bodies : Chitosan (CHT, 2.0% w/v) in molar ratios of 100:0, 80:20, 60:40, and 50:50, respectively, for MCT-CHT to prepare hybrid solutions Was dissolved in acetic acid (0.1% v/v), and slowly mixed with an aqueous collagen solution (MCT, 5% w/v). MCT-chitosan hybrid 3D-cavernous bodies were prepared by pouring each solution into glass molds, pouring a solvent, and freeze drying. The cavernous bodies were cut into small pieces (12 mm diameter, 3 mm thickness) for further characterization and cell proliferation studies and storage in a desiccator at controlled relative humidity.

Physical and chemical properties of MCT-chitosan hybrid 3D-cavernous bodies : Optical microscopic images of the prepared hybrid spongy bodies were collected in an inverted microscope (LIB-305, USA) at 4x magnification. The morphology of the MCT-chitosan hybrid 3D-cavernous bodies was observed on an electron microscope (SEM, JSM-5200, JEOL, USA) at a magnification of 20 kx. The tilt angle of each sample was 30 degrees. Thermogravimetric analysis (TGA) was performed on a TGA-7 instrument (Perkin Elmer, USA). Spongiform samples (5-10 mg) were poured into an aluminum holder and analyzed according to a temperature program set at 50 to 600° C. at a heating rate of 10° C./min under nitrogen atmosphere (10 mL/min). MCT/CHT hybrid 3D-sponges (n = 5 per condition) using a load cell of a universal tensile testing machine (Tensilon RTG, Japan) with a maximum tensile force of 250N at ambient conditions (20°C and 50% relative humidity, RH). ) Uniaxial mechanical compression test was performed. The compression (mm) and load (N) were collected at a crosshead speed of 5 mm/min. The compressive modulus was calculated as the tangential slope of the stress-strain curves within the initial linear region of the compression curve. The compressive strength was calculated at 15% strain (in the region where the stress-strain curve was linear in all samples). The dried 3D-cavernous body samples had a cylindrical shape with a diameter of 12 mm and a thickness of 3 mm as measured by an electron micrometer (DMH series 293, Mitotoyo, Japan).

Cell adhesion and proliferation studies : Adipose tissue-derived stem cells (ADSC) were isolated from live horses. The ADSCs (~10 ⁵ cells/cm ² ) were placed on each of the MCT-chitosan hybrid 3D-cavernous bodies. Tissue culture plate (polystyrene) wells were used as controls. The culture was placed in the incubator for 1 day, and when removed, washed with phosphate buffered saline (PBS) and treated with trypsin. Some samples of the resulting dissociated cell suspension were counted on a Coulter counter multisizer (model 0646, Coulter Electronics, Hialeah, FL, USA). Only those with a diameter of 8 to 32 μm were used. Cell proliferation was also measured as described above after 1, 3, 7 and 10 days by cell count. In this experiment, 6 replicate samples were examined. Adherent and/or ADSC cells were fixed with glutaraldehyde (2.5% v/v) in 0.1 M PBS (pH 7.4) for 30 min, then rinsed with 0.1 M PBS. The fixed cell samples were freeze-dried for morphology analysis by a scanning electron microscope (SEM, Hitachi Model S-2460N, Hitachi Ltd., Tokyo, Japan) and sputter coated with gold.

Data and Statistical Analysis : All data were reported as mean ± standard deviation of at least 3 repetitions. Statistical analysis was performed using JMP Pro (version 10.0.0; SAS Institute Inc., Cary, NC, USA) and was set to p = 0.05. To evaluate the significant difference, the results were analyzed with a two-way ANOVA model in which independent variables interact with each other between “sample” and “concentration”.

Physicochemical properties of MCT-chitosan hybrid 3D-cavernous bodies: Chitosan physically binds to collagen by hydrogen bonding interactions driven by available amine and hydroxyl groups. This interaction makes it possible to develop stable biomaterials such as nanoparticles, biofilms, biofoams, and tissue cavernous bodies (Madrigal-Carballo et al., electrostatic bioadsorption of natural polymers on soy lecithin liposomes). Polymer-liposome nanoparticles obtained by, Intl. J. Nanoparticles 5 (3) (2012) 196-209; Madrigal-Carballo et al., protein-loaded chitosan nanoparticles are egg egg white lysozymes by murine peritoneal macrophages. Of uptake and antigen presentation, Intl. J. Nanoparticles 3 (2) (2010) 179-191; Ma et al., Preliminary for the above synthesis and application of tissue engineering of a novel chitosan bilayer material as a cavernous body of human neonatal skin fibroblasts In vitro studies, Biomaterials, 22(4) (2001), pp. 331-336). 13A shows the prepared MCT-chitosan hybrid 3D-caverns. As shown in Fig. 13A, the hybrid 3D-cavernous bodies shown above are fabricated with different MCT/CHT mass ratios (50:50, 60:40, 80:20, and 100:0). 13B shows optical microscopic images of the cavernous bodies, and FIG. 13C is an SEM micrograph of each prepared 3D-cavernous body. In Fig. 13C, the scale bars are 500 mm.

The SEM micrograph of FIG. 13C shows the change in the surface morphology of the chitosan single cavernous body combined with MCT. This change is indicated by the decrease in the apparent pore size according to the addition of MCT in the MCT/CHT hybrid 3D-cavernous matrix. Thus, the interaction of chitosan and MCT appears to provide a greater crosslinking density, and the increased molecular alignment and miniaturization drive available between the two macromolecules by a greater number of potential hydrogen bonding interactions. .

14 is a thermal analysis graph of the MCT-chitosan hybrid 3D cavernous bodies by calorimetry (TG), and the thermal behavior of the MCT/CHT (50:50) hybrid 3D-cavernous body is MCT/CHT (100:0) and MCT /CHT(0:100) Shows the interaction between complex cavernous bodies. The hybrid 3D-cavernous body integrated with MCT shows superior thermal stability compared to the chitosan single cavernous body having an average decomposition temperature of 300 °C corresponding to a 15-fold increase in thermal stability when compared to the CHT single cavernous body.

Biological 3D-cavernous bodies require sufficient mechanical properties to remain intact after implantation. Therefore, a compression test was performed on the MCT-chitosan hybrid 3D-spongy body, the stress-strain mechanical curves were obtained, and the elastic modulus and compressive stress (15% strain) were calculated as shown in Table 2, respectively. The results show a positive effect on the mechanical properties by adding MCT to the chitosan 3D-cavernous matrix. The hybrid MCT/CHT (50:50) 3D-cavernous body showed an approximately 85-fold increase in compressive Young's modulus compared to the MCT/CHT (100:0) spongy body. In addition, the compressive strength (15% strain) was found to increase by about 78 times for the MCT/CHT (60:40) hybrid 3D-cavernous system. The observed improvement in mechanical strength is related to the internal structure, hydrogen bonding drive, polymer networks between collagen and chitosan, which promotes mechanical stabilization of the matrix, and can form a 3D-cavernous body suitable for injection purposes.

Table 2 shows the Young's mechanical modulus for different MCT-chitosan hybrid 3D-cavernous bodies, and the results are reported as ± SD (n = 5).

Sample Compression factor
(MPA) Compressive strength
(Mpa, 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

The swelling properties are important to promote the hydration and cell growth of the cavernous body. The MCT-chitosan 3D cavernous bodies showed an intermediate behavior between the two simple cavernous bodies of MCT and chitosan, and the cavernous body with the highest chitosan ratio showed the highest water absorption performance. These results can be explained as being present in the chitosan biomolecule rather than the collagen because of the reduction in rotation and mobility of the functional group by the quaternary structure of collagen.

15 shows the water absorption behavior obtained for different MCT-chitosan hybrid 3D-caverns. In Figure 15, the data are presented as mean±SD, n=3, and (*)=p<0.05 compared to the MCT/CHT(100:0) single 3D-cavernous body at the same time point. FIG. 15 also includes, for illustrative purposes, an inset figure to show the expansion behavior of the MCT/CHT (50:50) hybrid 3D-cavernous body. The graph of FIG. 15 shows the similarity of relative humidity of 70% or less for all other hybrid 3D-cavernous bodies. On the other hand, after reaching 85% relative humidity, as the ratio of the MCT in the hybrid 3D-cavernous matrix increased, significant differences were found between the systems. The MCT/CHT (50:50) hybrid 3D-cavernous body shows a difference in the range of 250 times, thereby showing the highest water absorption capacity of almost 300%, and the MCT/CHT (100:0) single 3D- The cavernous body exhibits the lowest water absorption capacity, a value close to 50%. MCT/CHT (80:20) and MCT/CHT (60:40) show the water absorption capacity between MCT/CHT (100:0) and MCT/CHT (50:50).

This ability of complex cavernous bodies to conserve water is an important aspect of evaluating its properties and suitability for skin tissue engineering. The water-binding ability of the MCT-chitosan cavernous body may be due to the maintenance of hydrophilicity and a three-dimensional structure. Chitosine and MCT have abundant hydrophilic groups such as hydroxy, amino, and carboxyl groups, which can retain moisture within the microstructure. MCT appears to promote an increase in hydrophilicity at higher relative humidity, leading to a higher water absorption capacity. The moisture absorption values obtained for the MCT-chitosan cavernous body agreed with similar experiments previously reported (Ma et al.,'Bio-stability improved chitosan porous cavernous bodies for skin tissue engineering', Biomaterials. Elsevier, 24(26). ) (2003), pp. 4833-4841; Chhabra et al.,'Optimization, characterization and evaluation of the efficacy of 2% chitosan cavernous bodies for tissue engineering and wound healing', Journal of Pharmacy and Biohomology, Medknow Publications, 8(4) ( 2016), p. 300).

Growth of adipose tissue-derived cells (ADSC) of MCT-chitosan hybrid 3D-cavernous bodies : To study the interactions between ADSC and MCT-chitosan hybrid 3D-cavernous bodies, porous structures with a diameter of about 12 mm and a thickness of about 3 mm were developed. Was used. After 72 hours incubation, the ADSC reached more than 90% concordance in the cavernous bodies. SEM images of the cross section of the MCT/CHT (100:0) 3D-cavernous body (Figs. 16A and 16B) showed that after 72 hours of cell transplantation, ADSC adhered to the porous MCT-chitosan (100:0) 3D-cavernous body surface and spread. Was shown, and when compared to the cavernous system in which the cells were not attached (FIG. 16A), the intercellular connections were not seen (FIG. 16B), and were completely merged with each other. In Figures 16A and 16B, the scale bar represents 10 mm. The surface of the porous cavernous bodies is filled with cells and films from which ECM precipitation can be secreted from the cells (Lin, Li and Su, '3D chitosan cavernous bodies influence the expression of extracellular matrix of Schwann cells', Materials Science and Engineering C, 42 (2014), pp. 474-478; Ji et al.,'Biocompatibility of silk fibroin-chitosan cavernous bodies using stem cells derived from adipose tissue', Experimental and Therapeutic Medicine, 6(2) (2013 ), pp. 513-518).

Figure 17 shows the proliferation level of the MCT-chitosan (50:50) complex 3D-cavernous body after a 15-day incubation period, and the legend (□) shows the MCT/CHT (100:0) 3D-cavernous body, the legend (o) represents the MCT/CHT(0:100) single 3D-cavernous body, the legend (Δ) represents the MCT/CHT(50:50) complex 3D-cavernous body, and the MCT/CHT( 0:100) Compared to a single 3D-cavernous body, the data are expressed as mean ± 　SD, n = 5. (*) = p <0.05. The three graphs show the 3D-cavernous MCT/CHT (0:100), MCT/CHT (100:0), and the MCT/CHT (50:50) complex 3D-cavernous body. When comparing MCT/CHT(0:100) and MCT/CHT(100:0) 3D-cavernous bodies, while incubating with ADSC for 3 days, the MCT/CHT (50:50) complex 3D-cavernous bodies are of cell adhesion and proliferation. Shows a significant increase.

The surface of the polystyrene dishes is known to facilitate cell adhesion and exhibit rapid cell confluence (Jeong Park et al.,'Platelet-derived growth factor that releases chitosan cavernous bodies for periodontal bone regeneration', Biomaterials, 21(2) ( 2000), pp.153-159). This degree of cell adhesion and proliferation means that the MCT-chitosan hybrid 3D-cavernous bodies have excellent cellular adaptability. As a result of examining cell proliferation in the cavernous body, it was found that there was a statistically significant difference between the experimental group and the control group after incubation with ADSC for 3 days. This may be due to the adaptation process that binds ADSC to the cavernous body. The fact that after 3 days incubation the difference between the samples started markedly is that the ADSCs exhibit a slow initial proliferation rate in the cavernous body, which may be due to the return to a more normal proliferation rate after 3 days. In addition, the MCT/CHT (50:50) complex 3D-cavernous body shows remarkably increased cell adhesion and proliferation. Compared to the MCT/CHT (0:100) and MCT/CHT (100:0) 3D-cavernous bodies during the 15-day culture period, the MCT/CHT (50:50) at a level of cell adhesion and proliferation as high as 50 times higher Was observed from MCT is known to stimulate proliferation, chemotaxis, and collagen protein synthesis of osteoblasts and ligament fibroblasts (Zhang et al.,'New chitosan/collagen corpuscles containing β1 DNA for periodontal tissue engineering', Biochemical and Biophysical Research Communications, 344(1) (2006), pp. 362-369). In addition, MCT has been reported to enhance the proliferation of progenitor cells (Costa-Pinto et al.,'Adhesion, proliferation, and proliferation of murine mesenchymal stem cell lines (BMC9) sprayed on new melt-based chitosan/polyester 3D porous cavernous bodies. Bone formation differentiation' tissue engineering. Part A, 14(6) (2008), pp. 1049-1057). This combination of MCT and chitosan can be very advantageous to increase the cell proliferative response.

Conclusion : Unlike the rapid degradation of collagen, chitosan is slowly biodegraded in vitro. Modification of collagen 3D-cavernous bodies with chitosan improves mechanical strength, thermal stability, biocompatibility, and biodegradability. The MCT-chitosan hybrid 3D cavernous bodies provide a multidimensional structure for ADSC on both the surface and the interior, and promote the cell growth and spatial features for cell adhesion, migration and proliferation. After 72 hours of incubation, ADSCs were combined to form a complete cell layer on the surface of the cavernous body, finding that the surface was almost covered and only a few pores were visible, and some cells migrated into the pores. The MCT-chitosan hybrid 3D-cavernous bodies supported ADSC attachment, proliferation and differentiation. SEM images showed that the large surface area of the porous cavernous bodies can attach, unfold, and grow the cavernous bodies. The flat morphology, and excellent diffusion around the interconnected porous structure, indicated strong cell adhesion and growth of cells. As a result, MCT-chitosan hybrid 3D-cavernous bodies show biocompatibility for ADSC attachment and are good candidates for potential applications in tissue engineering.

Experimental Example 3. Electrospun MCT-chitosan composite nanofibers as biocompatible cavernous bodies for cell proliferation in wound healing and tissue engineering applications

Electrospun nanofibers (ESNFs) were made from MCT-chitosan composite materials. Polyvinyl alcohol (PVA) was used as an auxiliary. MCT-chitosan/PVA mixed solutions of different volume ratios (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) were prepared and adjusted to approximate the appropriate viscosity and electrical conductivity for electrospinning. I did. The morphology of the ESNF was investigated using a scanning electron microscope (SEM), Fourier transform infrared spectrometer (FTIR), and differential scanning calorimetry (DSC). Studies have been used to characterize the chemical composition and thermal properties of nanofibers (NFs). The ability of the NF to support fibroblast proliferation was investigated in vitro using the optimized MCT-chitosan/PVA solution. The above results show that MCT-chitosan-based ESNF is suitable for fibroblast growth and is significantly superior to ESMF of PVA. The above results show that MCT-chitosan is better than supporting cell proliferation by chitosan alone.

Preparation of MCT-chitosan composites for electrospinning : MCT-chitosan powder was swelled in deionized water with vigorous stirring until a uniform dispersion (1% v/v) was obtained. The dispersion was left in a refrigerator (4ºC) overnight for complete hydration prior to characterization experiments. The MCT-chitosan composite dispersion was prepared with a cone-plate structure and electrical conductivity rheometer (C-VOR) at 25 ºC using a conductivity meter (Orion Star A215, ThermoFisher, Waltham MA, USA) with an electrical conductivity constant ^{of 0.7265 cm -1.} , Bohlin Instruments, Malvern, UK).

Electrospinning of MCT-chitosan composites : MCT-chitosan composites were mixed with aqueous acetone (30% v/v) with vigorous stirring until a homogeneous dispersion was obtained. The concentration of the complex solution was adjusted so that the viscosities and conductivities were similar and suitable for electrospinning. The dispersion was left in a refrigerator (4°C) overnight for complete hydration prior to electrospinning experiments. Volume flow rate (Q) is 2.78 x ^{10 -10} m ³ /s and the inner radius of the tube is 1 mm (R = 0.5 x 10 ^-3 m), according to the power law materials of the tubular shape, the maximum shear rate is each MCT -CHT was calculated for the composite sample.

To improve the composite properties for electrospinning, 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 hours. The mixed dispersions of the MCT-chitosan composite and PVA were mixed in a volume ratio of 100:00, 60:40, 50:50, 40:60 and 0:100. MCT-chitosan/PVA mixed samples (5 mL) were electrospun 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 needle tip and the collector was set to 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.

Characterization of MCT-chitosan ESNF : The ESNF was subjected to chemical profile and thermogravimetric analysis (TGA, using weakened total reflectance Fourier-modified infrared spectroscopy (Nicolet 4700 ATR FT-IR, Thermo Scientific, Grand Island, New York, USA)). Q100, TA Instruments, Lindon, Utah, USA). TGA analyzes were performed at 5°C/min over a temperature scan range of 100-400°C in a nitrogen atmosphere (20 mL/min). The morphology of the nanofibers was examined using 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 measured by MTT cell proliferation assay for the number of viable cells. Briefly, ESNF previously collected under sterile conditions was placed in other wells in a sterilized cell culture plate with a medium, and 3 mL of fibroblast suspension (L929, 1.5×105) was placed in each treatment well. The cell culture plate was left in an incubator at 37°C for 3, 7 and 14 days, respectively. After incubation, the medium was removed, and the MTT solution was diluted 1:10 using a new medium, and then added to each treatment well. The plates were incubated at 37 °C for 4 hours and absorbance was measured at 560 nm using a micro plate reader (SpectraMax Plus, Molecular Devices, Sunnyvale, CA, USA). After the cell growth experiment (7 days), ESNF was collected and washed with medium, fixed with glutaraldehyde (2.5% v/v) for 2 hours at 4°C and coated with gold before imaging by SEM.

Statistical Analysis : Statistical analysis was performed using AssistatVR software (Statistics, Arlington, TX). Experimental data are presented as mean±SD values. In order to compare the control and experimental groups, the data were analyzed as the least mean square (SAS; Cary, NC) by a generalized linear model. The differences were judged to be statistically significant at P <0.05.

Figure 18 is a graph of the ATR-FTIR spectra of ESNF of MCT/CHT (0:100) (indicated by continuous lines) and MCT/CHT (100:0) (indicated by "---" dotted lines) separately. , And also two other MCT/CHT composites (60:40, denoted by "..." and 40:60, "_._._." denoted by dashed lines). The arrows in the figure show the change in nanofiber FTIR spectrum associated with the addition of MCT to chitosan. In particular, an increased absorption trend can be seen in both ^{1650 cm -1} and 1000 cm ^-1. These correspond to the carbonyl (solid arrow) and carbon-oxygen (dotted arrow) stretching frequencies, respectively, related to the polysaccharide properties of chitosan. In general, the difference between CHT and MCT is presented in the above figure. In the case of the two composites, conversion occurs at certain locations in the graph, but significant overlap occurs at many locations, which is not surprising in view of the relatively similar ratio of CHT and MCT.

19 shows TGA thermal analysis for MCT/CHT composites (100:0, 60:40, 40:60 and 0:100), and shows a positive effect on chitosan thermal stability due to the addition of MCT. The MCT-chitosan (60:40) composite exhibits thermal stability compared to chitosan alone. The MCT-chitosan (60:40) composite material exhibits an average decomposition temperature of about 320°C, and chitosan alone exhibits an average decomposition temperature of 280°C.

20A-20F show SEM micrographs of chitosan electrospun nanofibers and MCT-chitosan electrospun nanofibers. 20A and 20D, the scale bar is 10 mm; In Figs. 21B and 20E the scale bar is 2 mm, and in Figs. 20C and 20F the scale bar is 200 nm. The dashed circles indicate the presence of droplets associated with an inadequate electrospinning process in chitosan ESNF, and indicate the improved ESNF of MCT-chitosan composites.

Figure 21 shows the proliferation of L929 fibroblasts cultured with chitosan, MCT, MCT-CHT complex (50:50) ESNF. The figure shows data from MTT cell proliferation assay using PVA ESNF as a control. The mean percent standard deviation of viable cells is derived from experiments conducted at three different times. 22A-22C are SEM micrographs showing cell adhesion to chitosan, MCT-chitosan composite, and MCT, respectively.

Conclusion : MCT-chitosan composite nanofibers were successfully prepared through electrospinning using PVS (10% w/v) as an adjuvant. Electrospinning parameters for the synthesis of MCT-chitosan composite nanofibers were set and the mass ratio of MCT to chitosan was optimized to be 50:50. ATR-FTIR analysis shows the presence of MCT-chitosan components in the ESNF. The thermal stability of the MCT-chitosan complex ESNF was compared with that of chitosan alone, suggesting that the addition of MCT to chitosan enhances the thermal stability. The proliferation results of fibroblasts indicate that MCT-chitosan ESNF is suitable for cell growth and is remarkably superior to chitosan or MCT ESNF alone after 7 days of culture. SEM images show that the large surface area of the MCT-chitosan ESNF allows excellent adhesion, diffusion and growth of L929 fibroblasts. The above results show that MCT-chitosan ESNF enhances biocompatibility and activation for cell adhesion of fibroblasts, develops wound dressing templates for application in tissue engineering and regenerative medicine, and is useful as a wound healing dressing for the treatment of tissue burns. Indicates that it is.

The above further discussion of Figures 36-38 is to provide a summary of the structure and application of GTR devices according to embodiments, as well as the advantages of such devices.

36 shows a 3D-cavernous body according to an embodiment. In the figure, the cavernous body includes an MCT composed of collagen and glycosaminoglycans, which are the main components of Neodermis. Neodermis is a new tissue that forms during wound healing. The structure of the collagen-GAG of the MCT promotes integrin binding during the healing process. As mentioned earlier, the MCT comes from a marine source including marine invertebrates in one point of view, echinoderms in a more specific point of view, and sea urchins and/or sea cucumbers in a more specific point of view. The microstructure of these MCTs is in part similar to human connective tissue, indicating beneficial GTR effects such as neodermis formation.

The cavernous body of Figure 36 also provides moisture management and promotes wound closure and healing. In one aspect, the cavernous body has a gelling effect, which improves patient comfort through cooling and moisturizing effects during treatment. Since the resulting structure exhibits low physical degradation and resistance to proteolytic enzymes, the crosslinking treatment of the MCT can make the cavernous body effective for up to 30 days. In particular, the resulting structure targets and inactivates excess enzymes such as matrix metalloproteinases (MMPs) that can degrade proteins. This targeting and inactivation promotes and improves wound closure and healing.

37 shows a 3D-cavernous body in accordance with some embodiments. The cavernous body may be composed of a matrix of MCT or MCT-CHT according to an embodiment. These substances in the cavernous body aid in the growth of tissues and blood vessels. Other aspects of the cavernous structure include hygroscopic and gel-forming compositions that maintain a humid environment and control exudate to promote healing and tissue regeneration.

38 shows a GTR device that may consist of a matrix of MCT or MCT-CHT sutured to an open portion of the skin as part of the treatment. The figure shows the sutures around the device as well as the neodermis formed under the device as part of the treatment process. Part of Figure 38 shows the neodermis, the MCT representation, a single or complex matrix of CHT with integrin binding sites.

Experimental Example 4. Cosmetic dosage forms

The following formulations illustrate representative pharmaceutical formulations. The formulations may be used for the therapeutic or prophylactic administration of the MCT and MCT-chitosan (MCT-CHT) compositions described herein, including, but not limited to, solutions, suspensions, gels, ointments, lotions, or creams. , Dermatological and/or pharmaceutically acceptable topical carriers may be used. In the following examples, a gel or cream is provided.

(i) GTR gel weight%

Part A

MCT or MCT-CHT Composite 2.0

Polyethylene oxide 0.4

Carbopol 0.4

Water 90.9

Part B

Ethoxylate glycerin 0.5

Polyethylene glycol oleyl ester 1.0

Diethanolamine oleth-3 phosphate 0.8

Crodamol PMP 0.5

Ethoxylated glucose methyl ester 0.7

Part C

Triethanolamine 0.4

Spices 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 in the other ingredients. All Part B ingredients were mixed together and heated to 70°C. Combine parts A and B and add triethanolamine and fragrance (Part C). The resulting cream is stable and smooth, has excellent moisturizing properties, and has excellent skin feel.

(ii) 1% by weight of GTR cream

MCT or MCT-CHT Composite 2.5

Polyethylene oxide 0.3

Glycerin 5.0

Ethanol 15.0

Oles-5 2.0

Ethoxylated glucose methyl ester 0.7

Preservative 0.3

Fragrance 0.15

Water up to 100.0

The formulation was prepared by mixing all of the above ingredients together.

(iii) 2% by weight of GTR cream

Part A

MCT or MCT-CHT Composite 2.5

Polyethylene oxide 1.5

Carbopol 0.5

Water 83.5

Part B

Vaseline 5.0

Polyethylene glycol stearic acid 2.5

Diethanolamine oleth-3 phosphoric acid 2.0

Silicone copolymer 1.5

Preservative 0.3

Part C

Triethanolamine 0.5

Spices 0.1

The formulation is prepared in separate steps as follows: Part A of the mixture was prepared by dispersing the carbopul in water and then stirring in the other ingredients. All of the above Part B ingredients were mixed together and heated to 70°C. Combine Parts A and B and add the above triethanolamine and fragrance (Part C). The resulting cream has excellent moisturizing properties and less oily skin.

(iv) scar cream composition

Preparation procedure:

1. Mix 150mg of vitamin E gently and mix it with the cream base dispersion.

2. MCT-CHT freeze-dried powder (formulated in MCT:CHT mass ratio 100:0 to 70:30.) Dissolve in 1000 mg and 5.00 mL of acetic acid (0.5M) prepared in (pH3.2).

3. Moringa/Vitamin C extract 200mg is dissolved in the aqueous solution of MCT-CHT.

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

5. 100 mg of royal jelly is mixed with the aqueous solution containing MCT-CHT, Moringa/Vitamin C extract and astaxanthin.

6. Mix MCT-CHT, Moringa/Vitamin C extract and astaxanthin continuously and gently until a homogeneous dispersion is reached. The astaxanthin has the cream base containing vitamin E.

7. Pour the homogenized scar cream formulation into an appropriate glass container and store at room temperature.

The formulations described above can be prepared by conventional procedures well known in the pharmaceutical field. It will be appreciated that the cosmetic compositions, as active ingredients, may vary according to well-known pharmaceutical techniques to accommodate different amounts and types of the MCT-CHT composite.

The scar cream composition described above has been applied to several patients. 30A and 30B show the before and after results for one patient. Fig. 30A is a photograph of a 5-year-old cesarean section before applying scar cream. 30B is a photograph of the same scar after applying the scar cream daily for 2 weeks. As is evident in Figure 30B, after treatment the scar is shorter and has a different color that is closer to the surrounding skin color.

31A and 31B show the before and after results for different patients. 31A is a picture of a 15 year old scar under the left knee joint before applying the scar creams listed above. 31B is a photograph of the same scar after application of the scar cream. As is evident from Figure 31B, the scar has a different color that is closer to the color of the surrounding skin.

32A-32C show before and after results for another patient. Figure 32A is a photograph of a 25 year old appendix surgery scar before applying the scar creams listed above. 32B is a photograph of the same scar after daily application, and FIG. 32C is a photograph of the same scar after daily application of the scar cream according to the embodiment for 8 days. As is evident in Figures 32B and 32C, the scar after treatment is shorter and has a different color that is closer to the surrounding skin color.

33A and 33B show before and after results for another patient. Fig. 33A is a photograph of a scar 6 months from knee surgery, and Fig. 33B is a photograph of the same scar after daily application of the above-listed scar cream for 7 days. As is evident from Figures 33A and 33B, the scar after treatment is much less noticeable and has a different color that is closer to the color of the surrounding skin. The patient did not experience side effects or hyperplasia.

Figures 34A-D show better results for another patient in the treatment of the scar. Fig. 34A shows a picture of a burn scar, Figs. 34B and 34C show the picture of the scar during daily application of the listed scar cream, and Fig. 34D shows the picture of the scar after 7 days of treatment. As is evident from these figures, compared to the appearance of the scar after treatment, the scar after treatment healed considerably with a color closer to the color of the surrounding skin.

In addition to the aforementioned treatment examples, scar formation can occur in various areas of the body, including around the eyelids, for example as a result of injury, surgery, plastic surgery, or other recovery and/or treatment procedure. The scar cream described above is also used and effective in this kind of treatment.

As part of the above-described work and produced examples, an operation was performed to determine and show structural differences between MCT extracted from sea cucumber and bovine collagen isolated from calf skin tissue. Analysis of the amino acid composition of collagen samples is described in the following data. (Cui F., Li Z., Zhang Y., Dong P., Fu X., Gao X., "Characteristics and auxiliary unit composition of collagen derived from the body wall of sea cucumber," Stichopus Japonicus, Food Chem. 100 (3) ) (2007): 1120-5). Briefly, collagen samples were hydrolyzed with 6M HCl at 110 ºC for 24 hours, and the major amino acid composition of the hydrolyzate was analyzed using SYKAM amino acid analyzer S433D (SYKAM, Munich, Germany).

Table 3 below shows the amino acid composition for different MCT samples isolated from sea cucumber and bovine (calf skin) collagen isolated from calfskin. Analysis of calf skin collagen samples 1 to 4 in the following table was performed in the studies listed in the following documents: X. Cheng, Z. Shao, C. Li, L. Yu, M.A. Raja, C. Liu, "Isolation, characterization and evaluation of collagen from the jellyfish Rhopilema esculentum Kishinouye for use in hemostasis," PLOS One, 2017, 0169731; Y. Han, J-R. Ahn, J-W. Woo, C-K. Jung, S-M. Cho, Y-B. Lee, S-B. Kim, "Process Optimization and Physical and Chemical Properties of Collagen from Scales of Yellowfin (Thunnus albacares)," Fisheries and Aquatic Sciences, Volume 13, Issue 2, 2010, pp.102-111; H. Li, B.L. Liu, L.Z. Gao, H.L. Chen, "Introduction to Sulfur Copper Skin Collagen Research," Food Chemistry, Volume 84, Issue 1, January 2004, pp. 65-69; P. Kittiphattanabawon, S. Nalinanon, S. Benjakul, and H. Kishimura. "Characteristics of pepsin-solubilized collagen in the skin of the gorgeous squid (Loligo formosana)," Journal of Chemistry, Volume 2015, Article ID 482354, 8 pages.

Table 3 is a table showing the amino acid composition.

Composition (%w/w) amino acid Code MCT 1 MCT 2 Calf Skin Collagen1 Calf Skin Collagen 2 Calf Skin Collagen 3 Calf Skin Collagen 4 CYS C 0.00 0.00 0.00 0.00 0.00 0.00 HYP Z 7.33 7.46 NR 9.06 8.60 9.40 ASP D 9.23 8.42 4.93 5.11 4.50 4.50 THR T 4.37 3.38 2.35 1.85 1.80 1.80 SER S 3.47 4.10 4.09 2.85 3.70 3.90 GLU E 13.46 14.01 7.84 10.69 7.10 7.50 PRO P 12.21 11.99 11.69 10.88 13.50 12.10 GLY G 19.13 19.64 32.56 16.28 32.50 33.00 ALA A 9.22 9.99 12.91 8.12 11.20 11.90 VAL V 2.53 2.60 3.97 2.27 2.20 2.10 MET M 0.65 0.61 0.86 NR 0.60 0.60 ILE I 1.72 1.64 2.35 1.37 1.10 1.10 LEU L 2.73 2.83 3.82 2.86 2.50 2.30 TYR Y 1.46 1.24 0.91 0.48 0.30 0.30 PHE F 1.42 1.19 1.94 1.84 1.30 0.30 HIS H 0.33 0.24 0.06 1.96 0.50 0.50 LYS K 0.65 0.50 4.27 0.27 2.70 2.60 ARG R 8.54 8.65 5.36 4.19 5.10 5.10

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

The primary structure of type 1 collagen is characterized by including a domain in which the Gly-XY sequence (X is mostly proline and Y is mostly hydroxyproline) is a continuous repeating domain, and telopeptides (15 to 26 amino acids) Residues) are characterized by including very short N- and C-terminal regions. The Gly-XY repeat sequence in the α1 chain plays an important role in the formation of the triple helix of the secondary structure. See Gelse K, Poschl E, Aigner T. 2003. Collagen-structure, function, and biosynthesis. Adv Drug Deliver Rev 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: review. Food Hydrocolloid 25(8):1813-27. As an amino acid with the smallest molecular weight, glycine residues arranged in the center of the triple helix can help the helix structure to fold easily. See Fraser R, MacRae T, Suzuki E. 1979. Chain structure of collagen molecules. J Mol Biol 129(3):463-81. Thus, glycine is the main amino acid of bovine collagen. According to previous literature, the glycine content of bovine collagen ranged from 14 to 33%, which is about 1/4 of the total amino acids, and this value was consistent with the MCT glycine content (19%).

Figures 23 and 24 show the results of the purity analysis of the collagen matrix for marine collagen compared to calf and chicken collagen, showing that marine collagen is as safe as calf and chicken collagen and therefore safe enough to apply as previously described. These figures show a triple helix structure, where the broad blue bond at the bottom is an alpha helix, above the beta or gamma helix. In these figures, MCT 1 and MCT 2 represent different MCT batches and represent the reproducibility of the results.

25A shows the spectrum of bovine collagen as indicated by continuous lines. Peaks marked with "+" in the figure show that calf collagen is hydrolyzed and dissolved. The comparison graph shows the MCT as a dotted line without a trace of the hydrolysis peak "o". FIG. 25B shows a comparable FTIR spectrum showing the reproducibility of the results regarding the efficiency of the MCT separation process for sea cucumbers, where MCT1 is indicated by a solid line and MCT2 is indicated by a dotted line. Figure 25B shows the high consistency of the MCT chemistry profile from batch to batch.

26 shows the results of heat capacity analysis (TGA) of collagen samples and shows the differences in thermal behavior for MCT samples. In Fig. 26, the reduced stability is displayed as the graph goes downward. The lines of bovine collagen increased instability at 280C, while the MCT1 and MCT2 lines showed stability at 400C. The graph shows that the thermal stability of MCT is better than that of bovine collagen, and the ability to store MCT-based products is improved.

Imino acids (proline and hydroxyproline) are important amino acids that make up the Gly-X-Y repeat sequence in the α chain because of their ability to maintain the stability of the pyrrolidine rings and the collagen triple helix. Wong, DW. See 1989. Mechanism and theory of food chemistry. New York: Van Nostrand Reinhold. The above contents of proline and hydroxyproline in MCT are 12.0 and 7.0%, respectively, for a total imino acid content of 20.0%, slightly lower than the value found in bovine collagen, as shown in FIG. 27.

This content of proline and hydroxyproline has been shown to be related to environmental 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 with bovine collagen, the ratio of the content of iminoic acid (Hyp/Pro) to Gly is low, suggesting a more efficient stability of the Gly-based triple helix by iminoic acid in MCT.

The stability of collagen can be influenced by its amino acid composition, especially imino acids. Proline (PRO) and hydroxyproline (HYP) can maintain the collagen spatial structure with pyrrolidine rings, while the hydroxy groups of hydroxyproline form hydrogen bonds with adjacent chains to improve the stability of the triple helix. . Analysis of the amino acid composition of MCT reveals a high presence of glycine and iminoic acid, which is suitable for the formation of stable collagen triple-helixes that exhibit lower degradation rates than bovine collagen.

Figure 27 is a bar graph of iminoic acid (sum of HYP and PRO) for MCT1 and MCT2 samples compared to several samples of calf collagen. One of the very advantageous aspects of MCT is an almost 1:1 ratio of imino acids to GLY, which is consistently reproducible. In comparison, even the best collagen sample in the middle of the figure is not close to a 1:1 ratio like the MCT1 and MCT2 samples. Compared to imino acids, a high content of GLY shows low stability. Although a high GLY content is advantageous, a 1:1 ratio of imino acids to GLY indicates greater stability.

Figures 28A and 28B and Figures 29A and 29B are scanning electron microscopy (SEM) pictures of MCTs formed in two different ways. 28A and 28B show the above results of forming an MCT-chitosan composite using a solvent casting technique. 28A shows this morphology of the structure of the MCT-chitosan dressing template (eg, 3D spongy). 28B shows a high degree of porosity for the structure. 29A and 29B show the results of forming the MCT-chitosan dressing template using electrospinning technique. 28A and 28B, respectively, FIG. 29A shows the shape of the structure, and FIG. 29B shows a high degree of porosity for the structure. Porosity is an important attribute in induced tissue regeneration to promote cell adhesion and growth.

All references, patents, and patent documents are incorporated herein by reference, as if individually incorporated by reference. In addition, various aspects of the present invention have been described with reference to various and specific, preferred embodiments and techniques. However, it should be understood that numerous variations and modifications may be made within the spirit and scope of the present invention.

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Claims (30)

A topical composition for treating skin conditions, wherein the composition comprises:
Variable collagen tissue (MCT) and chitosan; And
It includes a pharmaceutically acceptable topical carrier,
The MCT is isolated from echinoderm animals,
The chitosan is present in a mass percentage of 0 or more and 90 or less, and when the chitosan is present, it is electrostatically bonded to the MCT to form a matrix, and the matrix of chitosan and MCT is a topical composition in the form of an MCT-chitosan composite material . The topical composition of claim 1, wherein the topical composition is for treating scars. The topical composition of claim 1, wherein the topical composition is for promoting eyelid recovery. The topical composition of claim 1, wherein the topical composition is for treating skin discoloration. The topical composition of claim 4, wherein the topical composition is for treating melasma. The topical composition of claim 1, wherein the pharmaceutically acceptable topical carrier is selected from the group consisting of solutions, suspensions, liquids, gels, ointments, lotions, or creams. 7. The topical composition of claim 6, wherein the topical composition is a gel. 7. The topical composition of claim 6, wherein the topical composition is a cream. delete delete The topical composition of claim 1, wherein the MCT comprises collagen and glycosaminoglycan (GAG). The method of claim 1, wherein the topical composition comprises a mixture of two or more of type I, type II, type III, type V or type XI, or type I, type II, type III, type V or type XI. Topical composition consisting of fibrous collagen selected from the group. The topical composition of claim 11, wherein the glycosaminoglycan is selected from the group consisting of chondroitin sulfate, hyaluronic acid, or a mixture of chondroitin sulfate and hyaluronic acid. The method of claim 1, wherein the topical composition has a mass ratio of MCT to chitosan of 10:90, 20:80, 40:60, 50:50, 60:40, 80:20, 90:10, or 100:0. Topical composition selected from. The topical composition of claim 11, wherein the MCT-chitosan composite comprises a polyelectrolyte crosslinked structure between GAG and collagen in MCT having N-glucosamine units on chitosan. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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