Composition for tissue repair comprising high-molecular-weight collagen

Abstract

The present disclosure relates to a composition for tissue repair comprising high-molecular-weight collagen having a molecular weight of 22.4 kDa to 57 kDa, and a method for preparing high-molecular-weight collagen. The high-molecular-weight collagen prepared by the method of the present disclosure has properties similar to those of natural collagen and also has excellent physical properties, and thus can be usefully used for tissue repair in various fields.

Description

Tissue repair composition containing high molecular weight collagen

[0001] The present invention relates to a tissue repair composition comprising a high molecular weight collagen and a method for manufacturing a high molecular weight collagen.

[0002]

[0003] Collagen is a substance that fills the spaces between cells in the animal body. It is the fibrous structural protein present in the highest concentration within the body and is produced in multicellular animals rather than unicellular organisms. It is generally found in connective tissues, particularly as part of the extracellular matrix. Tendons, ligaments, cartilage, and bones are especially rich in collagen.

[0004] Collagen is known to aid in tissue regeneration when applied to damaged areas of the human body and is widely used in the medical field. This is because collagen possesses numerous functional advantages, including biocompatibility, biodegradability, and tissue repair capabilities. Currently, collagen is applied to almost all body tissues in the medical field, including bone, tissues, and biological membranes.

[0005] Existing medical devices are primarily manufactured using animal-derived collagen as a raw material. However, such animal-derived collagen carries the risk of viral contamination, and its molecules may trigger immune responses within the body due to specific structural characteristics and impurities. Additionally, there are disadvantages, such as potential limitations on raw material supply due to restricted animal populations or disease outbreaks, and the difficulty in guaranteeing quality consistency.

[0006] Therefore, many attempts are currently being made to artificially manufacture collagen. However, these collagens remain in the form of a two-dimensional matrix or a three-dimensional scaffold, and they also have problems such as lacking sufficient strength or failing to possess the characteristics of natural collagen (KR 10-2001-0090876, KR 10-2016-0032569).

[0007]

[0008] There is a growing need for synthetic collagen that possesses properties similar to natural collagen while also having excellent physical properties, for applications in various fields, particularly for therapeutic purposes, specifically for the prevention or treatment of conditions or diseases affecting the muscles, joints, bones, and skin of humans and animals.

[0009]

[0010] One objective of the present invention is to provide a high molecular weight collagen.

[0011] Another objective of the present invention is to provide a composition for manufacturing a medical device comprising the collagen.

[0012] Another objective of the present invention is to provide a tissue repair composition comprising the collagen.

[0013] Another objective of the present invention is to provide a method for manufacturing the collagen.

[0014] Another objective of the present invention is to provide a tissue repair method comprising the step of administering the tissue repair composition to an individual.

[0015] Another objective of the present invention is to provide a tissue repair use of the above tissue repair composition.

[0016]

[0017] The polymeric collagen produced by the method of the present invention has characteristics similar to natural collagen while also possessing excellent physical properties, making it useful for tissue repair in various fields.

[0018]

[0019] Figure 1 is a schematic diagram of a method for synthesizing a synthetic collagen polymer.

[0020] Figure 2 shows sample images of low / medium / high molecular weight synthetic collagen.

[0021] Figure 3 shows the viscosity of the mixed solution according to the polymerization time.

[0022] Figures 4, 5, and 6 show the results of confirming the molecular weight according to polymerization time using SEC (Size exclusive chromatography).

[0023] Figure 7 shows the results of confirming the molecular weight of low / medium / high molecular weight collagen polymers using SDS-PAGE.

[0024] Figure 8 shows the storage modulus of low / medium / high polymer collagen.

[0025] Figure 9 is a table showing the values ​​of storage modulus and complex viscosity of low / medium / high polymer collagen.

[0026] Figure 10 shows the storage modulus and composite viscosity of low / medium / high polymer collagen.

[0027] Figure 11 shows the results of volume measurements over time of synthetic collagen injection (injection) after subcutaneous injection in mice.

[0028] Figure 12 shows the results of observing the substance remaining in the subcutaneous tissue 6 weeks after subcutaneous injection in mice.

[0029] Figure 13 shows the volume measurement results of low / medium / high polymer collagen over time.

[0030] Figure 14 shows the expression results of collagen-producing factors, degradation factors, moisture-producing factors, and hyaluronic acid-producing factors of low / medium / high molecular weight polymer collagens.

[0031] Figure 15 shows the weight bearing results.

[0032] Figure 16 shows the results of the tensile strength test after the extraction of the Achilles tendon.

[0033] Figure 17 shows the histopathological results of the Achilles tendon.

[0034] Figure 18 shows the results of confirming the collagen density of the Achilles region through MT staining.

[0035]

[0036] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0037] Furthermore, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the present invention described in this invention using only ordinary experiments. In addition, such equivalents are intended to be included in the present invention.

[0038]

[0039] One aspect of the present invention provides a high molecular weight collagen.

[0040] The above-mentioned polymeric collagen may include tripeptides, specifically, may include tripeptides comprising glycine (Gly), hydroxyproline (Hyp), and / or proline (Pro) sequences as components regardless of order as repeating units, but is not limited thereto.

[0041] The term "tripeptide" above refers to a basic structure included in a collagen molecule, which forms a rope shape known as a triple helix structure, and possesses stability and function due to such structural characteristics. Additionally, collagen contains a large amount of hydroxyproline (Hyp), proline (Pro), and glycine (Gly). Specifically, the tripeptide of the present invention may be a tripeptide forming a triple helix structure in which the sequences of glycine (Gly), hydroxyproline (Hyp), and / or proline (Pro) are repeated regardless of order, and may be selected from the group consisting of Gly-Pro-Hyp, Pro-Hyp-Gly, and Hyp-Gly-Pro. In one embodiment, the polymeric collagen may form a triple helix structure characteristic of collagen by including the repeating unit multiple times.

[0042]

[0043] The term "collagen" above refers to a triple helix composed of three similar chains or two similar chains and an additional chain with a slightly different chemical composition. The amino acid composition of collagen is particularly characterized by a high glycine content, and common motifs are glycine-proline-X and glycine-X-hydroxyproline, where X may be an amino acid other than glycine, proline, or hydroxyproline. For the purposes of the present invention, "collagen" may be included without limitation as a form having a triple helix structure including tripeptides, whether synthetically, artificially, or recombinantly produced. Additionally, the "high molecular weight collagen" of the present invention may be used interchangeably with terms such as "artificial collagen," "synthetic collagen," and "recombinant collagen."

[0044] For the purposes of the present invention, the collagen is produced through step polymerization in which tripeptide (Pro-Hyp-Gly) monomers react repeatedly n times, and the molecular weight may depend on the polymerization time. When the monomer concentration and the amount of catalyst for polymerization are kept constant, the molecular weight of the collagen obtained may vary depending on the polymerization time. Therefore, collagen with different molecular weights can be produced by varying the polymerization time while maintaining the same synthesis method. The weight-average molecular weight (Mw) can be obtained using the molar mass and weight fraction of the synthesized collagen polymer chains, and the molecular weight is expressed based on the weight-average molecular weight.

[0045] Specifically, when n is 1 to 25, a low molecular weight collagen with a weight-average molecular weight of about 0.28 kDa to 7.1 kDa is formed; when n is 26 to 80, a medium molecular weight collagen with a weight-average molecular weight of about 7.1 kDa to 22.4 kDa is formed; and when n is 80 to 300 or more, a high molecular weight collagen with a weight-average molecular weight of about 22.4 kDa to 57 kDa is formed, but is not limited thereto. In one embodiment, the high molecular weight collagen may have a weight-average molecular weight of about 22.4 kDa to 57 kDa.

[0046] The lower limit of the above weight-average molecular weight may be, for example, about 22.4 kDa, about 25 kDa, about 28 kDa, about 30 kDa, about 32 kDa, about 34 kDa, about 36 kDa, or about 38 kDa, and the upper limit may be, for example, about 57 kDa, about 55 kDa, about 52 kDa, about 50 kDa, about 48 kDa, about 46 kDa, about 45 kDa, about 44 kDa, about 42 kDa, about 40 kDa, or about 35 kDa.

[0047] Specifically, the weight average molecular weight of the polymeric collagen is about 22.4 kDa to 57 kDa, about 30 kDa to 57 kDa, about 32 kDa to 57 kDa, about 34 kDa to 57 kDa, about 36 kDa to 57 kDa, about 38 kDa to 57 kDa, about 22.4 kDa to 52 kDa, about 22.4 kDa to 50 kDa, about 22.4 kDa to 48 kDa, about 22.4 kDa to 46 kDa, about 22.4 kDa to 45 kDa, about 22.4 kDa to 44 kDa, about 22.4 kDa to 43 kDa, about 22.4 kDa to 42 kDa, about 22.4 It may be kDa to 41 kDa, about 22.4 kDa to 40 kDa, about 30 kDa to 52 kDa, about 30 kDa to 50 kDa, about 30 kDa to 48 kDa, about 30 kDa to 46 kDa, about 30 kDa to 45 kDa, about 30 kDa to 44 kDa, about 30 kDa to 43 kDa, about 30 kDa to 42 kDa, about 30 kDa to 41 kDa, about 30 kDa to 40 kDa, about 30 kDa to 38 kDa, or about 30 kDa to 35 kDa, but is not limited thereto.

[0048] The term "about" above includes not only the exact number specified after the term, but also a range that is approximately that number or close to it. Whether the number is close to or nearly that specific number can be determined by considering the context in which it is presented. For example, the term "about" may refer to a range of -10% to +10% of a numerical value. For another example, the term "about" may refer to a range of -5% to +5% of a given numerical value. For yet another example, it may include, but is not limited to, a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc.

[0049]

[0050] The "collagen" of the present invention refers to a major matrix protein produced by fibroblasts of the skin as a major component of the extracellular matrix. Furthermore, as an important protein accounting for approximately 30% of the total weight of biological proteins, it has a rigid triple helix structure. Generally, collagen forms the majority of the organic material in the skin, tendons, bones, and teeth, with particularly high content in bones and skin (dermis). In most other internal structures, it exists as fibrous inclusions.

[0051] Additionally, collagen may refer to one of the biodegradable polymers. Biodegradable polymers include collagen, fibrigen, chitosan, gelatin, cellulose, hyaluronic acid, dextran, etc., and polymerized collagen, polymerized fibrigen, polymerized chitosan, polymerized gelatin, polymerized cellulose, polymerized hyaluronic acid, polymerized dextran, polycaprolactone, polylactic acid, polyglycolic acid, copolymers of lactic acid and glycolic acid, etc., which are polymerized and bonded to have a polymer structure, may be used.

[0052] These collagens are also known to be applicable to damaged areas of the human body, and can be classified into natural collagen isolated or derived from animals, recombinant collagen using genetic recombination technology, and artificial / synthetic collagen produced through the repetitive synthesis of amino acids.

[0053] There are various types of natural collagen derived from different animal species, body parts, and types; not only do they differ in their characteristics, but their constituent biomolecules and components, such as proteins, also vary. Animal-derived natural collagen presents issues such as concerns regarding immune reactions and the potential for viral and disease infections. In contrast, recombinant collagen produced using genetic recombination technology and synthetic collagen often exhibit problems compared to animal-derived natural collagen, such as lower regenerative capacity or issues with poor stability and physical properties.

[0054] To this end, the present invention provides a biocompatible material that can be used in medical devices, etc., by manufacturing a polymeric collagen that has characteristics similar to natural collagen while also possessing excellent physical properties.

[0055]

[0056] Another aspect of the present invention provides a composition for manufacturing a medical device comprising the polymeric collagen.

[0057] The term "medical device" above may refer to any product used for the purpose of diagnosing, treating, alleviating, managing, or preventing a disease. For the purposes of the present invention, the medical device may be one or more selected from the group consisting of wound dressings, biomaterials, injectables, infusions, medical threads, medical films, sutures, dental materials, tissue regeneration scaffolds, tissue substitutes, tissue repair materials, anti-adhesion agents, hemostatic agents, drug delivery systems, and reinforcing materials, but is not limited thereto.

[0058]

[0059] Another aspect of the present invention provides a tissue repair composition comprising the above-mentioned polymer collagen.

[0060] The term "tissue repair" above may mean improving or repairing tissues such as blood vessels, heart, diaphragm, fascia, skin, joints, joint cavities, cartilage, tendons, or ligaments temporarily or semi-permanently by injecting the composition, or regenerating tissues such as improving contours, forming tissue volume, or healing scars.

[0061] The tissue repair composition of the present invention can be used as a tissue repair material, and the tissue repair biomaterial refers to a material used for the replacement, repair, or reconstruction of human tissues and organs such as blood vessels, heart, diaphragm, fascia, skin, joints, joint cavities, cartilage, tendons, or ligaments, and is not limited to cosmetic fillers but may refer to a tissue repair material specified by the Ministry of Food and Drug Safety.

[0062] As one specific example, the composition may be for skin tissue repair.

[0063] As another specific example, the composition may be for joint cavity restoration.

[0064] In another specific example, the composition may be for tendon or ligament repair.

[0065]

[0066] The tissue repair composition of the present invention may be a liquid composition and may be an injectable or injectable composition, but is not limited thereto.

[0067] In the present invention, the terms "injectable" or "injectable" refer to a material having characteristics required to administer a composition to an individual using an injection (injection) device having a needle.

[0068] In the present invention, the term "administration" means introducing the composition of the present invention into an individual by any appropriate method, and the route of administration may be through various routes such as topical application, subcutaneous, dermal, blood vessels, biological membranes, tissue fibers, synovial fluid, etc., as long as the target tissue can be reached. The formulation may be, but is not limited to, a topical preparation, a subcutaneous injection, a dermal injection, a vascular injection, an intramuscular injection, an intra-articular injection, a tendon injection, and a ligament injection. The preferred dosage of the composition of the present invention may vary depending on the individual's condition.

[0069]

[0070] For example, when high molecular weight collagen prepared by the method of the present invention was injected subcutaneously into a mouse, it was confirmed that the volume of low molecular weight collagen decreased within 24 hours, whereas the volume of medium and high molecular weight collagens was maintained. However, it was confirmed that medium molecular weight collagen remained white after volume reduction, while high molecular weight collagen maintained its shape.

[0071]

[0072] In addition, the above-mentioned high molecular weight collagen is characterized by enhancing the expression of collagen-producing factors or hyaluronic acid-producing factors in vivo. For example, as a result of confirming the effects of high molecular weight collagen prepared by the method of the present invention on collagen production, water production, and hyaluronic acid production, it was confirmed that collagen-producing factors, water-producing factors, and / or hyaluronic acid-producing factors were expressed most highly in high molecular weight collagen compared to low molecular weight collagen and medium molecular weight collagen.

[0073] Thus, the polymeric collagen produced by the method of the present invention has excellent effects on tissue repair capacity and the expression of in vivo collagen-producing factors, water-producing factors, and / or hyaluronic acid-producing factors, suggesting that it can be used as a biocompatible tissue repair material.

[0074]

[0075] In addition, the collagen may have the characteristic of having a higher storage modulus (G′) compared to low molecular weight collagen and / or medium molecular weight collagen.

[0076] When the high molecular weight collagen of the present invention has a higher storage modulus compared to low molecular weight collagen and / or medium molecular weight collagen, it can maintain persistence in the injected material, the part requiring recovery, regeneration, or protection (e.g., tissue) when injected into the body. Additionally, materials with a relatively high storage modulus can reduce friction within tissues due to their buffering action. However, if the storage modulus is low, not only is it unable to buffer at the injection site, but a problem arises in that the material diffuses and disappears within the body within a short period after injection.

[0077] For example, the storage modulus can be measured using a rheometer, and the measurement temperature, frequency, strain, sample concentration, and measurement time can be adjusted according to the purpose. In another embodiment, the polymeric collagen may have a higher complex viscosity and storage modulus, lower fluidity, or better shape retention than low molecular weight collagen and / or medium molecular weight collagen under specific concentration conditions. In one embodiment, the polymeric collagen may be fluid during the injection or infusion process, while maintaining a desired shape and volume more stably within the tissue after administration.

[0078] Therefore, for the purposes of the present invention, it can be seen that the polymeric collagen can be used as a biocompatible medical device material.

[0079]

[0080] The above composition is characterized by excellent tissue regeneration ability. For example, when the regenerative effect of the ligament and tendon was confirmed after suturing the Achilles tendon of a rabbit with the polymeric collagen of the present invention, it was confirmed that there is a superior tissue regeneration effect even when compared to commercial tissue repair collagen (natural collagen).

[0081]

[0082] The tissue repair composition of the present invention may further comprise one or more compounds selected from the group consisting of anesthetics, vitamins, amino acids, metals, antioxidants, and mineral salts, but is not limited thereto.

[0083] The tissue repair composition of the present invention may additionally include any suitable excipients commonly used in the art, such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0084]

[0085] Another aspect of the present invention provides a tissue repair biomaterial comprising a tissue repair composition of the present invention.

[0086] The above tissue repair composition and tissue repair biomaterial are as described in other embodiments.

[0087]

[0088] Another aspect of the present invention provides a method for producing the polymeric collagen. Specifically, the method may comprise: (a) dissolving a polymer in a phosphate buffer (PB) solution; (b) dissolving a reaction promoter and a tripeptide in the PB solution; and (c) mixing the two solutions obtained in steps (a) and (b).

[0089] In the present invention, the PB (Phosphate buffer) solution may be used to dissolve a polymer, a reaction promoter, and / or a tripeptide, and the PB solution may be prepared by dissolving the PB (Phosphate buffer) in distilled water.

[0090] In the present invention, there may be no time interval between steps such as "(a), (b), (c)…", they may be performed simultaneously, or they may be performed at any interval, such as seconds, minutes, or hours.

[0091]

[0092] The specific details of each step of the method for manufacturing the above collagen are as follows. First, step (a) is the step of dissolving the polymer in a PB (Phosphate buffer) solution.

[0093] The term "polymer" above refers to a substance that can assist in the formation of repetitive structures of tripeptides as a chemical polymer that induces covalent bonds through various interactions with tripeptides.

[0094] For example, the polymer may be a natural polymer or a chemical polymer. Specifically, the polymer may be a chemical polymer. The chemical polymer may be, for example, one or more selected from the group consisting of DCC (Dicyclohexylcarbodiimide), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), and DIC (Diisopropylcarbodiimide). More specifically, the polymer may be EDC, but is not limited thereto. Additionally, the EDC may be used in combination with N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride.

[0095] In addition, the above polymer agent may be used alone or in combination with a polymerization promoter.

[0096] (b) Step is to dissolve a polymerization promoter and a tripeptide in a PB solution. The polymerization promoter may be one or more from the group consisting of, for example, HOBt (Hydroxybenzotriazole), HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate), HOAt (1-Hydroxy-7-azabenzotriazole), TBTU (O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), NHS (N-Hydroxysuccinimide), Sulfo-NHS (N-Hydroxysulfosuccinimide), and TMS (N-Hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid), and specifically, may be HOBt (Hydroxybenzotriazole), but is not limited thereto.

[0097] The above polymerization may be a single or multiple chemical reactions. It is not limited to the apparatus, tools, temperature, time, or location used for the polymerization, and any method capable of producing collagen of the desired molecular weight in the present invention through continuous chemical reactions occurring during the polymerization stage may be interpreted as multiple chemical reactions. The above polymerization means polymerizing without limitation, such as the number of chemical reactions, as long as it enables the production of collagen of the desired molecular weight in the present invention.

[0098]

[0099] In the above method for manufacturing collagen, step (c) is a step of mixing a solution containing a polymerizing agent and (b) a solution containing a tripeptide and a polymerization promoter using the PB (Phosphate buffer) solution obtained in step (a). The term 'tripeptide' may include a tripeptide having glycine (Gly), hydroxyproline (Hyp), and / or proline (Pro) as repeating units. The term "tripeptide" is as described above. In one embodiment, a method for manufacturing a polymeric collagen of the present invention may include: (a) a step of dissolving EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) in a PB (Phosphate buffer) solution; (b) a step of dissolving HOBt (Hydroxybenzotriazole) and a tripeptide in the PB solution; and (c) a step of mixing the two solutions obtained in steps (a) and (b).

[0100] In addition, the above method may additionally perform steps of grinding, ultrafiltration, dialysis bag purification, precipitation purification, sterilization filtration, heat sterilization, freeze-drying, or gamma sterilization after step (c), and may appropriately include or exclude some steps required for the manufacturing method in addition to the above steps. The sterilization filtration step may proceed sequentially from a large size filter to a small size filter, but is not limited thereto.

[0101] For the purposes of the present invention, the high molecular weight collagen produced by the method of the present invention may have a high molecular weight in the range of 22.4 kDa to 50 kDa.

[0102]

[0103] Another aspect of the present invention provides a tissue repair method comprising the step of administering a tissue repair composition of the present invention to an individual.

[0104] The above administration, tissue repair, tissue repair composition, etc. are as described in other embodiments.

[0105] In the present invention, the term "individual" refers to all animals, including humans, rats, mice, livestock, etc., that require or are likely to require tissue repair. Specifically, it may be mammals, including humans.

[0106]

[0107] Another objective of the present invention is to provide a tissue repair use of a composition comprising the tissue repair composition of the present invention.

[0108]

[0109] The present invention will be explained in more detail below through examples and experimental examples. However, these examples and experimental examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples and experimental examples.

[0110]

[0111] Example 1. Synthesis of synthetic collagen

[0112] As shown in Figure 1, 1) a phosphate buffer (PB solution) was prepared by dissolving 0.04 g KCl, 0.23 g Na2HPO4, and 0.04 g KH2PO4 in 200 mL of distilled water, and 2) an aqueous solution was prepared by completely dissolving 31.6 g of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, Sigma Aldrich) using 100 mL of the solution prepared in 1). 3) Subsequently, an aqueous solution was prepared by completely dissolving 10 g of Tripeptide (H-Pro-Hyp-Gly-OH, Aminologics) and 1 g of HOBt (Hydroxybenzotriazole, Sigma Aldrich) using 100 mL of the PB solution prepared in 1). After adding the aqueous solution of 2) to the obtained aqueous solution of 3), synthetic collagen was prepared by mixing at room temperature using a mechanical stirrer (300 rpm).

[0113] Mixed solutions were prepared according to the desired molecular weight, and the reaction times were varied. For low molecular weight samples, 1N sodium hydroxide was added to the mixed solution 1 minute later to adjust the pH to between 10 and 11 to terminate the reaction, and the precipitated material was washed with 500 mL of ethanol. Subsequently, the material was redispersed in distilled water and purified for 3 days using a dialysis bag (Cut-off: 1 kDa). The resulting solution was transferred to a square dish and dried using a freeze-dryer for 3 days. For medium molecular weight samples, preparation was carried out in the same manner, but the addition of sodium hydroxide was set to 15 minutes after the mixed solution was prepared; after washing with ethanol, the sample was dried using a freeze-dryer for 3 days. For high molecular weight synthetic collagen, the reaction was carried out for 48 hours, followed by purification using a dialysis bag without the addition of sodium hydroxide, and then dried using a freeze-dryer for 3 days.

[0114]

[0115] 1-1. Confirmation of molecular weight growth over time during polymerization

[0116] Since the viscosity of a solution depends on its molecular weight and increases as the molecular weight increases, we intended to observe the change in solution viscosity during polymerization.

[0117]

[0118] As a result, as shown in Figure 3, the viscosity increased significantly as the reaction time increased, and the increase in viscosity was greatest at a polymerization time of 60 min. After 60 min, the increase in viscosity was not significant, indicating that molecular growth proceeded at a very low rate or hardly at all.

[0119]

[0120] In the present invention, collagens with different molecular weights were prepared by introducing the variable of polymerization time. As shown in Fig. 4, it can be seen that the molecular weight increases with polymerization time, increasing rapidly in the initial stage and then showing no significant change thereafter. For example, the synthetic collagen low molecular weight of the present invention was prepared by a reaction time of 1 minute, the synthetic collagen medium molecular weight by 15 minutes, and the synthetic collagen high molecular weight by continuing until molecular growth ceased (48 hours) (Fig. 4). The weight-average molecular weight (Mw) and number-average molecular weight (Mn) were determined using Size Exclusive Chromatography (SEC) based on the molar mass and weight fraction of the prepared synthetic collagen polymer chains, and the polydispersity index (PDI) of the polymer was obtained using the values ​​of the weight-average molecular weight (Mw) and number-average molecular weight (Mn) (Figs. 5 and 6). The relative molecular weight of the synthetic collagen was also confirmed via SDS-PAGE by comparison with a general-purpose protein marker (Fig. 7).

[0121] As a result, as shown in Table 1 below, it was found that the synthetic collagen of the present invention exhibited a low molecular weight of 5.9 kDa, a medium molecular weight of 20 kDa, and a high molecular weight of 31 kDa. After proceeding with the synthetic collagen polymerization reaction for 48 hours, the reaction was further extended by 24 hours for a total of 72 hours (Sample name: 72h reaction), and it was confirmed that the molecular weight of the obtained collagen was 33 kDa. Considering that the change in molecular weight was not significant as the reaction time increased, this implies that after 48 hours, the additional polymerization reaction proceeded minimally or almost stopped.

[0122]

[0123] Classification Molecular Weight (Mw) (GPC Analysis) Polymerization Time Low Molecular Weight 5,890 1 min Medium Molecular Weight 20,100 1 min High Molecular Weight 30,700 48 min 72h Reaction Time 33,100 72h

[0124] However, since the rate of molecular weight increase depends on the concentrations of the polymerizing agent, polymerization accelerator, and tripeptide, it is not limited to the aforementioned reaction time. For example, if the concentrations of the polymerizing agent, polymerization accelerator, and tripeptide decrease, the reaction time may increase, and if the concentrations of the polymerizing agent, polymerization accelerator, and tripeptide increase, the reaction time may increase.

[0125]

[0126] Example 2. Measurement of physical properties of the prepared collagen

[0127] 2-1. Verification of Rheological Properties of Low / Medium / High Polymers

[0128] The storage modulus and complex viscosity of the low / medium / high molecular weight collagen prepared in Example 1 were to be measured. Specifically, the effect of concentration on the storage modulus and complex viscosity was to be determined. Low / medium molecular weight collagen samples were dissolved in distilled water to prepare target concentrations (0.5 wt%, 1 wt%, 3 wt%, 5 wt%, and 10 wt%). High molecular weight collagen samples were dissolved in distilled water to prepare target concentrations (0.5 wt%, 1 wt%, and 3 wt%). The prepared samples were injected between Rheometer (Kinexus Prime Ultra+, NETZSCH) plates, the strain was fixed at 5% and the temperature at 25 °C, and the storage modulus and viscosity of the solutions were measured at frequencies ranging from 0.01 Hz to 10 Hz.

[0129] As a result, as shown in Figure 8, it was confirmed that the synthetic collagen low molecular weight did not have a concentration dependency and showed a shape similar to water, while the synthetic collagen medium molecular weight had a concentration dependency on elasticity values ​​but showed a lower storage modulus compared to the synthetic collagen high molecular weight.

[0130] Additionally, as shown in Figures 9 and 10, it was observed that there were significant differences in viscosity values ​​depending on the molecular weight at the same concentration of 3 wt%. It was confirmed that as the molecular chain length of collagen increases in an aqueous solution, the intermolecular attractiveness and entanglement increase, and the physical crosslinking between molecules increases, thereby affecting elasticity and complex viscosity.

[0131]

[0132] Example 3: Confirmation of suitability as a tissue repair material

[0133] We intended to determine whether the collagen produced through the present invention is suitable as a material for tissue repair.

[0134]

[0135] 3-1: Confirmation of efficacy as an injectable (infusion) agent

[0136] To determine whether the low / medium / high molecular weight collagen produced through the present invention is suitable for tissue repair including fillers, the volume of synthetic collagen injections over time was measured after subcutaneous injection using mice. Low / medium / high molecular weight collagens were prepared as injection compositions at the same concentration of 3 wt% and injected into both upper thighs of hairless mice in equal volumes (150 μL). Photographs of each site were taken at specific intervals after injection, and the volume of the injected filler was measured using a vernier caliper.

[0137] When the same amount of 150 μL was injected subcutaneously into a Mice and the volume was observed, as shown in Figures 11 and 12, the volume of the synthetic collagen at the injection site was completely reduced after 6 weeks. In the case of the medium molecular weight, the volume was completely reduced after 6 weeks, but it remained white under the skin at the injection site, and in the case of the high molecular weight, it was confirmed that the volume at the injection site decreased compared to immediately after injection but maintained some shape.

[0138] As a result of measuring the volume over time, as shown in Figure 13, it was confirmed that the volume of synthetic collagen low and medium molecules decreased immediately after 2 days, whereas the volume of high molecules decreased gradually.

[0139]

[0140] Through this, it can be seen that the polymeric collagen produced in the present invention is suitable as a material for tissue repair.

[0141]

[0142] 3-2: Identification of Factors Affecting Collagen and Hyaluronic Acid Production

[0143] Forty-seven days after the injection of low / medium / high molecular weight polymer collagen in Example 3-1, skin tissue from the injection site was excised and gene expression analysis was performed. The excised skin tissue was completely homogenized using a high-speed homogenizer, and then extracted using an RNA extraction kit. The quality of the extracted RNA was evaluated by measuring the absorbance at 260 nm-280 nm and 230 nm-260 nm using a Nanodrop to confirm the concentration and purity of the RNA. cDNA was synthesized using cDNA Synthesis Master Mix.

[0144] Reverse Transcription Polymerase Chain Reaction (RT-PCR) was performed using the obtained cDNA, and changes in the expression of genes related to collagen production and water retention, as well as genes involved in hyaluronic acid synthesis, were evaluated. The expression levels of each gene were compared to appropriate internal reference genes (housekeeping genes) to calculate their relative expression levels.

[0145] As a result of confirming whether the low / medium / high molecular weight polymer collagen produced through the present invention affects collagen production, moisture production, and hyaluronic acid production, the factors affecting collagen production (COL1,3), collagen degradation factor (MMP1), moisture production factor (AQP3), or hyaluronic acid production factor (HAS) were observed in the order of high molecular weight > medium molecular weight > low molecular weight (Fig. 14).

[0146]

[0147] Example 4: Effect of improving osteoarthritis through animal experiments

[0148] This study was conducted to evaluate the effect of test substances on the improvement of osteoarthritis by administering low, medium, and high molecular weight polymer collagens, respectively, into the joint cavity as a single dose in a Monosodium Iodoacetate (MIA)-induced osteoarthritis model using Sprague-Dawley Rats.

[0149]

[0150] 4-1. Preparation of Experimental Animals

[0151] For the animal experiments, 7-week-old male Sprague-Dawley rats were purchased from Orientbio Inc. (Korea). Solid feed for laboratory animals (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2018C, Envigo RMS, Inc., USA) was placed in feeders for free intake, and tap water from Gyeonggi-do was filtered using a water sterilizer and irradiated with ultraviolet light for free intake. The environment of the animal housing room was maintained at a temperature of 19–25°C, relative humidity of 30–70%, ventilation rate of 10–15 times / hr, lighting time of 12 hours (7:00 AM–7:00 PM), and illuminance of 150–300 Lux. Environmental conditions such as temperature, humidity, ventilation rate, and illuminance in the animal room were measured regularly during the rearing period.

[0152]

[0153] 4-2. Composition of Test Group and Administration of Test Substance

[0154] After preparing experimental animals as in 4-1, test groups were formed as shown in Table 2 below, and body weight was measured on the first day of test substance administration (Day 0). Based on the ranked body weights, the average body weights of each group were distributed using a random method to ensure they were as uniform as possible.

[0155] Group Dosage (mg / kg) Administered Solution Volume (μL / kg) Number of Animals (Heads) G1 Normal Control Group - 2006 G2 Negative Control Group - 2006 G3 High Molecular Weight - 2006 G4 Medium Molecular Weight - 2006 G5 Low Molecular Weight - 2006

[0156] Test animals were induced under inhalation anesthesia with isoflurane, and the area around the knee joint of the right hind leg was shaved. Then, a single dose of the test substance was administered into the joint cavity using a 31G 1 mL syringe on the third day of inducing substance administration. General symptoms were observed once a day during the administration period, and the presence of morbid or deceased animals was checked. In addition, body weight was measured once a week starting from the day of group separation.

[0157]

[0158] 4-3: Checking weight-bearing results after arthritis induction

[0159] Prior to MIA administration, the Weight Bearing Index (WBI) was measured using a Static Weight Bearing test device (SWB-TOUCH-R, Bioseb, Boulogne, France) to measure static weight bearing. Subsequently, to induce osteoarthritis using MIA (Monosodium iodoacetate), the hair was removed from the knee joint area of ​​the right hind leg of experimental rats, and a single dose of MIA was administered into the joint cavity using a 1 mL syringe to induce osteoarthritis. On the third day of MIA induction, the WBI was measured to confirm the induction of osteoarthritis, and group separation within the induction group was performed based on the measurement results. Subsequently, a substance prepared as an injectable composition containing low, medium, and high molecular weight collagens at the same concentration of 3 wt% was administered into the joint cavity.

[0160] The weight-bearing index was evaluated a total of 6 times by measuring it before MIA induction (day 0), and on days 3, 7, 14, 21, and 28 after MIA administration.

[0161] It was confirmed that pain was significantly alleviated in the group administered synthetic collagen polymer (test group G3). In particular, on day 28 after MIA induction, the recovery of the weight-bearing index was significantly observed in the G3 administration group compared to the negative control group, suggesting that synthetic collagen polymer is an effective substance for pain relief and weight-bearing index improvement in an osteoarthritis induction model (Fig. 15).

[0162] Based on the overall results, it is determined that the synthetic collagen polymer test material is effective in pain relief in addition to the regeneration and recovery of damaged cartilage.

[0163]

[0164] Example 5: Confirmation of Achilles tendon tissue regeneration effect through animal experiments

[0165] This study aimed to confirm the effect of administering the test substance on the regeneration of damaged Achilles tendon tissue in a tenotomy model using NZW rabbits.

[0166]

[0167] 5-1. Preparation of Experimental Animals

[0168] For the animal experiment, 115 female New Zealand White Sam (NZW) rabbits weighing 2.2–3.6 kg were purchased from Coatec Co., Ltd. (181-21, Jinwi-ro, Jinwi-myeon, Pyeongtaek-si, Gyeonggi-do). Solid feed for laboratory animals (Purina Experimental Rabbit Feed 38302AF, Cargill Agri Purina Inc., Republic of Korea) was supplied once a day, and tap water from Gyeonggi-do was filtered using a water sterilizer and irradiated with ultraviolet light for free intake. The number of animals was verified upon entry, and general symptoms and body weight were measured. General symptoms were observed once daily during the quarantine and acclimatization period.

[0169]

[0170] 5-2. Composition of Test Group and Administration of Test Substance

[0171] After preparing experimental animals as in 5-1, the test group was formed as follows, and a complete defect was induced in the Achilles tendon area of ​​rabbits. After suturing, tissue repair collagen (natural collagen) and synthetic collagen were applied to regenerate the ligament and tendon, and the tissue healing and regeneration effects of the damaged area were evaluated through macroscopic, biomechanical, and histopathological methods.

[0172] - Control group 1: No treatment group

[0173] - Control Group 2: Treatment Group (Negative Control Group)

[0174] - Control Group 3: Tissue Repair Collagen (Natural Collagen, 3 wt% Atelocollagen)

[0175] - Experimental group: 3 wt% synthetic collagen polymer

[0176]

[0177] 5-3. Perform Achilles Tenotomy

[0178] The test animals were intramuscularly anesthetized in the left hind leg using a mixture of Zolletil (35 mg / kg) and Rumpun (5 mg / kg), and the midpoint of the Achilles tendon on the right hind leg was shaved. An antibiotic (Cefazolin sodium, 20 mg / kg) was administered intravenously prior to skin incision. The surgical site was disinfected twice with povidone, and after making an incision of approximately 5 cm in the skin, the fascia was incised to expose the Achilles tendon. The Achilles tendon was transversely severed 2 cm above the calcaneus. The severed Achilles tendon was sutured using the Krackow suture method (Ethibond 2-0), and the test substance was administered using the aforementioned administration method. After administration of the test substance, the fascia (Vicryl 4-0) and skin (Blue nylon 3-0) were tightly sutured. The suture site was disinfected twice with povidone. After surgery, antibiotics (Cefazolin sodium, 20 mg / kg) and analgesics (tramadol, 5 mg / kg) were administered intramuscularly, and the ankle was immobilized with a plaster cast at a 180-degree angle. Additional antibiotics and analgesics were administered intramuscularly for 3 days after surgery. Ten days after surgery, the plaster cast was removed, and the ankle was re-immobilized with a compression bandage. At this time, the ankle angle was fixed at 150°. On the 7th day of compression bandage immobilization, all bandages were removed, and the animal's condition was checked once a day for disinfection and treatment.

[0179]

[0180] 5-4. Observation and Measurement

[0181] General symptoms were observed once a day during the experiment period, and reflexes and the presence of deceased animals were checked. Body weight was measured once a week starting from the day of group separation. On the final day of each group, test animals for tensile strength measurement were sacrificed. The Achilles tendons of the right hind legs were extracted, wrapped in sterile gauze soaked in PBS, sealed in plastic, and stored at -80°C. The results obtained from the experiment were statistically analyzed using SPSS (Version 27.0, IBM Corporation, USA). Levene's test for equal variances and an independent t-test were performed on the control groups 1 to 3 and the experimental group (synthetic collagen polymer) administration group to determine significance (significance level: 0.05).

[0182]

[0183] 5-5. Tensile Strength Measurement Results

[0184] On the end date of each test group, the rabbit's Achilles tendon was extracted and stored at -80℃, and then moved to 4℃ the day before the tensile strength measurement to thaw before use.

[0185] On the day of the test, the tensile strength was measured by grasping both sides of the Achilles tendon using a tensile strength measuring device (Instron, 68SC-5, USA) and the value in Newtons was verified.

[0186] As a result, no inflammation or other adverse reactions were observed during visual inspection, and through tensile strength testing, it was confirmed that the experimental group (synthetic collagen polymer) exhibited relatively higher tensile strength compared to the treatment group (negative control group) and tissue repair collagen (natural collagen) (Fig. 16).

[0187] In addition, histopathological results showed that inflammatory cells and necrosis were identified in all experimental groups that underwent surgery, indicating no difference between groups due to the surgery (Fig. 17). Collagen density was confirmed by MT staining, and results at weeks 6 and 12 showed a significant increase in synthetic collagen polymers compared to the treatment group (negative control group) (Fig. 18).

[0188]

[0189] Based on the above results, it was confirmed that the polymeric collagen of the present invention, manufactured through the process of Example 1, has excellent tissue repair ability, collagen production ability, hyaluronic acid production ability, and regenerative effect, suggesting that it can be used as a biocompatible tissue repair material.

[0190]

[0191] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

A tissue repair composition comprising a high molecular weight collagen having a molecular weight of 22.4 kDa to 57 kDa.

2. In Paragraph 1, A composition in which the above-mentioned polymeric collagen promotes the expression of collagen-producing factors or hyaluronic acid-producing factors in vivo.

3. In Paragraph 1, A composition in which the above-mentioned high molecular weight collagen has a higher storage modulus compared to low molecular weight and medium molecular weight high molecular weight collagen.

4. The composition of claim 1, wherein the composition has excellent tissue regeneration ability.

5. The composition according to claim 1, wherein the composition is for skin tissue repair.

6. The composition of claim 1, wherein the composition is for restoring the joint cavity.

7. The composition of claim 1, wherein the composition is for tendon or ligament repair.

8. A tissue repair biomaterial comprising a composition of any one of claims 1 to 7.

9. A method for manufacturing the polymeric collagen of claim 1, comprising the following manufacturing process steps; (a) A step of dissolving the polymer in a PB (Phosphate buffer) solution; (b) a step of dissolving a polymerization promoter and a tripeptide in a PB solution; and (c) A step of mixing the two solutions obtained in steps (a) and (b) above.

10. In Paragraph 9, A method in which the polymer of step (a) above is one or more selected from the group consisting of DCC (Dicyclohexylcarbodiimide), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), and DIC (Diisopropylcarbodiimide).

11. In Paragraph 9, A method in which the polymerization promoter of step (b) above is one or more selected from the group consisting of HOBt(Hydroxybenzotriazole), HATU(O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate), HOAt(1-Hydroxy-7-azabenzotriazole), TBTU(O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), NHS(N-Hydroxysuccinimide), Sulfo-NHS(N-Hydroxysulfosuccinimide), and TMS(N-Hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid).

12. In Paragraph 9, A method in which the tripeptide of step (b) above comprises a repeating unit of a tripeptide having glycine (Gly), hydroxyproline (Hyp), and proline (Pro) as components.

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