Pharmaceutical composition for preventing or treating ramo sleeve traffic containing detectolicon collagen and induced pluripotent stem cell-derived

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating rotator cuff laceration comprising, as active ingredients, deteptided collagen and induced pluripotent stem cell-derived tendinocytes, and has confirmed that the induced pluripotent stem cell-derived tendinocytes have excellent adhesion and viability, can be used to produce a cell-support complex, and can be used to treat rotator cuff laceration. Especially, the compound with the detelopeptide collagen can enable cells to survive and maintain for a long time in the rotator cuff lacerated tissue. Furthermore, the apotelocollagen-induced pluripotent stem cell-derived tendinocyte complex exhibits a more excellent therapeutic effect than other complexes in terms of mechanical strength recovery and tissue regeneration promotion, and thus can be effectively used as a preparation for preventing or treating rotator cuff tear.

Description

Technical Field

[0001] This invention relates to a pharmaceutical composition for preventing or treating rotator cuff tears, comprising atelocollagen and induced pluripotent stem cell-derived tenocytes as active ingredients. Background Technology

[0002] A rotator cuff tear is a musculoskeletal disorder caused by damage to the four tendons that wrap around the shoulder (the rotator cuff). It leads to functional impairments such as pain and limited range of motion. Aging, trauma, and repetitive overuse are known to be the main causes, with a particularly high incidence in middle-aged and older adults. If treatment for a torn rotator cuff is missed, fibrosis and muscle atrophy can occur at the tear site, potentially making treatment and recovery difficult.

[0003] Rotator cuff tears are classified as partial or full-thickness tears, and treatment strategies vary depending on the extent and location of the injury. Initially, conservative treatments such as medication, physical therapy, and injections are primarily used to alleviate symptoms. If conservative treatment is ineffective or the tear worsens, surgical intervention is necessary. However, even with successful surgery, the rate of re-tears remains relatively high, which presents significant practical limitations to treatment.

[0004] The main causes of re-tearing have been identified as aging of the tissue at the tear site, decreased tissue regeneration capacity, and difficulties in postoperative rehabilitation and management. In recent years, to address these shortcomings, various tissue regeneration adjuvant technologies based on biomaterial scaffolds, growth factors, and stem cells have been developed and are being promoted. These technologies aim to improve the tissue healing environment at the suture site and enhance the structural stability of the fascia-bone junction.

[0005] Effective and fundamental treatment of rotator cuff tears is essential for restoring structural stability at the damaged tendon-bone junction and maintaining long-term shoulder joint function. To date, there are no reported examples of simultaneously applying a composite fusion of key components of the rotator cuff—collagen (telopeptide collagen) and cells (tendon cells derived from induced pluripotent stem cells)—to the treatment.

[0006] Prior technology literature [Patent Documents] (Korean Patent Publication No. 10-2018-0013664) Summary of the Invention

[0007] The technical problem to be solved The purpose of this invention is to provide an atelocollagen patch for the prevention or treatment of rotator cuff tears, the patch being composed of atelocollagen and containing induced pluripotent stem cell-derived tendon cells attached to the surface of the atelocollagen patch as an active ingredient.

[0008] Another object of the present invention is to provide a patch composition for the prevention or treatment of rotator cuff tears, the composition comprising determinate collagen and induced pluripotent stem cell-derived tendon cells as active ingredients.

[0009] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of rotator cuff tears, the pharmaceutical composition comprising determinate collagen and induced pluripotent stem cell-derived tendon cells as active ingredients.

[0010] Another object of the present invention is to provide a composition for making a patch for preventing or treating rotator cuff tears, the composition comprising determinate collagen and induced pluripotent stem cell-derived tendon cells as active ingredients.

[0011] Another object of the present invention is to provide a method for manufacturing the above-mentioned patch for preventing or treating rotator cuff tears, the method comprising the following steps: The steps of manufacturing a determinate collagen patch composed of determinate collagen; and treating the determinate collagen patch with induced pluripotent stem cell-derived tendon cells and culturing the tendon cells to attach to the surface of the determinate collagen patch.

[0012] Another object of the present invention is to provide a kit for manufacturing a patch for preventing or treating rotator cuff tears, the kit comprising the aforementioned composition and instructions.

[0013] However, the technical problems to be solved by the present invention are not limited to those mentioned above. Other problems not mentioned can be clearly understood by those skilled in the art through the following description.

[0014] Methods for solving technical problems The present invention provides a determinate collagen patch for the prevention or treatment of rotator cuff tears, the patch comprising a determinate collagen patch composed of determinate collagen and induced pluripotent stem cell-derived tendon cells attached to the surface of the determinate collagen patch as active ingredients.

[0015] In one embodiment of the invention, the determinate collagen patch for preventing or treating rotator cuff tears may contain more than 1×10^4 cells and less than 1×10^6 cells of induced pluripotent stem cell-derived tendon cells within its basic unit size, but is not limited thereto.

[0016] In one embodiment of the present invention, the basic unit size of the determinated peptide collagen patch is characterized in that its horizontal, vertical and thickness dimensions can be 1-10 mm, 1-10 mm and 0.1-1 mm respectively, but it is not limited thereto.

[0017] In one embodiment of the invention, the induced pluripotent stem cell-derived tendon cells are characterized by having more than one selected from the group consisting of, but not limited to: It is characterized by expressing CD73 and CD105, but not expressing CD34 and CD45; It expresses collagen type I, vimentin, SCXA (tendon cell-specific protein), tenomodulin, and MKX (Mohawk protein). And it exhibits a spindle-shaped morphological feature.

[0018] The present invention provides a patch composition for the prevention or treatment of rotator cuff tears, the composition comprising determinate collagen and induced pluripotent stem cell-derived tendon cells as active ingredients.

[0019] The present invention provides a pharmaceutical composition for the prevention or treatment of rotator cuff tears, the pharmaceutical composition comprising determinate collagen and induced pluripotent stem cell-derived tendon cells as active ingredients.

[0020] In one embodiment of the invention, the pharmaceutical composition may have the feature of increasing the toughness of torn rotator cuff tissue, but is not limited thereto.

[0021] The present invention provides a composition for manufacturing a patch for preventing or treating rotator cuff tears, the composition comprising determinate collagen and induced pluripotent stem cell-derived tendon cells as active ingredients.

[0022] This invention provides a method for manufacturing the patch for preventing or treating rotator cuff tears, the method comprising the following steps: The steps of manufacturing a telopeptide collagen patch composed of telopeptide collagen; and the steps of treating and culturing tendon cells derived from induced pluripotent stem cells on the telopeptide collagen patch to allow the tendon cells to attach to the surface of the telopeptide collagen patch.

[0023] The present invention provides a kit for manufacturing a patch for preventing or treating rotator cuff tears, the kit comprising the aforementioned composition and instructions.

[0024] In one embodiment of the invention, the specification may teach a method for manufacturing the patch for preventing or treating rotator cuff tears, but is not limited thereto.

[0025] Furthermore, the present invention provides a method for preventing or treating rotator cuff tears, the method comprising: applying or treating a pharmaceutically effective amount of the said telopeptide collagen patch for preventing or treating rotator cuff tears to an individual in need; telopeptide collagen and induced pluripotent stem cell-derived tendon cells; or a composition comprising telopeptide collagen and induced pluripotent stem cell-derived tendon cells as an active ingredient.

[0026] Furthermore, the present invention provides the use of the aforementioned determinate collagen patch and induced pluripotent stem cell-derived tendon cells; or determinate collagen and induced pluripotent stem cell-derived tendon cells; or a composition comprising determinate collagen and induced pluripotent stem cell-derived tendon cells as an active ingredient in the manufacture of an agent for the prevention or treatment of rotator cuff tears.

[0027] Furthermore, the present invention provides the use of a determinate collagen patch for the prevention or treatment of rotator cuff tears in the manufacture of an agent for the prevention or treatment of rotator cuff tears, the determinate collagen patch being composed of determinate collagen and containing induced pluripotent stem cell-derived tendon cells attached to the surface of the determinate collagen patch as an active ingredient.

[0028] Invention Effects Based on pharmaceutical compositions containing telopeptide collagen and induced pluripotent stem cell-derived tendon cells as active ingredients for the prevention or treatment of rotator cuff tears, it is known that induced pluripotent stem cell-derived tendon cells possess excellent adhesion and viability, enabling the formation of cell-support complexes. In particular, the complex formed with telopeptide collagen has been shown to allow cells to survive and maintain long-term survival within rotator cuff tear tissue. Furthermore, the telopeptide collagen-induced pluripotent stem cell-derived tendon cell complex, targeting the fundamental anatomical and histological structure of tendons, exhibits superior therapeutic effects compared to other complexes in restoring mechanical strength and promoting tissue regeneration, thus making it an effective agent for the prevention or treatment of rotator cuff tears. Attached Figure Description

[0029] Figure 1a The results show the expression of cell surface markers in induced pluripotent stem cells (iPSCs) and iPSC-derived tendon cells.

[0030] Figure 1b and Figure 1c The expression of various proteins in iPSCs and iPSC-derived tendon cells, as well as the morphological analysis results of each cell, are shown.

[0031] Figure 1d The results show the analysis of mRNA expression levels of markers in iPSCs and iPSC-derived tendon cells.

[0032] Figure 2a and Figure 2b Experimental results are shown to confirm the appropriate number and culture conditions of iPSC-derived tendon cells in various biomaterial-based supports.

[0033] Figure 3 The results of scanning electron microscopy (SEM) analysis of complexes formed by applying iPSC-derived tendon cells to various biomaterial-based supports are shown.

[0034] Figure 4a and Figure 4b This shows the results of in vivo tracking of fluorescently labeled iPSC-derived tendon cells 3 weeks (up to 30 days) post-transplantation in a rotator cuff tear model. Figure 4a The minimum color scale (Color Scale_Min) is 1.76e8, and the maximum color scale (Max) is 1.66e9.

[0035] Figure 5a and Figure 5b The in vitro fluorescence imaging results of supraspinatus muscle tissue isolated on day 30 after iPSC-derived tendon cells and determinated collagen patches were transplanted into a rotator cuff tear model are shown.

[0036] Figure 6 The results show the analysis of the stiffness level of the repaired rotator cuff tissue on day 30 after iPSC-derived tendon cells and determinated peptide collagen patches were transplanted into a rotator cuff tear model. Detailed Implementation

[0037] In this invention, "atelocollagen" is a collagen derivative that reduces immunogenicity and improves biocompatibility by removing the telopeptide region of type I collagen through pepsin treatment. Through physicochemical processing, it can be made into various forms such as sheets, gels, and sponges.

[0038] In this invention, "tenocytes" are spindle-shaped fibrous cells whose cytoplasm is connected by long, thin processes. They regulate the synthesis and breakdown of the extracellular matrix (ECM) and primarily express structural proteins such as collagen, elastin, and proteoglycans. Furthermore, they secrete various growth factors and cytokines, intervening in interactions with surrounding cells and regulating the microenvironment.

[0039] In this invention, the "rotator cuff" is a structure composed of four muscle groups (supraspinatus, infraspinatus, subscapularis, and teres minor) and their tendons surrounding the shoulder joint. These muscles stably position the humeral head within the glenoid cavity of the scapula, enabling the shoulder joint to perform fine movements such as external rotation, internal rotation, and abduction. Due to the high mechanical load and repetitive use of the rotator cuff, it is anatomically prone to micro-injuries and degenerative changes.

[0040] In this invention, "rotator cuff tear" refers to a partial or full-thickness injury to the continuous muscles or tendons that make up the rotator cuff. It can be caused by trauma, repetitive mechanical stress, or degenerative changes, and its pathological manifestations vary depending on the location and extent of the injury. When a tear occurs, the anatomical structure and mechanical stability of the muscle-tendon-bone junction decrease, leading to impaired normal shoulder joint movement and load transmission.

[0041] The present invention provides a determinate collagen patch for the prevention or treatment of rotator cuff tears, the patch being composed of determinate collagen and containing induced pluripotent stem cell-derived tendon cells attached to the surface of the determinate collagen patch as an active ingredient.

[0042] In this invention, a "patch" can refer to a single-support graft material that, by attaching to a defect or injury site, provides mechanical support, physical protection, and surface coverage. To ensure biocompatibility, it can be made from various materials such as collagen, synthetic polymers, and composite materials. In principle, it does not contain drugs or cells, but as needed, it can be impregnated with drugs, cells, or growth factors to impart functionality. Furthermore, its adhesion to tissue surfaces can be improved through physicochemical processing, and its stability in the in vivo environment can be maintained.

[0043] In one embodiment of the invention, induced pluripotent stem cell-derived tendon cells are treated with and cultured on a telopeptide-free collagen patch to manufacture a telopeptide-free collagen patch for the prevention or treatment of rotator cuff tears. Because telopeptide-free collagen has high cell affinity, and stem cells (especially tendon cell lines) typically attach to surfaces, the cells are able to attach to the substrate surface during culture. Furthermore, in another embodiment of the invention, microscopic examination has experimentally confirmed that tendon cells attach to the substrate surface of the telopeptide-free collagen patch.

[0044] Therefore, telopeptide collagen patches with induced pluripotent stem cell-derived tendon cells attached can function as an effective ingredient in agents for the prevention or treatment of rotator cuff tears.

[0045] In one embodiment of the invention, it has been demonstrated that, regardless of the type of patch, when 1×10^5 cells of induced pluripotent stem cell-derived tendon cells are processed, the tendon cells are uniformly distributed and attached to the patch, thereby maintaining colonization capacity. Conversely, this phenomenon was not observed when there are fewer than 1×10^4 cells or 1×10^6 cells. For example, under conditions of fewer than 1×10^4 cells, despite increased culture time, the OD value is low, indicating poor coverage of the patch; while at the level of 1×10^6 cells, cell death and functional decline occur due to over-density. Therefore, the inventors have confirmed that when induced pluripotent stem cell-derived tendon cells are contained within a basic unit size of the substrate layer in a range of more than 1×10^4 cells and less than 1×10^6 cells, cell colonization capacity can be stably maintained, while avoiding the adverse effects of over-density. Therefore, in one embodiment of the present invention, the telopeptide-free collagen patch for preventing or treating rotator cuff tears may contain more than 1×10^4 cells and less than 1×10^6 cells of induced pluripotent stem cell-derived tendon cells within its basic unit size, but is not limited thereto. For example, it may contain more than 1×10^4 cells and less than 1×10^6 cells, more than 1×10^4 cells and less than 9×10^5 cells, more than 1×10^4 cells and less than 8×10^5 cells, and more than 1×10^4 cells. cells and less than 7×10^5 cells, more than 1×10^4 cells and less than 6×10^5 cells, more than 1×10^4 cells and less than 5×10^5 cells, more than 1×10^4 cells and less than 4×10^5 cells, more than 1×10^4 cells and less than 3×10^5 cells, more than 1×10^4 cells and less than 2×10^5 cells, more than 1×10^4 cells and less than 1×10^5 cells, 3×10^4 cells and less than 1×10^6 cells, 3×10^4 cells to less than 9×10^5 cells, 3×10^4 cells to less than 8×10^5 cells, 3×10^4 cells to less than 7×10^5 cells, 3×10^4 cells toBelow 6×10^5 cells, 3×10^4 cells to below 5×10^5 cells, 3×10^4 cells to below 4×10^5 cells, 3×10^4 cells to below 3×10^5 cells, 3×10^4 cells to below 2×10^5 cells, 3×10^4 cells to 1×10^5 cells, 5×10^4 cells to at least 1×10^6 cells, 5×10^4 cells to below 9×10^5 cells, 5×10^4 cells to below 8×10^5 cells, 5×10^4 cells to below 7×10^5 cells, 5×10^4 cells to below 6×10^5 cells, 5×10^4 cells to less than 5×10^5 cells, 5×10^4 cells to less than 4×10^5 cells, 5×10^4 cells to less than 3×10^5 cells, 5×10^4 cells to less than 2×10^5 cells, 5×10^4 cells to less than 1×10^5 cells, 7×10^4 cells to at least 1×10^6 cells, 7×10^4 cells to less than 9×10^5 cells, 7×10^4 cells to less than 8×10^5 cells, 7×10^4 cells to less than 7×10^5 cells, 7×10^4 cells to less than 6×10^5 cells, 7×10^4 cells to less than 5×10^5 cells, 7×10^4 cells to less than 4×10^5 cells, 7×10^4 cells to less than 3×10^5 cells 7×10^4 cells to less than 2×10^5 cells, 7×10^4 cells to less than 1×10^5 cells, 9×10^4 cells to at least 1×10^6 cells, 9×10^4 cells to less than 9×10^5 cells, 9×10^4 cells to less than 8×10^5 cells, 9×10^4 cells to less than 7×10^5 cells, 9×10^4 cells to less than 6×10^5 cells, 9×10^4 cells to less than 5×10^5 cells, 9×10^4 cells to less than 4×10^5 cells, 9×10^4 cells to less than 3×10^5 cells, 9×10^4 cells to less than 2×10^5 cells, 9×10^4 cells to less than 1×10^5 cells, or 1×10^5 cells, but not limited to these.

[0046] As described above, in one embodiment of the present invention, when 1×10^5 cells of tendon cells were attached to a telopeptide-free collagen patch, their excellent colonization and maintenance capabilities were confirmed. Furthermore, considering that the OD value at the 24-hour time point showed a level similar to that of a cell culture dish (approximately 0.6), the range of tendon cells that can ensure technically stable and highly reproducible cell attachment, survival, and maintenance capabilities can be from 5×10^4 cells to 5×10^5 cells.

[0047] In one embodiment of the present invention, the basic unit size of the determinated peptide collagen patch is characterized in that the length, width and thickness can be 1-10 mm, 1-10 mm and 0.1-1 mm respectively, but are not limited thereto.

[0048] In one embodiment of the invention, the telopeptide-free collagen patch used may be a patch composed of 100% telopeptide-free collagen. In this case, the telopeptide-free collagen patch may be a bioabsorbable collagen material with a square matrix morphology (membrane layer, absorbent layer) composed of Type I collagen, wherein antigenic telopeptides have been removed to minimize immune responses. Depending on the rate (duration) of absorption, it may consist of two surfaces named the membrane layer and the absorbent layer. Furthermore, since collagen fibers form a flexible network with surrounding tissues, it can be used as an absorbable collagen material for coating wounds of damaged biological tissues (soft or hard tissues) to replenish missing or damaged ligaments, tendons, muscles, (bio)membranes, etc., during (internal and surgical) treatments and surgeries.

[0049] In one embodiment of the present invention, the induced pluripotent stem cell-derived tendon cells may have one or more selected from the group consisting of, but are not limited to: It is characterized by expressing CD73 and CD105, but not expressing CD34 and CD45; It expresses collagen type I, vimentin, SCXA, tenomodulin, and MKX (Mohawk); and It exhibits a spindle-shaped morphological feature.

[0050] The present invention provides a patch composition for the prevention or treatment of rotator cuff tears, the composition comprising determinate collagen and induced pluripotent stem cell-derived tendon cells as active ingredients.

[0051] In this invention, "patch composition" refers to a composition comprising telopeptide collagen and induced pluripotent stem cell-derived tendon cells as active ingredients, used as components of a patch for the prevention or treatment of rotator cuff tears. Examples have demonstrated that telopeptide collagen not only provides a physical and biological environment for cell attachment and survival but also plays a preventative or therapeutic role in rotator cuff tears. Furthermore, the tendon cells attached to the telopeptide collagen patch serve as the active ingredient for therapeutic effects. The composition is characterized by promoting tissue regeneration and functional recovery at the target site through the interaction of the two active ingredients.

[0052] Therefore, the present invention provides a pharmaceutical composition comprising determinate collagen and induced pluripotent stem cell-derived tendon cells as active ingredients for the prevention or treatment of rotator cuff tears.

[0053] In one embodiment of the invention, the pharmaceutical composition may have the characteristic of increasing the toughness of torn rotator cuff tissue, but is not limited thereto. Implantation of the patch in an animal model of rotator cuff tear resulted in a significant increase in mechanical stiffness (N / mm) compared to the control group. This increase in stiffness directly implies restoration of the mechanical stability and load-bearing capacity of the tendon-bone junction, demonstrating that the rotator cuff tear has been treated. Therefore, the patch treatment can be used as an effective means of prevention, treatment, or improvement for achieving structural and functional restoration of the rotator cuff tear site. Therefore, in this invention, "for prevention or treatment" can be used interchangeably with "for prevention or improvement."

[0054] The pharmaceutical compositions according to the present invention can be used in conventional dosage forms such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, elixirs, emulsions, suspensions, tinctures, lozenges, aromatic waters, lemon waters, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, ointments, creams, dry ointments, liquid ointments, injections, capsules, irrigation solutions, film-forming agents, emulsions, pastes, sprays, inhalers, patches, sterile injectable solutions, or aerosols, etc., for external use. The external preparations may be in dosage forms such as creams, gels, patches, sprays, ointments, film-forming agents, emulsions, liniments, pastes, or mud dressings.

[0055] The pharmaceutical compositions according to the present invention may include carriers, excipients, and diluents such as lactose, glucose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0056] In the formulation process, conventional diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc., can be used for manufacturing.

[0057] The additives used in the tablets, powders, granules, capsules, pills, and lozenges according to the present invention may include: corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, dimannitol, precipitated calcium carbonate, synthetic aluminum silicate, dicalcium phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, refined lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC1928, HPMC 2208, HPMC 2906, and HPMC. 2910. Excipients such as propylene glycol, casein, calcium lactate, and Primojel; gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium casein, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethyl cellulose, refined shellac, starch paste, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, and polyvinylpyrrolidone; adhesives such as hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium carbonate, sodium dodecyl sulfate, anhydrous silicate, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginate, amyl starch, and guar gum. Disintegrants include: gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, disorbitol solution, and light anhydrous silicic acid; lubricants include: calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, asbestos, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil, aluminum stearate, zinc stearate, sodium dodecyl sulfate, magnesium oxide, polyethylene glycol (Macrogol), synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ethers, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid.

[0058] The additives for the liquid formulation according to the present invention may include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (Tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, protamine sulfate, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc.

[0059] The syrups according to the present invention may use a solution of sucrose, other sugars or sweeteners, and may also use flavoring agents, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. as needed.

[0060] Purified water can be used in the emulsions according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may also be used as needed.

[0061] The suspending agents according to the present invention may include gum arabic, tragacanth gum, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910 and other suspending agents. Surfactants, preservatives, stabilizers, colorants, fragrances and other agents may also be used as needed.

[0062] The injectable formulations according to the present invention may include: distilled water for injection, 0.9% sodium chloride injection, Ringer's solution, glucose injection, glucose + sodium chloride injection, polyethylene glycol (PEG), lactated Ringer's solution, ethanol, propylene glycol, non-volatile oils such as sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl isostearate, sodium benzoate, and benzene solvents; such as sodium benzoate, sodium salicylate, sodium acetate, urea, ethyl carbamate, monoethylacetamide, phenylbutazone, propylene glycol, Tween derivatives, nicotinamide, etc. Hexamethylenetetramine and dimethylacetamide are cosolvents; buffers for weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; isotonic agents such as sodium chloride; stabilizers for sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen (N2), and ethylenediaminetetraacetic acid; sulfation agents such as 0.1% sodium bisulfite, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and sodium acetone sulfite; local anesthetics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium carboxymethyl cellulose, sodium alginate, Tween 80, and aluminum monostearate.

[0063] The base of the suppositories according to the present invention may be cocoa butter, lanolin, Witopsol, polyethylene glycol, glycerin gelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, Lanette wax, glyceryl monostearate, Tween or Span, Imhausen, Monolen (propylene glycol monostearate), glycerin, stearin (Adeps solidus), Buytyrum Tego-G, Cebes Pharma 16, Hexalide Base95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrokote 711. 711), Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neo-Supostal-EN, Paramound-B, Supocillo (OSI, OSIX, A, B, C, D, H, L), Suppository Matrix Type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supocillo (N, Es), Vicoby (W, R, S, M, Fs), Tezester Triglyceride Matrix (TG-95, MA, 57), etc.

[0064] Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid dosage forms are manufactured by mixing the above-mentioned extracts with at least one excipient (such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.). In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0065] Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. Besides commonly used simple diluents (such as water and liquid paraffin), they may also contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for non-oral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0066] The pharmaceutical compositions according to the present invention are administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an adequate amount for treating a disease at a reasonable benefit / risk ratio suitable for medical treatment, and the effective dosage level can be determined based on factors including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the excretion rate, the treatment period, the factors of concurrently used drugs, and other factors known in the medical field.

[0067] The pharmaceutical compositions according to the invention can be administered alone or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents, either alone or in multiple applications. Importantly, considering all the foregoing factors, the dosage that yields the maximum effect without side effects can be readily determined by those skilled in the art to which this invention pertains.

[0068] The pharmaceutical compositions of the present invention can be administered to an individual via a variety of routes. All methods of administration are contemplated, such as oral administration, subcutaneous injection, intraperitoneal administration, intravenous administration, intramuscular injection, perispinal space (intradural) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, intraauricular administration, nasal administration, inhalation, oral or nasal spray, skin administration, transdermal administration, etc.

[0069] The pharmaceutical composition of the present invention depends on a variety of relevant factors, such as the disease to be treated, the route of administration, the patient's age, gender, weight, and disease severity, as well as the type of drug as the active ingredient.

[0070] In this invention, "individual" refers to the object that needs to be treated for a disease, and more specifically, to humans or non-human primates, mice, rats, dogs, cats, horses, cattle and other mammals.

[0071] In this invention, "application" means providing an individual with a certain amount of the composition of this invention by any suitable method.

[0072] In this invention, "prevention" refers to all actions that inhibit or delay the onset of a target disease; "treatment" refers to all actions that improve or benefit the target disease and its resulting metabolic abnormalities by applying a pharmaceutical composition according to the invention; and "improvement" refers to all actions that reduce parameters (such as symptom severity) associated with the target disease by applying a composition according to the invention.

[0073] In this invention, specific embodiments demonstrate that when induced pluripotent stem cell-derived tendon cells are cultured and attached to a patch composed of telopeptide-derived collagen, a patch for the prevention or treatment of rotator cuff tears can be manufactured. Therefore, this invention provides a composition for manufacturing a patch for the prevention or treatment of rotator cuff tears, the composition comprising telopeptide-derived collagen and induced pluripotent stem cell-derived tendon cells as active ingredients.

[0074] This invention provides a method for manufacturing a patch for preventing or treating rotator cuff tears, the method comprising the following steps: The steps for manufacturing a determinated peptide collagen patch composed of determinated peptide collagen; and The determinate collagen patch is treated with induced pluripotent stem cell-derived tendon cells and cultured to allow the tendon cells to attach to the surface of the determinate collagen patch.

[0075] At this point, the determinated peptide collagen patch, composed of determinated peptide collagen, can be manufactured according to conventional methods in the art, so that it has the basic unit size (horizontal × vertical × thickness) produced in one embodiment of the present invention.

[0076] The present invention provides a kit for manufacturing a patch for preventing or treating rotator cuff tears, the kit comprising the aforementioned composition and instructions.

[0077] In one embodiment of the invention, the specification may teach a method for manufacturing the patch for preventing or treating rotator cuff tears, but is not limited thereto.

[0078] In this invention, "kit" refers to a tool containing determinate collagen and tendon cells, thereby enabling the manufacture of the patch of this invention for the prevention or treatment of rotator cuff tears.

[0079] In addition to the substances mentioned above, the kit of the present invention may also include other components, compositions, solutions, devices, etc., typically required during its storage and processing. Specifically, there is no limit to the number of times each component can be used (it can be used more than once), no restriction on the order in which each substance is used, and each substance can be used simultaneously or at different times.

[0080] In this invention, the kit may include a container, instructions, etc. The container serves to package the substance and also to store and secure it. The container may be made of materials such as bottles, tubes, sachets, envelopes, tubing, ampoules, etc., and may be partially or wholly made of plastic, glass, paper, foil, wax, etc. The container may be equipped with a fully or partially detachable cap, which may initially be part of the container or may be attached to the container mechanically, adhesively, or by other means; in addition, it may be equipped with a stopper that allows access to the contents through an injection needle. The kit may include an external component package, which may contain instructions on the use of the components.

[0081] To aid in understanding the present invention, preferred embodiments are provided below. However, these embodiments are provided merely for the purpose of facilitating a clearer understanding of the invention, and the scope of the invention is not limited to these embodiments.

[0082]

Example

[0083] First, differentiation into tendon cells was induced from iPSCs (Human iPScell ​​Line 1231A3, yipscell Co., Ltd.). Specifically, to induce differentiation of human induced pluripotent stem cells (iPSCs) into mesodermal lineages, the initial culture phase (days 0-4) was performed using StemFit AK01 medium (AJINOMOTO) containing SB431542, CHIR99021, DMH1, and FGF2. Subsequently, to induce somite formation at the presomitic mesoderm stage, the cells were continuously cultured in SB431542 and CHIR99021 until day 8. Next, to induce osteorotome formation, the medium was changed to contain SAG (Smoothed Agonist) and LDN193189 between days 8 and 11. Finally, to induce differentiation into the syndetome, the cells were cultured in a medium containing FGF8, TGF-β3, and BMP7 from day 11 to day 19. Each step was performed approximately 3-5 days apart, and the entire differentiation process took approximately 19 days.

[0084] After harvesting tendon cells that had differentiated from iPSCs, they were prepared at a concentration of 1×10^6 cells / mL, washed with PBS, and then suspended in 1 mL single-cell suspensions. The prepared cells were centrifuged (1300 RPM, 5 min), the supernatant was removed, and the cells were treated with fluorescently labeled antibodies specific to tendon cell markers (CD34 Monoclonal Antibody-APC (Invitrogen, cat no_17-0349-42), CD45 Monoclonal Antibody-PE (Invitrogen, cat no_12-0451-82), CD73 Monoclonal Antibody-FITC (Invitrogen, cat no_11-0739-42), CD105 Monoclonal Antibody-PE-Cyanine7 (Invitrogen, cat no_25-1057-42)) at room temperature for 30 min. Subsequently, the cells were washed twice more with PBS, and the expression of surface markers was analyzed using a flow cytometer.

[0085] Flow cytometry analysis showed that the surface marker patterns of iPSCs and iPSC-derived tendon cells were as follows: Figure 1a As shown in the figure, iPSC-derived tendon cells specifically expressed CD73+, CD105+, CD34-, and CD45- as surface markers. Conversely, iPSCs that did not differentiate into tendon cells were characterized by CD73- and CD105-. Furthermore, to investigate reproducibility, iPSC-derived tendon cells underwent three repeated differentiation induction experiments, and the results were analyzed using the same method, confirming that they exhibited a consistent marker expression pattern.

[0086] Examples 1-2. Confirmation of tendon cell-specific characteristics of iPSC-derived tendon cells This embodiment uses immunofluorescence staining to analyze the protein expression and morphological changes of stem cell markers and tendon cell markers to confirm whether iPSC-derived tendon cells possess tendon cell-specific characteristics. Specifically, iPSCs and iPSC-derived tendon cells were smeared onto glass slides and fixed with 4% paraformaldehyde. Then, the cells were permeated with Triton X-100, and non-specific binding was blocked with bovine serum albumin (BSA). Primary antibodies (anti-OCT4 (abcam, cat no. ab19857), anti-Collagen type I (abcam, cat no. ab6308), anti-Vimentin (Invitrogen, cat no. MA5-11883), anti-SCXA (Invitrogen, cat no. PA5-23943), and anti-Tenomodulin (Santa Cruz, cat no. sc-518131)) were diluted 1:200–1:50 according to the manufacturer's recommended conditions and treated at 4°C for 16 hours. Then, fluorescently labeled secondary antibodies were diluted 1:500 with PBS at room temperature and stained for 1 hour. (The secondary antibodies used at this time were: 1. Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488, Invitrogen cat no. A-11001; 2. Goat anti-Rabbit...) 1. IgG(H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488, Invitrogen cat no_A-11008; 2. Goat anti-Mouse IgG(H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594, Invitrogen cat no_A-11005; 3. Goat anti-Rat IgG(H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594, Invitrogen cat no_A-11007. Subsequently, the nucleus was stained with DAPI, and the expression location and intensity of each marker were compared using a fluorescence microscope.

[0087] The results (Figures 1b and 1c) showed that OCT4 was expressed only in iPSCs, while in iPSC-derived tendon cells, tendon cell-specific markers, namely collagen type I, vimentin, SCXA, and tenomodulin (TNMD), were all strongly expressed. Furthermore, it was observed that during the differentiation of iPSCs into tendon cells, the cell morphology changed from the round and small induced pluripotent stem cell morphology to the slender spindle-shaped morphology characteristic of tendon cells.

[0088] This indicates that during the differentiation of iPSCs into tendon cells, stem cell characteristics are lost and tendon cell-specific phenotypes are acquired. It also confirms that the differentiation induction method of the present invention can induce the transformation into functional tendon cells.

[0089] Examples 1-3. Gene expression analysis to confirm the differentiation characteristics of iPSC-derived tendon cells. This embodiment uses reverse transcription polymerase chain reaction (RT-PCR) to analyze at the gene level whether differentiation induction can actually induce the expression of tendon cell-specific genes. Specifically, total RNA was extracted from iPSCs and iPSC-derived tendon cells using TRIzol reagent (Invitrogen™, Cat. No. 15596026). Subsequently, complementary deoxyribonucleic acid (cDNA) was synthesized using the SensiFAST™ cDNA Synthesis Kit (Meridian Bioscience). Using the synthesized cDNA as a template, the gene expression of OCT4, collagen type I, Mohawk protein (MKX), and tenomodulin (TNMD) was analyzed. Real-time quantitative PCR was performed using the CFX96 TouchReal-Time PCR Detection System (Bio-Rad). Amplification was performed using SensiFAST™ SYBR. ® The Hi-ROX Kit (Meridian Bioscience) was used for initial denaturation at 95°C for 10 minutes. Subsequently, denaturation was performed for a total of 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Gene expression levels were quantified using the 2^-ΔΔCt method, with the expression level of the intrinsic contrast gene GAPDH as a baseline.

[0090] The results (Figure 1d) showed that the stem cell-specific gene OCT4 was expressed only in iPSCs; while in iPSC-derived tenocytes, the expression levels of collagen type I, MKX and TNMD genes were relatively high.

[0091] The above results indicate that as iPSCs differentiate into tendon cells, tendon cell specificity is conferred at the gene level, which corresponds to the protein expression results confirmed by immunofluorescence staining performed in Examples 1-2.

[0092] Example 2. Confirmation of the concentration of iPSC-derived tendon cells demonstrating therapeutic efficacy in rotator cuff tears. The optimal cell number and culture conditions for treating iPSC-derived tendon cells, whose characteristics were analyzed in Example 1, onto biomaterial-based supports to form a complex were pre-identified. Specifically, the Cell Counting Kit-8 (Dojindo) was used. To confirm the extent of cell survival and proliferation after attachment to each support, absorbance (OD value) was measured at 450 nm at various initial cell densities of 1×10^3 cells, 1×10^4 cells, 1×10^5 cells, and 1×10^6 cells after culturing for 24, 48, and 72 hours.

[0093] result( Figure 2a , Figure 2b Under the condition of 1×10^5 cells cultured for 24 hours, all three supports (RegenPatch, PLGA / Col, and pig dermis (PigSkin)) showed OD values ​​similar to those of the cell culture dish (approximately 0.6). This indicates that this number of cells stably adhered to each support.

[0094] Conversely, under conditions below 1×10^4 cells, the measured OD values ​​were generally low, which was attributed to the low initial cell density, which failed to adequately cover the support surface, resulting in a limited number of attached cells. Therefore, even after extending the culture time to 48 hours and 72 hours, the OD values ​​did not increase but decreased, indicating that cell proliferation and surface adaptation failed to proceed smoothly.

[0095] On the other hand, under the condition of 1×10^6 cells, although the initial OD value was high, the OD value showed a decreasing trend after 24 hours of culture. It is speculated that this is due to the excessive density between cells, which leads to competition for nutrients and local cell death; at the same time, it was observed that the heterogeneity of the cell layer caused by the excessive density culture hindered the diffusion and stable attachment of cells on the surface of the support.

[0096] Based on the above results, the condition of attaching 1×10^5 tendon cells for 24 hours was set for the experiment in the following embodiments.

[0097] Example 3. Adhesion and surface adaptation analysis of iPSC-derived tendon cells on various biomaterial supports The iPSC-derived tendon cells with the characteristics analyzed in Example 1 were attached to different biomaterial supports to form a composite, and the surface adaptability of the composite was analyzed.

[0098] First, iPSC-derived tendon cells are attached to three biomaterial supports to form a composite: ① telopeptide-free collagen patch (100% telopeptide-free collagen, 5×5×0.5mm, RegenPatch, Sewon Cellontech); ② PLGA / collagen membrane (PLGA: type I Col = 3:7, thickness 0.01~0.05mm); ③ supercritical drying decellularized dermis (thickness 0.5~0.8mm). The manufacturing process of supercritical drying decellularized dermis involves separating the dermis from animals (such as cattle, rabbits, pigs, etc.), washing and degreasing it, then immersing it step-by-step in a concentrated dehydrating solvent to remove moisture, followed by solvent replacement and drying under supercritical carbon dioxide conditions.

[0099] Next, each biomaterial support (5×5 mm) was inserted into a 12-chamber slide (SPL, Cat. No. 301012), and iPSC-derived tendon cells were seeded in each well at a concentration of 1×10^5 cells and cultured at 37°C for 24 hours. The culture medium consisted of 1x DMEM (welgene, cat no: LM001-01) supplemented with 10% FBS (welgene, cat no: S101-01) and 1% antibiotic-antimycotic (welgene, cat no: LS203-01). Cells were then fixed with 2.5% glutaraldehyde solution at 4°C for 24 hours, followed by a series of dehydration and metal coating processes to prepare the samples. Scanning electron microscopy (SEM) analysis was performed using a Hitachi Regulus 8220 instrument to observe cell attachment and diffusion on the supports.

[0100] Comparing the states where no cells were applied to each support (support only; implant only) and the states where cells were applied (support + induced pluripotent stem cells-tendon cells, 24 hours; implant + iPSC-TC, 24 hours), the results (Figure 3) showed that cells were uniformly distributed and attached to the surface of all supports where cells were applied; however, the diffusion morphology and distribution pattern of cells varied depending on the microscopic surface structure of the support.

[0101] This indicates that the iPSC-derived tendon cells according to the present invention can stably attach and survive on various types of biological supports, serving as a cell-support complex and a potential graft for tissue regeneration. Furthermore, it is hypothesized that the induced activity may differ depending on the type of support.

[0102] Example 4. Confirmation of the excellent survival and maintenance capacity of iPSC-derived tendon cells and determinated peptide collagen complex in a rotator cuff tear model. This embodiment analyzed the survival and maintenance capacity of iPSC-derived tendon cells (hereinafter referred to as the "complex") combined with various biomaterial supports in tissues. Specifically, 54 12-week-old Sprague Dawley rats with an average weight of 200 to 300 g were used in the experiment. The iPSC-derived tendon cells were fluorescently labeled with PKH26 Red Fluorescent Cell Linker (Sigma-Aldrich, MINI26) and then attached to the supports.

[0103] The experimental groups in this embodiment are divided into the following 9 complex groups (n=6): 1. Control (No treatment) 2. Surgery only (tendon suturing only) 3. TC only (transplantation of TC with PKH26 marker alone 1×10 5 cells) 4. RegenPatch (Terminal peptide-free collagen patch) 5. RegenPatch + TC (1×10) 5 cells) 6. PLGA / Collagen Film (Polylactic acid-glycolic acid copolymer / collagen film) 7. Polyester PLGA / Collagen Film + TC (1×10) 5 cells) 8. Pig Skin 9. Genuine pigskin + TC (1×10) 5 cells) On the other hand, physical surgery was performed to create an animal model of rotator cuff tear, and the specific surgical procedure is as follows: After anesthetizing the rats with ketamine (75-95 mg / kg) and toluenethiazide (5 mg / kg) intramuscularly, the shoulder area was incised under aseptic conditions, and the deltoid muscle was dissected. Subsequently, the supraspinatus tendon was severed at the greater tuberosity, inducing a full-thickness rotator cuff tear. The severed tendon was reconstructed using the Mason-Allen suture technique and Ethibond 5-0, creating the rotator cuff tear animal model. Afterwards, depending on the group settings, fluorescently labeled individual TCs (tendon cells) or a scaffold complex (containing TCs) (5×5 mm) were transplanted to the suture site (tendon-to-bone interface).

[0104] PKH26 fluorescence signals were analyzed every 7 days post-transplantation using an in vivo fluorescence imaging system (IVIS) to assess cell survival, location, and maintenance characteristics for a total of 3 weeks (up to 30 days).

[0105] The results (Figure 4a) showed that the RegenPatch + TC group maintained the highest fluorescence intensity, and compared with other complex groups mixed with biomaterial supports, this group exhibited a significantly sustained fluorescence signal over time. In particular, it maintained a strong fluorescence signal at week 3, demonstrating excellent results in terms of transplanted cell viability and tissue maintenance.

[0106] Furthermore, quantitative analysis results also showed that the RegenPatch + TC group had the highest average radiant efficiency (Figure 4b), which means that under the same conditions, the colonization rate and survival rate of iPSC-derived tendon cells were significantly improved compared with other biomaterial supports.

[0107] In summary, the iPSC-derived tendon cells compound of the present invention, applied to the RegenPatch support, is most effective as a cell delivery carrier and cell therapy agent, and has been shown to have excellent therapeutic effects on rotator cuff tears by ensuring stable distribution and survival of cells in the tissue, which promotes tissue regeneration.

[0108] Example 5. Confirming the possibility of long-term colonization of iPSC-derived tendon cells and telopeptide collagen complex. This embodiment evaluated the colonization and tissue maintenance of a complex composed of iPSC-derived tendon cells and a biomaterial support. To this end, after rotator cuff tear surgery, rotator cuff tissue from each experimental group was harvested, and fluorescence intensity was quantified using an in vitro fluorescence imaging system (IVIS). At this point, the experimental groups were the same as in Example 4, and nine groups were compared: Control, Surgery only, TC only, RegenPatch (telopeptide-free collagen patch), RegenPatch+TC (telopeptide-free collagen patch + tendon cells), PLGA / Col film (polylactic acid-glycolic acid copolymer / collagen film), PLGA / Col film+TC (polylactic acid-glycolic acid copolymer / collagen film + tendon cells), pig skin, and pig skin+TC.

[0109] The results (Figures 5a and 5b) showed that the RegenPatch + TC group had the highest fluorescence intensity, averaging 4.07 × 10⁻⁶. 7 p / s / cm² / sr [μW / cm²]; and confirmed that the porcine dermal + TC group and the PLGA / Col Film + TC group were 4.16×10⁻⁶. 7 and 4.13×10 7 .

[0110] In particular, the RegenPatch + TC group and the RegenPatch alone group (3.42 × 10⁻⁶) 7 ), only TC group (3.25×10 7 ) and the surgery-only group (3.45×10 7 Compared to the previous method, it exhibited a significantly higher average fluorescence intensity, confirming that binding to the support had a positive effect on the maintenance of iPSC-derived cells in tissues.

[0111] Furthermore, the RegenPatch + TC group exhibited the lowest variability in standard deviation, at 1.85 × 10⁻⁶. 6 This confirms that the determinate peptide collagen patch of the present invention is a platform that can stably ensure the fixation and transmission efficiency of tendon cells.

[0112] In summary, experiments have demonstrated that, compared to other complexes, the complex of the present invention, composed of iPSC-derived tendon cells and determinate collagen support, ensures high survival and maintenance rates when transplanted into tissues; furthermore, the delivered cells can be maintained within the tissue for a long period, thus enabling it to function as a highly effective cell delivery system for biosupport-based cell therapy that contributes to tissue regeneration.

[0113] Example 6. Confirmation of the excellent therapeutic effect of iPSC-derived tendon cells and determinated peptide collagen complex on rotator cuff tears. This embodiment compares and analyzes whether applying iPSC-derived tendon cells to various supports (including patches composed of determinate collagen) demonstrates therapeutic efficacy. Specifically, using the same experimental groups as in Examples 4 and 5, each complex was transplanted into the torn rotator cuff of the mouse model of rotator cuff tear from Example 4. Subsequently, at a time point of 4 weeks, the recovery of mechanical strength of the tissue was assessed using a tensile test. The tensile test used stiffness (N / mm) as a baseline, which is the most consistent and reliable indicator of tissue biomechanical recovery, to analyze the degree of mechanical recovery between groups. Quantitative analysis results were used as a benchmark to assess the contribution of biomaterials and cell complexes to tissue recovery.

[0114] The results (Figure 6) show that the Surgery Only group exhibited relatively low mechanical strength values ​​across all parameters, indicating insufficient tissue repair. Conversely, the composite transplantation group using iPSC-TC showed high values ​​overall, with only the RegenPatch + TC group demonstrating statistically significant high values, showcasing optimal performance in terms of mechanical recovery.

[0115] These results demonstrate that the RegenPatch-based composite graft of the present invention can effectively support the colonization and functional expression of transplanted tendon cells, thereby contributing to the structural and mechanical recovery of rotator cuff tissue and thus exhibiting excellent therapeutic activity for rotator cuff injuries.

[0116] The above description of the present invention is merely exemplary. Those skilled in the art should understand that it can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, the embodiments described above are exemplary in all respects and not restrictive.

Claims

1. A determinate peptide collagen patch for preventing or treating rotator cuff tears, characterized in that, It will consist of a determinate collagen patch made of determinate collagen, and Induced pluripotent stem cell-derived tendon cells attached to the surface of the determinate collagen patch are included as an active ingredient.

2. The patch for preventing or treating rotator cuff tears according to claim 1, characterized in that, The determinate collagen patch for the prevention or treatment of rotator cuff tears contains more than 1×10^4 cells and less than 1×10^6 cells of induced pluripotent stem cell-derived tendon cells within a basic unit size of the determinate collagen patch.

3. The patch for preventing or treating rotator cuff tears according to claim 1, characterized in that, The basic unit dimensions of the determinated peptide collagen patch are 1 to 10 mm in width, 1 to 10 mm in height, and 0.1 to 1 mm in thickness.

4. The patch for preventing or treating rotator cuff tears according to claim 1, characterized in that, The induced pluripotent stem cell-derived tendon cells have at least one characteristic selected from the group consisting of: It is characterized by expressing CD73 and CD105, but not expressing CD34 and CD45; Expression of type 1 collagen, vimentin, SCXA, tendon regulatory protein, and MKX; and It exhibits a spindle-shaped morphological feature.

5. A patch composition for preventing or treating rotator cuff tears, characterized in that, It contains determinate collagen and tendon cells derived from induced pluripotent stem cells as active ingredients.

6. A pharmaceutical composition for the prevention or treatment of rotator cuff tears, characterized in that, It contains determinate collagen and tendon cells derived from induced pluripotent stem cells as active ingredients.

7. The pharmaceutical composition for preventing or treating rotator cuff tears according to claim 6, characterized in that, The pharmaceutical composition can increase the toughness of torn rotator cuff tissue.

8. A composition for manufacturing a patch for preventing or treating rotator cuff tears, characterized in that, It contains determinate collagen and tendon cells derived from induced pluripotent stem cells as active ingredients.

9. The method of manufacturing the patch for preventing or treating rotator cuff tears as described in claim 1, characterized in that, The manufacturing method includes the following steps: The steps for manufacturing a determinated peptide collagen patch composed of determinated peptide collagen; and The step of treating the determinate collagen patch with induced pluripotent stem cell-derived tendon cells and culturing them to allow the tendon cells to attach to the surface of the determinate collagen patch.

10. A kit for manufacturing a patch for preventing or treating rotator cuff tears, characterized in that, It includes the composition according to claim 8 and the specification.

11. The kit for manufacturing a patch for preventing or treating rotator cuff tears according to claim 10, characterized in that, The specification teaches a method for manufacturing the patch for preventing or treating rotator cuff tears as described in claim 9.

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