A composition for retarding the in vivo degradation rate and enhancing the rheological properties of a hyaluronic acid hydrogel for biological transplantation

Abstract

This invention relates to a composition for delaying the in vivo degradation rate of hyaluronic acid hydrogels for bio-implantation and enhancing their rheological properties. Specifically, it relates to a composition that can be used in the medical field to mix allogeneic decellularized dermal matrix particles with hyaluronic acid hydrogels for tissue repair bio-implantation with a storage modulus (G') of 300 Pa or less. The effect is to delay the in vivo degradation rate of hyaluronic acid, relatively increase the storage modulus (G') without the need for cross-linking agents, and provide convenient injection thrust.

Description

Technical Field

[0001] This invention relates to a composition for delaying the in vivo degradation rate of hyaluronic acid hydrogels for bio-implantation and enhancing their rheological properties. Specifically, it relates to a composition that can be used in the medical field to mix allogeneic decellularized dermal matrix particles with hyaluronic acid hydrogels for bio-implantation of tissue repair with a storage modulus (G') of 300 Pa or less. Background Technology

[0002] Hyaluronic acid (HA) is a naturally biosynthesized substance found in various tissues of the body, such as the skin. Due to its excellent biocompatibility, moisturizing properties and physical characteristics, it is widely used in biomedical fields, such as facial fillers and intra-articular injections, in the form of hydrogels.

[0003] However, hyaluronic acid-based hydrogels degrade rapidly in vivo, making it difficult to maintain their function as biomaterials. To compensate for this deficiency, chemical cross-linking agents such as 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), and divinyl sulfone (DVS) are typically used for cross-linking to improve in vivo stability and enable commercial applications. The rheological properties of cross-linked hyaluronic acid hydrogels as tissue repair biomaterials are significantly affected by the cross-linking density.

[0004] When injecting hyaluronic acid hydrogels into the skin, different rheological properties are required for each site. For example, areas such as the forehead and bridge of the nose require compositions with relatively high storage modulus (G'). Currently, commercially available products meet this requirement by increasing the concentration of hyaluronic acid in the finished product or using more cross-linking agents during preparation. Increasing the concentration of hyaluronic acid may increase the injection force during application or make it difficult to control the content after the washing process, while increasing the amount of cross-linking agent used during preparation may raise issues related to the safety of biomaterials.

[0005] Therefore, there is a need for a hyaluronic acid-based hydrogel composition and its preparation method that can delay the in vivo degradation of hyaluronic acid without adding more crosslinking agents during preparation.

[0006] On the other hand, skin grafts are categorized into human allogeneic dermal grafts, animal xenogeneic dermal grafts, and synthetic materials. While human acellular dermal matrix-based skin grafts are relatively expensive, they are considered the superior alternative because the decellularization process prevents immune rejection or inflammation after transplantation. The extracellular matrix (ECM) components in the dermis, such as collagen, elastin, and fibronectin, are essential intracellular elements that promote cell adhesion and proliferation, thereby achieving skin regeneration.

[0007] "Sheet-type human acellular dermal matrix" refers to the dermis obtained by chemically treating isolated allogeneic dermis to remove cells that could trigger an immune rejection response. Its main components are collagen and elastin. Sheet-type acellular dermal matrix tissue is prepared into various sizes according to the target lesion site and used by grafting or implantation.

[0008] "Particulated human acellular dermal matrix (phADM)" is obtained by crushing sheet-like acellular dermal matrix into granular structures. In existing technologies, freeze-dried granular acellular dermal matrix is ​​directly hydrated with physiological saline or distilled water and used as a skin substitute. Because it remains fluid, it offers the advantage of being non-surgical and injectable directly with a syringe. Therefore, granular acellular dermal matrix tissue is used not only to treat skin tissue defects caused by accidents but also for tissue repair or reconstruction, such as treating chronic diseases like diabetic ulcers. Currently, most commercially available granular allogeneic dermal matrix has an average particle size of 500 μm or larger. Therefore, injections for plastic and cosmetic purposes require larger diameter needles, and it is difficult to homogeneously mix and disperse with existing commercially available tissue repair biomaterials such as hyaluronic acid hydrogel fillers (HA fillers), presenting certain limitations.

[0009] Korean Invention Patent No. 10-1523878 discloses a method for preparing a biological transplant composition consisting of a cross-linked granular acellular dermal matrix and hyaluronic acid. However, the granular allogeneic dermal matrix used in this invention has a particle size of 300 μm to 800 μm, making it difficult to use for injection applications for plastic and cosmetic purposes. Furthermore, this invention employs a method of cross-linking hyaluronic acid onto the surface of acellular dermal matrix particles, which differs significantly from the method of physically mixing commercially available hyaluronic acid hydrogels with acellular dermal matrix.

[0010] To date, there is no precedent for mixing granular allogeneic decellularized dermal matrix into smaller particles and then mixing it with hyaluronic acid hydrogel, making it readily injectable as a biomaterial for tissue repair. Furthermore, no studies have been reported on improving the rheological properties of compositions by addressing the shortcomings of commercially available hyaluronic acid hydrogel products without cross-linking or other reactions.

[0011] Although some literature discloses finished compositions containing allogeneic decellularized dermal matrix and hyaluronic acid hydrogel, there are currently no reports on research regarding methods for directly preparing decellularized dermal matrix mixtures in the medical setting with the aim of delaying the in vivo degradation of commercially available hyaluronic acid hydrogel products and improving their rheological properties.

[0012] Therefore, the inventors conducted a specific study on a method for mixing decellularized dermal matrix particles with commercially available hyaluronic acid hydrogel products in a medical setting. This led to the discovery of conditions that delay the in vivo degradation of hyaluronic acid and improve its rheological properties without the addition of a cross-linking agent, thus completing this invention. Specifically, this was achieved by determining the particle size of the decellularized dermal matrix, the relative ratio of hyaluronic acid particle size to the decellularized dermal matrix particle size, the mixing concentration of the decellularized dermal matrix, and commercially available...

[0013] The storage modulus (G') of hyaluronic acid hydrogel itself revealed the conditions for delaying the in vivo degradation of hyaluronic acid and improving its rheological properties. Summary of the Invention

[0014] Technical issues

[0015] The first objective of this invention is to provide a composition for delaying the in vivo degradation rate of hyaluronic acid hydrogels used in tissue repair bio-implants and enhancing their rheological properties.

[0016] A second objective of this invention is to provide a biotransplant composition for on-site preparation of tissue repair.

[0017] A third objective of this invention is to provide a finished biological transplant composition for tissue repair.

[0018] Technical solution

[0019] This invention specifically investigates a method for using decellularized dermal matrix particles mixed with commercially available hyaluronic acid hydrogel products in a medical setting. This revealed conditions for delaying the in vivo degradation of hyaluronic acid and improving its rheological properties without the addition of a cross-linking agent. Specifically, by determining the particle size of the decellularized dermal matrix, the relative ratio of hyaluronic acid particle size to the decellularized dermal matrix particle size, the mixing concentration of the decellularized dermal matrix, and the storage modulus (G') of the commercially available hyaluronic acid hydrogel, the conditions for delaying the in vivo degradation of hyaluronic acid and improving its rheological properties were discovered.

[0020] When applied to sites requiring a relatively high storage modulus (G') (such as the forehead, bridge of the nose, etc.), this invention can effectively improve application satisfaction and is expected to extend the retention time of tissue repair biomaterial implants in the body.

[0021] Composition for delaying the in vivo degradation rate of hyaluronic acid hydrogels and enhancing their rheological properties

[0022] This invention provides a composition for delaying the in vivo degradation rate of hyaluronic acid hydrogels and enhancing their rheological properties, comprising allogeneic decellularized dermal matrix particles and a medical diluent.

[0023] The hyaluronic acid hydrogel is used for biological transplantation for tissue repair, and its energy storage elastic modulus (G') is 300 Pa or less.

[0024] In the composition of the present invention, the hyaluronic acid hydrogel is a hyaluronic acid hydrogel that needs to slow down the in vivo degradation rate or enhance rheological properties. It can be a commercially available product that is already in use in the medical field or a product that is about to be commercially available.

[0025] In this invention, allogeneic decellularized dermal matrix particles can be selectively mixed with hyaluronic acid hydrogels with a storage modulus (G') of 300 Pa or less to improve their rheological properties (storage modulus, G'). If hyaluronic acid hydrogels with a storage modulus (G') exceeding 300 Pa are used, the storage modulus (G') may actually decrease (see Figure 3 and Table 4).

[0026] In the compositions of the present invention, the average particle size of the allogeneic acellular dermal matrix can be 300 μm or smaller, specifically 200 μm or smaller, more specifically 100 μm or smaller. If an allogeneic acellular dermal matrix with a particle size exceeding 300 μm is used, the allogeneic acellular dermal matrix particles may not be uniformly mixed in the hyaluronic acid hydrogel (see Table 1 and Figure 1).

[0027] Furthermore, the particle size of the allogeneic acellular dermal matrix can be 1 / 3 or smaller than the average particle size of the hyaluronic acid, preferably 1 / 5 or smaller. If it exceeds 1 / 3, there may be a problem that the allogeneic acellular dermal matrix particles cannot be uniformly mixed in the hyaluronic acid hydrogel (see Table 1 and Figure 1).

[0028] In the composition of the present invention, the medical diluent can be normal saline, distilled water, phosphate-buffered saline (PBS), or a combination of two or more of them. Preferably, normal saline, which is most commonly used in medical settings, can be used.

[0029] In the compositions of the present invention, the content of the allogeneic acellular dermal matrix particles in the mixture of the composition and hyaluronic acid hydrogel can be 10% by weight or less, specifically 1% to 10% by weight, and more specifically 1% to 5% by weight. If it exceeds 10% by weight, there may be problems with application due to a sharp increase in injection force (see Table 2). If it is less than 1% by weight, the effect of delaying the in vivo degradation of hyaluronic acid and improving rheological properties may not be significant. In addition, after using the composition of the present invention in combination with hyaluronic acid hydrogel, the degradation rate of hyaluronidase on hyaluronic acid in vivo is significantly reduced. If the content of allogeneic acellular dermal matrix particles in the mixture exceeds 10% by weight, the effect of delaying the degradation rate of hyaluronic acid may gradually weaken (see Table 3).

[0030] For reference, when using hyaluronic acid hydrogel as a biomaterial for tissue repair in cosmetic surgery, due to issues such as ease of injection, the injection thrust is generally set to 40 N or less when connecting the syringe needle (Guidelines for Licensing Review of Biomaterials for Tissue Repair in Cosmetic Surgery, Ministry of Food and Drug Safety, Korea, 2020).

[0031] The rheological properties refer to an increase in the storage modulus (G'). This also implies a decrease in the loss factor (tan delta, tan δ) and an increase in complex viscosity.

[0032] On-site prepared biological transplantation compositions for tissue repair

[0033] This invention provides a biological transplant composition for tissue repair, comprising allogeneic acellular dermal matrix particles.

[0034] The application involves using a mixed composition prepared by mixing a solution with hyaluronic acid hydrogel on-site, the solution being prepared by mixing the allogeneic decellularized dermal matrix particles in a medical diluent.

[0035] The energy storage modulus (G') of the hyaluronic acid hydrogel is 300 Pa or less.

[0036] In the biological transplant composition for tissue repair of the present invention, the medical diluent can be physiological saline, distilled water, phosphate-buffered saline, etc., or a mixture of two or more of them. Preferably, physiological saline, which is most commonly used in medical settings, can be used.

[0037] Specifically,

[0038] The size of the allogeneic decellularized dermal matrix particles can be 300 μm or smaller.

[0039] The content of allogeneic decellularized dermal matrix particles in the mixture can be from 0.5% to 10% by weight.

[0040] The content of hyaluronic acid hydrogel in the mixture can be from 0.5% to 2% by weight.

[0041] The size of the allogeneic decellularized dermal matrix particles can be 1 / 3 or smaller than the average particle size of the hyaluronic acid.

[0042] Specifically,

[0043] The size of the allogeneic decellularized dermal matrix particles can be 200 μm or smaller.

[0044] The content of allogeneic decellularized dermal matrix particles in the mixture can be from 1% to 10% by weight.

[0045] The content of hyaluronic acid hydrogel in the mixture can be from 0.7% to 1.5% by weight.

[0046] The size of the allogeneic decellularized dermal matrix particles can be 1 / 3 or smaller than the average particle size of the hyaluronic acid.

[0047] Specifically,

[0048] The size of the allogeneic acellular dermal matrix particles can be 100 μm or smaller.

[0049] The content of allogeneic decellularized dermal matrix particles in the mixture can be from 1% to 5% by weight.

[0050] The content of hyaluronic acid hydrogel in the mixture can be from 0.8% to 1.4% by weight.

[0051] The size of the allogeneic decellularized dermal matrix particles can be 1 / 3 or smaller than the average particle size of the hyaluronic acid.

[0052] Specifically,

[0053] The size of the allogeneic decellularized dermal matrix particles can be 100 μm or smaller.

[0054] The content of allogeneic decellularized dermal matrix particles in the mixture can be from 1% to 5% by weight.

[0055] The content of hyaluronic acid hydrogel in the mixture can be from 0.9% to 1.3% by weight.

[0056] The size of the allogeneic decellularized dermal matrix particles can be 1 / 3 or smaller than the average particle size of the hyaluronic acid.

[0057] Specifically,

[0058] The size of the allogeneic acellular dermal matrix particles can be 100 μm or smaller.

[0059] The content of allogeneic decellularized dermal matrix particles in the mixture can be from 1% to 5% by weight.

[0060] The hyaluronic acid hydrogel content in the mixture is from 1% to 1.2% by weight.

[0061] The size of the allogeneic decellularized dermal matrix particles can be 1 / 3 or smaller than the average particle size of the hyaluronic acid.

[0062] Pre-made biological transplantation compositions for tissue repair

[0063] Obviously, the conditions for delaying the in vivo degradation of hyaluronic acid and improving its rheological properties as determined by the present invention can also be applied to finished compositions including allogeneic decellularized dermal matrix and hyaluronic acid hydrogel.

[0064] Therefore, the present invention provides a biological transplant composition for tissue repair, comprising: a solution prepared by mixing allogeneic decellularized dermal matrix particles with a medical diluent; and

[0065] Hyaluronic acid hydrogel with a storage modulus (G') of 300 Pa or less.

[0066] In the tissue repair composition according to the present invention

[0067] The size of the allogeneic decellularized dermal matrix particles can be 300 μm or smaller.

[0068] The content of allogeneic decellularized dermal matrix particles in the composition can be from 0.5% to 15% by weight.

[0069] The content of hyaluronic acid hydrogel in the composition can be from 0.5% to 2% by weight.

[0070] The size of the allogeneic decellularized dermal matrix particles can be 1 / 3 or smaller than the average particle size of the hyaluronic acid.

[0071] Beneficial effects

[0072] The present invention relates to a composition for delaying the in vivo degradation rate of hyaluronic acid hydrogels with a storage modulus (G') of 300 Pa or less used in bio-transplantation for tissue repair and enhancing their rheological properties. By mixing allogeneic decellularized dermal matrix particles with hyaluronic acid hydrogel, the composition has the effects of delaying the in vivo degradation rate of hyaluronic acid, relatively increasing the storage modulus (G') without the addition of crosslinking agents, and providing convenient injection thrust.

[0073] Brief description of the attached diagram

[0074] Figure 1A shows images of the average particle size of the decellularized dermal matrix prepared into a mixture of allogeneic decellularized dermal matrix particles and hyaluronic acid, with the particle size being 100 μm or smaller, 100 μm to 300 μm, 300 μm to 500 μm, and 500 μm or larger, and the characteristics of each sample observed.

[0075] Figure 1B shows the results of preparing the average particle size of the decellularized dermal matrix in a mixture of allogeneic decellularized dermal matrix particles and hyaluronic acid to 100 μm or smaller, 100 μm to 300 μm, 300 μm to 500 μm and 500 μm or larger, respectively, and observing the morphology of phADM particles in each sample using an optical microscope.

[0076] Figure 1C is a graph comparing the particle size distribution of the hyaluronic acid sample (sample name: HA1, manufacturer: Hugel, South Korea) with four different allogeneic decellularized dermal matrix powders with average particle sizes of 100 μm or smaller, 100 μm to 300 μm, 300 μm to 500 μm and 500 μm or larger in a mixture of allogeneic decellularized dermal matrix particles and hyaluronic acid.

[0077] Figure 2A shows the results of filling a sample of a mixture of allogeneic decellularized dermal matrix particles (1 wt%, 5 wt%, 10 wt%, 20 wt%) and hyaluronic acid (sample name: HA2, manufacturer: Hugel, South Korea) (1 wt%) into a syringe equipped with a conventional 23G (thin-wall) injection needle to measure the injection thrust.

[0078] Figure 2B shows the degradation rate of hyaluronic acid in the mixture after 24 hours of treatment with hyaluronidase on a sample of allogeneic decellularized dermal matrix particles (0 wt%, 1 wt%, 5 wt%, 10 wt%) and hyaluronic acid (sample name: HA2, manufacturer: Hugel, South Korea) (1 wt%).

[0079] Data in Figure 2 = mean ± standard deviation; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0080] Figure 3A shows the measurement results of the storage modulus (G') before and after mixing phADM with an average particle size of 100 μm or smaller into HA3 or HA4 hyaluronic acid (sample name: HA3, manufacturer: Medytox, South Korea; sample name: HA4, manufacturer: Hugel, South Korea).

[0081] Figure 3B shows the measurement results of loss modulus (G'') before and after mixing phADM with an average particle size of 100 μm or smaller into HA3 or HA4 hyaluronic acid (sample name: HA3, manufacturer: Medytox, South Korea; sample name: HA4, manufacturer: Hugel, South Korea).

[0082] Figure 3C shows the measurement results of the loss factor (tandelta) before and after mixing phADM with an average particle size of 100 μm or smaller into HA3 or HA4 hyaluronic acid (sample name: HA3, manufacturer: Medytox, South Korea; sample name: HA4, manufacturer: Hugel, South Korea).

[0083] Figure 3D shows the measurement results of complex viscosity before and after mixing phADM with an average particle size of 100 μm or smaller into HA3 or HA4 hyaluronic acid (sample name: HA3, manufacturer: Medytox, South Korea; sample name: HA4, manufacturer: Hugel, South Korea).

[0084] Data in Figure 3 = mean ± standard deviation; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0085] Figure 4A shows the measurement results of the storage modulus (G') before and after mixing phADM with an average particle size of 100 μm or smaller into HA5 to HA7 hyaluronic acid (sample name: HA5, manufacturer: Medytox, South Korea; sample name: HA6, manufacturer: Jetema, South Korea; sample name: HA7, manufacturer: Medytox, South Korea).

[0086] Figure 4B shows the measurement results of loss modulus (G'') before and after mixing phADM with an average particle size of 100 μm or smaller into HA5 to HA7 hyaluronic acid (sample name: HA5, manufacturer: Medytox, South Korea; sample name: HA6, manufacturer: Jetema, South Korea; sample name: HA7, manufacturer: Medytox, South Korea).

[0087] Figure 4C shows the measurement results of the loss factor (tan delta) before and after mixing phADM with an average particle size of 100 μm or smaller into HA5 to HA7 hyaluronic acid (sample name: HA5, manufacturer: Medytox, South Korea; sample name: HA6, manufacturer: Jetema, South Korea; sample name: HA7, manufacturer: Medytox, South Korea).

[0088] Figure 4D shows the measurement results of complex viscosity before and after mixing phADM with an average particle size of 100 μm or smaller into HA5 to HA7 hyaluronic acid (sample name: HA5, manufacturer: Medytox, South Korea; sample name: HA6, manufacturer: Jetema, South Korea; sample name: HA7, manufacturer: Medytox, South Korea).

[0089] The data in Figure 4 = mean ± standard deviation; . Detailed Implementation

[0090] The present invention will now be described in detail.

[0091] The term "sheet-type humanacellular dermal matrix" as used in this invention refers to the dermis obtained by chemically treating isolated allogeneic dermis to remove cells that could trigger an immune rejection response; its main components are collagen and elastin. Sheet-type humanacellular dermal matrix tissue is prepared into various area sizes according to the target lesion site and used via grafting or implantation.

[0092] The term "particulated human acellular dermal matrix (phADM)" used in this invention refers to a granular structure obtained by crushing sheet-like acellular dermal matrix. Currently, freeze-dried particulated acellular dermal matrix is ​​directly hydrated with physiological saline or distilled water and used as a skin substitute. Because it maintains a fluid state, it has the advantage of being non-surgical and can be directly injected using a syringe. Therefore, particulated acellular dermal matrix tissue is not only used to treat skin tissue defects caused by accidents, but also for tissue repair or reconstruction, such as treating chronic diseases like diabetic ulcers. Most commercially available particulated allogeneic dermal matrix has an average particle size of 500 μm or larger. Therefore, injections for plastic and cosmetic purposes require the use of larger diameter needles, and it is difficult to homogeneously mix and disperse with existing commercially available tissue repair biomaterials such as hyaluronic acid hydrogel fillers (HA fillers), which presents certain limitations.

[0093] The term "hyaluronic acid (HA)" used in this invention refers to a naturally occurring biosynthetic substance abundant in the skin, synovial fluid, cartilage, and other tissues of animals. Due to its high hydroxyl group content, it is a hydrophilic mucopolysaccharide. When combined with water, it forms a gel, participating in joint lubrication and skin flexibility. Its high viscosity also plays a crucial role in preventing bacterial invasion and toxin penetration into the skin. When hyaluronic acid in the skin is insufficient, the skin becomes dry, lacks elasticity, and wrinkles appear. Therefore, hyaluronic acid is widely used as a biomaterial for tissue repair to prevent wrinkles or fill in sunken areas. Depending on the cross-linking method using BDDE or similar cross-linking agents and the degree of cross-linking, commercially available hyaluronic acid hydrogel products vary in their physicochemical and biological properties.

[0094] The term "injection force" as used in this invention refers to the force applied when force is applied through a syringe, measured in N. When hyaluronic acid hydrogel is used as a biomaterial for tissue repair in cosmetic surgery, due to issues such as ease of injection, the injection force is generally set to 40 N or less when connecting the syringe needle (Guidelines for Licensing Review of Biomaterials for Tissue Repair in Cosmetic Surgery, Korean Ministry of Food and Drug Safety, 2020).

[0095] The term "elastic modulus (G')" used in this invention refers to the value representing the elastic response of an object when force is applied. It is an indicator reflecting the characteristic of an object to change shape after force is applied and to return to its original shape after the force is removed, and the unit is [Pa]. For the physical properties of biomaterials used in tissue repair, a higher elastic modulus indicates stronger elasticity and a firmer feel after bio-injection. This is especially beneficial for fillers used in cosmetic surgery, as they provide a fuller feel and better shape retention.

[0096] The term "viscous modulus (G): loss modulus" as used in this invention refers to an indicator of the degree of energy dissipation due to friction or shear force, measured in Pa. In biomaterials for tissue repair, a higher loss modulus indicates stronger viscosity, especially for fillers used in cosmetic surgery, which is related to their diffusion characteristics in the dermis after injection.

[0097] The term "loss factor (tan delta, tan δ)" used in this invention represents the ratio of elasticity to viscosity, calculated from the loss modulus of elasticity / storage modulus of elasticity (G" / G'), indicating its degree of approximation to a solid or liquid state. A loss factor greater than 1 indicates approximation to a liquid state, while a loss factor less than 1 indicates approximation to a solid state. The smaller the loss factor, the higher the likelihood of the biomaterial moving as a whole. For biomaterials used in tissue repair for cosmetic surgery, a near-solid state means a reduced likelihood of morphological deformation after application and displacement of the implanted filler contents within the tissue. For example, fillers with a lower loss factor are better able to withstand the forces generated by facial muscle movements.

[0098] The term "complex viscosity" as used in this invention, as a function of various measurement frequencies, is a flow resistance value obtained from the geometric mean of the storage elastic modulus and the loss elastic modulus, measured in Pa·s. Higher complex viscosity means that the contents of the tissue repair biomaterial are more rigid; such fillers are suitable for injection into deeper layers of skin tissue or for providing a fuller appearance. Conversely, lower complex viscosity results in a softer, more fluid filler. These fillers are suitable for applications where deformation is not affected or for intradermal injection. Furthermore, higher complex viscosity reduces the diffusion of hyaluronic acid hydrogel fillers within tissues, which is an important factor in maintaining a clear contour after application.

[0099] The present invention will now be described in more detail through the following embodiments. However, the following embodiments are for illustrative purposes only, and the content of the present invention is not limited to the following embodiments.

[0100] <Preparation Example 1> Preparation of freeze-pulverized allogeneic decellularized dermal matrix particles

[0101] Skin tissue (from cadavers donated for non-profit patient treatment) was treated with 1.0 units / mL of neutral protease dispersant. The tissue was then shaken in a shaking incubator at 37°C for 60 to 120 minutes, followed by washing three times with sterile distilled water to separate the dermis and epidermis, after which the epidermis was removed. For the tissue with the epidermis removed, it was treated with a 1% Triton X-100 solution at 30°C for 100 minutes to remove cells from the dermis. The tissue was washed three or more times with sterile distilled water to remove the processing materials. For lyophilization, the allogeneic decellularized dermal matrix was frozen at -40°C or lower for 2 hours or more, followed by lyophilization for 12 hours or more to remove moisture.

[0102] The allogeneic acellular dermal matrix was cut into approximately 1×1 cm² pieces using a scalpel. 100 g of the allogeneic acellular dermal matrix was placed in a micro-pulverizer and pulverized for 3 minutes at 5,000 rpm (optimal for preventing collagen and elastin denaturation) under sterile conditions, producing allogeneic acellular dermal matrix particles that could pass through 500 μm and 300 μm sieves, respectively. To prepare even smaller allogeneic acellular dermal matrix particles, the particles were pulverized using a cryogenic pulverizer, producing allogeneic acellular dermal matrix particles that could pass through a 100 μm sieve. In summary, through this preparation process, phADM particles with an average size less than 100 μm, particles with an average size in the range of 100 μm to 300 μm, particles with an average size in the range of 300 μm to 500 μm, and particles with an average size greater than 500 μm were obtained, respectively.

[0103] <Preparation Example 2> On-site preparation of a mixture of allogeneic decellularized dermal matrix particles and hyaluronic acid hydrogel

[0104] (1) Preparation of a solution containing allogeneic decellularized dermal matrix particles

[0105] Place 5 mL to 10 mL of normal saline into a syringe, and dispense the allogeneic acellular dermal matrix particles prepared in Preparation Example 1 into another syringe at the desired concentration. After installing the Luer-lock connector, reciprocate the plungers on both sides 5 to 10 times to prepare an allogeneic acellular dermal matrix solution containing the allogeneic acellular dermal matrix particles and normal saline.

[0106] (2) Preparation of a mixed composition of allogeneic decellularized dermal matrix solution and hyaluronic acid hydrogel

[0107] The allogeneic decellularized dermal matrix solution and commercially available hyaluronic acid hydrogel were loaded into different syringes according to the required concentration and volume. After installing the Luer lock connector, the plunger was reciprocated 5 to 10 times to prepare a mixed composition of hyaluronic acid hydrogel and allogeneic decellularized dermal matrix solution.

[0108] Injection thrust evaluation

[0109] Injection force was evaluated using a ZwickiLine (Zwick / Roell, Germany) instrument. A pre-filled syringe containing the sample was inserted into the zig and adjusted so that the syringe piston was centered on the fixed plate. The injection force was measured at a rate of 12 mm / min.

[0110] Particle size measurement

[0111] The particle size measurement and analysis of this invention were performed using a particle size analyzer (LS13320XR, Beckman Coulter, USA). The sample to be tested was diluted in physiological saline at a concentration of 0.1% to 1% by weight, and the measurement was performed in wet mode.

[0112] Measurement of the enzymatic degradation rate of hyaluronic acid

[0113] The degradation assay of this invention was performed according to the method described in JL Reissig et al. (A modified colorimetric method for the estimation of N-acetylamino sugar, J. Biol. Chem. 1955, 217:959-966). Equal masses of hyaluronic acid hydrogel were loaded into each test tube, followed by the addition of PBS (pH 7.4) containing 10 IU / mL of hyaluronidase (Sigma-Aldrich, USA). The mixture was incubated at 37 °C for 24 hours. The enzymatic reaction was terminated with 0.1 N HCl (40 vol%), and after centrifugation, only the supernatant was collected. The amount of N-acetylglucosamine (NAG) degraded was measured using the carbazole assay.

[0114] To perform the carbazole method, 0.5 mL of sample was added to H₂SO₄ reagent containing 0.025 M sodium tetraborate·10H₂O, and heated at 99 °C for 10 minutes. After slow cooling, 0.1 mL of 0.125% carbazole reagent was added, and the reaction was terminated by heating for another 15 minutes. For samples that completed the carbazole colorimetric reaction, the absorbance was measured at 530 nm using a UV-Vis spectrophotometer. The degree of degradation of the crosslinked material was analyzed for each sample with the product label content set at 100%.

[0115] Viscoelastic property analysis

[0116] The viscoelastic properties analysis of this invention was performed using a vibratory-rotational rheometer equipped with parallel plates. The sample to be tested was placed between the plates, one plate was rotated, and a constant horizontal shear stress was applied while the viscoelasticity was measured. The experimental equipment was a rheometer (MCR302e, Anton Paar Ltd., Austria), and the experimental items recorded were storage modulus (G', Pa·s), loss modulus (G", Pa·s), loss factor (G" / G', dimensionless), and complex viscosity (Pa·s). During the measurement, the vibration frequency was set to 0.1 Hz, the temperature to 25 ℃, the shear strain to 1%, the measurement interval to 1.0 mm, and the parallel plates were 25 mm circular plates.

[0117] <Experimental Example 1> Evaluate the physical properties of the mixed composition based on the average particle size of allogeneic decellularized dermal matrix particles.

[0118] After preparing allogeneic acellular dermal matrix powder according to the method of Preparation Example 1, it was filtered through a sieve according to the average particle size. The average particle size range of the filtered particles was 100 μm or smaller, 100 μm to 300 μm, 300 μm to 500 μm, and 500 μm or larger. The four powders were each mixed in physiological saline at a concentration of 10%, and then 1 mL of the solution was mixed with 1 mL of commercially available hyaluronic acid hydrogel (sample name: HA1, manufacturer: Hugel, South Korea) according to Preparation Example 2. Accordingly, the content of particulate allogeneic acellular dermal matrix (phADM) in the mixed composition was 5% by weight. The comparison results of the properties of the various mixed compositions are shown in Figure 1 and Table 1.

[0119] Figure 1A shows images of the particle sizes of decellularized dermal matrix prepared to be 100 μm or smaller, 100 μm to 300 μm, 300 μm to 500 μm, and 500 μm or larger in a mixture of allogeneic decellularized dermal matrix particles and hyaluronic acid, and the characteristics of each sample observed.

[0120] Figure 1B shows the results of preparing the particle size of the decellularized dermal matrix in a mixture of allogeneic decellularized dermal matrix particles and hyaluronic acid to 100 μm or smaller, 100 μm to 300 μm, 300 μm to 500 μm and 500 μm or larger, respectively, and observing the morphology of phADM particles in each sample using an optical microscope.

[0121] Figure 1C is a graph comparing the particle size distribution of a hyaluronic acid sample (sample name: HA1, manufacturer: Hugel, South Korea) with four different allogeneic decellularized dermal matrix powders of varying average particle sizes in a mixture of allogeneic decellularized dermal matrix particles and hyaluronic acid, where the particle sizes of the decellularized dermal matrix were prepared to be 100 μm or smaller, 100 μm to 300 μm, 300 μm to 500 μm, and 500 μm or larger.

[0122] The results of Figure 1C are summarized in Table 1 below.

[0123] Table 1

[0124]

[0125] As shown in Figures 1A and 1B, the mixture prepared from decellularized dermal matrix particles with an average particle size of 100 μm or smaller exhibits good uniform distribution of the particles within the mixture. The mixture prepared from particles with an average particle size in the range of 100 μm to 300 μm shows slight particle aggregation. In solutions prepared from particles with an average particle size in the range of 300 μm to 500 μm and those with an average particle size greater than 500 μm, particle aggregation is more pronounced.

[0126] Therefore, it can be seen that when the average size of phADM particles is 300 μm or less, they can be uniformly mixed with existing hyaluronic acid hydrogels. When the average size is 300 μm or greater, the particle distribution is relatively wide and cannot form a uniform mixed composition.

[0127] As shown in Figure 1C and Table 1, the mean particle size of HA1 was 541 ± 6.62 μm (mean ± standard deviation, n=3), and the mode particle size was 534 ± 29.2 μm (mean ± standard deviation, n=3). Based on measurements across sieve aperture ranges, the mean particle sizes of the four powders (100 μm or smaller, 100 μm to 300 μm, 300 μm to 500 μm, and 500 μm or larger) were measured to be 45.1 ± 1.25 μm, 152 ± 0.569 μm, 663 ± 34.6 μm, and 829 ± 43.6 μm (mean ± standard deviation, n=3), respectively, and the mode particle sizes were measured to be 40.4 ± 2.14 μm, 140 ± 0.066 μm, 434 ± 80.7 μm, and 605 ± 32.0 μm (mean ± standard deviation, n=3), respectively. Therefore, when the average size of the mixed phADM particles is about 1 / 3 or less of the average size of the hyaluronic acid hydrogel particles, a more uniform mixed composition can be formed.

[0128] <Experimental Example 2> Evaluate the changes in physical properties of the mixed composition based on the concentration of allogeneic decellularized dermal matrix particles in the solution.

[0129] After preparing the allogeneic acellular dermal matrix powder according to the method of Preparation Example 1, the particles were filtered through a 100 μm pore size sieve, and only the particles that passed through the sieve were collected. The powder was mixed in physiological saline at concentrations of 2 wt%, 10 wt%, 20 wt%, and 40 wt%, respectively. According to Preparation Example 2, 1 mL of the solution was mixed with 1 mL of commercially available hyaluronic acid hydrogel (sample name: HA2, manufacturer: Hugel, South Korea), and the concentrations were adjusted to achieve a final concentration of 1 wt%, 5 wt%, 10 wt%, and 20 wt% for the allogeneic acellular dermal matrix powder and a final concentration of 1 wt% for hyaluronic acid (HA2) to prepare the sample.

[0130] The mixture was filled into a syringe fitted with a conventionally used 23G (thin-wall) injection needle to measure the injection thrust. The results are shown in Figure 2A and Table 2.

[0131] Figure 2A shows the results of filling a sample of a mixture of allogeneic decellularized dermal matrix particles (1 wt%, 5 wt%, 10 wt%, 20 wt%) and hyaluronic acid (HA2) (1 wt%) into a syringe equipped with a conventional 23G (thin-wall) injection needle to measure the injection thrust (n=3).

[0132] The results of Figure 2A are summarized in Table 2 below.

[0133] Table 2

[0134]

[0135] As shown in Figure 2A and Table 2, the mixed composition prepared at a phADM concentration of 1 wt% had the lowest injection thrust of 19.5 N, while the mixed compositions prepared at concentrations of 5 wt% and 10 wt% had injection thrusts of 24.9 N and 30.3 N, respectively. Conversely, the mixed composition prepared at a concentration of 20 wt% required an injection thrust exceeding 40 N, reaching 115.1 N, confirming difficulties at injection.

[0136] Next, the enzymatic degradation results of hyaluronic acid in the allogeneic decellularized dermal matrix particles and hyaluronic acid (HA2) mixture are shown in Figure 2B and Table 3.

[0137] Figure 2B shows the degradation rate of hyaluronic acid in the mixed composition after 24 hours of treatment with hyaluronidase on a sample of allogeneic decellularized dermal matrix particles (0 wt%, 1 wt%, 5 wt%, 10 wt%) and hyaluronic acid (HA2) (1 wt%). (n=3)

[0138] The results of Figure 2B are summarized in Table 3 below.

[0139] Table 3

[0140]

[0141] As shown in Figure 2B and Table 3, after 24 hours of treatment with hyaluronidase, the degradation rate of the original hyaluronic acid hydrogel (HA2) without phADM was approximately 93%, while the degradation rate of the composition mixed with 1% to 10% phADM was significantly reduced to 56% to 64%, confirming an improved enzyme resistance. Therefore, mixing particulate phADM into hyaluronic acid hydrogel can enhance its resistance to hyaluronidase, thereby slowing the degradation rate and significantly prolonging the duration of the therapeutic effect when the developed tissue repair mixture is administered in vivo.

[0142] Compositions with phADM content ranging from 1% to 10% by weight all showed a trend of significantly reduced degradation rate, which can be considered as good effect. However, as the phADM content increases, the degradation rate of hyaluronic acid also shows a slow upward trend. Therefore, it is expected that the mixed composition should not contain more than 10% by weight of phADM.

[0143] <Experimental Example 3> Evaluate the physical properties of the mixed composition based on the storage modulus (G') of the hyaluronic acid hydrogel.

[0144] PhADM with an average particle size of 100 μm or smaller, prepared in Preparation Example 1, was mixed in physiological saline at a concentration of 10% for later use. To measure the physical properties of the mixed composition, two commercially available hyaluronic acid hydrogels were prepared: one with a pre-mixing storage modulus (G') of 300 Pa or less, and the other with a pre-mixing storage modulus (G') exceeding 300 Pa (G'≤300 Pa sample name: HA3, manufacturer: Medytox, South Korea, n=3; G'>300 Pa sample name: HA4, manufacturer: Hugel, South Korea). After preparing the mixture according to Preparation Example 2 by mixing 1 mL of phADM solution with 1 mL of hyaluronic acid hydrogel, viscoelastic properties were analyzed (n=3). At this point, the final concentration of phADM was 5.0 wt%, and the concentration of hyaluronic acid was 1.0 wt%.

[0145] Figure 3A shows the results of measuring the storage modulus (G') before and after mixing phADM of 100 μm or smaller into HA3 or HA4 hyaluronic acid (n=3).

[0146] Figure 3B shows the results of measuring the loss modulus (G'') before and after mixing phADM of 100 μm or smaller into HA3 or HA4 hyaluronic acid (n=3).

[0147] Figure 3C shows the results of measuring the loss factor (tan delta) before and after mixing phADM of 100 μm or smaller into HA3 or HA4 hyaluronic acid (n=3).

[0148] Figure 3D shows the results of complex viscosity measurements (n=3) before and after mixing phADM of 100 μm or smaller into HA3 or HA4 hyaluronic acid.

[0149] The results of Figures 3A to 3D are summarized in Table 4 below.

[0150] Table 4

[0151]

[0152] As shown in Figure 3 and Table 4.

[0153] For mixtures of hyaluronic acid (HA3) hydrogels and decellularized dermal matrix powder solutions with an initial storage modulus (G') of 300 Pa or less, the average storage modulus increased from 289 Pa to 338 Pa after mixing, an increase of 17.0%. Conversely, for mixtures of hyaluronic acid (HA4) hydrogels with an initial G' exceeding 300 Pa, the average storage modulus decreased from 396 Pa to 371 Pa after mixing, a decrease of 6.3%.

[0154] Regarding the loss modulus, regardless of the physical properties of the hyaluronic acid hydrogel before mixing, it decreases after mixing. Specifically, when the hyaluronic acid hydrogel G'≤300 Pa before mixing, it decreases from 73 Pa to 67 Pa; when the hyaluronic acid hydrogel G'>300 Pa before mixing, it decreases from 60 Pa to 36 Pa.

[0155] Regarding the loss factor (tan delta), after mixing with phADM solution, the tan delta decreased from 0.25 to 0.20 when the initial hyaluronic acid hydrogel G' ≤ 300 Pa, and from 0.15 to 0.12 when the initial hyaluronic acid hydrogel G' > 300 Pa. Therefore, this confirms that mixing with phADM can improve the relative elasticity ratio of existing hyaluronic acid hydrogels.

[0156] Regarding the average complex viscosity, when the unmixed hyaluronic acid hydrogel G' is 300 Pa or less, it increases from 469 Pa·s to 548 Pa·s; when the unmixed hyaluronic acid hydrogel G' exceeds 300 Pa, it decreases from 615 Pa·s to 595 Pa·s.

[0157] <Experimental Example 4> Evaluation of the physical property changes of the mixed composition using various hyaluronic acid hydrogels with a storage modulus (G') of 300 Pa or less.

[0158] The decellularized dermal matrix powder of 100 μm or smaller prepared in Preparation Example 1 was mixed in physiological saline at a concentration of 10% by weight for later use. To verify the measurement results in Figure 3 and Table 4, three other hyaluronic acid hydrogels (n=3) with a pre-mixing storage modulus (G') of 300 Pa or smaller were prepared. Then, 1 mL of phADM solution and 1 mL of hyaluronic acid hydrogel were mixed according to Preparation Example 2. After preparation, analysis was performed (Sample name: HA5, manufacturer: Medytox, South Korea; Sample name: HA6, manufacturer: Jetema, South Korea; Sample name: HA7, manufacturer: Medytox, South Korea). The final concentration of phADM was 5% by weight, and the concentration of hyaluronic acid was 1.0% by weight.

[0159] Figure 4A shows the results of measuring the storage modulus (G') before and after mixing phADM with an average size of 100 μm or smaller into HA5 to HA7 hyaluronic acid (n=3).

[0160] Figure 4B shows the results of measuring the loss modulus (G'') before and after mixing phADM with an average size of 100 μm or smaller into HA5 to HA7 hyaluronic acid (n=3).

[0161] Figure 4C shows the results of measuring the loss factor (tan delta) before and after mixing phADM of 100 μm or smaller into HA5 to HA7 hyaluronic acid (n=3).

[0162] Figure 4D shows the results of complex viscosity measurements (n=3) before and after mixing phADM with an average size of 100 μm or smaller into HA5 to HA7 hyaluronic acid.

[0163] The results of Figures 4A to 4D are summarized in Table 5 below.

[0164] Table 5

[0165]

[0166] As shown in Figure 4 and Table 5.

[0167] The average storage elastic moduli of HA5, HA6 and HA7 before mixing were 138 Pa, 167 Pa and 289 Pa, respectively. After mixing with phADM solution, the average values ​​of the samples increased to 216 Pa, 191 Pa and 338 Pa, respectively, with increases of 56.5%, 14.4% and 17.0%.

[0168] The average loss elastic moduli of hyaluronic acid hydrogels before mixing HA5, HA6 and HA7 were 40 Pa, 40 Pa and 73 Pa, respectively. After mixing with phADM solution, they were measured to be 41 Pa, 27 Pa and 67 Pa, respectively.

[0169] The measured loss factors of HA5, HA6 and HA7 before mixing were 0.29, 0.24 and 0.25 respectively. After mixing with phADM solution, they decreased to 0.19, 0.14 and 0.20 respectively, all showing a decrease.

[0170] The average complex viscosities of HA5, HA6 and HA7 before mixing were 243 Pa·s, 271 Pa·s and 469 Pa·s, respectively. After mixing with phADM solution, they increased to 350 Pa·s, 307 Pa·s and 548 Pa·s, respectively, showing an increase in all of them.

[0171] The results above show that when the energy storage modulus of the hyaluronic acid hydrogel before mixing is 300 Pa or less, mixing it with particulate allogeneic decellularized dermal matrix with an average particle size of 300 μm or less at a concentration of 10% by weight or less can improve the elasticity and complex viscosity of the mixed composition for tissue repair and reduce the loss factor.

[0172] The above description of the present invention is for illustrative purposes only. Those skilled in the art should understand that modifications can be easily made in other specific forms without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims

1. A composition for delaying the in vivo degradation rate of hyaluronic acid hydrogels and enhancing their rheological properties, comprising: Allogeneic acellular dermal matrix particles and medical diluents The hyaluronic acid hydrogel is used for biological transplantation for tissue repair, and its energy storage elastic modulus (G') is 300 Pa or less.

2. The composition according to claim 1, wherein, The average particle size of the allogeneic decellularized dermal matrix is ​​300 μm or smaller.

3. The composition according to claim 2, wherein, The average particle size of the allogeneic decellularized dermal matrix is ​​100 μm or smaller.

4. The composition according to claim 1, wherein, The size of the allogeneic decellularized dermal matrix particles is 1 / 3 or smaller than the average particle size of the hyaluronic acid.

5. The composition according to claim 1, wherein, The medical diluent is selected from one or more of the following groups: physiological saline, distilled water, and phosphate-buffered saline.

6. The composition according to claim 1, wherein, In the mixture of the composition and hyaluronic acid hydrogel, the content of the allogeneic decellularized dermal matrix particles is 10% by weight or less.

7. The composition according to claim 1, wherein, The rheological properties refer to the increase in the energy storage elastic modulus (G').

8. The composition according to claim 1, wherein, The composition is used to reduce the in vivo degradation rate of hyaluronic acid hydrogel.

9. A biological transplant composition for tissue repair, comprising: Allogeneic acellular dermal matrix particles The application involves using a mixed composition prepared by mixing a solution with hyaluronic acid hydrogel on-site, the solution being prepared by mixing the allogeneic decellularized dermal matrix particles in a medical diluent. The energy storage modulus (G') of the hyaluronic acid hydrogel is 300 Pa or less.

10. The composition according to claim 9, wherein, The medical diluent is selected from one or more of the following groups: physiological saline, distilled water, and phosphate-buffered saline.

11. The composition according to claim 9, wherein, The size of the allogeneic acellular dermal matrix particles is 300 μm or smaller. The content of allogeneic decellularized dermal matrix particles in the mixture is 0.5% to 10% by weight. The hyaluronic acid hydrogel content in the mixture is from 0.5% to 2% by weight. The size of the allogeneic decellularized dermal matrix particles is 1 / 3 or smaller than the average particle size of the hyaluronic acid.

12. The composition according to claim 11, wherein, The size of the allogeneic decellularized dermal matrix particles is 200 μm or smaller. The content of allogeneic decellularized dermal matrix particles in the mixture is 1% to 10% by weight. The hyaluronic acid hydrogel content in the mixture is from 0.7% to 1.5% by weight. The size of the allogeneic decellularized dermal matrix particles is 1 / 3 or smaller than the average particle size of the hyaluronic acid.

13. The composition according to claim 12, wherein, The size of the allogeneic decellularized dermal matrix particles is 100 μm or smaller. The content of allogeneic decellularized dermal matrix particles in the mixture is 1% to 5% by weight. The hyaluronic acid hydrogel content in the mixture is from 0.8% to 1.4% by weight. The size of the allogeneic decellularized dermal matrix particles is 1 / 3 or smaller than the average particle size of the hyaluronic acid.

14. The composition according to claim 13, wherein, The size of the allogeneic decellularized dermal matrix particles is 100 μm or smaller. The content of allogeneic decellularized dermal matrix particles in the mixture is 1% to 5% by weight. The hyaluronic acid hydrogel content in the mixture is from 0.9% to 1.3% by weight. The size of the allogeneic decellularized dermal matrix particles is 1 / 3 or smaller than the average particle size of the hyaluronic acid.

15. The composition according to claim 14, wherein, The size of the allogeneic decellularized dermal matrix particles is 100 μm or smaller. The content of allogeneic decellularized dermal matrix particles in the mixture is 1% to 5% by weight. The hyaluronic acid hydrogel content in the mixture is from 1% to 1.2% by weight. The size of the allogeneic decellularized dermal matrix particles is 1 / 3 or smaller than the average particle size of the hyaluronic acid.

16. A biological transplant composition for tissue repair, comprising: The solution is prepared by mixing allogeneic decellularized dermal matrix particles with a medical diluent; as well as Hyaluronic acid hydrogel with a storage modulus (G') of 300 Pa or less.

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