Stimuli-responsive hydrogel, tissue expander comprising same, and method for using tissue expander comprising same

Abstract

The present invention relates to a stimuli-responsive hydrogel in which the timing of swelling is controllable by external stimuli, to a tissue expander using same, and to a method for using the tissue expander comprising same.

Description

Stimulus-responsive hydrogel, tissue expander comprising the same, and method of using a tissue expander comprising the same

[0001] The present invention claims the benefit of the filing date of Korean Patent Application No. 10-2024-0156788 filed with the Korean Intellectual Property Office on November 7, 2024, the entire contents of which are incorporated into the present invention.

[0002] The present invention relates to a stimulus-responsive hydrogel, a tissue expander containing the same, and a method of using a tissue expander containing the same.

[0003] A tissue expander is a device inserted subcutaneously to induce the expansion of skin tissue. Tissue expanders are primarily used to be temporarily inserted prior to implantation to create or maintain the space required for the implant. Alternatively, tissue expanders can be used to promote the formation of new tissue by keeping living tissue taut.

[0004] Silicone is generally used as a tissue expander. Since silicone lacks self-expanding capabilities, silicone-based tissue expanders are used by periodically injecting physiological saline through an infusion port after implantation to expand the tissue. However, this method necessitates periodic hospital visits, and damage to the surrounding skin is inevitable due to the over-inflation and stabilization phases. Furthermore, there are issues such as inflammation and fibrosis caused by foreign body reactions resulting from silicone's low biocompatibility, as well as the inconvenience of having to separately manufacture a silicone balloon identical in shape to the tissue to be expanded.

[0005] Therefore, there is a need for the development of a tissue expander that can spontaneously expand to match the physical properties of the surrounding skin after being implanted in the body and continuously expand to a set target swelling level.

[0006] Meanwhile, hydrogels are materials in which hydrophilic polymers are cross-linked to form a three-dimensional network structure and retain water within the network structure. The degree to which hydrogels can retain water and their physical and chemical properties vary depending on the type of polymer and the network structure. Due to their flexibility and biocompatibility, hydrogels are currently being used in the medical and cosmetic fields.

[0007] The technical problem that the present invention aims to solve is to provide a stimulus-responsive hydrogel whose expansion timing can be controlled by external stimulation, and a tissue expander using the same.

[0008] Another technical objective of the present invention is to provide a method for using a tissue expander comprising a stimulus-responsive hydrogel that can be applied not only in the form of an implant that is incised and transplanted into skin tissue, but also in the form of an injectable.

[0009] However, the problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0010] One embodiment of the present invention provides a stimulus-responsive hydrogel comprising a first network structure comprising a stimulus-responsive polymer and a second network structure comprising a hydrogel-forming polymer, wherein the first network structure and the second network structure form an interpenetrating polymer network (IPN).

[0011] Another embodiment of the present invention provides a tissue expander comprising a stimulus-responsive hydrogel according to one embodiment of the present invention.

[0012] Another embodiment of the present invention provides a method for manufacturing a stimulus-responsive hydrogel, comprising: a step of forming a first network structure comprising a stimulus-responsive polymer; and a step of forming a second network structure comprising a hydrogel-forming polymer in a solution comprising the first network structure to form an interpenetrating polymer network structure.

[0013] A stimulus-responsive hydrogel according to one embodiment of the present invention can control the timing of expansion by external stimulation.

[0014] A method of using a tissue expander implemented by a tissue expander comprising a stimulus-responsive hydrogel according to one embodiment of the present invention can be applied to diseases such as diabetes, in which wound healing time is delayed due to reduced immunity and hemostasis is difficult due to altered platelet function, by applying the stimulus-responsive hydrogel in an injectable form by crushing the skin tissue and in addition to an implant form by cutting and transplanting the skin tissue.

[0015] A tissue expander according to one embodiment of the present invention can adjust the timing of expansion by external stimulation and can reach a target expansion rate through continuous expansion.

[0016] The effects of the present invention are not limited to those described above, and unmentioned effects will be clearly understood by those skilled in the art from the present specification and the accompanying drawings.

[0017] FIG. 1 is a diagram illustrating the principle by which the interpenetrating polymer network structure of a stimulus-responsive hydrogel according to one embodiment of the present invention inhibits volume expansion.

[0018] FIG. 2 is a diagram showing that after an external stimulus is applied to a stimulus-responsive hydrogel according to one embodiment of the present invention, the hydrogel-forming polymer absorbs water within a second network structure as the structure of the stimulus-responsive polymer changes, thereby expanding in volume.

[0019] FIG. 3 is a schematic diagram of a conventional tissue expander (a) using silicone and a tissue expander (b) according to one embodiment of the present invention.

[0020] Figure 4 is a schematic diagram of the process for manufacturing a stimulus-responsive hydrogel according to Example 1.

[0021] Figure 5 is a diagram showing the volume expansion of the hydrogel of Example 1 in a laboratory environment.

[0022] Figure 6 shows the appearance of the hydrogel of Example 1 implanted in the subcutaneous tissue of a mouse (a), the appearance of the sutured site healed (b), and the appearance of the skin expanded due to the expansion of the hydrogel after enzyme injection (c).

[0023] Figure 7 is a graph showing the expansion rate evaluated for the hydrogels of Example 1 and Example 4 in a laboratory environment (a) and an in-vivo environment (b).

[0024] FIG. 8 is a graph showing the expansion rate evaluated for the hydrogels of Examples 1 to 3 in a laboratory environment (a) and an in-vivo environment (b).

[0025] Figure 9 shows fluorescence microscope images of control skin fibroblasts and skin fibroblasts cultured in the eluent from the hydrogel of the example.

[0026] FIG. 10 is a figure showing the cell proliferation rate (a) and TGF-β expression level (b) evaluated for the hydrogels of Examples 1 to 3.

[0027] Figure 11 is a microscopic image of control skin tissue (a) and surrounding skin tissue (b) on which the hydrogel of Example 1 was implanted.

[0028] Figure 12 shows the epidermal layer thickness measured for the control skin tissue and the surrounding skin tissue where the hydrogel of Example 1 was implanted.

[0029] FIG. 13 is a microscopic image of a control skin tissue (a), a surrounding skin tissue where the hydrogel of Example 1 was implanted without enzyme injection (b), and a skin tissue (c) 4 days after enzyme was injected into the surrounding skin tissue where the hydrogel of Example 1 was implanted.

[0030] Figure 14 is a graph of the quantitative analysis of YAP measured for the control group, the skin tissue surrounding the implantation of the hydrogel of Example 1, and the skin tissue 4 days after the enzyme was injected into the skin tissue surrounding the implantation of the hydrogel of Example 1.

[0031] Figure 15 is a graph of the quantitative analysis of YAP cytoplasmic MFI measured for the control group, the skin tissue surrounding the implantation of the hydrogel of Example 1, and the skin tissue 4 days after the enzyme was injected into the skin tissue surrounding the implantation of the hydrogel of Example 1.

[0032] Figure 16 is a photograph showing whether skin tissue adheres to the hydrogel of Example 1 (a), the hydrogel of pure hyaluronic acid (b), and the hydrogel of pure polyacrylic acid (c).

[0033] FIG. 17 shows fluorescence microscope images of skin fibroblasts cultured in the hydrogel of Example 1 (a), the hydrogel of pure hyaluronic acid (b), and the hydrogel of pure polyacrylic acid (c), respectively.

[0034] Figure 18 is a graph showing the number of skin fibroblasts that have grown out of the hydrogel of Example 1, the hydrogel of pure hyaluronic acid, and the hydrogel of pure polyacrylic acid, respectively.

[0035] FIG. 19 shows the hydrogel of Example 1 implanted in the subcutaneous tissue of a mouse (a), the enzyme injected after 10 days (b), the skin expanded due to the expansion of the hydrogel after 24 days and local flap surgery performed (c), and the skin recovered after 60 days (d).

[0036] FIG. 20 is a graph (a) showing the swelling ratio and extra swelling ratio over time after injecting the hydrogel of Example 1 with the enzyme hyaluronidase at concentrations of 1500, 3000, and 6000 IU, and a graph (b) showing the extra swelling ratio over time from the point in time when the hydrogel was implanted in the subcutaneous tissue and injected with the enzyme hyaluronidase at concentrations of 375, 750, 1125, and 1500 IU.

[0037] FIG. 21 is a hydrogel in the form of the hydrogel of Example 1 ground for 60 seconds (a), 90 seconds (b), and 120 seconds (c).

[0038] FIG. 22 shows the hydrogel of Example 1, which was ground for 120 seconds and injected into the subcutaneous tissue of a mouse using a syringe (a), the expansion of the hydrogel 1 day after the injection of the enzyme hyaluronidase (b), the expansion of the hydrogel 6 days after injection (c), and a graph of the change in volume of the mouse over time after the enzyme injection (d).

[0039] Figure 23 shows images immediately after injecting a hydrogel in the form of the hydrogel of Example 1, ground for 120 seconds, into a mouse with type 1 diabetes, and images after injecting the enzyme hyaluronidase.

[0040] Throughout this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0041] Throughout this specification, when a component is described as being located "on" another component, this includes not only cases where a component is in contact with another component, but also cases where another component exists between the two components.

[0042] Throughout the entire specification, the unit "parts by weight" may refer to the ratio of weight between each component.

[0043] Throughout this specification, "A and / or B" means "A and B, or A or B".

[0044] Stimulus-responsive hydrogel

[0045] One embodiment of the present invention provides a stimulus-responsive hydrogel comprising a first network structure including a stimulus-responsive polymer and a second network structure including a hydrogel-forming polymer, wherein the first network structure and the second network structure form an interpenetrating polymer network structure.

[0046] In the present invention, the network structure is understood to mean a three-dimensional net-shaped polymer structure formed by cross-linking between polymer chains.

[0047] In the present invention, a stimulus-responsive polymer is understood to mean a polymer whose physical or chemical properties change reversibly or irreversibly in response to a specific external stimulus. More specifically, the stimulus-responsive polymer may be one in which the composition of polymer chains or the cross-linking between polymer chains changes in response to an external stimulus.

[0048] According to one embodiment of the present invention, the first network structure comprising the stimulus-responsive polymer may be decomposed by an external stimulus.

[0049] According to one embodiment of the present invention, the first network structure comprising the stimulus-responsive polymer is decomposed by an external stimulus, and the stimulus-responsive hydrogel may begin to expand by an external stimulus. In addition, the expansion of the stimulus-responsive hydrogel according to one embodiment of the present invention may be continuous.

[0050] According to one embodiment of the present invention, the expansion of the second network structure may be inhibited by the first network structure. By inhibiting the expansion of the second network structure containing the hydrogel-forming polymer by the first network structure containing the stimulus-responsive polymer, the expansion of the volume of the hydrogel-forming polymer by absorbing water within the second network structure can be inhibited until an external stimulus is applied.

[0051] According to one embodiment of the present invention, the first network structure and the second network structure may include a crosslinked structure. The crosslinked structure may be a chemical crosslink or a physical crosslink.

[0052] FIG. 1 is a diagram illustrating the principle by which the interpenetrating polymer network structure of a stimulus-responsive hydrogel according to one embodiment of the present invention inhibits volume expansion.

[0053] As shown in FIG. 1, the first network structure containing a polymer of glycidyl methacrylate hyaluronic acid (GMHA) can inhibit the expansion of the second network structure containing a polymer of acrylic acid, and specifically, the expansion may be inhibited by the twisting between the polymer chains of the first network structure and the second network structure.

[0054] FIG. 2 is a diagram showing that after an external stimulus is applied to a stimulus-responsive hydrogel according to one embodiment of the present invention, the hydrogel-forming polymer absorbs water within a second network structure as the structure of the stimulus-responsive polymer changes, thereby expanding in volume.

[0055] As shown in Figure 2, the stimulus-responsive polymer is degraded by external stimuli such as enzyme injection, causing the first network structure to collapse, and consequently, the force inhibiting the expansion of the second network structure disappears, allowing the hydrogel-forming polymer to absorb water and expand.

[0056] According to one embodiment of the present invention, the hydrogel-forming polymer may comprise one or more of polyacrylic acid (PAA), polymethacrylic acid, polyacrylic acetate, polyethylene glycol (PEG), polyethylene oxide (PEO), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), polyacrylamide, polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), alginate, cellulose, gelatin, collagen, hyaluronic acid, carrageenan, chitosan, albumin, agarose, and copolymers thereof.

[0057] Preferably, the hydrogel-forming polymer may include one or more of polyacrylic acid, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, copolymers thereof, alginate, cellulose, gelatin, collagen, hyaluronic acid, carrageenan, chitosan, albumin, and agarose. When the hydrogel-forming polymer includes the above-mentioned substances, the expansion rate of the hydrogel in response to external stimuli may be high, and adhesion with surrounding tissues may not occur when implanted into subcutaneous tissue.

[0058] According to one embodiment of the present invention, the external stimulus may be one or more of an enzyme, temperature, and pH. More specifically, the external stimulus may be an enzyme.

[0059] According to one embodiment of the present invention, the stimulus-responsive polymer may include one or more of hyaluronic acid, gelatin, and collagen.

[0060] When the above stimulus-responsive polymer contains hyaluronic acid, the suitable stimulus may be the injection of hyaluronidase. Specifically, when the first network structure contains hyaluronic acid, the stimulus-responsive hydrogel does not expand and maintains its shape until the injection of hyaluronidase, at which point the glycosidic bonds of the hyaluronic acid are cleaved by the injection of hyaluronidase, causing the hyaluronic acid to decompose into low molecular weight degradation products, and thus the first network structure may decompose.

[0061] When the above stimulus-responsive polymer contains gelatin, the suitable form of stimulus may be a change in temperature. Specifically, when the first network structure contains gelatin, it maintains its shape without expanding at a temperature below the gelation temperature, but when a stimulus is applied at a temperature above the gelation temperature, the gelatin becomes sol, and the first network structure can be decomposed.

[0062] When the above stimulus-responsive polymer contains collagen, the suitable stimulus may be the injection of collagenase. Specifically, when the first network structure contains collagen, the peptide bonds of the collagen may be cleaved by the injection of collagenase, causing the collagen to be broken down into low molecular weight degradation products, and thus the first network structure may be broken down.

[0063] In the present specification, the degree of crosslinking refers to the ratio of crosslinks formed for all positions capable of forming crosslinks included in the polymer. The positions capable of forming crosslinks may be positions capable of introducing crosslinkable functional groups or positions capable of forming crosslinks by reacting with a crosslinking agent.

[0064] According to one embodiment of the present invention, the degree of crosslinking of the first network structure comprising the stimulus-responsive polymer may be 5% or more, 7.5% or more, 10% or more, 12.5% ​​or more, or 15% or more, and may be 100% or less, 95% or less, 90% or less, 85% or less, or 80% or less. The higher the degree of crosslinking of the first network structure, the more stably the shape of the hydrogel comprising it can maintain its shape without swelling until an external stimulus is applied. Preferably, the degree of crosslinking of the first network structure may be 10% or more. A hydrogel comprising the first network structure with a degree of crosslinking of 10% or more can maintain its shape stably until an external stimulus is applied, and has excellent moldability, so it can be easily molded into the shape required for a tissue expander implanted in the body.

[0065] According to one embodiment of the present invention, the degree of crosslinking of the second network structure comprising the hydrogel-forming polymer is preferably 0.025% or more and 1% or less, more preferably 0.5% or less, and more preferably 0.1% or less. By satisfying the degree of crosslinking of the second network structure at 0.025% or more, the hydrogel can maintain its shape even after an external stimulus is applied, and by satisfying 1% or less, the degree of swelling after an external stimulus is applied can be high, and within the above range, the lower the degree of crosslinking, the greater the degree of swelling can be improved.

[0066] Tissue expander comprising stimulus-responsive hydrogel

[0067] Another embodiment of the present invention provides a tissue expander comprising a stimulus-responsive hydrogel according to the aforementioned embodiment of the present invention. The tissue expander according to the embodiment of the present invention, by including a stimulus-responsive hydrogel, can be implanted in the body and its expansion can be suppressed until an external stimulus is applied, and expansion can be initiated by the external stimulus. The expansion can be continuous.

[0068] FIG. 3 is a schematic diagram of a conventional tissue expander (a) using silicone and a tissue expander (b) according to one embodiment of the present invention.

[0069] As shown in Fig. 3, a conventional tissue expander using silicone includes an injection port and a connecting line separately from the expander, and expands the expander by injecting saline solution into the injection port through a tube or syringe, and the expander undergoes an over-inflation-stabilization phase. Conventional tissue expanders using silicone cause pain due to the injection of saline solution and require frequent hospital visits.

[0070] On the other hand, the tissue expander according to the present invention does not expand until the surgical site is sutured after implantation surgery, but undergoes continuous expansion from the moment an external stimulus, such as enzyme injection, is applied, thereby reaching a target time and target volume swelling degree. Through continuous expansion, the tissue expander does not require an over-inflation-stabilization phase, and pain associated with tissue expansion can be alleviated. Furthermore, since the tissue expander self-inflates without the need for saline injection, hospital visits can be minimized.

[0071] In addition, the expansion of the hydrogel proceeds until thermodynamic equilibrium is reached between the force attempting to expand by absorbing water due to osmotic pressure and the force attempting to inhibit expansion by the elastic force of the network structure of the hydrogel-forming polymer. When such expansion of the hydrogel occurs beneath the skin, the elastic force generated as the skin expands additionally acts, causing the expansion of the hydrogel to continue until the three forces are in equilibrium. Therefore, the tissue expander of the present invention has the effect of being able to self-regulate the degree of expansion to match the physical properties of the surrounding skin where it is implanted.

[0072] A tissue expander according to one embodiment of the present invention can control the maximum expansion rate reached after stimulation by controlling the degree of crosslinking of a second network structure containing a hydrogel-forming polymer included in the stimulation-responsive hydrogel.

[0073] A tissue expander according to one embodiment of the present invention may have a maximum expansion rate after stimulation of 10% or more, 20% or more, or 30% or more compared to before stimulation. Here, the expansion rate refers to the rate of change in the diameter of the tissue expander before and after stimulation in an in-vivo environment.

[0074] A tissue expander according to one embodiment of the present invention may have excellent biocompatibility.

[0075] According to one embodiment of the present invention, the stimulus-responsive hydrogel may further include an aqueous liquid.

[0076] According to one embodiment of the present invention, the aqueous liquid may be water or a physiological buffer.

[0077] For example, the above physiological buffer may use one or more of phosphate buffer solution (PBS), Dulbecco phosphate buffered saline (DPBS), normal saline, Tris buffered saline (TBS), and Hank's Balanced Salt Solution (HBSS).

[0078] According to one embodiment of the present invention, the stimulus-responsive polymer included in the stimulus-responsive hydrogel includes hyaluronic acid, and the tissue expander can begin to expand upon the injection of hyaluronidase. Specifically, when hyaluronidase is injected into the stimulus-responsive hydrogel, the hyaluronic acid is decomposed, and accordingly, the force inhibiting the expansion of the second network structure disappears, allowing the hydrogel-forming polymer to absorb water and the stimulus-responsive hydrogel to expand.

[0079] In addition, the degradation product of the hyaluronidase can play a role in promoting skin regeneration in the subcutaneous layer. Specifically, the degradation product of the hyaluronidase can increase the expression level of transforming growth factor beta (TGF-β).

[0080] According to one embodiment of the present invention, the hydrogel-forming polymer included in the stimulus-responsive hydrogel may include polyacrylic acid. By including polyacrylic acid in the hydrogel-forming polymer, adhesion with surrounding tissues may not occur after implantation in the body.

[0081] How to use a tissue expander

[0082] Another embodiment of the present invention provides a method of using a tissue expander comprising the aforementioned stimulus-responsive hydrogel.

[0083] A method of using a tissue expander comprising a stimulus-responsive hydrogel according to one embodiment of the present invention comprises: a step of inserting a tissue expander comprising a stimulus-responsive hydrogel into a tissue; and a step of expanding the tissue expander by an external stimulus after inserting the tissue expander.

[0084] According to one embodiment of the present invention, the step of inserting the tissue expander into the tissue may be applied in the form of an implant that implants the tissue expander containing a stimulus-responsive hydrogel into the incised skin tissue, or in the form of an injectable that is inserted into the skin tissue using a syringe or the like.

[0085] More specifically, according to one embodiment of the present invention, the step of applying and inserting a tissue expander comprising the stimulation-responsive hydrogel in the form of an implant may include: the step of implanting the tissue expander into an incised skin tissue; and the step of suturing the incised skin; thereby enabling the tissue expander according to the present invention to be implemented.

[0086] The method of inserting a tissue expander according to the present invention into a tissue in the form of an implant eliminates the need for an over-inflation-stabilization phase through continuous expansion, can alleviate pain associated with tissue expansion, and minimizes hospital visits as it self-inflates without the injection of saline solution.

[0087] According to one embodiment of the present invention, the step of applying and inserting a tissue expander comprising the stimulation-responsive hydrogel in an injectable form may include: a step of crushing the tissue expander comprising the stimulation-responsive hydrogel; and a step of inserting the crushed tissue expander into the tissue; thereby enabling the tissue expander according to the present invention to be implemented.

[0088] The grinding step described above may be applied by grinding the stimulus-responsive hydrogel to a size of 50 to 1000 μm for 50 to 200 seconds at a speed of 10,000 to 20,000 rpm. More specifically, the stimulation-responsive hydrogel can be applied by grinding it to a size of 50 μm or more, 100 μm or more, 150 μm or more, or 200 μm or more, 1000 μm or less, 950 μm or less, 900 μm or less, 850 μm or less, 800 μm or less, 750 μm or less, 700 μm or less, 650 μm or less, 600 μm or less, 550 μm or less, or 500 μm or less, under a speed of 10000 μm or more, 11000 μm or more, 12000 μm or more, or 13000 μm or more, and 20000 μm or less, and 19000 μm or less, or 18000 μm or less, or 17000 μm or less.

[0089] In addition, the stimulus-responsive hydrogel and water are mixed together and ground during the grinding process, and the stimulus-responsive hydrogel ground in water is freeze-dried to obtain a stimulus-responsive hydrogel in a ground form.

[0090] Additionally, in the step of inserting into the tissue in the above-mentioned injectable form, the pulverized tissue expander may be inserted into the tissue using an injection device. Specifically, the injection device may be a syringe.

[0091] The method of inserting a tissue expander according to the present invention into a tissue by applying it in an injectable form can be applied to subjects with diseases such as diabetes, in which wound healing time is delayed due to reduced immunity and hemostasis is difficult due to altered platelet function.

[0092] According to one embodiment of the present invention, the step of expanding the tissue expander by the external stimulus may be carried out by one or more external stimuli among an enzyme, temperature, and pH, and preferably may be an enzyme.

[0093] Method for manufacturing stimulus-responsive hydrogel

[0094] Another embodiment of the present invention provides a method for manufacturing the aforementioned stimulus-responsive hydrogel.

[0095] A method for manufacturing a stimulus-responsive hydrogel according to one embodiment of the present invention comprises: a step of forming a first network structure comprising a stimulus-responsive polymer; and a step of forming a second network structure comprising a hydrogel-forming polymer in a solution comprising the first network structure to form an interpenetrating polymer network structure.

[0096] An interpenetrating polymer network structure can be obtained by forming the second network structure in a solution containing the first network structure.

[0097] According to one embodiment of the present invention, the step of forming the first network structure may be performed by: introducing a photocrosslinkable functional group into the stimulus-responsive polymer; and photopolymerizing a first composition comprising the stimulus-responsive polymer into which the photocrosslinkable functional group has been introduced, a reactive monomer, a photoinitiator, and a solvent.

[0098] According to one embodiment of the present invention, the stimulus-responsive polymer having the photocrosslinkable functional group introduced may be included in the first composition in an amount of 3 to 5 parts by weight per 100 parts by weight of solvent. By satisfying the above range, the crosslinking density of the first network structure formed is appropriate, so that the stimulus-responsive hydrogel produced can maintain its shape without expanding until an external stimulus is applied, and the first network structure can be decomposed by the external stimulus.

[0099] According to one embodiment of the present invention, forming the second network structure may be performed by the step of photopolymerizing a second composition comprising the first network structure, a monomer of the hydrogel-forming polymer, a crosslinking agent, a photoinitiator, and a solvent.

[0100] According to one embodiment of the present invention, the molar concentration of the monomer of the hydrogel-forming polymer in the second composition may be 1.5 M or more, 2 M or more, 3 M or more, 4 M or more, 5 M or more, and 10 M or less. Preferably, the molar concentration of the monomer of the hydrogel-forming polymer in the second composition may be 3 M to 5 M. By satisfying the ranges described above for the molar concentration of the monomer of the hydrogel-forming polymer, the prepared hydrogel can maintain its shape before stimulation is applied, and the maximum swelling degree of the hydrogel after stimulation can be increased.

[0101] According to one embodiment of the present invention, the crosslinking agent in the second composition may be 0.01 mol% or more and 0.5 mol% or less with respect to the molar amount of the monomer of the hydrogel-forming polymer. More preferably, the crosslinking agent in the second composition may be 0.03 mol% or more and 0.1 mol% or less, 0.03 mol% or more and 0.08 mol% or less, or 0.03 mol% or more and 0.07 mol% or less with respect to the molar amount of the monomer of the hydrogel-forming polymer. By satisfying the above-described range for the ratio of the crosslinking agent in the second composition, the prepared hydrogel maintains its shape before stimulation and the maximum swelling degree of the hydrogel after stimulation can be increased.

[0102] Hereinafter, the present invention will be described in detail with reference to examples to specifically explain the invention. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention is not to be interpreted as being limited to the embodiments described below. The embodiments of this specification are provided to more completely explain the present invention to those with average knowledge in the art.

[0103] Example 1. Stimulus-responsive hydrogel

[0104] (1) Formation of the first network structure

[0105] First, to introduce methacrylate groups, which are photocrosslinkable functional groups, into hyaluronic acid, 3.505 mL of triethylamine (TEA), 3.505 mL of glycidyl methacrylate (GMA), and 3.505 g of tetrabutylammonium bromide (TBAB) were added to 100 mL of an aqueous hyaluronic acid solution (water:hyaluronic acid = 100:1, w / w) at 1-hour intervals, and the mixture was thoroughly stirred. Then, the mixture was stirred at 55 ℃ for 1 hour, cooled, and the polymer obtained by precipitating in acetone was dried to synthesize glycidyl methacrylate hyaluronic acid (GMHA). The chemical formula for the reaction in which methacrylate groups are introduced into hyaluronic acid is as follows.

[0106]

[0107] After dissolving the synthesized glycidyl methacrylate hyaluronic acid in water at 3 w / v%, a mixture was prepared by adding 0.1 parts by weight of a photoinitiator (Irgacure 2959) and 0.6 parts by weight of N-vinylpyrrolidone as a reactive monomer to 100 parts by weight of the glycidyl methacrylate hyaluronic acid aqueous solution.

[0108] The above mixture was placed in an acrylic mold and photopolymerization was performed by irradiating with UV light for 60 minutes to form a first network structure containing cross-linked hyaluronic acid.

[0109] The ratio of methacrylate functional groups introduced into the synthesized glycidyl methacrylate hyaluronic acid was calculated through NMR analysis, and the degree of crosslinking of the first network structure was calculated using the following formula and found to be 17.8%.

[0110]

[0111] (2) Formation of a second network structure

[0112] To form cross-linked polyacrylic acid as a second network structure, acrylic acid was used as a monomer, N',N'-methylenebisacrylamide (MBAAm) as a crosslinking agent, and 2-hydroxy-2-methyl-1-phenyl-propan-1-one (HMPP) as a photoinitiator.

[0113] First, a 4 M aqueous acrylic acid solution was prepared, and 0.05 mol% of a crosslinking agent (MBAAm) and 0.1 mol% of a photoinitiator (HMPP) were added to the aqueous solution and stirred.

[0114] Then, the first network structure formed above was introduced into the acrylic acid aqueous solution and left for 2 days, and then photopolymerization was performed by irradiating with UV for 120 minutes to form a second network structure containing cross-linked polyacrylic acid, thereby producing a stimulus-responsive hydrogel.

[0115] The stimulus-responsive hydrogel prepared as described above was washed with Dulbecco phosphate-buffered saline (DPBS) and fabricated into a disc shape with a diameter of 8 mm and a thickness of 2 mm using a biopsy punch.

[0116] Figure 4 is a schematic diagram of the process for manufacturing a stimulus-responsive hydrogel according to Example 1.

[0117] Example 2.

[0118] In the first network structure formation step, a stimulus-responsive hydrogel was prepared in the same manner as in Example 1, except that the synthesized glycidyl methacrylate hyaluronic acid was dissolved in water at 4 w / v%.

[0119] Example 3.

[0120] In the first network structure formation step, a stimulus-responsive hydrogel was prepared in the same manner as in Example 1, except that the synthesized glycidyl methacrylate hyaluronic acid was dissolved in water at 5 w / v%.

[0121] Example 4.

[0122] In the second network structure formation step, a stimulus-responsive hydrogel was prepared in the same manner as in Example 1, except that 0.1 mol% of MBAAm and 0.1 mol% of HMPP were added.

[0123] Experimental Example 1. Confirmation of swelling by enzyme injection

[0124] For the stimulus-responsive hydrogel prepared in Example 1, the degree of volume expansion in a laboratory environment and an iv-vivo environment was confirmed.

[0125] Specifically, in a laboratory setting, the hydrogel was immersed in 5 ml of DPBS solution mixed with 6000 IU of hyaluronidase, and the weight of the hydrogel was measured daily at a temperature of 37 ℃.

[0126] In an iv-vivo environment, the dorsal skin tissue of a mouse was incised, a hydrogel was implanted into the subcutaneous tissue, and the incision was sutured to allow the sutured area to heal for about 10 days. When it was determined that the area had healed, 1500 IU of enzyme solution was directly injected into the implanted hydrogel, and the diameter of the hydrogel was measured daily after the injection.

[0127] Figure 5 is a diagram showing the volume expansion of the hydrogel of Example 1 in a laboratory environment. Specifically, part (a) of Figure 5 shows the appearance of the hydrogel prepared in Example 1, part (b) of Figure 5 shows the hydrogel prepared in Example 1 pre-swelled by being supported in DPBS, and part (c) of Figure 5 shows the hydrogel prepared in Example 1 fully swollen after 28 days of injecting hyaluronidase.

[0128] Figure 6 shows the hydrogel of Example 1 implanted in the subcutaneous tissue of a mouse (a), the sutured area healed (b), and the skin expanded due to the expansion of the hydrogel after enzyme injection (c). As shown in Figure 6, the hydrogel of Example 1 did not start to expand and maintained its shape during the implantation process in the subcutaneous tissue and the wound healing process, but was able to self-expand after an external stimulus, such as enzyme injection, was applied.

[0129] Experimental Example 2. Verification of changes in expansion rate according to network structure formation conditions

[0130] In order to confirm the effect according to the ratio of acrylic acid and crosslinking agent in the formation of the second network structure, the expansion rate of the hydrogels of Example 1 and Example 4 was evaluated in a laboratory environment and an in-vivo environment (rat subcutaneous tissue environment).

[0131] FIG. 7 is a graph showing the expansion rate evaluated for the hydrogels of Example 1 and Example 4 in a laboratory environment (a) and an in-vivo environment (b). Specifically, in the graph, day 0 corresponds to immediately after the injection of hyaluronidase enzyme.

[0132] As shown in Figure 7, it was confirmed that as the ratio of crosslinking agent to acrylic acid decreased in the formation of the second network structure, the initial expansion rate and maximum expansion rate increased in both laboratory and in-vivo environments.

[0133] In order to confirm the effect according to the concentration of GMHA, a polymer with photocrosslinkable functional groups introduced in the formation of the first network structure, the expansion rate of the hydrogels of Examples 1 to 3 was evaluated in a laboratory environment and in a mouse subcutaneous tissue environment.

[0134] FIG. 8 is a graph showing the expansion rate evaluated for the hydrogels of Examples 1 to 3 in a laboratory environment (a) and an in-vivo environment (b).

[0135] As shown in Figure 8, in a laboratory environment, it was observed that as the concentration of GMHA decreased, the initial expansion rate of the prepared hydrogel increased, but the maximum expansion rate decreased. In an in-vivo environment, it was observed that as the concentration of GMHA decreased, both the initial expansion rate and the maximum expansion rate of the prepared hydrogel increased. This is believed to be because, in the formation of the first network structure, the higher the concentration of glycidyl methacrylate hyaluronic acid contained in the composition prior to photocrosslinking, the greater the amount of glycidyl methacrylate hyaluronic acid present per unit volume of the composition, and thus the first network structure obtained by photocrosslinking becomes denser.

[0136] Experimental Example 3. Evaluation of Biocompatibility of Hydrogel

[0137] To evaluate biocompatibility, human dermal fibroblasts (HDF) were cultured in the eluent obtained by digesting the hydrogel of the example with hyaluronidase and in the eluent obtained from the hydrogel of the example. Biocompatibility was evaluated by measuring cell proliferation using a Live / Dead assay and a CCK (cell counting kit). In addition, the expression level of transforming growth factor beta (TGF-β) was confirmed for each cell sample using the PCR method.

[0138] Figure 9 shows fluorescence microscope images of control skin fibroblasts and skin fibroblasts cultured in the eluent from the hydrogel of the example.

[0139] As shown in Fig. 9, it was confirmed that skin fibroblasts cultured in the eluent from the hydrogel of the example were non-toxic.

[0140] FIG. 10 is a figure showing the cell proliferation rate (a) and TGF-β expression level (b) evaluated for the hydrogels of Examples 1 to 3.

[0141] As shown in Fig. 10, all hydrogels of Examples 1 to 3 exhibited cell proliferation rates of approximately 95–150% in their own eluents and in the decomposition eluents by hyaluronidase, confirming their biocompatibility. In addition, it was confirmed that TGF-β was expressed more under conditions where hyaluronic acid was eluted in large quantities, proving that the eluted hyaluronic acid helps in the regeneration of skin tissue.

[0142] Experimental Example 4. Evaluation of Biological Characteristics in Mouse Subcutaneous Tissue

[0143] The immune response occurring after implanting the hydrogel of Example 1 into the subcutaneous tissue of a mouse was confirmed through hematoxylin and eosin staining (H&E) tissue analysis. Figure 11 shows microscopic images of the control skin tissue (a) and the surrounding skin tissue (b) where the hydrogel of Example 1 was implanted. As shown in Figure 11, it was confirmed that no immune response occurred in the surrounding skin tissue where the hydrogel of Example 1 was implanted, compared to the control group. The thickness of the epidermis was measured through analysis of the microscopic images. Figure 12 shows the measured thickness of the epidermis for the control skin tissue and the surrounding skin tissue where the hydrogel of Example 1 was implanted. As shown in Figure 12, it was confirmed that the thickness of the epidermis increased due to skin expansion caused by the expansion of the hydrogel.

[0144] In addition, another reason for the increase in the thickness of the epidermal layer as described above is due to a mechanism called mechanotransduction. As the skin tissue surrounding the hydrogel of Example 1 undergoes an immune response to external stimuli, the nuclear pores of the tissue cell nuclear membrane open, and as the YAP (Yes-Associated Protein) factor is injected into the nuclear pores, the YAP factor binds to the TEAD (TEA Domain Family Member) transcription factor within the nuclear membrane, thereby activating skin tissue cell proliferation and increasing the thickness of the epidermal layer.

[0145] Experimental Example 5. Verification of YAP Localization Characteristics

[0146] The YAP localization characteristics occurring after implanting the hydrogel of Example 1 into the subcutaneous tissue of a mouse were confirmed through DAPI (nuclear staining, blue) and YAP1 (YAP protein staining, green) staining analysis and YAP signal intensity analysis (ImageJ program).

[0147] Figure 13 shows microscopic images of a control skin tissue (a), skin tissue surrounding the implantation of the hydrogel of Example 1 without enzyme injection (b), and skin tissue 4 days after enzyme injection into the skin tissue surrounding the implantation of the hydrogel of Example 1 (c). As shown in Figure 13, it was confirmed that the skin tissue surrounding the implantation of the hydrogel of Example 1 with enzyme injection showed an overall increase in YAP (green) localization characteristics from the tissue cytoplasm to the nucleus (DAPI, blue) compared to the control group.

[0148] Figure 14 is a graph of the quantitative analysis of YAP measured for the control, the skin tissue surrounding the implantation of the hydrogel of Example 1 (3% w / o HYAL), and the skin tissue 4 days after the enzyme was injected into the skin tissue surrounding the implantation of the hydrogel of Example 1 (3% w HYAL). As shown in Figure 14, it can be seen that the skin tissue 4 days after the enzyme was injected into the skin tissue surrounding the implantation of the hydrogel of Example 1 has a significantly large portion of the overlapping area (N>C) between the nucleus (N, blue) and the cytoplasm (C, green).

[0149] Figure 15 is a graph of the quantification of YAP cytoplasmic MFI measured for the control, the skin tissue surrounding the implantation of the hydrogel of Example 1 (3% w / o HYAL), and the skin tissue 4 days after the enzyme was injected into the skin tissue surrounding the implantation of the hydrogel of Example 1 (3% w HYAL). As shown in Figure 15, it can be observed that the YAP signal intensity in the cytoplasm decreases as the skin tissue expands 4 days after the enzyme was injected into the skin tissue surrounding the implantation of the hydrogel of Example 1.

[0150] Through the above results, it can be confirmed that the expansion of the hydrogel induces mechanotransduction at the actual cellular level. More specifically, it can be anticipated that skin tissue was generated through an increase in the epidermal layer during the tissue expansion process, and this suggests that skin defects can be effectively covered through subsequent local flap procedures to form skin tissue identical or similar to normal tissue, thereby proving the reason for the increase in epidermal layer thickness as in Experimental Example 4.

[0151] Experimental Example 6. Confirmation of anti-adhesion properties

[0152] In order to easily remove a tissue expander implanted subcutaneously after it has expanded, it is required that the degree of adhesion to surrounding tissue be low. The hydrogel of Example 1, the hydrogel of pure hyaluronic acid, and the hydrogel of pure acrylic acid were each implanted into the dorsal skin tissue of mice, and the incisions were sutured to allow the sutures to heal for about 10 days. When it was determined that the site had healed, the implantation site was incised and observed visually.

[0153] Figure 16 is a photograph showing the adhesion of skin tissue to the hydrogel of Example 1 (a), the hydrogel of pure hyaluronic acid (b), and the hydrogel of pure polyacrylic acid (c). As shown in Figure 16, the hydrogel of Example 1 had no adhesion to the surrounding skin tissue at all. On the other hand, the hydrogel of pure hyaluronic acid had severe adhesion to the surrounding skin tissue, making it difficult to remove the hydrogel. In addition, it was confirmed that the hydrogel of pure polyacrylic acid also had almost no adhesion to the surrounding skin tissue.

[0154] In addition, for quantitative evaluation, skin fibroblasts were cultured for one day on the hydrogel of Example 1, the hydrogel of pure hyaluronic acid, and the hydrogel of pure polyacrylic acid, respectively. Then, the number of skin fibroblasts was counted using a fluorescence microscope.

[0155] FIG. 17 shows fluorescence microscope images of skin fibroblasts cultured in the hydrogel of Example 1 (a), the hydrogel of pure hyaluronic acid (b), and the hydrogel of pure polyacrylic acid (c), respectively, and FIG. 18 shows the number of skin fibroblasts that have extended their legs. As can be seen in FIG. 17 and FIG. 18, skin fibroblasts cultured in the pure hyaluronic acid hydrogel have extended their legs and are asymmetrically attached to each other, whereas such a phenomenon was not observed in the hydrogel of Example 1.

[0156] Experimental Example 7. Confirmation of Applicability of Local Flap Surgery

[0157] To solve the problem of wound dehiscence caused by the immediate expansion of conventional hydrogels implanted subcutaneously, the possibility of applying local flap surgery to effectively cover the lost skin is required. After implanting the hydrogel of Example 1 into the subcutaneous tissue of a mouse and suturing it, an enzyme was injected into the suture site about 10 days later. Then, after confirming that the hydrogel of Example 1 had expanded after 24 days, local flap surgery was performed on the site, and the recovery of the mouse's skin was observed visually after 60 days.

[0158] FIG. 19 shows the hydrogel of Example 1 implanted in the subcutaneous tissue of a mouse (a), the enzyme injected after 10 days (b), the skin expanded due to the expansion of the hydrogel after 24 days and local flap surgery performed (c), and the skin recovered after 60 days (d). As shown in FIG. 19, it can be confirmed that the skin of the mouse recovered after local flap surgery was performed on the skin that had expanded due to the hydrogel of Example 1, and through this, it can be seen that the hydrogel of the present invention can be applied to local flap surgery.

[0159] Experimental Example 8. Confirmation of Volume Expansion Rate Characteristics According to Enzyme Amount

[0160] Swelling controllability is required to prevent over-expansion of the skin for stable tissue expansion. Conventional hydrogel tissue expanders do not possess swelling controllability because swelling occurs immediately from a dry state to a maximum swelling state without control. Therefore, the ability to control the volume expansion rate by adjusting the amount of enzyme is a very important characteristic.

[0161] 8.1. In vitro characteristics

[0162] After completely drying the hydrogel of Example 1 (V dry), and immersed in DPBS (Dulbecco's Phosphate Buffered Saline) for 4 days. Then, the enzyme hyaluronidase was injected at concentrations of 1500, 3000, and 6000 IU, and the swelling ratio and extra swelling ratio were calculated using the following formula.

[0163] [Swelling Ratio Formula]

[0164] Swelling ratio = V / V dry

[0165] [Extra swelling ratio formula]

[0166] Extra swelling ratio = V / V 초기팽창

[0167] In the above swelling ratio formula, V is the volume at the time of measurement of Example 1, and V dry is the volume of the hydrogel of Example 1 after complete drying, and V 초기팽창 is the volume at the point when the completely dried hydrogel is placed in DPBS before enzyme injection and expansion is complete.

[0168] FIG. 20(a) shows a graph representing the swelling ratio and extra swelling ratio according to the time of enzyme injection for the hydrogel of Example 1 at concentrations of 1500, 3000, and 6000 IU, in addition to the control group (no enzyme injection). As shown in FIG. 20(a), the initial swelling rate increased as the enzyme concentration increased, and it can be confirmed that no additional swelling occurred in the control group without enzyme injection.

[0169] 8.2. In vivo characteristics

[0170] The hydrogel of Example 1 was implanted into the subcutaneous tissue of the back of a mouse and sutured. After the wound site had healed about 10 days later, the enzyme hyaluronidase was injected into the wound site at concentrations of 375, 750, 1125, and 1500 IU. Then, the extra swelling ratio according to the concentration of the injected enzyme was measured. The extra swelling ratio of the in vivo hydrogel was calculated as the cube of the increase in the diameter of the injected hydrogel.

[0171] Figure 20(b) shows a graph representing the extra swelling ratio over time from the time the hydrogel was implanted in the subcutaneous tissue. As shown in Figure 20(b), it can be seen that the initial swelling rate increases as the enzyme concentration increases.

[0172] Experimental Example 9. Verification of application of injectable form

[0173] Conventional hydrogels have mostly been applied in the form of implants that are transplanted by surgically incising the subcutaneous tissue; however, due to the problems of insufficient convenience and the long time required for skin recovery, there is a demand for the possibility of applying them in an injectable form. The hydrogel of Example 1 was ground using a grinding mixer at a speed of 15,000 rpm for 60 seconds, 90 seconds, and 120 seconds, respectively. At this time, the micro-sized hydrogel of Example 1, ground for 120 seconds, was injected into the subcutaneous tissue of a mouse using a syringe, and then the enzyme hyaluronidase was additionally injected using a syringe to check whether the hydrogel expanded.

[0174] FIG. 21 shows the hydrogel of Example 1 after grinding for 60 seconds (a), 90 seconds (b), and 120 seconds (c) (Scale bar: 500 μm). At this time, it can be confirmed that a hydrogel of uniform micro-size of 1000 μm or less was formed in the hydrogel (c) after grinding for 120 seconds.

[0175] FIG. 22 shows the hydrogel of Example 1, which was ground for 120 seconds in micro-size, injected into the subcutaneous tissue of a mouse using a syringe (a), the expansion of the hydrogel 1 day after enzyme injection (b), the expansion of the hydrogel 6 days after enzyme injection (c), and a graph of the change in the volume of the mouse's subcutaneous tissue over time after enzyme injection (d). As shown in FIG. 22, it can be observed that the hydrogel of Example 1, applied in an injectable form, expands over time. This is because the surface area of ​​the hydrogel increased through grinding, thereby increasing the reactivity of the injected enzyme.

[0176] Experimental Example 10. Confirmation of application characteristics of injectable tissue expanders in a diabetes model

[0177] In diabetic patients, wound healing time is delayed due to reduced immunity, and implantation of implant-type tissue expanders is difficult due to difficulty in hemostasis caused by altered platelet function, so the application of an injectable type is required.

[0178] First, Streptozotocin (STZ) was injected intraperitoneally into mice daily for 5 days (150 µl at 10 mg / mL) to selectively destroy pancreatic β-cells. At this time, mice were fed 10% sucrose water to model mice with type 1 diabetes with a blood glucose level of 375 mg / dL. Then, a micro-sized hydrogel obtained by grinding the hydrogel of Example 1 through a mixer for 120 seconds was injected into the back of the mice with type 1 diabetes using a syringe, and after 3 days, an additional enzyme was injected to measure the extra swelling ratio of the hydrogel.

[0179] [Table 1]

[0180]

[0181] Figure 23 shows images immediately after injecting a hydrogel in the form of the hydrogel of Example 1, ground for 120 seconds, into a mouse with type 1 diabetes, and images after enzyme injection. As shown in Figure 23 and Table 1, it can be confirmed that tissue expansion occurs in mice with type 1 diabetes after enzyme injection.

[0182] From the above results, it can be seen that the stimulation-responsive hydrogel according to the present invention can be applied to diabetic patients by enabling tissue expansion without a surgical step through injection.

[0183] Although the present invention has been described above by limited embodiments, the present invention is not limited thereto, and it is obvious that various modifications and variations are possible within the scope of the technical spirit of the present invention and the equivalent scope of the claims described below by those skilled in the art to which the present invention belongs.

Claims

1. A first network structure comprising a stimulus-responsive polymer and It comprises a second network structure including a hydrogel-forming polymer, and The first network structure and the second network structure above are stimulus-responsive hydrogels that form an interpenetrating polymer network (IPN).

2. In Claim 1, The above-mentioned second network structure is a stimulus-responsive hydrogel in which expansion is inhibited by the above-mentioned first network structure.

3. In Claim 1, A stimulus-responsive hydrogel comprising a first network structure containing the above stimulus-responsive polymer, which decomposes upon external stimulation.

4. In Claim 1, The above stimulus-responsive hydrogel is a stimulus-responsive hydrogel that begins to expand upon external stimulation.

5. In claim 3 or 4, The above external stimulus is one or more of enzymes, temperature, and pH, in a stimulus-responsive hydrogel.

6. In Claim 1, The above stimulus-responsive polymer is a stimulus-responsive hydrogel comprising one or more of hyaluronic acid, gelatin, and collagen.

7. In Claim 1, The above-mentioned hydrogel is a stimulus-responsive hydrogel comprising one or more of polyacrylic acid (PAA), polymethacrylic acid, polyacrylic acetate, polyethylene glycol (PEG), polyethylene oxide (PEO), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), polyacrylamide, polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), alginate, cellulose, gelatin, collagen, hyaluronic acid, carrageenan, chitosan, albumin, agarose, and copolymers thereof.

8. In Claim 4, The expansion of the above stimulus-responsive hydrogel is a continuous stimulus-responsive hydrogel.

9. A tissue expander comprising a stimulus-responsive hydrogel according to claim 1.

10. In Claim 9, The above tissue expander is a tissue expander in which expansion begins upon external stimulation.

11. In Claim 9, The above stimulus-responsive polymer contains hyaluronic acid, and the tissue expander is a tissue expander in which expansion begins upon injection of hyaluronidase.

12. In Claim 11, The breakdown product of the above hyaluronidase is a tissue expander that promotes skin regeneration in the subcutaneous tissue.

13. A method of using a tissue expander implemented by a tissue expander comprising a stimulus-responsive hydrogel according to claim 9.

14. In Claim 13, The method of using the above tissue expander is, A step of inserting a tissue expander comprising a stimulus-responsive hydrogel into the tissue; and A method for using a tissue expander comprising the step of expanding the tissue expander by external stimulation after inserting the tissue expander.

15. In Claim 14, The step of inserting the above tissue expander into the tissue is a method of using a tissue expander in which it is inserted in the form of an implant or an injectable.

16. In Claim 15, The step of inserting in the form of the above-mentioned implant is, The step of implanting the above tissue expander into the incised skin tissue; and A method of using a tissue expander, performed by the step of suturing the incised skin.

17. In Claim 15, The step of inserting in the above-mentioned injection form is, A step of crushing a tissue expander comprising the stimulus-responsive hydrogel; and A method of using a tissue expander, performed by the step of injecting the above-mentioned crushed tissue expander into the tissue.

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