Medical hydrogel composition, medical hydrogel and method and use thereof

Abstract

The present application provides a kind of medical hydrogel composition, medical hydrogel and its preparation method and application.The medical hydrogel composition includes: carrier matrix, the carrier matrix is derived from degradable biomaterial, and the degradable biomaterial includes collagen;And, active ingredient, the active ingredient is modified on the surface and / or inside of the carrier matrix, and the active ingredient is polypeptide substance.The medical hydrogel composition of the present application can not only effectively release polypeptide, but also promote the formation of epithelial attachment by different signal pathways, improve the closure of soft tissue around implant, and provide a new idea for preventing inflammation around implant.

Description

Technical Field

[0001] This invention relates to a medical hydrogel composition, a medical hydrogel, a method for preparing the same, and its applications, belonging to the field of medical materials. Background Technology

[0002] Implant surgery is a safe and predictable method for treating complete and partial tooth loss, but peri-implant disease is one of the biggest factors affecting its survival rate. Peri-implant disease is inflammation of the surrounding soft and hard tissues caused by bacteria, often leading to implant failure, so strategies to prevent peri-implant disease are urgently needed. In recent years, evidence has shown that the soft tissue seal formed around the implant can effectively resist physical and pathological stimuli. By strengthening the bond between epithelial cells and the titanium surface, it can prevent the spread of bacteria and their metabolites, increase implant stability during healing, and thus reduce the likelihood of inflammation and marginal bone loss.

[0003] Both healthy teeth and the soft tissue seal around implants are essentially maintained by the binding epithelium (JE). The JE consists of a basal plate and hemidesmosomes (HDs). JE attachment primarily relies on the anchoring effect of HDs, which are highly specialized epithelial attachment structures on the basal plate. However, due to the ischemic state of the peri-implant tissue and the slow release of metal ions from the implant, only the peri-root third of the attachment epithelium around the implant forms HDs, and their number is significantly reduced compared to that around natural teeth. This results in a significantly poorer biological seal around implants compared to natural teeth. Therefore, increasing the quality and quantity of HDs around implants is crucial for achieving a good peri-implant soft tissue seal.

[0004] Histone-rich 1 (Hst1) is a polypeptide derived from human salivary glands such as the submandibular and parotid glands. It can affect epithelial cell extension, enhance cell adhesion and migration, and has potential in maintaining the integrity of soft tissues. Studies have shown that Hst1 can promote cell adhesion to implant materials (titanium, hydroxyapatite, etc.) and participate in the formation of the host's innate immune system. van Dijk et al. systematically studied the dynamic adhesion process of epithelial cells to titanium surfaces promoted by Hst1 and speculated that this process is generated by the activation of integrin-mediated cell adhesion. Integrin α6 and integrin β4, as transmembrane portions of hematoxylin and eosin (HDs), initiate the assembly of HDs, thereby mediating cell adhesion and epithelial attachment to improve peri-implant soft tissue closure. Therefore, the application of Hst1 in peri-implant soft tissues should have good prospects. However, direct application of Hst1 may lead to the inhibition or enzymatic degradation of its effects, and also means that a short dosing interval is required. Therefore, in order to achieve good in vivo application of Hst1, it is necessary to find a carrier material to achieve sustained release of Hst1 in vivo.

[0005] Hydrogels are a widely used carrier material in clinical practice. They can mimic the tissue environment of the human body and provide structural support for defect sites, allowing the encapsulated active ingredients to be released slowly into the surrounding tissues. However, current hydrogels do not provide sufficient sustained release of active ingredients, and they require a certain level of strength to function as carriers. Furthermore, existing hydrogels have poor epithelial adhesion, resulting in poor sealing of the soft tissue around the implant and leading to peri-implant inflammation.

[0006] Therefore, researching a medical hydrogel with excellent sustained-release effect of active ingredients and a certain strength, which can promote the formation of epithelial attachment and improve the sealing of soft tissue around implants, has become an urgent technical problem to be solved. Summary of the Invention

[0007] The problem the invention aims to solve

[0008] In view of the technical problems existing in the prior art, the present invention first provides a medical hydrogel composition and a medical hydrogel prepared from the medical hydrogel composition. The medical hydrogel of the present invention can not only effectively release peptides; at the same time, it can also promote the formation of epithelial attachment through mutual compensation of different signaling pathways, improve the sealing of peri-implant soft tissue, and provide a new approach to prevent peri-implant inflammation.

[0009] Furthermore, the present invention also provides a method for preparing medical hydrogel, which is simple and easy to implement, uses readily available raw materials, and is suitable for mass production.

[0010] In addition, the present invention also provides a medical hydrogel kit and the use of medical hydrogels.

[0011] Solution for solving the problem

[0012] This invention provides a medical hydrogel composition comprising:

[0013] A carrier matrix derived from biodegradable biomaterials, wherein the biodegradable biomaterials contain collagen; and,

[0014] The active ingredient, which is modified on the surface and / or inside the carrier matrix, and,

[0015] The active ingredient is a polypeptide.

[0016] Furthermore, the carrier matrix is ​​obtained by digesting the degradable biomaterial using an acidic protease.

[0017] Furthermore, the polypeptide substance includes one or more of histone-rich 1, notoginsenoside R1, histone-rich 5, and antimicrobial peptide LL-37.

[0018] The present invention also provides a medical hydrogel, which is formed by modifying the active ingredients in the medical hydrogel composition on the surface and / or inside a carrier matrix;

[0019] Preferably, the active ingredient is modified on the surface and / or inside the carrier matrix by cross-linking.

[0020] The present invention also provides a method for preparing a medical hydrogel, which includes the step of modifying an active ingredient on the surface and / or inside a carrier matrix.

[0021] Furthermore, the preparation method includes the following steps:

[0022] After digesting the biodegradable biological material using acidic protease, a carrier digestion solution was obtained.

[0023] The carrier digest solution was freeze-dried to obtain the carrier matrix;

[0024] The carrier matrix is ​​mixed with a polypeptide compound in a solvent to obtain a reaction precursor;

[0025] In the presence of a crosslinking agent, the carrier matrix and the polypeptide compound undergo a crosslinking reaction to obtain a reaction product;

[0026] After removing the crosslinking agent, the pH is adjusted to neutral to form a gel product.

[0027] Furthermore, the digestion process is carried out under acidic conditions, preferably by using an acidic substance to make the digestion process under acidic conditions; and / or, the digestion process lasts for 24-72 hours and the digestion process is carried out at a temperature of 23-28°C.

[0028] Further, in the reaction precursor, the concentration of the carrier matrix can be 1-100 mg / ml, and the mass concentration of the polypeptide compound is 0.0001 mg / ml-0.1 mg / ml; and / or,

[0029] The crosslinking agent includes one or more of N-hydroxysuccinimide, carbodiimide, carbodiimide / N-hydroxysuccinimide, and genipin; preferably, the crosslinking reaction takes 3-9 hours.

[0030] Furthermore, the crosslinking agent is removed by dialysis; preferably, the dialysis time is 12-36 hours.

[0031] The present invention also provides a medical hydrogel kit comprising the medical hydrogel composition described herein; preferably, the medical hydrogel kit further comprises a crosslinking agent.

[0032] Furthermore, the carrier matrix and active ingredient of the medical hydrogel composition are stored separately; preferably, the crosslinking agent is stored separately from both the carrier matrix and the active ingredient.

[0033] The present invention also provides the use of the medical hydrogel composition according to the present invention in the preparation of implant sealant or implant lubricant.

[0034] The effects of the invention

[0035] The medical hydrogel prepared by the medical hydrogel composition of the present invention can not only effectively release peptides, but also promote the formation of epithelial attachment through different signaling pathways, improve the sealing of peri-implant soft tissue, and provide a new approach to prevent peri-implant inflammation.

[0036] The preparation method of the medical hydrogel of the present invention is simple and easy to implement, the raw materials are readily available, and it is suitable for mass production. Attached Figure Description

[0037] Figure 1 The synthesis of the medical hydrogel of the present invention and its potential applications around implants are illustrated.

[0038] Figure 2 A schematic diagram illustrating the basic performance testing of the medical hydrogel of the present invention is shown; wherein,

[0039] A shows scanning electron microscope (SEM) images of the surface and cross-section of the medical hydrogels and SIS matrix of Examples 1 and 2;

[0040] B shows the morphology of the medical hydrogel of Example 1 under different conditions; (a) is the morphology of the medical hydrogel at room temperature, (b) is the morphology of the medical hydrogel at 37°C, (c) is the morphology of the medical hydrogel after being fluorescently labeled with histone 1, and (d) is the morphology of the medical hydrogel after 2 weeks of sustained release of histone 1.

[0041] C shows the degradation rates of the medical hydrogels and SIS matrix of Examples 1 and 2;

[0042] D shows the swelling ratios of the medical hydrogels and SIS matrix of Examples 1 and 2;

[0043] E shows the sustained-release curves of histone-rich 1 in the medical hydrogels and SIS matrix of Examples 1 and 2;

[0044] F shows the storage modulus (G') and loss modulus (G”) of the medical hydrogel of Example 1 under strain amplitude scan (γ1 / 40.1%–1000%) at a fixed angular frequency;

[0045] G shows the stress-strain curve of the medical hydrogel of Example 1.

[0046] Figure 3 The diagrams show relevant schematics of cell compatibility and antibacterial experiments; among them,

[0047] A shows a schematic diagram of live / dead cell staining of cells cultured in medical hydrogels and SIS matrix for 12h and 24h in Examples 1 and 2, respectively, with the corresponding live cell count on the right.

[0048] B shows the cell proliferation curves after culturing cells in medical hydrogels, histone-1-rich substrates, and SIS matrix in Examples 1 and 2, respectively, analyzed by CCK-8 counting.

[0049] C shows the inhibition zones of co-cultured Streptococcus Gordonii strains, where (a) blank, (b) SIS; (c) SIS-Hst1(L); and (d) SIS-Hst1(H).

[0050] D shows photographs of colony formation on BHI agar plates, where (a) blank, (b) SIS, (c) SIS-Hst1(L), and (d) SIS-Hst1(H).

[0051] E shows fluorescent live / dead staining images of *Streptococcus Gordonii* strains after culturing cells in the medical hydrogels and SIS matrix of Examples 1 and 2, respectively.

[0052] Figure 4 A schematic diagram illustrating cell adhesion and migration is shown; in which,

[0053] A shows the cross-well assay of cell migration ability after culturing cells in the medical hydrogels and SIS matrix of Examples 1 and 2, respectively.

[0054] B shows the cell counting analysis of migrating cells after culturing cells in the medical hydrogel and SIS matrix of Examples 1 and 2, respectively.

[0055] C shows cell morphology observed under a scanning electron microscope; the inset is a high-power image with a white frame.

[0056] D shows the cytoskeleton staining after culturing cells in the medical hydrogels and SIS matrix of Examples 1 and 2, respectively.

[0057] Figure 5The diagram illustrates the immunofluorescence analysis of relevant protein localization in the medical hydrogels and SIS matrix of Examples 1 and 2; specifically,

[0058] The localization and expression of laminin-α6 (A), laminin-β4 (B), and laminin 5 (C) in oral epithelial cells were observed and analyzed using CLSM (red represents proteins, and blue represents cell nuclei and the result of synthesis).

[0059] Figure 6 The diagram illustrates the detection of laminin 5, integrin-α6, and integrin-β4 expression in cells using Western blotting with medical hydrogels and SIS matrix as described in Examples 1 and 2; wherein,

[0060] A shows the Western blot analysis of hemidesmosome (HDs) related proteins;

[0061] B shows the quantitative analysis of hemidesmosome (HDs) related proteins;

[0062] C shows the qRT-PCR analysis of the relevant genes.

[0063] Figure 7 The diagram shows relevant animal experiments of the medical hydrogel and SIS matrix of Example 1; wherein,

[0064] A shows the observation of peri-implant epithelial formation after HE staining (blue represents basal cells, orange represents suprabasal cells);

[0065] B shows the expression and distribution of proteins detected by immunofluorescence staining (green represents proteins, and blue represents the cell nucleus and the results of synthesis). Detailed Implementation

[0066] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0067] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0068] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0069] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0070] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0071] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0072] In this instruction manual, when "room temperature" or "room temperature" is used, the temperature can be 15-25℃.

[0073] <First Aspect>

[0074] A first aspect of the present invention provides a medical hydrogel composition comprising:

[0075] A carrier matrix derived from biodegradable biomaterials, wherein the biodegradable biomaterials contain collagen; and,

[0076] The active ingredient, which is modified on the surface and / or inside the carrier matrix, and,

[0077] The active ingredient is a polypeptide.

[0078] The medical hydrogel prepared by the medical hydrogel composition of the present invention can not only effectively release peptides, but also promote the formation of epithelial attachment through different signaling pathways and improve the sealing of peri-implant soft tissue.

[0079] carrier matrix

[0080] In this invention, the carrier matrix is ​​derived from biodegradable biomaterials, and the biodegradable biomaterials contain collagen. The collagen may be of natural origin, synthetically produced, modified, or cross-linked, and includes, but is not limited to, the submucosa, dermis, pericardium, collagen, gelatin, etc.

[0081] In some embodiments of the present invention, the carrier matrix is ​​derived from the submucosa of the small intestine, preferably from a decellularized submucosa of the small intestine, meaning that the carrier matrix of the present invention can be prepared using the submucosa of the small intestine. In some embodiments of the present invention, the carrier matrix is ​​derived from the dermis, preferably from a decellularized dermis, meaning that the carrier matrix of the present invention can be prepared using the dermis. In some embodiments of the present invention, the carrier matrix is ​​derived from the submucosa of the bladder, preferably from a decellularized submucosa of the bladder, meaning that the carrier matrix of the present invention can be prepared using the submucosa of the bladder. In some embodiments of the present invention, the carrier matrix is ​​derived from the pericardium, preferably from a decellularized pericardium, meaning that the carrier matrix of the present invention can be prepared using the pericardium. The submucosa (e.g., the submucosa of the small intestine), dermis, and pericardium are preferably derived from mammals, such as pigs, cattle, sheep, dogs, and cats.

[0082] In one embodiment of the present invention, the carrier matrix is ​​derived from decellularized porcine small intestinal submucosa. This porcine small intestinal submucosa is commercially available. It is widely available, economically accessible, and easy to process. Furthermore, the decellularized porcine small intestinal submucosa exhibits excellent biocompatibility, is rich in collagen and growth factors, and can induce cell diffusion, adhesion, growth, and proliferation, promoting the repair and regeneration of tissue at the site of tissue loss. Therefore, it is suitable for use as a carrier matrix in the preparation of the biological patch of the present invention. Thus, the present invention preferably uses porcine small intestinal submucosa as the raw material to prepare the porcine small intestinal submucosa material as the carrier matrix.

[0083] The present invention does not specifically limit the preparation method of the porcine small intestinal submucosal material, and it can be some commonly used preparation methods in the art. Of course, the porcine small intestinal submucosal material can also be purchased commercially. Preferably, the porcine small intestinal submucosal material of the present invention can be prepared according to the preparation method in CN107007886A.

[0084] The inventors of this invention discovered that the main components of the porcine small intestinal submucosa material are type I and type III collagen, exhibiting good biocompatibility and producing a medical hydrogel with excellent physical properties. Furthermore, SIS contains various active factors such as proteoglycans, elastin, glycosaminoglycans, and growth factors, which promote cell proliferation. As a raw material for hydrogels, the porcine small intestinal submucosa material can not only serve as a carrier for the sustained release of Hst1, but also promote epithelial attachment formation through mutual compensation of different signaling pathways, thereby improving the sealing of peri-implant soft tissue.

[0085] In another embodiment of the invention, the carrier matrix may also be derived from a fibrous membrane prepared using chemicals containing collagen. The invention does not particularly limit the type of fibrous membrane; it can be any fibrous membrane commonly used in the art, such as a nonwoven fibrous membrane or a spun fibrous membrane.

[0086] The nonwoven fiber membrane of the present invention can be obtained by processing with one or more of the following: a chemical substance containing collagen and optionally other polymers or their derivatives, through a nonwoven process. The spun fiber membrane of the present invention can be obtained by processing with one or more of the following: a chemical substance containing collagen and optionally other polymers or their derivatives, through processes such as electrospinning, centrifugal spinning, hot melt spinning, and melt electrospinning.

[0087] Other polymers or their derivatives may be various commonly used polymers or their derivatives in this field. For example, they may be selected from one or more combinations of natural polymers. Commonly used polymers or their derivatives may be one or more combinations of cellulose, chondroitin sulfate, chitosan, modified chitosan, fibroin, silk fibroin, elastin-mimicking peptide polymers, heparin, agar, dextran, alginic acid, cellulose, alginic acid, and starch.

[0088] Furthermore, in this invention, the carrier matrix can be obtained by digesting the degradable biomaterial using an acidic protease. This digestion process facilitates the preparation of a medical hydrogel.

[0089] Active ingredients

[0090] The active ingredient of this invention is modified on the surface and / or interior of the carrier matrix, and the active ingredient is a polypeptide. Polypeptides can affect the extension of epithelial cells, enhance intercellular adhesion and migration, and have the potential to maintain the integrity of soft tissues.

[0091] Furthermore, the present invention does not specifically limit the polypeptide material, and it can be any feasible polypeptide material in the art. Preferably, in order to maximize the efficacy of the medical hydrogel, the polypeptide material can include one or more of histone 1, notoginsenoside R1, histone 5 and antimicrobial peptide LL-37, etc., preferably histone 1.

[0092] <Second aspect>

[0093] A second aspect of the present invention provides a medical hydrogel. The medical hydrogel is formed by modifying the active ingredient of the medical hydrogel composition described in the first aspect onto the surface and / or interior of a carrier matrix.

[0094] In some specific embodiments, the active ingredient is modified on the surface and / or inside the carrier matrix through cross-linking. The inventors of this invention have discovered that cross-linking allows the active ingredient to bind more tightly to the carrier and enables the medical hydrogel to possess a certain degree of mechanical strength.

[0095] Furthermore, in this invention, the medical hydrogel is flowable at room temperature (15-25°C) and semi-solid at 36-38°C, at which point it generally does not flow. A semi-solid is a substance between a solid and a liquid. A semi-solid can support its own weight and maintain its shape, but it also possesses some properties of liquids, such as changing shape under pressure and flowing at low temperatures. Therefore, the medical hydrogel of this invention has temperature-sensitive properties and is a temperature-sensitive hydrogel.

[0096] <Third aspect>

[0097] A third aspect of the present invention provides a method for preparing a medical hydrogel according to a second aspect of the present invention, comprising the step of modifying an active ingredient on the surface and / or inside a carrier matrix.

[0098] In some specific implementations, the preparation method includes the following steps:

[0099] After digesting the biodegradable biological material using acidic protease, a carrier digestion solution was obtained.

[0100] The carrier digest solution was freeze-dried to obtain the carrier matrix;

[0101] The carrier matrix is ​​mixed with a polypeptide compound in a solvent to obtain a reaction precursor;

[0102] In the presence of the crosslinking agent, the carrier matrix and the polypeptide compound undergo a crosslinking reaction to obtain the reaction product;

[0103] After removing the cross-linking agent from the reaction product and adjusting the pH value, a gel product is formed.

[0104] Specifically, the digestion process is carried out under acidic conditions, preferably by using an acidic substance to achieve this; and / or, the digestion process lasts for 24-72 hours, for example: 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, etc.; the digestion process is carried out at a temperature of 23-28°C, for example: 24°C, 25°C, 26°C, 27°C, etc.

[0105] The present invention does not specifically limit the acidic protease, and can be any acidic protease commonly used in the art, such as pepsin.

[0106] The present invention does not impose any particular limitation on the amount of acidic substances and acidic proteases used, as long as the digestion process can be completed.

[0107] In some specific implementations, the biodegradable biomaterials can be pulverized before digestion, specifically using a cryogenic mill.

[0108] Preferably, the acidic substance can be either an inorganic acid or an organic acid. Considering that the protein should not be denatured, the present invention preferably uses an organic acid as the acidic substance, such as oxalic acid, acetic acid, propionic acid, etc.

[0109] The present invention does not impose particular limitations on the freeze-drying conditions, as long as the carrier digestion solution is dried to obtain the carrier matrix. Specifically, the freeze-drying can be carried out in a freeze dryer at a temperature of -100 to -50°C to obtain the carrier matrix.

[0110] Further, the carrier matrix is ​​mixed with the peptide compound in a solvent to obtain a reaction precursor. Specifically, the concentration of the carrier matrix in the reaction precursor can be 1-100 mg / ml, for example: 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml. The concentrations of the polypeptide compounds are 0.0001 mg / ml to 0.1 mg / ml, for example: 0.001 mg / ml, 0.005 mg / ml, 0.01 mg / ml, 0.02 mg / ml, 0.03 mg / ml, 0.04 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, etc.

[0111] Furthermore, the crosslinking reaction is carried out in the presence of a crosslinking agent. Preferably, the crosslinking agent includes one or more of N-hydroxysuccinimide, carbodiimide, carbodiimide / N-hydroxysuccinimide, and genipin. Preferably, the carbodiimide includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0112] To enable sustained release of peptides in hydrogels, carbodiimide / N-hydroxysuccinimide can be used as a cross-linking agent. Specifically, carbodiimide and N-hydroxysuccinimide are used in combination to promote amide cross-linking, and both cross-linking agents can be removed by dialysis to avoid cell damage. Specifically, the mass ratio of carbodiimide to N-hydroxysuccinimide can be 1:0.5-3, for example: 1:0.8, 1:1, 1:1.5, 1:2, 1:2.5, etc. Preferably, the cross-linking agent comprises a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0113] Furthermore, the cross-linking reaction takes 3-9 hours, for example: 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, etc. When the cross-linking reaction takes 3-9 hours, the desired medical hydrogel can be obtained.

[0114] In some specific implementations, the crosslinking agent is removed by dialysis; preferably, the dialysis time is 12-36 hours, for example: 16 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, etc.

[0115] To adjust the pH to neutral, an alkaline substance can be used, such as sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, or potassium hydroxide. Neutralization will yield the desired gel product. Specifically, the pH can be adjusted to 7.2-7.6 to obtain the gel product.

[0116] Afterwards, the obtained gel product can be subjected to post-processing operations such as washing to obtain the final medical hydrogel. For washing, a buffer solution, such as PBS buffer solution, can generally be used.

[0117] <Fourth Aspect>

[0118] A fourth aspect of the present invention provides a medical hydrogel kit comprising the medical hydrogel composition described in the first aspect. To improve the shelf life of the medical hydrogel, the present invention can be presented in the form of a medical hydrogel kit, allowing for on-demand preparation. Preferably, the medical hydrogel kit further comprises a cross-linking agent, thereby modifying the active ingredient on the surface and / or interior of a carrier matrix.

[0119] Specifically, the medical hydrogel kit may include a carrier matrix and an active ingredient. Preferably, the medical hydrogel kit may also include a cross-linking agent. The carrier matrix and the active ingredient may be separate. During use, the active ingredient is modified onto the surface and / or interior of the carrier matrix, preferably using a cross-linking agent. In practical applications, the carrier matrix and the active ingredient can be stored separately. This invention does not limit the storage method of each component in the medical hydrogel composition. Those skilled in the art can choose specific storage methods as needed, all of which are within the scope of this invention.

[0120] <Fifth Aspect>

[0121] A fifth aspect of the present invention provides the use of the medical hydrogel composition according to the first aspect of the present invention in the preparation of implant sealant or implant lubricant.

[0122] The medical hydrogel of this invention can simulate the tissue environment of the human body and provide structural support for damaged areas, allowing the encapsulated active ingredients to be slowly released into the surrounding tissue. The medical hydrogel of this invention remains flowable at low temperatures; it transforms into a semi-solid state at 36-38°C, particularly 37°C, and generally does not flow after this transformation.

[0123] Furthermore, the medical hydrogel of this invention acts as a lubricant when it is flowable, reducing interfacial friction and thus increasing the preload between the implant and the abutment. When it transforms into a semi-solid state, it seals the micro-gaps around the implant and abutment, preventing the invasion of bacteria and their metabolic products, while simultaneously improving the stability of the abutment tooth. The application of the medical hydrogel around the implant allows its excellent biocompatibility and unique phase transition properties to work together effectively.

[0124] Example

[0125] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0126] In the examples and comparative examples, the porcine small intestinal submucosa material carrier was prepared according to the following preparation method.

[0127] (1) Initial treatment of raw materials:

[0128] Porcine small intestinal submucosal tissue material (also known as porcine small intestinal submucosal SIS) was taken and cut into specified dimensions of 8 cm wide and 15 cm long. Unnecessary tissue (such as lymphoid tissue) was removed. The material was rinsed twice with tap water, and then rinsed with purified water until the surface was free of stains. The rinsed porcine small intestinal submucosal tissue material was placed in a filter or similar filtration device for at least 5 minutes to drain the water. When taking the porcine small intestinal submucosal tissue material, the volume of the filtered material was measured using a graduated cylinder or similar measuring device.

[0129] (2) Virus inactivation:

[0130] Virus inactivation was performed by immersing porcine small intestinal submucosal tissue material in a peracetic acid-ethanol solution. This process could be carried out in a stainless steel tank. The peracetic acid concentration in the peracetic acid-ethanol solution was 1% (volume percentage), and the ethanol concentration was 24% (volume percentage). The ratio (volume ratio) of the peracetic acid-ethanol solution to the porcine small intestinal submucosal tissue material was 5:1. The inactivation time was 2 hours, and the inactivation temperature (i.e., the temperature at which the peracetic acid-ethanol solution was used to immerse the porcine small intestinal submucosal tissue material) was within the range of 20°C.

[0131] (3) Cleaning process:

[0132] The submucosal tissue material of porcine small intestine was cleaned with a PBS solution with a pH of 7.2-7.4 at 20°C. The ratio of PBS solution to porcine submucosal tissue material (volume ratio) was 30:1. It was preferably cleaned 3 times, 20 minutes each time. Then, purified water was used for cleaning, with a purified water ratio of 30:1 to porcine submucosal tissue material, until the conductivity was detected to be below 10 μS / cm. The cleaning process was carried out in an ultrasonic cleaner at a frequency of 40 kHz and a power of 3000 W.

[0133] (4) Decellularization:

[0134] The decellularization solution was a PBS solution containing 0.025% trypsin and 0.5 mmol / L EDTA-2Na; the pH of the decellularization solution was 7.2-7.4; the mixing ratio (volume ratio) of the decellularization solution and porcine small intestinal submucosal tissue material was 30:1; the decellularization process was carried out in a dual-frequency ultrasound device, including low frequency and high frequency, with the low frequency being 20 kHz and the high frequency being 80 kHz, and the ultrasound power being 5 kW; the low frequency treatment was 10 min and the high frequency treatment was 10 min, with the temperature being 30 ℃; the ultrasound power was 5000 W.

[0135] (5) Cleaning process:

[0136] Cleaning was performed using a cleaning solution in an ultrasonic cleaner with a power of 3000W. The cleaning solution was a PBS solution with a pH of 7.2-7.4, at a temperature of 20°C. The ratio of PBS solution to porcine small intestinal submucosal tissue material (volume ratio) was 30:1. Ideally, the cleaning was performed three times, each time for 20 minutes. Then, the tissue was cleaned with cooled water for injection at 20°C, with a volume ratio of water for injection to porcine small intestinal submucosal tissue material of 30:1. The cleaning was terminated when the difference in conductivity between the cleaned and uncleaned water for injection was less than 1 μS / cm. The frequency was 40 kHz and the power was 3000W.

[0137] (6) Vacuum freeze drying:

[0138] Porcine small intestinal submucosal tissue material was freeze-dried at -80°C in a freeze dryer (GAMMA 2–16LSC; Christ, Germany), and then pulverized in a freeze mill (MM400; Retsch, Germany) to obtain porcine small intestinal submucosal material (SIS) carrier.

[0139] Example 1

[0140] The obtained porcine small intestinal submucosa material (SIS) carrier was stirred in an aqueous solution containing 3% acetic acid and 0.1% pepsin (25°C) for 48 hours to obtain SIS digestion solution;

[0141] The obtained SIS digest was freeze-dried again to obtain a soluble SIS matrix, which was then sterilized with ethylene oxide gas.

[0142] 100 μg of histone-rich 1 was mixed with 10 ml of soluble SIS matrix with a concentration of 10 mg / ml to obtain the reaction precursor;

[0143] 8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6 mg of N-hydroxysuccinimide (NHS) were added to the reaction precursor, and the mixture was stirred for 6 hours to obtain the reaction product.

[0144] The reaction product was dialyzed in the dark for 24 hours to remove 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, yielding the dialyzed product.

[0145] Subsequently, 2.5 mol / L NaOH was added to the dialysis product to neutralize it to pH 7.4, and the neutralized solution formed a gel product at 37°C.

[0146] Finally, the gel product was washed twice with PBS buffer solution at pH 7.4 and twice with DMEM medium to obtain a medical hydrogel, denoted as SIS-Hst1(H).

[0147] Example 2

[0148] The obtained porcine small intestinal submucosa material (SIS) carrier was stirred in an aqueous solution containing 3% acetic acid and 0.1% pepsin (25°C) for 48 hours to obtain SIS digestion solution;

[0149] The obtained SIS digest was freeze-dried again to obtain a soluble SIS matrix, which was then sterilized with ethylene oxide gas.

[0150] 10 μg of histone-rich 1 and 10 ml of soluble SIS matrix with a concentration of 10 mg / ml were mixed to obtain the reaction precursor;

[0151] 8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6 mg of N-hydroxysuccinimide (NHS) were added to the reaction precursor, and the mixture was stirred for 6 hours to obtain the reaction product.

[0152] The reaction product was dialyzed in the dark for 24 hours to remove 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, yielding the dialyzed product.

[0153] Subsequently, 2.5 mol / L NaOH was added to the dialysis product to neutralize it to pH 7.4, and the neutralized solution formed a gel product at 37°C.

[0154] Finally, the gel product was washed twice with PBS buffer solution at pH 7.4 and twice with DMEM medium to obtain the medical hydrogel, denoted as: SIS-Hst1(L).

[0155] Example 3

[0156] The obtained porcine small intestinal submucosa material (SIS) carrier was stirred in an aqueous solution containing 3% acetic acid and 0.1% pepsin (25°C) for 48 hours to obtain SIS digestion solution;

[0157] The obtained SIS digest was freeze-dried again to obtain a soluble SIS matrix, which was then sterilized with ethylene oxide gas.

[0158] 100 μg of histone-rich 1 and 10 ml of soluble SIS matrix (10 mg / ml) were mixed to obtain a mixed product;

[0159] Subsequently, 2.5 mol / L NaOH was added to the mixed product to neutralize it to pH 7.4, and the neutralized solution formed a gel product at 37°C.

[0160] Finally, the gel product was washed twice with PBS buffer solution at pH 7.4 and twice with DMEM medium to obtain a medical hydrogel, denoted as SIS-Hst1.

[0161] Performance testing

[0162] 1. Basic performance testing

[0163] 1.1 Morphology

[0164] The medical hydrogels of Example 1 (SIS-Hst1(H) group), Example 2 (SIS-Hst1(L) group), and the SIS matrix were freeze-dried at -80°C (GAMMA2-16LSC, Christ, Germany) for 24 hours. After adding a gold scanning electron conductive layer, the surface and cross-section of the freeze-dried samples were observed. The arrows indicate peptides. Microscopic images were obtained using an emission scanning electron microscope (SEM, Gemini300; Zeiss, Germany). The results are shown below. Figure 2 As shown.

[0165] 1.2 Liquidity

[0166] The sol-gel transition behavior was observed using the vial tilting method. The medical hydrogel from Example 1 was added to a vial and incubated at 37°C to observe its flowability in real time. The results are as follows: Figure 2 As shown.

[0167] 1.3 Degradation performance:

[0168] The medical hydrogels from Example 1 (SIS-Hst1(H) group), Example 2 (SIS-Hst1(L) group), and the SIS matrix group were respectively placed in PBS buffer solution with a pH of 7.4 containing collagenase (Sigma). 1 ml of each sample was lyophilized and accurately weighed (W0). The samples were then immersed in 15 mL of enzyme buffer at 37°C for degradation. The remaining sample mass (W1) was weighed daily for two weeks, after which degradation was terminated. The degradation rate was calculated using the formula: Degradation rate (%) = (1 - W1 / W0) × 100. The results are shown below. Figure 2 As shown.

[0169] 1.4 Swelling rate

[0170] Weigh out the medical hydrogel (SIS-Hst1(H) group) from Example 1 and place it in artificial saliva. In a water bath at 37°C, remove the hydrogel periodically, wipe off surface moisture with damp filter paper, weigh it, and then return it to the solution. Calculate the swelling rate based on the mass before and after swelling at different time points. The swelling rate (Q) is calculated using the formula: Q = (m t -m0) / m0; where m t The mass of the medical hydrogel at swelling time t is given, and m0 is the initial mass of the medical hydrogel. The results are as follows: Figure 2 As shown.

[0171] 1.5 Histone 1 (Hst1) enriched sustained release in vitro

[0172] The medical hydrogels of Example 1 (SIS-Hst1(H) group), Example 2 (SIS-Hst1(L) group), and Example 3 (SIS-Hst1 group) formed using FITC-labeled histone 1-rich hydrogels simulated the sustained release of histone 1 in medical hydrogels and were stabilized and incubated in a shaking water bath at 37°C and 100 rpm. The medical hydrogels were immersed in PBS buffer solution at pH 7.4, and 1 ml of the sustained-release solution was collected daily, with 1 ml of fresh, equal-volume PBS buffer solution added. The absorbance was measured at 480 nm using a BIOTEK UV spectrophotometer, and the sustained-release amount was calculated. The results are shown below. Figure 2 As shown. The standard curve was obtained by measuring FITC-Hst1 in a gradient dilution in PBS buffer.

[0173] 1.6 Rheology

[0174] The rheological properties of the medical hydrogel of Example 1 were measured using an HR-2 rheometer (AR-G2; TA Instrument, USA) with a 20 mm cone plate. The storage modulus (G') of the SIS matrix and the medical hydrogel was monitored over time at a frequency of 1 Hz and a stress-strain ratio of 1% at a constant temperature of 37°C. The storage modulus (G') and loss modulus (G”) of the medical hydrogel were measured at a fixed angular frequency (10 rad / s) under strain amplitude sweeps (γ1 / 40.1%–1000%). Furthermore, a compression test was performed on the medical hydrogel of Example 1 at 37°C using a load cell from a PC-type compressor (Minnesota, USA). A cylindrical medical hydrogel (Φ10 mm × 5 mm) on the lower plate was compressed at a compression rate of 5 mm / min to obtain stress-strain curves, the results of which are shown below. Figure 2 As shown.

[0175] 1.7 Basic Performance Test Results

[0176] Medical hydrogels are tissue-adaptive structures; after freeze-drying, their loose, porous structure and the loading of peptides can be observed. Figure 2 A). Cross-linking creates small through-pore structures around macropores, increasing the porosity of the structure. This not only provides more cell adhesion sites but also promotes the co-supply of nutrients and the excretion of metabolites.

[0177] Medical hydrogels possess a certain degree of fluidity before gelation and can change their shape to adapt to the shape of tissues through temperature changes. At room temperature, medical hydrogels can exhibit a flowable form. Figure 2 B(a)). At 36-38℃, the SIS matrix transforms into a gel, allowing the medical hydrogel to exhibit a semi-solid form. Figure 2 B(b)) generally does not flow. The degradation curve of medical hydrogels is basically proportional to time. Figure 2 C). The degradation rate of the histone 1-rich medical hydrogel was slightly lower, possibly due to increased resistance to enzymatic degradation after cross-linking. It is speculated that the amide bonds of histone 1 are broken as it diffuses or degrades within the SIS matrix, thereby allowing it to be released slowly into the surrounding tissue.

[0178] The swelling of the medical hydrogel reached equilibrium in approximately 400 minutes, while the swelling rates of the medical hydrogels in Examples 1 and 2 were lower than those of the SIS matrix. Figure 2 D). This is related to the cross-linking effect of the cross-linking agent on collagen, which makes the relative structure of medical hydrogels more stable.

[0179] The sustained-release results showed that in the medical hydrogel of Example 3, histone-rich 1 did not crosslink with the SIS matrix; therefore, histone-rich 1 was completely released after 2 days. In the medical hydrogels of Examples 1 and 2, histone-rich 1 was released sustainably for about 1 week. Figure 2 E). In the second week, there was almost no difference between the medical hydrogel of Example 3 and the medical hydrogels of Examples 1 and 2. Furthermore, the concentration of histone-rich 1 had no statistically significant effect on sustained release. Also, the color of histone-rich 1 in the medical hydrogel after fluorescent labeling gradually faded with the sustained release of histone-rich 1. Figure 2 B(c,d)).

[0180] In addition, the structure of medical hydrogels provides certain mechanical properties. Medical hydrogels possess a certain degree of fluidity before gelation and can change their shape through temperature changes to adapt to the shape of tissues. Specifically, the SIS matrix transforms into a gel at 37°C, forming a semi-solid state. This is achieved through rheological testing (…). Figure 2 F) and compression test ( Figure 2G) Its mechanical properties were tested, specifically the stress-strain relationship was studied using a compression testing machine. When the strain was 79%, the tensile strength of the medical hydrogel reached 81 kPa. Therefore, the medical hydrogel has temperature sensitivity and certain mechanical strength, and can sustain release after cross-linking, thus showing great promise for injection applications.

[0181] 2. Cell compatibility test and antibacterial test

[0182] 2.1 Staining of live and dead cells

[0183] To assess biocompatibility with human oral epithelial cells, 100 μL of solidified medical hydrogel from Example 1 (SIS-Hst1(H) group), medical hydrogel from Example 2 (SIS-Hst1(L) group), and SIS matrix were placed in 24-well plates and DMEM medium was added. The control group consisted of DMEM medium and 100 μL of PBS buffer solution (pH 7.4). Each well was seeded with 1 × 10⁶ cells / well. 4 Personal oral epithelial cells (OECs) were cultured for 12 and 24 hours. After 12 and 24 hours of culture, the cells in 24-well plates were stained for 20 minutes with a live / dead staining agent (AO / EB staining agent, Beijing Solarbio Science & Technology Co., Ltd.), and images were acquired using an inverted fluorescence microscope (Olympus IX71, Japan). Five fields of view were randomly selected for photography, and the number of cells was counted within the microscope field of view. CCK-8 cell counting was performed using ImageJ software. The results are shown below. Figure 3 As shown.

[0184] 2.2 CCK-8 Experiment

[0185] To further evaluate cell proliferation, 10 μL of solidified medical hydrogel from Example 1 (SIS-Hst1(H) group), medical hydrogel from Example 2 (SIS-Hst1(L) group), histone-rich 1, and SIS matrix were placed in 96-well plates and seeded with 5 × 10⁶ cells per well using titanium sheet medium without fetal bovine serum. 3 Personal oral epithelial cells were used, with the control group receiving titanium-coated culture medium without fetal bovine serum and 10 μL of PBS buffer (pH 7.4). LDH levels in the cell supernatant were measured on days 1, 3, 5, and 7 using a lactate dehydrogenase kit (LDH kit; Solarbio). Relative optical density (OD) values ​​were measured using a microplate reader (Multiskan FC, USA) to determine relative cell viability. Results are shown below. Figure 3 As shown.

[0186] 2.3 Antibacterial test

[0187] Three circular molds of fixed size were used to load the medical hydrogel of Example 1 (SIS-Hst1(H) group), the medical hydrogel of Example 2 (SIS-Hst1(L) group), and the SIS matrix, respectively, to form three experimental groups. 100 μL of 1×10 6 A CFU / mL solution of *Gnaphalium affine* was inoculated onto BHI agar plates and incubated at 37°C for 30 minutes. After bacterial colonization, 100-1000 μL pipettes were used to drill wells in the agar plates, and equal volumes of the pre-prepared experimental groups were placed in different wells. The plates were then incubated at 37°C for 24 hours. PBS buffer solution at pH 7.4 was used as a blank control.

[0188] 1 ml of the medical hydrogel from Example 1, the medical hydrogel from Example 2, and the SIS matrix were respectively immersed in different test tubes containing 2 mL of *S. gordonii* and incubated at 37°C for 12 hours. Then, 100 μL of bacterial solution was removed from the test tubes and stained with a live / dead staining kit. Live bacteria (green) and dead bacteria (red) were observed using CLSM. A bacterial suspension in sterile PBS buffer (pH 7.4) served as a control.

[0189] 2.4 Bacterial Inoculation Experiment

[0190] 20 μL of *Streptococcus Gordonii* suspension was coated onto the surfaces of the medical hydrogels of Example 1, Example 2, and the SIS matrix in the wells of a bacterial growth plate for 12 h. 20 μL of *Streptococcus Gordonii* suspension in 1 mL of sterile PBS buffer (pH 7.4) was used as a control. The bacterial suspension was diluted with sterile PBS buffer (pH 7.4) in the wells of the bacterial growth plate, and 10 μL of the diluted suspension was evenly spread on a BHI agar plate. After 24 h of inoculation, photographs were taken and the bacteria were counted.

[0191] 2.4 Results of cell compatibility and antibacterial experiments

[0192] Depend on Figure 3 As can be seen, cell viability / death staining analysis was used to evaluate the cytotoxicity of each group. The fields of view included the blank group, SIS group, SIS-Hst1(L) group, and SIS-Hst1(H) group. After co-culturing for 12 / 24 h, almost no cell death was observed in the SIS group, SIS-Hst1(L) group, and SIS-Hst1(H) group. Figure 3 A). Counting the number of cells in the field of view showed that the number of cells in the SIS group, SIS-Hst1(L) group, and SIS-Hst1(H) group was statistically different from that in the control group, but there was no statistically significant difference in the number of cells in the SIS group, SIS-Hst1(L) group, and SIS-Hst1(H) group.

[0193] CCK-8 cell counting analysis is used to assess cell proliferation over 7 days. Figure 3 B). The proliferation of other cells was enhanced compared with the control group, but there was no statistically significant difference in cell proliferation between the SIS-Hst1(L) group, the SIS-Hst1(H) group and the SIS matrix group.

[0194] The positive stimulatory effect of the carrier matrix on cell proliferation may be related to the growth factors present in the carrier matrix, including transforming growth factor β, basic fibroblast growth factor (FGF), hepatocyte growth factor, and vascular endothelial growth factor (VEGF), which are retained in the carrier matrix in their bioactive form after sterilization. These growth factors, along with collagen, proteins, and other components, enhance cell adhesion and activate signaling to influence cell differentiation and regeneration potential. There was no statistically significant difference in the effect of introducing histone-1-rich protein on cell proliferation between the SIS-Hst1(L) and SIS-Hst1(H) groups and the control group containing only the SIS matrix.

[0195] The antibacterial activity experiment showed that the SIS group, SIS-Hst1(L) group, and SIS-Hst1(H) group all had certain antibacterial effects. Figure 3 (C, D, E) The antibacterial area of ​​the SIS-Hst1(L) and SIS-Hst1(H) groups was slightly larger than that of the SIS group, but there was no statistically significant difference. BHI agar plates ( Figure 3 D) and fluorescent live / dead staining images ( Figure 3 The staining results of colony formation on E) were similar, with both the SIS-Hst1(L) and SIS-Hst1(H) groups exhibiting antibacterial activity. It can be inferred that histone-rich groups may have a certain synergistic effect, but this synergistic effect is weak. In conclusion, the results indicate that the medical hydrogel is biocompatible.

[0196] 3. Cell adhesion and migration

[0197] 3.1 Adhesion ability

[0198] Human oral epithelial cells were resuspended in serum-free culture medium, and the cell suspension density was adjusted to 1×10⁻⁶. 6 Cells / mL. 100 μL of human oral epithelial cell suspension was placed in the upper chamber of a Transwell. The medical hydrogel of Example 1 (SIS-Hst1(H) group), the medical hydrogel of Example 2 (SIS-Hst1(L) group), and the SIS matrix were added to the wells of the lower chamber along with DMEM and 10% fetal bovine serum, respectively. The control group used only DMEM and 10% fetal bovine serum. After 24 hours, migrating cells were collected from the lower chamber, stained with crystal violet, and observed under a microscope. Cells were then randomly selected in the field of view for counting using ImageJ. The results are shown below. Figure 4 As shown.

[0199] 3.2 Cytoskeleton and Cell Morphology

[0200] Human oral epithelial cells were cultured in 24-well plates containing titanium sheets and fixed with 4% paraformaldehyde. They were then dehydrated in a gradient of ethanol solutions (30%, 50%, 75%, 90%, 95%, and 100%). Cell morphology on the titanium sheets was imaged using scanning electron microscopy (SEM), and the results are shown below. Figure 4 As shown.

[0201] After 12 hours of culture, the cytoskeleton actin was stained, fixed with 4% paraformaldehyde at 4°C, and infiltrated with 0.25% Triton X-100 (Solarbio). Under light protection, the cell nuclei were stained with DAPI (Thermo Fisher Scientific) and actin labeled with rhodamine B (Thermo Fisher Scientific) and phalloidin. Images were taken using a confocal laser scanning microscope (CLSM; Zeiss, Baden-Württemberg, Germany).

[0202] 3.3 Results of Cell Adhesion and Migration

[0203] Medical hydrogels promote cell migration ( Figure 4 (A, B). Through counting and observation, the SIS-Hst1(H) group showed a stronger effect on cell migration compared to other groups. Histone 1 enrichment enhanced cell migration by activating the RIN2 / Rab5 / Rac1 signaling pathway, promoting effective healing of oral wounds, which may be related to vascular endothelial growth factor receptor. Histone 1 enrichment promoted cell migration on tissues and titanium sheets, which is beneficial for tissue regeneration and subsequent cell adhesion on titanium.

[0204] Compared with the control group, the SIS matrix group showed significantly more pseudopodia. Figure 4 C). This indicates that the SIS matrix has a certain promoting effect on cell adhesion. The SIS matrix is ​​a hydrogel with collagen as its main component, which can effectively improve cell affinity and promote early cell adhesion and elongation. Cells on the surface of the SIS-Hst1(H) and SIS-Hst1(L) groups showed more pronounced plate-like and filamentous feet. Figure 4 C), exhibiting stronger cell biological activity. This indicates that histone-rich 1 can promote the extension of pseudopodia, which may be a dynamic change in cell surface expansion and an early change in cell migration, demonstrating that histone-rich 1 also has a certain influence on cell motility. Therefore, cell adhesion in the SIS-Hst1(H) group and the SIS-Hst1(L) group was superior to that in the SIS matrix group, confirming the synergistic effect of the two materials. In addition, cytoskeleton morphology ( Figure 4 D) Consistent with scanning electron microscope (SEM) data.

[0205] 4. Protein Expression 4.1 Immunofluorescence Staining

[0206] Human oral epithelial cells were seeded in 24-well plates containing the medical hydrogel of Example 1 (SIS-Hst1(H) group), the medical hydrogel of Example 2 (SIS-Hst1(L) group), and SIS matrix. The control group served as a blank. Cells were fixed in 4% formaldehyde and infiltrated with 0.5% (v / v) Triton X-100. After blocking with 5 mg / mL BSA solution, the cells were mixed with primary antibody (Abcam, UK) and incubated overnight. The cells were protected from light, and then incubated with secondary antibody (Abcam, UK) at 37°C for 2 hours. Cell nuclei were stained with DAPI (Abcam, UK) solution. The stained cells were then observed and analyzed using CLSM to determine the localization and expression of laminin 5, integrin α6, and integrin β4 in human oral epithelial cells. The results are as follows: Figure 5 As shown.

[0207] 4.2 Western blot detection

[0208] The expression levels of laminarin 5, integrin-α6, and integrin-β4 in human oral epithelial cells were detected using Western blotting. Total protein content was determined using a Bradford assay kit after protein sample preparation. Samples were adjusted for concentration, subjected to polyacrylamide gel electrophoresis, transferred to a membrane, blocked, and incubated at 4°C with 1:1000 primary antibody (Abcam, UK) and the corresponding 1:5000 secondary antibody (Abcam, UK). Proteins were finally detected using Pierce ECL Western blot substrate (Mersey & Company, USA). Quantitative analysis was performed using ImageJ software, and the relative expression levels of each target protein were normalized to the intensity of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band. Results are shown below. Figure 6 As shown.

[0209] 4.3 Real-time quantitative polymerase chain reaction (qRT-PCR)

[0210] To target the expression of laminin 5, integrin-α6, and integrin-β4, human oral epithelial cells were seeded in six-well plates containing medical hydrogels (SIS-Hst1(H) group from Example 1), medical hydrogels (SIS-Hst1(L) group from Example 2), and SIS matrix. A control group served as the blank. Human oral epithelial cell suspensions were extracted, and RNA was extracted using TRIzol. The RNA was then dissolved in an appropriate amount of enzyme-free (DEPC) water, and the RNA concentration was measured. A GoScript reverse transcriptase reaction system was prepared, and reverse transcription was performed at 50°C followed by inactivation at 85°C. cDNA was obtained and subjected to real-time quantitative PCR using a Roche LC480II system (Roche, Switzerland). The results were calculated using the ΔΔCt method, and the results are shown below. Figure 6As shown in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control.

[0211] Table 1

[0212]

[0213] 4.4 Protein Expression Results

[0214] like Figure 5 As shown, immunofluorescence was used to detect the expression of hemidesmosome (HDs)-related proteins laminin 5, integrin-α6, and integrin-β4 in human oral epithelial cells. Laminin 5 was mainly distributed in the cytoplasm, while integrin-α6 was mainly distributed on the cell membrane surface. Specifically, CLSM was used to observe and analyze laminin-α6 (…). Figure 5 A) Integrin-β4 ( Figure 5 B), Adhesionin 5 ( Figure 5 C) Localization and expression in oral epithelial cells (red represents proteins, blue represents cell nuclei and synthesis results). Immunofluorescence staining revealed that laminin 5, integrin-α6, and integrin-β4 were widely distributed in the cells of the SIS-Hst1(H) and SIS-Hst1(L) groups, with higher intensities than the control group. This indicates that the medical hydrogel can effectively promote the expression of laminin 5, integrin-α6, and integrin-β4 in epithelial cells.

[0215] Western blotting (US) Figure 6 A, B) also confirmed this. Therefore, we examined the expression of laminin 5, integrin-α6, and integrin-β4 in the lamina of human oral epithelial cells. The expression of laminin 5, integrin-α6, and integrin-β4 in both the SIS-Hst1(H) and SIS-Hst1(L) groups was higher than that in the control group, with the SIS-Hst1(H) group showing the best expression, indicating that the medical hydrogel can promote the expression of hemidesmosome (HDs)-related factors. Integrin-α6 and integrin-β4 are key components of transmembrane hemidesmosomes (HDs), and the intermediate adhesin connects the basal and basal layers of keratinocytes, forming the main adhesion structure and regulating laminin 5. The ligand (LG) domain of laminin 5 interacts with integrin-α6 and integrin-β4, and basal keratinocytes adhere to the inner basal layer (IBL) connecting the enamel through this structure.

[0216] Upregulated expression levels of integrin α6 and integrin-β4 in human oral epithelial cells demonstrate enhanced cell adhesion. Integrin-α6 and integrin-β4 not only support the adhesion of basal keratinocytes but also maintain cell proliferation. Simultaneously, the combined signaling of integrin and growth factors activates focal adhesion kinase (FAK), whose function is related to wound healing, including the formation, migration, proliferation, survival, and expression of the polarized lamellipodia layer.

[0217] Furthermore, the results of real-time quantitative PCR showed increased expression of laminin 5, integrin-α6, and integrin-β4, consistent with the results of immunofluorescence and Western blotting. Figure 6 C).

[0218] 6. Animal experiments

[0219] Animal experiments were approved by the Animal Ethics Committee of Tianjin Medical University. The medical hydrogel SIS-Hst1(H) and SIS matrix from Example 1 were selected as experimental groups. Eight-week-old SD rats were anesthetized by inhalation and intraperitoneal anesthesia, and the right upper first molar was extracted. Twenty-four rats were randomly divided into three groups: a blank implant group, a SIS matrix group, and a SIS-Hst1(H) group, and implants were then implanted in each group. Immediately after implantation, the SIS matrix group and the SIS-Hst1(H) group were injected with SIS matrix and SIS-Hst1(H) into the interface between the implant and soft tissue using a syringe. Injections were given every two days, and gingival specimens were collected at the implant-epithelial junction one week later. Sections were then stained with hematoxylin and eosin (HE) and immunofluorescence. The stained sections were photographed using a fully automated quantitative pathological imaging system (Vectra Polaris) (PerkinElmer, USA) and visualized using NDP.view 2 Inc. software. Results... Figure 7 As shown.

[0220] If the peri-implant epithelium does not integrate well with the implant, inflammation will quickly affect surrounding tissues, including the underlying bone. Simultaneously, the mucosa will become painful and discolored, doubling the damage to function and aesthetics after implantation. The peri-implant epithelium is more similar to the connective epithelium of a natural tooth in many ways; the tighter its integration with the implant, the stronger its resistance to external toxic substances and the better its soft tissue seal.

[0221] Depend on Figure 7 It can be seen that after the first molar is extracted and the implant is placed, the morphology of the oral junctional epithelium (JE) and peri-implant epithelium (PIE) in rats was initially observed. Figure 7A). Epithelial regeneration was observed one week post-surgery, and a thin epithelial layer was observed to form around the implants in all groups. The histological morphology of the SIS-Hst1(H) group was closer to that of natural teeth. The oral junctional epithelium of natural teeth is a stratified squamous non-keratinized epithelium, composed only of the basal layer and the suprabasal layer. The basal cell layer of the SIS-Hst1(H) group consisted of 3-5 layers of flattened cells, more than the SIS group and the control group, and its morphology was similar to that of the oral junctional epithelium. All remaining cells in the suprabasal layer were flattened, parallel to the tooth surface, and very similar to each other. The suprabasal cells of the implant periimplantation group in the control group were not flat enough, possibly due to some inflammatory infiltration. The SIS group and the SIS-Hst1(H) group were superior to the control group, which may be related to the antibacterial properties of the SIS matrix. The suprabasal cells of the SIS-Hst1(H) group were closest to those of natural teeth. In addition, in terms of overall morphology, both the SIS-Hst1(H) group and the oral junctional epithelium gradually tapered towards the apex. Based on in vitro results, the medical hydrogel of the present invention promotes healing and epithelial adhesion while reducing inflammatory infiltration, playing a synergistic role in healing and enabling the peri-implant epithelium of rats treated with the medical hydrogel to have a shape closest to that of natural teeth.

[0222] Immunofluorescence staining was used to detect the expression and distribution of peri-implant hemidesmosomes (HDs)-related proteins. Figure 7 B). Hemidesmosomes (HDs) are crucial for epithelial attachment stability. Laminin 5 is a non-extracellular component of hemidesmosomes (HDs), while integrin-α6 and integrin-β4 are primarily transmembrane proteins. Hemidesmosome (HD) formation can be inferred by measuring the expression of these key proteins. After one week, laminin 5 expression was detected in both the basal and supra-basal layers of the peri-implant epithelium, with SIS-Hst1(H) showing significant expression in both the inner and outer layers. Laminin 5 plays a role in guiding epithelial healing, and its higher expression also represents increased epithelial migration and re-epithelialization. Fluorescent bands were generally observed in the SIS-Hst1(H) group, with integrin-α6 showing more pronounced expression in the middle region and integrin-β4 showing more pronounced expression in the lower middle region. Overall, the SIS-Hst1(H) group showed higher protein expression levels. Therefore, it can be concluded that medical hydrogels can promote the formation of peri-implant hemidesmosomes (HDs), thus providing a potential therapeutic opportunity to encourage peri-implant soft tissue closure.

[0223] All experiments in this invention included at least three replicates (n≥3), and each experiment was repeated at least three times. One-way ANOVA and independent samples t-tests were used to analyze the data for each group. Variables are expressed as mean standard deviation (SD), and p<0.05 was considered statistically significant.

[0224] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0225] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A medical hydrogel, characterized in that, include: The carrier matrix is ​​derived from the submucosa of the small intestine; as well as, The active ingredient, which is modified on the surface and / or inside the carrier matrix, and, The active ingredient is a polypeptide compound, which includes one or both of histone-rich 1 and histone-rich 5. The medical hydrogel is formed by cross-linking the active ingredients onto the surface and / or interior of a carrier matrix; The medical hydrogel is in a flowable state at 15-25℃; It exhibits a semi-solid state at 36-38℃; The preparation method of the medical hydrogel includes the following steps: After digesting the submucosa of the small intestine with acidic protease, a carrier digestion solution was obtained. The carrier digest solution was freeze-dried to obtain the carrier matrix; The carrier matrix is ​​mixed with a polypeptide compound in a solvent to obtain a reaction precursor; In the presence of a crosslinking agent, the carrier matrix and the polypeptide compound undergo a crosslinking reaction to obtain a reaction product; After removing the crosslinking agent, the pH value is adjusted to neutral to form a gel product; The crosslinking agent includes one or more of N-hydroxysuccinimide, carbodiimide, carbodiimide / N-hydroxysuccinimide, and genipin.

2. A method for preparing a medical hydrogel according to claim 1, characterized in that, The preparation method includes the following steps: After digesting the submucosa of the small intestine with acidic protease, a carrier digestion solution was obtained. The carrier digest solution was freeze-dried to obtain the carrier matrix; The carrier matrix is ​​mixed with a polypeptide compound in a solvent to obtain a reaction precursor; In the presence of a crosslinking agent, the carrier matrix and the polypeptide compound undergo a crosslinking reaction to obtain a reaction product; After removing the crosslinking agent, the pH value is adjusted to neutral to form a gel product; The crosslinking agent includes one or more of N-hydroxysuccinimide, carbodiimide, carbodiimide / N-hydroxysuccinimide, and genipin.

3. The preparation method according to claim 2, characterized in that, The digestion process is carried out under acidic conditions.

4. The preparation method according to claim 3, characterized in that, The digestion process is carried out under acidic conditions by using an acidic substance; and / or the digestion process lasts for 24-72 hours and is carried out at a temperature of 23-28°C.

5. The preparation method according to any one of claims 2-4, characterized in that, In the reaction precursor, the concentration of the carrier matrix is ​​1-100 mg / ml, and the mass concentration of the polypeptide compound is 0.0001 mg / ml-0.1 mg / ml.

6. The preparation method according to any one of claims 2-4, characterized in that, The cross-linking reaction takes 3-9 hours.

7. The preparation method according to any one of claims 2-4, characterized in that, The crosslinking agent is removed by dialysis.

8. The preparation method according to claim 7, characterized in that, The dialysis time is 12-36 hours.

9. Use of the medical hydrogel according to claim 1 in the preparation of implant sealant or implant lubricant.

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