Modified collagen, precursor thereof, collagen gel containing same, composite hydrogel containing same and application thereof

Abstract

The invention discloses modified collagen, a precursor of the modified collagen, collagen gel containing the modified collagen, composite hydrogel containing the modified collagen and application of the modified collagen. Wherein the modified collagen has a structure as shown in the following formula: (3, 4-L-dihydroxyphenylalanine) n-collagen; wherein n is an integer greater than 1. The composite hydrogel prepared from the modified collagen and the biological bone meal has biocompatibility and a repairing effect.

Description

Technical Field

[0001] The present application relates to the field of biomedical materials, and in particular to a modified collagen, its precursor, a collagen gel containing the modified collagen, a composite hydrogel containing the modified collagen, and uses thereof. Background Art

[0002] The repair of oral tissue defects is a common problem in clinical practice. Known oral repair products, such as oral repair membranes composed of an upper membrane, a middle membrane and a lower membrane that are bonded in sequence, and oral bone regeneration and repair systems that rely on three-dimensional models and 3D printing technology, have many limitations, such as 1) Poor biocompatibility: Many existing materials may cause inflammatory reactions or foreign body reactions in host tissues, limiting their long-term stability and success rate; 2) Insufficient mechanical properties: Some repair materials may lack the mechanical strength and toughness that match natural teeth or periodontal tissues, resulting in fragile or easy-to-wear restorations; 3) Limited regeneration capacity: Traditional materials often only play a filling role and lack the ability to promote the regeneration of periodontal tissues (such as alveolar bone and periodontal membrane); 4) Adaptability issues: Existing repair materials may be difficult to adapt to different oral environments and patient needs, and lack personalized treatment plans; 5) Operational complexity: Some materials are complex to operate in clinical applications and require specific techniques or equipment, which increases the difficulty and time of treatment and is difficult to mass produce.

[0003] The emerging oral regenerative injectable gel is a biomaterial used for periodontal tissue repair and regeneration. It belongs to the field of injectable self-healing hydrogel technology. Its development and application is an important branch of tissue engineering. It is known that some of these products use aldehyde-modified hyaluronic acid to prepare hydrogels, and use hydrogels to regulate the release of drugs to treat periodontal tissue defects. However, this method needs to be used in combination with other drugs and does not have much repair and regeneration effect.

[0004] Therefore, there is an urgent need for oral repair and regeneration products that are both biocompatible and have a restorative effect. Summary of the invention

[0005] Based on this, it is necessary to provide at least a modified collagen, its precursor, a collagen gel containing it, a composite hydrogel containing it and its use.

[0006] In the first aspect of the present application, a modified collagen precursor is provided, which has a structure as shown in the following formula:

[0007] Tyrosine m-collagen; wherein m is an integer greater than 1.

[0008] In a second aspect of the present application, a modified collagen is provided, which has a structure as shown in the following formula: (3,4-L-dihydroxyphenylalanine)n-collagen; wherein n is an integer greater than 1.

[0009] In the third aspect of the present application, a nucleic acid molecule is provided, which encodes the modified collagen precursor as described in the first aspect.

[0010] In the fourth aspect of the present application, a gene expression cassette is provided, which contains a promoter, the nucleic acid molecule as described in the third aspect, and a terminator.

[0011] In the fifth aspect of the present application, a recombinant vector is provided, which comprises a backbone vector and one or more of the following items:

[0012] (I) the nucleic acid molecule as described in the third aspect; and

[0013] (II) The gene expression cassette as described in the fourth aspect.

[0014] In the sixth aspect of the present application, a recombinant cell is provided, which comprises one or more of the nucleic acid molecule described in the third aspect, the gene expression cassette described in the fourth aspect, and the recombinant vector described in the fifth aspect.

[0015] In a seventh aspect of the present application, a method for preparing a collagen gel is provided, comprising the following steps:

[0016] Mixing the modified collagen described in the second aspect with a solution containing magnesium ions, stirring and reacting to obtain a collagen peptide magnesium chelate;

[0017] The solution containing the collagen peptide magnesium chelate is mixed with a cross-linking agent to perform a cross-linking reaction, thereby obtaining a solution containing a collagen gel.

[0018] In an eighth aspect of the present application, a collagen gel prepared by the preparation method described in the seventh aspect is provided.

[0019] In a ninth aspect of the present application, a method for preparing a composite hydrogel is provided, comprising the following steps:

[0020] The collagen gel described in the eighth aspect is mixed with biological bone powder.

[0021] In the tenth aspect of the present application, a composite hydrogel prepared by the preparation method described in the ninth aspect is provided.

[0022] In the eleventh aspect of the present application, there is provided use of the modified collagen precursor of the first aspect, the modified collagen of the second aspect, or the collagen gel of the eighth aspect in the preparation of a product for oral repair and regeneration.

[0023] In the twelfth aspect of the present application, a method for repairing the oral cavity is provided, the method comprising: applying the composite hydrogel described in the tenth aspect to the oral cavity of a subject.

[0024] The inventors independently developed a novel oral repair and regeneration gel prepared by using DOPA-modified recombinant collagen, magnesium ions and biological bone powder:

[0025] (1) Improve biocompatibility: By using DOPA-modified recombinant collagen and magnesium ions, the compatibility of the material with the host tissue is improved.

[0026] (2) Enhanced mechanical properties: Combined with bone powder material components, the mechanical strength and toughness of the repair material are enhanced to better withstand the pressure of the oral environment.

[0027] (3) Promote tissue regeneration: The use of DOPA-modified recombinant collagen combined with magnesium ions and bone powder can effectively promote the repair and regeneration of periodontal tissues, especially the alveolar bone and periodontal ligament.

[0028] (4) Improve adaptability and personalization: Develop restorative materials that are easy to adjust and adapt to the needs of different patients and provide personalized treatment plans.

[0029] (5) Simplify clinical operations: Design materials that are easy to operate and apply to reduce the complexity and time of clinical treatment.

[0030] (6) Improve stability and durability: Improve stability and durability in the oral environment by optimizing material formulation and cross-linking technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0032] Figure 1 Display product photos.

[0033] Figure 2 This is the cell morphology diagram of cytotoxicity detection experimental group 1.

[0034] Figure 3 This is the cell morphology diagram of cytotoxicity detection experimental group 2.

[0035] Figure 4 This is the cell morphology diagram of group 3 of the cytotoxicity detection experiment.

[0036] Figure 5 This is the cell morphology diagram of group 4 of the cytotoxicity detection experiment.

[0037] Figure 6 This is the cell morphology diagram of group 5 in the cytotoxicity detection experiment.

[0038] Figure 7 This is the cell morphology diagram of group 6 in the cytotoxicity detection experiment.

[0039] Figure 8 This is the cell morphology diagram of group 7 in the cytotoxicity detection experiment.

[0040] Fig. 9 The mandibular periodontal defect model was established in rats (experimental group).

[0041] Fig.10 These are the results of Masson staining of the test group (NB refers to new bone) in the 6th week of the mandibular periodontal defect experiment in rats (experimental group).

[0042] Fig.11 These are the results of Masson staining of the control group (control group) obtained after 6 weeks of the mandibular periodontal defect experiment in rats. DETAILED DESCRIPTION

[0043] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0045] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0046] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0047] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0048] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0049] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0050] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0051] In this application, unless otherwise specified, the term "between" used in numerical ranges does not include the number itself. For example, "between 1 and 9" does not include the numbers 1 and 9.

[0052] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0053] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0054] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0055] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0056] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0057] As a natural polymer material, collagen has good biocompatibility and biodegradability and has been widely studied for use in tissue engineering. The mechanism of action of type I collagen on bone repair in oral repair involves multiple aspects, including its role as a component of the extracellular matrix, promotion of cell proliferation and differentiation, and interaction with growth factors. However, simple collagen gels have limitations in terms of mechanical strength and promotion of hard tissue regeneration.

[0058] Bone powder is also one of the important materials for periodontal regeneration treatment, playing a key role in repairing alveolar bone defects, promoting osteogenic differentiation of stem cells, and improving treatment effects. Studies have shown that bone powder is effective in indications such as vertical alveolar bone resorption, but has limited effects on patients with horizontal or severe resorption. Using bone powder alone for treatment requires doctors to conduct a comprehensive evaluation to determine the feasibility.

[0059] In addition, in recent years, research on magnesium ion repair has increased year by year, and studies have shown that magnesium ions play an important role in periodontal repair and regeneration. According to research from the Li Ka Shing Faculty of Medicine at the University of Hong Kong, magnesium ions significantly promote new bone growth in the early stages of bone repair (1 to 7 days after surgery), which can promote osteoblast activity and immune response.

[0060] Novel hydrogels combining type I collagen, bone powder, and magnesium ions (e.g., injectable hydrogels) show great potential for application in periodontal tissue repair and regeneration.

[0061] In a first aspect of the present application, a modified collagen precursor is provided, which has a structure as shown in the following formula: tyrosine m-collagen; wherein m is an integer greater than 1.

[0062] In some embodiments, m is an integer between 1 and 9, such as 2, 3, 4, 5, 6, 7 or 8.

[0063] In the present application, m tyrosines are operably linked to the collagen.

[0064] Unless otherwise specified, the term "operably linked" in the present application refers to the functional relationship between two regions of the modified collagen precursor, namely m tyrosines and the collagen; wherein the two regions are linked to produce the modified collagen precursor.

[0065] In some embodiments, different regions are connected by a linker, such as a flexible linker, to reduce mutual interference between the regions.

[0066] In some embodiments, the amino acid sequence of the modified collagen precursor comprises the sequence shown in SEQ ID NO: 6. In some embodiments, the amino acid sequence of the modified collagen precursor is shown in SEQ ID NO: 6.

[0067] In some embodiments, the amino acid sequence of the modified collagen precursor comprises the sequence shown in SEQ ID NO: 7. In some embodiments, the amino acid sequence of the modified collagen precursor is shown in SEQ ID NO: 7.

[0068] In some embodiments, the amino acid sequence of the modified collagen precursor comprises the sequence shown in SEQ ID NO: 8. In some embodiments, the amino acid sequence of the modified collagen precursor is shown in SEQ ID NO: 8.

[0069] In a second aspect of the present application, a modified collagen is provided, which has a structure as shown in the following formula: (3,4-L-dihydroxyphenylalanine)n-collagen; wherein n is an integer greater than 1.

[0070] In some embodiments, the modified collagen is obtained by oxidizing the modified collagen precursor described in the first aspect.

[0071] In some embodiments, the oxidation method comprises mixing the modified collagen precursor with tyrosinase to obtain the modified collagen.

[0072] 3,4-L-dihydroxyphenylalanine (3,4-dihydroxy-L-phenylalanine), referred to as DOPA amino acid, is widely present in the adhesive proteins secreted by mussels. The catechol (i.e., catechol) in DOPA can be covalently and non-covalently bound to the surface of various materials, and is an important means for mussels to attach to surfaces such as reefs and ships. The catechol group and metal ions quickly form a stable coordination binding effect when they are simply in contact in water. Therefore, DOPA-modified collagen quickly forms a stable coordination binding effect with magnesium ions and calcium ions in biological bone powder when they are simply in contact in water, promoting the enrichment of magnesium ions and calcium ions on the surface of the material, so that the concentration of magnesium ions and calcium ions reaches a supersaturated state, providing nucleation sites for apatite, which is conducive to its formation on the surface of bone repair materials, thereby achieving a triple-effect bone repair effect.

[0073] In some embodiments, n is an integer between 1 and 9, such as 2, 3, 4, 5, 6, 7 or 8.

[0074] The collagen in the present application may be a collagen commonly used in the art. Exemplarily, the collagen is type I collagen, which is the most widely distributed collagen in tissues, secreted by osteoblasts, and participates in the development, differentiation and activity regulation of osteoblasts, and plays an important role in maintaining the biomechanical properties and structural integrity of bone tissue. At present, type I collagen has been widely used in bone tissue engineering repair, such as guided bone tissue regeneration, guided periodontal tissue regeneration, hard bone, cartilage repair materials and tissue engineering scaffold structures. Type I collagen is the main component of the extracellular matrix of healthy bone tissue, and plays a vital role in the osteogenic differentiation, proliferation of bone marrow mesenchymal stem cells and the development of bones.

[0075] In some embodiments, the collagen is a collagen with a molecular weight of 10KD to 50KD. For example, the molecular weight of the collagen in the present application may be 10KD, 15KD, 20KD, 25KD, 30KD, 35KD, 40KD, 45KD, 50KD, or a range or value between any two values.

[0076] In some embodiments, the amino acid sequence of the collagen comprises the sequence shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the collagen comprises the sequence shown in SEQ ID NO: 1.

[0077] In some embodiments, the nucleic acid sequence encoding the collagen protein comprises a sequence as shown in SEQ ID NO: 5. In some embodiments, the nucleic acid sequence encoding the collagen protein is as shown in SEQ ID NO: 5.

[0078] In the present application, n 3,4-L-dihydroxyphenylalanine is operably linked to the collagen.

[0079] In some embodiments, different regions are connected by a linker, such as a flexible linker, to reduce mutual interference between the regions.

[0080] The flexible linker in the present application may be, for example, GGGGS (Gly-Gly-Gly-Gly-Ser) or GGGGG (Gly-Gly-Gly-Gly-Gly).

[0081] In the third aspect of the present application, a nucleic acid molecule is provided, which encodes the modified collagen precursor as described in the first aspect.

[0082] In some embodiments, the nucleic acid sequence of the nucleic acid molecule has a nucleic acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0083] In some embodiments, the nucleic acid molecule encodes the same modified collagen precursor as the nucleic acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, but has a nucleic acid sequence different from the nucleic acid sequence shown in SEQ ID NO: 2 due to the degeneracy of the genetic code.

[0084] In some embodiments, the nucleic acid molecule hybridizes with the nucleic acid molecule described above under stringent conditions and encodes the modified collagen precursor described in the first aspect.

[0085] In some embodiments, the nucleic acid molecule is a nucleic acid sequence obtained by replacing, deleting or adding one or more nucleotides of the above nucleic acid molecule, and has the same or similar functions as the above nucleic acid molecule.

[0086] In the present application, unless otherwise specified, "nucleic acid" has the commonly known meaning in the technical field, also known as "polynucleotide", which is a molecule formed by multiple nucleotide monomers.

[0087] In the fourth aspect of the present application, a gene expression cassette is provided, which contains a promoter, the nucleic acid molecule as described in the third aspect, and a terminator.

[0088] In the fifth aspect of the present application, a recombinant vector is provided, which comprises a backbone vector and one or more of the following items:

[0089] (I) the nucleic acid molecule as described in the third aspect; and

[0090] (II) The gene expression cassette as described in the fourth aspect.

[0091] In the sixth aspect of the present application, a recombinant cell is provided, which comprises one or more of the nucleic acid molecule described in the third aspect, the gene expression cassette described in the fourth aspect, and the recombinant vector described in the fifth aspect.

[0092] In a seventh aspect of the present application, a method for preparing a collagen gel is provided, comprising the following steps:

[0093] Mixing the modified collagen described in the second aspect with a solution containing magnesium ions, stirring and reacting to obtain a collagen peptide magnesium chelate;

[0094] The solution containing the collagen peptide magnesium chelate is mixed with a cross-linking agent to perform a cross-linking reaction, thereby obtaining a solution containing a collagen gel.

[0095] In the present application, the magnesium source of the magnesium ion can be conventional in the art.

[0096] In some embodiments, the magnesium source is selected from the group consisting of magnesium chloride, magnesium sulfate, magnesium citrate, magnesium glycinate and magnesium malate. Exemplarily, the magnesium source is magnesium chloride or magnesium sulfate.

[0097] In some embodiments, the stirring reaction time is 24h~48h, and the stirring reaction temperature is 0°C~4°C. In some embodiments, the stirring reaction time is 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, 48h, or a range or value between any two values.

[0098] In some embodiments, the concentration of magnesium ions in the solution containing magnesium ions is less than 100mM. In some embodiments, the concentration of magnesium ions in the solution containing magnesium ions is 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, or a range or value between any two values. In some embodiments, the concentration of magnesium ions in the solution containing magnesium ions is 10mM~70mM.

[0099] In some embodiments, the pH value of the solution containing magnesium ions is controlled to be 7 to 8. Exemplarily, the pH value is 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, or a range or value between any two values. In some embodiments, the pH value is 7.4.

[0100] In some embodiments, the preparation of the solution containing magnesium ions comprises dissolving a magnesium source in a buffer such as water, PBS or sodium citrate.

[0101] After the modified collagen is mixed with a solution containing magnesium ions, the concentration of the modified collagen in the mixed solution is 2 wt% to 5 wt%. In some embodiments, the concentration of the modified collagen in the mixed solution is 2 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, or any range or value between any two values.

[0102] The cross-linking agent used in the present application may be conventional in the art. The use of the cross-linking agent can further modify the modified collagen, and its function is at least to improve the stability and durability of the collagen in the oral environment.

[0103] Exemplary cross-linking agents include, but are not limited to, formaldehyde, glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS), and genipin, etc.

[0104] Depending on the cross-linking agent, the relative amount of the cross-linking agent and the solution containing the magnesium chelate of collagen peptides may vary. In some embodiments, the relative amount of the cross-linking agent and the solution containing the magnesium chelate of collagen peptides is (0.05-0.2):100 (weight ratio).

[0105] In some embodiments, the cross-linking agent used is EDC / NHS.

[0106] In some embodiments, the amount of EDC / NHS is:

[0107] The weight ratio of the EDC to the solution containing the collagen peptide magnesium chelate is 0.1:100, and the weight ratio of the EDC to the NHS is 1:0.05.

[0108] In some embodiments, the cross-linking agent used is glutaraldehyde.

[0109] In some embodiments, the weight ratio of the glutaraldehyde to the solution containing the collagen peptide magnesium chelate is 0.05:100.

[0110] In some embodiments, the cross-linking agent used is genipin.

[0111] In some embodiments, the weight ratio of the genipin to the solution containing the collagen peptide magnesium chelate is 0.15:100.

[0112] The temperature and time for the cross-linking reaction can be conventional in the art, as long as the cross-linking agent can exert its effect.

[0113] In some embodiments, the cross-linking reaction time is 2 hours to 6 hours. For example, the cross-linking reaction time can be 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, or any range or value between any two values.

[0114] In some embodiments, the temperature of the cross-linking reaction is 15° C. to 35° C. For example, the temperature of the cross-linking reaction can be 15° C., 20° C., 25° C., 30° C., 35° C., or any range or value between any two values.

[0115] To ensure the purity of the obtained collagen gel, the method may optionally further include dialyzing the solution containing the collagen gel obtained after cross-linking.

[0116] In some embodiments, the method comprises using water in a volume 10 to 30 times the volume of the solution containing the collagen gel as a dialysate for agitated dialysis; optionally, the agitated dialysis is performed at 0 to 4° C. for 24 to 48 hours.

[0117] Depending on the dialysis effect, the stirring time is also different. In some embodiments, the stirring time is 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, 48h, or any range or value between any two values.

[0118] In an eighth aspect of the present application, a collagen gel prepared by the preparation method described in the seventh aspect is provided.

[0119] In a ninth aspect of the present application, a method for preparing a composite hydrogel is provided, comprising the following steps:

[0120] The collagen gel described in the eighth aspect is mixed with biological bone powder.

[0121] Without wishing to be bound by any theory, the use of biological bone powder can at least enhance the mechanical strength and toughness of the composite hydrogel, allowing the composite hydrogel to better withstand the pressure of the oral environment.

[0122] The biological bone powder used in the present application may be conventional in the art.

[0123] In some embodiments, the biological bone powder is prepared by referring to the method disclosed in CN112587729B.

[0124] In some embodiments, the method for preparing the biological bone powder comprises the following steps:

[0125] i) taking animal bone particles, and washing them with isopropanol and purified water by repeated alternating shaking for 3 to 6 times to obtain defatted bone particles, wherein the mass ratio of the animal bone particles to isopropanol is 1:2.5;

[0126] ii) adding the defatted bone particles to hydrogen peroxide, allowing the particles to stand for 12 to 24 hours, discarding the hydrogen peroxide, washing the particles in purified water for 3 to 6 times by shaking, drying the particles at 75 to 100° C. for 5 to 7 hours, and passing the particles through a 14-mesh sieve to obtain sieved bone particles; the particle size distribution range of the bone particles may be 100 to 250 μm.

[0127] iii) adding the sieved bone particles to a 70% to 100% ethylenediamine solution and heating to a slight boil, then maintaining the solution at 100° C. to 125° C. for 7 h to 9 h, discarding the waste liquid, adding the particles to purified water and shaking and washing for 3 times, and repeating step iii) twice;

[0128] iv) The bone particles obtained in step iii) are washed 15 times with purified water and washed twice with injection water, with the washing process taking 3 to 6 minutes per time. During the washing process, the container is turned over and the waste liquid is discarded to obtain biological bone powder.

[0129] In some embodiments, the particle size of the biological bone powder is 100 μm to 250 μm, for example, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, or a range or value between any two values.

[0130] In some embodiments, the weight ratio of the biological bone powder to the collagen gel is 1:2 to 1:10. Exemplarily, the weight ratio may be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or a range or value between any two values. In some embodiments, the weight ratio of the biological bone powder to the collagen gel is 1:5 to 1:8.

[0131] In the step of mixing the collagen gel and the biological bone powder, the biological bone powder is dispersed in the collagen gel and homogenized for 5 minutes (min) to 15 minutes. The homogenization time depends on factors such as the particle size of the biological bone powder and the relative amount of the collagen gel. In some embodiments, the homogenization time is 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, or a range or value between any two values.

[0132] The biological source of the biological bone meal can be conventional in the art, usually mammals, such as large mammals. In some embodiments, the mammal is selected from the group consisting of cattle, sheep, pigs, horses and donkeys. In some embodiments, the biological bone meal is derived from cattle. In some embodiments, the biological bone meal is derived from pigs. In some embodiments, the biological bone meal is derived from sheep.

[0133] In the tenth aspect of the present application, a composite hydrogel prepared by the preparation method described in the ninth aspect is provided.

[0134] The present application creatively combines the original modified collagen, magnesium ions and biological bone powder together, and the prepared composite hydrogel has significant advantages over known similar products, and has both biocompatibility and repair effects. The modified collagen of the present application has a unique amino acid sequence. The researchers unexpectedly found that adding magnesium ions to the modified collagen of the present application before the cross-linking step can form a unique spatial structure of the product, thereby improving the performance of the product. The modified collagen quickly forms a stable coordination binding effect with magnesium ions and calcium ions in biological bone powder in simple contact in water, promoting the enrichment of magnesium ions and calcium ions on the surface of the material, so that the concentration of magnesium ions and calcium ions reaches an oversaturated state, providing nucleation sites for apatite, which is conducive to its formation on the surface of bone repair materials. Combined with a variety of materials that promote periodontal tissue regeneration, it can effectively promote the repair and regeneration of periodontal tissue.

[0135] The composite hydrogel in the present application can be developed into various product forms, which are easy to operate, can reduce the complexity and time of clinical treatment, and can easily adjust the formula ratio to meet the needs of different patients, thereby providing personalized treatment plans. For example, the product form can be an injectable gel.

[0136] In addition, the main components of the composite hydrogel include modified collagen, magnesium ions and biological bone powder, etc., and combined with its gel-like product form, it has good biocompatibility and can effectively promote the adhesion and proliferation of oral tissue cells.

[0137] In the eleventh aspect of the present application, there is provided use of the modified collagen variant as described in the first aspect, the modified collagen as described in the second aspect, the collagen gel as described in the eighth aspect, or the composite hydrogel as described in the tenth aspect in the preparation of a product for oral repair and regeneration.

[0138] The oral repair product may be, for example, a drug containing a composite hydrogel.

[0139] In some embodiments, the product is a composite hydrogel.

[0140] In the twelfth aspect of the present application, a method for repairing the oral cavity is provided, the method comprising: applying the composite hydrogel described in the tenth aspect to the oral cavity of a subject.

[0141] Some examples are provided below.

[0142] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0143] Example 1

[0144] This embodiment provides a method for preparing type I collagen gel for injection containing biological bone powder and magnesium ions, and verifies the effect of the prepared product.

[0145] 1. The preparation method mainly comprises the following steps:

[0146] 1) The nucleic acid encoding the target sequence (M-SEQ ID NO: 7-HHHHHHHH) and the proline hydroxylase gene (P4H) were co-transduced into Pichia pastoris cells (host cells), the fermentation broth was centrifuged and the supernatant was separated and collected, the supernatant was affinity purified by a nickel column to obtain a high-purity target protein with a molecular weight of 30KD, the target protein was mixed with tyrosinase, tyrosinase catalyzed to generate (DOPA) 4 using tyrosine as a substrate, and the protein containing the (DOPA) 4 sequence was obtained after affinity purification by a nickel column again, and finally the DOPA-modified recombinant collagen was obtained after freeze-drying.

[0147] 2) The above-mentioned DOPA-modified recombinant collagen was dissolved in a magnesium chloride solution at pH 7.4 to prepare a 5 wt% DOPA-modified recombinant collagen solution. The stirring temperature during the preparation process was 4° C. and the stirring was continued for 24 h.

[0148] Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) crosslinker and N-hydroxysuccinimide (NHS) catalyst were added to the DOPA-modified recombinant collagen solution, wherein EDC: collagen solution = 0.1:100 (weight ratio), EDC:NHS = 1:0.05 (weight ratio), and the crosslinking reaction was carried out at room temperature for 4 hours to obtain a crude modified recombinant collagen gel.

[0149] The crude modified recombinant collagen gel was placed in a dialysis bag, and 30 times the volume of purified water was used as the dialysis fluid. The bag was stirred at 4° C. for 48 hours to remove the EDC residue, thereby preparing the modified recombinant collagen gel.

[0150] 3) Mix with biological bone meal:

[0151] Biological bone powder with a particle size of 100 microns was selected, and the surface of the bone powder was treated again. The biological bone powder was ultrasonically cleaned in 75% (v / v) ethanol for 10 minutes to remove impurities, and then rinsed with PBS for 3 times and finally dried at 60°C.

[0152] The treated biological bone powder was added to the modified collagen gel in proportion, the weight ratio of biological bone powder to modified recombinant collagen gel was 1:5, and homogenized for 15 minutes to ensure that the bone powder was evenly dispersed to obtain a composite hydrogel.

[0153] Wherein, the preparation method of biological bone powder comprises the following steps:

[0154] i) taking animal bone particles, and washing them with isopropanol and purified water by repeated alternating shaking for 3 to 6 times to obtain defatted bone particles, wherein the mass ratio of the animal bone particles to isopropanol is 1:2.5;

[0155] ii) adding the defatted bone particles to hydrogen peroxide, allowing the particles to stand for 12 to 24 hours, discarding the hydrogen peroxide, placing the particles in purified water for 3 to 6 times of shaking washing, drying the particles at 75 to 100° C. for 5 to 7 hours, and sieving the particles to obtain sieved biological bone particles;

[0156] iii) adding the sieved biological bone particles to a 70%-100% ethylenediamine solution and heating to a slight boil, then maintaining at 100-125° C. for 7-9 hours, discarding the waste liquid, adding purified water and shaking and washing for 3 times, and repeating step iii) twice;

[0157] iv) The bio-bone particles obtained in step iii) are washed 15 times with purified water and washed twice with injection water, with the washing process taking 3 to 6 minutes per time. During the washing process, the container is turned over and the waste liquid is discarded to obtain bio-bone powder.

[0158] The biological bone powder in the following examples is prepared by this method.

[0159] 4) Filling and sterilization

[0160] The composite hydrogel is filled into a pre-filled syringe and then sterilized to prepare a multifunctional oral repair and regeneration gel.

[0161] Part of the multifunctional oral repair and regeneration gel was isolated and prepared into an injectable form ( Figure 1 ).

[0162] Example 2

[0163] 1. The preparation method mainly comprises the following steps:

[0164] 1) The nucleic acid encoding the target sequence (M-SEQ ID NO: 6-HHHHHHHH) and the proline hydroxylase gene (P4H) were co-transduced into Pichia pastoris cells (host cells), the fermentation broth was centrifuged and the supernatant was separated and collected, the supernatant was affinity purified by a nickel column to obtain a high-purity target protein with a molecular weight of 30KD, the target protein was mixed with tyrosinase, tyrosinase catalyzed to generate (DOPA) 2 using tyrosine as a substrate, and the protein containing the (DOPA) 2 sequence was obtained after affinity purification by a nickel column again, and finally the DOPA-modified recombinant collagen was obtained after freeze-drying.

[0165] 2) The DOPA-modified recombinant collagen was dissolved in a magnesium sulfate solution at pH 7.0 to prepare a 4 wt% DOPA-modified recombinant collagen solution. The stirring temperature was 0° C. and the stirring time was 36 h.

[0166] Then, glutaraldehyde cross-linking agent was added to the DOPA-modified recombinant collagen solution, wherein glutaraldehyde: collagen solution = 0.05:100 (weight ratio), and the cross-linking reaction was carried out at room temperature for 6 hours to obtain a crude modified recombinant collagen gel.

[0167] The crude modified recombinant collagen gel was placed in a dialysis bag, and 20 times the volume of purified water was used as the dialysis fluid. The bag was stirred at 0° C. for 36 hours to remove the residual glutaraldehyde, thereby preparing the modified recombinant collagen gel.

[0168] 3) Mix with biological bone meal:

[0169] Biological bone powder with a particle size of 150 μm was selected and the surface of the bone powder was treated again. The biological bone powder was ultrasonically cleaned in 75% (v / v) ethanol for 10 minutes to remove impurities, then rinsed with PBS for 3 times and finally dried at 60°C.

[0170] The treated biological bone powder was added to the modified collagen gel in proportion, the weight ratio of biological bone powder to modified recombinant collagen gel was 1:8, and homogenization was performed for 10 minutes to ensure that the bone powder was evenly dispersed to obtain a composite hydrogel.

[0171] 4) Filling and sterilization

[0172] The composite hydrogel is filled into a pre-filled syringe and then sterilized to prepare a multifunctional oral repair and regeneration gel.

[0173] Example 3

[0174] 1. The preparation method mainly comprises the following steps:

[0175] 1) The nucleic acid encoding the target sequence (M-SEQ ID NO: 8-HHHHHHHH) and the proline hydroxylase gene (P4H) were co-transduced into Pichia pastoris cells (host cells), the fermentation broth was centrifuged and the supernatant was separated and collected, the supernatant was affinity purified by a nickel column to obtain a high-purity target protein with a molecular weight of 30KD, the target protein was mixed with tyrosinase, tyrosinase catalyzed to generate (DOPA) 7 using tyrosine as a substrate, and the protein containing the (DOPA) 7 sequence was obtained after affinity purification by a nickel column again, and finally the DOPA-modified recombinant collagen was obtained after freeze-drying.

[0176] 2) The above-mentioned DOPA-modified recombinant collagen was dissolved in a magnesium sulfate solution at pH 7.0 to prepare a DOPA-modified recombinant collagen solution with a concentration of 4 wt %. During the preparation process, the stirring temperature was 4° C. and the stirring was performed for 48 h.

[0177] Then, glutaraldehyde cross-linking agent was added to the DOPA-modified recombinant collagen solution, wherein genipin:collagen solution=0.15:100 (weight ratio), and the cross-linking reaction was carried out at 20°C for 4 hours to obtain a crude modified recombinant collagen gel.

[0178] The crude modified recombinant collagen gel was placed in a dialysis bag, and 15 times the volume of purified water was used as the dialysis fluid. The mixture was stirred at 2° C. for 30 h to remove the residual genipin, thereby preparing the modified recombinant collagen gel.

[0179] 3) Mix with biological bone meal:

[0180] Biological bone powder with a particle size of 250 μm was selected and the surface of the bone powder was treated again. The biological bone powder was ultrasonically cleaned in 75% (v / v) ethanol for 10 minutes to remove impurities, then rinsed with PBS for 3 times and finally dried at 60°C.

[0181] The treated biological bone powder was added to the modified collagen gel in proportion, the weight ratio of biological bone powder to modified recombinant collagen gel was 1:10, and homogenization was performed for 12 minutes to ensure that the bone powder was evenly dispersed to obtain a composite hydrogel.

[0182] 4) Filling and sterilization

[0183] The composite hydrogel is filled into a pre-filled syringe and then sterilized to prepare a multifunctional oral repair and regeneration gel.

[0184] Effect verification:

[0185] 1. In vitro cytotoxicity test of gel samples with different magnesium ion concentrations: According to steps 1 to 3 in Example 1, different magnesium ion concentrations (magnesium ion concentrations are 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mM) are prepared to obtain different gel samples, 1 g of each sample is taken, the extraction ratio is 0.2 g / ml, the extraction medium is MEM culture medium, and the operation is performed according to the provisions of the Pharmacopoeia 2020 edition to obtain formula gels containing different concentrations of magnesium ions. Compare the relative proliferation rate of fibroblasts in gels with different formulas.

[0186] (1) Experimental steps: L929 (ATCC CCL 1, NCTC clone 929, clone of Strain L) cells in the logarithmic growth phase were digested and collected, and the cell density was adjusted to 1.0×10 5 / mL, seeded into 96-well plates, and cultured in different formulas of gel respectively. The growth of fibroblasts and osteoblasts was observed and compared, and the relative cell proliferation rate was calculated based on the cell proliferation of the blank control.

[0187] (2) Experimental results: The safe survival concentrations of magnesium ions for fibroblasts and osteoblasts are 0 to 100 mM and 0 to 70 mM, respectively. This experiment shows that the growth and development of fibroblasts and osteoblasts can be regulated by regulating the magnesium ion concentration. The total data of the two types of cells show that the cell activity level is higher when the magnesium ion concentration is 10-70 mM. The cell morphology is shown in the figure. Figure 2~Figure 8 .

[0188] Table 1 Summary of in vitro cell experiment results on magnesium ion concentration

[0189]

[0190] 2. Intradermal irritation test

[0191] Samples from experimental group 5 were taken for intradermal reaction test according to the test methods in Part 10: Irritation and skin sensitization test of national standard GB / T 16886.10-2017 “Biological Evaluation of Medical Devices”.

[0192] (1) Preparation of experimental animals: 4 to 24 hours before the test, remove a sufficient area of ​​hair (approximately 10 × 15 cm area) on both sides of the animal's spine on the back to be used as the test and observation site.

[0193] (2) Sample preparation: Take 1 g of sample as the test group and absorbent gauze as the blank control.

[0194] (3) Test steps: Place the test material sample in direct contact with the skin on both sides of the rat spine, and apply the control sample in the same way. Use a bandage to fix the patch for at least 4 hours. Use three rats of similar size and weight for parallel experimental verification.

[0195] (4) Observation results: Observe the skin reaction under natural light or full-spectrum light for 24h, 48h, and 72h. The specific test results are shown in Table 2. The samples in the test group and the control group did not cause erythema and edema on the animal skin surface, and their irritation index was 0, indicating that the samples in the experimental group had high biosafety.

[0196] Table 2 Summary of intradermal irritation test results

[0197]

[0198] 3. Mandibular periodontal defect test in rats

[0199] Six 6-week-old male rats (300g-320g) were divided into two groups. Sample 5 of the experimental group was used as the experimental group, and the control group was the sham operation group. After the rats were anesthetized with 4-5% (v / v) isoflurane, a defect model of 2×3mm (height×width) and 1mm depth was made in the mandibular bone of the rats. Sample 5 of the experimental group was injected into the mandibular periodontal defect of the rats, and the defect of the control group was not treated. After the operation, the regeneration of periodontal tissue was evaluated histologically after 6 weeks of healing. Figure 9~Figure 11 As shown in the figure, according to the Masson staining results of the injured site after 6 weeks, the experimental group had obvious new bone formation compared with the control group, which can effectively promote the repair of the injured site.

[0200] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0201] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A modified collagen precursor, characterized in that: It has the structure shown below: Tyrosine m-collagen; wherein m is an integer greater than 1.

2. A modified collagen, characterized in that: It has the structure shown below: (3,4-L-dihydroxyphenylalanine)n-collagen; wherein n is an integer greater than 1; Optionally, the modified collagen is obtained by oxidizing the modified collagen precursor according to claim 1.

3. The modified collagen precursor according to claim 1 or the modified collagen according to claim 2, characterized in that: It satisfies one or more of the following conditions (1) to (3): (1) m and / or n is an integer between 1 and 9; (2) The collagen is type I collagen; and (3) The collagen has a molecular weight of 10KD to 50KD; Optionally, the amino acid sequence of the collagen comprises the sequence shown in SEQ ID NO: 1; Further optionally, the amino acid sequence of the modified collagen precursor comprises a sequence as shown in any one of SEQ ID NOs: 6 to 8.

4. A nucleic acid molecule encoding the modified collagen precursor according to claim 1 or 3; Optionally, the nucleic acid molecule has one of the following conditions 1) to 4): 1) The nucleic acid sequence thereof comprises the sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; 2) The nucleic acid molecule encodes the same modified collagen precursor as the nucleic acid molecule shown in 1), but has a different nucleic acid sequence from the nucleic acid molecule shown in 1) due to the degeneracy of the genetic code; 3) hybridizes with the nucleic acid molecule defined in 1) or 2) under stringent conditions and encodes the modified collagen precursor as described in claim 1 or 3; as well as, 4) is a nucleic acid sequence obtained by replacing, deleting or adding one or more nucleotides of the nucleic acid sequence shown in any one of 1) to 3), and has the same or similar function as the nucleic acid molecule shown in any one of 1) to 3).

5. A method for preparing collagen gel, characterized in that: It includes the following steps: Mixing the modified collagen as claimed in claim 2 or 3 with a solution containing magnesium ions, stirring and reacting to obtain a collagen peptide magnesium chelate; mixing a solution containing the collagen peptide magnesium chelate with a cross-linking agent to perform a cross-linking reaction to obtain a solution containing a collagen gel; Optionally, the method satisfies one or more of the following conditions (i) to (iii): The magnesium source of the magnesium ion is selected from the group consisting of magnesium chloride, magnesium sulfate, magnesium citrate, magnesium glycinate and magnesium malate; The stirring reaction time is 24h~48h, and the stirring reaction temperature is 0℃~4℃; The concentration of magnesium ions in the solution containing magnesium ions is less than or equal to 100 mM; The pH of the solution containing magnesium ions is controlled to be 7-8; After the modified collagen is mixed with a solution containing magnesium ions, the concentration of the modified collagen is 2wt% to 5wt%; The cross-linking agent is selected from the group consisting of formaldehyde, glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide and genipin; The cross-linking reaction time is 2h~6h, and the cross-linking reaction temperature is 15°C~35°C; and The method also includes dialyzing the solution containing the collagen gel; optionally, the dialysis includes using water with a volume 10 to 30 times the volume of the solution containing the collagen gel as a dialysate for stirred dialysis; the conditions of the stirred dialysis are optionally stirring at 0 to 4°C for 24 to 48 hours.

6. A collagen gel, characterized in that: It is prepared by the preparation method as claimed in claim 5.

7. A method for preparing a composite hydrogel, characterized in that: It includes the following steps: The collagen gel as claimed in claim 6 is mixed with biological bone powder.

8. The preparation method according to claim 7, characterized in that: It meets one or more of the following conditions from i) to iii): i) the particle size of the biological bone powder is 100 μm to 250 μm; ii) the weight ratio of the biological bone powder to the collagen gel is 1:2 to 1:10; and iii) the mixing comprises dispersing the biological bone powder in the collagen gel and homogenizing for 5 minutes to 15 minutes; and iv) The biological bone meal is derived from mammals; optionally, the mammals are selected from the group consisting of cattle, sheep, pigs, horses and donkeys.

9. A composite hydrogel, characterized in that: It is prepared by the preparation method according to claim 7 or 8.

10. Use of the modified collagen precursor according to claim 1 or 3, the modified collagen according to claim 2 or 3, or the collagen gel according to claim 9 in the preparation of a product for oral repair and regeneration; Optionally, the product comprises a composite hydrogel.

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