Maxillofacial hard tissue functional regeneration and repair mineralized hydrogel and preparation thereof

Abstract

The application provides a jaw and face hard tissue functional regeneration and repair mineralized hydrogel and a preparation method thereof, and belongs to the field of bone repair materials. 2+ The mineralized precursor is self-assembled on a template. The application successfully constructs a mineralized precursor which is stable in structure and excellent in biomimetic mineralization activity, and provides a new material for the regeneration and repair of jaw and face hard tissues.

Description

Technical Field

[0001] This invention relates to the field of bone repair materials technology, specifically to a functional regenerative repair mineralized hydrogel for maxillofacial hard tissues, its preparation method, and its application. Background Technology

[0002] The hard tissues of the maxillofacial region (including dental tissues, alveolar bone, and jawbone) are the core support for oral function and facial morphology. Damage to these tissues (such as tooth defects caused by dental caries, alveolar bone defects caused by trauma, and jawbone defects after tumor surgery) not only affects physiological functions such as chewing and speech but can also lead to facial deformities, severely reducing patients' quality of life. Currently, commonly used restorative materials in clinical practice mainly include metal alloys, zirconia ceramics, bone substitutes (such as hydroxyapatite and β-tricalcium phosphate), and natural biomaterials (such as collagen sponges). However, these materials still have significant limitations in their application. Insufficient biocompatibility and tissue integration: Although inorganic materials such as metals and ceramics have high mechanical strength, their chemical composition and structure differ greatly from those of natural hard tissues. Long-term implantation can easily trigger interfacial inflammatory reactions, making it difficult to achieve "bone-like integration" with the host tissue. The degradation rate of some synthetic bone substitutes does not match the rate of new bone formation, which can easily lead to insufficient mechanical support in the repair area in the long term.

[0003] Weak mineralization induction ability: The repair of maxillofacial hard tissues is essentially a process of "mineralization regeneration" (such as hydroxyapatite deposition in dentin and osteoblast-mediated mineralization in alveolar bone). However, existing materials can only provide "physical scaffolds" and cannot simulate the mineralization microenvironment of natural hard tissues. They are difficult to actively induce mineralization regeneration in the defect area, and their repair effect is limited, especially for large-area defects.

[0004] Poor clinical adaptability: Maxillofacial injuries such as tooth defects and alveolar bone defects are often irregular in shape. Traditional prefabricated restorative materials (such as porcelain inlays and metal implant abutments) are difficult to accurately match the contour of the defect, requiring multiple clinical adjustments, which not only prolongs the treatment period but may also damage the surrounding healthy tissue. Some materials (such as collagen sponge) have low mechanical strength and cannot withstand the dynamic load during the chewing process in the maxillofacial region, making them prone to collapse or displacement in the early stages of restoration.

[0005] Insufficient retention of bioactive components: The regeneration of natural hard tissues depends on bioactive molecules (such as dentin sialophosphoprotein and bone morphogenetic protein) in the dentin matrix and bone matrix. However, existing materials either lack such active components or the active components are deactivated during the preparation process due to high temperature, chemical cross-linking and other processes, which cannot effectively regulate the proliferation and differentiation of odontoblasts and osteoblasts.

[0006] Therefore, developing new restorative materials that combine "precise mineralization induction capability, good biocompatibility, adaptation to maxillofacial anatomy, and ability to withstand dynamic loads" has become a key direction for solving the challenges of maxillofacial hard tissue repair. Summary of the Invention

[0007] In view of this, the present invention provides a functional regenerative and repairing mineralized hydrogel for maxillofacial hard tissues and its preparation method, aiming to provide a new material for the regeneration and repair of maxillofacial hard tissues.

[0008] In a first aspect, the present invention provides a functional regenerative and repairing mineralized hydrogel for maxillofacial hard tissues, which is prepared from γ-polyglutamic acid (γ-PGA), succinic anhydride modified chitosan (SCS) and dentin matrix (TDM) as the main raw materials, and is therefore referred to below as γ-PGA-SCS-TDM hydrogel.

[0009] In the aforementioned γ-PGA-SCS-TDM hydrogel, the dentin matrix, as an autologous biomineral material, is rich in collagen and mineralization-associated non-collagenous proteins (NCPs), which can effectively induce mesenchymal stem cells to differentiate into osteoblast / dentinous lineages. γ-Polyglutamic acid is a natural microbial fermentation product rich in carboxyl groups. After degradation into glutamic acid, it is non-cytotoxic and can precisely mimic the structure of γ-carboxyglutamic acid (Gla) in the bone matrix to guide the orderly deposition of hydroxyapatite (HAp). It can also activate integrin signaling (αvβ3) and promote osteoblast differentiation. Chitosan (CS) is a natural cationic polysaccharide polymer extracted from chitosan shells. In this invention, it is prepared into a soluble chitosan derivative by nucleophilic acylation reaction with succinic anhydride, thereby promoting osteoblast migration.

[0010] In the aforementioned γ-PGA-SCS-TDM hydrogel, succinic anhydride-modified chitosan can be prepared as follows: methanol is added to chitosan and lactic acid, followed by the addition of succinic anhydride for reaction. After the reaction is complete, the product is separated using a mixed solution of sodium hydroxide and ethanol. The obtained product is then dialyzed and freeze-dried to obtain powdered succinic anhydride-modified chitosan. In this preparation reaction, lactic acid and methanol do not directly participate in the esterification reaction between succinic anhydride and chitosan; they mainly function as solvents or reaction media. Specifically, the role of lactic acid includes: providing H+... +The process involves protonating the amino groups on the chitosan molecular chain, breaking the intermolecular hydrogen bonds, and dissolving the chitosan into a homogeneous solution, providing a well-dispersed reaction environment for the subsequent reaction with succinic anhydride. Simultaneously, lactic acid, as a weakly acidic medium, maintains the system's pH within an appropriate range, ensuring the chitosan remains dissolved while preventing excessive acidity that could lead to hydrolysis of the chitosan backbone, and promoting the ring-opening reaction of succinic anhydride. Methanol, on the one hand, ensures the uniform dispersion of succinic anhydride in the reaction system, and on the other hand, reduces the aggregation degree of chitosan molecules, allowing the active groups to be more fully exposed, thereby improving the reaction efficiency with succinic anhydride.

[0011] Preferably, in the preparation of the above-mentioned succinic anhydride modified chitosan, the mass ratio of chitosan to succinic anhydride is 1:(0.5~3).

[0012] Secondly, the present invention provides a method for preparing γ-PGA-SCS-TDM hydrogel, comprising the following steps: S1. Succinic anhydride-modified chitosan is dissolved in Na2HP4 solution to obtain a uniform suspension, and then dentin matrix is ​​added and mixed well. S2. Under vigorous stirring, the mixture obtained in step S1 is added dropwise to the γ-polyglutamic acid solution until a uniform white suspension is formed; S3. Add CaCl2 solution to the reaction solution obtained in step S2 and continue stirring until a hydrogel is formed; S4. Wash the hydrogel obtained in step S3 with deionized water, freeze-dry and sterilize.

[0013] In the above preparation method, the preferred mass ratio of dentin matrix, succinic anhydride modified chitosan, γ-polyglutamic acid and CaCl2 is 1:(2~5):(1~10):(0.3~10).

[0014] In the above preparation method, the stirring speed is preferably 150-500 rpm.

[0015] In the preparation method of this invention, γ-polyglutamic acid solution is used as a solution rich in carboxyl groups (-COO). ﹣ Anionic polyelectrolytes, whose negatively charged sites on the molecular chain are related to the introduced Ca2+. 2+ There are strong electrostatic interactions; most importantly, Ca 2+As a divalent cation, it can bridge carboxyl groups on multiple γ-polyglutamic acid chains. Even under controlled reaction concentration conditions, this interaction easily induces gelation or precipitation in the system, hindering the formation of a homogeneous and stable composite solution, and thus affecting the efficiency of subsequent mineralization template construction. To ensure that γ-polyglutamic acid, succinic anhydride-modified chitosan, and CaCl2 solution can fully and uniformly participate in the reaction and be effectively loaded onto the dentin matrix template, this invention effectively alleviates the problem of CaCl2 bridging by adjusting the feeding sequence and optimizing the mixing rate. 2+ The problem of premature gelation caused by induction is effectively addressed by promoting the synergistic self-assembly of various components on the dentin matrix template.

[0016] Thirdly, this invention provides the application of γ-PGA-SCS-TDM hydrogel in the preparation of products for repairing hard tissue defects in the maxillofacial region. This is due to the high compatibility of the core components with the physiological composition of the maxillofacial hard tissues (dentin, alveolar bone) and surrounding soft tissues: succinic anhydride-modified chitosan retains the natural biocompatibility of chitosan, and the modification with succinic anhydride further enhances its water solubility and cell affinity, avoiding the generation of acidic substances that irritate the maxillofacial soft tissues during the degradation process of traditional synthetic polymers (such as polylactic acid); γ-polyglutamic acid, as a naturally occurring amino acid polymer in the human body (produced by microbial fermentation), does not conflict with proteins, polysaccharides, and other components in saliva and tissue fluid, thus reducing inflammatory reactions at the repair site; the dentin matrix is ​​directly derived from natural dentin and contains bioactive components such as collagen, hydroxyapatite, and non-collagenous proteins (such as dentin sialophosphoprotein), which are consistent with the chemical composition of the maxillofacial hard tissues and can be recognized by the body as an "autologous tissue analog," significantly reducing the risk of immune rejection, and is particularly suitable for the repair of sensitive areas of the maxillofacial region (such as subgingival and alveolar sockets). Furthermore, during the preparation process, the hydrogel is in a uniform suspension state before the addition of CaCl2, which can be precisely injected into irregular defect areas of the maxillofacial region (such as alveolar sockets and root surface defects) through a syringe, and then rapidly cross-links into a gel in vivo; at the same time, the suspension before gel formation can be shaped according to the defect morphology, which solves the problem that traditional block-shaped restorative materials are difficult to adapt to complex defects of the maxillofacial region, and greatly improves the convenience of clinical operation and the accuracy of restoration.

[0017] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0018] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 This is a technical route diagram for preparing γ-PGA-SCS-TDM hydrogel according to the present invention; Figure 2 Strain scanning curve of the γ-PGA-SCS-TDM hydrogel prepared in this invention; Figure 3 Dynamic time-scan curve of the γ-PGA-SCS-TDM hydrogel prepared in this invention; Figure 4 The swelling ratio of the γ-PGA-SCS-TDM hydrogel prepared in this invention. Detailed Implementation

[0020] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0022] To address the shortcomings of existing maxillofacial hard tissue repair materials, this invention, based on a biomimetic mineralization strategy, provides a regenerative and repairing mineralized hydrogel suitable for maxillofacial hard tissues and its preparation method. Through the supramolecular self-assembly of γ-polyglutamic acid, succinic anhydride-modified chitosan, and calcium ions, a γ-PGA-SCS-TDM composite system with dentin matrix as the mineralization core template is constructed, which exhibits excellent biomimetic mineralization activity.

[0023] Reference Figure 1 The preparation method of γ-PGA-SCS-TDM hydrogel provided in this embodiment of the invention includes the following steps: (1) Preparation of succinic anhydride modified chitosan: Methanol was added to chitosan and lactic acid, and then succinic anhydride was added to react, wherein the mass ratio of chitosan to succinic anhydride was 1:(0.5~3); after the reaction was completed, the product was separated by a mixed solution of sodium hydroxide and ethanol, and the obtained product was dialyzed and freeze-dried to obtain powdered succinic anhydride modified chitosan. (2) Preparation of γ-PGA-SCS-TDM hydrogel.

[0024] Succinic anhydride-modified chitosan was dissolved in Na2HP4 solution to obtain a homogeneous suspension. Dentin matrix was then added and mixed thoroughly. Under vigorous stirring, the resulting mixture was added dropwise to γ-polyglutamic acid solution until a homogeneous white suspension was formed. CaCl2 solution was then added to the reaction solution and stirring was continued until a hydrogel was formed. The hydrogel was washed with deionized water, lyophilized, and sterilized.

[0025] In some embodiments of the present invention, the mass ratio of dentin matrix, succinic anhydride modified chitosan, γ-polyglutamic acid and CaCl2 is 1:(2~5):(1~10):(0.3~10).

[0026] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0027] Example 1 This example provides a γ-PGA-SCS-TDM hydrogel, the preparation of which includes the following: (1) Preparation of succinic anhydride modified chitosan.

[0028] A chitosan aqueous solution (50 mL, 2 wt%) and lactic acid (20 mL, 5 wt%) were added to a round-bottom flask, followed by 60 mL of methanol. Then, 2.5 g of succinic anhydride was added to the mixture, and the mixture was stirred continuously at room temperature for 24 h. The reaction mixture was then precipitated in a mixture of NaOH and ethanol to separate the formed product. The product was then dissolved in distilled water, dialyzed continuously for 3 days, and freeze-dried to obtain powdered succinic anhydride-modified chitosan.

[0029] (2) Preparation of γ-PGA-SCS-TDM hydrogel.

[0030] 0.1 g of succinic anhydride-modified chitosan was dissolved in Na2HP4 aqueous solution (0.2 mol / L, 10 mL) to obtain a uniform white suspension. Then, 0.02 g of dentin matrix was added and mixed well. Under vigorous stirring (stirring speed 300 rpm), the resulting mixture was added dropwise to 1 wt% 10 mL of γ-polyglutamic acid solution until a uniform white suspension was formed. Then, CaCl2 solution (1 mol / L, 1 mL) was added to the reaction solution and stirring was continued until a hydrogel was formed. The hydrogel was washed three times with deionized water and sterilized with ethylene oxide after lyophilization for 12 h.

[0031] Example 2 This example provides a γ-PGA-SCS-TDM hydrogel. Unlike Example 1, in the preparation process of this example, the mass ratio of dentin matrix, succinic anhydride modified chitosan, γ-polyglutamic acid and CaCl2 is 1:4:6:4.

[0032] The following performance tests were performed on the γ-PGA-SCS-TDM hydrogel prepared in this invention: 1. Mechanical properties.

[0033] Take 0.75g of lyophilized γ-PGA-SCS-TDM hydrogel powder and 10mL of aqueous solution to prepare a cylindrical sample with a diameter of 8mm and a thickness of 2mm. The sample is placed between the parallel plate clamps of the DMA and gently compacted (ensuring complete contact between the clamps and the sample with no air bubbles remaining), and equilibrated for 10 minutes.

[0034] Control conditions: Temperature 37℃, Frequency 1Hz; Strain range: 0.01%-100%; Scan rate: 0.5% / min; Monitoring indicators: Real-time recording of the variation curves of G' (energy storage modulus) and G'' (loss modulus) with strain.

[0035] Data repeatability verification: The same sample is tested in parallel 3 times, and the data deviation is calculated (G' deviation is required to be <5%) to eliminate the influence of uneven sample preparation or instrument error.

[0036] The detection results of the γ-PGA-SCS-TDM prepared in Example 1 are as follows: Figure 2 As shown in the figure, within the strain range of 0.01%-10%, G' is stable and higher than G'', indicating that the hydrogel is mainly elastic under physiological strain, can withstand chewing loads and quickly recover its deformation, avoiding permanent deformation caused by excessive viscosity (such as material collapse in alveolar bone repair). The upper limit of the linear region is much higher than the maximum physiological strain in the oral cavity (5%), indicating that the hydrogel will not exceed the linear range during normal use, has stable mechanical properties, and has no risk of structural damage.

[0037] 2. Self-healing performance.

[0038] Take 2 mL of the mixture and inject it into the DMA parallel plate clamp.

[0039] Parameter settings: temperature 37℃, frequency 1Hz, strain 0.1-100%, test time 1000s.

[0040] Dynamic time scans were performed over a strain range of 0.1% to 100% to evaluate the repeatable self-healing properties of the hydrogel.

[0041] The detection results of the γ-PGA-SCS-TDM prepared in Example 1 are as follows: Figure 3 As shown in the figure, when small-amplitude oscillatory shear (strain = 0.1%) was applied to all three hydrogels, their G' values ​​slightly exceeded the G'' value. Subsequently, under large-amplitude oscillatory shear (strain = 100%) lasting 200 s, the G'' values ​​of all hydrogels exceeded the G' value, indicating that the hydrogels exhibited fluid-like behavior and failed; however, when the shear force decreased, their properties immediately recovered to the original solid-like behavior. This characteristic was maintained in multiple repeatable cycles, indicating that the hydrogels have good self-healing properties.

[0042] 3. Swelling rate.

[0043] Using PBS buffer at pH 7.4 as the medium, the samples were dried to constant weight at 37°C and tested for 24 hours to calculate the mass swelling rate. Three parallel samples were used in each group to ensure that the data were consistent with the biological application scenario and material characteristics.

[0044] Test results as follows Figure 4 As shown in the figure, TDM has no significant effect on the swelling coefficient of the gel, indicating that TDM does not change the cross-linking backbone and swelling channels of the gel, and that the biological function of TDM and the physical swelling properties of the gel can be achieved independently. Figure 4 The preparation of γ-PGA-SCS was carried out in accordance with Example 1, without the addition of 0.02g of dentin matrix. Figure 4 The preparation of γ-PGA-SCS-0.025g TDM was also carried out in accordance with Example 1, except that the amount of dentin matrix added was adjusted from 0.02g to 0.025g.

[0045] In summary, the repair material provided by this invention has the advantages of mineralization induction ability, good biocompatibility, and ability to withstand dynamic loads, providing a new material for the repair of hard tissues in the maxillofacial region.

[0046] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A method for preparing a functional regenerative and repairing mineralized hydrogel for maxillofacial hard tissue, characterized in that, The hydrogel uses dentin matrix as a mineralization core template and is modified with γ-polyglutamic acid, succinic anhydride, chitosan, and Ca. 2+ The preparation method, which involves self-assembly on the template, includes the following steps: S1. Succinic anhydride-modified chitosan is dissolved in Na2HP4 solution to obtain a uniform suspension, and then dentin matrix is ​​added. S2. While stirring, add the mixture obtained in step S1 dropwise to the γ-polyglutamic acid solution; S3. Add CaCl2 solution to the reaction solution obtained in step S2 and continue stirring until a hydrogel is formed; The mass ratio of the dentin matrix, succinic anhydride-modified chitosan, γ-polyglutamic acid, and CaCl2 is 1:(2~5):(1~10):(0.3~10).

2. The preparation method according to claim 1, characterized in that, The preparation method of the succinic anhydride modified chitosan is as follows: methanol is added to a mixed solution of chitosan and lactic acid, and then succinic anhydride is added to react. After the reaction is completed, the product is separated and purified and dried.

3. The preparation method according to claim 2, characterized in that, The product was separated using a mixed solution of sodium hydroxide and ethanol, and then purified by dialysis.

4. The preparation method according to claim 2, characterized in that, The mass ratio of chitosan to succinic anhydride is 1:(0.5~3).

5. The preparation method according to claim 1, characterized in that, In step S2, the stirring speed is 150-500 rpm.

6. The preparation method according to claim 1, characterized in that, It also includes the following steps: S4. Wash the hydrogel obtained in step S3 with deionized water, freeze-dry and sterilize.

7. The maxillofacial hard tissue functional regeneration and repair mineralized hydrogel prepared according to any one of claims 1-6.

8. The application of the maxillofacial hard tissue functional regenerative repair mineralized hydrogel as described in claim 7 in the preparation of maxillofacial hard tissue defect repair products.

Images