A growth factor-loaded oral repair hydrogel and a method of making the same

Abstract

The application belongs to the technical field of oral cavity repairing materials, and particularly relates to a growth factor loaded oral cavity repairing hydrogel and a preparation method thereof. The oral cavity repairing hydrogel comprises the following raw materials in parts by weight: modified protein 10-22 parts, HA-PBA 30-70 parts, Wnt3a recombinant protein 0.01 part, growth factor 0.03 part, antioxidant 1-3 parts and crosslinking agent 8-20 parts. The modified protein prepared by taking ovalbumin high phosphoprotein as a raw material is used as a mineralization promoting framework. High-density phosphoric acid groups are used to induce local calcium and phosphorus deposition and accelerate mineralization. Meanwhile, dopamine catechol groups are used to provide tissue adhesion and crosslinking reaction sites. The oral cavity repairing hydrogel is phase change solidified under the double triggers of body temperature and physiological pH, covalent crosslinking of the gel is promoted in situ, a three-dimensional interpenetrating network hydrogel with certain mechanical strength is formed, and the sustained slow release of active ingredients is controlled.

Description

Technical Field

[0001] This invention belongs to the field of oral restoration materials technology, specifically relating to an oral restoration hydrogel loaded with growth factors and its preparation method. Background Technology

[0002] In the fields of oral and maxillofacial surgery and periodontal treatment, the repair and regeneration of alveolar bone defects has always been a major clinical challenge. In recent years, hydrogel materials, due to their three-dimensional network structure highly similar to the natural extracellular matrix (ECM) and their ability to perfectly fill irregular bone defect areas through minimally invasive injection, have become a research hotspot for bone tissue engineering scaffolds and drug delivery systems. With the development of regenerative medicine, loading various bone growth factors and signaling proteins that regulate stem cell proliferation and differentiation into hydrogels has proven to be one of the most effective ways to accelerate bone mineralization repair.

[0003] There are still some technical shortcomings in the actual research, development, production, and clinical translation of oral restorative hydrogels: First, the active drugs in conventional hydrogels are mostly in a simple physical encapsulation state, and are prone to "burst release" after implantation. This not only leads to low drug utilization, but also the excessively high local drug concentration can inhibit the alkaline phosphatase activity and osteogenic differentiation of dental pulp stem cells. Second, existing in-situ cross-linked hydrogels mostly use light curing molding methods, which are prone to problems such as uneven mixing, high curing hardness leading to poor patient compliance, or incomplete curing during clinical operation. In addition, the oral microenvironment is filled with flowing saliva, and conventional hydrogels lack specific tissue adhesion to moist bone surfaces. They are easily detached and lost under chewing stress or saliva erosion, making it difficult to maintain a long-term bone repair microenvironment. Summary of the Invention

[0004] To address the above issues, this invention provides a growth factor-loaded oral repair hydrogel and its preparation method. It employs a combination of macromolecular dynamic covalent modification and microenvironment-responsive phase transition to construct a hydrogel that maintains stable single-phase flow in vitro and achieves adhesion and curing in situ at living lesions triggered by multiple environmental factors. This aims to solve the problems of poor adhesion, easy inactivation and burst release of biological proteins in existing oral repair gels under humid conditions, and to provide a biocompatible dental repair material that can achieve synergistic repair of mineralization and signaling pathways.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides an oral repair hydrogel loaded with growth factors, the oral repair hydrogel comprising the following raw materials in parts by weight: 10-22 parts modified protein, 30-70 parts HA-PBA (phenylboronic acid modified hyaluronic acid), 0.01 parts Wnt3a recombinant protein, 0.03 parts growth factors, 1-3 parts antioxidants, and 8-20 parts crosslinking agents.

[0006] Furthermore, the growth factor is selected from one or more of EGF (epidermal growth factor), FGF (fibroblast growth factor), BMP-2 (bone morphogenetic protein 2), PRGF (growth factor-rich plasma), and VEGF (vascular endothelial growth factor).

[0007] Furthermore, the antioxidant is selected from ascorbic acid or glutathione.

[0008] Furthermore, the crosslinking agent is selected from poloxamer 407 or sodium β-glycerophosphate.

[0009] Furthermore, the modified protein comprises the following raw materials: egg yolk high-phosphorus protein, dopamine hydrochloride, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide), wherein the mass ratio of egg yolk high-phosphorus protein, dopamine hydrochloride, EDC and NHS is 100:12:4:2.

[0010] Furthermore, the modified protein is prepared as follows: S1: Dissolve egg yolk high-phosphoprotein in MES (2-morpholinoethanesulfonic acid) buffer containing sodium chloride, then add dopamine hydrochloride and stir to dissolve, to obtain the reaction solution; S2: After purging nitrogen into the reaction solution, EDC and NHS are added and dissolved in it under an ice-water bath. The mixture is stirred and reacted for 24 hours to achieve the amidation grafting of dopamine and obtain the reaction solution. S3: Dialyze and purify the reaction solution to remove unreacted raw materials and salts, and then freeze-dry to obtain the modified protein.

[0011] This invention also provides a method for preparing an oral repair hydrogel loaded with growth factors, the specific steps of which are as follows: Step 1: Dissolve the modified protein and antioxidant together in PBS (phosphate buffer) at pH 5.5 to obtain a pre-solution. Under weakly acidic conditions, the catechol group of the modified protein is inhibited from deprotonation and cannot spontaneously oxidize to the highly active o-quinone structure, thus blocking the intermolecular polymerization of dopamine groups. Under ice-water bath conditions, add Wnt3a recombinant protein and growth factor to the pre-solution and stir until completely dissolved to obtain a premix. Step 2: While stirring continuously, slowly add HA-PBA and crosslinking agent to the premix, and let it stand and incubate. The phenylboronic acid on the side chain of HA-PBA lacks sufficient electrophilicity in a weakly acidic environment and cannot undergo dehydration condensation with catechol on the modified protein, thereby ensuring that the system does not undergo a violent covalent crosslinking reaction during the preparation process, maintaining a certain fluidity, and promoting the dispersion of active protein and growth factors in the three-dimensional structure of HA-PBA to obtain a pregel solution. Step 3: Sterilize and dispense the pregel solution, and store it in the dark and refrigerated to obtain oral repair hydrogel loaded with growth factors.

[0012] The beneficial effects achieved by this invention are as follows: The oral repair hydrogel loaded with growth factors prepared in this invention uses natural egg yolk high-phosphorus protein as its backbone substrate. Its inherent high-density phosphate groups can highly mimic the mineralization-promoting function of natural osteophyte protein, acting as a powerful calcium ion trap at the microscopic level and inducing homologous nucleation of hydroxyapatite. Simultaneously, by grafting dopamine onto the protein chain, abundant catechol groups are introduced, which not only endow the hydrogel with extremely strong tissue adhesion in the moist oral environment but also provide high-density reactive sites for chemical cross-linking between the gel backbone and the protein, achieving a combination of promoting bone mineralization and strong wet adhesion. A dual-environmental stimulus-responsive system was constructed using HA-PBA, modified protein, and a temperature-sensitive crosslinking agent. Under in vitro weakly acidic conditions and cold chain storage and transportation, the hydrogel maintains good fluid properties, facilitating dispensing, filling, and use. Upon injection into the lesion area, the oral temperature triggers a phase transition in the crosslinking agent, forming a robust physical gel framework. Simultaneously, the physiological pH environment activates the phenylboronic acid groups, causing them to rapidly esterify with the catechol groups of the modified protein, achieving rapid dynamic covalent crosslinking of the gel and quickly forming an in-situ cured repair gel with mechanical strength. The introduced antioxidant not only inhibits the premature oxidative self-polymerization of dopamine groups in the weakly acidic substrate but also effectively protects the cysteine ​​structure in the Wnt3a recombinant protein from damage, ensuring its bioactivity. When the hydrogel undergoes pH-responsive curing in situ, the phenylboronic acid groups on HA-PBA not only participate in framework crosslinking but also dynamically covalently bind to the glycan chains on the surface of glycoproteins such as Wnt3a. This allows growth factors to resist early swelling and release, reducing the risk of mineralization inhibition caused by high-concentration drug side effects. Attached Figure Description

[0013] Figure 1 The results of the gel curing time and storage modulus of the oral repair hydrogels prepared in Examples 1-4 and Comparative Examples 1-2 are as follows: Figure 2 The results of the appearance morphology examination of the oral repair hydrogel prepared in Example 3 before and after curing; Figure 3 The results of the cumulative drug release rate of the oral repair hydrogels prepared in Comparative Example 1 and Example 3 are presented. Figure 4 The results of the tissue adhesion performance study of the oral repair hydrogels prepared in Examples 1-4 and Comparative Examples 1-2 are as follows; Figure 5 The results of staining for stem cell mineralization nodules were observed in the control group, comparative example 2, and example 3. Detailed Implementation

[0014] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0016] Unless otherwise specified, all methods used in the following examples are conventional; all quantities are by weight; unless otherwise specified, all materials used in the following examples are new materials purchased from the market; wherein, the phosphoprotein used contains 10% phosphorus, catalog number P1253; the dopamine hydrochloride used has a purity ≥98%, CAS number 62-31-7; the hyaluronic acid used in HA-PBA has a molecular weight of 200 kDa, model EFL-HA-PBA001; the recombinant Wnt3a protein used is recombinant human Wnt3a protein, with a molecular weight of 60-75 kDa, tag C-Fc, purity ≥95%, expression system HEK293.

[0017] In the following examples and comparative examples, the modified protein comprises the following raw materials in parts by weight: 100 parts egg yolk phosphatidylcholine, 12 parts dopamine hydrochloride, 4 parts EDC, and 2 parts NHS. The preparation method is as follows: S1: Dissolve 100 parts of egg yolk high phosphoprotein in 20 mM, pH 6.0 MES buffer containing 1.7 M sodium chloride, then add 12 parts of dopamine hydrochloride and stir to dissolve. Stir at 400 rpm for 30 min in the dark to obtain the reaction solution. S2: After purging nitrogen into the reaction solution, add 4 parts of EDC and 2 parts of NHS to dissolve in an ice-water bath, stir at 300 rpm for 24 h to achieve amidation grafting of dopamine and obtain the reaction solution. S3: Dialyze and purify the reaction solution. The molecular weight cutoff of the dialysis bag is 20 kDa. The dialysis time is 48 hours. The dialysis solution is changed every 12 hours to remove unreacted raw materials and salts. After dialysis, the solution is lyophilized to obtain the modified protein.

[0018] Example 1: This example provides an oral repair hydrogel loaded with growth factors. The oral repair hydrogel comprises the following raw materials in parts by weight: 10 parts modified protein, 30 parts HA-PBA, 0.01 parts Wnt3a recombinant protein, 0.03 parts PRGF, 1 part ascorbic acid, and 20 parts poloxamer 407.

[0019] This embodiment also provides a method for preparing an oral repair hydrogel loaded with growth factors, the specific steps of which are as follows: Step 1: Dissolve 10 parts of modified protein and 1 part of ascorbic acid together in 1000 parts of pH 5.5 PBS buffer under ice water bath to obtain a pre-solution. Add 0.01 parts of Wnt3a recombinant protein and 0.03 parts of PRGF to the pre-solution and stir at 200 rpm until completely dissolved to obtain a premixed solution. Step 2: Under continuous stirring at 200 rpm, first add 20 parts of poloxamer 407 to the premixed solution to dissolve it, then slowly add 30 parts of HA-PBA. After the addition is complete, incubate at 4°C for 4 h to obtain the premixed solution. Step 3: Sterilize the pregel solution with ultraviolet irradiation, remove air bubbles, dispense into portions, and store in the dark and refrigerated to obtain oral repair hydrogel loaded with growth factors.

[0020] Example 2: This example provides an oral repair hydrogel loaded with growth factors. The oral repair hydrogel comprises the following raw materials in parts by weight: 15 parts modified protein, 40 parts HA-PBA, 0.01 parts Wnt3a recombinant protein, 0.03 parts EGF, 2 parts ascorbic acid, and 15 parts sodium β-glycerophosphate.

[0021] This embodiment also provides a method for preparing an oral repair hydrogel loaded with growth factors, the specific steps of which are as follows: Step 1: Dissolve 15 parts of modified protein and 2 parts of ascorbic acid together in 1000 parts of pH 5.5 PBS buffer under ice water bath to obtain a pre solution. Add 0.01 parts of Wnt3a recombinant protein and 0.03 parts of EGF to the pre solution and stir at 200 rpm until completely dissolved to obtain a premixed solution. Step 2: Under continuous stirring at 200 rpm, first add 15 parts of sodium β-glycerophosphate to the premixed solution to dissolve it, and then slowly add 40 parts of HA-PBA. After the addition is complete, let it stand at 4°C for 4 hours to obtain the premixed solution. Step 3: Sterilize the pregel solution with ultraviolet irradiation, remove air bubbles, dispense into portions, and store in the dark and refrigerated to obtain oral repair hydrogel loaded with growth factors.

[0022] Example 3: This example provides an oral repair hydrogel loaded with growth factors. The oral repair hydrogel comprises the following raw materials in parts by weight: 18 parts modified protein, 55 parts HA-PBA, 0.01 parts Wnt3a recombinant protein, 0.01 parts BMP-2, 0.02 parts VEGF, 2 parts glutathione, and 12 parts poloxamer 407.

[0023] This embodiment also provides a method for preparing an oral repair hydrogel loaded with growth factors, the specific steps of which are as follows: Step 1: Dissolve 18 parts of modified protein and 2 parts of glutathione together in 1000 parts of pH 5.5 PBS buffer under ice water bath to obtain a pre-solution. Add 0.01 parts of Wnt3a recombinant protein, 0.01 parts of BMP-2 and 0.02 parts of VEGF to the pre-solution and stir at 200 rpm until completely dissolved to obtain a premixed solution. Step 2: Under continuous stirring at 200 rpm, first add 12 parts of poloxamer 407 to the premixed solution to dissolve it, and then slowly add 55 parts of HA-PBA. After the addition is complete, incubate at 4°C for 4 h to obtain the premixed solution. Step 3: Sterilize the pregel solution with ultraviolet irradiation, remove air bubbles, dispense into portions, and store in the dark and refrigerated to obtain oral repair hydrogel loaded with growth factors.

[0024] Example 4: This example provides an oral repair hydrogel loaded with growth factors. The oral repair hydrogel comprises the following raw materials in parts by weight: 22 parts modified protein, 70 parts HA-PBA, 0.01 parts Wnt3a recombinant protein, 0.01 parts FGF, 0.02 parts BMP-2, 3 parts glutathione, and 8 parts sodium β-glycerophosphate.

[0025] This embodiment also provides a method for preparing an oral repair hydrogel loaded with growth factors, the specific steps of which are as follows: Step 1: Dissolve 22 parts of modified protein and 3 parts of glutathione together in 1000 parts of pH 5.5 PBS buffer under ice water bath to obtain a pre-solution. Add 0.01 parts of Wnt3a recombinant protein, 0.01 parts of FGF and 0.02 parts of BMP-2 to the pre-solution and stir at 200 rpm until completely dissolved to obtain a premixed solution. Step 2: Under continuous stirring at 200 rpm, first add 8 parts of sodium β-glycerophosphate to the premixed solution to dissolve it, then slowly add 70 parts of HA-PBA. After the addition is complete, incubate at 4°C for 4 h to obtain the premixed solution. Step 3: Sterilize the pregel solution with ultraviolet irradiation, remove air bubbles, dispense into portions, and store in the dark and refrigerated to obtain oral repair hydrogel loaded with growth factors.

[0026] Comparative Example 1: The difference from Example 3 is that an equal weight of unmodified egg yolk phosphoprotein was used instead of modified protein for the preparation of hydrogels, while the rest of the process was the same as in Example 3.

[0027] Comparative Example 2: The difference from Example 3 is that the Wnt3a recombinant protein was not added, and the rest is the same as Example 3.

[0028] Rheological investigation: The oral restorative hydrogels prepared in Examples 1-4 and Comparative Examples 1-2 were placed on a rotational rheometer for steady-state shear testing. The initial viscosity was recorded. The test stage temperature was set to 37°C, and the pH of the hydrogel was rapidly adjusted to 7.0. The gel curing time and the storage modulus G' of the cured hydrogel were recorded. The results are shown in [Figure number missing]. Figure 1 The gel morphology before and after curing is shown in the figure. Figure 2 .

[0029] Tissue adhesion test: Fresh detached pig skin was soaked in artificial simulated saliva at 37°C for 30 min to simulate the moist surface environment of the oral cavity. The oral repair hydrogels prepared in Examples 1-4 and Comparative Examples 1-2 were applied to different surfaces of the same tissue, ensuring the application area was the same. After in-situ curing for 5 min, the gels were placed on a universal testing machine and stretched at a constant rate of 5 mm / min. The adhesion strength between the gel and bone tissue was recorded. The results are shown in […]. Figure 3 .

[0030] Drug release study: After the repair hydrogels prepared in Example 3 and Comparative Example 1 were completely cured, they were placed in 100 kDa dialysis bags and dialyzed. Samples were taken at 0.5 days, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days. The concentration of recombinant Wnt3a protein in the release solution was determined using a Wnt3a ELISA kit, and the cumulative release rate was calculated. The results are shown in [Figure number missing]. Figure 4 .

[0031] Mineralization promotion study: DPSC (dental pulp stem cells) were collected at a rate of 2×10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 24-well plates. After cell adhesion, osteogenic induction medium containing hydrogel extracts from Examples 3 and 2 (Comparative Example 2) was added for culture. On day 28, cells were fixed with 4% paraformaldehyde and stained with alizarin red for mineralization nodules. The control group received osteogenic induction medium without hydrogel extracts. Results are shown in [details omitted]. Figure 5 .

[0032] Figure 1 The results showed that the hydrogels prepared in Examples 1-4 all cured within 1 minute, indicating the synergistic effect of responsive crosslinking and dynamic covalent bonds. In contrast, Comparative Example 1 lacked crosslinking sites provided by dopamine and lacked the conditions for the formation of dynamic covalent bonds of borate esters, resulting in extremely slow curing and decreased strength.

[0033] Figure 2 The results showed that the oral repair hydrogel prepared in Example 3 had a certain fluidity before curing, which was beneficial for in-situ injection and production. After curing, it had good adhesion and shaping properties.

[0034] Figure 3 The results showed that although the final release rates of the recombinant proteins were similar, the repair hydrogel protein prepared in Comparative Example 1 had a faster release rate, and the hydrogel prepared in Example 3 could maintain a longer therapeutic concentration at the treatment site.

[0035] Figure 4 The results showed that the adhesion performance of Examples 1-4 and Comparative Example 2 was significantly improved compared with Comparative Example 1 due to the presence of dopamine. This indicates that dopamine modification can not only improve the degree of gel crosslinking, but also promote the efficient adhesion of gel to tissue sites and reduce loss.

[0036] Figure 5 The results showed that the number and area of ​​mineralized nodules in cells incubated with osteogenic induction medium containing the hydrogel extract prepared in Example 3 increased significantly, indicating that it has a good effect on promoting stem cell proliferation, differentiation and mineralization, which is beneficial to the self-repair of oral tooth defects and improves the therapeutic effect.

[0037] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0038] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A hydrogel for oral repair loaded with growth factors, characterized in that, The oral repair hydrogel comprises the following raw materials in parts by weight: 10-22 parts modified protein, 30-70 parts HA-PBA, 0.01 parts Wnt3a recombinant protein, 0.03 parts growth factor, 1-3 parts antioxidant and 8-20 parts crosslinking agent; The modified protein comprises the following raw materials: egg yolk phosphoprotein, dopamine hydrochloride, EDC, and NHS; The modified protein is prepared as follows: S1: Dissolve the egg yolk high-phosphorus protein, add dopamine hydrochloride to it to obtain the reaction solution; S2: Add EDC and NHS to the reaction solution to dissolve and react, and obtain the reaction solution; S3: Dialyze, purify, and freeze-dry the reaction solution to obtain the modified protein.

2. The oral repair hydrogel loaded with growth factors according to claim 1, characterized in that, The mass ratio of egg yolk phosphoprotein, dopamine hydrochloride, EDC and NHS is 100:12:4:

2.

3. The oral repair hydrogel loaded with growth factors according to claim 2, characterized in that, The growth factor is selected from one or more of EGF, FGF, BMP-2, PRGF and VEGF.

4. The oral repair hydrogel loaded with growth factors according to claim 2, characterized in that, The antioxidant is selected from ascorbic acid or glutathione.

5. The oral repair hydrogel loaded with growth factors according to claim 2, characterized in that, The crosslinking agent is selected from poloxamer 407 or sodium β-glycerophosphate.

6. A method for preparing an oral repair hydrogel loaded with growth factors according to any one of claims 1-5, characterized in that, The specific steps are as follows: Step 1: Dissolve the modified protein and antioxidant together to obtain a pre-solution. Add Wnt3a recombinant protein and growth factor to the pre-solution and stir to dissolve to obtain a premixed solution. Step 2: Add HA-PBA and crosslinking agent to the premix and incubate to obtain a pregel solution; Step 3: Sterilize and dispense the pregel solution to obtain oral repair hydrogel loaded with growth factors.

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