Double cross-linked viscoelastic hydrogels, methods of preparation and uses

Abstract

The application discloses a kind of double crosslinking viscoelastic hydrogel, preparation method and purposes, double crosslinking viscoelastic hydrogel is composed of following substance: phenylboronic acid modified alginic acid (Alg-PBA) 20-40mg / mL, spermidine (SPD) 10mg / mL.The application is combined by borate ester and B-N coordination bond, and a kind of double crosslinking viscoelastic hydrogel (Alg-PBA / Spd) is prepared, and spermidine is released in the process of hydrogel degradation, and Alg-PBA / Spd hydrogel has biocompatibility, injectability, and can be adapted to complex periodontal structure by dynamic crosslinking.

Description

Technical Field

[0001] This invention belongs to the field of regenerative medicine technology, specifically relating to a double crosslinked viscoelastic hydrogel, its preparation method, and its uses. Background Technology

[0002] Periodontitis can cause irreversible damage to the periodontal ligament (PDL), and its regeneration is a major obstacle in the clinical treatment of periodontitis. Hydrogel implantation to release anti-inflammatory drugs and promote PDL regeneration is a promising treatment approach. However, existing hydrogel systems cannot mimic the typical viscoelastic characteristics of native periodontal tissue, which may play an important role in tissue regeneration. At the same time, the synergistic effect of mechanical cues and biochemical agents on PDL regeneration remains elusive. Summary of the Invention

[0003] In view of this, the main objective of the present invention is to provide a double crosslinked viscoelastic hydrogel, its preparation method, and its uses.

[0004] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0005] This invention provides a double crosslinked viscoelastic hydrogel composed of the following substances: 20-40 mg / mL of phenylboronic acid-modified alginic acid (Alg-PBA) and 10 mg / mL of spermidine (SPD).

[0006] This invention also provides a method for preparing a double-crosslinked viscoelastic hydrogel, the method comprising:

[0007] Step 1: Synthesize phenylboronic acid modified alginic acid (Alg-PBA) by esterification reaction of alginic acid (Alg) and 3-aminophenylboronic acid (PBA);

[0008] Step 2: Mix the solutions of Alg-PBA and Spd at room temperature to obtain a double crosslinked viscoelastic hydrogel (Alg-PBA / Spd).

[0009] In the above scheme, step 1 specifically involves: firstly, dissolving 1g of Alg in 0.1M MES buffer to form a 10mg / mL (w / v) Alg solution; then, adding 500mg of PBA dissolved in 5mL of dimethyl sulfoxide, 0.5g of EDC, and 0.7g of NHS to the Alg solution to obtain a mixed solution; maintaining the pH of the mixed solution at 4.5-5 for 24 hours at 25°C using 1M NaOH; after the reaction is complete, centrifuging at 7500rpm / min for 0.5 hours to remove unreacted PBA; finally, dialysis of the supernatant with deionized water (mwco3500) for 5 days, followed by lyophilization to obtain phenylboronic acid-modified alginate (Alg-PBA).

[0010] In the above scheme, step 2 specifically involves mixing Alg-PBA solutions of 40 mg / mL, 30 mg / mL, and 20 mg / mL and Spd solutions of 10 mg / mL at a volume ratio of 20:1 in a 1 ml syringe at room temperature using a Luer lock to obtain double crosslinked viscoelastic hydrogels (Alg-PBA / Spd) of different concentrations.

[0011] This invention also provides an application of a double crosslinked viscoelastic hydrogel for periodontal ligament regeneration.

[0012] Compared with the prior art, the present invention combines phenylboronic acid-modified alginate with the anti-inflammatory agent spermidine through borate ester and BN coordination bonds to prepare a double crosslinked viscoelastic hydrogel (Alg-PBA / Spd). Speridine is released during the hydrogel degradation process. The Alg-PBA / Spd hydrogel has biocompatibility and injectability, and can quickly adapt to complex periodontal structures through dynamic crosslinking. Attached Figure Description

[0013] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0014] Figure 1 Synthesis process and material characterization of Alg-PBA;

[0015] Figure 2 Schematic diagram of Alg-PBA / Spd hydrogel structure;

[0016] Figure 3 Mechanical properties and structure of Alg-PBA / Spd hydrogel;

[0017] Figure 4 Characterization of the injectability, self-healing properties, tensile properties, remodeling and degradation properties of Alg-PBA / Spd hydrogel;

[0018] Figure 5 To ensure the biocompatibility of Alg-PBA / Spd hydrogels;

[0019] Figure 6 Synthesis and mechanical property characterization of Alg-MA-PBA / Spd elastic hydrogel;

[0020] Figure 7 To evaluate the in vivo therapeutic effect of Alg-PBA / Spd hydrogel on an animal model of periodontal defects. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, article, or apparatus that includes that element.

[0023] This invention provides a double crosslinked viscoelastic hydrogel composed of the following substances: 20-40 mg / mL of phenylboronic acid-modified alginic acid (Alg-PBA) and 10 mg / mL of spermidine (SPD).

[0024] The viscoelastic Alg-PBA / Spd hydrogel of this invention significantly promotes periodontal collagen deposition and accelerates the repair of periodontal damage, making it a promising mechano-biochemical synergistic treatment for periodontal regeneration.

[0025] This invention also provides a method for preparing a double-crosslinked viscoelastic hydrogel, the method comprising:

[0026] Step 1: Synthesize Alg-PBA via esterification of Alg and PBA;

[0027] Specifically, firstly, 1 g of Alg was dissolved in 0.1 MME S buffer to form a 10 mg / mL (w / v) Alg solution; then, 500 mg of PBA dissolved in 5 mL of methyl sulfoxide, 0.5 g of EDC and 0.7 g of NHS were added to the Alg solution to obtain a mixed solution; the pH of the mixed solution was maintained in the range of 4.5-5 at 25 °C using 1 M NaOH for 24 h. After the reaction was completed, unreacted PBA was removed by centrifugation at 7500 rpm / min for 0.5 h. Finally, the supernatant was dialyzed with deionized water (mwco3500) for 5 days and then lyophilized to obtain Alg-PBA.

[0028] Step 2: Mix the solutions of Alg-PBA and Spd at room temperature to obtain a double crosslinked viscoelastic hydrogel (Alg-PBA / Spd).

[0029] Specifically, solutions of Alg-PBA (40 mg / mL, 30 mg / mL, 20 mg / mL in PBS) and Spd (10 mg / mL in PBS) were mixed at a volume ratio of 20:1 in a 1 ml syringe using a Luer lock at room temperature to obtain hydrogels of different concentrations of Alg-PBA.

[0030] This invention also provides an application of a double crosslinked viscoelastic hydrogel for periodontal ligament regeneration.

[0031] Sodium alginate modified with phenylboronic acid was prepared. Figure 1 A and Figure 2 A) and Spd( Figure 2 B), the successful grafting of PBA onto alginate was verified by proton nuclear magnetic resonance (NMR) spectroscopy. Figure 1 B).

[0032] This hydrogel network is cross-linked via dynamic boron esters between boric acid and cis-diol in the alginate backbone, and further cross-linked via dynamic BN coordination bonds between boric acid and the amino groups of Spd. Figure 2 CE).

[0033] To verify the gelling properties of the hydrogel, a 40 mg / mL Alg-PBA solution and a 10 mg / mL Spd solution were mixed at a ratio of 20:1. Due to the rapid cross-linking and dissociation of boronic acid bonds and BN coordination bonds, the mixed precursor gel and hydrogel did not flow when placed horizontally or upside down. Figure 2 F).

[0034] To evaluate the effect of Alg-PBA concentration on the storage modulus (G') and loss modulus (G”) of Alg-PBA / Spd hydrogels, different concentrations (20 mg / mL, 30 mg / mL, 40 mg / mL) of Alg-PBA were mixed with 10 mg / mL Spd solution at a ratio of 20:1. It was observed that both G' and G” increased with increasing Alg-PBA concentration, but decreased with decreasing angular frequency. Figure 3 A) This is because the Alg-PBA / Spd hydrogel, composed of dynamic bonds, is viscoelastic and does not have a frequency range with stable modulus.

[0035] Therefore, to evaluate the stiffness of hydrogels of different concentrations, the storage modulus with an angular frequency of 1 rad / s was selected as a reference. The storage modulus of hydrogels of different concentrations differed significantly. Figure 3 B).

[0036] To compare the effects of different concentrations of Alg-PBA on the porosity of hydrogels, the microstructure of the hydrogels was characterized using SEM, and the porosity was quantitatively analyzed. It was observed that hydrogels of all concentrations exhibited irregular porous microstructures with pore sizes greater than 200 μm and porosities between 50% and 80%. Figure 3 CD).

[0037] The porous structure of these hydrogels ensures the transport of nutrients and oxygen, which is conducive to cell penetration and proliferation, thereby promoting the formation of new tissues.

[0038] To maximize the drug loading capacity of the hydrogel and improve its hardness, 40 mg / mL Alg-PBA / Spd was selected as the material concentration for subsequent experiments.

[0039] To demonstrate the interaction between spermidine and Alg-PBA, the ratio of 40 mg / mL Alg-PBA to 10 mg / mL Spd was varied (20:1, 20:3, 20:5), and the gel point of the hydrogel (loss tangent = 1) was investigated as the loss tangent changed with angular frequency. Different ratios resulted in different trends in the curves, demonstrating the existence of intermolecular interactions between Spd and Alg-PBA. Figure 3 E). The results show that different concentrations of Spd alter the gel point of the hydrogel, and at low shear frequencies, the loss modulus of hydrogels with different concentrations differs significantly, indicating that the concentration of Spd changes the dynamic crosslinking network of the hydrogel; therefore, these results suggest that BN coordination bonds are formed between Spd and Alg-PBA.

[0040] To characterize the viscoelastic properties of Alg-PBA / Spd hydrogels, stress relaxation of the hydrogels under different strains was tested. Figure 3 F) The hydrogel has a short stress relaxation time and can be completely relaxed within 100s under different strain conditions (1%, 5%, 10%). These results indicate that Alg-PBA / Spd hydrogel has good viscoelasticity and can buffer periodontal stress through rapid relaxation.

[0041] To characterize the self-healing properties of the hydrogel, alternating cyclic strain measurements and macroscopic and microscopic hydrogel segmentation self-healing experiments were conducted. It was observed that the Alg-PBA / Spd hydrogel exhibited yielding behavior at a high strain level of 500% (G”>G’), but its mechanical properties recovered rapidly at a low strain level of 5% (G’>G”). Figure 4 A) This indicates that the self-healing process can be repeated multiple times without affecting the mechanical properties of the hydrogel.

[0042] From a macroscopic perspective, upon contact with two hydrogels of different colors, it was observed that the two hydrogels quickly fused into a single unit, easily resisting their own weight. Figure 4 B).

[0043] Further cutting a section of the hydrogel in the middle revealed that the incision rapidly decreased in size and completely healed within 120 seconds. Figure 4 C) These results demonstrate the self-healing ability and stability of the dual dynamic cross-linking network of BN coordination bonds and borate ester bonds. The self-healing properties of Alg-PBA / Spd hydrogel can greatly improve its application scenarios. When the hydrogel fills the periodontal tissue defect area, external force may cause the hydrogel to break. However, the rapid self-healing ability of the hydrogel will help improve the hydrogel's adaptability to complex defect morphologies.

[0044] Due to the narrow and complex spatial structure of periodontal defects, injection is a crucial method for delivering medications to periodontal tissues. To evaluate the injectability of the Alg-PBA / Spd hydrogel, a frequency-viscosity test was performed; the viscosity of the hydrogel decreased significantly with increasing shear rate. Figure 4 D); In addition, hydrogels of any shape can be continuously injected using a #12 needle. Figure 4 These results indicate that dynamic BN coordination bonds and borate ester bonds can endow hydrogels with good shear thinning properties.

[0045] To characterize the tensile and remodeling properties of the hydrogel, the Alg-PBA / Spd hydrogel was stretched, and it was observed that the hydrogel could resist a wide range of tensile strains. Figure 4 G) and reshaped into different shapes ( Figure 4 These results indicate that hydrogels can adapt to irregularly shaped periodontal defects, achieve perfect filling of the defects, and maintain shape stability under the compression of surrounding soft tissues.

[0046] To test the degradability of the Alg-PBA / Spd hydrogel, the hydrogel was immersed in PBS solution. We observed that the hydrogel rapidly expanded to its maximum volume within 4 hours, began to degrade after 4 hours, and was completely degraded after 5 days. Figure 4 I) indicates that the Alg-PBA / Spd hydrogel can be degraded due to the presence of dual dynamic crosslinking; since spermidine is crosslinked with the dynamic bonds in the hydrogel, the dynamic bonds are broken during the hydrogel degradation process, thereby achieving the synchronous and slow release of spermidine.

[0047] To assess the cytotoxicity of the hydrogel, the cytotoxicity of 40 mg / mL Alg-PBA / Spd hydrogel against periodontal ligament fibroblasts (PDLFs) was determined using the CCK-8 assay. It was observed that cell viability remained stable (cell viability >100%) when the hydrogel concentration was below 10 μL / mL (volume fraction, v / v), while cell viability decreased rapidly when the concentration was above 10 μL / mL. Figure 5 A) Based on this result, the concentration of the hydrogel solution (below 10 μL / mL) was selected to further detect the cell proliferation level. PDLFs were maintained for proliferation for 5 days in hydrogel solutions of different concentrations. Figure 5 B).

[0048] To further verify the biocompatibility of the hydrogel, a live / dead staining experiment was conducted. It was observed that the PDLF in both the experimental groups (5 μL / mL and 2.5 μL / mL) and the control group remained essentially live (green) and morphologically normal. After 5 days of culture, the proliferation process was unaffected. Figure 5 C), there were no significant differences between the 5 μL / mL, 2.5 μL / mL and blank groups at the same time points. Figure 5 D), consistent with the CCK-8 results; the above results confirm that the Alg-PBA / Spd hydrogel has good biocompatibility.

[0049] To demonstrate the role of viscoelasticity in periodontal regeneration, methacrylic anhydride groups were first grafted onto Alg, followed by phenylboronic acid groups, to synthesize Alg-MA-PBA (…). Figure 6 A); Alg-MA-PBA / Spd hydrogels were obtained by mixing with 10 mg / mL Spd at a volume ratio of 20:1 and adding 50 mg / mL LAP as a photoinitiator at a volume ratio of 100:1, followed by crosslinking under 490 nm blue light for 30 s. To compare the stiffness of Alg-MA-PBA / Spd hydrogels with those of Alg-PBA / Spd hydrogels, G' and G'' were measured using frequency scanning. Figure 6 B), and compared the stiffness of the two at a frequency of 1 Hz. Figure 6 C) No significant difference was found. To evaluate the viscoelastic difference between Alg-MA-PBA / Spd hydrogel and Alg-PBA / Spd hydrogel, the stress relaxation rate of the two hydrogels was tested. It was found that the stress relaxation rate of Alg-MA-PBA / Spd hydrogel was significantly slower than that of the hydrogel. Figure 6 D) can be used as an elastic control group for viscoelastic Alg-PBA / Spd hydrogels.

[0050] To evaluate the in vivo therapeutic effect of Alg-PBA / Spd hydrogel, a periodontal defect 1.5 mm wide and 2 mm deep was created around the maxillary first molar in rats, and the defect was completely filled with hydrogel. Figure 7 AB).

[0051] To assess the histological and regeneration status of PDL in each group, Masson staining and hematoxylin and eosin (H&E) staining were performed. With prolonged tissue recovery time, the collagen fibers in the PDL interstitial space showed an increasing trend among the four groups. Figure 7 C); In the first week, the total amount of periodontal ligament and gingival tissue in the Alg-PBA and Alg-PBA / Spd hydrogel groups was greater than that in the control group and the Alg-MA-PBA / Spd hydrogel group. In the control group, numerous red-stained muscle fibers were visible in the periodontal ligament spaces, while in the Alg-PBA / Spd group, only a small amount of collagen fiber bundles were arranged. The other three groups did not exhibit similarly well-arranged fiber structures. In the second week, the periodontal ligament spaces in all four groups were filled with new periodontal collagen fibers. The Alg-PBA and Alg-PBA / Spd groups showed regular fiber arrangement and dense fiber bundles, while the control group and the Alg-MA-PBA / Spd group showed irregular fiber arrangement, with the control group even having more muscle fibers. In the third week, the proportion of collagen fibers increased in all four groups. Magnified images of the control group showed irregular local muscle fibers, while the collagen fibers in the other three groups were well-arranged, forming fiber bundle structures.

[0052] Next, the volume fraction of collagen and the amount of collagen fibers produced were quantified. Figure 7 D); In the early stage of tissue recovery, the collagen volume of each group after hydrogel treatment was higher than that of the blank control group, with the Alg-PBA / Spd group having the highest volume fraction; In the middle and late stages of tissue recovery, there was no significant difference in collagen production between the Alg-PBA / Spd group and the Alg-MA-PBA / Spd group, and both groups were significantly higher than the Alg-PBA group and the blank group.

[0053] Furthermore, the arrangement of periodontal ligament cells was assessed using H&E staining. Figure 7 E); As shown by the arrows in the figure, the cells in the blank group were disordered and alveolar bone tissue resorption was obvious; the cells in the Alg-PBA group were mostly concentrated near the alveolar bone, with dense and regularly arranged fiber structures; the cells in the Alg-MA-PBA / Spd group and the Alg-PBA / Spd group were evenly distributed along the fibers, and the fibers were neatly arranged; these results indicate that viscoelastic hydrogels may promote the generation of periodontal ligament fibers in the early stage of repair, and synergistic treatment with spermidine can even promote collagen production in PDLFs, maximizing the treatment effect.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. A method for preparing a double crosslinked viscoelastic hydrogel, characterized in that, The preparation method includes: Step 1: Phenyboronic acid-modified alginate Alg-PBA was synthesized by esterification reaction of 10 mg / mL alginate Alg with 500 mg of 3-aminophenylboronic acid PBA in 5 mL of dimethyl sulfoxide. Step 2: Mix Alg-PBA solutions of 40 mg / mL, 30 mg / mL, and 20 mg / mL with Spd solutions of 10 mg / mL in a 1 mL syringe at a volume ratio of 20:1 using a Luerlock syringe at room temperature to obtain Alg-PBA / Spd hydrogels of different concentrations.

2. The method for preparing the double crosslinked viscoelastic hydrogel according to claim 1, characterized in that, Step 1 specifically involves: firstly, dissolving 1g of Alg in 0.1M MES buffer to form an Alg solution with a weight-volume concentration of 10mg / mL; then, adding 500mg of PBA dissolved in 5mL of dimethyl sulfoxide, 0.5g of EDC, and 0.7g of NHS to the Alg solution to obtain a mixed solution; maintaining the pH of the mixed solution at 4.5-5 using 1M NaOH at 25℃ for 24 h; after the reaction is complete, centrifuging at 7500rpm for 0.5h to remove unreacted PBA; finally, dialyzing the supernatant through deionized water with a MWCO of 3500Da for 5 days, followed by lyophilization to obtain phenylboronic acid-modified alginate Alg-PBA.

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