Injectable composite collagen dispersion liquid, hydrogel of injectable composite collagen dispersion liquid, preparation method of injectable composite collagen dispersion liquid and application of injectable composite collagen dispersion liquid in preparation of medicine for treating periodontitis

Abstract

The invention discloses injectable composite collagen dispersion liquid and hydrogel thereof, a preparation method and application of the injectable composite collagen dispersion liquid in preparation of drugs for treating periodontitis, the injectable composite collagen dispersion liquid comprises freeze-dried SIS collagen, itaconic anhydride and a photoinitiator, the mass ratio of the freeze-dried SIS collagen to the itaconic anhydride is 1: (1-5), a solvent is a phosphate buffer solution, the injectable composite collagen dispersion liquid is subjected to photoinitiation, and the injectable composite collagen is obtained. To form a hydrogel. The injectable composite collagen dispersion liquid has certain flowability, directly acts on a periodontal lesion of a patient through an injector, and forms hydrogel in situ through short-time photo-initiation, the mechanical properties of the injectable composite collagen dispersion liquid and the hydrogel can meet the mechanical requirements of periodontal tissues, and the injectable composite collagen dispersion liquid has the effect of promoting osteogenesis; therefore, the compound can be applied to preparation of drugs for treating periodontitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogels, and more specifically, relates to an injectable composite hydrogel for osteogenesis after inflammatory bone defects and a preparation method thereof. Background Art

[0002] Periodontitis is a persistent inflammatory response caused by bacterial invasion of periodontal tissues. It can lead to severe loss of oral soft and hard tissues, making it difficult to treat periodontal disease and repair missing teeth. The occurrence of periodontitis is often accompanied by the appearance of periodontal pockets, and the formation of periodontal pockets is often accompanied by the loss of alveolar bone. Due to the difficulty of drug delivery caused by the complexity of the periodontal pocket morphology, traditional treatment methods such as periodontal scaling, filling with antibacterial drugs, etc. are often difficult to achieve antibacterial effects and cannot effectively promote bone regeneration. Among traditional bone regeneration-promoting materials, the classic periosteum / powder combination often lacks effective antibacterial ability when dealing with complex bone resorption with the risk of infection, and systemic antibiotics must be used to deal with the possibility of local infection. Therefore, the industry generally hopes to construct a bioactive material with both morphological plasticity, antibacterial and osteogenic capabilities, in the hope that it can play a role in guiding tissue regeneration in a periodontitis environment.

[0003] As a natural extracellular matrix, porcine small intestinal submucosa (SIS) hydrogels are non-immunogenic and have strong potential to promote proliferation and osteogenesis. SIS also possesses a porous microstructure, which provides conditions for the colonization and growth of osteoblasts. Previous studies have demonstrated the biocompatibility and anti-inflammatory properties of SIS materials, promoting the repair and regeneration of native tissue in defect areas. In vivo, bioactive factors within SIS partially inhibit the proliferation of Th1 cells and the secretion of interleukin 12 (IL-12) and interferon g (IFN-g) in a TGF-β-dependent manner, alleviating inflammation. Furthermore, SIS can upregulate the expression of osteogenic factors such as BMP-2 and CD-31, activate the BMPs / SMAD signaling pathway, and promote osteogenesis, bone regeneration, differentiation, and angiogenesis. However, as an osteogenic material for periodontal defects, SIS undergoes a slow self-assembly process under the complex stress conditions of the oral cavity, as its collagen molecules rely solely on van der Waals forces and hydrogen bonds. Furthermore, it easily collapses under external stress, affecting its function within the tissue. Therefore, a method is needed to modify SIS collagen to enable rapid gelation while also enhancing its mechanical properties, allowing it to quickly demonstrate good mechanical stability after implantation. Summary of the Invention

[0004] The present invention aims to overcome the deficiencies of the prior art and to provide an injectable composite hydrogel for post-inflammatory bone defect osteogenesis and a method for its preparation. SIS photosensitive hydrogels are prepared by modification using itaconic anhydride. Itaconic acid is a biogenic dibasic acid containing one carbon-carbon unsaturated double bond and can be obtained through fungal fermentation. Changing the crosslinking density of polyols and itaconate-containing materials can control their mechanical profile, hydrophobicity, and other macromolecular properties. Itaconic acid undergoes photolysis under ultraviolet light to produce acidic intermediates, thereby promoting the photocuring reaction of photosensitive materials. Itaconic anhydride can directly react with amino groups in collagen to form amide-grafted collagen. Free radical polymerization of carbon-carbon double bonds is initiated by a cell-compatible photoinitiator to reduce the solubility of the polymer and form a mesh structure, ultimately losing fluidity and forming a gel. At the same time, the mechanical effect is improved by strengthening the chemical bond strength between collagens.

[0005] The technical purpose of the present invention is achieved through the following technical solutions.

[0006] An injectable composite collagen dispersion comprises freeze-dried SIS collagen, itaconic anhydride and a photoinitiator, wherein the mass ratio of the freeze-dried SIS collagen to the itaconic anhydride is 1:(1-5), the solvent is phosphate buffer, and the ratio of the sum of the masses (mg) of the freeze-dried SIS collagen and the itaconic anhydride to the volume (ml) of the solvent is (15-20):(1-5).

[0007] In the above technical solution, the mass ratio of freeze-dried SIS collagen to itaconic anhydride is 1:(3-5).

[0008] In the above technical solution, the ratio of the sum of the masses (mg) of freeze-dried SIS collagen and itaconic anhydride to the volume (ml) of the solvent is (15-20):(1-2).

[0009] In the above technical solution, the photoinitiator is 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

[0010] In the above technical solution, the amount of photoinitiator used is 0.1%-0.2% of the total mass of the dispersion.

[0011] In the above technical solution, photosensitive collagen composed of freeze-dried SIS collagen and itaconic anhydride is dispersed in a solvent.

[0012] In the above technical solution, an acetic acid solution of freeze-dried SIS collagen and an acetone solution of itaconic anhydride are uniformly mixed and dialyzed, and then freeze-dried to obtain photosensitive collagen; the mass ratio of freeze-dried SIS collagen to itaconic anhydride is 1:(1-5), preferably 1:(3-5). During the preparation, a certain amount of freeze-dried SIS collagen is taken and uniformly dispersed with a 1% by mass acetic acid aqueous solution to form an acetic acid solution of freeze-dried SIS collagen; a certain amount of itaconic anhydride is uniformly dispersed in acetone to form an acetone solution of itaconic anhydride, and the mass percentage is consistent with that of the acetic acid solution of freeze-dried SIS collagen; according to the mass ratio of the two solutions, different volumes of the two solutions are selected, mixed uniformly and dialyzed, and then freeze-dried to obtain photosensitive collagen.

[0013] In the above technical solution, the pH of the solvent phosphate buffer is 7.4-7.6.

[0014] The hydrogel obtained by using the above-mentioned injectable composite collagen dispersion is subjected to photoinitiation to form a hydrogel.

[0015] Use 365nm ultraviolet light for initiation, and the illumination time is 60-180s, preferably 120-150s.

[0016] The injectable composite collagen dispersion of the present invention has a certain fluidity and acts directly on the patient's periodontal lesion through a syringe. It forms a hydrogel in situ through short-term light initiation. The mechanical properties of both can meet the mechanical requirements of periodontal tissue and have the effect of promoting osteogenesis, so it can be used in the preparation of drugs for treating periodontitis.

[0017] Compared to other collagen modification methods, the preparation of photocross-linked biomatrix gels (Photocross-linked hydrogels) by SIS collagen has the characteristics of being simpler and easier to implement. However, the methacrylic acid-modified collagen currently used to prepare collagen-based photocurable hydrogels has certain irritation and allergic properties, and also has certain toxicity. Therefore, in the present invention, itaconic anhydride is selected to modify and prepare SIS photosensitive hydrogels. Itaconic acid is a biogenic dibasic acid containing a carbon-carbon unsaturated double bond and can be obtained by fungal fermentation. Changing the cross-linking density of polyols and itaconate-containing materials can control their mechanical profile, hydrophobicity and other macromolecular properties. As an antibacterial metabolite and immune cell metabolism regulator of organisms, itaconic acid has biocompatibility, degradability, antibacterial properties, anti-inflammatory properties and the ability to release drugs slowly, and also has antibacterial activity. Itaconic acid undergoes a photolysis reaction under ultraviolet light to produce acidic intermediates, thereby promoting the photocuring reaction of photosensitive materials. Itaconic anhydride can directly react with the amino groups in collagen to generate amide bond-grafted collagen. The free radical polymerization of carbon-carbon double bonds is initiated by a cell-compatible photoinitiator to reduce the solubility of the polymer and form a mesh structure, which eventually loses fluidity and forms a gel. At the same time, the mechanical effect is improved by enhancing the strength of the chemical bonds between collagens. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a photograph of the collagen dispersion and hydrogel of the present invention (1).

[0019] Figure 2 This is a photograph of the collagen dispersion and hydrogel of the present invention (2).

[0020] Figure 3 is the infrared spectrum of the hydrogel of the present invention.

[0021] Figure 4 It is a schematic diagram of the gelation time test results of hydrogels with different cross-linking degrees prepared in the present invention.

[0022] Figure 5 Schematic diagram of SEM and pore size test results of hydrogels with different cross-linking degrees in the present invention.

[0023] Figure 6 Schematic diagram of the porosity test results of hydrogels with different cross-linking degrees in the present invention.

[0024] Figure 7 This is a schematic diagram of the results of thermomechanical DMA performance testing of the injectable composite collagen of different compositions in the present invention after irradiation.

[0025] Figure 8 Schematic diagram of the hemolytic effect test results of hydrogels with different cross-linking degrees in the present invention.

[0026] Figure 9 It is a schematic diagram of the test results of the effect of the injectable composite hydrogel prepared by the present invention on cell proliferation.

[0027] Figure 10 This is a photograph of the osteogenic induction test results of the injectable composite hydrogel prepared by the present invention (Alizarin Red ARS staining).

[0028] Figure 11 This is a photograph of the osteogenic induction test results of the injectable composite hydrogel prepared by the present invention (alkaline phosphatase ALP staining).

[0029] Figure 12 This is a test result diagram of osteogenic gene expression of the injectable composite hydrogel prepared by the present invention.

[0030] Figure 13 This is a photo of the protein expression test results of the osteogenic gene of the injectable composite hydrogel prepared by the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below with reference to specific embodiments.

[0032] Extraction references for SIS hydrogel: Giobbe, GG; Crowley, C.; Luni, C.; Campinoti, S.; Khedr, M.; Kretzschmar, K.; De Santis, MM; Zambaiti, E.; Michielin, F.; Meran, L.; Hu, Q.; van Son, G.; Urbani, L.; Manfredi, A.; Giomo, M.; Eaton, S.; Cacchiarelli, D.; Li, VSW; Clevers, H.; Bonfanti, P.; Elvassore, N.; De Coppi, P. Extracellular Matrix Hydrogel Derived from Decellularized Tissues Enables Endodermal Organoid Culture. Nat Commun 2019, 10 (1). The obtained SIS hydrogel is freeze-dried to obtain freeze-dried SIS collagen, and the hydrogel of the present invention is prepared. The chemical formula of itaconic anhydride is shown below:

[0033]

[0034] A certain amount of freeze-dried SIS collagen was uniformly dispersed in a 1% by mass aqueous acetic acid solution to form an acetic acid solution of freeze-dried SIS collagen; a certain amount of itaconic anhydride was uniformly dispersed in acetone to form an itaconic anhydride / acetone solution, the mass percentage of which was consistent with that of the acetic acid solution of freeze-dried SIS collagen; different volumes of the acetic acid solution of freeze-dried SIS collagen and the itaconic anhydride / acetone solution were mixed in a mass ratio of freeze-dried SIS collagen to itaconic anhydride of 1:(1-5), the mixture was thoroughly mixed and stirred at 4°C for 24 hours, and the mixture was transferred to an 18-44 kDa dialysis bag and dialyzed with ddH2O for 72 hours, and photosensitive collagen was obtained after freeze-drying.

[0035] The photosensitive collagen was uniformly dispersed in phosphate buffer (pH = 7.4) (20 mg of photosensitive collagen was uniformly dispersed in 1 mL of buffer). The photoinitiator Irgacure 2959 (2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone) was then added to the uniformly dispersed photosensitive collagen buffer to obtain the injectable composite collagen dispersion of the present invention (the amount of photoinitiator added was 0.2% of the total mass of the dispersion). After centrifugation and defoaming, the dispersion was allowed to stand for 2 hours to allow the collagen to fully swell, and then transferred to a syringe for later use.

[0036] The itaconic anhydride (IA) / pig small intestinal submucosa (SIS) collagen dispersion prepared by the present invention can be injected into the composite collagen dispersion (hereinafter referred to as SIS / IA collagen dispersion) through the needle of the syringe. Figure 1 In the figure, the leftmost part is the collagen dispersion in the bottle, which is in a flowing state; the middle part is the collagen dispersion irradiated by ultraviolet light, the initiator Irgacure2959 is a photoinitiator: 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, the excitation wavelength is 365nm ultraviolet light, and the reaction time is 180s; the rightmost part is after the ultraviolet light irradiation ends, the bottle is turned upside down, and the substance in the bottle is no longer in a flowing state, but has formed a gel. The flowing collagen dispersion is transferred to a syringe, and words can be written in the container by squeezing it out through the syringe, such as Figure 2 As shown in the right side of the figure; the gel formed after UV irradiation is as follows: Figure 2 Lyophilized hydrogels (i.e., irradiated with 365 nm UV light for 180 s to form hydrogels and then freeze-dried) with different cross-linking degrees (i.e., different mass ratios of lyophilized SIS collagen to itaconic anhydride) were ground and pressed with potassium bromide and subjected to Fourier transform infrared spectroscopy. Figure 3 As shown in the figure, the FTIR results show that compared with the pure SIS collagen, the spectrum of the functionalized collagen is at 3432 cm -1 The absorption peak near the amine group is slightly red-shifted and the peak is broadened. This is because itaconic anhydride consumes the amino group and generates an amide bond, so the stretching vibration of the NH in the primary amine in the amino group (3446 cm -1) is weakened, while the secondary amide NH stretching vibration (3300cm -1 At the same time, 1541cm -1 The peak absorption intensity at 47° increased significantly, which is due to the bending vibration of the CH bond in the CH2=CR1R2 structure. The changes in the infrared spectrum prove that itaconic anhydride is grafted onto SIS collagen.

[0037] The hydrogels with different cross-linking degrees prepared in the present invention were treated with 365nm ultraviolet light to measure the gelation time of the hydrogels. The experiment was repeated 3 times. The gelation time of each group of samples is detailed in Figure 4 , basically controlled within 150s.

[0038] After freeze-drying, scanning electron microscopy was used to observe the pore size and distribution. The porosity of the hydrogel was measured by soaking the freeze-dried gel in anhydrous ethanol. Figure 5 and 6 As shown in the figure, the hydrogel presents a porous structure with pore size concentrated in the micron level. With the increase of IA, the pore size of the hydrogel gradually decreases from 110-120 μm to 50-70 μm, and the porosity also gradually decreases.

[0039] The rheological properties of the SIS / IA hydrogel prepared in the present invention were tested under alternating strain test conditions: a fixed test temperature of 37°C, high shear strain (100%) and low shear strain (0.1%), four cycles of action, and a frequency of 1-100 Hz. Figure 7 As can be seen, the injectable composite collagen dispersion prepared by the present invention has good rheological properties and is injectable. Combined with the previous gelation after UV irradiation, the hydrogel has good self-healing properties. Considering the use environment of periodontal treatment, the injectable composite collagen dispersion is injected into the periodontal lesion and solidifies after a short period of UV irradiation. It also has good mechanical properties, which can adapt to the changing mechanical environment of the periodontal lesion (such as occlusion and soft tissue tensile stress in the mouth).

[0040] The hydrogels in different cross-linking groups were placed in a 4% red blood cell suspension to evaluate their hemolytic effect. The results showed that the hydrogels in all cross-linking groups had low hemolytic effects, such as Figure 8 As shown in the figure, the 3IA:SIS group showed better overall results in the above tests and this hemolysis experiment, so this group was used in the following experiments.

[0041] The cross-linked hydrogel and high-glucose cell culture medium DMEM were mixed at a mass ratio of 1:10 and immersed in water. After 24 hours, the mixture was centrifuged and sterilized by filtration with a 0.22 μm filter membrane, and then co-cultured with BMSC cells.

[0042] The effect of the injectable composite hydrogel prepared by the present invention on cell proliferation was tested. The composite hydrogel was co-cultured with rat bone marrow mesenchymal stem cells (BMSCs) for 3 days. Figure 9 As can be seen, compared with the number of cells in the control group without hydrogel, the cells in the experimental group with injectable composite hydrogel showed proliferation, proving that the injectable composite hydrogel can be used for tissue repair. Using acridine orange / ethidium bromide solution (AO / EB) for cell live and dead staining, the cell survival rate was no significantly different from the blank group, and the cell number was significantly increased.

[0043] The cross-linked hydrogel and high-glucose cell culture medium DMEM were mixed and soaked at a mass ratio of 1:10, centrifuged after 24 hours, filtered through a 0.22 μm filter membrane for sterilization, and co-cultured with BMSC cells. After the cells adhered to the wall, they were cultured to a confluence of 70-80%, and osteogenic induction components (dexamethasone, sodium β-glycerophosphate, ascorbic acid) were added. The cells were cultured for another 14 days and then stained with Alizarin Red ARS ( Figure 10 ) and alkaline phosphatase (ALP) staining Figure 11 Alizarin red (ARS) staining can stain calcified nodules of osteoblastic differentiation and can be used as a late marker of osteogenic induction. Alkaline phosphatase (ALP) staining is used as an early quantitative indicator of osteogenic markers. Figure 10 —11 The results demonstrated that the cross-linked hydrogel can be used for osteogenic induction.

[0044] BMACs were seeded in 6-well plates and cultured for 14 days. Total RNA was extracted from cells cultured on each sample using Trizol reagent. RNA was then reverse transcribed into cDNA using a reverse transcription kit (Takara, Japan). RT-PCR was performed using Maxima™ SYBRGreen / ROX qPCR premix (Thermo). All reactions were performed in triplicate. Figure 12 The primer sequences are as follows:

[0045]

[0046] BMSC cells were seeded on a 24-well plate cell slide and induced for 72 hours using osteogenic induction solution containing hydrogel extract. They were then incubated with anti-BMP-2, anti-OPN, and anti-RUNX-2 primary antibodies, washed with PBST overnight, and incubated with secondary antibodies and DAPI. After incubation, the expression of various proteins was observed using a laser confocal microscope, such as Figure 13 From the above tests, it can be seen that the hydrogel of the present invention promotes the expression of osteogenic genes, which is also confirmed at the protein expression level.

[0047] By adjusting the process parameters according to the present invention, hydrogels can be prepared, and testing has shown properties substantially consistent with those of the present invention. The above description of the present invention is illustrative, and it should be noted that any simple variations, modifications, or equivalent substitutions that can be made by those skilled in the art without inventive effort, without departing from the core of the present invention, fall within the scope of protection of the present invention.

Claims

1. An injectable composite collagen dispersion, characterized in that: The invention comprises freeze-dried SIS collagen, itaconic anhydride and a photoinitiator, wherein the mass ratio of the freeze-dried SIS collagen to the itaconic anhydride is 1:(1-5), the solvent is phosphate buffer, and the ratio of the sum of the mass (mg) of the freeze-dried SIS collagen and the itaconic anhydride to the volume (ml) of the solvent is (15-20):(1-5).

2. The injectable composite collagen dispersion according to claim 1, characterized in that: The mass ratio of freeze-dried SIS collagen to itaconic anhydride is 1:(3-5).

3. The injectable composite collagen dispersion according to claim 1 or 2, characterized in that: The ratio of the sum of the mass of freeze-dried SIS collagen and itaconic anhydride (mg) to the volume of the solvent (ml) is (15-20):(1-2).

4. The injectable composite collagen dispersion according to claim 1 or 2, characterized in that: The photoinitiator is 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, and the dosage is 0.1%-0.2% of the total mass of the dispersion; the pH value of the solvent phosphate buffer is 7.4-7.

6.

5. The injectable composite collagen dispersion according to claim 1 or 2, characterized in that: Photosensitive collagen composed of freeze-dried SIS collagen and itaconic anhydride is dispersed in a solvent, and then a photoinitiator is added.

6. A method for preparing an injectable composite collagen dispersion, characterized in that: The acetic acid solution of freeze-dried SIS collagen and the acetone solution of itaconic anhydride are evenly mixed and dialyzed, and then freeze-dried to obtain photosensitive collagen. The photosensitive collagen is then dispersed in a solvent and a photoinitiator is added.

7. The method for preparing an injectable composite collagen dispersion according to claim 6, characterized in that: The mass ratio of freeze-dried SIS collagen to itaconic anhydride is 1:(1-5), preferably 1:(3-5). During the preparation, freeze-dried SIS collagen is taken and uniformly dispersed with a 1% by mass aqueous acetic acid solution to form an acetic acid solution of freeze-dried SIS collagen; the itaconic anhydride is uniformly dispersed in acetone to form an acetone solution of itaconic anhydride, and the mass percentage is consistent with that of the acetic acid solution of freeze-dried SIS collagen; according to the mass ratio of the two solutions, different volumes are selected, mixed uniformly, dialyzed, and then freeze-dried to obtain photosensitive collagen.

8. A hydrogel obtained by using the injectable composite collagen dispersion according to any one of claims 1 to 5, characterized in that: The injectable composite collagen dispersion was photo-initiated to form a hydrogel.

9. The hydrogel according to claim 8, characterized in that Use 365nm ultraviolet light for initiation, and the illumination time is 60-180s, preferably 120-150s.

10. Use of the injectable composite collagen dispersion according to any one of claims 1 to 5 or the hydrogel according to any one of claims 8 to 9 in the preparation of a drug for treating periodontitis.

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