Bone substitute material containing thermally-inactivated Ackerman muciniphila and application of bone substitute material

Abstract

The invention relates to a bone substitute material containing thermally-inactivated Ackerman muciniphila and application thereof, and belongs to the technical field of oral medical materials. The invention provides a novel probiotic-modified hydroxyapatite microsphere which can resist colonization of pathogenic bacteria, reduce infection, activate functional change of macrophages, cause a novel biological effect, improve a bone immune microenvironment state and accelerate bone tissue repair and regeneration, so that the novel probiotic-modified hydroxyapatite microsphere can be used for repairing and regenerating bone tissues. The compound can be applied to preparation of related bone regeneration filling materials and biological regulators. The immunoregulation active material prepared by the invention has good biocompatibility, resists the formation of biological membranes and has better bone formation characteristics.

Description

Technical Field

[0001] The invention relates to a bone substitute material containing heat-killed Akkermansia muciniphila and application thereof, belonging to the technical field of oral medicine materials. Background Art

[0002] Bone regeneration surgery is one of the key technologies for reconstructing the supporting function of periodontal tissue for affected teeth. The combined application of guided tissue regeneration (GTR) and bone grafting is a commonly used regenerative treatment technique in clinical practice. Usually, after flap debridement, bone substitute materials are filled into the subbone pocket and covered with a barrier membrane to promote periodontal bone regeneration. Infection control and regulation of osteoblast differentiation are the key to bone regeneration. The bone substitute materials currently used mainly focus on the direct regulation of osteoblasts, while ignoring the resistance to pathogen colonization infection and the construction of an immune microenvironment conducive to bone regeneration. Summary of the invention

[0003] In view of the shortcomings of the prior art, the object of the present invention is to provide a hydroxyapatite microsphere modified with the probiotic Akkermansia muciniphila as a bone replacement material, which has anti-pathogen adhesion and immunomodulatory activity to promote periodontal bone repair.

[0004] A bone replacement material containing heat-killed Akkermansia muciniphila comprises a bone repair material, and the bone repair material is loaded with probiotics.

[0005] The bone repair material is hydroxyapatite.

[0006] The hydroxyapatite has a diameter of 80-100 μm.

[0007] The probiotic is Akkermansia muciniphila.

[0008] The probiotics may or may not be inactivated.

[0009] The method for preparing the above-mentioned bone substitute material comprises the following steps: Step 1, placing a bone substitute material in a bacterial solution containing probiotics for culturing, so that the probiotics adhere to the surface of the bone substitute material; Step 2: washing and centrifugation.

[0010] In the step 1, after the cultivation is completed, an inactivation treatment is performed.

[0011] The culture conditions are: 2-3 days under anaerobic conditions, the OD600 value of the bacterial solution is in the range of 0.5-1.5, and the concentration of the bone substitute material in the bacterial solution is 10-50 mg / mL.

[0012] Phosphate buffer solution was used for washing.

[0013] Use of bone substitute materials containing heat-killed Akkermansia muciniphila in the preparation of medical materials for periodontal bone regeneration. Beneficial Effects

[0014] The Akkermansia muciniphila used in the present invention Akkermansia muciniphila ) is a probiotic belonging to the phylum Verrucomicrobia that can inhibit the periodontal pathogen Porphyromonas gingivalis ( P. gingivalis )、Fusobacterium nucleatum( F. nucleatum ) colonization and growth, regulate macrophage inflammatory response and promote the secretion of osteogenic related cytokines, thereby enhancing osteogenesis. In addition, A. muciniphila The effective molecules can still be retained after inactivation, which greatly enhances the safety of in vivo application. A. muciniphila It has the characteristics of wide therapeutic effect, stable colonization, and still effective after inactivation, and is considered to be the next generation of star probiotics. In terms of safety assessment, it has been conducted in many animal and clinical trials, demonstrating good biosafety.

[0015] The present invention selects A. muciniphila The currently clinically available hydroxyapatite (HA) is modified to construct a new bone replacement material, which is pasteurized to resist pathogenic bacterial colonization and infection while creating a more effective bone regeneration microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 . Prepared A. muciniphila Scanning electron microscopy images of modified HA microspheres.

[0017] Figure 2 . Prepared A. muciniphila Modified HA microspheres ( Akk SYTO9 / PI fluorescence staining of modified HA and unmodified HA.

[0018] Figure 3 . A. muciniphila Modified HA microspheres resist periodontal pathogens P. gingivalis , F. nucleatum Adhesion and colonization.

[0019] Figure 4 . A. muciniphila Comparison of modified HA microspheres regulating the production of Raw264.7 factors in the mouse macrophage cell line and promoting OSM production.

[0020] Figure 5 . A. muciniphila Modified HA microspheres are used in periodontitis bone defects to resist the adhesion of periodontal pathogens.

[0021] Figure 6 . A. muciniphilaModified HA microspheres promote the repair of periodontitis bone defects. DETAILED DESCRIPTION

[0022] The Akkermansia muciniphila used in the following examples A. muciniphila It is deposited in China Industrial Microbiological Culture Collection Center with the accession number CICC 24917.

[0023] The hydroxyapatite microspheres used had a diameter of 80-100 μm and were obtained from Shanghai Yipuri Biotechnology Co., Ltd.

[0024] Hydroxyapatite microspheres (20 mg / mL) were mixed with A. muciniphila Co-cultivation under anaerobic conditions for 3 days promoted A. muciniphila Adhesion to the surface of hydroxyapatite A. muciniphila -HA. Maintain at 70°C for 30 minutes to inactivate. Centrifuge at 4000rpm for 5 minutes to fully remove the culture supernatant. Add phosphate buffer for washing, centrifuge at 4000rpm for 5 minutes to remove the upper liquid to remove free bacteria and other substances in the culture medium, repeat twice. The obtained probiotic-modified bone substitute material is stored at 4°C for use.

[0025] Hydroxyapatite microspheres and OD600 = 1 A. muciniphila The bacteria were co-cultured under anaerobic conditions for 1, 3 and 7 days. Through the pathogen colonization resistance test experiment and macrophage viability test, it was found that under the condition of co-culture for 1 day, akk had less adhesion on the HA surface and failed to form a continuous biofilm, which could not effectively resist periodontal pathogens. P. gingivalis and F. nucleatum Colonization; co-cultivation for 7 days, A. muciniphila -HA has a certain inhibitory effect on macrophage activity. The following experiments used microspheres obtained by culturing for 3 days.

[0026] 3. Application The hydroxyapatite microspheres modified by the probiotic Akkermansia muciniphila of the present invention are used in medicines for treating bone defects, bone trauma, etc., including but not limited to periodontal bone defects, maxillofacial bone defects and other trunk bone defects.

[0027] The present invention has been proven in vitro to be effective against periodontal pathogen Porphyromonas gingivalis P. gingivalis Fusobacterium nucleatum F. nucleatum Colonization can promote macrophages to secrete anti-inflammatory and osteogenic-related cytokines such as IL-10 and OSM to promote bone defect repair. This was demonstrated in vivo in periodontal bone defects in mice, indicating that it has the ability to resist pathogenic bacteria colonization and promote bone repair.

[0028] The present invention provides a new probiotic-modified hydroxyapatite microsphere, which can resist the colonization of pathogenic bacteria and reduce the occurrence of infection, while activating the functional changes of macrophages, causing new biological effects, improving the state of bone immune microenvironment, accelerating bone tissue repair and regeneration, and can be used in the preparation of related bone regeneration filling materials and bioregulators. The immunomodulatory active material prepared by the present invention has good biocompatibility, resists the formation of biofilm, and has better bone formation characteristics. Figure 1 It is prepared A. muciniphila Scanning electron micrograph of modified HA microspheres. The selected HA microspheres are about 80-100 μm in diameter. After modification, the surface is coated with oval A. muciniphila .

[0029] Figure 2 It is prepared A. muciniphila Modified HA microspheres ( Akk -HA) and unmodified HA were stained with SYTO9 / PI. A. muciniphila The modified HA surface adheres to a large amount of A. muciniphila and is fully inactivated (red fluorescent label).

[0030] Figure 3 yes A. muciniphila Modified HA microspheres resist periodontal pathogens P. gingivalis , F. nucleatum The quantitative method is: unmodified HA and A. muciniphila Modified HA microspheres ( Akk -HA) respectively with P. gingivalis , F. nucleatum After co-cultivation at 37°C in an anaerobic environment for 24 h, the bacterial genomic DNA on the HA surface was extracted, and the pathogenic bacteria adhering to its surface were quantified by real-time fluorescence quantitative PCR.

[0031] Figure 4 yes A. muciniphila Modified HA microspheres promoted the production of higher levels of IL-10 and OSM by the mouse macrophage cell line Raw264.7. The quantitative method was: RAW264.7 cells were given equal amounts of A. muciniphila Modified HA microspheres ( Akk-HA ) and unmodified HA, and the group with culture medium alone was used as the CON group. After culturing in an incubator at 37°C and 5% CO2 for 24 h, the cell culture supernatant was collected, and the expression levels of IL-10 and OSM in the supernatant were detected by enzyme-linked immunosorbent assay.

[0032] Figure 5 A. muciniphilaModified HA microspheres were used in periodontitis bone defects to resist the adhesion of periodontal pathogens. C57BL / 6 mice were used to construct a periodontitis bone defect model by silk ligation and local application of periodontal pathogens. Akk -HA group filled the periodontal defect area A. muciniphila The CON group was given an equal amount of solvent, and the HA group was filled with an equal amount of unmodified HA microspheres. After 1 week, periodontal tissues were collected for oscillation, and the liquid was plated for bacterial counts, and bacterial DNA was extracted for quantitative analysis. The results showed: A. muciniphila Modified HA microspheres effectively resist periodontal pathogens P. gingivalis and F. nucleatum of colonization.

[0033] Figure 6 Shown A. muciniphila Modified HA microspheres promote the repair of periodontal infection-induced bone defects. C57BL / 6 mice were used to establish a periodontitis bone defect model by silk ligation and local application of periodontal pathogens. Akk -HA group filled the periodontal defect area A. muciniphila The CON group was given an equal amount of solvent, and the HA group was filled with an equal amount of unmodified HA microspheres. After 4 weeks, the tissue repair was evaluated by microCT and histopathological sections, and the results showed: A. muciniphila Modified HA microspheres can significantly promote the repair of periodontal bone tissue and the formation of new periodontal attachment.

Claims

1. A bone substitute material containing heat-killed Akkermansia muciniphila, characterized in that: The invention comprises bone repair materials, and the bone repair materials are loaded with probiotics.

2. The bone substitute material containing heat-killed Akkermansia muciniphila according to claim 1, characterized in that: The bone repair material is hydroxyapatite.

3. The bone substitute material containing heat-killed Akkermansia muciniphila according to claim 1, characterized in that: The hydroxyapatite has a diameter of 80-100 μm.

4. The bone substitute material containing heat-killed Akkermansia muciniphila according to claim 1, characterized in that: The probiotics are Akkermansia muciniphila; the probiotics are heat-inactivated.

5. The method for preparing a bone substitute material containing heat-killed Akkermansia muciniphila according to claim 1, characterized in that: The steps include: Step 1, placing a bone substitute material in a bacterial solution containing probiotics for culturing, so that the probiotics adhere to the surface of the bone substitute material; Step 2: washing and centrifugation.

6. The preparation method according to claim 5, characterized in that: In the step 1, after the cultivation is completed, an inactivation treatment is performed.

7. The preparation method according to claim 5, characterized in that: The culture conditions are: 2-3 days under anaerobic conditions, the OD600 value of the bacterial solution is in the range of 0.5-1.5, and the concentration of the bone substitute material in the bacterial solution is 10-50 mg / mL.

8. The preparation method according to claim 5, characterized in that: Phosphate buffer solution was used for washing.

9. Use of the bone substitute material containing heat-killed Akkermansia muciniphila according to claim 1 in preparing medical repair materials for bone defects or bone trauma.

10. The use according to claim 9, characterized in that Bone defects include, but are not limited to, periodontal bone defects, maxillofacial bone defects, and other trunk bone defects.

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