An injectable bone powder having anti-inflammatory biological activity and a preparation method and application thereof

Abstract

This invention discloses an injectable bone powder with anti-inflammatory biological activity, its preparation method, and its application. The bone powder is a hydrogel internally loaded with a hydroxyapatite composite material, wherein the hydroxyapatite composite material is hydroxyapatite with membrane vesicles coated on its surface; the membrane vesicles are derived from pulp-derived suppressor cells; the mass-to-volume ratio of the hydroxyapatite composite material to the hydrogel is 20-30%. This injectable bone powder with anti-inflammatory biological activity, containing membrane vesicles derived from pulp-derived suppressor cells coated on the surface of the hydroxyapatite, has biological effects that inhibit T cell proliferation and macrophage differentiation into osteoclasts, thus blocking the inflammatory environment, suppressing the inflammatory response, and promoting alveolar bone repair and regeneration.

Description

Technical Field

[0001] This invention belongs to the technical field of anti-inflammatory and bone repair materials. More specifically, it relates to an injectable bone powder with anti-inflammatory bioactivity, its preparation method, and its application. Background Technology

[0002] The immune system plays a crucial role in maintaining tissue homeostasis. Overactive innate and adaptive immune cells often trigger a series of inflammatory activities, leading to tissue damage and disease progression. Current treatments, such as systemic administration of broad-spectrum immunosuppressants, inevitably suffer from rapid clearance and systemic side effects, and may even cause opportunistic infections and malignancies. Meanwhile, anti-inflammatory drugs targeting single targets, due to the complex and heterogeneous inflammatory networks, only exert weak inflammatory suppression effects. Therefore, overcoming these limitations and finding new strategies to improve overactive immune cells is essential.

[0003] The rapid development of nanotechnology and bioengineering has provided new opportunities to improve the safety and effectiveness of immunomodulation, enabling precise anti-inflammatory interventions. Nanotechnology-based delivery of immunosuppressants or anti-inflammatory agents can reduce off-target effects and systemic toxicity. However, nanoplatform-based delivery often requires frequent and prolonged high-dose administration. On the other hand, modified nanoparticles often act as decoys to neutralize pro-inflammatory factors and reduce inflammatory responses through specific ligand-receptor interactions. Although this reduces pro-inflammatory factors, nanoparticles cannot directly interact with overactivated cells, allowing pro-inflammatory factors to be continuously produced, thus failing to eliminate them at their source. Furthermore, current engineered nanosystems aim to target single immune cell types, while inflammation often results from the combined action of multiple immune cells. Therefore, finding new strategies to regulate the behavior of multiple dysregulated immune cells is crucial for effectively suppressing excessive immune activation.

[0004] Myeloid-derived suppressor cells (MDSCs) originate from bone marrow hematopoietic progenitor cells and are a collective term for a heterogeneous group of cells with immunosuppressive functions. MDSCs possess immunosuppressive functions, including inhibiting the secretion of amino acids (such as arginine and cysteine) and the production of reactive oxygen species, nitric oxide, and peroxynitrites. These cytokines have immunosuppressive effects. Furthermore, MDSCs express a series of CD protein molecules on their cell membrane surface, which can also exert immunosuppressive effects through signal transduction.

[0005] Periodontitis is a multifactorial disease primarily caused by plaque. It is usually characterized by poor oral hygiene, leading to local inflammation from excessive tartar buildup, accompanied by varying degrees of alveolar bone resorption. Currently, clinical treatment mainly involves basic periodontal therapy to remove plaque and tartar to reduce external irritants, followed by surgical implantation of bone grafts to improve alveolar bone resorption. Since existing bone grafts (composed of hydroxyapatite) lack anti-inflammatory biological effects, postoperatively, patients are routinely instructed to take oral antibiotics for anti-inflammatory treatment, with or without local anti-inflammatory medication applied to the periodontal inflammation sites. Despite this, bone repair failure is commonly observed after treatment due to recurrence of inflammation.

[0006] Therefore, developing a bone powder based on hydroxyapatite that can both promote bone regeneration and exert anti-inflammatory biological effects is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] To address the aforementioned problems in the prior art, the primary objective of this invention is to provide an injectable bone powder with anti-inflammatory biological activity. This bone powder possesses advantages such as high biocompatibility, injectability, inhibition of inflammation (e.g., periodontitis), and promotion of bone regeneration.

[0008] A second objective of this invention is to provide a method for preparing injectable bone powder with anti-inflammatory biological activity.

[0009] A third objective of this invention is to provide the use of the above-mentioned injectable bone powder with anti-inflammatory biological activity in the preparation of a drug that inhibits T cell proliferation.

[0010] A fourth objective of this invention is to provide the use of the above-mentioned injectable bone powder with anti-inflammatory biological activity in the preparation of a drug that inhibits macrophage differentiation into osteoclasts.

[0011] A fifth objective of this invention is to provide the use of the above-mentioned injectable bone powder with anti-inflammatory biological activity in the preparation of medicaments for inhibiting inflammation and / or promoting the repair and regeneration of bone defects.

[0012] The above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0013] An injectable bone powder with anti-inflammatory bioactivity, wherein the bone powder is a thermosensitive hydrogel internally loaded with a hydroxyapatite composite material, wherein the hydroxyapatite composite material is hydroxyapatite with membrane vesicles on its surface; the membrane vesicles are membrane vesicles derived from myeloid-derived suppressor cells; and the mass-to-volume ratio of the hydroxyapatite composite material to the thermosensitive hydrogel is 20-30%.

[0014] This invention provides an injectable bone powder with anti-inflammatory biological activity, possessing advantages such as high biosafety, injectability, anti-inflammatory properties, and promotion of bone regeneration. The invention involves coating the surface of hydroxyapatite with a layer of membranous vesicles derived from pulp-derived suppressor cells. In the microenvironment of periodontitis, these membranous vesicles coated on the nano-hydroxyapatite surface have a biological effect of inhibiting T-cell proliferation, thus blocking the inflammatory environment and suppressing the inflammatory response. Simultaneously, the vesicles also have a biological effect of inhibiting macrophage differentiation into osteoclasts, thereby promoting alveolar bone repair and regeneration. Furthermore, the bone powder prepared by this invention also possesses the characteristics of a thermosensitive hydrogel and is injectable. The bone powder prepared by this invention exhibits good fluidity at 4°C, but gradually gels and loses fluidity at 37°C as the temperature rises, thus providing shaping and retention effects in vivo. We also found that the ratio of the hydroxyapatite composite material to the thermosensitive hydrogel is crucial; if the content of the hydroxyapatite composite material is less than 20%, it will alter the thermosensitive properties of the hydrogel, hindering rapid molding.

[0015] Preferably, the mass ratio of hydroxyapatite to membrane vesicles is 1:1 to 2.

[0016] Preferably, the myeloid-derived suppressor cells are myeloid-derived suppressor cells derived from animals.

[0017] Preferably, the myeloid-derived suppressor cells are myeloid-derived suppressor cells derived from animal bone marrow.

[0018] More preferably, the animal is a mouse.

[0019] Preferably, the hydroxyapatite has a particle size range of 20–100 nm. Injectable bone powder prepared using hydroxyapatite within this particle size range exhibits more suitable osteogenic effects.

[0020] Preferably, the surface of the membrane vesicle contains CTLA4 protein, LAG-3 protein, CD39 protein, CD73 protein and CD27 protein.

[0021] Preferably, the thermosensitive hydrogel is selected from chitosan hydrogels, cellulose hydrogels, polyethylene glycol-polyester hydrogels, and polyethylene glycol-polyamide hydrogels.

[0022] More preferably, the temperature-sensitive hydrogel is a polyethylene glycol-polyamide hydrogel. Commonly used polyethylene glycol-polyamide hydrogels, such as Pluronic F127 hydrogel, are already used clinically and have strong biocompatibility and safety.

[0023] More preferably, the Pluronic F127 hydrogel is prepared by mixing Pluronic F127 with a buffer solution at 2–6°C with stirring. The mass-to-volume ratio of Pluronic F127 to the buffer solution is 20% (m / v). The buffer solution can be selected from conventional buffer solutions in the art; in this invention, it is specifically selected as phosphate-buffered saline (PBS).

[0024] Preferably, the method for preparing the membrane vesicles is as follows: purifying myeloid-derived suppressor cells from an animal, homogenizing them, centrifuging them, and taking the supernatant; centrifuging the supernatant again and taking the centrifuged precipitate, washing it, and obtaining the purified myeloid-derived suppressor cell membrane; the myeloid-derived suppressor cell membrane is then filtered through a microporous membrane to obtain the membrane vesicles.

[0025] Further preferably, as a specific embodiment of the present invention, the method for preparing the membrane vesicles is as follows: Mouse femurs are obtained, and the bone marrow contents are extracted by irrigation with PBS buffer. The bone marrow contents are then filtered sequentially through 70μm and 40μm filters to obtain a cell suspension. The cell suspension is then purified into mouse myeloid-derived suppressor cells (MDSCs) using a mouse myeloid-derived suppressor cell sorting kit. The purified mouse myeloid-derived suppressor cells are homogenized and then centrifuged at 18000–22000g for 30 minutes. The supernatant is collected and centrifuged at 75000–85000g for 2 hours. The supernatant is discarded, and the precipitate is washed with EDTA and Tris-HCl to obtain purified myeloid-derived suppressor cell membranes. These membranes are then sequentially passed through 400nm and 200nm microporous membranes to obtain membrane vesicles derived from myeloid-derived suppressor cells.

[0026] Furthermore, this invention also claims protection for a method for preparing injectable bone powder with anti-inflammatory biological activity, comprising the following steps:

[0027] (1) Purify myeloid-derived suppressor cells from animal bodies, homogenize, centrifuge, and collect the supernatant; centrifuge the supernatant again, collect the centrifuged precipitate, wash, and obtain the purified myeloid-derived suppressor cell membrane; the myeloid-derived suppressor cell membrane is then filtered through a microporous membrane to obtain the membrane vesicles;

[0028] (2) The hydroxyapatite is mixed with the membrane vesicles, and then extruded using a microporous membrane and centrifuged to obtain the hydroxyapatite composite material; the hydroxyapatite composite material is mixed with a thermosensitive hydrogel and stirred to obtain injectable bone powder with anti-inflammatory biological activity.

[0029] Preferably, in step (2), after mixing the hydroxyapatite with the membrane vesicles, the mixture is extruded multiple times through 400nm and 200nm microporous membranes. Preferably, the number of extrusions is 8 to 15.

[0030] Preferably, in step (2), the stirring temperature is 2-6°C; the stirring speed is 600-1000 rpm; and the stirring time is 1-3 hours.

[0031] Furthermore, the present invention also claims protection for the use of the above-mentioned injectable bone powder with anti-inflammatory biological activity in the preparation of a drug for inhibiting T cell proliferation.

[0032] Furthermore, the present invention also claims protection for the use of the above-mentioned injectable bone powder with anti-inflammatory biological activity in the preparation of a drug that inhibits macrophage differentiation into osteoclasts.

[0033] Furthermore, the present invention also claims protection for the use of the above-mentioned injectable bone powder with anti-inflammatory biological activity in the preparation of medicaments for inhibiting inflammation and / or promoting the repair and regeneration of bone defects.

[0034] Furthermore, the inflammation is caused by T cell proliferation.

[0035] Furthermore, the inflammation is periodontitis.

[0036] Furthermore, the bone defect is caused by macrophages differentiating into osteoclasts.

[0037] Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides an injectable bone powder with anti-inflammatory biological activity, which has the advantages of high biosafety, injectability, inhibition of inflammation, and promotion of bone regeneration. The membrane vesicles derived from medullary suppressor cells encapsulated on the surface of hydroxyapatite have biological effects of inhibiting T cell proliferation and macrophage differentiation into osteoclasts, thus blocking the inflammatory environment, inhibiting the inflammatory response, and promoting alveolar bone repair and regeneration. Furthermore, the bone powder prepared by the present invention has the characteristics of a thermosensitive hydrogel, exhibiting shaping and retention effects in vivo. It has good fluidity at 4°C and gradually gels and loses fluidity at 37°C. Attached Figure Description

[0038] Figure 1 The image shows a scanning electron microscope (SEM) image of the Pluronic F127 hydrogel and nHMF from Example 1.

[0039] Figure 2 This is a schematic diagram showing the flow properties of nHMF at different temperatures in Example 1.

[0040] Figure 3 The degradation of nHMF in Example 1 varies at different times.

[0041] Figure 4 The rheological curves of Pluronic F127 hydrogel and nHMF in Example 1 are shown at different temperatures.

[0042] Figure 5 The elastic modulus and loss modulus of the Pluronic F127 hydrogel in Example 1 are shown to change under different stresses.

[0043] Figure 6 The elastic modulus and loss modulus of nHMF in Example 1 are shown to change under different stresses.

[0044] Figure 7 The protein expression of MDSCs, mMDSCs, and nHM in Example 1 is shown.

[0045] Figure 8 This is a quantitative analysis diagram of the differentiation of mouse macrophages into osteoclasts under different treatment groups.

[0046] Figure 9 This is a graph showing the quantitative analysis of mouse T cell proliferation under different treatment groups.

[0047] Figure 10 Micro-CT images of bone repair after treatment with nHF and nHMF in Example 1 in a rat periodontitis model.

[0048] Figure 11 The CEJ-ABC values ​​of the two groups of rats under different treatment groups are shown. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0050] Example 1: Preparation of an injectable bone powder with anti-inflammatory bioactivity

[0051] (1) Add 2 mg of polyoxyethylene polypropylene triblock polymer (Pluronic F127) powder to 10 mL of phosphate buffer (PBS), stir in a cold room at 4 °C (800 rpm) for 4 hours to obtain 20% (m / v) Pluronic F127 hydrogel, and store at 4 °C.

[0052] (2) Take the femur of a mouse, cut off both ends of the femur with scissors, draw PBS with a syringe, and remove the bone marrow contents by irrigation. The bone marrow contents are filtered through 70μm and 40μm filters to obtain cell suspension. The myeloid-derived suppressor cells are purified using a mouse myeloid-derived suppressor cell (MDSCs) sorting kit, and the sorting effect is detected by flow cytometry.

[0053] (3) The purified cells were processed using a Dunns homogenizer, and the resulting solution was centrifuged at 20,000 g for 30 minutes. The supernatant was collected and centrifuged at 80,000 g for 2 hours. The supernatant was discarded, and the precipitate was washed once with 1 mM EDTA and 10 mM Tris-HCl to obtain a purified MDSCs membrane. Then, the MDSCs membrane was passed through 400 nm and 200 nm microporous membranes multiple times using a microextruder to obtain membrane vesicles derived from myeloid repressive cells. Subsequently, the membrane vesicles derived from myeloid repressive cells were mixed with nano-hydroxyapatite (nHA) with a particle size range of 20–100 nm at a mass ratio of 1:1, and extruded sequentially through 400 nm and 200 nm microporous membranes 11 times each. After centrifugation, excess membrane vesicles were removed to obtain a hydroxyapatite composite material (i.e., hydroxyapatite with MDSCs membrane vesicles coated on the surface, nHA@MDSCs, abbreviated as nHM).

[0054] (4) Add 2.5 mg of nHM to 10 ml of the Pluronic F127 hydrogel prepared in step (1) and stir with a magnetic stirrer in a cold room at 4°C (800 rpm) for 2 hours to obtain the final product, injectable bone powder with anti-inflammatory biological activity (Pluronic F127 hydrogel loaded with nHM, nHA@MDSCs@F127, abbreviated as nHMF).

[0055] Example 2: Preparation of an injectable bone powder with anti-inflammatory bioactivity

[0056] The difference between this embodiment and Example 1 is that: in step (3), the membrane vesicles derived from myeloid-derived suppressor cells are mixed with nano-hydroxyapatite (nHA) at a mass ratio of 1.5:1; in step (4), 2.5 mg of nHM is added to 8.34 ml of the Pluronic F127 hydrogel prepared in step (1).

[0057] Example 3: Preparation of an injectable bone powder with anti-inflammatory bioactivity

[0058] The difference between this embodiment and Example 1 is that: in step (3), the membrane vesicles derived from myeloid-derived suppressor cells are mixed with nano-hydroxyapatite (nHA) at a mass ratio of 2:1; in step (4), 2.5 mg of nHM is added to 12.5 ml of the Pluronic F127 hydrogel prepared in step (1).

[0059] Test case

[0060] (1) Microscopic morphology characterization of the injectable bone powder prepared in Example 1

[0061] The morphology of the Pluronic F127 hydrogel (F127hydrogel) prepared in step (1) and the nHMF prepared in step (4) of Example 1 were observed using scanning electron microscopy. Figure 1 As shown, scanning electron microscopy revealed that the hydroxyapatite composite material (nHM) in nHMF (nHA@MDSCs@F127) is uniformly distributed within the Pluronic F127 hydrogel.

[0062] (2) Thermosensitive properties test of the injectable bone powder prepared in Example 1

[0063] The Pluronic F127 hydrogel prepared in step (1) of Example 1 and the nHMF prepared in step (4) were observed at 4°C and 37°C, respectively. Figure 2 The diagram shows the flow properties of nHMF at 4℃ and 37℃. The behavior of nHMF is consistent with Pluronic F127 hydrogel, exhibiting good flowability at 4℃, but gelling and losing flowability at 37℃. This indicates that nHMF can be used as bone powder for injection applications and has shaping and retention effects in vivo.

[0064] (3) Degradation characteristics test of the injectable bone powder prepared in Example 1

[0065] The degradation characteristics of nHMF (nHA@MDSCs@F127) prepared in Example 1 were tested, and the degradation characteristics of nHMF were detected. The results are as follows: Figure 3 As shown, nHMF gradually degrades over time, and is basically degraded after 72 hours, which indicates that nHMF can provide space for new bone growth.

[0066] (4) Dynamic mechanical property test of the injectable bone powder prepared in Example 1

[0067] Rheometers were used to analyze the Pluronic F127 hydrogel (F127hydrogel) prepared in step (1) of Example 1 and the nHMF (F127@nHA@mMDSCs) prepared in step (4). The results are as follows: Figure 4 As shown, the addition of nHM does not change the temperature-sensitive properties of Pluronic F127, and also demonstrates that nHMF possesses a certain degree of mechanical strength. Figure 5 and Figure 6 The figures shown are the storage modulus and loss modulus of Pluronic F127 hydrogel and nHMF, respectively. Figure 5 and Figure 6The comparison shows that when the strain is less than 10%, the storage modulus and loss modulus of nHMF are much higher than those of Pluronic F127 hydrogel. However, as the strain gradually increases, the storage modulus and loss modulus of both nHMF and Pluronic F127 hydrogel gradually decrease and approach 0.

[0068] (5) Western blot analysis of protein composition on the surface of nHM in Example 1

[0069] The protein composition on the surface of the nHM prepared in Example 1 was detected using Western blotting. The results are as follows: Figure 7 As shown in the figure, the nHM prepared in Example 1 expresses CTLA4, LAG-3, CD39, CD73, and CD27 on its surface. This indicates that the nHMF in Example 1 also expresses these proteins and can play an immunomodulatory role.

[0070] (6) Detection of the regulatory effect of nHM on the differentiation of macrophages into osteoclasts in Example 1

[0071] The nHM prepared in Example 1 was co-incubated with mouse macrophages RAM264.7, with MCSF (30 ng / mL) and RANKL (50 ng / mL) added. After 7 days of culture, the cells were stained with TRAP staining kit, and the number of osteoclasts was counted under a microscope. The above nHM was replaced with nano-hydroxyapatite (nHA) as a control group, and the cells without nHM were used as a blank control group.

[0072] The results are as follows Figure 8 As shown, the number of osteoclasts in the nHM group was significantly less than that in the nHA group and the blank control group, indicating that nHM inhibits the differentiation of macrophages into osteoclasts, blocks osteocytes, and promotes bone repair.

[0073] (7) Detection of the regulatory effect of injectable bone powder prepared in Example 1 on T cells

[0074] Mouse spleen cell single-cell suspensions were prepared and activated for 4 days with anti-CD3 (1.5 μg / mL), anti-CD28 (1 μg / mL), and IL-2 (200 U / mL). The activated spleen cell single-cell suspensions (containing T cells) were co-incubated with nHM from Example 1, and T cell proliferation was detected by CFSE. The above-mentioned nHM was replaced with nano-hydroxyapatite (nHA) as a control group, and the group without nHM served as a blank control group.

[0075] The results are as follows Figure 9 As shown, the number of T cells proliferating in the nHM group was significantly less than that in the nHA group and the blank control group, indicating that nHM can inhibit T cell proliferation and that nHMF can suppress the inflammatory response.

[0076] (8) To test the effect of the injectable bone powder prepared in Example 1 on bone regeneration in periodontitis.

[0077] A suture was embedded in the gingiva of the second molar of a rat to induce periodontitis as an exogenous stimuli. The rats were implanted with the suture for 7 days to establish a periodontitis model. The rats were then divided into two groups. One group was given F127 hydrogel (nHF) without MDSC membrane vesicles (nHA@F127), and the other group was given nHMF treatment as described in Example 1. The rats were euthanized after 1 month, and the repair morphology of bone defects was examined by micro-CT.

[0078] The results are as follows Figure 10 and Figure 11 As shown, compared with the nHF treatment group, the CEJ-ABC value of the nHMF group was significantly smaller, and the amount of regenerated bone was better in the nHMF group. This indicates that nHMF can better promote bone regeneration.

[0079] The injectable bone powder prepared in Examples 2 and 3 was used in the same animal experiments and showed the same results.

[0080] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An injectable bone powder with anti-inflammatory biological activity, characterized in that, The bone powder is a thermosensitive hydrogel internally loaded with a hydroxyapatite composite material, wherein the hydroxyapatite composite material is hydroxyapatite with membrane vesicles coated on its surface; the membrane vesicles are membrane vesicles derived from myeloid-derived suppressor cells; the mass-to-volume ratio of the hydroxyapatite composite material to the thermosensitive hydrogel is 20-30%. The mass ratio of hydroxyapatite to membrane vesicles is 1:1 to 2; The particle size range of the hydroxyapatite is 20–100 nm; The surface of the membrane vesicles contains CTLA4 protein, LAG-3 protein, CD39 protein, CD73 protein and CD27 protein; The thermosensitive hydrogel is Pluronic F127.

2. The injectable bone powder according to claim 1, characterized in that, The myeloid-derived suppressor cells are myeloid-derived suppressor cells derived from animal bodies.

3. The method for preparing the injectable bone powder with anti-inflammatory biological activity as described in any one of claims 1 to 2, characterized in that, Includes the following steps: (1) Purify myeloid-derived suppressor cells from animals, homogenize, centrifuge, and collect the supernatant; After centrifuging the supernatant again, the precipitate was collected, washed, and purified myeloid-derived inhibitory cell membrane was obtained; the myeloid-derived inhibitory cell membrane was then filtered through a microporous membrane to obtain the membrane vesicles; (2) The hydroxyapatite is mixed with the membrane vesicles, and then extruded using a microporous membrane and centrifuged to obtain the hydroxyapatite composite material; The hydroxyapatite composite material was mixed with a thermosensitive hydrogel and stirred to obtain injectable bone powder with anti-inflammatory bioactivity.

4. The use of the injectable bone powder with anti-inflammatory biological activity as described in any one of claims 1 to 2 in the preparation of a drug that promotes the repair and regeneration of bone defects.

Images