Allicin-loaded platelet-rich plasma gel system and preparation method thereof

Abstract

The invention relates to an allicin-loaded platelet rich plasma (PRP) gel system and a preparation method thereof. The preparation method comprises the following steps: S1, adding sodium citrate into a phosphate buffer solution to obtain an anticoagulant solution; centrifuging the whole blood, sucking bottom layer red blood cells, centrifuging again, sucking upper layer plasma, and shaking to obtain pretreated platelet-rich plasma; injecting platelet-deficient plasma into the pretreated platelet-rich plasma to enable the platelet content to reach a preset threshold value to obtain platelet-rich plasma; s2, dissolving thrombin into a calcium ion solution to obtain a coagulant solution; adding the allicin injection into the coagulant solution to obtain an allicin / coagulant solution; and mixing the allicin / coagulant solution with the platelet-rich plasma, and stirring. The allicin-loaded PRP gel system prepared by the invention is compact and uniform in fibrin, has the effect of obviously inhibiting inflammatory mediators and cytokines in infection, and can effectively promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and prevent and treat inflammatory bone destruction.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a platelet-rich plasma gel system loaded with allicin and a preparation method thereof. Background Art

[0002] Traumatic osteomyelitis has a long treatment cycle, many surgeries, many complications, high amputation rate and mortality rate, which is a global problem that plagues the treatment of war injuries. The mechanism of inflammatory bone destruction and infected bone defect formation caused by traumatic osteomyelitis is: pathogens activate the body's natural and innate immune response to cause systemic and local inflammatory response, and produce a large number of inflammatory mediators and cytokines at the lesion site. These cytokines can destroy the dynamic balance of bones, inhibit osteoblast differentiation, and activate osteoclasts, thereby accelerating bone loss. At present, the mainstream Western medicine technology for traumatic osteomyelitis is repeated debridement, irrigation, antibiotics, negative pressure drainage, external fixation bracket fixation of fracture ends, and after the bone and soft tissue infection is controlled, the second stage is Papineau bone grafting technology, Ilizarov bone transfer technology or membrane-induced osteogenesis technology (Masquelet technology). The internal treatment of traditional Chinese medicine adopts syndrome differentiation and treatment, and treats according to different syndromes. According to different syndromes, oral Chinese medicine treatments such as dispelling wind and dampness, clearing heat and detoxifying, tonifying spleen and kidney, and replenishing qi and blood are given; the external treatment method is to give Chinese medicine in different dosage forms, external application, fumigation, or debridement to remove pus and rot, remove blood stasis and regenerate new. Since inflammatory bone destruction is mainly the result of host response rather than the direct effect of pathogen response, surgical debridement and antibiotic treatment to control infection cannot completely prevent the development of inflammatory bone loss. The mainstream view that inflammatory cytokines inhibit osteoblast differentiation and induce osteoclast differentiation and formation in inflammatory, autoimmune and infectious diseases, leading to bone loss, has been widely recognized. Therefore, preventing the expression of inflammatory cytokines or RANKL (osteoclast differentiation factor) is a problem that cannot be bypassed and must be overcome in medicine. However, in the entire treatment process, both Chinese and Western medicine have no drugs to inhibit inflammatory bone destruction caused by traumatic osteomyelitis. Therefore, how to control osteomyelitis while reducing inflammatory bone destruction and infectious defects has become one of the focuses of medical research at home and abroad.

[0003] With the rise of tissue engineering, developing materials with dual properties of both antibacterial effects and promoting bone healing has become a new direction, and at the same time, it has also made biomaterials an effective way to treat infectious bone defects. Among them, platelet-rich plasma (PRP) is an important achievement in tissue engineering. Research reports show that it has good biocompatibility and certain antibacterial properties. On the other hand, platelets are rich in various growth factors and cytokines, including PDGF-BB (platelet-derived growth factor BB) and TGF-β (transforming growth factor β), etc. These growth factors play important roles in promoting tissue repair and regeneration, including stimulating the proliferation and differentiation of osteoblasts, thus promoting the formation of new bone tissue. Moreover, PRP is considered to have potential benefits for bone regeneration and shows very broad clinical application potential.

[0004] Currently, cytokine inhibitors used to treat inflammatory diseases include targeted IL-1, IL-6R, IL-17, IL-12 / 23 inhibitors, etc. However, the above-mentioned drugs all have defects to varying degrees, such as not yet being developed and applied clinically, being expensive, having a short action time, requiring high storage conditions, poor stability, and not being able to be used locally for osteomyelitis, etc. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a platelet-rich plasma gel system loaded with allicin and its preparation method.

[0006] In the first aspect, the present invention provides a preparation method of a platelet-rich plasma gel system loaded with allicin, which is characterized by including the following steps:

[0007] S1. Add sodium citrate to phosphate buffer solution to obtain an anticoagulant solution;

[0008] After sucking the anticoagulant solution with a syringe, draw whole blood. After centrifuging the whole blood, discard the bottom layer of red blood cells, and after centrifuging again, suck the upper layer of plasma (i.e., platelet-poor plasma), and shake it to obtain pretreated platelet-rich plasma;

[0009] Inject platelet-poor plasma into the pretreated platelet-rich plasma to make the platelet content reach a preset threshold to obtain platelet-rich plasma;

[0010] S2. Dissolve thrombin in calcium ion solution to obtain a coagulant solution;

[0011] Add allicin injection to the coagulant solution to obtain an allicin / coagulant solution;

[0012] Mix the allicin / coagulant solution with the platelet-rich plasma and stir to prepare the platelet-rich plasma gel system loaded with allicin.

[0013] Optionally, in the anticoagulant solution described in step S1, the concentration of sodium citrate is 3-4 g / 100 mL, preferably 3.5-4 g / 100 mL.

[0014] Optionally, in step S1, aspirate the bottom layer of red blood cells to a position 0.5-1.5 cm below the platelet layer.

[0015] Optionally, in step S1, during the second centrifugation process, the rotation speed is 2500-3500×g, and the centrifugation duration is 8-15 min.

[0016] Optionally, in step S1, during the centrifugation process, the rotation speed is 2000-3000×g, and the centrifugation duration is 8-15 min.

[0017] Optionally, in step S1, the preset threshold is greater than or equal to 3000×10 9 / L.

[0018] Optionally, in the coagulant described in step S2, the concentration of thrombin is 500-2000 U / 0.5-2 mL.

[0019] Optionally, in step S2, the concentration of allicin injection is 3 mg / mL.

[0020] Optionally, in step S2, stir at a rotation speed of 200-500 r / min.

[0021] In a second aspect, the present invention provides a platelet-rich plasma gel system loaded with allicin prepared according to the method described above.

[0022] The beneficial effects of the present invention are as follows:

[0023] Due to the weak anticoagulant effect of allicin, the platelet-rich plasma gel system loaded with allicin prepared by the present invention neutralizes with thrombin, making the fibrin formed in PRP have a more uniform and dense network structure.

[0024] The platelet-rich plasma gel system loaded with allicin prepared by the present invention has an obvious effect of inhibiting the expression of inflammatory mediators and cytokines in infection.

[0025] The platelet-rich plasma gel system loaded with allicin prepared by the present invention has the ability to effectively promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, and can prevent and treat inflammatory bone destruction.

[0026] The allicin-loaded platelet-rich plasma gel system prepared by the present invention is applied to the site of inflammatory bone destruction, and releases allicin from the inside out within a certain period of time, which can inhibit the release of inflammatory mediators and cytokines in the body, filling the gap that there is no locally applied drug for inflammatory bone diseases on the current market and solving the technical problem of local application of inflammatory mediator and cytokine inhibitors.

[0027] Allicin and its main component diallyl disulfide (DADS) have been proven to have a variety of pharmacological properties and chemopreventive effects such as anti-inflammatory, antioxidant, anti-tumor, anticoagulant, etc. The applicant first discovered that the main component DADS in allicin can effectively inhibit the formation, fusion and bone resorption function of osteoclasts, reduce LPS-induced infectious bone destruction, and the platelet-rich plasma loaded with allicin delivery system (PADS) can promote the proliferation of bone marrow mesenchymal stem cells and improve the osteogenic inhibition of bone marrow mesenchymal stem cells by inflammatory mediators, suggesting that PADS has significant application value and clinical significance in the treatment of traumatic osteomyelitis.

[0028] In summary, the allicin-loaded platelet-rich plasma gel system prepared by the present invention has a relatively uniform microscopic three-dimensional grid structure, and allicin that inhibits inflammatory mediators and cytokines is loaded on this three-dimensional grid structure, which can assist in the treatment of inflammatory bone destruction including osteomyelitis. Brief Description of the Drawings

[0029] Figure 1 It is a scanning electron microscope image of ordinary platelet-rich plasma and the allicin-loaded platelet-rich plasma gel system prepared by the present invention placed on a cell slide (in the form of a membrane). Among them, 1A, 1B, and 1C are the ordinary platelet-rich plasma group magnified 1000, 2000, and 5000 times, and 1D, 1E, and 1F are the allicin-loaded platelet-rich plasma gel system magnified 1000, 2000, and 5000 times.

[0030] Figure 2 It is a result diagram of the protective effect of the effective component diallyl disulfide monomer (DADS) of allicin on LPS-induced inflammatory bone destruction of mouse skulls. 2A is the microscopic CT scan and three-dimensional reconstruction diagram of the skull; 2B is the percentage quantitative detection result diagram of bone volume / total volume (BV / TV); 2C is the quantitative analysis result diagram of the porosity ratio in the mouse skull;

[0031] Figure 3Results graphs of histological and histomorphometric analysis of the effect of DADS on LPS-induced inflammatory bone destruction in mouse skulls and results graphs of serum inflammatory factor levels. 3A is the H&E staining result graph of skull sections, 3B is the TRAP staining result graph of skull sections, 3C is the quantitative analysis result graph of the percentage of bone area in the border area of H&E staining of skull sections, 3D is the quantitative analysis result graph of the number of TRAP(+) cells after TRAP staining of skull sections, and 3E is the result graph of serum TNF-α, IL-1β, and IL-6 levels.

[0032] Figure 4 Results graph of the proliferation effect of platelet-rich plasma loaded with allicin delivery system (PADS) on bone marrow mesenchymal stem cells in an LPS-induced inflammatory microenvironment studied by CCK8 assay. 4A is the cell proliferation result at 24 hours; 4B is the cell proliferation result at 48 hours; 4C is the cell proliferation result at 72 hours.

[0033] Figure 5 Results graph of the osteogenic differentiation effect of platelet-rich plasma loaded with allicin delivery system (PADS) on bone marrow mesenchymal stem cells in an LPS-induced inflammatory microenvironment studied by ALP alkaline phosphatase staining.

[0034] Figure 6 Results graph of the osteogenic mineralization effect of platelet-rich plasma loaded with allicin delivery system (PADS) on bone marrow mesenchymal stem cells in an LPS-induced inflammatory microenvironment studied by ARS staining.

[0035] Figure 7 Results graph of the effect of PADS on the expression of osteogenic differentiation-related marker genes in bone marrow mesenchymal stem cells in an LPS-induced inflammatory microenvironment studied by qPCR assay. 7A is the Col1a1 expression result, 7B is the Alp expression result, and 7C is the Runx2 expression result.

[0036] Figure 8 Results graph of the effect of PADS on the expression of osteogenic differentiation-related marker proteins in bone marrow mesenchymal stem cells in an LPS-induced inflammatory microenvironment studied by Western blot assay. 8A is the WB result graph, and 8B, 8C, and 8D are the quantitative analyses of COL1A1, RUNX2, and ALP proteins, respectively. Detailed implementation mode

[0037] The examples given are for better explaining the content of the present invention, but the content of the present invention is not limited to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation scheme according to the above-mentioned content of the present invention still fall within the protection scope of the present invention.

[0038] The following will specifically illustrate the present invention through specific examples. Similarly, it should be understood that the following examples are only used for specifically illustrating the present invention and cannot be understood as limiting the protection scope of the present invention. Those skilled in the art who make some non-essential improvements and adjustments based on the above content of the present invention fall within the protection scope of the present invention. The specific process parameters and the like in the following examples are also only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description in this article, rather than being limited to the specific values in the following examples.

[0039] (1) Synthesis of allicin-loaded platelet-rich plasma gel system

[0040] A preparation method of an allicin-loaded platelet-rich plasma gel system is as follows:

[0041] S1. Dissolve 3.8 g of sodium citrate in 100 mL of phosphate buffer solution (i.e., PBS buffer solution), shake and mix well to obtain an anticoagulant solution with a sodium citrate concentration of 3.8 g / 100 mL.

[0042] Place whole blood (a total of 10 tubes, about 15 mL per tube, and perform full-automatic blood cell counting in a blood cell counter, and the results are shown in Table 1) in a centrifuge, centrifuge at 2500×g for 10 min. After centrifugation, use a catheter to aspirate the bottom layer of red blood cells to about 1 cm below the platelet layer. Place the centrifuged centrifuge tube in the centrifuge again and centrifuge at 3000×g for another 10 min. Use a catheter to aspirate the upper layer of plasma (PPP), place the aspirated upper layer of plasma in another 15 mL centrifuge tube, and shake the centrifuged centrifuge tube for 30 s. Each centrifuge tube obtains about 2 mL of pretreated platelet-rich plasma.

[0043] Mix the pretreated platelet-rich plasma in each centrifuge tube.

[0044] Take about 200 μL of pretreated platelet-rich plasma and place it in a blood cell counter for full-automatic blood cell counting, and record the number of platelets in the pretreated platelet-rich plasma. According to the data results, inject platelet-poor plasma (i.e., PPP, which is placed in a blood cell counter for full-automatic blood cell counting, and the results are shown in Table 1) into the pretreated platelet-rich plasma to make the theoretical value of the number of platelets 3000×10 9 / L, and then placed in a hemocytometer again to record the number of platelets, white blood cells, and red blood cells in the PRP (the results are shown in Table 1), obtaining platelet-rich plasma (i.e., PRP);

[0045] Table 1 The number of various blood cells and platelets in whole blood, PRP, and PPP

[0046] Sample Platelet White blood cell Red blood cell Whole blood (n = 31) <![CDATA[(490±96)×10 9 / L]]> <![CDATA[(6.6±3)×10 9 / L]]> <![CDATA[(6±1)×10 12 / L]]> PRP (n = 23) <![CDATA[(3078±70)×10 9 / L]]> <![CDATA[(26±8)×10 9 / L]]> <![CDATA[(4±1)×10 12 / L]]> PPP (n = 20) <![CDATA[(28±2)×10 9 / L]]> <![CDATA[(0.22 ± 0.1) × 10 9 / L]]> 0

[0047] As can be seen from Table 1, the number of platelets in the platelet-rich plasma (i.e., PRP) is much higher than that in whole blood. This result indicates that the platelet-rich plasma (i.e., PRP) prepared by the present invention is indeed platelet-rich plasma.

[0048] S2. Dissolve 1000 U of thrombin in 1 mL of calcium chloride solution with a concentration of 10%, mix evenly to obtain a coagulant solution;

[0049] Add allicin injection (concentration: 30 mg / 2 mL) to 1 mL of the coagulant solution, mix evenly to obtain an allicin / coagulant solution;

[0050] Mix the allicin / coagulant solution with the platelet-rich plasma obtained in step S1, and stir at a speed of 200 r / min to prepare an allicin-loaded platelet-rich plasma gel system.

[0051] Perform electron microscopy scanning on the prepared allicin-loaded platelet-rich plasma gel system, and the results are as Figure 1 shown in D-F.

[0052] From Figure 1 it can be seen that the allicin-loaded platelet-rich plasma gel system prepared by the present invention has a relatively uniform microscopic three-dimensional network structure.

[0053] (2) Experimental study on the protective effect of the active ingredient DADS of allicin on LPS-induced inflammatory bone destruction in mouse skulls

[0054] Establish a mouse cranial inflammatory bone loss model. Twenty-four healthy 6-week-old C57 / BL6 female mice were randomly divided into 4 groups: ① Sham operation group (injected with PBS); ② LPS group (treated with 5 mg / kg LPS); ③ Low-dose DADS group (injected with 20 mg / kg DADS after LPS treatment); ④ High-dose DADS group (injected with 40 mg / kg DADS after LPS treatment). All animals were anesthetized by intraperitoneal injection of 4% chloral hydrate (5 μl / g). The heads of the anesthetized mice were shaved to receive subperiosteal injection of PBS, LPS, or LPS + DADS. The total volume of the injected liquid each time was 100 μl. After weighing the mice, a 1 ml syringe was used to inject at the point of the sagittal midline suture of the skull between the ear and the eye. Next, a skin bleb was formed, and then the needle was slowly withdrawn to prevent liquid overflow. Subsequently, injections were performed every other day for 14 days. After the experiment, the mice were subjected to micro-CT scanning of the skull and three-dimensional reconstruction, and the percentage of bone volume to total volume (BV / TV) and the porosity ratio in the mouse skull were quantitatively analyzed.

[0055] The results of micro-CT scanning and three-dimensional reconstruction are as Figure 2 shown in

[0056] The quantitative analysis results of the percentage of bone volume to total volume (BV / TV) in the mouse skull are as Figure 2 shown in

[0057] The quantitative analysis results of the porosity ratio in the mouse skull are as Figure 2 shown in

[0058] Figure 2 In, the data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (based on one-way ANOVA).

[0059] It can be seen from Figure 2 that DADS treatment (low-dose group and high-dose group) significantly prevented the decrease in bone volume to total volume after LPS induction and reduced the percentage of porosity. This result indicates that the active ingredient of garlicin, DADS, can protect against inflammatory bone destruction in vivo.

[0060] (III) DADS improves LPS-induced cranial inflammatory response in vivo and reduces osteoclastogenesis

[0061] The mouse experiment and grouping method were the same as in (II). The mice were sacrificed and the calvaria were isolated for histological analysis. The isolated skull samples were successively fixed with 4% paraformaldehyde, decalcified with 10% EDTA, and made into 5-μm-thick sections for hematoxylin and eosin (H&E) staining and TRAP staining. The blood of the mice was drawn to detect the levels of pro-inflammatory factors in the serum. The obtained data were used to describe the inflammatory response and osteoclast formation in vivo.

[0062] Skull sections of each group (specifically, the Sham control group, LPS group, LPS + DADS (20 mg / kg group), and LPS + DADS (40 mg / kg group)) were stained with H&E. The specific steps were as follows: Place 5-μm-thick paraffin sections in an oven at 60 °C for 2 hours to enhance adhesion, dewax in gradients and hydrate. Stain with Harris hematoxylin solution (containing 0.5% mercury oxide) for 8 minutes, rinse with running water for 10 minutes to remove surface stain, differentiate with 1% hydrochloric acid ethanol for 5 seconds (controlled under the microscope until the contrast between the nucleus and the background is clear), blue with 0.2% ammonia water for 30 seconds, and rinse with running water for 15 minutes. Stain with 0.5% eosin Y aqueous solution (pH 4.5 - 5.0) for 1.5 minutes, quickly terminate staining with distilled water, and then dehydrate in gradients. Clear with xylene I and II for 5 minutes each, seal with neutral gum, and cure the sealed slides in an oven at 37 °C overnight. The results are as Figure 3 shown in

[0063] Skull sections of each group were stained with TRAP. The specific steps were as follows: Immerse the sections in the acid phosphatase staining solution and stain at room temperature for 1 hour; rinse the sections 3 times with PBS buffer; immerse the sections in the alkaline phosphatase staining solution and stain at room temperature for 1 hour; rinse the sections 3 times with PBS buffer. Immerse the sections in the TRAP chromogenic solution and develop color at room temperature for 15 - 30 minutes; terminate the reaction with distilled water; counterstain the cell nuclei with hematoxylin solution for 3 - 5 minutes; rinse the sections with tap water to make the cell nuclei blue; dehydrate successively with 75%, 85%, 95%, and 100% ethanol for 1 minute each, and then clear with xylene; finally, seal with neutral resin. The results are as Figure 3 shown in

[0064] The percentage of bone area in the border region of the cranial H&E staining was quantitatively analyzed using Image J software. The results are as Figure 3 shown in

[0065] The number of TRAP(+) cells after TRAP staining of the skull sections of each group was quantitatively analyzed using Image J software. The results are as Figure 3 shown in

[0066] Mouse blood was drawn, and the mouse blood was allowed to stand at room temperature for 30 min and centrifuged at 3000 g for 10 min at 4 °C. Mouse serum was obtained from the whole blood. Mouse ELISA kits were used to detect the levels of TNF-α, IL-1β, and IL-6 in the serum. The absorbance of each standard and sample was measured at 450 nm. The standard concentration gradient was used as the standard curve. The results are as Figure 3 shown in

[0067] Figure 3Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (based on one-way ANOVA).

[0068] It can be seen from Figure 3 that DADS treatment significantly prevented bone loss in the bone area, significantly reduced the number of TRAP-positive multinucleated osteoclasts (red arrows), and DADS significantly decreased the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in mice. These results indicate that DADS can treat LPS-induced inflammatory bone destruction and reduce the inflammatory response.

[0069] (IV) PRP loaded with allicin alleviates the toxicity of inflammation to BMSCs and promotes osteoblast proliferation

[0070] BMSC cells were seeded into 96-well plates at a density of 3×10 3 / well. During the seeding process, the cells were repeatedly pipetted to ensure an equal number of cells in each well. After culturing in an incubator for 24 hours, complete medium containing LPS (25 μg / ml) and PRP loaded with allicin (5%) without anticoagulant was added to the 96-well plates according to the grouping as follows: ① control group (without adding drugs); ② LPS group; ③ LPS + PRP loaded with allicin group; ④ PRP loaded with allicin group. The 96-well plates were placed in a cell incubator and incubated for 24 - 72 hours. Fresh medium was replaced. 10 μL of CCK-8 solution was added to each well (note that no bubbles should be generated), and the plates were placed in a cell incubator and incubated for 2 h. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. Then, the cell proliferation rate was calculated according to the following formula: Cell proliferation rate = (absorbance value of the experimental group - absorbance value of the blank control) / (absorbance value of the control group - absorbance value of the blank control) × 100%. The results are as Figure 4 shown.

[0071] Figure 4 Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (based on one-way ANOVA).

[0072] It can be seen from Figure 4 that PRP loaded with allicin can improve the reduced viability of BMSC cells induced by LPS (25 μg / ml), and PRP loaded with allicin can also increase the viability of BMSC cells under normal conditions. These results indicate that PRP loaded with allicin can alleviate the toxicity of LPS-induced inflammation to BMSCs and promote osteoblast proliferation.

[0073] (V) PADS improves ALP expression induced by osteogenic induction of BMSCs in normal and inflammatory microenvironments

[0074] ALP expression is considered an indicator of osteoblast differentiation and activity. Therefore, ALP levels were measured 14 days after osteogenic induction of BMSC cells. BMSC cells were seeded into 24-well plates at a density of 2×10 4 / well. During the seeding process, the cells were repeatedly pipetted to resuspend them and ensure an equal number of cells in each well. After culturing the cells to 80 - 90% confluence using complete medium, the medium was replaced with osteogenic differentiation induction medium and divided into the following groups: ① control group; ② LPS group; ③ LPS + platelet-rich plasma loaded with allicin delivery system (PADS) group; ④ PADS group. According to the grouping, LPS (25 μg / ml) and cell chambers were added to the 24-well plates, and PADS (2 mm 3 / well) was added to the cell chambers. The cells were co-cultured with PADS for 14 days, and the medium was changed every 2 - 3 days during this period. After the culture was completed, the cells in the 24-well plates were washed twice with 4°C pre-cooled PBS and fixed with paraformaldehyde at 4°C for 30 min. Then, they were washed 3 - 5 times with PBS, 3 - 5 minutes each time. The following solutions were added in sequence and mixed evenly to prepare the BCIP / NBT staining working solution:

[0075] Alkaline phosphatase chromogenic buffer 10ml BCIP solution (300X) 33 μl NBT solution (150X) 66 μl BCIP / NBT staining working solution 10.1ml

[0076] After the last wash, the washing solution was removed, an appropriate amount of BCIP / NBT staining working solution was added, and the samples were incubated at room temperature in the dark for 30 minutes. Ensure that the staining working solution fully covers the samples. After removing the BCIP / NBT working solution, wash with distilled water 1 - 2 times to terminate the color reaction, and take pictures of each well for recording. The results are as Figure 5 shown.

[0077] As Figure 5 can be seen, compared with the control group induced normally, the number of osteoblasts induced by BMSCs positive for ALP decreased in the LPS inflammatory microenvironment. Compared with the LPS group, PADS treatment increased the number of ALP-positive cells. In addition, compared with the control group, the number of ALP-positive cells increased in the PADS treatment group. This result indicates that PADS can rescue the inhibition of osteogenic differentiation in the inflammatory environment, and PADS can promote the osteogenic differentiation of BMSCs under normal conditions.

[0078] (VI) PADS improves the formation of mineralized nodules in the osteogenic induction of BMSCs under normal and inflammatory microenvironments

[0079] In the later stage of osteoblast development, due to calcium accumulation, the extracellular matrix gradually mineralizes, leading to the formation of bone nodules. Therefore, alizarin red mineralization staining was used to evaluate the effect of PADS on the mineralization ability of BMSCs after osteogenic induction. The cell culture method and grouping of BMSCs were the same as in (5). After 21 days of osteogenic differentiation induction of the cells, alizarin red staining was performed. The cells in the 24-well plate were washed twice with pre-cooled PBS at 4°C and fixed with paraformaldehyde at 4°C for 30 min. Then, they were washed 3 - 5 times with PBS, 3 - 5 minutes each time. After removing the PBS, 0.5 ml of alizarin red staining solution was added to each well and incubated at room temperature for 15 minutes, ensuring that the staining working solution could fully cover the samples. After removing the alizarin red staining solution, the color development reaction was terminated by washing 1 - 2 times with distilled water, and each well was photographed and recorded. The results are as Figure 6 shown.

[0080] As Figure 6 can be seen, the normally induced control group showed the most obvious calcium nodule formation, while a significant reduction in calcium deposition was observed in the inflammatory environment induced by LPS. At the same time, PADS could rescue the reduced mineralization of osteoblasts in the inflammatory environment. In addition, the formation of mineralization nodules in the PADS treatment group was comparable to that in the normally induced control group. This result indicates that PADS can rescue the inhibition of osteogenic calcium nodules in the inflammatory environment, and PADS can promote the osteogenic mineralization of BMSCs under normal conditions.

[0081] (7) PADS improves the specific mRNA expression of BMSC osteogenic induction in normal and inflammatory microenvironments

[0082] To verify the promoting effect of PADS on osteogenic induction and differentiation in normal and inflammatory microenvironments, the expression of osteogenic genes in BMSC cells after 14 days of induction was measured by qPCR experiment. BMSC cells were seeded into 6-well plates at a density of 2×10 5 / well. During the seeding process, the cells were repeatedly pipetted to ensure that the number of cells in each well was the same. After culturing the cells to 80 - 90% confluence with complete medium, the medium was replaced with osteogenic differentiation induction medium and divided into the following groups: ① control group; ② LPS group; ③ LPS + PADS group; ④ PADS group. According to the different groups, LPS (25 μg / ml) and cell chambers were added to the 6-well plates, and PADS (5 mM 3(Holes), the cells were co-cultured with PADS for 14 days, and the medium was changed every 2-3 days during this period. Then, RNA was extracted using the SuperFastPure Cell RNA Isolation Kit (Vazyme, China). 500 μl of Buffer CRL was added to each well of cells to fully cover the cell surface, and the cells were repeatedly pipetted to make them detached. All the lysis products were transferred into RNAColumns I (each in a 2 ml Collection Tube), and centrifuged at 12,000 rpm (13,400×g) for 30 seconds, and the waste liquid was discarded. 500 μl of Buffer RWA (anhydrous ethanol had been added) was added to the RNA Columns I, and centrifuged at 12,000 rpm (13,400×g) for 30 seconds, and the waste liquid was discarded. 500 μl of Buffer RW (anhydrous ethanol had been added) was added to the RNA Columns I, and centrifuged at 12,000 rpm (13,400×g) for 1 min, and the waste liquid was discarded. The RNA Columns I were placed back into the collection tubes, centrifuged at 12,000 rpm (13,400×g) for 1 min without sample, and carefully transferred the RNA Columns I into a new 1.5 ml RNase-free centrifuge tube (prepared by oneself), and 20-100 μl of RNase-free ddH 2 O was added dropwise in the middle of the adsorption column membrane and incubated at room temperature for 1 min, and then centrifuged at 12,000 rpm (13,400×g) for 1 min to elute the RNA. Using the IIOne Step qRT-PCR SYBR Green Kit (Vazyme, China) to perform quantitative PCR to detect the expression of osteogenic genes Col1a1, Runx2, and Alp, and using Graphpad Prism 10.4 software for data analysis and graphing. The results are as Figure 7 shown.

[0083] As Figure 7 can be seen, the expressions of osteogenic differentiation-specific genes Col1a1, Runx2, and Alp were decreased in the inflammatory environment induced by LPS (25 μg / ml), and could be rescued by PADS. In addition, under the normal osteogenic induction environment, PADS could promote the expressions of Col1a1, Runx2, and Alp. This result further indicates that PADS has a significant promoting effect on osteoblast differentiation in both inflammatory and normal environments.

[0084] (VIII) PADS improves the expression of osteogenic induction marker proteins in BMSCs under normal and inflammatory microenvironments

[0085] To verify the promoting effect of PADS on osteogenic induction and differentiation in normal and inflammatory microenvironments, Western blot experiments were used to measure the expression of osteogenic proteins in BMSC cells after 14 days of induction. The culture method and grouping of BMSC cells were the same as in (VII). Then, after rinsing the well plates with PBS, RIPA lysis buffer (Beyotime P0013B): protease inhibitor was mixed at a ratio of 50:1, and 100 μl of the mixed complete lysis buffer was added to each well. The plate was shaken repeatedly to ensure complete contact between the lysis buffer and the cells. The cells were collected using a cell scraper and then sonicated using an ultrasonic machine. Subsequently, centrifugation was performed at 12,000 rpm and 4 °C for 10 minutes, and the supernatant was the total protein solution. A BCA kit was used to equalize the protein concentration in each group. WB experiments were conducted. 20 μg of protein samples were loaded in each group, and electrophoresis was carried out at a voltage of 80 V (upper gel) and 120 V (lower gel). Then, transfer to a PVDF membrane was performed at 400 mA for 40 min (rapid transfer). Invitrogen TM No-Stain TM A stain-free protein labeling reagent was used to detect the total protein and image it. After blocking with 5% non-fat milk at 60 r / min for 1 h, the primary antibodies (COL1A1, ALP, RUNX2) were incubated overnight at 4 °C, and the secondary antibody was incubated at room temperature for 1 h. TBST solution was used for rinsing between steps. Finally, exposure and development were performed in a Chemidoc XRS+ imaging system (Bio-RAD), and Image J and Graphpad Prism 10.4 software were used for quantification, data analysis, and graphing. The results are as Figure 8 shown.

[0086] As Figure 8 can be seen, the expression of osteogenic differentiation marker proteins COL1A1, RUNX2, and ALP decreased in the inflammatory environment induced by LPS (25 μg / ml), and could be rescued by PADS. In addition, in the normal osteogenic induction environment, PADS could promote the expression of COL1A1, RUNX2, and ALP proteins. This result further indicates that PADS has a significant promoting effect on osteoblast differentiation in both inflammatory and normal environments.

[0087] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a platelet-rich plasma gel system loaded with allicin, characterized in that: The following steps are involved: S1. adding sodium citrate to a phosphate buffer solution to obtain an anticoagulant solution; After the anticoagulant solution is drawn with a syringe, whole blood is drawn, the red blood cells at the bottom are drawn after centrifugation, the plasma at the top is drawn after centrifugation again, and the blood is shaken to obtain pre-treated platelet-rich plasma; injecting platelet-poor plasma into the pretreated platelet-rich plasma so that the platelet content reaches a preset threshold value to obtain platelet-rich plasma; S2. dissolving thrombin in a calcium ion solution to obtain a coagulant solution; Adding the allicin injection into the coagulant solution to obtain an allicin / coagulant solution; The allicin / coagulant solution is mixed with the platelet-rich plasma and stirred to obtain the allicin-loaded platelet-rich plasma gel system.

2. The preparation method according to claim 1, characterized in that In the anticoagulant solution of step S1, the concentration of sodium citrate is 3-4 g / 100 mL.

3. The preparation method according to claim 1, characterized in that: In step S1, the bottom layer of red blood cells is aspirated to 0.5-1.5 cm below the platelet layer.

4. The preparation method according to claim 1, characterized in that: In step S1, during the second centrifugation, the rotation speed is 2500-3500×g, and the centrifugation time is 8-15 min.

5. The preparation method according to claim 1, characterized in that: In step S1, during the centrifugation process, the rotation speed is 2000-3000×g, and the centrifugation time is 8-15 min.

6. The preparation method according to claim 1, characterized in that: In step S1, the preset threshold is greater than or equal to 3000×10 9 / L.

7. The preparation method according to claim 1, characterized in that: In the coagulant of step S2, the concentration of thrombin is 500-2000U / 0.5-2mL.

8. The preparation method according to claim 1, characterized in that: In step S2, the concentration of the allicin injection is 3 mg / mL.

9. The preparation method according to claim 1, characterized in that: In step S2, the mixing ratio of allicin injection: thrombin: PRP is 3 mg: 500-2000 U: 10 ml.

10. The platelet-rich plasma gel system loaded with allicin prepared according to the method according to any one of claims 1 to 9.

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