A bioceramic preparation for treating lower limb ischemia and its application

Abstract

A bioceramic preparation and application for treating lower limb ischemia diseases. The active ions silicon ions and strontium ions in the bioceramic have excellent abilities to enhance angiogenesis, regulate inflammation and inhibit muscle necrosis, and have great application potential in tissue engineering. It is proved that silicon ions and strontium ions in the range of 0.12 to 25 μg / mL can specifically regulate the activity of vascular endothelial cells, smooth muscle cells, macrophages and myoblasts. The material prepared according to this concentration can be used to treat lower limb ischemia diseases and has great application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a bioceramic preparation for treating lower limb ischemic diseases and its application. Background Art

[0002] Ischemic diseases of the lower limbs, such as arteriosclerosis obliterans (ASO), are common geriatric diseases that affect more than 230 million people worldwide and are increasing year by year. About 10-15% of ASO patients will progress to resting pain or even amputation. More worryingly, the cardiovascular morbidity and mortality of ASO patients are higher than those of healthy people. At present, endovascular stent surgery or artificial vascular bypass surgery are the only surgical treatments, but these methods have disadvantages such as low long-term patency and large trauma. Although repeated surgeries can keep blood vessels patency, the difficulty and risk of surgery will increase rapidly with the increase in the number of surgeries. Therefore, for patients who cannot undergo surgery, they can only rely on drug treatment. However, the current clinical drugs for the treatment of ASO are mainly drugs with anticoagulant, antiplatelet and lipid-lowering functions. They mainly delay vascular occlusion to relieve the symptoms of ASO, but cannot really treat it. Therefore, it is of certain clinical significance to develop a biomaterial that can specifically treat lower limb ischemic diseases. Summary of the invention

[0003] In order to solve the technical defects of the prior art, the present invention provides a bioceramic preparation and application for treating lower limb ischemic diseases, which has excellent ability to enhance angiogenesis, regulate inflammation and inhibit muscle necrosis, and has great application potential in tissue engineering.

[0004] The technical solution adopted by the present invention is: a bioceramic preparation for treating lower limb ischemic diseases, wherein the bioceramic preparation is a bioceramic hydrogel obtained by mixing bioceramics containing silicon and strontium elements with a water-soluble polymer solution.

[0005] The bioceramics containing silicon and strontium elements are one or more of the bioceramics containing calcium silicate, strontium carbonate, strontium silicate, bioglass, zinc silicate, magnesia feldspar, and copper silicate.

[0006] The water-soluble polymer solution is one or more of a sodium alginate solution, a hyaluronic acid solution and a chitosan solution.

[0007] The content of the bioceramic containing silicon and strontium elements in the bioceramic preparation is 0.5% to 30% w / v.

[0008] The content of the bioceramic containing silicon and strontium elements in the bioceramic preparation is 1% w / v.

[0009] An application of the bioceramic preparation in preparing medicines for treating lower limb ischemic diseases.

[0010] The effective concentration of silicon ions and strontium ions in the medicine for treating lower limb ischemic diseases is 0.12-25 μg / mL.

[0011] The lower limb ischemic diseases include lower limb arteriosclerosis obliterans, thromboangiitis obliterans, and diabetic lower limb vascular disease.

[0012] The strontium ion is Sr 2+ Ion, silicon ion is SiO3 2- ion.

[0013] The beneficial effects of the present invention are as follows: the present invention provides a bioceramic preparation and application for treating lower limb ischemic diseases. The active ions silicon ions and strontium ions in the bioceramic have excellent abilities to enhance angiogenesis, regulate inflammation and inhibit muscle necrosis, and have great application potential in tissue engineering. It is proved that silicon ions and strontium ions in the range of 0.12 to 25 μg / mL can specifically regulate the activity of vascular endothelial cells, smooth muscle cells, macrophages and myoblasts. The material prepared according to this concentration can be used to treat lower limb ischemic diseases and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The effects of different concentrations of calcium silicate ion solution and strontium carbonate ion solution on the viability of endothelial cells (HUVECs) and smooth muscle cells (SMCs).

[0015] Figure 2 The effect of hydrogels containing different concentrations of bioceramics on the viability of endothelial cells (HUVECs). Figure 3 Preparation and characterization of hydrogels containing 1% bioceramics. (A) Photographs of hydrogels (SA: blank sodium alginate; SA / Sr: strontium carbonate hydrogel; SA / Si: calcium silicate hydrogel; SA / Sr / Si: strontium carbonate / calcium silicate composite hydrogel). (B) Gel time of hydrogels. (C) SEM images and element distribution of hydrogels. (D) Ion release of hydrogels in phosphate buffered saline (PBS) (Sr2+: strontium ions; SiO32-: silicon ions).

[0016] Figure 4Effects of bioceramic hydrogels on mouse myoblasts (C2C12). (A) Effects of bioceramic hydrogels on C2C12 cell viability under normal culture conditions. (B, C) Effects of bioceramic hydrogels on C2C12 cell migration under normal culture conditions. (B) Representative images of C2C12 migration. (C) Quantitative statistical analysis of C2C12 migration rate. (D) Protective effect of bioceramic hydrogels on C2C12 under glucose-oxygen deprivation conditions. (E, F) Effects of bioceramic hydrogels on C2C12 myogenic differentiation under glucose-oxygen deprivation conditions. Expression of muscle regulatory factors (muscle regulatory factors: MyoG and MyoD) was analyzed by PCR. Figure 5 Effects of bioceramic hydrogels on human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs). (A, B) Effects of bioactive hydrogels on the cell viability of HUVECs and SMCs. (C, D) Representative images of HUVECs and SMCs migration. (E, F) Mobility of HUVECs and SMCs.

[0017] Figure 6 The therapeutic effect of bioceramic hydrogel on lower limb ischemia. (A) Schematic diagram of the construction and treatment of mouse lower limb ischemia model. (B) Representative images of mouse ischemic limbs detected by laser Doppler perfusion imager (red areas indicate blood flow, blue areas indicate no blood flow). (CE) Quantitative analysis of blood perfusion ratio (ischemic / normal) on days 7, 14, and 28.

[0018] Figure 7 Bioceramic hydrogels can protect skeletal muscle from necrosis in the early stage of lower limb ischemia. (A) Photograph of mouse gastrocnemius muscle on day 28. (B) H&E tissue section staining of gastrocnemius muscle on days 3, 7, and 28. (C) Masson tissue section staining of gastrocnemius muscle on days 3, 7, and 28. (DF) Quantitative analysis of collagen fiber distribution based on Masson tissue section staining. (G) TUNEL tissue section staining of gastrocnemius muscle on days 3 and 7. (H) Quantitative analysis of the number of apoptotic cells based on TUNEL tissue section staining.

[0019] Figure 8 Bioceramic hydrogels can promote the formation of collateral capillaries in the ischemic area of ​​the lower limb. (A) Angiography images of the ischemic limb after 28 days of treatment. (B) Immunofluorescence staining images of α-SMA (green) and CD31 (red). (C) Quantitative statistical analysis of the number of new blood vessels based on α-SMA (green) and CD31 (red) staining. (DF) Western Blot analysis of the protein expression levels of VEGF and HIF-1α in the gastrocnemius muscle at days 3, 7, and 28.

[0020] Fig. 9The effect of bioceramic hydrogel on the early stage of inflammatory regulation (day 3) of lower limb ischemia. (A) Effect of bioceramic hydrogel on macrophages in the ischemic part of the lower limb (CD206: macrophage M2 marker, green; CD11C: macrophage M1 marker, yellow; F4 / 80: total macrophage marker, red). (B, C) The percentage of M1 and M2 polarized macrophages in the total number of macrophages. (DG) ELISA analysis of the expression of inflammatory-related factors such as TNF-a, IL-6, Arg-1 and IL-10 in the gastrocnemius muscle. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] In the present disclosure, the calcium silicate preparation (SA / Si) contains a calcium silicate concentration of 0.5% to 30% w / v, preferably 1% w / v. The strontium carbonate preparation (SA / Sr) contains a calcium silicate concentration of 0.5% to 30% w / v, preferably 1% w / v. The strontium carbonate / calcium silicate composite preparation (SA / Sr / Si) contains a calcium silicate concentration of 0.5% to 30% w / v, preferably 0.5% w / v, contains a calcium silicate concentration of 0.5% to 30% w / v, preferably 0.5% w / v, and the total bioceramic concentration is preferably 1% w / v. The strontium carbonate ion solution contains 0.12ppm to 25ppm of strontium ions. The calcium silicate ion solution contains 0.12ppm to 25ppm of silicon ions.

[0023] In one embodiment of the present invention, a strontium carbonate ion solution and a calcium silicate ion solution are prepared. Specifically, a series of gradient diluted ion extracts of strontium carbonate ion solution and calcium silicate ion solution are prepared. First, the strontium carbonate ion solution or the calcium silicate ion solution is added to the serum-free high-glucose DMEM cell culture medium, incubated at 37°C for 24 hours at a solid / liquid ratio of 200 mg / mL, and the mixture is centrifuged at 4000 rpm for 10 minutes, and then the supernatant is sterilized and collected through a filter membrane to obtain a stock solution. Subsequently, the stock solution is diluted with serum-free high-glucose DMEM cell culture medium to prepare a suitable ion extract, and all ion extracts are stored at 4°C. Among them, the concentrations of silicon ions and strontium ions are 0.12-100 μg / mL

[0024] In another embodiment of the present invention, calcium silicate hydrogel preparations (SA / Si), strontium carbonate hydrogel preparations (SA / Sr) and strontium carbonate / calcium silicate composite hydrogel preparations (SA / Sr / Si) are prepared. Specifically, calcium silicate, strontium carbonate and strontium carbonate / calcium silicate mixture powders, and at least one of water-soluble polymer solutions such as sodium alginate solution, hyaluronic acid solution, chitosan solution, etc. are fully mixed with a syringe and a connecting tube to obtain the desired preparation. In an optional embodiment, the concentration of the bioceramic powder is 0.5% to 30% w / v, preferably 1% w / v.

[0025] In the present disclosure, the prepared bioceramic ion solution and hydrogel preparation can target vascular endothelial cells, smooth muscle cells, macrophages and myoblasts, while enhancing angiogenesis, regulating inflammation and inhibiting muscle necrosis, thereby achieving the purpose of treating lower limb ischemic diseases.

[0026] Effect of bioceramic ion solution on the viability of venous endothelial cells and smooth muscle cells. In one embodiment of the present invention, calcium silicate and strontium carbonate ion solutions were used to evaluate the cell viability of venous endothelial cells and smooth muscle cells. Take the previously prepared stock solution and add fetal bovine serum, penicillin and streptomycin in proportion. The cells were inoculated in a 96-well culture plate at an initial density of 1×10 per well. 3 After culturing for 24 hours, the cell culture medium was replaced with the prepared Si ion solution. Ordinary cell culture medium was used as a control. At 48 hours, the absorbance of all samples was measured at 450nm on a microplate reader using CCK-8 (cell counting kit-8) to evaluate the cell viability.

[0027] Effect of bioceramic hydrogel preparations on the cell viability of venous endothelial cells, smooth muscle cells and myoblasts. In one embodiment of the present invention, calcium silicate and strontium carbonate and their composite hydrogels were used to evaluate the cell viability of venous endothelial cells, smooth muscle cells and myoblasts. Cells (5×10 4 Cells) were seeded in 6-well plates. At the same time, the bioceramic hydrogel was placed in a 6-well culture Transwell chamber (1 g / well). At a specific time, the absorbance of all samples was measured by CCK-8 (cell counting kit-8) at 450 nm on a microplate reader to evaluate the cell viability.

[0028] Effect of bioceramic hydrogel preparations on cell migration of venous endothelial cells, smooth muscle cells and myoblasts. In one embodiment of the present invention, calcium silicate and strontium carbonate and their composite hydrogels were used to evaluate the cell viability of venous endothelial cells, smooth muscle cells and myoblasts. Cells (5×10 3Cells) were seeded in 24-well plates. At the same time, the bioceramic hydrogel was placed in a 24-well culture Transwell chamber (1g / well). At the corresponding time, the absorbance of all samples was measured by CCK-8 (cell counting kit-8) at 450nm on a microplate reader to evaluate the viability of the cells. Then, the cell monolayer in each well was scraped off using a pipette (200μL) and washed with PBS (0 hours), and the Transwell chamber containing the bioactive hydrogel was placed on a 6-well plate with cells, and optical images were obtained using an optical microscope at 0 and 12 hours. After 12 hours, the cells were stained with crystal violet for 1 minute, and optical images were obtained using the above optical microscope. The mobility was analyzed by ImageJ software, and the mobility was determined by the ratio of the initial scratch area to the final scratch area.

[0029] The protective effect of bioceramic hydrogel preparations on myoblasts under glucose and oxygen deprivation, as well as their effect on myogenic differentiation. Mouse myoblasts were cultured in a hypoxic incubator (1% O2, 5% CO2) at 37°C and in sugar-free DMEM medium to construct an injury model. The hydrogel was placed in a Transwell chamber (1g / well) in a 24-well culture plate. After 8 hours, the cells were stained with a live and dead cell staining kit, in which green fluorescence marked live cells and red fluorescence marked dead cells. The fluorescence intensity was observed by confocal laser scanning microscopy. In addition, RNAsio was used to extract RNA from myoblasts. Then, the expression of intracellular myogenin MyoD and MyoG was determined using a PCR system.

[0030] Test method for treating lower limb ischemia. The bioceramic hydrogel preparation prepared above was used for corresponding tests. The specific steps include: selecting adult male C57BL / 6 mice (6-8 weeks old) to establish a femoral aorta ligation lower limb ischemia model. Randomly divided into five groups: blank group (Ctrl), hydrogel group (SA), strontium carbonate hydrogel group (SA / Sr), calcium silicate hydrogel group and strontium carbonate / calcium silicate gel group (SA / Sr / Si). The recovery of lower limb blood supply was recorded on days 1, 3, 7, 14, and 28, and wound tissue samples were taken at the corresponding time for H&E, Masson, tissue section staining, Tunel immunohistochemistry staining, CD31, α-SMA, CD206, CD11C, F4 / 80 immunofluorescence staining, vascular CT angiography imaging, Western Blot protein expression, and PCR cytokine expression.

[0031] Embodiment 1:

[0032] Effects of calcium silicate and strontium carbonate ionic liquids and hydrogels on cell viability of vascular-associated cells.

[0033] (1) Take the previously prepared stock solution and add fetal bovine serum, penicillin and streptomycin in proportion. Inoculate the cells in a 96-well culture plate at an initial density of 1×10 cells per well. 3 After culturing for 24 hours, the cell culture medium was replaced with the prepared Si ion solution. Ordinary cell culture medium was used as a control. At 48 hours, the absorbance of all samples was measured at 450nm on a microplate reader using CCK-8 (cell counting kit-8) to evaluate the cell viability.

[0034] (2) The viability of venous endothelial cells was evaluated using calcium silicate, strontium carbonate and their composite hydrogels. Cells (5×10 4 Cells) were seeded in 6-well plates. At the same time, the bioceramic hydrogel was placed in a 6-well culture Transwell chamber (1 g / well). At 48 hours, the cell viability was evaluated by measuring the absorbance of all samples at 450 nm using a microplate reader using CCK-8 (cell counting kit-8). Figure 1 It was shown that the ionic solution of calcium silicate and strontium carbonate could significantly promote the cell viability of venous endothelial cells and smooth muscle cells, and the optimal concentration of silicon ions and strontium ions was 0.5-12 μg / mL.

[0035] Figure 2 It is shown that the hydrogel of calcium silicate and strontium carbonate can significantly promote the cell viability of venous endothelial cells, wherein the optimal loading concentration of the bioceramic is 1-5%, preferably 1%.

[0036] In subsequent examples, the optimal ratio is selected to prepare materials and conduct subsequent tests.

[0037] Embodiment 2:

[0038] Preparation and performance characterization of strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogels.

[0039] (1) First, 3 g of sodium alginate was weighed and dissolved in 100 mL of deionized water. Then, 0.1 g of strontium carbonate, 0.1 g of calcium silicate, and 0.1 g of gluconolactone were added to 10 mL of the sodium alginate solution (3% w / v) and mixed thoroughly.

[0040] (2) Allow the mixed suspension to stand at room temperature for 10 minutes to obtain the desired bioceramic hydrogel.

[0041] (3) The hydrogel was freeze-dried for 24 hours. The microscopic morphology of the hydrogel was observed using a scanning electron microscope, and the dispersion of the ceramic powder in the hydrogel was observed using an energy dispersive spectrometer.

[0042] (4) 1 g of the hydrogel was added to 10 mL of PBS buffer solution and placed for different time periods (1, 2, 3, 4, 7, 14, 21 and 28 days). Then, the suspension was centrifuged at 4000 rpm for 10 minutes at a preset time. The release medium was collected and replaced with 10 mL of fresh PBS solution. The collected release medium was analyzed by ICP-AES to determine the concentration of strontium ions and silicon ions.

[0043] Figure 3 The results show that the desired bioceramic composite hydrogel was successfully prepared. Among them, calcium silicate and strontium carbonate were evenly distributed in the hydrogel. In addition, the composite hydrogel can release the required bioactive strontium ions and silicon ions for a long time for 28 days.

[0044] Embodiment 3:

[0045] Effects of strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogels on the proliferation and migration of vascular-related cells.

[0046] (1) Cells (5×10 per well) were added 4 Cells were seeded in 6-well plates. Meanwhile, the bioceramic hydrogel was placed in a 6-well culture Transwell chamber (1 g / well). On days 1, 3, and 5, the viability of the cells was assessed by measuring the absorbance of all samples at 450 nm using a microplate reader using CCK-8 (cell counting kit-8).

[0047] (2) Cells (5×10 per well) were added 3 Cells) were seeded in 24-well plates. At the same time, the bioceramic hydrogel was placed in a 24-well culture Transwell chamber (1g / well). At the corresponding time, the absorbance of all samples was measured by CCK-8 (cell counting kit-8) at 450nm on a microplate reader to evaluate the viability of the cells. Then, the cell monolayer in each well was scraped off using a pipette (200μL) and washed with PBS (0 hours), and the Transwell chamber containing the bioactive hydrogel was placed on a 6-well plate with cells, and optical images were obtained using an optical microscope at 0 and 12 hours. After 12 hours, the cells were stained with crystal violet for 1 minute, and optical images were obtained using the above optical microscope. The mobility was analyzed by ImageJ software, and the mobility was determined by the ratio of the initial scratch area to the final scratch area.

[0048] Figure 4The results showed that cell viability assays showed that strontium carbonate hydrogel, calcium silicate hydrogel, and strontium carbonate / calcium silicate composite hydrogel significantly promoted the viability of endothelial cells and smooth muscle on the 3rd and 5th days. In particular, the strontium carbonate / calcium silicate composite hydrogel group had a significantly higher regulatory effect on cell viability than the strontium carbonate hydrogel and calcium silicate hydrogel groups. In addition, the cell viability of endothelial cells in the strontium carbonate / calcium silicate composite hydrogel group was 1.62 times higher than that in the blank hydrogel group, and the cell viability of smooth muscle cells in the strontium carbonate / calcium silicate composite hydrogel group was 1.31 times that in the blank hydrogel group. The cell scratch test showed that all composite hydrogels promoted cell migration compared with the blank hydrogel group, and the stimulating effect of the strontium carbonate / calcium silicate composite hydrogel was significantly higher than that of the strontium carbonate hydrogel and calcium silicate hydrogel groups, indicating that strontium ions and silicon ions have a synergistic effect on cell migration.

[0049] Embodiment 4:

[0050] Effects of strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogels on the proliferation, migration and differentiation of myoblasts.

[0051] (1) Cells (5×10 per well) were added 4 Cells were seeded in 6-well plates. Meanwhile, the bioceramic hydrogel was placed in a 6-well culture Transwell chamber (1 g / well). On days 1, 3, and 5, the viability of the cells was assessed by measuring the absorbance of all samples at 450 nm using a microplate reader using CCK-8 (cell counting kit-8).

[0052] (2) Cells (5×10 per well) were added 3 Cells) were seeded in 24-well plates. At the same time, the bioceramic hydrogel was placed in a 24-well culture Transwell chamber (1g / well). At the corresponding time, the absorbance of all samples was measured by CCK-8 (cell counting kit-8) at 450nm on a microplate reader to evaluate the viability of the cells. Then, the cell monolayer in each well was scraped off using a pipette (200μL) and washed with PBS (0 hours), and the Transwell chamber containing the bioactive hydrogel was placed on a 6-well plate with cells, and optical images were obtained using an optical microscope at 0 and 12 hours. After 12 hours, the cells were stained with crystal violet for 1 minute, and optical images were obtained using the above optical microscope. The mobility was analyzed by ImageJ software, and the mobility was determined by the ratio of the initial scratch area to the final scratch area.

[0053] (3) Mouse myoblasts were cultured in a hypoxic incubator (1% O2, 5% CO2) at 37°C and in sugar-free DMEM medium to construct an injury model. The hydrogel was placed in a Transwell chamber (1 g / well) in a 24-well culture plate. After 8 hours, the cells were stained with a live-dead cell staining kit, in which green fluorescence marked live cells and red fluorescence marked dead cells. The fluorescence intensity was observed by confocal laser scanning microscopy. In addition, RNAsio was used to extract RNA from myoblasts. Then, a PCR system was used to determine the expression of intracellular myogenin MyoD and MyoG.

[0054] Figure 5 It was shown that calcium silicate hydrogel and strontium carbonate / calcium silicate composite hydrogel could effectively enhance the viability and migration ability of stimulated myoblasts.

[0055] Under glucose-oxygen deprivation conditions, the number of cell deaths in the strontium carbonate hydrogel and calcium silicate hydrogel groups was significantly reduced compared with the blank hydrogel group, indicating that the two hydrogels have a protective effect on myoblasts under glucose-oxygen deprivation conditions. More interestingly, the strontium carbonate / calcium silicate composite hydrogel not only significantly reduced the number of dead cells, but also significantly increased the number of living cells under glucose-oxygen deprivation conditions. PCR results showed that the strontium carbonate / calcium silicate composite hydrogel group significantly promoted the expression of MyoG and MyoD. This result indicates that the combination of strontium ions and silicon ions has a synergistic effect on the activation of myoblasts.

[0056] Embodiment 5:

[0057] The efficacy of strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogel on blood supply restoration in the lower limb ischemia model.

[0058] (1) Adult male C57BL / 6 mice (6-8 weeks old) were selected to establish the femoral aorta ligation lower limb ischemia model and randomly divided into five groups: blank group (Ctrl), hydrogel group (SA), strontium carbonate hydrogel group (SA / Sr), calcium silicate hydrogel group, and strontium carbonate / calcium silicate gel group (SA / Sr / Si).

[0059] (2) On days 1, 3, 7, 14, and 28 after surgery, blood flow in the lower limbs was measured by laser Doppler perfusion imaging to evaluate the blood supply to the ischemic and healthy lower limbs.

[0060] (3) The limb flow volume ratio was calculated by the blood flow of the left hind limb (ligated) / the blood flow of the right hind limb (unligated).

[0061] from Figure 6It can be found that the lower limbs of mice in the blank group and the blank hydrogel group were necrotic. In contrast, strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogels avoided necrosis of the lower limbs of mice. Laser Doppler blood flow analysis showed that the blood supply to the lower limbs in the strontium carbonate / calcium silicate hydrogel group was almost completely restored on the 7th day, while the blood supply to the lower limbs in the strontium carbonate hydrogel and calcium silicate hydrogel groups was slightly restored on the 14th day. In the control group and the blank hydrogel group, since the lower limbs were already necrotic on the 28th day, even if the blood supply was gradually restored, the lower leg was almost completely lost. Quantitative analysis of the blood flow ratio showed that the blood flow ratio of the strontium carbonate / calcium silicate bioceramic hydrogel group to the healthy limb reached 53% on the 7th day, 87% on the 14th day, and 97% on the 28th day, which was almost the same as the healthy limb.

[0062] Embodiment 6:

[0063] Effects of strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogels on muscle protection in a lower limb ischemia model.

[0064] (1) Adult male C57BL / 6 mice (6-8 weeks old) were selected to establish the femoral aorta ligation lower limb ischemia model and randomly divided into five groups: blank group (Ctrl), hydrogel group (SA), strontium carbonate hydrogel group (SA / Sr), calcium silicate hydrogel group, and strontium carbonate / calcium silicate gel group (SA / Sr / Si).

[0065] (2) On the 28th day after surgery, the gastrocnemius muscles of the lower limbs were obtained and optical photographs were taken.

[0066] (3) The gastrocnemius muscles of the lower limbs were obtained on days 3, 7, and 28 after surgery for H&E and Masson's tissue section staining.

[0067] (4) The gastrocnemius muscles of the lower limbs were obtained on days 3 and 7 after surgery for Tunel immunohistochemical staining.

[0068] from Figure 7 It was shown that strontium carbonate / calcium silicate hydrogels can significantly inhibit the atrophy of muscles in the ischemic part of the lower limbs. To further verify the role of strontium carbonate / calcium silicate bioceramic hydrogels in protecting skeletal muscle from necrosis in the early stage of lower limb ischemia, we analyzed the gastrocnemius muscles of mice on days 3, 7, and 28. The results of H&E staining showed that a large number of cavities were visible in the muscle tissues of the blank and hydrogel groups on days 3 and 7, indicating that this part of the muscle was necrotic. In contrast, the cavities found in the strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogel groups were significantly less than those in the blank and blank hydrogel groups, especially in the strontium carbonate / calcium silicate bioceramic hydrogel group.

[0069] Fibrosis in the tissue was analyzed by Masson staining. It was found that blue collagen fibers were observed in all groups on day 3. Interestingly, on day 7, all bioceramic composite hydrogels showed an inhibitory effect on collagen deposition, among which the inhibitory effect of the strontium carbonate / calcium silicate hydrogel group was significantly higher than that of the strontium carbonate hydrogel and calcium silicate hydrogel groups. On day 28, a large number of blue collagen fibers were still observed in the blank group and the blank hydrogel group, indicating the formation of fibrous tissue. In contrast, fewer collagen fibers were observed in the strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogel groups, especially in the strontium carbonate / calcium silicate hydrogel group. Tunel staining was used to observe cell apoptosis in muscle tissue. On day 3, both the strontium carbonate hydrogel and calcium silicate hydrogel groups significantly inhibited cell apoptosis, especially the Tunel-positive cells in the strontium carbonate hydrogel group were significantly less than those in the calcium silicate hydrogel. In addition, the strontium carbonate / calcium silicate bioceramic hydrogel showed a stronger anti-apoptotic effect, indicating that strontium ions and silicon ions have a unique synergistic effect.

[0070] Embodiment 7:

[0071] Effects of strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogels on angiogenesis in a lower limb ischemia model.

[0072] (1) Adult male C57BL / 6 mice (6-8 weeks old) were selected to establish the femoral aorta ligation lower limb ischemia model and randomly divided into five groups: blank group (Ctrl), hydrogel group (SA), strontium carbonate hydrogel group (SA / Sr), calcium silicate hydrogel group, and strontium carbonate / calcium silicate gel group (SA / Sr / Si).

[0073] (2) The lower limbs were obtained on the 28th day after surgery, and CT imaging was used to analyze the remodeling of the lower limb vessels.

[0074] (3) On the 28th day after surgery, the gastrocnemius muscles of the lower limbs were obtained and immunofluorescence staining of CD31 and α-SMA was performed.

[0075] (4) The gastrocnemius muscles of the lower limbs were obtained on days 3, 7, and 28 after surgery, and the expressions of VEGF and HIF-1α in the lower limbs were analyzed by Western Blot.

[0076] Figure 8 The results of CT imaging of the lower limbs of mice on day 28 clearly showed that the strontium carbonate / calcium silicate hydrogel group significantly promoted the formation of capillary networks in the lower limbs compared with the blank group and the blank hydrogel group. In addition, strontium carbonate hydrogel and calcium silicate hydrogel also promoted angiogenesis. Immunofluorescence sections further proved that bioceramics can effectively promote angiogenesis.

[0077] Furthermore, Western Blot was used to analyze the expression of angiogenesis-related cytokines (VEGF and HIF-1α) in the ischemic lower limbs of mice at different time points. Interestingly, we found that on days 3 and 7, the protein expression of VEGF and HIF-1α in the lower limbs of mice treated with strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogels was significantly higher than that in the blank group and the hydrogel group.

[0078] Embodiment 8:

[0079] Effects of strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogels on inflammation regulation in the lower limb ischemia model.

[0080] (1) Adult male C57BL / 6 mice (6-8 weeks old) were selected to establish the femoral aorta ligation lower limb ischemia model and randomly divided into five groups: blank group (Ctrl), hydrogel group (SA), strontium carbonate hydrogel group (SA / Sr), calcium silicate hydrogel group, and strontium carbonate / calcium silicate gel group (SA / Sr / Si).

[0081] (2) On the third day after surgery, the gastrocnemius muscles of the lower limbs were obtained for immunofluorescence staining of CD206, CD11C, and F4 / 80. (3) ELISA was used to evaluate the levels of IL-6, TNF-a, IL-10, and Arg-1 in the gastrocnemius muscles of the lower limbs.

[0082] Fig. 9 The results showed that compared with the blank group and blank hydrogel group, the CD11C staining intensity of the strontium carbonate or calcium silicate or strontium carbonate / calcium silicate bioceramic hydrogel group was reduced, and CD206 staining was significantly increased. The quantitative analysis results showed that in the strontium carbonate / calcium silicate hydrogel group, the proportion of M1 macrophages was significantly lower, while the proportion of M2 macrophages was significantly higher than that in the other groups. In addition, the ELISA test results showed that strontium carbonate / calcium silicate hydrogel had the best effect in reducing the expression of pro-inflammatory factors (TNF-α and IL-6) and promoting the expression of anti-inflammatory factors (Arg-1 and IL-10).

[0083] Technical personnel should note that although the present invention has been described according to the above specific implementation methods, the inventive concept of the present invention is not limited to this invention, and any modification using the inventive concept will be included in the scope of protection of this patent.

[0084] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. Application of a bioceramic preparation in the preparation of a drug for treating lower limb ischemia, characterized in that The effective concentration of silicon ions in the drug for treating lower limb ischemic diseases is 0.5~12μg / mL, the effective concentration of strontium ions is 0.5~12μg / mL, and the bioceramic preparation is a bioceramic hydrogel obtained by mixing bioceramics containing silicon and strontium elements with a water-soluble polymer solution.

2. The use according to claim 1, characterized in that The bioceramics containing silicon and strontium elements are one or more of the bioceramics containing calcium silicate, strontium carbonate, strontium silicate, bioglass, zinc silicate, magnesia feldspar, and copper silicate.

3. The use according to claim 1, characterized in that The water-soluble polymer solution is one or more of sodium alginate solution, hyaluronic acid solution, and chitosan solution.

4. The use according to claim 1, characterized in that The content of bioceramics containing silicon and strontium elements in the bioceramic preparation is 0.5%~30%w / v.

5. The use according to claim 4, characterized in that , the content of bioceramics containing silicon and strontium elements in the bioceramic preparation is 1% w / v.

6. The use according to claim 1, characterized in that , the lower limb ischemic diseases include lower limb arteriosclerosis obliterans, thromboangiitis obliterans, and diabetic lower limb vascular disease.

7. The use according to claim 1, characterized in that: The strontium ion is Sr 2+ Ion, silicon ion is SiO3 2- ion.

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