Anti-osteoporosis drug-loaded slow-release degradable bone cement and application thereof

Abstract

The invention belongs to the technical field of slow-release degradable bone cement for bone defect repair, and particularly relates to slow-release degradable bone cement loaded with an anti-osteoporosis drug and application. According to the bone cement disclosed by the invention, calcium phosphate bone cement powder is used as a solidification material, and the psoralen and / or isopsoralen-loaded PLGA microspheres are added, so that the bone cement obtains a drug slow release duration of at least 16 weeks. By further adding chitosan, the early release of the medicine can be reduced, and the accumulation release rate of the medicine is increased. After the sodium caprylate is added to fill the bone defect part, the osteoblast activity and the whole-body bone mineral density can be improved. The bone cement is long in slow release time, low in early release rate and suitable for bone defect repair of osteoporosis patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of sustained-release biodegradable bone cement for bone defect repair, and particularly relates to a sustained-release biodegradable bone cement loaded with anti-osteoporosis drugs and its uses. Background Art

[0002] 1. Market demand and technical demand analysis of bone cement loaded with anti-osteoporosis drugs

[0003] Bone defects caused by fractures or formed during surgeries are relatively common in clinical practice. The incidence of fractures alone is higher than 2%, and the incidence of fractures in the middle-aged and elderly population is higher than 6%, while the incidence of fractures in the osteoporosis population is higher than 10%. The healing after fracture is divided into 5 stages. Around 2 weeks is the fibrotic tissue formation stage, around 3 weeks is the fibrous callus stage, and around 4 weeks is the mature callus stage. The new bone growth rate after bone defect is related to age, gender, concomitant diseases, etc. New bone growth in children and adolescents takes about 4 - 6 weeks; in the middle-aged and elderly population, it takes about 4 - 8 weeks; in the population with osteoporosis, it takes about 8 - 12 weeks (Gao Haiyan, et al. Study on the effect of Yangyuan Jiangu Decoction on the curative effect, bone mineral density and fracture healing rate of senile osteoporosis fractures. Chinese Archives of Traditional Chinese Medicine, 2023). The above are only the healing times in terms of morphology, and the recovery of indicators such as bone mineral density requires a longer time.

[0004] Bone cement is a commonly used Class III medical device for bone defects. Currently, there are mainly several categories on the market, such as Polymethyl methacrylate (PMMA) bone cement, Calcium Phosphate Cement (CPC), and Calcium Sulfate bone cement. Among them, PMMA bone cement is difficult to degrade and absorb, new bone is difficult to grow into, and there is an exothermic reaction that is prone to cause damage; Calcium Sulfate bone cement can induce bone growth and is easy to degrade and absorb, but the degradation rate is often faster than the bone growth rate during osteoporosis, which is prone to cause surgical failure. The degradation rate can be delayed by adding CPC components (CN107638593A). The degradation rate of CPC bone cement is slower than that of Calcium Sulfate bone cement, but it has low hardness and high brittleness. When bone defects are accompanied by osteoporosis, the implantation of bone cement only plays a role in supporting or inducing bone repair, and cannot play a therapeutic effect on osteoporosis itself. Therefore, the use of bone cement still needs to be combined with anti-osteoporosis drugs. Even for non-osteoporosis patients, it is beneficial for bone recovery at the bone defect site and promotes the increase of bone mineral density.

[0005] Currently, there are few varieties of bone cement loaded with drugs on the market, and the drugs loaded are antibiotics such as vancomycin, aiming to prevent and treat recent infections after surgery. For example, the bone cement loaded with gentamicin sulfate approved by the National Medical Products Administration in 2022 and the bone cement loaded with anti-tumor drugs or antibiotics announced in CN104288833A. Among them, the early drug burst release amount of the drug-loaded bone cement disclosed in CN104288833A accounts for a relatively high proportion, close to 20% - 80% of the cumulative release amount, and the total sustained release time is about 15 days, which is not suitable for the bone growth characteristics and the medication cycle of anti-osteoporosis drugs in osteoporosis bone defects.

[0006] Several kinds of bone cements for loading anti-osteoporosis drugs are under research. CN110075351A provides a dual-drug release bone cement. Gelatin microspheres are used to load antibiotics and alendronate sodium. Among them, the early release proportion of alendronate sodium is low and the later release proportion is high, with a sustained release effect of 70 days, and the release behavior of antibiotics is opposite. However, it uses PMMA bone cement, which has problems such as non-absorbability, non-biodegradability, exothermic damage, and difficult bone ingrowth. And the sustained release for 70 days still does not adapt to the bone growth cycle of the osteoporosis bone defect site. Houdt et al. prepared a poly(lactic-co-glycolic acid) (PLGA)-CPC bone cement loaded with alendronate, and the sustained release can be up to 140 days, but the drug release rate is low. This bone cement does not increase bone formation in the defect area, but promotes bone formation in the area around the defect (Fang Xuancheng et al. Research progress of calcium phosphate bone cement drug sustained release system. Journal of Guangzhou Medical University, 2023.).

[0007] Combined with the above situation, it can be seen that there is a need for bone cement loaded with anti-osteoporosis drugs on the current market. And bone cement has the disadvantages that it is difficult to replace or repeatedly implant and administer drugs once implanted, and reasonable drug loading and reasonable sustained release characteristics are required. However, the existing drug-loaded bone cements have problems such as too high burst release proportion or insufficient sustained release time, which are not sufficient to meet the technical and market demands of the subdivision field of bone defect repair in osteoporosis patients; especially, there is a lack of degradable bone cement whose drug release behavior meets the bone ingrowth cycle.

[0008] To achieve the dual purposes of repairing bone defects and drug treatment, Fang Xuancheng et al. believe that the drug-loaded bone cement should have the following characteristics: it should be degradable and the degradation rate should be as synchronous as possible with the new bone formation rate; it should have a large porosity to adapt to the ingrowth of new tissues; the drug burst release time should be short, the burst release proportion should be low, and it should have a long sustained release time; it should have good mechanical strength and other characteristics. And when additional materials or drugs are added to bone cement, especially CPC bone cement, there are complex interactions between them, resulting in difficult-to-predict changes in various aspects such as drug release behavior, bone cement degradation, and bone cement mechanical strength (Fang Xuancheng et al. Research progress of calcium phosphate bone cement drug sustained release system. Journal of Guangzhou Medical University, 2023.), which further leads to great difficulties in the research and development of drug-loaded bone cement.

[0009] 2. Characteristics of the effects of psoralen and isopsoralen, and analysis of the technology and market demand for drug-loaded sustained-release degradable bone cement

[0010] Both psoralen and isopsoralen can directly act on osteoblasts, osteoclasts, and bone marrow mesenchymal stem cells to play a role in bone growth regulation, either by acting on cells in close proximity through contact or by acting on surrounding and other bone tissues throughout the body after entering the bloodstream (Hao Yifan. Research on the effects and mechanisms of psoralen on osteogenic differentiation of rat bone marrow mesenchymal stem cells [D]. Liaoning University of Traditional Chinese Medicine, 2018; Yuan X et al. Psoralen and isopsoralen ameliorate sex hormone deficiency-induced osteoporosis in female and male mice. Biomed research international, 2016.). Moreover, psoralen and isopsoralen have reversible hepatotoxicity and cytotoxicity (Yang Kuo et al. Research progress on the pharmacological effects and hepatotoxicity mechanism of psoralen. Chinese Traditional and Herbal Drugs, 2021; Yang Kuo et al. Research progress on the pharmacological effects and liver and kidney injury mechanisms of isopsoralen. Evaluation and Analysis of Drug Use in Hospitals of China, 2021), and slow release and bone-targeted drug delivery are required to reduce toxicity, such as the bone-targeted preparation in CN107028885A (bone-targeted liposome for the treatment of osteoporosis and its preparation method). However, the bone-targeted liposome still cannot distinguish between bone defect locations and non-bone defect locations, and it is difficult to exert a targeted local effect on bone defect locations. Summary of the Invention

[0011] In the above background, aiming at the drug release problem of bone cement loaded with anti-osteoporosis drugs, the purpose of the present invention is to provide a sustained-release degradable bone cement loaded with anti-osteoporosis drugs, which is prepared by mixing a solid phase of bone cement and a liquid phase of bone cement. The solid phase of the bone cement contains poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with anti-osteoporosis drugs and calcium phosphate bone cement powder as a setting material, and PLGA is used as a sustained-release capsule material. The anti-osteoporosis drugs are psoralen (CAS: 66-97-7) and / or isopsoralen (Angelicin / Isopsoralen; CAS: 523-50-2).

[0012] The bone cement is prepared by the following method:

[0013] S1: Dissolve the anti-osteoporosis drug in absolute ethanol to make an active ingredient solution, dissolve PLGA in dichloromethane to make a capsule material solution, and mix the active ingredient solution and the capsule material solution to make a PLGA microsphere oil phase.

[0014] S2: Use the PVA aqueous solution as the aqueous phase of the PLGA microspheres. Mix the oil phase of the PLGA microspheres with the aqueous phase of the PLGA microspheres, and prepare an O / W emulsion by ultrasonic treatment. Stir and volatilize the organic solvent from the O / W emulsion to obtain a suspension of drug-loaded microspheres. Centrifuge the suspension of drug-loaded microspheres, wash the precipitate with deionized water, and freeze-dry to obtain the drug-loaded microspheres.

[0015] Sn1: Take the drug-loaded microspheres and mix them with the calcium phosphate cement powder as the solid phase of the cement. Mix and stir the solid phase of the cement with the liquid phase of the cement to prepare a sustained-release and degradable bone cement loaded with anti-osteoporosis drugs.

[0016] Preferably, in S1: the weight ratio of the anti-osteoporosis drug to PLGA is 1:(3 - 12), the concentration of PLGA in the capsule material solution is 7.5 mg / mL - 30 mg / mL, and the volume ratio of the active ingredient solution to the capsule material solution is 1:(2 - 4); in S2: the mass-volume concentration of the PVA aqueous solution is 0.5% - 2%, the volume ratio of the oil phase of the PLGA microspheres to the aqueous phase of the PLGA microspheres is 1:(5 - 15), the ultrasonic power is 20 W - 80 W (20 - 80 watts), the ultrasonic duration is 1 min - 3 min (1 - 3 minutes), the stirring speed is 300 rpm - 800 rpm, and the stirring duration is 4 h - 6 h (4 - 6 hours); in Sn1: the weight ratio of the drug-loaded microspheres to the calcium phosphate cement powder is (2 - 5):6 based on the weight ratio of PLGA to the calcium phosphate cement powder.

[0017] The bone cement prepared by encapsulating psoralen or isopsoralen in PLGA microspheres, adding calcium phosphate cement powder, and mixing with the liquid phase of the cement shows a two-phase drug release characteristic and a sustained-release duration of at least 16 weeks, which is in line with the slow bone defect repair characteristics of osteoporosis patients.

[0018] Furthermore, the object of the present invention is to provide a sustained-release and degradable bone cement loaded with anti-osteoporosis drugs that reduces the initial cumulative release rate of the drug and increases the total cumulative release rate of the drug. It is composed of a solid phase of the cement and a liquid phase of the cement. The solid phase of the cement contains PLGA microspheres loaded with anti-osteoporosis drugs, chitosan, and calcium phosphate cement powder as a setting material, where PLGA is used as a sustained-release capsule material. The anti-osteoporosis drug is psoralen and / or isopsoralen.

[0019] The bone cement is prepared by the following method:

[0020] S1: Dissolve the anti-osteoporosis drug in absolute ethanol to prepare an active ingredient solution, dissolve PLGA in dichloromethane to prepare a capsule material solution, and mix the active ingredient solution and the capsule material solution to prepare the oil phase of the PLGA microspheres.

[0021] S2: Use the PVA aqueous solution as the aqueous phase of the PLGA microspheres. Mix the oil phase of the PLGA microspheres with the aqueous phase of the PLGA microspheres, and prepare an O / W emulsion by ultrasonic treatment. Stir and volatilize the organic solvent from the O / W emulsion to obtain a suspension of drug-loaded microspheres. Centrifuge the suspension of drug-loaded microspheres, wash the precipitate with deionized water, and freeze-dry to obtain the drug-loaded microspheres.

[0022] S31: Dissolve chitosan in an acetic acid aqueous solution to prepare a chitosan acetate solution. Add the drug-loaded microspheres to the chitosan acetate solution, stir and mix to submerge the drug-loaded microspheres in the chitosan acetate solution, centrifuge, and freeze-dry the precipitate to obtain the drug-loaded microsphere chitosan powder.

[0023] Sn2: Mix the drug-loaded microsphere chitosan powder with the calcium phosphate cement powder to prepare the solid phase of the cement. Mix and stir the solid phase of the cement with the liquid phase of the cement to prepare a sustained-release and degradable cement loaded with anti-osteoporosis drugs.

[0024] Preferably, in S1: the weight ratio of the anti-osteoporosis drug to the PLGA feed is 1:(3 - 12), the PLGA concentration in the capsule material liquid is 7.5 mg / mL - 30 mg / mL, and the volume ratio of the active ingredient liquid to the capsule material liquid is 1:(2 - 4); in S2: the mass-volume concentration of the PVA aqueous solution is 0.5% - 2%, the volume ratio of the oil phase of the PLGA microspheres to the aqueous phase of the PLGA microspheres is 1:(5 - 15), the ultrasonic power is 20 W - 80 W (20 - 80 watts), the ultrasonic duration is 1 min - 3 min (1 - 3 minutes), the stirring speed is 300 rpm - 800 rpm, and the stirring duration is 4 h - 6 h (4 - 6 hours); in S31: the mass-volume concentration of chitosan in the chitosan acetate solution is 0.01% - 0.05%; in Sn2: the weight ratio of the drug-loaded microsphere chitosan powder to the calcium phosphate cement powder, calculated based on the weight ratio of PLGA to the calcium phosphate cement powder feed, is (2 - 5):6.

[0025] The PLGA microspheres encapsulate psoralen or isopsoralen. The drug-loaded microspheres are coated with chitosan, and calcium phosphate cement powder is added. The cement prepared by mixing with the liquid phase of the cement shows a three-phase release characteristic in drug release, with a sustained-release duration of at least 16 weeks. Moreover, the cumulative drug release rate is reduced within the first 0 - 2 weeks and increased until the 16th week. This conforms to the characteristics of slow early bone ingrowth and long repair time in osteoporotic patients with bone defects.

[0026] Furthermore, the present invention provides another sustained-release and degradable bone cement loaded with anti-osteoporosis drugs, which can reduce the initial cumulative release rate of the drug and increase the total cumulative release rate of the drug. It is prepared by mixing a solid phase of bone cement and a liquid phase of bone cement. The solid phase of bone cement contains PLGA microspheres loaded with anti-osteoporosis drugs, chitosan, sodium caprylate (CAS: 1984-06-1), and calcium phosphate bone cement powder as a setting material, wherein PLGA is used as a sustained-release capsule material. The anti-osteoporosis drug is psoralen and / or isopsoralen.

[0027] The bone cement is prepared by the following method:

[0028] S1: Dissolve the anti-osteoporosis drug in absolute ethanol to prepare an active ingredient solution, dissolve PLGA in dichloromethane to prepare a capsule material solution, and mix the active ingredient solution and the capsule material solution to prepare an oil phase of PLGA microspheres.

[0029] S2: Take an aqueous PVA solution as the aqueous phase of PLGA microspheres, mix the oil phase of PLGA microspheres and the aqueous phase of PLGA microspheres, and prepare an O / W emulsion by ultrasonic treatment. The O / W emulsion is stirred and volatilized to remove the organic solvent to obtain a suspension of drug-loaded microspheres. The suspension of drug-loaded microspheres is centrifuged, the precipitate is washed with deionized water, and freeze-dried to obtain drug-loaded microspheres.

[0030] S32: Dissolve chitosan in an aqueous acetic acid solution to prepare a chitosan acetic acid solution, add the drug-loaded microspheres and sodium caprylate to the chitosan acetic acid solution, stir and mix to submerge the drug-loaded microspheres in the chitosan acetic acid solution, centrifuge, and freeze-dry the precipitate to obtain a powder of drug-loaded microspheres chitosan sodium caprylate.

[0031] Sn3: Mix the powder of drug-loaded microspheres chitosan sodium caprylate and calcium phosphate bone cement powder to prepare the solid phase of bone cement, and mix and stir the solid phase of bone cement and the liquid phase of bone cement to prepare a sustained-release and degradable bone cement loaded with anti-osteoporosis drugs.

[0032] Preferably, in S1: the weight ratio of the anti-osteoporosis drug to the PLGA feedstock is 1:(3 - 12), the PLGA concentration in the capsule material solution is 7.5 mg / mL - 30 mg / mL, and the volume ratio of the active ingredient solution to the capsule material solution is 1:(2 - 4); in S2: the mass-volume concentration of the PVA aqueous solution is 0.5% - 2%, the volume ratio of the PLGA microsphere oil phase to the PLGA microsphere water phase is 1:(5 - 15), the ultrasonic power is 20 W - 80 W (20 - 80 watts), the ultrasonic duration is 1 min - 3 min (1 - 3 minutes), the stirring speed is 300 rpm - 800 rpm, and the stirring duration is 4 h - 6 h (4 - 6 hours); in S32: the mass-volume concentration of chitosan in the chitosan acetate solution is 0.01% - 0.05%, and the feed amount of sodium octanoate by weight is 1 / 6 - 1 / 2 of the feed amount of the anti-osteoporosis drug; in Sn3: the weight ratio of the drug-loaded microsphere chitosan sodium octanoate powder to the calcium phosphate cement powder, calculated based on the weight ratio of PLGA to the calcium phosphate cement powder, is (2 - 5):6.

[0033] The PLGA microspheres encapsulate psoralen or isopsoralen. After being coated with chitosan for the drug-loaded microspheres and adding sodium octanoate, they are mixed with the calcium phosphate cement powder and blended with the bone cement liquid phase to prepare the bone cement. Sodium octanoate does not affect the in vitro drug release, compressive strength, cytotoxicity and other characteristics of the bone cement. Implanting into the bone defect site significantly increases the blood drug concentration, the activity of osteoblasts throughout the body and the bone density at the non-bone defect site. In addition to being beneficial to the bone defect site, it is more conducive to improving the osteoporosis problem of the whole body in osteoporosis patients.

[0034] The aforementioned PLGA is divided into specifications such as 50:50, 65:35, 75:25, 85:15 according to the ratio of lactide:glycolide, and is sold with a molecular weight of 20 kDa - 240 kDa. It is known to those skilled in the art that as the lactide content increases, the degradation rate of PLGA slows down; as the molecular weight of PLGA increases, the degradation rate of PLGA slows down. Therefore, selecting PLGA with a higher lactide ratio or a larger molecular weight is more conducive to further improving the sustained release effect of PLGA microspheres. The aforementioned PVA is polyvinyl alcohol.

[0035] The aforementioned calcium phosphate cement (CPC) is a commonly used bone cement material in the market. It is a mixture of one or several calcium phosphate salt powders, namely a powder system mainly composed of calcium phosphate, such as a mixture of one or several of tricalcium phosphate, tetracalcium phosphate, calcium hydrogen phosphate, hydroxyapatite, calcium carbonate, calcium dihydrogen phosphate, etc. Specifically, such as a mixture of α-tricalcium phosphate, tetracalcium phosphate, calcium hydrogen phosphate, and hydroxyapatite; a mixture of α-tricalcium phosphate and tetracalcium phosphate; a mixture of α-tricalcium phosphate, tetracalcium phosphate, and calcium carbonate; a mixture of β-tricalcium phosphate, tetracalcium phosphate, and calcium dihydrogen phosphate; a mixture of α-tricalcium phosphate, calcium dihydrogen phosphate, and calcium carbonate, etc. The common feature of CPC is that the hydration reaction finally forms hydroxyapatite identical to human bone tissue; its calcium-phosphorus ratio is usually 1.5 - 1.6.

[0036] The liquid phase of the aforementioned CPC bone cement is usually water or a salt solution, such as an aqueous solution of sodium dihydrogen phosphate.

[0037] The aforementioned chitosan, also known as deacetylated chitin, soluble chitin, and polyglucosamine, is a kind of off-white powder. The deacetylation degree of chitosan sold in the market is usually 70% - 90%. The repeating units of chitosan in this field have also been chemically modified to improve its solubility. The chitosan described in the present invention is cationic chitosan, including but not limited to natural chitosan (deacetylation degree 70% - 90%) sold in the market or cationic chitosan such as chitosan chloride.

[0038] The present invention also provides the application of the aforementioned sustained-release and degradable bone cement loaded with anti-osteoporosis drugs in the preparation of bone defect filling materials for osteoporosis patients.

[0039] Advantages of the present invention:

[0040] (1) The PLGA microsphere bone cement loaded with anti-osteoporosis drugs of the present invention can extend the drug sustained-release time. The in vitro drug release curve shows a two-phase release and the sustained release can reach 16 weeks, which is in line with the slow bone defect repair speed of osteoporosis patients.

[0041] (2) For the PLGA microsphere bone cement loaded with anti-osteoporosis drugs of the present invention, after adding chitosan to the solid phase of the bone cement, the coating effect on the PLGA microspheres delays the release of the drugs on the outer layer of the PLGA microspheres; the certain moisture absorption property of chitosan and the residual acetic acid slowly promote the degradation of the PLGA microspheres. Thereby, it reduces and slows down the drug cumulative release rate and release speed in the first 2 weeks of the PLGA drug-loaded microsphere bone cement, and improves the total drug cumulative release rate within 16 weeks. The in vitro drug release curve shows a three-phase release and the sustained release can reach 16 weeks, which is in line with the characteristics of slow early bone ingrowth and slow bone defect repair speed of osteoporosis patients, and is beneficial to reducing the drug loss caused by excessive drug release in the early stage.

[0042] (3) The chitosan-loaded anti-osteoporosis drug PLGA microsphere bone cement of the present invention is precipitated with chitosan through anion-cation interaction after adding sodium caprylate. Sodium caprylate does not affect the compressive strength, setting time, in vitro drug release characteristics, etc. of the bone cement, nor does it cause additional cytotoxicity, but improves the improvement effect of the bone cement on systemic osteoblast-related indexes and bone density at non-bone defect sites in vivo. Brief Description of the Drawings

[0043] Figure 1 It is the in vitro drug release curve of the drug-loaded PLGA microsphere bone cement sample of Example 3;

[0044] Figure 2 It is the in vitro drug release curve of the drug-loaded PLGA microsphere bone cement sample of Example 4;

[0045] Figure 3 It is the in vitro drug release curve of the drug-loaded PLGA microsphere bone cement sample of Example 5;

[0046] Figure 4 It is the result of the in vitro cytotoxicity test of the bone cement sample of Example 6. Detailed Description of the Invention

[0047] Unless otherwise specified, the temperature condition in the following examples is 25°C ± 5°C; the indicated W / V is the mass-volume concentration in g / mL; when expressed in %, it is g / 100 mL; for example, 1% (W / V) is 1 g / 100 mL. All the chitosan used is cationic chitosan without surface chemical cross-linking modification. Materials used in the examples: PLGA (lactide:glycolide = 50:50; Mw: 30 - 60 kDa), PVA (Mw: 13 - 23 kDa; degree of hydrolysis 87 - 89%), chitosan (degree of deacetylation > 80%), Sigma reagent; psoralen, isopsoralen, reference standards from the National Institutes for Food and Drug Control. The mouse embryonic osteoblast precursor cell line MC3T3-E1 is numbered ATCC: CRL-2593. Other reagents, solvents, reagent kits, etc. are all common consumables and are available on the market. The concentration of isopsoralen / psoralen in the active ingredient solution of the examples is an exemplary concentration. Those skilled in the art can adjust it within the solubility range based on the solubility characteristics of isopsoralen, so it should not be regarded as a limitation to the protection scope. The solid-liquid ratio of the solid phase and liquid phase of the bone cement in the examples is an exemplary solid-liquid ratio. Those skilled in the art can adjust it within the common solid-liquid ratio ranges such as 2 g / mL, 3 g / 2 mL, 1 g / mL, etc. based on the common solid-liquid ratio range of calcium phosphate bone cement, and it should not be regarded as a limitation to the protection scope.

[0048] Example 1: Isopsoralen-Loaded PLGA Microspheres and Their Characteristics

[0049] 1 Preparation method (emulsion-solvent evaporation method, preparation parameters and feeding information are shown in Table 1)

[0050] (1) Dissolve psoralen in absolute ethanol to prepare an active ingredient solution (I2 and I3 are 10 mg / mL, and I1, I4 - I9 are 5 mg / mL). Dissolve PLGA in dichloromethane to prepare a capsule material solution. Mix the active ingredient solution and the capsule material solution to prepare an oil phase of PLGA microspheres.

[0051] (2) Take an aqueous PVA solution as the water phase of PLGA microspheres. Mix the oil phase of PLGA microspheres and the water phase of PLGA microspheres, and use 40 W ultrasonic waves for 2 min to prepare an O / W emulsion. Stir the O / W emulsion at 300 rpm for 5 h to volatilize and remove the organic solvent to obtain a suspension of drug-loaded microspheres. Centrifuge the suspension of drug-loaded microspheres, wash the precipitate with deionized water, and freeze-dry to obtain drug-loaded microspheres, which are stored at -80 °C for standby.

[0052] 2 Detection method

[0053] (1) Encapsulation efficiency:

[0054] Determine the content of psoralen by ultraviolet spectrophotometry at a wavelength of 246 nm. Establish a standard curve of absorbance - drug concentration in the solvent. Take the drug-loaded microspheres, add dichloromethane (10 mg / mL), ultrasonically dissolve the microspheres, add 2 volumes of absolute ethanol, vortex and shake, centrifuge at 2000 rpm for 10 min, take the supernatant, filter through a 0.45 μm microporous filter membrane, record the absorbance, and calculate the drug concentration according to the standard curve. Encapsulation efficiency = drug content in drug-loaded microspheres / drug dosage × 100%.

[0055] (2) Average particle size

[0056] Observe the morphology of the sample microspheres under an electron microscope, and use Nano Measurer software to statistically analyze the average particle size of the sample microspheres.

[0057] Table 1 shows the determination results of 9 kinds of psoralen-loaded PLGA microspheres. The average particle size is in the range of 60 μm - 210 μm, and the microsphere encapsulation efficiency is in the range of 80% - 96%. When the PLGA concentration in the capsule material solution is 7.5 - 30 mg / mL and the PVA concentration (W / V) in the water phase of PLGA microspheres is 0.5 - 2%, the microsphere morphology and encapsulation efficiency results are relatively ideal, meeting the requirements for use in bone cement.

[0058] Table 1 Preparation parameters and microsphere characteristics of psoralen-loaded PLGA microspheres

[0059]

[0060] Note: A is the feeding ratio (W:W) in the preparation of the oil phase of PLGA microspheres: psoralen:PLGA; B is the feeding ratio (V:V) in the preparation of the oil phase of PLGA microspheres: active ingredient solution: capsule material solution; C is the feeding ratio (V:V) in the preparation of the O / W emulsion of PLGA microspheres: oil phase of PLGA microspheres: aqueous phase of PLGA microspheres; D is the PVA concentration (W / V) of the aqueous phase of PLGA microspheres.

[0061] Example 2: PLGA Microspheres Loaded with Psoralen or Isopsoralen and Their Characteristics

[0062] The preparation parameters and feeding information are shown in Table 2.

[0063] PLGA Microspheres Loaded with Psoralen:

[0064] (1) Dissolve psoralen in absolute ethanol to prepare an active ingredient solution (5 mg / mL), dissolve PLGA in dichloromethane to prepare a capsule material solution (30 mg / mL), and mix the active ingredient solution and the capsule material solution according to a volume ratio of 1:2 to prepare the oil phase of PLGA microspheres (i.e., psoralen:PLGA = 1:12);

[0065] (2) Take an aqueous PVA solution (1%, W / V) as the aqueous phase of PLGA microspheres, mix the oil phase of PLGA microspheres and the aqueous phase of PLGA microspheres according to a volume ratio of 1:15, sonicate for 2 min at 40 W to prepare an O / W emulsion, stir the O / W emulsion at 300 rpm for 5 h to volatilize and remove the organic solvent to obtain a suspension of drug-loaded microspheres. Centrifuge the suspension of drug-loaded microspheres, wash the precipitate with deionized water, and lyophilize to obtain drug-loaded microspheres, which are stored at -80 °C for later use.

[0066] PLGA Microspheres Loaded with Isopsoralen:

[0067] (1) Dissolve isopsoralen in absolute ethanol to prepare an active ingredient solution (10 mg / mL), dissolve PLGA in dichloromethane to prepare a capsule material solution (10 mg / mL), and mix the active ingredient solution and the capsule material solution according to a volume ratio of 1:3 to prepare the oil phase of PLGA microspheres (i.e., isopsoralen:PLGA = 1:3);

[0068] (2) Take an aqueous PVA solution (1%, W / V) as the aqueous phase of PLGA microspheres, mix the oil phase of PLGA microspheres and the aqueous phase of PLGA microspheres according to a volume ratio of 1:10, sonicate to prepare an O / W emulsion, and stir the O / W emulsion to volatilize and remove the organic solvent to obtain a suspension of drug-loaded microspheres. Centrifuge the suspension of drug-loaded microspheres, wash the precipitate with deionized water, and lyophilize to obtain drug-loaded microspheres, which are stored at -80 °C for later use.

[0069] Particle Size and Encapsulation Efficiency Detection:

[0070] Determine isopsoralen / psoralen in the supernatant by ultraviolet spectrophotometry at a wavelength of 246 nm. Establish a standard curve of absorbance-drug concentration in the solvent. Take the prepared drug-loaded microspheres, add dichloromethane (10 mg / mL), dissolve the microspheres by ultrasonic treatment, add 2 volumes of absolute ethanol, vortex and shake, centrifuge at 2000 rpm for 10 min, take the supernatant, filter it through a 0.45-μm microporous membrane, record the absorbance, and calculate the drug concentration according to the standard curve. Encapsulation efficiency = drug content in drug-loaded microspheres / dosage of drug input × 100%.

[0071] Observe the morphology of the sample microspheres under an electron microscope, and use Nano Measurer software to statistically analyze the average particle size of the sample microspheres.

[0072] Table 2 Preparation parameters and microsphere characteristics of psoralen and isopsoralen-loaded PLGA microspheres

[0073]

[0074]

[0075] Note: For the active ingredient item, P is psoralen, and ISP is isopsoralen.

[0076] Table 2 shows the preparation parameters, microsphere morphology, and encapsulation parameters of the above psoralen-loaded PLGA microspheres and isopsoralen-loaded PLGA microspheres.

[0077] By comparison, the preparation conditions of the I7 isopsoralen-loaded PLGA microspheres in Example 1 and the psoralen-loaded PLGA microspheres in this example are the same. The microsphere morphology and encapsulation parameters of the psoralen-loaded PLGA microspheres are similar to those of the isopsoralen-loaded PLGA microspheres. The encapsulation efficiency of the psoralen-loaded PLGA microspheres is slightly lower than that of the isopsoralen-loaded PLGA microspheres, which may be related to the slightly higher solubility of psoralen in water than that of isopsoralen.

[0078] By comparison, the I2 isopsoralen-loaded PLGA microspheres in Example 1 and the isopsoralen-loaded PLGA microspheres in this example are the same except for the ultrasonic power, duration, stirring speed, and time. The ultrasonic power and duration for preparing the O / W emulsion and the stirring speed and time for volatilizing the organic solvent to prepare the drug-loaded microsphere suspension have a certain impact on the microsphere morphology and encapsulation parameters. However, when the ultrasonic power for preparing the O / W emulsion is 20 W - 80 W, the ultrasonic duration is 1 min - 3 min; the stirring speed of the O / W emulsion solvent is 300 rpm - 800 rpm, and the stirring duration is 4 h - 6 h; the encapsulation efficiency of the drug-loaded microspheres is higher than 75%, and the microsphere particle size is greater than 60 μm, meeting the requirements for use in bone cement.

[0079] Example 3 Isopsoralen-loaded PLGA microspheres-calcium phosphate bone cement and characteristics

[0080] 1. Drug-loaded microspheres: Take the I1, I7, I10, and P1 drug-loaded microspheres prepared in Examples 1 and 2 as representative drug-loaded microspheres with different particle sizes, encapsulation efficiencies, and loaded drugs.

[0081] 2. Preparation of blank microspheres: Follow the preparation methods of I1, I7, I10, and P1 drug-loaded microspheres in Examples 1 and 2 respectively. Without adding psoralen and isopsoralen, and keeping other operations unchanged, prepare the blank microspheres corresponding to the drug-loaded microspheres. For example, the preparation method of the blank microspheres of P1 drug-loaded microspheres is as follows:

[0082] (1) Dissolve PLGA in dichloromethane to make the capsule material solution (30 mg / mL), take absolute ethanol as the blank solution of the active ingredient (without adding psoralen), and mix the active ingredient solution and the capsule material solution according to a volume ratio of 1:2 to make the oil phase of PLGA microspheres;

[0083] (2) Take the PVA aqueous solution (1%, W / V) as the water phase of PLGA microspheres. Mix the oil phase of PLGA microspheres and the water phase of PLGA microspheres according to a volume ratio of 1:15, and ultrasonicate at 40 W for 2 min to make an O / W emulsion. Stir the O / W emulsion at 300 rpm for 5 h to volatilize and remove the organic solvent to obtain a blank microsphere suspension. Centrifuge the microsphere suspension, wash the precipitate with deionized water, and freeze-dry to obtain blank microspheres, which are stored at -80 °C for later use.

[0084] 3. Calcium phosphate cement powder:

[0085] Every 10 g of calcium phosphate cement powder contains 7.5 g of α-tricalcium phosphate (α-TCP), 1.8 g of tetracalcium phosphate (TTCP), 0.5 g of calcium hydrogen phosphate, and 0.2 g of hydroxyapatite.

[0086] 4. Cement liquid phase: Sodium dihydrogen phosphate aqueous solution (1 mmol / L).

[0087] 5. Preparation of PLGA microsphere cement:

[0088] (1) Drug-loaded PLGA microsphere cement: Take the I1, I7, I10, and P1 drug-loaded microspheres prepared in Examples 1 and 2, and mix them with calcium phosphate cement powder respectively as the solid phase of the cement (the feeding ratio is shown in Table 3). Add the cement liquid phase according to a solid-liquid ratio of 2 g / mL and mix and stir to prepare different drug-loaded PLGA microsphere calcium phosphate cement slurries.

[0089] (2) Blank microsphere-drug-cement: Take the blank microspheres corresponding to I1, I7, I10, and P1 drug-loaded microspheres, and mix them with calcium phosphate cement powder and the corresponding drugs respectively as the solid phase of the cement (the feeding ratio is shown in Table 3). Add the cement liquid phase according to a solid-liquid ratio of 2 g / mL and mix and stir to prepare different blank microsphere-drug-calcium phosphate cement slurries.

[0090] For example, the preparation method of P1 blank microspheres-drug-calcium phosphate cement is as follows:

[0091] (1) Take P1 blank microspheres, and mix them with calcium phosphate cement powder and psoralen according to the feeding ratio of PLGA:calcium phosphate cement powder (W:W)=1:3 and the feeding ratio of psoralen:PLGA (W:W)=1:12 as the solid phase of the cement. Add the liquid phase of the cement and stir at a solid-liquid ratio of 2 g / mL to prepare different blank microspheres-drug-calcium phosphate cement slurries.

[0092] Before measurement, inject the obtained microspheres-drug-calcium phosphate cement slurry into a stainless-steel mold for curing. The specifications of the stainless-steel mold for the cement are: diameter and height are 6 mm and 12 mm respectively.

[0093] 6. Measurement of setting time difference

[0094] Inject the calcium phosphate cement slurry into the cement mold, and measure the initial setting time and final setting time of the sample at 37 °C using the Gillmore double-needle method (Gillmore) according to the ASTM C2661999 standard, and calculate the setting time difference. The formula is: setting time difference (min)=final setting time - initial setting time.

[0095] 7. Measurement of compressive strength

[0096] After the sample is cured and demolded, measure the compressive strength of the sample at 37 °C and a humidity of 100% according to the T / CSBM 0027-022 standard.

[0097] 8. Measurement of drug release characteristics

[0098] Put the cured and demolded bone cement solids into centrifuge tubes of the same specification, add an equal volume of phosphate buffer solution (pH 7.2) to submerge them, and perform in vitro release in a 37 °C constant temperature water bath. Samples are taken at the same time points (for the method, see: Mao Lu, Zhang Bosong, Dong Di, et al. Study on the local release of vancomycin and meropenem-loaded bone cement in the human body [J]. Chinese Journal of Hospital Pharmacy, 2022). The sample solution is filtered through a microporous filter membrane, and the concentration of the active ingredient is measured by HPLC method, and the cumulative drug release percentage is calculated. Plot the time against the cumulative drug release percentage to draw the drug release curve.

[0099] HPLC chromatographic conditions: Waters SunFire C18 column (250 mm x 4.6 mm, 5 μm); mobile phase: acetonitrile-water (32:68), flow rate: 1.0 mL / min, detection wavelength: 246 nm.

[0100] 9. Results

[0101] The measurement results are shown in Table 3. The initial setting time of each drug-loaded PLGA microsphere bone cement is within the range of 3 - 6 min. As shown in Table 3, the setting time difference of the drug-loaded PLGA microsphere bone cement is 6.9 - 14.2 min, providing sufficient time for intraoperative bone cement mixing and injection at the bone defect site. The compressive strength of the drug-loaded PLGA microsphere bone cement is 34 - 60 Mpa.

[0102] The instruction manual appendix Figure 1 is the drug release curve of each bone cement sample. Figure 1 A, Figure 1 B, Figure 1 C, Figure 1 D are respectively the drug release curves of I1, I7, 110, P1 blank microsphere-drug-bone cement (i.e., the control bone cement, such as I1CCPC) and drug-loaded PLGA microsphere bone cements with different PLGA / calcium phosphate bone cement powder feeding ratios (such as I1CPC1, I1CPC2). I1CCPC, I7CCPC, 110CCPC, P1CCPC are control bone cements. The PLGA / calcium phosphate bone cement powder feeding ratios of I1CPC1, I7CPC1, I10CPC1, P1CPC1 are 2:6 (W:W), and those of I1CPC2, I7CPC2, I10CPC2, P1CPC2 are 5:6 (W:W).

[0103] As shown in the instruction manual appendix Figure 1 , the cumulative drug release percentage of the control bone cement is significantly lower than that of the corresponding drug-loaded PLGA microsphere bone cement. Each drug-loaded PLGA microsphere bone cement shows a two-phase release characteristic, consisting of a burst release phase and a sustained release phase. Except for the relatively fast drug release from 0 (24 h) to 2 weeks, it is in the sustained release phase from 2 to 16 weeks, with a long sustained release time, which is in line with the slow bone defect repair characteristics of osteoporosis patients.

[0104] During the 16-week detection period, the cumulative drug release percentages of the drug-loaded PLGA microsphere bone cements I7CPC1, I7CPC2, P1CPC1, P1CPC2, I10CPC1, I10CPC2, I1CPC1, I1CPC2 are 19.7%, 21.4%, 24.9%, 30.1%, 31.5%, 42.7%, 39.5%, 57.2% respectively, which are significantly higher than those of the corresponding blank microsphere-drug-bone cements. The cumulative drug release percentage is positively correlated with the drug content and PLGA content (microspheres) in the bone cement.

[0105] Table 3 Physical properties of different PLGA microsphere calcium phosphate bone cements

[0106]

[0107] Note: Sample number items: CCPC is blank microsphere-drug-calcium phosphate bone cement; CPC1 and CPC2 are corresponding drug-loaded PLGA microsphere bone cements. Drug items: P is psoralen, and ISP is isopsoralen. A is the feeding ratio (W:W) in the preparation of the oil phase of PLGA microspheres, drug:PLGA. B is the feeding ratio (W:W) in the preparation of the solid phase of bone cement: PLGA: calcium phosphate bone cement powder. For example, when preparing the corresponding drug-loaded microspheres in Example 1, if the feeding amount of PLGA is 5 g, then the feeding amount of calcium phosphate bone cement powder is 15 g or 6 g. ST: Setting time difference (final setting time - initial setting time), min. CS: Compressive strength, Mpa. a : Three samples were measured for each sample. * is the estimated content of isopsoralen in the bone cement according to the feeding ratio (including the solid phase and liquid phase of the bone cement).

[0108] Other preparation parameters of I1CPC1 and I1CPC2: The concentration of isopsoralen in the active ingredient solution in the preparation of PLGA microspheres is 5 mg / mL, the concentration of PLGA in the capsule material solution in the preparation of PLGA microspheres is 7.5 mg / mL, the feeding ratio (V:V) of the active ingredient solution to the capsule material solution in the preparation of the oil phase of PLGA microspheres: active ingredient solution: capsule material solution = 1:2, the PVA concentration (W / V) of the aqueous phase of PLGA microspheres is 0.5%, the ultrasonic emulsification condition of the O / W emulsion is 40 W x 2 min, the solvent stirring and volatilization condition of the O / W emulsion is 300 rpm x 5 h, the feeding ratio (V:V) in the preparation of the O / W emulsion of PLGA microspheres: oil phase of PLGA microspheres: aqueous phase of PLGA microspheres = 1:5, and the solid-liquid ratio in the preparation of bone cement is 2 g / mL.

[0109] Other preparation parameters of I10CPC1 and I10CPC2: The concentration of isopsoralen in the active ingredient solution in the preparation of PLGA microspheres is 10 mg / mL, the concentration of PLGA in the capsule material solution in the preparation of PLGA microspheres is 10 mg / mL, the feeding ratio (V:V) of the active ingredient solution to the capsule material solution in the preparation of the oil phase of PLGA microspheres: active ingredient solution: capsule material solution = 1:3, the PVA concentration (W / V) of the aqueous phase of PLGA microspheres is 1%, the ultrasonic emulsification condition of the O / W emulsion is 20 W x 3 min, the solvent stirring and volatilization condition of the O / W emulsion is 800 rpm x 4 h, the feeding ratio (V:V) in the preparation of the O / W emulsion of PLGA microspheres: oil phase of PLGA microspheres: aqueous phase of PLGA microspheres = 1:10, and the solid-liquid ratio in the preparation of bone cement is 2 g / mL.

[0110] I7CPC1, I7CPC2, P1CPC1, P1CPC2 and other preparation parameters: The concentration of psoralen or isopsoralen in the active ingredient solution during the preparation of PLGA microspheres is 5 mg / mL, the concentration of PLGA in the capsule material solution during the preparation of PLGA microspheres is 30 mg / mL, the feeding ratio (V:V) of the active ingredient solution to the capsule material solution in the preparation of the oil phase of PLGA microspheres: active ingredient solution: capsule material solution = 1:2, the concentration of PVA in the aqueous phase of PLGA microspheres (W / V) is 1.0%, the ultrasonic emulsification conditions for the O / W emulsion are 40 W x 2 min, the solvent stirring and volatilization conditions for the O / W emulsion are 300 rpm x 5 h, the feeding ratio (V:V) in the preparation of the O / W emulsion of PLGA microspheres: oil phase of PLGA microspheres: aqueous phase of PLGA microspheres = 1:15, and the solid-liquid ratio for the preparation of bone cement is 2 g / mL.

[0111] Example 4 Drug-loaded Microsphere Chitosan Bone Cement and Its Characteristics

[0112] The drug-loaded PLGA microsphere bone cement in Example 3 has the advantage of a long sustained-release period. However, by analyzing the drug release curve, it is found that among the drugs cumulatively released from the drug-loaded PLGA microsphere bone cement, the release amount in the 0-1 week accounts for about 50-70% of the total release within 16 weeks, and the release amount in the 0-2 weeks accounts for about 62-84% of the total release within 16 weeks. Compared with the bone defect repair process of osteoporosis patients, there is a deficiency of a relatively high early release amount. Figure 1

[0113] The content of this study is the effect of chitosan on the drug release of isopsoralen-loaded PLGA microsphere calcium phosphate bone cement. Since the average particle size of the obtained PLGA microspheres is relatively large, it indirectly leads to relatively large voids formed in the bone cement during the degradation process of PLGA microspheres. Although it is beneficial for bone ingrowth, it also significantly reduces the compressive strength of the bone cement. Therefore, it is investigated whether chitosan can further improve drug release at a low feeding amount.

[0114] Drug-loaded Microsphere Chitosan Bone Cement:

[0115] Taking I1CPC2 and I7CPC1 prepared in Example 3 as representatives of isopsoralen-loaded PLGA microsphere bone cements with different cumulative release percentages, chitosan is added to prepare the corresponding drug-loaded microsphere chitosan bone cement. The preparation method is as follows:

[0116] (1) Take chitosan and dissolve it in an acetic acid aqueous solution (W / V) with a concentration of 1%, and dilute it according to the required concentration in Table 4 to prepare a chitosan (W / V) acetic acid solution with a concentration of 0.01% or 0.05% for standby.

[0117] (2) Take different concentrations of chitosan acetic acid solution, add the I1 or I7 drug-loaded microspheres prepared in Example 1 according to Table 4, stir and mix for 1-2 h to immerse the drug-loaded microspheres in the chitosan acetic acid solution, centrifuge, and freeze-dry the precipitate to obtain drug-loaded microsphere chitosan powder, which is stored at -80 °C for standby.

[0118] (3) Take the drug-loaded microsphere chitosan powder and prepare the bone cement solid phase containing drug-loaded microsphere chitosan according to the same feeding ratio of PLGA:calcium phosphate bone cement powder as that of I1CPC2 and I7CPC1 (see Table 4) by the method of Example 3. Then mix and stir it with the bone cement liquid phase to prepare the bone cement slurry. Measure the setting time difference, compressive strength and drug release characteristics of each drug-loaded microsphere chitosan bone cement by the method of Example 3.

[0119] Results:

[0120] For each drug-loaded PLGA microsphere-chitosan-bone cement, the initial setting time is 3 - 6 min, the setting time difference is 8.2 - 12.6 min, and the compressive strength is 33.5 - 50.8 Mpa. It is suitable for intraoperative bone cement mixing and injection.

[0121] Table 4 Physical properties of different drug-loaded microsphere chitosan bone cements

[0122] Sample number Drug loading A B C <![CDATA[ST a > <![CDATA[CS a <!-- 10 -->]]> I1CPC2 ISP 1:3 5:6 - 10.1±2.7 52.5±6.4 I1CPC2K1 ISP 1:3 5:6 0.01 12.6±4.1 50.8±7.1 I1CPC2K2 ISP 1:3 5:6 0.05 8.2±1.5 46.2±6.9 I7CPC1 ISP 1:12 2:6 - 7.3±2.0 36.4±2.7 I7CPC1K1 ISP 1:12 2:6 0.01 10.8±3.9 37.1±6.2 I7CPC1K2 ISP 1:12 2:6 0.05 8.5±2.4 33.5±1.8

[0123] Note: Sample number item K is the chitosan-containing bone cement; Drug-loading item: ISP is isopsoralen. A is the feeding ratio (W:W) in the preparation of the PLGA microsphere oil phase, drug:PLGA. B is the feeding ratio (W:W) in the preparation of the bone cement solid phase: PLGA:calcium phosphate bone cement powder. For example, when the PLGA feeding amount is 5 g in the preparation of the corresponding drug-loaded microspheres in Example 1, the calcium phosphate bone cement powder feeding amount is 15 g or 6 g. C is the concentration of the chitosan acetic acid solution (W / V, %), such as 0.01 (W / V, %) which is 0.01 g / 100 mL. ST: Setting time difference (final setting time - initial setting time), min. CS: Compressive strength, Mpa. a : Three samples were measured for each sample.

[0124] Other preparation parameters of I1CPC2, I1CPC2K1, I1CPC2K2: The concentration of the active ingredient liquid isopsoralen in the preparation of PLGA microspheres is 5 mg / mL, the concentration of the encapsulating material liquid PLGA in the preparation of PLGA microspheres is 7.5 mg / mL, the feeding ratio (V:V) of the active ingredient liquid to the encapsulating material liquid in the preparation of the PLGA microsphere oil phase: active ingredient liquid:encapsulating material liquid = 1:2, the concentration of PVA in the PLGA microsphere aqueous phase (W / V) is 0.5%, the O / W emulsion ultrasonic emulsification condition is 40 W x 2 min, the O / W emulsion solvent stirring and volatilization condition is 300 rpm x 5 h, the feeding ratio (V:V) in the preparation of the PLGA microsphere O / W emulsion: PLGA microsphere oil phase:PLGA microsphere aqueous phase = 1:5, and the solid-liquid ratio of the bone cement preparation is 2 g / mL.

[0125] Other preparation parameters of I7CPC1, I7CPC1K1, and I7CPC1K2: The concentration of psoralen in the active ingredient solution during PLGA microsphere preparation is 5 mg / mL, the concentration of PLGA in the capsule material solution during PLGA microsphere preparation is 30 mg / mL, the feeding ratio (V:V) of the active ingredient solution to the capsule material solution in the preparation of the oil phase of PLGA microspheres: active ingredient solution: capsule material solution = 1:2, the concentration of PVA in the aqueous phase of PLGA microspheres (W / V) is 1%, the ultrasonic emulsification conditions for the O / W emulsion are 40 W x 2 min, the solvent stirring and volatilization conditions for the O / W emulsion are 300 rpm x 5 h, the feeding ratio (V:V) in the preparation of the O / W emulsion of PLGA microspheres: oil phase of PLGA microspheres: aqueous phase of PLGA microspheres = 1:15, and the solid-liquid ratio for bone cement preparation is 2 g / mL.

[0126] As shown in the attached instruction Figure 2 , Figure 2 A, Figure 2 B are I1CPC2, I1CPC2K1, and I1CPC2K2 respectively; and the drug release characteristics of the bone cements of I7CPC1, I7CPC1K1, and I7CPC1K2. The cumulative release percentage of the drug-loaded microsphere chitosan bone cement within 16 weeks is significantly higher than that of the corresponding chitosan-free bone cement; by 16 weeks, the cumulative release percentage increases by 13% - 29%. And the drug release amount of the drug-loaded microsphere chitosan bone cement from week 0 to week 2 accounts for 12 - 33% of the total release within 16 weeks, significantly slowing down the early release rate.

[0127] Further analysis found that the drug-loaded microsphere chitosan bone cement showed obvious three-phase release characteristics (three-stage step-like elevation). In the early stage (week 0 - week 2), the cumulative drug release rate increased slowly, and the release amount accounted for a low proportion of the total release amount within 16 weeks; in the middle stage (week 2 - week 4 or week 2 - week 6), the cumulative drug release rate increased the fastest and the cumulative release rate was high; in the later stage (week 4 - week 16 or week 6 - week 16), the cumulative drug release rate increased slowly. The drug release characteristics of the drug-loaded microsphere chitosan bone cement are in line with the characteristics of slow bone defect healing, long cycle, especially slow early bone ingrowth in osteoporosis patients.

[0128] According to the drug release curve, it is speculated that the change in drug release behavior is related to the addition method of chitosan. Chitosan is coated on the outer layer of the drug-loaded microspheres, restricting the release of the drug on the outer layer of the PLGA microspheres; the hygroscopicity of chitosan provides an accelerating condition for the hydrolysis of the PLGA microspheres. The acetic acid remaining in the chitosan solution also helps to promote the degradation of the PLGA microspheres. The cumulative drug release rate is increased, thus achieving the effect of both reducing the early release of the PLGA microspheres and increasing the later cumulative release rate of the PLGA microspheres.

[0129] Example 5 Drug-loaded Microsphere Chitosan Sodium Octanoate Bone Cement and Its Characteristics

[0130] Medium-chain fatty acids may be added to partial calcium phosphate bone cement and remain in the bone cement. In this study, sodium octanoate was used as a representative to prepare drug-loaded microsphere chitosan sodium octanoate bone cement containing medium-chain fatty acids, and the effects of sodium octanoate on the physical properties and drug release characteristics of bone cement were investigated.

[0131] Taking I1CPC2K2 and I7CPC1K1 prepared in Example 4 as representatives of drug-loaded microsphere chitosan bone cements with different cumulative release percentages, different drug-loaded microsphere chitosan sodium octanoate bone cements were prepared. The preparation method of the drug-loaded microsphere chitosan sodium octanoate bone cement referred to Example 4. During the preparation stage of the drug-loaded microsphere chitosan, sodium octanoate was added, and other steps were the same. Specifically as follows:

[0132] (1) Take chitosan and dissolve it in an acetic acid aqueous solution (W / V) with a concentration of 1%, and dilute it according to the required concentration in Table 5 to prepare a chitosan (W / V) acetic acid solution with a concentration of 0.01% or 0.05% for standby.

[0133] (2) Take different concentrations of chitosan acetic acid solution, add the I1 or I7 drug-loaded microspheres prepared in Example 1 and sodium octanoate according to the feeding ratio in Table 5, stir and mix for 1 - 2 h to submerge the drug-loaded microspheres in the chitosan acetic acid solution, centrifuge, and freeze-dry the precipitate to obtain drug-loaded microsphere chitosan sodium octanoate powder, which is stored at -80 °C for standby.

[0134] (3) Take the drug-loaded microsphere chitosan sodium octanoate powder, and according to the method of Example 3, prepare the bone cement solid phase containing the drug-loaded microsphere chitosan sodium octanoate powder respectively according to the same PLGA: calcium phosphate bone cement powder feeding ratio as I1CPC2 and I7CPC1 (see Table 5), and mix and stir with the bone cement liquid phase to prepare the bone cement slurry. According to the method of Example 3, measure the setting time difference, compressive strength and drug release characteristics of each drug-loaded microsphere chitosan bone cement respectively.

[0135] Results:

[0136] The setting time differences and compressive strengths of I1CPC2K2O1 and I1CPC2K2O2 were 8.1 - 8.4 min and 47.5 - 48.1 Mpa respectively; the setting time differences and compressive strengths of I7CPC1K1O1 and I7CPC1K1O2 were 9.2 - 11.3 min and 35.4 - 36.0 Mpa respectively. There were no obvious changes compared with I1CPC2K2 and I7CPC1K1.

[0137] The description appendix Figure 3 shows the drug release characteristics of the bone cement. The drug release characteristics of I1CPC2K2O1, I1CPC2K2O2 and I7CPC1K1O1, I7CPC1K1O2 were compared with those of I1CPC2K2 and I7CPC1K1, and there were no obvious changes.

[0138] Table 5 Feed ratios of drug-loaded microspheres chitosan sodium octanoate bone cement

[0139] Sample number Drug loading A B C D I1CPC2K2 ISP 1:3 5:6 0.05 - I1CPC2K2O1 ISP 1:3 5:6 0.05 2:1 I1CPC2K2O2 ISP 1:3 5:6 0.05 6:1 I7CPC1K1 ISP 1:12 2:6 0.01 - I7CPC1K1O1 ISP 1:12 2:6 0.01 2:1 I7CPC1K1O2 ISP 1:12 2:6 0.01 6:1

[0140] Note: In the sample number item, CPC is calcium phosphate bone cement powder, K is chitosan, and O is sodium octanoate; in the drug-loaded item, ISP is psoralen. A is the feed ratio (W:W) in the preparation of the PLGA microsphere oil phase, drug:PLGA. B is the feed ratio (W:W) in the preparation of the bone cement solid phase: PLGA:calcium phosphate bone cement powder. For example, when the corresponding drug-loaded microspheres in Example 1 are prepared, if the feed amount of PLGA is 5 g, then the feed amount of calcium phosphate bone cement powder is 15 g or 6 g. C is the concentration of the chitosan acetate solution (W / V, %), such as 0.01 (W / V, %) which is 0.01 g / 100 mL. D is the feed ratio (W:W), drug:sodium octanoate. ST: Setting time difference (final setting time - initial setting time), min. CS: Compressive strength, Mpa. a : Three samples were measured for each sample.

[0141] Other preparation parameters for I1CPC2K2, I1CPC2K2O1, I1CPC2K2O2: The concentration of psoralen in the active ingredient solution in the preparation of PLGA microspheres is 5 mg / mL, the concentration of PLGA in the capsule material solution in the preparation of PLGA microspheres is 7.5 mg / mL, the feed ratio (V:V) of the active ingredient solution to the capsule material solution in the preparation of the PLGA microsphere oil phase: active ingredient solution:capsule material solution = 1:2, the PVA concentration (W / V) of the PLGA microsphere aqueous phase is 0.5%, the O / W emulsion ultrasonic emulsification condition is 40 W x 2 min, the O / W emulsion solvent stirring and volatilization condition is 300 rpm x 5 h, the feed ratio (V:V) in the preparation of the PLGA microsphere O / W emulsion: PLGA microsphere oil phase:PLGA microsphere aqueous phase = 1:5, and the solid-liquid ratio in the preparation of the bone cement is 2 g / mL.

[0142] Other preparation parameters for I7CPC1K1, I7CPC1K1O1, I7CPC1K1O2: The concentration of psoralen in the active ingredient solution in the preparation of PLGA microspheres is 5 mg / mL, the concentration of PLGA in the capsule material solution in the preparation of PLGA microspheres is 30 mg / mL, the feed ratio (V:V) of the active ingredient solution to the capsule material solution in the preparation of the PLGA microsphere oil phase: active ingredient solution:capsule material solution = 1:2, the PVA concentration (W / V) of the PLGA microsphere aqueous phase is 1.0%, the O / W emulsion ultrasonic emulsification condition is 40 W x 2 min, the O / W emulsion solvent stirring and volatilization condition is 300 rpm x 5 h, the feed ratio (V:V) in the preparation of the PLGA microsphere O / W emulsion: PLGA microsphere oil phase:PLGA microsphere aqueous phase = 1:15, and the solid-liquid ratio in the preparation of the bone cement is 2 g / mL.

[0143] Cytotoxicity and cell proliferation rate of the bone cement in Example 6

[0144] 1. Drug-loaded bone cement samples:

[0145] I1CPC2 and P1CPC2 of Example 3 (bone cements representing different drugs, different drug loadings, and PLGA contents);

[0146] I1CPC2K2 and I7CPC1K2 of Example 4 (psoralen bone cements representing different drug loadings, PLGA contents, and different chitosan concentrations);

[0147] I1CPC2K2O1 and I7CPC1K1O2 of Example 5 (psoralen bone cements representing different drug loadings, PLGA contents, different chitosan concentrations, and different sodium caprylate contents).

[0148] 2. PLGA bone cement reference:

[0149] According to the preparation method of I1 drug-loaded microspheres in Example 1, blank PLGA microspheres were prepared without psoralen and psoralen. Then, according to the preparation method of I1CCPC bone cement in Example 3, blank PLGA microsphere bone cement was prepared without psoralen and psoralen.

[0150] (1) Dissolve PLGA in dichloromethane to make a 7.5 mg / mL capsule material solution, and add 1 / 2 volume of anhydrous ethanol for feeding and mixing to make the oil phase of PLGA microspheres;

[0151] (2) Take 5 times the volume of PVA aqueous solution (0.5%, W / V) as the aqueous phase of PLGA microspheres. Mix the oil phase of PLGA microspheres with the aqueous phase of PLGA microspheres, and make an O / W emulsion by ultrasonic treatment at 40 W for 2 min. Stir the O / W emulsion at 300 rpm for 5 h to volatilize and remove the organic solvent to obtain a blank microsphere suspension. Centrifuge the blank microsphere suspension, wash the precipitate with deionized water, and freeze-dry to obtain blank microspheres, which are stored at -80 °C for later use.

[0152] (3) Take the blank microspheres and mix them with calcium phosphate bone cement powder in an amount 3 times the feeding amount of PLGA as the solid phase of the bone cement. Add the bone cement liquid phase according to a solid-liquid ratio of 2 g / mL for mixing and stirring to prepare a blank microsphere-calcium phosphate bone cement slurry.

[0153] Inject the above bone cement slurry into a stainless steel mold with specifications of diameter and height of 6 mm and 12 mm respectively.

[0154] 3. Sample extraction and cell culture:

[0155] The bone cement samples and the reference samples were ground into powder after curing and demolding, passed through a 200-mesh sieve, and sterilized by ultraviolet light. The sample and reference powder were added to DMEM culture medium (0.2 g / mL) containing 10% fetal bovine serum, and extracted in an incubator at 37 °C and 5% CO2 for 3 days. The extract was collected by filtration through a 0.45-μm filter membrane and stored at 4 °C.

[0156] After digestion of MC3T3-E1 cells in the logarithmic growth phase, the cell concentration was adjusted to 2.5×10 4 cells / mL with DMEM culture medium containing 10% fetal bovine serum, inoculated into a multi-well plate, and cultured in an incubator at 37 °C and 5% CO2. After cell adhesion, the culture medium was discarded and replaced with the extract. After culturing for 1, 4, and 7 days respectively, 5 mg / mL MTT PBS solution was added to the multi-well plate and incubated for 4 h, and cell proliferation was detected by the MTT method. The absorbance value was measured at 540 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The negative control did not add the extract and was measured in the same way.

[0157] 4. Results

[0158] The drug loading, chitosan concentration, sodium caprylate feeding ratio, etc. of each drug-loaded bone cement sample are shown in Table 6, and the results are shown in the appendix of the specification. Figure 4 .

[0159] The cells in each group grew well within 1-7 days after adding the sample. Compared with the DEME culture medium (negative control) and the PLGA bone cement control, the proliferation rate of each drug-loaded bone cement group increased on the 7th day and p<0.05. The drug-loaded bone cement sample did not show in vitro cytotoxicity and had a promoting effect on the proliferation of MC3T3-E1.

[0160] Table 6 Information on the feeding of bone cement samples used in this study

[0161] Sample number Drug loading A B C D PLGA bone cement control - According to 1:3 2:6 - - I1CPC2 ISP 1:3 5:6 - - P1CPC2 P 1:12 5:6 - - I1CPC2K2 ISP 1:3 5:6 0.05 - I7CPC1K2 ISP 1:12 2:6 0.05 - I1CPC2K2O1 ISP 1:3 5:6 0.05 2:1 I7CPC1K1O2 ISP 1:12 2:6 0.01 6:1

[0162] Note: Drug loading item: ISP is isopsoralen; P is psoralen. A is the feeding ratio (W:W) in the preparation of the oil phase of PLGA microspheres, drug:PLGA; B is the feeding ratio (W:W) in the preparation of the solid phase of bone cement: PLGA: calcium phosphate bone cement powder. For example, when the feeding amount of PLGA is 5 g, the feeding amount of calcium phosphate bone cement powder is 15 g (2:6, i.e., 1:3) or 6 g (5:6); C is the concentration of chitosan acetate solution (W / V, %), such as 0.01% (W / V) i.e., 0.01 g / 100 mL; D is the feeding ratio (W:W), drug:sodium caprylate.

[0163] P1 CPC2, I7 CPC1K2, I7 CPC1K1O2 Other preparation parameters: The concentration of the active ingredient psoralen or isopsoralen in the preparation of PLGA microspheres is 5 mg / mL, the concentration of PLGA in the encapsulating material solution in the preparation of PLGA microspheres is 30 mg / mL, the feeding ratio (V:V) of the active ingredient solution to the encapsulating material solution in the preparation of the PLGA microsphere oil phase: active ingredient solution: encapsulating material solution = 1:2, the PVA concentration (W / V) of the PLGA microsphere aqueous phase is 1.0%, the ultrasonic emulsification condition of the O / W emulsion is 40 W x 2 min, the stirring and volatilization condition of the O / W emulsion solvent is 300 rpm x 5 h, the feeding ratio (V:V) in the preparation of the PLGA microsphere O / W emulsion: PLGA microsphere oil phase: PLGA microsphere aqueous phase = 1:15, and the solid-liquid ratio of the bone cement preparation is 2 g / mL.

[0164] I1 CPC2, I1 CPC2K2, I1 CPC2K2O1 Other preparation parameters: The concentration of the active ingredient isopsoralen in the preparation of PLGA microspheres is 5 mg / mL, the concentration of PLGA in the encapsulating material solution in the preparation of PLGA microspheres is 7.5 mg / mL, the feeding ratio (V:V) of the active ingredient solution to the encapsulating material solution in the preparation of the PLGA microsphere oil phase: active ingredient solution: encapsulating material solution = 1:2, the PVA concentration (W / V) of the PLGA microsphere aqueous phase is 0.5%, the ultrasonic emulsification condition of the O / W emulsion is 40 W x 2 min, the stirring and volatilization condition of the O / W emulsion solvent is 300 rpm x 5 h, the feeding ratio (V:V) in the preparation of the PLGA microsphere O / W emulsion: PLGA microsphere oil phase: PLGA microsphere aqueous phase = 1:5, and the solid-liquid ratio of the bone cement preparation is 2 g / mL.

[0165] Example 7 In vivo investigation of isopsoralen-loaded bone cement

[0166] 1. Drug-loaded bone cement samples:

[0167] Select two bone cements I1 CPC2K2 and I1 CPC2K2O1 with high cell proliferation rates on the 7th day in Example 6 as the research objects. The solid phase and liquid phase of the bone cement are mixed and adjusted into a slurry immediately before use and then injected.

[0168] 2. PLGA bone cement control

[0169] Same as Example 6, the solid phase and liquid phase of the bone cement are mixed and adjusted into a slurry immediately before use and then injected.

[0170] 3. Osteoporotic bone defect modeling and grouping

[0171] Three-month-old healthy adult female Sprague-Dawley rats, weighing 220±20 g, were randomly divided into 4 groups, with 8 rats in each group. The normal group, i.e., the sham operation group, was not modeled. Only the adipose tissue near the bilateral ovaries was excised, and the rats were fed a diet-type feed. The remaining 3 groups were induced with osteoporosis by excising bilateral ovaries combined with 8-week low-calcium diet according to the literature method (van Houdt CIA.et al.Alendronaterelease from calcium phosphate cement for bone regeneration in osteoporoticconditions.Scientific reports,2018,8(1):15398.). The bone mineral density (BMD) of the whole body of the rats was measured by a dual-energy X-ray absorptiometry. After comparing with the sham operation group and confirming that the whole-body BMD was significantly reduced, a bone defect with a diameter of 2.5 mm and a depth of 5 mm was created at the femoral condyle of the ipsilateral hind limb. The bone defect site was cleaned with normal saline. After debridement with sterile gauze, the PLGA bone cement control, I1CPC2K2 bone cement, and I1CPC2K2O1 bone cement homogenates were filled into the bone defect site using a syringe barrel. After the bone cement was cured, the wound was sutured. Postoperatively, antibiotics were routinely injected to prevent infection, and the low-calcium diet was maintained for 6 weeks.

[0172] 4. Blood and imaging analysis

[0173] At the 12th week after surgery, the body weight of the rats was weighed and calculated. There was no significant difference in body weight among the bone cement groups. The BMD of the 5th and 6th lumbar vertebrae of the rats was measured by dual-energy X-ray absorptiometry (g / m 2) After the measurement, the rats were fasted for 8 h, blood was collected, and then the rats were sacrificed. Plasma and serum were separated by centrifugation. The levels of serum alkaline phosphatase (ALP) and osteocalcin (OC) were detected by ELISA according to the kit instructions. The plasma drug concentration of isopsoralen was determined by HPLC (Gu Y, et al. Simultaneous quantification of psoralen and isopsoralen in rat plasma by ultra-performance liquid chromatography / tandem mass spectrometry and its application to a pharmacokinetic study after oral administration of Haigou Pill[J]. Journal of Chromatography B Analytical Technologies in the Biomedical&Life Sciences, 2009, 877(27): 3137-3143.). The bone defect site was dissected, fixed in paraformaldehyde, dehydrated and decalcified, embedded in paraffin to prepare 5-mm sections, stained with HE, observed under a microscope, and the new bone formation rate was measured and analyzed by Image-Pro Plus in the target area of the bone defect. The new bone formation rate = (newly formed bone area / bone defect area) x 100%.

[0174] 5. Results

[0175] The detection indexes and results of the rats with osteoporosis and bone defects are summarized in Table 7. The feeding information and preparation parameters of the bone cement samples used in this study are summarized in Table 8.

[0176] At 12 weeks after bone cement filling: The OC value and ALP value in the PLGA bone cement group were higher than those in the sham operation group (p < 0.05), and the BMD of L5-6 lumbar vertebra was lower than that in the sham operation group (p < 0.05). It indicated that osteoporosis still existed in the modeled animals.

[0177] The ALP value and OC value in the drug-loaded bone cement groups (I1CPC2K2, I1CPC2K2O1) were lower than those in the PLGA bone cement group (p < 0.05), and the BMD of L5-6 lumbar vertebra and the new bone formation rate were higher than those in the PLGA bone cement group (p < 0.05). It indicated that the drug-loaded bone cement could improve the local bone repair of bone defects and the related indexes of systemic osteoporosis. Among them, the ALP value in the I1CPC2K2O1 group was lower than that in the I1CPC2K2 (p < 0.05), and the BMD of L5-6 lumbar vertebra was higher than that in the I1CPC2K2. It indicated that the osteoblast activity in the I1CPC2K2O1 group was higher than that in the I1CPC2K2 group and the improvement of osteoporosis in the non-defect site was better than that in the I1CPC2K2.

[0178] Since the preparation parameters of I1CPC2K2 and I1CPC2K2O1 bone cements are the same, the psoralen content is similar (see Table 7), and their promoting effects on cell proliferation in vitro are similar (see the attached Figure 4 ). Considering the difference in blood drug concentration between the two groups, it is speculated that the improvement of ALP and bone BMD at non-defective sites by I1CPC2K2O1 is associated with its significant increase in plasma psoralen concentration.

[0179] Table 7 Detection indexes and results of rats with osteoporosis bone defects

[0180]

[0181] Table 8 Feeding information and preparation parameters of bone cement samples used in this study

[0182] PLGA bone cement I1CPC2K2 I1CPC2K2O1 A 0 5 5 B According to 1:3 1:3 1:3 C 7.5 7.5 7.5 D 1:2 1:2 1:2 E 0.5 0.5 0.5 F 1:5 1:5 1:5 G 40w, 2min 40w, 2min 40w, 2min H 300rpm, 5h 300rpm, 5h 300rpm, 5h I 2:6 5:6 5:6 J 2 2 2 K - 0.05 0.05 L - - 2:1 M - 10.6 10.1

[0183] Note: A is the concentration of psoralen in the active ingredient solution (mg / mL); B is the feeding ratio (W:W) in the preparation of the oil phase of PLGA microspheres, drug:PLGA; C is the concentration of PLGA in the capsule liquid in the preparation of PLGA microspheres (mg / mL); D is the feeding ratio (V:V) of the active ingredient solution to the capsule liquid in the preparation of the oil phase of PLGA microspheres: active ingredient solution: capsule liquid; E is the concentration of PVA in the aqueous phase of PLGA microspheres (W / V, %); F is the feeding ratio (V:V) in the preparation of the O / W emulsion of PLGA microspheres: oil phase of PLGA microspheres: aqueous phase of PLGA microspheres; G is the ultrasonic emulsification condition of the O / W emulsion; H is the condition for stirring and volatilizing the solvent of the O / W emulsion; I is the feeding ratio (W:W) in the preparation of the solid phase of bone cement: PLGA: calcium phosphate bone cement powder. For example, when the feeding amount of PLGA is 5 g, the feeding amount of calcium phosphate bone cement powder is 15 g (2:6, i.e., 1:3) or 6 g (5:6); J is the solid-liquid ratio (g / mL) of bone cement preparation; K is the concentration of chitosan acetate solution (W / V, %), such as 0.01 (W / V, %) which is 0.01 g / 100 mL; L is the feeding ratio (W:W), drug: sodium caprylate; M is the estimated content of psoralen in bone cement (including the solid phase and liquid phase of bone cement, W / W, %).

Claims

1. A sustained-release and degradable bone cement loaded with anti-osteoporosis drugs, which is prepared by mixing a solid phase of bone cement and a liquid phase of bone cement. The solid phase of bone cement contains PLGA microspheres loaded with anti-osteoporosis drugs and calcium phosphate bone cement powder as a setting material, and is characterized in that, The anti-osteoporosis drug is psoralen and / or isopsoralen.

2. The bone cement according to claim 1, characterized in that, The preparation method of the bone cement comprises the following steps: S1: Dissolve the anti-osteoporosis drug in absolute ethanol to prepare an active ingredient solution, dissolve PLGA in dichloromethane to prepare a capsule material solution, and mix the active ingredient solution and the capsule material solution by feeding to prepare an oil phase of PLGA microspheres; S2: Take an aqueous PVA solution as the water phase of PLGA microspheres, mix the oil phase of PLGA microspheres and the water phase of PLGA microspheres by feeding, and ultrasonically prepare an O / W emulsion. The O / W emulsion is stirred to volatilize and remove the organic solvent to obtain a suspension of drug-loaded microspheres. The suspension of drug-loaded microspheres is centrifuged, the precipitate is washed with deionized water, and freeze-dried to obtain drug-loaded microspheres; S41: Take the drug-loaded microspheres and calcium phosphate bone cement powder and mix them by feeding as the solid phase of the bone cement. The solid phase of the bone cement and the liquid phase of the bone cement are mixed and stirred by feeding to prepare a sustained-release and degradable bone cement loaded with anti-osteoporosis drugs.

3. The bone cement according to claim 2, characterized in that, In the above S1: The weight part ratio of the anti-osteoporosis drug to PLGA in the feed is 1:(3 - 12), the concentration of PLGA in the capsule material solution is 7.5 mg / mL - 30 mg / mL, and the volume ratio of the active ingredient solution to the capsule material solution in the feed is 1:(2 - 4); In the above S2: The mass-volume concentration of the aqueous PVA solution is 0.5% - 2%, the volume ratio of the oil phase of PLGA microspheres to the water phase of PLGA microspheres in the feed is 1:(5 - 15), the ultrasonic power is 20 W - 80 W, the ultrasonic time is 1 min - 3 min, the stirring speed is 300 rpm - 800 rpm, and the stirring time is 4 h - 6 h; In the above S41: The weight part ratio of the drug-loaded microspheres to the calcium phosphate bone cement powder in the feed, calculated by the weight part ratio of PLGA to the calcium phosphate bone cement powder in the feed, is (2 - 5):

6.

4. The bone cement according to claim 1, characterized in that The solid phase of the bone cement contains chitosan. The chitosan is made into an acetic acid solution, mixed with the drug-loaded microspheres, centrifuged, freeze-dried, and then mixed with the calcium phosphate bone cement powder.

5. The bone cement according to claim 4, characterized in that, The preparation method of the bone cement comprises the following steps: S1: Dissolve the anti-osteoporosis drug in absolute ethanol to prepare an active ingredient solution, dissolve PLGA in dichloromethane to prepare a capsule material solution, and mix the active ingredient solution and the capsule material solution by feeding to prepare an oil phase of PLGA microspheres; S2: Take an aqueous PVA solution as the water phase of PLGA microspheres, mix the oil phase of PLGA microspheres and the water phase of PLGA microspheres by feeding, and ultrasonically prepare an O / W emulsion. The O / W emulsion is stirred to volatilize and remove the organic solvent to obtain a suspension of drug-loaded microspheres. The suspension of drug-loaded microspheres is centrifuged, the precipitate is washed with deionized water, and freeze-dried to obtain drug-loaded microspheres; S31: Dissolve chitosan with an aqueous acetic acid solution to prepare a chitosan acetic acid solution. Add the drug-loaded microspheres to the chitosan acetic acid solution, stir and mix to submerge the drug-loaded microspheres in the chitosan acetic acid solution, centrifuge, and freeze-dry the precipitate to obtain drug-loaded microsphere chitosan powder; S42: Take the drug-loaded microsphere chitosan powder and calcium phosphate bone cement powder and mix them by feeding to prepare the solid phase of the bone cement. The solid phase of the bone cement and the liquid phase of the bone cement are mixed and stirred by feeding to prepare a sustained-release and degradable bone cement loaded with anti-osteoporosis drugs.

6. The bone cement according to claim 5, characterized in that, In S1: the weight ratio of the anti-osteoporosis drug to PLGA in the feed is 1:(3 - 12), the concentration of PLGA in the capsule material solution is 7.5 mg / mL - 30 mg / mL, and the volume ratio of the active ingredient solution to the capsule material solution in the feed is 1:(2 - 4); in S2: the mass-volume concentration of the PVA aqueous solution is 0.5% - 2%, the volume ratio of the oil phase of the PLGA microspheres to the aqueous phase of the PLGA microspheres in the feed is 1:(5 - 15), the ultrasonic power is 20 W - 80 W, the ultrasonic duration is 1 min - 3 min, the stirring speed is 300 rpm - 800 rpm, and the stirring duration is 4 h - 6 h; in S31: the mass-volume concentration of chitosan in the chitosan acetate solution is 0.01% - 0.05%; in S42: the weight ratio of the drug-loaded microsphere chitosan powder to the calcium phosphate cement powder in the feed, calculated based on the weight ratio of PLGA to the calcium phosphate cement powder in the feed, is (2 - 5):

6.

7. The bone cement according to claim 4, characterized in that, The bone cement solid phase contains sodium caprylate, and the sodium caprylate is mixed, centrifuged, and freeze-dried with the chitosan acetate solution and the drug-loaded microspheres, and then mixed with the calcium phosphate cement powder.

8. The bone cement according to claim 7, wherein The preparation method of the bone cement includes the following steps: S1: Dissolve the anti-osteoporosis drug in absolute ethanol to prepare an active ingredient solution, dissolve PLGA in dichloromethane to prepare a capsule material solution, and mix the active ingredient solution and the capsule material solution in the feed to prepare an oil phase of PLGA microspheres; S2: Take the PVA aqueous solution as the aqueous phase of the PLGA microspheres, mix the oil phase of the PLGA microspheres and the aqueous phase of the PLGA microspheres in the feed, ultrasonically prepare an O / W emulsion, stir and volatilize the O / W emulsion to remove the organic solvent to obtain a suspension of drug-loaded microspheres, centrifuge the suspension of drug-loaded microspheres to remove the supernatant, wash the precipitate with deionized water, and freeze-dry to obtain drug-loaded microspheres; S32: Take chitosan and prepare a chitosan acetate solution with an acetic acid aqueous solution, add the drug-loaded microspheres and sodium caprylate in the feed to the chitosan acetate solution, stir and mix to submerge the drug-loaded microspheres with the chitosan acetate solution, centrifuge, and freeze-dry the precipitate to obtain a drug-loaded microsphere chitosan sodium caprylate powder; S43: Take the drug-loaded microsphere chitosan sodium caprylate powder and the calcium phosphate cement powder in the feed and mix them to prepare a bone cement solid phase, and mix and stir the bone cement solid phase and the bone cement liquid phase in the feed to prepare a sustained-release and degradable bone cement loaded with the anti-osteoporosis drug.

9. The bone cement according to claim 8, wherein, In S1: the weight ratio of the anti-osteoporosis drug to PLGA in the feed is 1:(3 - 12), the concentration of PLGA in the capsule material solution is 7.5 mg / mL - 30 mg / mL, and the volume ratio of the active ingredient solution to the capsule material solution in the feed is 1:(2 - 4); in S2: the mass-volume concentration of the PVA aqueous solution is 0.5% - 2%, the volume ratio of the oil phase of the PLGA microspheres to the water phase of the PLGA microspheres in the feed is 1:(5 - 15), the ultrasonic power is 20 W - 80 W, the ultrasonic duration is 1 min - 3 min, the stirring speed is 300 rpm - 800 rpm, and the stirring duration is 4 h - 6 h; in S32: the mass-volume concentration of chitosan in the chitosan acetate solution is 0.01% - 0.05%, and the feeding amount of sodium octanoate by weight is 1 / 6 - 1 / 2 of the feeding amount of the anti-osteoporosis drug; in S43: the weight ratio of the drug-loaded microsphere chitosan sodium octanoate powder to the calcium phosphate cement powder in the feed, calculated based on the weight ratio of PLGA to the calcium phosphate cement powder in the feed, is (2 - 5):

6.

10. Use of the bone cement according to any one of claims 1 to 9 in the preparation of a bone defect filling material for osteoporosis patients.

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