阅读说明：本技术 一种锶掺杂改性的天然羟基磷灰石支架材料的制备方法和应用 (Preparation method and application of strontium-doped modified natural hydroxyapatite scaffold material ) 是由 陈大福 万奔 曹晶晶 王宏刚 郭建勋 王芊 黎广平 王任先 聂井君 吴新宝 蒋协 于 2021-07-23 设计创作，主要内容包括：本发明公开了一种锶掺杂改性的天然羟基磷灰石支架材料的制备方法和应用,本发明制备得到的锶掺杂改性的天然羟基磷灰石支架材料具有优异的生物相容性、生物活性、溶解性以及促进骨组织再生的能力,且能以一种持续而缓慢的方式释放锶离子,释放的锶离子既能刺激成骨分化,又能抑制破骨细胞的功能,加速骨缺损的修复,在骨缺损修复治疗中极具临床转化应用前景。(The strontium-doped modified natural hydroxyapatite scaffold material prepared by the invention has excellent biocompatibility, bioactivity, solubility and capability of promoting bone tissue regeneration, and can release strontium ions in a continuous and slow manner, and the released strontium ions can stimulate osteogenic differentiation, inhibit the function of osteoclasts, accelerate the repair of bone defects, and have great clinical transformation application prospects in the repair and treatment of bone defects.)

1. A preparation method of a strontium-doped modified natural hydroxyapatite scaffold material is characterized by comprising the following steps:

(1) sequentially degreasing and heating and calcining the cancellous bone at a gradient temperature to prepare a calcined bovine bone hydroxyapatite scaffold material;

(2) placing the calcined bovine bone hydroxyapatite scaffold material obtained in the step (1) into a strontium nitrate reaction solution for reaction by adopting a high-temperature ion exchange method, and calcining at a high temperature to obtain a strontium-doped modified natural hydroxyapatite scaffold material;

Preferably, the strontium-doped modified natural hydroxyapatite scaffold material comprises nano-scale particles, submicron particles, micron-scale particles and millimeter-scale particles;

more preferably, the submicron particles have a particle size of 100nm to 1 μm;

more preferably, the particle size of the millimeter-sized particles is 1.25 to 1.6 mm;

more preferably, the proportion of calcium in the strontium-substituted hydroxyapatite in the strontium-doped modified natural hydroxyapatite scaffold material is 4.6-9.8%;

most preferably, the proportion of calcium in the strontium-substituted hydroxyapatite in the strontium-doped modified natural hydroxyapatite scaffold material is 4.6 percent and 6.8 percent;

most preferably, the proportion of calcium in the strontium-substituted hydroxyapatite in the strontium-doped modified natural hydroxyapatite scaffold material is 6.8%.

2. The method according to claim 1, wherein the cancellous bone source in step (1) is the lower cancellous bone of bovine femoral bone;

preferably, the lower cancellous bone of the bovine femur is the lower cancellous bone of the adult bovine femur of 3 years old;

more preferably, the cancellous bone is a 30mm by 20mm by 15mm block.

3. The method according to claim 1, wherein the degreasing in the step (1) comprises the steps of:

(a) Soaking cancellous bone for 24-48h by using 1-2M NaOH, and rinsing by using deionized water;

(b)2％-20％H₂O₂soaking the cancellous bone obtained in the step (a) for 24-48h, and rinsing with deionized water;

(c) dehydrating the cancellous bone obtained in the step (b) by 75-100% series of alcohol for 24-48h, and naturally drying;

(d) drying the cancellous bone obtained in the step (c) at 60-100 ℃ for 4-24 h;

preferably, the method further comprises rinsing, soaking and rinsing the cancellous bone to remove impurities before the step (a).

4. The method according to claim 1, wherein the temperature control procedure of the gradient temperature-rising calcination in the step (1) is to set 12-18 temperature procedure segments from 100 ℃ to 1000 ℃, wherein the fastest temperature-rising rate is 15-30 ℃/min, the slowest temperature-rising rate is 2-5 ℃, and the temperature procedure segments are specifically allocated as follows:

(a) the room temperature is raised to 100 ℃ and 130 ℃ for 10-20min, and the temperature is kept for 10-20 min;

(b) heating from 100 ℃ to 130 ℃ to 200 ℃ to 220 ℃ for 10-30min, and keeping the temperature for 60-90 min;

(c) raising the temperature of 200 ℃ and 220 ℃ to the temperature of 250 ℃ and 260 ℃ for 15-25min, and keeping the temperature for 20-40 min;

(d) raising the temperature from 250 ℃ to 260 ℃ to 280 ℃ to 310 ℃ for 5-15min, and keeping the temperature for 10-20 min;

(e) raising the temperature of 280-310 ℃ to 320-330 ℃ for 8-15min, and keeping the temperature for 20-60 min;

(f) raising the temperature of 320-330 ℃ to the temperature of 360-400 ℃ for 5-10min, and keeping the temperature for 20-35 min;

(g) raising the temperature from 400 ℃ at 360 ℃ to 500 ℃ at 450 ℃ for 10-20min, and keeping the temperature for 10-30 min;

(h) raising the temperature from 500 ℃ to 560 ℃ at 450 ℃ to 540 ℃ for 5-20min, and keeping the temperature for 10-20 min;

(i) Raising the temperature of 540-560 ℃ to 580-610 ℃ for 15-25min, and keeping the temperature for 30-60 min;

(j) raising the temperature of 580-610 ℃ to 660-680 ℃ for 6-15min, and keeping the temperature for 10-20 min;

(k) heating 660-680 ℃ to 750-800 ℃ for 10-15min, and keeping the temperature for 15-20 min;

(l) Raising the temperature of 750-;

(m)880-910 ℃ is raised to 940-960 ℃ for 10-20min, and the temperature is maintained for 90-180 min.

5. The method according to claim 1, wherein the concentration of the strontium nitrate reaction solution in step (2) is 0.5 to 1 mol/L;

preferably, the concentration of the strontium nitrate reaction solution is 0.5mol/L and 0.75 mol/L;

more preferably, the concentration of the strontium nitrate reaction solution is 0.75 mol/L;

most preferably, the calcined bovine bone hydroxyapatite scaffold material in the step (2) is in an inflated state in the strontium nitrate reaction solution.

6. The method according to claim 1, wherein the strontium-doped modified natural hydroxyapatite scaffold material solution obtained by the reaction in the step (2) is left for 2 hours, dried and then calcined at high temperature;

preferably, the temperature control procedure of the high-temperature calcination is 10min at room temperature to 200 ℃, 10min at 200 ℃ to 200 ℃, 40min at 200 ℃ to 600 ℃, 5h at 600 ℃ to 600 ℃ and 600 ℃ to room temperature;

more preferably, after the material obtained by high-temperature calcination is cooled to room temperature, the material is soaked in deionized water for 24-48h, cleaned by ultrasonic waves and dried at 80-100 ℃ for 4-8h, and then the strontium-doped modified natural hydroxyapatite scaffold material is obtained.

7. The method of claim 1, wherein step (2) further comprises subjecting the material obtained by the high-temperature calcination to a pH-lowering treatment;

preferably, the pH-lowering treatment comprises the steps of:

(a) 1g (material obtained by high temperature calcination): 4mL (diluted phosphoric acid) for 1-2 h;

(b) after washing the material with deionized water, the mixture was washed with 1g (material after washing): soaking for 1h at the ratio of 30mL (deionized water), and drying at 80-100 ℃;

more preferably, the concentration of the dilute phosphoric acid in the step (a) is 0.1 moL/L.

8. A strontium-doped modified natural hydroxyapatite scaffold material for bone defect repair is characterized in that the proportion of calcium in strontium-substituted hydroxyapatite in the material is 4.6-9.8%;

preferably, the proportion of calcium in the strontium-substituted hydroxyapatite in the material is 4.6%, 6.8%;

more preferably, the proportion of calcium in the strontium-substituted hydroxyapatite in the material is 6.8%;

preferably, the strontium-doped modified natural hydroxyapatite scaffold material comprises nano-scale particles, submicron particles, micron-scale particles and millimeter-scale particles;

more preferably, the submicron particles have a particle size of 100nm to 1 μm;

more preferably, the particle size of the millimeter-sized particles is 1.25 to 1.6 mm;

Most preferably, the material is prepared by the method of any one of claims 1 to 7.

9. A strontium-doped modified natural hydroxyapatite composite material for bone defect repair, characterized in that the composite material comprises the material of claim 8 and a carrier solution;

preferably, the mass percentage of the material in the composite material is 1-90%;

preferably, the mass percentage content of the carrier solution in the composite material is 0.5% -3%;

more preferably, the carrier solution in the composite material comprises blood, serum-free culture medium, physiological saline, deionized water, polylactic acid, collagen, chitosan, dextran, gelatin, glycerol, poly-L-lactic acid, polycaprolactone, polyethylene, polyamide, cellulose, calcium polyphosphate fiber, an antioxidant, a wetting agent, a solubilizer and a pH regulator.

10. The use of any one of the following aspects, wherein said use comprises:

(1) use of the material of claim 8 for the preparation of a strontium doped modified natural hydroxyapatite composite for bone defect repair;

(2) use of the material of claim 8 in the preparation of a bone defect repair material;

(3) Use of the composite material of claim 9 for the preparation of a bone defect repair material;

(4) use of the material of claim 8 in the repair of bone defects;

(5) use of the composite material of claim 9 in the repair of a bone defect.

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a strontium-doped bovine bone-derived hydroxyapatite scaffold material, and more particularly relates to a preparation method and application of a strontium-doped modified natural hydroxyapatite scaffold material.

Background

Bone defects refer to diseases in which the intact structure of the bone is destroyed due to trauma, inflammation, bone disease, or surgery. Bone is a metabolically active connective tissue with the ability to fully regenerate remodelling, but in cases of large bone defects, such as segmental lesions, tumour resection, reconstructive procedures require additional support. Clinically, autografting remains the most common strategy for supporting and stimulating bone growth, and although desirable from a biological standpoint, patients have a limited number of grafts offered in their body and can cause secondary damage. In addition, allografts from bone banks or other animals have poor osteointegration and risk of disease transmission, and therefore, the study of novel bone repair materials is of great interest.

Calcium phosphate ceramics (CaPs) are a basic bone repair material with excellent biocompatibility, bone conduction and bone induction properties, including Hydroxyapatite (HAP), β -tricalcium phosphate (β -Tri-Calcium phosphate, β -TCP), Calcium polyphosphate (SCPP), and Biphase Calcium Phosphate (BCP), etc. they are widely used in the fields of bone surgery and dentistry in the form of implant surface coatings, bone cements and scaffold materials.

Hydroxyapatite (HAP) can be synthesized directly from chemical reagents or extracted from various natural sources. At present, hydroxyapatite which is purely derived from bovine bone and is used for assisting in repairing bone defects has been reported, and although the hydroxyapatite has good biocompatibility and excellent osteoconductivity, the hydroxyapatite mostly lacks surface reaction activity, capability of stimulating and inducing regeneration of bone tissues and degradation capability, and the solubility of the material is low. The existing bone repair scaffold materials also have the defects of over-quick drug release, unsatisfactory mechanical property of the scaffold, poor degradability and osteogenesis capacity and the like, so the development of the bone repair scaffold material which has long-acting slow release, strong surface reactivity and degradability, strong mechanical property and osteogenesis capacity and can accelerate bone defect repair has very important significance in the field of bone repair treatment.

Disclosure of Invention

The invention aims to overcome the defects in the preparation of the hydroxyapatite scaffold material with a biological structure from bovine bones, and provides a strontium-doped modified natural hydroxyapatite scaffold material for bone repair and a preparation method thereof.

The above object of the present invention is achieved by the following technical solutions:

the invention provides a preparation method of a strontium-doped modified natural hydroxyapatite scaffold material.

Further, the method comprises the steps of:

(1) sequentially degreasing and heating and calcining the cancellous bone at a gradient temperature to prepare a calcined bovine bone hydroxyapatite scaffold material;

(2) placing the calcined bovine bone hydroxyapatite scaffold material obtained in the step (1) into a strontium nitrate reaction solution for reaction by adopting a high-temperature ion exchange method, and calcining at a high temperature to obtain a strontium-doped modified natural hydroxyapatite scaffold material;

preferably, the strontium-doped modified natural hydroxyapatite scaffold material comprises nano-scale particles, submicron particles, micron-scale particles and millimeter-scale particles;

More preferably, the submicron particles have a particle size of 100nm to 1 μm;

more preferably, the particle size of the millimeter-sized particles is 1.25 to 1.6 mm;

more preferably, the proportion of calcium in the strontium-substituted hydroxyapatite in the strontium-doped modified natural hydroxyapatite scaffold material is 4.6-9.8%;

most preferably, the proportion of calcium in the strontium-substituted hydroxyapatite in the strontium-doped modified natural hydroxyapatite scaffold material is 4.6 percent and 6.8 percent;

most preferably, the proportion of calcium in the strontium-substituted hydroxyapatite in the strontium-doped modified natural hydroxyapatite scaffold material is 6.8%.

Further, the cancellous bone in the step (1) is sourced from lower cancellous bone of the bovine femoral bone;

preferably, the lower cancellous bone of the bovine femur is the lower cancellous bone of the adult bovine femur of 3 years old;

more preferably, the cancellous bone is a 30mm by 20mm by 15mm block.

Further, the degreasing in the step (1) comprises the following steps:

(a) soaking cancellous bone for 24-48h by using 1-2M NaOH, and rinsing by using deionized water;

(b)2％-20％H₂O₂soaking the cancellous bone obtained in the step (a) for 24-48h, and rinsing with deionized water;

(c) dehydrating the cancellous bone obtained in the step (b) by 75-100% series of alcohol for 24-48h, and naturally drying;

(d) drying the cancellous bone obtained in the step (c) at 60-100 ℃ for 4-24 h;

Preferably, the method further comprises rinsing, soaking and rinsing the cancellous bone to remove impurities before the step (a).

Further, the temperature control procedure of the gradient temperature rise calcination in the step (1) is to set 12-18 temperature procedure sections from 100 ℃ to 1000 ℃, wherein the fastest temperature rise rate is 15-30 ℃/min, the slowest temperature rise rate is 2-5 ℃, and the temperature procedure sections are specifically distributed as follows:

(a) the room temperature is raised to 100 ℃ and 130 ℃ for 10-20min, and the temperature is kept for 10-20 min;

(b) heating from 100 ℃ to 130 ℃ to 200 ℃ to 220 ℃ for 10-30min, and keeping the temperature for 60-90 min;

(c) raising the temperature of 200 ℃ and 220 ℃ to the temperature of 250 ℃ and 260 ℃ for 15-25min, and keeping the temperature for 20-40 min;

(d) raising the temperature from 250 ℃ to 260 ℃ to 280 ℃ to 310 ℃ for 5-15min, and keeping the temperature for 10-20 min;

(e) raising the temperature of 280-310 ℃ to 320-330 ℃ for 8-15min, and keeping the temperature for 20-60 min;

(f) raising the temperature of 320-330 ℃ to the temperature of 360-400 ℃ for 5-10min, and keeping the temperature for 20-35 min;

(g) raising the temperature from 400 ℃ at 360 ℃ to 500 ℃ at 450 ℃ for 10-20min, and keeping the temperature for 10-30 min;

(h) raising the temperature from 500 ℃ to 560 ℃ at 450 ℃ to 540 ℃ for 5-20min, and keeping the temperature for 10-20 min;

(i) raising the temperature of 540-560 ℃ to 580-610 ℃ for 15-25min, and keeping the temperature for 30-60 min;

(j) raising the temperature of 580-610 ℃ to 660-680 ℃ for 6-15min, and keeping the temperature for 10-20 min;

(k) heating 660-680 ℃ to 750-800 ℃ for 10-15min, and keeping the temperature for 15-20 min;

(l) Raising the temperature of 750-;

(m)880-910 ℃ is raised to 940-960 ℃ for 10-20min, and the temperature is maintained for 90-180 min.

Further, the concentration of the strontium nitrate reaction solution in the step (2) is 0.5-1 mol/L;

preferably, the concentration of the strontium nitrate reaction solution is 0.5mol/L and 0.75 mol/L;

more preferably, the concentration of the strontium nitrate reaction solution is 0.75 mol/L;

most preferably, the calcined bovine bone hydroxyapatite scaffold material in the step (2) is in an inflated state in the strontium nitrate reaction solution.

Further, standing the strontium-doped modified natural hydroxyapatite scaffold material solution obtained by the reaction in the step (2) for 2 hours, drying, and then calcining at high temperature;

preferably, the temperature control procedure of the high-temperature calcination is 10min at room temperature to 200 ℃, 10min at 200 ℃ to 200 ℃, 40min at 200 ℃ to 600 ℃, 5h at 600 ℃ to 600 ℃ and 600 ℃ to room temperature;

more preferably, after the material obtained by high-temperature calcination is cooled to room temperature, the material is soaked in deionized water for 24-48h, cleaned by ultrasonic waves and dried at 80-100 ℃ for 4-8h, and then the strontium-doped modified natural hydroxyapatite scaffold material is obtained.

Further, the step (2) also comprises the step of carrying out pH reduction treatment on the material obtained by high-temperature calcination;

preferably, the pH-lowering treatment comprises the steps of:

(a) 1g (material obtained by high temperature calcination): 4mL (diluted phosphoric acid) for 1-2 h;

(b) After washing the material with deionized water, the mixture was washed with 1g (material after washing): soaking for 1h at the ratio of 30mL (deionized water), and drying at 80-100 ℃;

more preferably, the concentration of the dilute phosphoric acid in the step (a) is 0.1 moL/L.

The invention provides a strontium-doped modified natural hydroxyapatite scaffold material for bone defect repair.

Further, the proportion of calcium in the strontium substituted hydroxyapatite in the material is 4.6-9.8%;

preferably, the proportion of calcium in the strontium-substituted hydroxyapatite in the material is 4.6%, 6.8%;

more preferably, the proportion of calcium in the strontium-substituted hydroxyapatite in the material is 6.8%;

preferably, the strontium-doped modified natural hydroxyapatite scaffold material comprises nano-scale particles, submicron particles, micron-scale particles and millimeter-scale particles;

more preferably, the submicron particles have a particle size of 100nm to 1 μm;

more preferably, the particle size of the millimeter-sized particles is 1.25 to 1.6 mm;

most preferably, the material is prepared by the method of the first aspect of the invention.

In a third aspect of the invention, a strontium-doped modified natural hydroxyapatite composite material for bone defect repair is provided.

Further, the composite material comprises the material according to the second aspect of the present invention and a carrier solution;

preferably, the mass percentage of the material of the second aspect of the present invention in the composite material is 1% to 90%;

preferably, the mass percentage content of the carrier solution in the composite material is 0.5% -3%;

more preferably, the carrier solution in the composite material comprises blood, serum-free culture medium, physiological saline, deionized water, polylactic acid, collagen, chitosan, dextran, gelatin, glycerol, poly-L-lactic acid, polycaprolactone, polyethylene, polyamide, cellulose, calcium polyphosphate fiber, an antioxidant, a wetting agent, a solubilizer and a pH regulator.

Further, the antioxidants include (but are not limited to): sulfite, ascorbate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), vitamin E, vitamin C, sodium metabisulfite, and dibutylhydroxytoluene.

Further, the wetting agents include (but are not limited to): starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose.

Further, the solubilizing agents include (but are not limited to): poloxamer, sodium dodecyl sulfate, tween-80, sodium carboxymethylcellulose, polyvinylpyrrolidone and lecithin.

Further, the pH adjusting agents include (but are not limited to): citric acid, phosphoric acid, succinic acid, sodium hydroxide, hydrochloric acid, boric acid, acetic acid, potassium hydroxide, ammonium hydroxide, magnesium oxide, calcium carbonate, magnesium aluminum silicate, malic acid, potassium citrate, sodium phosphate, lactic acid, gluconic acid, tartaric acid, diethanolamine, monoethanolamine, sodium carbonate, 1,2,3, 4-butanetetracarboxylic acid, fumaric acid, sodium bicarbonate, triethanolamine.

A fourth aspect of the invention provides the use of any one of the following:

(1) the material of the second aspect of the invention is applied to the preparation of the strontium-doped modified natural hydroxyapatite composite material for bone defect repair;

(2) the material of the second aspect of the invention is applied to the preparation of bone defect repair materials;

(3) the composite material of the third aspect of the invention is applied to preparing bone defect repairing materials;

(4) use of a material according to the second aspect of the invention in the repair of a bone defect;

(5) use of a composite material according to the third aspect of the invention in the repair of a bone defect.

Compared with the prior art, the invention has the advantages and beneficial effects that:

the invention provides a preparation method and application of a strontium-doped modified natural hydroxyapatite scaffold material for bone repair, and compared with a repair material disclosed in the prior art, the bone repair material has better biocompatibility, bioactivity and solubility and the capability of promoting bone tissue regeneration; in addition, the strontium replaces calcium in the hydroxyapatite, so that the crystal lattice of the hydroxyapatite is distorted, the crystallization degree is reduced, and the solubility of the material is improved. The bone repair material can release strontium ions in a continuous and slow mode, and the released strontium ions can stimulate osteogenic differentiation, inhibit the function of osteoclasts and accelerate the repair of bone defects. The bone repair material has wide raw material sources and simple preparation method, and has great clinical transformation application prospect in bone defect repair treatment.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1 shows an appearance and characterization result chart of a strontium-doped modified natural hydroxyapatite scaffold material, wherein, a chart: appearance, B diagram: appearance of the granular packing material obtained by grinding through the mesh screen, panel C: appearance, panel D: SEM result chart, E chart: EDX results statistical plot, panel F: EDX results plot, G plot: appearance of 6.8% SrHAP in bulk, panel H: SEM results of powdered 6.8% SrHAP;

Fig. 2 shows a graph of the results obtained from the characterization of three different molar ratios of strontium-doped hydroxyapatite material (9.8% SrHAP, 6.8% SrHAP, 4.6% SrHAP), where graph a: XRD, pattern B: FTIR, Panel C: XPS, panel D: the cumulative release amount of Sr after 6.8 percent of SrHAP is soaked in PBS for different days;

FIG. 3 is a diagram showing the results of statistics of pH and strontium doping ratio before and after acid reduction, wherein, A is a diagram: pH before and after deacidification, n is 6, panel B: the doping proportion of strontium elements before and after deacidification is 6;

FIG. 4 is a graph showing the appearance of a direct hemolysis experiment;

FIG. 5 is a graph showing the results of cell adhesion at day 3, day 7 with hMSCs, wherein, Panel A: HAP, B diagram: 4.6% SrHAP, Panel C: 6.8% SrHAP, Panel D: 9.8% SrHAP;

FIG. 6 shows the results of the survival status of hMSCs on scaffold material, wherein Panel A: HAP, B diagram: 4.6% SrHAP, Panel C: 6.8% SrHAP, Panel D: 9.8% SrHAP;

FIG. 7 is a graph showing the results of cell proliferation status of hMSCs on scaffold material, wherein, Panel A: CCK-8 detection result graph, B-E graph: a live and dead cell staining result graph;

fig. 8 is a graph showing the results of osteogenic differentiation promoted by scaffold material leaching solution, wherein, a graph: ALP viability assay, panel B: ALP staining, panel C: the expression of osteogenesis related genes indicates that the ratio P to the common induction group is less than 0.05;

FIG. 9 is a graph showing the results of a rabbit femoral lateral condyle bone defect repair experiment, wherein, A-a: rabbit femur distal lateral condyle was exposed, metaphyseal line was revealed, graph a-bc: the defect is cylindrical, the diameter is 5cm, the depth is 8cm, and an A-d diagram is as follows: post-filling state, panel B: verifying the 1W filling effect after operation by X rays;

FIG. 10 is a graph showing the results of X-ray plain films of the effect of defect repair at different time points, wherein, A is a graph: 6W, B diagram: 12W, Panel C: 26W, Panel D: 52W;

fig. 11 shows a graph of the results of 6.8% SrHAP particle repair effect at different time points, wherein, graph a: 6W, B diagram: 12W, Panel C: 26W, Panel D: 52W, p <0.01, ns. indicates no significant difference;

fig. 12 shows a graph of the results of 6.8% SrHAP particle repair effect at different time points, panel a: toluidine blue staining result chart, panel B: statistical plots, NB for new bone, P for material particles, P <0.05, ns. for no significant difference.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 preparation of strontium-doped modified Natural hydroxyapatite scaffold Material

1. Experimental Material

Bovine bone (cancellous bone) was purchased from fuhua meat ltd (china) of the national municipality of major factories; strontium chloride hexahydrate (analytically pure) was purchased from alatin reagent (shanghai) ltd (china); a live and dead cell staining kit (cat # C2015M) was purchased from petunia (china); MSC FBS (cat # 04-400-1A) was purchased from Biological Industries (Israel); L-DMEM (cat # SH30021.01) was purchased from Hyclone (USA); crystal violet stain (cat # C0121) was purchased from bi yun tian (china); CCK-8 (cat # CK04) was purchased from Homophilus chemical (Japan).

2. Preparation of calcined cow bone hydroxyapatite scaffold material

(1) Sources of cancellous bone: taking cancellous bone of the lower part of the femur of a 3-year-old cattle, and cutting a block material of 30mm by 20mm by 15 mm;

(2) degreasing: washing raw materials, soaking in warm water, rinsing with deionized water for 2-5 times respectively to remove impurities; soaking in 1-2M NaOH solution for 24-48h, and rinsing with deionized water for 2-4 times; 2% -20% of H₂O₂Soaking for 24-48h, rinsing for 2-4 times with deionized water, and air drying; dehydrating with 75-100% alcohol for 24-48h, and naturally drying; drying at 60-100 deg.C for 4-24 hr;

(3) and (3) gradient temperature rise calcination: high-temperature treatment, setting a temperature control program, setting 12-18 temperature program sections from 100 ℃ to 1000 ℃, wherein the fastest heating rate is 15-30 ℃/min, the slowest heating rate is 2-5 ℃, and the temperature program sections are specifically distributed as follows:

1) The room temperature is raised to 100 ℃ and 130 ℃ for 10-20min, and the temperature is kept for 10-20 min;

2) heating from 100 ℃ to 130 ℃ to 200 ℃ to 220 ℃ for 10-30min, and keeping the temperature for 60-90 min;

3) raising the temperature of 200 ℃ and 220 ℃ to the temperature of 250 ℃ and 260 ℃ for 15-25min, and keeping the temperature for 20-40 min;

4) raising the temperature from 250 ℃ to 260 ℃ to 280 ℃ to 310 ℃ for 5-15min, and keeping the temperature for 10-20 min;

5) raising the temperature of 280-310 ℃ to 320-330 ℃ for 8-15min, and keeping the temperature for 20-60 min;

6) raising the temperature of 320-330 ℃ to the temperature of 360-400 ℃ for 5-10min, and keeping the temperature for 20-35 min;

7) raising the temperature from 400 ℃ at 360 ℃ to 500 ℃ at 450 ℃ for 10-20min, and keeping the temperature for 10-30 min;

8) raising the temperature from 500 ℃ to 560 ℃ at 450 ℃ to 540 ℃ for 5-20min, and keeping the temperature for 10-20 min;

9) raising the temperature of 540-560 ℃ to 580-610 ℃ for 15-25min, and keeping the temperature for 30-60 min;

10) raising the temperature of 580-610 ℃ to 660-680 ℃ for 6-15min, and keeping the temperature for 10-20 min;

11) heating 660-680 ℃ to 750-800 ℃ for 10-15min, and keeping the temperature for 15-20 min;

12) raising the temperature of 750-;

13) heating 880-910 ℃ to 940-960 ℃ for 10-20min, and keeping the temperature for 90-180 min;

14) naturally cooling to room temperature;

(4) soaking in deionized water for 24-48 h;

(5) ultrasonic cleaning for 3-5 times, and drying at 80-100 deg.C for 4-8 hr.

3. Preparation of strontium-doped modified natural hydroxyapatite scaffold material (SrHAP)

The strontium-doped modified natural hydroxyapatite scaffold material is prepared by adopting a high-temperature ion exchange method, and the specific method comprises the following steps:

(1) Preparing strontium nitrate solutions with different concentrations (the concentrations are 0.5mol/L, 0.75mol/L and 1mol/L respectively) and taking the solutions as reaction liquid;

(2) at normal temperature, placing the calcined bovine bone hydroxyapatite scaffold material in strontium nitrate reaction solutions with different concentrations for reaction (in a filling state);

(3) standing for 2h, and drying at 80-100 ℃;

(4) high temperature calcination (room temperature-200 ℃ for 10 minutes, 200-600 ℃ for 40 minutes, 600-600 ℃ for 5 hours, 600-room temperature);

(5) cooling to room temperature, and soaking in deionized water for 24-48 h;

(6) ultrasonic cleaning for 3-5 times, and drying at 80-100 deg.C for 4-8 hr.

4. Treatment of strontium-doped modified natural hydroxyapatite scaffold material (SrHAP) for reducing pH

Phosphoric acid (concentration 14.74moL/L) was diluted to an application solution (diluted phosphoric acid) having a concentration of 0.1moL/L, as 1g (material): soaking for 1h at the ratio of 4mL (diluted phosphoric acid), and adjusting the soaking time (soaking for 2h with strontium) according to different materials; deionized Water (DDW) washing 6 times; 1g (material): soaking for 1h at the ratio of 30mL (DDW); drying at 80-100 ℃, and finally detecting the pH value of the strontium-containing calcined bovine bone scaffold and the difference of the strontium content of the strontium-containing calcined bovine bone scaffold before and after treatment.

5. Preparation of strontium-doped modified natural hydroxyapatite scaffold material leaching liquor

Preparing four material groups of 0, 4.8%, 6.8% and 9.8% SrHAP, smashing the cleaned blocky material into small blocks of granular materials by using an agate mortar, sieving the small blocks of granular materials by using a steel sieve with the pore diameter of 1.25-1.6mm, placing the granules in a 10cm dish, subpackaging 4g of the granules in a 50mL centrifuge tube, carrying out double-layer packaging, and carrying out Co-60 irradiation sterilization (12kGy, overnight); the preparation process of the leaching liquor refers to the GB16886.5 standard; firstly, adding 20mL of serum-free medium (L-DMEM) into a centrifuge tube containing 4g of granular materials, and laying the centrifuge tube in a shaking table; leaching conditions were then set, including a frequency of 120rpm, a temperature of 37 ℃ and a duration of 24h, and the leachate was finally filtered in an ultra clean bench using a 0.22 μ M filter (Millipore) and uniformly adjusted to a pH of around 8.4.

6. Characterization of strontium-doped modified Natural hydroxyapatite scaffold Material

All prepared calcined bovine bone hydroxyapatite scaffold materials and strontium-doped modified natural hydroxyapatite scaffold materials are characterized by being completed by the analysis and test center of southern China university, and mainly adopted instruments comprise an X-ray photoelectron spectrometer (Kratos Axis Mlra DLD.), a Fourier infrared spectrometer (Thermo-Nicolet) and an X-ray diffractometer (Brukerco.). The concentration of strontium, calcium and phosphorus ions in the material and the leaching solution of the material is finished by the spectronier test group ltd, and the test is finished by adopting a PS1000-AT type inductively coupled plasma emission spectrometer of the Leeman company in the United states.

7. Release of strontium ions in phosphate buffer

Selecting 6.8% SrHAP block materials, grinding the SrHAP block materials to 1.25-1.6mm of particle size, setting 6 groups for parallel soaking experiments, immersing samples into phosphate buffer solution (pH 7.4) without calcium and magnesium in a ratio of 0.2g/mL for corresponding time (1d, 3d, 5d, 7d, 2w, 3w, 6w and 12w), placing the samples in an environment at 37 ℃ for incubation, shaking for 1-2 times per day, taking liquid at fixed time intervals after shaking, centrifuging the samples at high speed at each time point, carefully absorbing about 7.5mL of supernatant, storing the supernatant in a refrigerator at-20 ℃, supplementing 7.5mL of PBS into a centrifuge tube, placing the centrifuge tube back in a constant temperature shaking table at 37 ℃, waiting for sampling at the next time point, detecting the strontium ion solubility of each time period by adopting ICP after the sampling is finished, and finally calculating the release amount.

8. Results of the experiment

The strontium-doped modified natural hydroxyapatite scaffold material prepared by an ion exchange reaction (shown in figure 1A) is subjected to grinding and screening to prepare a 1.25-1.6mm particle filling material (shown in figure 1B), and the 6.8% SrHAP particle material is subjected to SEM and EDX detection, so that the surface morphology and components of the particle material are shown, the main components of the particle material are Ca, Sr, P and O (shown in figures 1D-H), and the particle size of the strontium-doped modified natural hydroxyapatite scaffold material is uniform (shown in figure 1C), and the strontium is successfully doped into the 6.8% SrHAP particles;

In order to understand the influence on the hydroxyapatite crystal after doping strontium, XRD, FT-IR and XPS tests are carried out on strontium-doped hydroxyapatite materials with three different molar ratios (9.8 percent SrHAP, 6.8 percent SrHAP and 4.6 percent SrHAP), and the results show that the diffraction peak of SrHAP is basically consistent with the diffraction peak position and relative intensity of a hydroxyapatite PDF standard card, a specific diffraction peak with a left deviation is formed at 30-35 degrees, and the intensity is increased along with the increase of the content of strontium (see figure 2A), thereby proving that the strontium is successfully doped into the hydroxyapatite crystal and forming a new crystal phase; 3696. 3572cm^-1The peak is the hydroxyl-OH stretching vibration peak at 1092 and 1031cm^-1The peak of (A) is assigned to PO₄Middle antisymmetric telescopic vibration peak, 965cm^-1Peak of (b) is PO₄Symmetric stretching vibration peak, 563cm^-1Peak of (b) is PO₄The asymmetric variable angle vibration is the characteristic absorption peak of the apatite, the apatite with different Sr contents in three groups of 4.6%, 6.8% and 9.8% has a structure similar to that of pure HAP, and the characteristic peaks of P-O bonds in 4.6%, 6.8% and 9.8% are respectively reduced to 565 cm and 1029cm^-1(see FIG. 2B), these changes are due to Sr²⁺Substituted Ca²⁺Entering phosphor ashStone lattice of Sr²⁺Radius of 0.112nm, Ca²⁺0.100nm of Sr ²⁺The larger the radius, the lower the bonding strength of O-H and P-O, confirming Sr²⁺Can replace Ca²⁺Entering an apatite lattice; the results of XPS tests show that three groups of SrHAP materials mainly consist of three elements of Ca, P and O, and the Sr element content in pure HAP is extremely low (see figure 2C); 6.8% SrHAP particles with a particle size of 1.25-1.6mm are soaked in PBS for 84 days, and the cumulative strontium release amount can obtain a curve with steep early stage and gentle later stage (see FIG. 2D) through ICP detection, which shows that strontium ions are released from the material in a continuous and slow manner;

in order to examine the change of calcium-phosphorus content after strontium ion doped hydroxyapatite, ICP element content detection was performed on SrHAP (see table 1), and the results showed that strontium was successfully incorporated into hydroxyapatite, and three hydroxyapatite scaffolds with different strontium substitution molar ratios (Sr/(Sr + Ca)) were prepared, including 4.6% SrHAP, 6.8% SrHAP, and 9.8% SrHAP; the strontium-doped hydroxyapatite support is subjected to secondary high-temperature calcination, metal oxides are attached to the surface of the strontium-doped hydroxyapatite support and are easily dissolved after contacting water, the alkalinity of the surface of the material can be improved, the SrHAP subjected to secondary high-temperature calcination is subjected to cleaning and diluted phosphoric acid treatment, the pH values of 4.6% SrHAP and 6.8% SrHAP materials are remarkably reduced (p is less than 0.05), an acceptable slightly alkaline environment (pH is 8-9) is reached, but the pH value of the material is not remarkably reduced (p is 0.0524) when 9.8% SrHAP of a high-concentration strontium-containing group is subjected to the acid reduction step (see fig. 3A); the strontium content of the material after acid treatment is detected, and the result shows that the proportion of the substituted calcium of the strontium before and after acid reduction is not significantly changed (p is more than 0.05) (see figure 3B), which indicates that the acid reduction treatment can be adopted to reduce the pH value of the material, but does not influence the doping proportion of the strontium.

TABLE 1 statistical results of ICP elemental content testing for SrHAP

Example 2 direct hemolysis assay

1. Experimental methods

The direct hemolysis experiment is a method for evaluating the biological compatibility of medical instruments by the national standard GB/T16886.4, so the direct hemolysis experiment is respectively carried out on four groups of materials (0, 4.8%, 6.8%, 9.8% SrHAP) in the embodiment;

respectively weighing 4 groups of sieved material particles, 0.2g of each material particle, and adding the material particles into a centrifugal tube containing 10mL of physiological saline to serve as an experimental group; the positive control of the experiment is deionized water; the negative control was normal saline; placing the mixture in a constant temperature shaking table, keeping the temperature at 37 ℃ for 30min, adding 0.2mL of diluted rabbit blood (normal saline: rabbit blood is 5:4), then placing the mixture in the constant temperature shaking table at 37 ℃ again, keeping the temperature for 60min, centrifuging the mixture at room temperature for 5min at 2500r/min, taking the supernatant, and measuring the absorbance at 545nm on an enzyme labeling instrument, wherein the hemolysis rate (%) is (D sample-D negative control)/(D positive control-D negative control). times.100%. And judging that the material has hemolysis when the hemolysis evaluation standard hemolysis rate is greater than 5% according to GB/T16886, and judging that the material meets the requirements of the hemolysis experiment of the medical material when the hemolysis rate is less than or equal to 5%.

2. Results of the experiment

The results are shown in table 2 and fig. 4, and show that the hemolysis rates of HAP, 4.6% SrHAP and 6.8% SrHAP are less than 5%, which meet the national standard and show that the biocompatibility is good, while the hemolysis rate of 9.8% SrHAP is 7.96%, the erythrocyte is broken and the contact hemolysis is generated.

TABLE 2 statistics of the direct hemolysis experiment

Example 3 cell adhesion and survival status on strontium-doped modified native hydroxyapatite scaffolds

1. Cell culture

Human telomere immortalized mesenchymal stem cells (hMSCs) are from the donation of Zhongshan university Huanong subject group, are immortalized cells transfected with telomerase, have no tumorigenicity, can maintain the physiological characteristics of normal cells, have multidirectional differentiation potential, are normally cultured in L-DMEM containing 10% FBS and 1% PS, and are subcultured for about 3-4 days under the culture condition of 37 DEG C，5％CO₂(ii) a An induction culture medium for differentiation experiments is additionally added with an osteoinduction culture solution (containing 10mmol/L of beta-sodium glycerophosphate, 10-7mol/L of dexamethasone and 50 mu g/L of vitamin C) on the basis of a common culture medium.

2. Cell adhesion experiment

Cutting four groups of materials (0, 4.8%, 6.8%, 9.8% SrHAP) into small blocks of 5mm x 3mm, cleaning, soaking, drying, sterilizing, and making into cell experiment, wherein the prepared hMSC cell suspension is 3 × 10⁵Dripping 50 μ L of cell suspension into the suspension per mL, standing for 4 hr, supplementing complete culture medium, and adding 5% CO at 37 deg.C₂And culturing for 3 days and 7 days at saturated humidity, pouring out the culture medium, washing for three times by PBS (phosphate buffer solution), finally changing into a tissue fixing solution, spraying gold to prepare a sample, and observing under a scanning electron microscope.

3. Fluorescein Diacetate fluorescent staining

Cutting four groups of materials (0, 4.8%, 6.8%, 9.8% SrHAP) into small blocks of 5mm x 3mm, cleaning, soaking, drying, sterilizing, and making into hMSC cell suspension of 3 × 10⁵Adding 75 μ L cell suspension dropwise, standing for 4 hr, supplementing complete culture medium, and adding 5% CO at 37 deg.C₂Culturing for 24h at saturated humidity, adopting a fluoroescein di-O-acetate (F.D.A) staining method to prepare F.D.A mother solution, wherein a solvent is DMSO or Acetone (Acetone) to 5mg/mL, adding 4 microliter of the mother solution into 4mL of PBS with the pH value of 7.2 to prepare F.D.A application solution, dropwise adding 2mL of the prepared F.D.A application solution onto a material, placing the material into a water bath at 37 ℃ for 1-2min, and quickly observing and photographing under a fluorescence microscope.

4. Results of the experiment

The results are shown in fig. 5A-D and fig. 6A-D, and show that the mesenchymal stem cells are better adhered and stretched on the surfaces of different strontium-doped hydroxylapatite on day 3, the cells are fully stretched on the surface of the material, the platy pseudopodium and the filamentous pseudopodium are rich in multiple polarities and are anchored and adhered on the surface of the material, and the cells are full and adhered on the surface of the material on day 7, so that the surface of the material is difficult to see in the displayed visual field; the result of viable cell staining after the hMSCs are directly inoculated on the surfaces of four groups of scaffold materials for 24 hours shows that the cells are uniformly distributed on the surfaces of HAP, 4.6 percent SrHAP and 6.8 percent SrHAP, the cell activity is high, while the bright fluorescence of the viable cells of the 9.8 percent SrHAP scaffold is less than that of the other three groups, which indicates that the biocompatibility of the 9.8 percent SrHAP scaffold is poor.

Example 4 cell proliferation status on strontium-doped modified native hydroxyapatite scaffolds

1. Cell proliferation assay

In vitro experiments all adopt an ion leaching solution mode to indirectly evaluate the effect of materials of different groups on mesenchymal stem cells, and ICP is adopted to detect the content of each ion in different material leaching culture media according to the leaching method of GB 16886.5. Inoculating hMSC to a 96-well plate at the density of 2-3000/well, changing the culture solution into a material leaching stock solution after 24h, co-culturing each group with hMSC for 1, 4 and 7d (changing the solution every other day), taking out the well plate to terminate the culture, taking the cells without the leaching solution as a blank control group, and detecting the cell proliferation activity by adopting a CCK-8 kit, wherein the specific operation steps are as follows: after the medium was aspirated, 100. mu.L of a medium containing 10% CCK-8 was added to each well, incubated in an incubator for 1 hour in the dark, and then placed in a microplate reader to measure the light absorbance at 450 nm.

2. Staining observation of live and dead cells

Before cell plating, a 12-hole plate is plated with 0.5% gelatin, and the gelatin is sucked and removed and then is washed by PBS; at 3-5X 10⁴Plating per well cell amount, after 8h cell adherence, incubating for 24h by changing material leaching liquor, then, changing 500 mu L/well living and dead cell staining solution application liquid (the final concentration of Calcein-AM is 2 mu M, the PI is 4.5 mu M), incubating for 15min at 37 ℃, and finally, simultaneously detecting living cells (yellow green fluorescence) and dead cells (red fluorescence) by using a 490 +/-10 nm excitation filter disc under a fluorescence microscope.

3. Results of the experiment

The results show that strontium is contained in the L-DMEM and HAP groups in an extremely low content, and the concentration of Ca ions in SrHAP leaching solution is greatly reduced compared with that in the L-DMEM group, which is related to the formation of strontium apatite on the surface of SrHAP, and partial Ca ions in the culture medium are consumed on the basis (see Table 3);

the results showed that the 9.8% SrHAP extract group was not conducive to cell proliferation, whereas HAP and 4.6% SrHAP material extract stock groups were not significantly different on days 1, 4, and 7 compared to the normal medium group, whereas the 6.8% SrHAP extract group was different from the normal medium on day 7 (p <0.05) (see fig. 7A); the results in fig. 7B-E show that the 9.8% SrHAP leached group had the most dead cell fluorescence spots, followed by the 6.8% SrHAP group, which indicates that the 4.6% SrHAP material was superior to the 6.8% SrHAP, 9.8% SrHAP.

TABLE 3 statistics of the results of ion content in leach liquors

Example 5 strontium-doped modified Natural hydroxyapatite scaffolds to promote osteogenic differentiation of cells

1. Real-time quantitative PCR experiment for promoting osteogenic differentiation of material leaching liquor in vitro

(1) RNA extraction: extracting RNA by using Trizol reagent, taking a six-hole plate as an example, sucking away a culture medium, washing for 2 times by PBS, adding 1mL Trizol, blowing and uniformly beating cells, sucking into a 1.5mL EP tube, and storing in a refrigerator at-80 ℃;

1) Unfreezing: adding 200 mu L of chloroform into 1mL of TRIZOL, manually and violently shaking for 30s, and standing for 5min at room temperature;

2) centrifuging: centrifuging at 4 deg.C for 15min at 12,000g, and separating into three layers, wherein the uppermost layer contains RNA in water phase;

3) carefully transfer the upper aqueous phase to a new EP tube, unabsorbable the intermediate phase;

4) adding isopropanol with equal volume, mixing, and standing at room temperature for 5 min;

5) centrifuging: 12,000g for 10min at 4 ℃, the supernatant is discarded, and a small amount of white RNA precipitate can be seen at the bottom of the tube;

6) preparing 75% ethanol by using DEPC water, adding 1mL of the prepared ethanol, gently oscillating the centrifugal tube, and carrying out suspension precipitation;

7) centrifuging: 8,000g at 4 ℃ for 5min, and abandoning the supernatant as much as possible;

8) drying at room temperature for 10 min;

9) dissolving the RNA precipitate, and dissolving the RNA sample by using 20-50 mu L DEPC water;

10) measuring an O.D value by using the Nanodrop, recording and quantifying the RNA concentration;

(2) RNA reverse transcription process:

1) genomic DNA removal, configured as in table 4;

TABLE 4 preparation of solutions

Gently blowing and beating by using a pipette, and mixing uniformly at 42 ℃ for 2 min;

2) configuring a reverse transcription reaction system, and showing in a table 5;

TABLE 5 reverse transcription reaction System

Gently blowing and beating the mixture by using a pipettor, and uniformly mixing the mixture, and setting the reaction condition at 37 ℃ for 15 min; 5sec at 85 ℃;

3) RT-qPCR (real-time quantitative PCR), according to Novoweb ChamQ Universal SYBR qPCR Master Mix instruction to detect the expression of related genes, configuration reaction system (10 uL system) is shown in Table 6;

TABLE 6 reaction System

The PCR reaction program was set up as follows: pre-denaturation: 3min at 95 ℃; and (3) cyclic reaction: circulating for 40 times at 95 ℃ for 10sec and 60 ℃ for 30 sec; quantitative analysis is carried out by using Bio-Rad CFX Manager software of Bio-Rad company, and the sequence of the hMSC osteogenesis related gene PCR primer used in the experiment is shown in Table 7;

TABLE 7 PCR primer sequences

2. Alkaline phosphatase (ALP) staining

After removing the culture medium liquid, rinsing with PBS once, fixing with 4% paraformaldehyde for 30min, washing with PBS for 3 times, preparing ALP staining working solution according to the proportion in the specification, adding 200 μ L (aiming at a 24-hole plate, ensuring that the sample can be fully covered) of prepared BCIP/NBT staining working solution into each hole, incubating at room temperature in a dark place for 30min (expected depth), removing the staining solution, washing with DDW for 1-2 times to terminate the color reaction, observing under an inverted microscope and taking a picture.

3. Alkaline phosphatase (ALP) Activity assay

(1) Collecting cells, cracking on ice, collecting in an EP tube after about 10min, shaking for 1min, and centrifuging at the highest rotation speed of a centrifuge for 5 min; (2) BCA protein concentration determination, wherein after the concentration of each histone is calculated, each histone sample is diluted to a uniform concentration according to the standard of the lowest protein concentration; (3) ALP enzyme activity experiment reagent preparation, a, chromogenic substrate solution: dissolving a tube of chromogenic substrate in 2.5mL of detection buffer solution, fully dissolving and uniformly mixing, and placing on ice; b. standard working solution: 10 μ L p-nitrophenol solution (10mM) was diluted to 0.2mL with assay buffer to a final concentration of 0.5 mM; (4) adding samples according to the specification, adding a chromogenic substrate, starting the reaction, paying attention to avoid bubbles, putting the mixture into a 37 ℃ oven, incubating for 10-30min, and taking out the mixture after obvious yellow appears; (5) terminating the reaction, and adding 100 mu L of termination solution into each well; (6) absorbance was measured spectrophotometrically at 405nm and 620nm, and relative ALP enzyme activity was calculated.

4. Statistical analysis

The data for statistical analysis at least comprises three parallel samples, statistical processing adopts statistical software built in GraphPad Prism 8, data statistical description is expressed by mean +/-standard deviation, statistical inference adopts t test between independent samples, and difference is considered to have statistical significance when P is less than 0.05.

5. Results of the experiment

The leaching solutions of HAP, 4.6% SrHAP and 6.8% SrHAP were cultured for 3 days, 6 days and 9 days, respectively, and tested for ALP activity and staining thereof, and the results showed that the 6.8% SrHAP leaching solution had significant effects of promoting ALP expression and increasing ALP activity, as compared with the induction medium (see fig. 8A and B); the PCR results after 6 days of culture showed that both strontium-containing material leachate had a promoting effect on osteogenesis-related gene expression (ALP, Col1a1, Runx2, OCN, OPN) (P <0.05) (see FIG. 8C), and 4.6% SrHAP group > 6.8% SrHAP > HAP material leachate group. From the above results, it can be seen that 6.8% of the material performs better in ALP staining and its viability, and that 6.8% SrHAP is a better molar ratio of strontium-doped hydroxyapatite material, taking into account the ability to release strontium continuously.

Example 6 Experimental animal study of bone Defect repair

1. Animal experiment grouping and operation procedure

In order to verify the bone promoting performance of the strontium-doped modified natural hydroxyapatite scaffold material in vivo, an animal model of the lateral condyle bone defect of the femur of a new zealand big ear white rabbit is constructed, and HAP and a 6.8% SrHAP scaffold are respectively implanted into the bone defect part of an experimental animal, wherein the size of the bone defect part is a cylindrical defect with the diameter of 5mm and the height of 5 mm. The animal experiments are divided into three groups: blank defect group (defect no implant material); HAP group (defect implanted non-strontium-doped bovine bone hydroxyapatite); 6.8% SrHAP group (defect implanted with strontium-doped 6.8% bovine bone hydroxyapatite). Observation time points were 6 weeks, 12 weeks, 26 weeks, 52 weeks after surgery;

the experimental animals are selected from big ear New Zealand rabbits with average 2.6 kg, and are adaptively raised for more than one week before the experiment, and 42 New Zealand rabbits are combined in the embodiment. The operation is performed for 8 hours without food or water, monitoring and supportive care are not needed in the operation process, and the used aseptic methods are ultraviolet irradiation sterilization and high-pressure treatment sterilization. The rabbit was supine, tied and fixed with its two hind legs and mouth on a rabbit frame special for animal experiment with a bandage, and skin-prepared with a razor special for animal (electric razor hair and skin preparation) with a skin preparation area of 10 × 10cm, with the incision at the center and a distance of 5cm from the periphery. The anesthesia adopts compound ketamine injection with the dosage being calculated by weight (0.12mL/kg), and intramuscular injection anesthesia is carried out. The white rabbit eyelashes are slow in reflection, and no clear reaction is caused to the skin in the operation area clamped by the toothed forceps, so that the success of anesthesia is indicated;

The operation process comprises the following steps: sterilizing by conventional operation, and spreading. The skin of lidocaine is cut after surface anesthesia, subcutaneous tissues are separated layer by layer in an inactive manner, blood vessels are avoided as much as possible, and the operation area is fully exposed by hemostatic forceps; the surgical site is the lateral condyle of the distal femur, and the physiological marker is a raised white growth line; using a knife to draw an H near the growth line, and using a periosteal spatula to separate the covered periosteum (keeping the integrity as much as possible); the tip drill perforates, positions near the growth line, deflects towards the distal end, and then is expanded using a rough drill. The resulting defect was cylindrical, 5mm in diameter and 5mm in height. The model preparation process is continuously washed by 0.9% physiological saline, and the visual field is clean. Filling the granular graft and mixing with the exuded blood, and filling and compacting into the defect part. The filled granular graft is composed of 1.25-1.6mm (particle diameter) HAP, 6.8% SrHAP granules mixed with rabbit blood, and then intermittently sewing layer by layer, smearing on the surface with Bauduobang (mupirocin ointment-Zhongmeishake), injecting postoperative penicillin 80 ten thousand units of muscle for 1 time/day for 3 days, preventing infection, and feeding in cages. After operation, the general living conditions of the experimental animals are observed, and whether the inflammatory reaction exists at the operation part or not, whether the limb movement is limited or not and the like are noticed.

2. Mirco-CT scan analysis

After operation, 6W, 12W, 26W and 52W, cross-joint material collection and X-ray plain film photographing are carried out. After trimming the specimen, in formalin fixation for 2 days, a high-resolution mio-CT scanning system (Skyscan 1172, n.v., Aartselaar, Belgium) was followed by a circular sweep at 89kV and 80 μ a current, with a scan angle of 180 ° and a scan step size of 0.40 °. CTAn software (Bruker micro CT, Belgium) was used for image processing.

3. Histological analysis of specimens

In order to more visually observe the integration relationship between the material of the defect part and the host, the lateral condyle of the femur is cut along the sagittal long axis. The specimens were prepared into non-decalcified sections using the EXAKT cutting and milling system (EXAKT 310CP, Germany) as follows: firstly, immersing a specimen subjected to gradient dehydration into a photopolymerization monomer for curing under an ultraviolet condition; then cut and milled with EXAKT to reveal the section of interest; washing with 30% hydrogen peroxide for 5min, and washing with distilled water; and finally, dyeing the toluidine blue for 30-40min, sealing the sheet after drying, and scanning and drawing the sheet in a panoramic scanning system. Bone histomorphometry was analyzed using Image J (version 1.8.0).

4. Results of the experiment

The operation flow chart is shown in fig. 9A, HAP of 1.25-1.6mm and SrHAP particles of 6.8% are mixed with rabbit blood and then implanted into the defect part, and the result of X-ray examination of the filling effect is shown in fig. 9B;

In order to research the repair condition of bone tissues, the specimen is subjected to X-ray plain film shooting (see fig. 10A-D), wherein a white bright area in the X-ray plain film shows high density, a dark area in the X-ray plain film shows a low-density part, and filled bovine hydroxyapatite particles show high density shadow in the external condyle part of the femur and have higher density than rabbit cortical bone, and the comparison of 6W and 52W X ray plain films shows that the relative density of a defect part is reduced and the white color becomes fuzzy as relatively low-density new bone tissues grow in;

in order to research the osteogenesis difference between HAP and 6.8% SrHAP, a sample is subjected to micro-CT scanning after being taken, the micro-CT tomography can show different gray levels (GV) on a single-layer picture according to different densities of tissues, soft tissues and normal rabbit bone tissues can be screened out by reconstructing and setting the upper limit and the lower limit of GV, then the gray value ratio of new bone formation to a scaffold material is calculated within a certain threshold value, so that the Percent bone volume of a new bone can be obtained, the result is shown in FIGS. 11A-D, the two implanted material particles are both near a growth plate, and the filled material forms a plurality of gaps at a defect part, so that the migration of cells is facilitated, and the formation of the new bone is guided; in the first 12 weeks, 6.8% SrHAP is significantly more than the percentage of new bone mass produced by HAP material filling (P <0.01), and after 26 weeks, there is no statistical difference in the new bone formed by the two materials, indicating that the strontium-doped hydroxyapatite has the ability to promote new bone formation early and the incorporation of strontium accelerates bone defect repair;

Non-decalcified sections and toluidine blue staining were performed on the grafts harvested at weeks 6, 12, 26 and 52, and the results are shown in fig. 12A, which shows that both grafts can form new bone-like mineralized tissues on their surfaces (indicated by arrows) running along the surface of the implant; taking image J to perform histomorphometric measurements on the tissue of the slices, the results show that 6.8 percent of SrHAP surface bone-like tissues at the early stage of 6 weeks after implantation are obviously more than HAP groups (P <0.05), and the measurement results at 12 weeks show that the forming area of new bones on the surface of 6.8 percent of SrHAP is different from that of the HAP groups; in the later period of repair (26 weeks and 52 weeks), the difference between the strontium-doped group and the strontium-undoped group is not significant, and in addition, compared with 6 weeks, the two groups of filling materials, namely 6.8% SrHAP and HAP, are not degraded obviously in the later period of repair (see figure 12B), further showing that the strontium-doped hydroxyapatite (6.8% SrHAP) has the capacity of promoting new bone formation in the early period, the doping of strontium accelerates the repair of bone defects, and has excellent biocompatibility, osteogenic differentiation promotion, relatively stable crystal structure, and low in vitro solubility and degradability.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

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<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cccagtatga gagtaggtgt cc 22

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gggtaagact ggtcatagga cc 22

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gctcactggc atggccttcc g 21

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtgggccatg aggtccacca c 21