AU2021100655A4

AU2021100655A4 - POLY γ-GLUTAMIC ACID/CHITOSAN/CALCIUM CITRATE BIOMATERIAL AND PREPARATION METHOD THEREO

Info

Publication number: AU2021100655A4
Application number: AU2021100655A
Authority: AU
Inventors: Chengchuan Che; Zhijin Gong; Jinfeng Liu; Yifei Liu; Ge Yang
Original assignee: Qufu Normal University
Current assignee: Qufu Normal University
Priority date: 2020-11-27
Filing date: 2021-02-02
Publication date: 2021-04-22
Anticipated expiration: 2029-02-02
Also published as: CN112354010A

Abstract

The present disclosure relates to the field of bone biomaterials, in particular to a poly y-glutamic acid/chitosan/calcium citrate biomaterial and a preparation method thereof. The method includes firstly preparing y-polyglutamic acid/chitosan gel particles by a spray freeze-drying method, loading recombinant human bone morphogenetic protein 2 growth factors, premixing with a solid phase of calcium citrate by a wet method, grinding and freeze-drying, adding a curing liquid according to a proportion, and carrying out injection molding and pressing molding. The poly-y glutamic acid and chitosan used in the present disclosure are colorless, non-toxic, tasteless and easily degradable microbial fermentation extracts, and the carboxyl group of the y-PGA and the amino group of CS have high coordination coefficient and stable structures. According to the present disclosure, the recombinant human bone morphogenetic protein 2 and calcium ions can be slowly released into a human body to help promote bone tissue repair. The components of the present disclosure are all biological agents having good biological affinity and small stimulation, which are degradable in organisms, green and safe. 100 90 80 70 60 50 40 3 - Release rate of Ca 20 - Release rate of the recombinant 10 human bone morphogenetic protein 2 0 0 24 48 72 96 120 144 168 192 216 240 264 288 312 Time (h) Fig. 1 1/1

Description

3 - Release rate of Ca - Release rate of the recombinant human bone morphogenetic protein 2 0 0 24 48 72 96 120 144 168 192 216 240 264 288 312 Time (h)

Fig. 1

1/1

Poly y-Glutamic Acid/Chitosan/Calcium Citrate Biomaterial and Preparation Method Thereof

Technical Field

The present disclosure relates to the field of bone biomaterials, in particular to a poly y-glutamic acid/chitosan/calcium citrate biomaterial and a preparation method thereof.

Background of the Invention

Chitosan is a natural polymer compound, which is a natural alkaline polysaccharide with positive charge obtained by deacetylation of chitin, and is abundant in the shells of shrimp and crabs. The chitosan not only has good biocompatibility, biodegradability and non-toxicity, but also has good antibacterial property and can inhibit the growth of certain pathogenic microorganisms such as Escherichia coli and Staphylococcus aureus. The biodegradability of the chitosan also helps to prevent environmental pollution caused by waste products of the chitosan, and improves the microecological property of soil. Chitosan is positively charged and cross-linked with negatively charged poly-y-glutamic acid in a liquid medium.

Poly-y-glutamic acid (abbreviated as y-PGA) is a non-ribosomal peptide composed of L-and D-glutamic acid monomers linked by y-glutamyl bond under the catalytic action of enzyme, the molecular chain has a large number of side chain carboxyl groups, which can form hydrogen bonds within or between molecules. It has biodegradability, ease of modification, water retention property, solubilization property and slow release property.

At present, the treatment of massive and refractory bone defects caused by various reasons is a big problem that plagues the medical field. Increasing the activity of artificial bone materials, improving the speed and ability of bone tissue repair, and shortening the healing/integration time are the main line and goals throughout the whole research of bone tissue repair materials. Bone tissue repair is a complex process involving a series of cascade processes such as differentiation and proliferation of pluripotent stem cells, recognition of extracellular matrix and signaling molecules, expression and targeting of related factors and development and maturation of new bones. The application of various osteogenic factors, particularly controlled release carriers loaded with osteogenic factors, to bone defect areas to stimulate bone formation has proven to be significantly effective. Although the research history of local controlled release of growth factors is not long, it has shown its vitality and unique advantages. In bone tissue engineering, an ideal drug carrier not only has a three-dimensional and microporous scaffold structure, but also has a bone-guiding property while releasing osteogenic factors regularly and quantitatively, and is beneficial to cell attachment and vascular ingrowth. In addition, it also has the characteristics that the degradation is matched with the osteogenesis speed, the mechanical strength is proper, and the like. Among many of these indexes, the controlled release rate is the most important, which mainly depends on the combination of factors and carriers, physical and chemical properties of carriers and the microenvironment of tissues around carriers. There is currently no ideal materials to meet the above requirements.

Summary of the Invention

Aiming at technical problems existing in the prior art, the present disclosure aims to provide a poly-y-glutamic acid/chitosan/calcium citrate biological material which can slowly release calcium citrate into a body, thereby accelerating healing by serving as a supporting structure for bone growth and helping to accelerate bone regeneration.

The present disclosure also provides a preparation method of the biomaterial prepared from poly y-glutamic acid/chitosan/calcium citrate. According to the method, poly y-glutamic acid/chitosan is compounded with recombinant human bone morphogenetic protein 2 and calcium citrate, so that the purpose of slowly releasing the calcium citrate into a human body is achieved.

The technical solution adopted by the present disclosure for achieving the purpose is as follows.

The present disclosure provides a poly-y-glutamic acid/chitosan/calcium citrate biomaterial prepared by the following method: firstly preparing y-polyglutamic acid/chitosan gel particles by a spray freeze-drying method, loading recombinant human bone morphogenetic protein 2 growth factors, premixing with a solid phase of calcium citrate by a wet method, grinding and freeze-drying, adding a curing liquid according to a proportion, and carrying out injection molding and pressing molding to prepare the poly-y-glutamic acid/chitosan/calcium citrate biomaterials.

The present disclosure also provides a preparation method of the polyy-glutamic acid/chitosan/calcium citrate biomaterial, which specifically comprises the following steps: (1) dissolving chitosan in an acetic acid solution, stirring uniformly at room temperature, and diluting with deionized water to obtain the chitosan solution; dropwise adding the poly-y-glutamic acid solution into an equal volume of chitosan solution, magnetically stirring, fully mixing, spraying and freeze-drying to obtain the poly-y-glutamic acid/chitosan dry powder; (2) dissolving the recombinant human bone morphogenetic protein 2 in double distilled water to obtain a recombinant human bone morphogenetic protein 2 solution, then fully swelling the poly y-glutamic acid/chitosan dry powder therein, and uniformly emulsifying by using an ultrasonic homogenizer to obtain the poly y-glutamic acid/chitosan dry powder composite carrier; (3) mixing the composite material carrier with calcium citrate powder, grinding and triturating with an agate, preparing powder by a freeze-drying method, and uniformly mixing the powder with a curing liquid; (4) injecting into a mold, pressing a cylinder at 600 kPa for 5s to remove large bubbles formed when the slurry is filled, demolding to obtain a cylindrical sample, curing, taking out, immersing same into an ethanol solution, and air-drying.

Further, in step (1), the volume percentage concentration of acetic acid in the acetic acid solution is 1%; the concentration of the poly-y-glutamic acid solution is 2 mg/mL; the concentration of the chitosan solution is 2 mg/mL; and the deacetylation degree of the chitosan is 80.0 to 95.0.

Further, in step (2), the concentration of the recombinant human bone morphogenetic protein 2 solution is 20 g/mL; the swelling is performed by adding 100 mg of poly y-glutamic acid/chitosan dry powder per 5 mL of the recombinant human bone morphogenetic protein 2 solution.

Further, in step (3), a mass ratio of the poly y-glutamic acid/chitosan dry powder composite carrier to the calcium citrate powder is 1:9; and a mixing ratio of the poly y-glutamic acid/chitosan/calcium citrate powder to the curing liquid is 3:1 (g/mL).

The curing liquid used in the present disclosure is composed of the following raw materials in percentage by mass:2-9% of citric acid, 1-6% of VP, 5-25% of phosphate, 1-5% of PVA, 0.5-0.8% of squalene and the balance of water.

Further, in step (4), the curing is carried out in a constant temperature and

humidity box with the temperature of 37C and the humidity of 97%, and taken out

after 3 days; The time of immersion in the ethanol solution was 2 h.

Compared with the prior art, the present disclosure has the following beneficial effects. (1) The poly-y glutamic acid and chitosan used in the present disclosure are colorless, non-toxic, tasteless and easily degradable microbial fermentation extracts, and the carboxyl group of the y-PGA and the amino group of CS have high coordination coefficient and stable structures. (2) According to the present disclosure, the recombinant human bone morphogenetic protein 2 and calcium ions can be slowly released into a human body to help promote bone tissue repair. (3) The components of the present disclosure are all biological agents having good biological affinity and small stimulation, which are degradable in organisms, green and safe.

Brief Description of the Drawings

Fig. 1 is a graph showing the release rates of Ca+ and recombinant human bone morphogenetic protein 2 in the biomaterial prepared in Example 1.

Detailed Description of the Embodiments

Specific embodiments of the present disclosure are illustrated by the following examples, but the content of protection of the present disclosure is not limited thereto.

Example 1 (1) Chitosan was dissolved in an acetic acid solution, uniformly stirred at room temperature, and diluted with deionized water to obtain the chitosan solution; 2 mg/mL of the poly-y-glutamic acid solution was dropwise added into an equal volume of 2 mg/mL of chitosan solution, magnetically stirred, fully mixed, sprayed and freeze-dried to obtain the poly-y-glutamic acid/chitosan dry powder; (2) 100 g of the recombinant human bone morphogenetic protein 2 was dissolved in 5 mL of double distilled water, then 100 mg of the polyy-glutamic acid/chitosan dry powder was fully swelled therein, and uniformly emulsified by using an ultrasonic homogenizer to obtain the poly y-glutamic acid/chitosan dry powder composite carrier; (3) the above-mentioned carrier material was mixed with calcium citrate powder in a mass ratio of 1: 9, ground and triturated with an agate, and made into powder by a freeze-drying method, and mixed with a curing liquid in a ratio of 3: 1 (g/mL); the curing liquid was consisted of: 8% of citric acid, 4% of VP, 20% of phosphate, 5% of PVA, 0.5% of squalene and the balance of water; (4) it was injected into a mold with a height of 7 mm and a diameter of 10 mm, respectively; then it was pressed with a cylinder having a diameter of 6.6 mm at 600 kPa for 5 s to remove large bubbles formed when the slurry was filled, and then a cylindrical sample was demolded and cured in a constant temperature and humidity chamber at 37°C and 97% humidity, taken out after 3 days, immersed in an ethanol solution for 2 h to terminate the hydration reaction, and then air-dried to obtain a novel biomaterial prepared from poly-y-glutamic acid/chitosan/calcium citrate.

Comparative Example 1 (1) Chitosan was dissolved in an acetic acid solution, stirred uniformly at room temperature, and diluted with deionized water to obtain the chitosan solution; 2 mg/mL of the poly-y-glutamic acid solution was dropwise added into an equal volume of 2 mg/mL of chitosan solution, magnetically stirred, fully mixed, sprayed and freeze-dried to obtain the poly-y-glutamic acid/chitosan dry powder; (2) 100 g of the recombinant human bone morphogenetic protein 2 was dissolved in 5 mL of double distilled water, then 100 mg of the polyy-glutamic acid/chitosan dry powder was fully swelled therein, and uniformly emulsified by using an ultrasonic homogenizer to obtain the poly y-glutamic acid/chitosan dry powder composite carrier; (3) the above-mentioned carrier material was mixed with calcium citrate powder in a mass ratio of 1: 9, ground and triturated with an agate, and made into powder by a freeze-drying method, and mixed with the solidified liquid in a ratio of 3: 1 (g/mL); the curing liquid was consisted of: 8% of citric acid, 4% of VP, 20% of phosphate, 5% of PVA, and the balance of water; (4) it was injected into a mold with a height of 7 mm and a diameter of 10 mm, respectively; then, it was pressed with a cylinder having a diameter of 6.6 mm at 600 kPa for 5 s to remove large bubbles formed when the slurry was filled, and then a cylindrical sample was demolded and cured in a constant temperature and humidity chamber at 37°C and 97% humidity, taken out after 3 days, immersed in an ethanol solution for 2 h to terminate the hydration reaction, and then air-dried to obtain a novel biomaterial prepared from poly-y-glutamic acid/chitosan/calcium citrate.

Comparative Example 2 (1) Chitosan was dissolved in an acetic acid solution, stirred uniformly at room temperature, and diluted with deionized water to obtain the chitosan solution; 1.5mg/mL of the poly-y-glutamic acid solution was dropwise added into an equal volume of 2 mg/mL of chitosan solution, magnetically stirred, fully mixed, sprayed and freeze-dried to obtain the poly-y-glutamic acid/chitosan dry powder; (2) 100 g of the recombinant human bone morphogenetic protein 2 was dissolved in 5 mL of double distilled water, then 100 mg of the polyy-glutamic acid/chitosan dry powder was fully swelled therein, and uniformly emulsified by using an ultrasonic homogenizer to obtain the poly y-glutamic acid/chitosan dry powder composite carrier; (3) the above-mentioned carrier material was mixed with calcium citrate powder in a mass ratio of 1: 9, ground and triturated with an agate, and made into powder by a freeze-drying method, and mixed with a curing liquid in a ratio of 3: 1 (g/mL); (4) it was injected into a mold with a height of 7 mm and a diameter of 10 mm, respectively; then it was pressed with a cylinder having a diameter of 6.6 mm at 600 kPa for 5 s to remove large bubbles formed when the slurry was filled, and then a cylindrical sample was demolded and cured in a constant temperature and humidity chamber at 37°C and 97% humidity, taken out after 3 days, immersed in an ethanol solution for 2 h to terminate the hydration reaction, and then air-dried to obtain a novel biomaterial prepared from poly-y-glutamic acid/chitosan/calcium citrate.

Performance Testing: (I) Drug release studies were conducted by placing poly-y-glutamic acid/chitosan/calcium citrate in 20 mL of Simulated Body Fluid (SBF) in a 37°C water bath environment. At regular intervals, 1 mL of supernatant was removed and supplemented with fresh simulated body fluid. The release amount of the recombinant human bone morphogenetic protein 2 in 1 mL of supernatant was measured by ELISA kit and the release rate was calculated. The release amount of the calcium ions in 1 mL of supernatant was measured by EDTA titration method and the release rate was calculated.

As shown in Fig. 1, the release rates of calcium ion and the recombinant human bone morphogenetic protein 2 in the simulated body fluid of Example 1 reached 70% and 60% at 192 hours, respectively, demonstrating that they have good release capability in the simulated body fluid. The trend of the first half of the curve is gentle, which proves that the material indeed has the capability of slowly releasing calcium ions and the recombinant human bone morphogenetic protein 2. In Comparative Example 2, when the ratio of poly-y-glutamic acid to chitosan was changed, the prepared biomaterial had poor capability of slow release, and the release rate of calcium ions could reach 85% at 120 h; in Comparative Example 1, the loading rate of calcium citrate was reduced by about 15% compared with Example 1.

Claims

1. A poly-y-glutamic acid/chitosan/calcium citrate biomaterial, characterized in that, it is prepared by the following method: firstly preparing y-polyglutamic acid/chitosan gel particles by a spray freeze-drying method, loading recombinant human bone morphogenetic protein 2 growth factors, premixing with a solid phase of calcium citrate by a wet method, grinding and freeze-drying, adding a curing liquid according to a proportion, and carrying out injection molding and pressing molding to prepare the poly-y-glutamic acid/chitosan/calcium citrate biomaterials.

2. A preparation method of the poly-y-glutamic acid/chitosan/calcium citrate biomaterial of claim 1, characterized by specifically comprising the following steps: (1) dissolving chitosan in an acetic acid solution, stirring uniformly at room temperature, and diluting with deionized water to obtain the chitosan solution; dropwise adding the poly-y-glutamic acid solution into an equal volume of the chitosan solution, magnetically stirring, fully mixing, spraying and freeze-drying to obtain the poly-y-glutamic acid/chitosan dry powder; (2) dissolving the recombinant human bone morphogenetic protein 2 in double distilled water to obtain a recombinant human bone morphogenetic protein 2 solution, then fully swelling the poly y-glutamic acid/chitosan dry powder therein, and uniformly emulsifying by using an ultrasonic homogenizer to obtain the poly y-glutamic acid/chitosan dry powder composite carrier; (3) mixing the composite material carrier with calcium citrate powder, grinding and triturating with an agate, preparing powder by a freeze-drying method, and uniformly mixing the powder with a curing liquid; (4) injecting into a mold, pressing a cylinder at 600 kPa for 5s to remove large bubbles formed when the slurry is filled, demolding to obtain a cylindrical sample, curing, taking out, immersing same into an ethanol solution, and air-drying.

3. The preparation method according to claim 2, characterized in that, in step (1), the volume percentage concentration of acetic acid in the acetic acid solution is 1% ; the concentration of the poly-y-glutamic acid solution is 2 mg/mL; the concentration of the chitosan solution is 2 mg/mL; and the deacetylation degree of the chitosan is 80.0 to 95.0.

4. The preparation method according to claim 2, characterized in that in step (2), the concentration of the recombinant human bone morphogenetic protein 2 solution is 20 [g/mL; the swelling is performed by adding 100 mg of poly y-glutamic acid/chitosan dry powder per 5 mL of the recombinant human bone morphogenetic protein 2 solution.

5. The preparation method according to claim 2, characterized in that in step (3), a mass ratio of the poly y-glutamic acid/chitosan dry powder composite carrier to the calcium citrate powder is 1:9; and a mixing ratio of the poly y-glutamic acid/chitosan/calcium citrate powder to the curing liquid is 3:1 (g/mL).

6. The preparation method according to claim 2 or 5, characterized in that the curing liquid is composed of the following raw materials in percentage by mass: 2-9% of citric acid, 1-6% of VP, 5-25% of phosphate, 1-5% of PVA, 0.5-0.8% of squalene and the balance of water.

7. The preparation method according to claim 2 to 6, characterized in that in step (4), the curing is performed in a constant temperature and humidity chamber at 37°C and 97% humidity, and taken out after 3 days; and the time of immersion in the ethanol solution is 2 h.