CN113425896A - Polyamino acid microcarrier for alveolar bone defect repair and preparation method thereof - Google Patents

Polyamino acid microcarrier for alveolar bone defect repair and preparation method thereof Download PDF

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CN113425896A
CN113425896A CN202110498620.2A CN202110498620A CN113425896A CN 113425896 A CN113425896 A CN 113425896A CN 202110498620 A CN202110498620 A CN 202110498620A CN 113425896 A CN113425896 A CN 113425896A
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microcarrier
polyamino acid
alveolar bone
molar ratio
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尹静波
卜帅
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University of Shanghai for Science and Technology
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    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

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Abstract

The invention relates to a polyamino acid multifunctional microcarrier for repairing alveolar bone defects and a preparation method thereof. Compounding Hydroxyapatite (HA), hydroxyl Copper Phosphate (CP) inorganic nanoparticles and polyamino acid macromolecules (such as poly L-benzyl glutamate (PBLG), poly L-methyl glutamate (PMLG) and poly L-ethyl glutamate (PELG)) by a chemical grafting method to obtain organic-inorganic hybrid polyamino acid macromolecules, further blending the macromolecules and small molecular drug Icariin (ICA) according to a certain molar ratio, and obtaining the polyamino acid multifunctional microcarrier by a double emulsion method, wherein the size of the microcarrier is 150-300 mu m, and the ranges of the pore diameter and the porosity are respectively 25-45 mu m and 87.5% +/-3.6%. In vitro cell experiments and in vivo animal experiments show that the polyamino acid multifunctional microcarrier prepared by the invention has good osteogenesis promoting, angiogenesis promoting and antibacterial effects, and can accelerate the repair of rabbit alveolar bone defects. The invention provides a novel repair material for alveolar bone defect repair and a preparation method thereof.

Description

Polyamino acid microcarrier for alveolar bone defect repair and preparation method thereof
Technical Field
The invention relates to an alveolar bone defect repair material and a preparation method thereof, in particular to a polyamino acid microcarrier and a preparation method thereof.
Background
Periodontal disease, trauma, etc. are the major causes of alveolar bone defects and resorption. Clinically, having healthy and bone-mass adequate alveolar bone is crucial for the implantation of dental implants. To restore functional and aesthetic characteristics to the defect area, the most common methods currently used clinically include Distraction Osteogenesis (DO) and direct grafting of Bone Graft Material (BGM) to the defect site, regeneration of the lost bone tissue using guided tissue/bone regeneration (GTR/GBR) techniques.
The Distraction Osteogenesis (DO) is mainly characterized in that on the premise of reserving original bone tissues and soft tissues, the DO is fixed at two ends of the original bone through a distraction device, distraction is carried out in a required direction at a proper speed and frequency, and the characteristic that the alveolar bone has high plasticity is utilized, so that new bone tissues are generated in gaps of alveolar bone defects. However, this technique has the disadvantage of an excessively long overall repair cycle, and the patient is required to wear the retractor continuously, which, apart from affecting the aesthetics and the daily pronunciation, does not guarantee oral hygiene and is susceptible to infections.
However, it is not suitable for the irregular and small-sized alveolar bone defect Distraction Osteogenesis (DO), and the problem can be solved by directly filling the alveolar bone defect with a Bone Graft Material (BGM), and thus it is widely used in clinical practice. Bone Graft Materials (BGM) can be classified into autologous bone, allogeneic bone, xenogeneic bone, and synthetic bone substitutes according to the raw material. Autologous bone grafts are the "gold standard" for bone defect reconstruction because of their extremely strong osteogenic properties and non-immunogenic characteristics during bone healing, shaping and remodeling. However, the problems of limited supply of bone, the need for additional donor sites, etc. are significant drawbacks of autologous bone grafting. While the allograft bone graft material has osteoinductivity and osteoconductivity, its osteogenic capacity is far inferior to that of the autologous bone graft due to the lack of living cells. While heterogeneous bone graft materials are widely available, for example, Bio-Os (Geistlieh AG, Switzerland) is a bone powder material processed from calf bone, which has a highly porous structure and good osteoconductivity. Although Bio-Os are advantageous for vascular invasion and osteoblast migration, the clinical application of Bio-Os is limited by the poor osteoinductive properties of bone powder, which is found by Kbler et al, and by the lower proliferation and differentiation capacity of osteoblasts compared to other alternatives. (K ü bler A, et al, growth and promotion of human osteo plasts on differential bone graft substituents: an in vitro study [ J ]. Implant Dent,2004,13: 171-.
Blood vessels are of vital importance in the bone repair process, not only are the nutrient-transmitting organs for nutrient diffusion, cell proliferation and new bone tissue growth, but also play a key role in regulating cells and signaling molecules involved in bone regeneration. Aiming at the special environment of the oral cavity, the antibacterial property in the alveolar bone defect repairing process is also very important. Therefore, the clinically ideal alveolar bone defect repair material should have several functions: promoting bone tissue regeneration, promoting blood vessel regeneration and resisting bacteria. However, at present, no alveolar bone defect repair material with the functions integrated in clinic is reported.
Disclosure of Invention
It is an object of the present invention to overcome the problems of the prior art and to provide a polyamino acid microcarrier for alveolar bone defect repair.
The second object of the present invention is to provide a method for preparing the microcarrier.
The invention compounds Hydroxyapatite (HA), hydroxyl Copper Phosphate (CP) inorganic nano particles and polyamino acid macromolecules (such as poly L-benzyl glutamate (PBLG), poly L-methyl glutamate (PMLG) and poly L-ethyl glutamate (PELG)) by a chemical grafting method to obtain organic and inorganic hybrid polyamino acid macromolecules, the macromolecules are further blended with small molecular drug Icariin (ICA) according to a certain molar ratio, and the polyamino acid multifunctional microcarrier is obtained by a double emulsion method.
Firstly, carrying out surface amination modification on two nano particles of copper hydroxy phosphate (CP) and Hydroxyapatite (HA) to respectively initiate the ring-opening polymerization of gamma-benzyl-L-glutamic acid-N-carboxylic anhydride (BLG-NCA), L-glutamic acid methyl ester-N-carboxyl-cyclic anhydride (NCAs) and L-glutamic acid ethyl ester-N-carboxyl-cyclic anhydride to respectively obtain chemically grafted CP-g-PBLG and HA-g-PBLG; CP-g-PMLG, HA-g-PMLG, CP-g-PELG and HA-g-PELG. And then the three groups of raw materials are respectively mixed with small molecular drug Icariin (ICA) according to a certain molar ratio to prepare the polyamino acid multifunctional microcarrier by a double emulsion method.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a polyamino acid microcarrier for repairing alveolar bone defects is characterized in that the microcarrier is prepared from organic and inorganic hybridized polyamino acid macromolecules and icariin according to the weight ratio of 1: 1-1: 5 in a molar ratio; the structural formula of the organic-inorganic hybrid polyamino acid macromolecule is as follows:
Figure BDA0003055523250000021
a mixture according to a molar ratio of 1:1, wherein x is 2000-3000, y is 2500-3500, and R is: benzyl, methyl or ethyl; the structural formula of the icariin is respectively as follows:
Figure BDA0003055523250000031
the molecular weight distribution PDI of the polyamino acid macromolecules is 1.25-1.32.
The size of the microcarrier is 150-300 mu m, and the ranges of the pore diameter and the porosity are respectively 25-45 mu m and 87.5% +/-3.6%.
The preparation method of the polyamino acid microcarrier for repairing the alveolar bone defect is characterized by comprising the following specific steps:
a. ammoniation of hydroxyapatite HA and hydroxyl copper phosphate CP to obtain CP-NH2And HA-NH2Nanoparticles;
b. c, adding the CP-NH obtained in the step a2Dissolving amino acid and the mixed solution in a 1: 2-1: 10 molar ratio in a 1, 4-dioxane solution, stirring and reacting for 3-6 days at 25-50 ℃ in an inert atmosphere, settling by absolute ethyl alcohol, and performing post-treatment to obtain CP-g-polyamino acid;
c. subjecting the HA-NH obtained in the step a2Dissolving amino acid and the amino acid in a 1: 2-1: 10 molar ratio in a 1, 4-dioxane solution, stirring and reacting for 3-6 days at 25-50 ℃ in an inert atmosphere, settling by absolute ethyl alcohol, and performing post-treatment to obtain HA-g-polyamino acid;
d. dissolving the CP-g-polyamino acid obtained in the step b, the HA-g-polyamino acid obtained in the step c and icariin into dichloromethane according to a molar ratio of 1:1: 1-1: 1:5 to obtain a first mixed solution system;
e. and d, mixing 1-10 wt% of gelatin aqueous solution with the first mixed solution system obtained in the step d according to the weight ratio of gelatin: emulsifying the CP-g-polyamino acid at a molar ratio of 1: 1.2-1: 2.4 for 30-150 s to obtain a second mixed system;
f. and e, mixing 0.1-1% of polyvinyl alcohol PVA aqueous solution with the second mixed system obtained in the step e according to the weight ratio of polyvinyl alcohol: CP-g-polyamino acid 1: 0.1-1: mixing at a molar ratio of 0.5, and stirring at a rotating speed of 1000-2000 r/min until dichloromethane in a mixed system is completely volatilized;
g. and f, sieving the mixed system obtained in the step f through a sieve to obtain the polyamino acid microcarrier, then placing the obtained microcarrier in a constant-temperature shaking table at 37 ℃, changing water every 30 minutes, repeatedly washing for many times until gelatin in the microcarrier is removed completely, freezing, drying and storing to obtain the polyamino acid microcarrier for repairing the alveolar bone defect.
The fifth specific step of the ammoniation in the step a is as follows:
a. adding 3-aminopropyltriethoxysilane into 75-100 wt% ethanol water solution to prepare a solution with the concentration of 0.03-0.06 moL/L;
b. respectively dissolving 10-20 mmol of hydroxyapatite HA and copper hydroxyphosphate CP in the solution a, stirring for 3-6 hours at 25-50 ℃, adjusting the pH value of the system to 9-12 by using ammonia water, continuing to react for 3-6 hours, washing, removing supernatant, and freeze-drying to obtain CP-NH2And HA-NH2Nanoparticles.
The microcarrier of the invention is filled into the alveolar bone defect site (critical dimension: 5x6x10mm) by means of injection.
The microcarrier provided by the invention realizes integration of multiple functions (osteogenesis promotion, vascularization promotion and antibiosis) and repairs specific environments of alveolar bone defects. Wherein the polyamino acid-based material is used as a high molecular material with a main chain connected by peptide bonds, has a unique secondary structure and good performanceThe biodegradability and biocompatibility of the composite material are widely applied to the field of tissue engineering. The small molecule drugs ICA and Hydroxyapatite (HA) in the microcarrier have good osteoinductivity and osteoconductivity; and Cu in copper hydroxyphosphate in microcarrier2+It can stimulate the vascular endothelial cells to form blood vessels and simultaneously endow the microcarrier with antibacterial property. Experiments show that the polyamino acid microcarrier prepared by the invention has good effects of promoting bone formation, promoting blood vessel and resisting bacteria, and can accelerate the repair of rabbit alveolar bone defects.
Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:
1. the polyamino acid multifunctional microcarrier for repairing the alveolar bone defect has the advantage of injectability, and is used for regeneration and reconstruction of the alveolar bone;
2. according to the invention, inorganic components of copper hydroxy phosphate (CP) and Hydroxyapatite (HA) are combined with the polyamino acid component in a chemical grafting manner, so that the dispersibility of the inorganic components and the binding force with a matrix material are greatly improved;
3. the polyamino acid multifunctional microcarrier provided by the invention has multiple functions of promoting osteogenesis, promoting vascularization and resisting bacteria, and meets the specific microenvironment for repairing the alveolar bone defect in the oral cavity.
Drawings
FIG. 1(a) is a TEM photograph of CP-g-PBLG in accordance with an embodiment of the present invention.
FIG. 1(b) is a TEM photograph of HA-g-PBLG in accordance with an embodiment of the present invention.
FIG. 2(a) is a scanning electron microscope photograph of a polyamino acid multifunctional microcarrier according to an embodiment of the present invention.
FIG. 2(b) is an energy spectrum of inorganic distribution in a polyamino acid multifunctional microcarrier according to an embodiment of the present invention.
FIG. 3 is a statistical graph of the expression of alkaline phosphatase (ALP) gene in rabbit bone marrow mesenchymal stem cells (BMSCs) cultured on the polyamino acid multifunctional microcarrier for 7 days and 14 days in accordance with an embodiment of the present invention (. p < 0.05).
FIG. 4 is a statistical graph of Vascular Endothelial Growth Factor (VEGF) gene expression (. p. < 0.05) in rabbit bone marrow mesenchymal stem cells (BMSCs) after 7 days and 14 days of culture on microcarriers according to an example of the present invention.
FIG. 5(a) is a pair of bacteriostatic rings of Staphylococcus aureus in accordance with an embodiment of the present invention.
FIG. 5(b) is a graph showing the bacteriostatic rate of Staphylococcus aureus in accordance with the example of the present invention.
FIG. 6 is a micro-CT image of a one-month repair of a rabbit alveolar bone defect according to an embodiment of the present invention, wherein (a) is a blank control group; (b) is an experimental group after injection filling repair by using the composite porous microcarrier.
Detailed Description
The above-described scheme is further illustrated below with reference to specific embodiments, which are detailed below:
the first embodiment is as follows:
in this embodiment, a method for preparing CP-g-PBLG and HA-g-PBLG comprises the following steps:
a. adding 3-aminopropyltriethoxysilane into 75-100 wt% ethanol water solution to prepare the following components: 0.03-0.06 moL/L of solution;
b, performing amination modification on CP and HA, namely orienting the ethanol aqueous solution by using two single-mouth bottles, adding 0.5-3 g of CP and 0.4-4.0 g of HA into the ethanol aqueous solution respectively, stirring the mixture at the temperature of 25-50 ℃ for 3-6 hours, adjusting the pH value of the system to 9-12 by using ammonia water, continuing reacting for 3-6 hours, washing, pouring out supernatant, and freeze-drying to obtain CP-NH2And HA-NH2And (4) placing the nano particles in a vacuum oven for storage.
Preparation of CP-g-PBLG and HA-g-PBLG 0.64-3.2 g of CP-NH2Adding the mixture into a 400-600 mL 1, 4-dioxane solution of 8-16 g BLG-NCA, and adding the mixture into N2Stirring and reacting for 3-6 d at 25-50 ℃ in the atmosphere, settling through absolute ethyl alcohol, filtering, washing twice, and drying at room temperature to obtain CP-g-PBLG; adding 0.32-1.6 g of HA-NH2Adding 8-16 g of BLG-NCA in 1, 4-dioxane solution in N2Stirring and reacting for 3-6 d at 25-50 ℃ in the atmosphere, settling by absolute ethyl alcohol, and filteringFiltering, washing twice, and drying at room temperature to obtain the HA-g-PBLG.
In this embodiment, a method for preparing a multifunctional poly-amino acid microcarrier material comprises the following steps:
(1) respectively dissolving 0.3-3 mg of CP-g-PBLG, 100-300 mg of HA-g-PBLG and 0.1-0.5 mg of ICA into 10-30 mL of dichloromethane to obtain a first mixed liquid system;
(2) adding 5-10 mL of 1-10 wt% gelatin aqueous solution into the step (1), and emulsifying for 30-150 s by using an emulsifying machine to obtain a second mixed system;
(3) quickly adding the second mixed system prepared in the step (2) into 500-1000 mL of 0.1-1% polyvinyl alcohol (PVA) aqueous solution, and stirring at the rotating speed of 1000-2000 r/min until dichloromethane in the mixed system is completely volatilized;
(4) and (4) sieving the mixed system in the step (3) through a sieve with a specific size to obtain the polyamino acid multifunctional microcarrier with the size of 150-300 microns, placing the obtained microcarrier in a constant-temperature shaking table at 37 ℃, changing water every 30 minutes, repeatedly washing for multiple times until gelatin in the microcarrier is removed, freezing, drying and storing.
In this embodiment, the cell assay for the polyamino acid multifunctional microcarrier comprises the following steps:
a. and (3) disinfection of the polyamino acid multifunctional microcarrier: 0.1-0.5 g of the polyamino acid multifunctional microcarrier is sterilized by gamma ray irradiation for 6-12 h.
b. Inoculation of rabbit BMSCs in polyamino acid multifunctional microcarriers: rabbit BMSCs were assigned 1x105～1x106The cell density of the culture medium is inoculated on the sterilized polyamino acid multifunctional microcarrier, and the cell is changed every 3 days.
Detection of genes in bmscs: BMSCs cultured for 7 days and 14 days respectively are taken, and the expression of an osteogenic specific gene ALP and a vascularization specific gene VEGF in cells is detected by a PCR technology.
In this embodiment, an antibacterial assay for a multifunctional microcarrier of polyamino acid comprises the following steps:
a. preparation of a staphylococcus aureus suspension: mixing Staphylococcus aureus with liquid culture medium to obtain a mixture with bacterial concentration of 1 × 107～1x109cfu/ml staphylococcus aureus suspension.
b. And (3) detecting the bacteriostatic ring of staphylococcus aureus: placing PBLG microcarrier and polyamino acid multifunctional microcarrier on filter paper with diameter of 1cm, respectively, inverting and inoculating Staphylococcus aureus (5x 10)5～1x108cfu/ml) at 37 ℃ for 24 hours, and the size of the zone of inhibition was measured.
c. Detecting the bacteriostatic rate of staphylococcus aureus: 1ml of microcarriers was mixed with 1X107～1x109And (3) co-culturing cfu/ml of staphylococcus aureus suspension for 6-24 hours, and then measuring the OD value of the suspension.
In this embodiment, an animal experiment with the polyamino acid multifunctional microcarrier comprises the following steps:
a. establishing an alveolar bone defect model: after the second lower molar of the rabbit is pulled out, the rabbit is ground into 5x6x10mm defects by a handheld machine, and the whole process needs to be continuously washed by normal saline, so that the overhigh temperature caused by friction is avoided.
b. Filling the polyamino acid multifunctional microcarrier: 0.1-1 g of the polyamino acid multifunctional microcarrier is injected into the alveolar bone defect part together with normal saline by using an injector.
c. And (3) gum suturing: pressing gelatin sponge above the multifunctional micro-carrier filled with polyamino acid, suturing the stripped gingival tissue with absorbable surgical suture, and sterilizing the wound with iodophor and 75% ethanol after operation.
Experimental test analysis:
cell experiments on the polyamino acid multifunctional microcarriers prepared in this example: the rabbit BMSCs are respectively inoculated on the PBLG microcarrier and the polyamino acid multifunctional microcarrier, and after being respectively cultured for 7 days and 14 days, the expression of the osteogenesis specific gene ALP and the vascularization specific gene VEGF in the cells is detected by the PCR technology. The result shows that the polyamino acid multifunctional microcarrier can promote osteogenic differentiation and vascularization of the rabbit bone marrow mesenchymal stem cells in the early stage.
Bacteriostasis experiment on the composite porous microcarrier prepared in this example: placing PBLG microcarrier and polyamino acid multifunctional microcarrier on filter paper with diameter of 1cm, respectively, inverting and inoculating Staphylococcus aureus (5x 10)5cfu/ml) at 37 ℃ for 24 hours, and the size of the zone of inhibition was measured. The antibacterial rate of the polyamino acid multifunctional microcarrier is realized by mixing 1ml of microcarrier and 1x108And determining the OD value of the suspension after cfu/ml staphylococcus aureus suspension is co-cultured for 24 hours.
Animal experiments on the polyamino acid multifunctional microcarriers prepared in this example: the polyamino acid multifunctional microcarrier is injected into the defect part (defect size: 5x6x10mm) of the rabbit alveolar bone, and after being compacted by gelatin sponge, the gingiva is sutured. After 4 weeks of operation, the alveolar bone at the operation site was removed for micro-CT analysis. The results show that compared with a blank control group, the experimental group has obvious new bone formation, and the composite system is proved to have good bone regeneration capability and can accelerate the repair of alveolar bone defect.
FIG. 1 is a TEM photograph of CP-g-PBLG and HA-g-PBLG nanoparticles prepared in this example. FIGS. 1A and 1B are TEM images of CP-g-PBLG and HA-g-PBLG nanoparticles, in which CP-g-PBLG nanoparticles are spherical and have a size of 50-80 nm, and the presence of a ring of PBLG polymer is clearly visible on the periphery of the inorganic material; the HA-g-PBLG nano-particles are in a rod-shaped structure, the size is 80-100 nm, and the periphery similar to the CP-g-PBLG also obviously shows the existence of a circle of graft.
FIG. 2 is a scanning electron micrograph and an elemental distribution of the polyamino acid multifunctional microcarrier prepared in this example. FIG. 2A is a SEM of a polyamino acid multifunctional microcarrier, which shows that the microcarrier is spherical, has a size of 150-300 μm and a through pore structure, and has a pore diameter of 25-45 μm; FIG. 2B is an EDS chart showing the distribution and content of Ca, P, C and Cu elements in the multifunctional microcarrier of polyamino acid, and it can be seen that the distribution of inorganic substances in the microcarrier is very uniform.
FIG. 3 shows the results of the present example, in which BMSCs were cultured with PBLG microcarriers and polyamino acid multifunctional microcarriers for 7 days and 14 days, respectively, and the expression of alkaline phosphatase (ALP) gene in cells was detected by PCR (p < 0.05), respectively. The results show that the polyamino acid multifunctional microcarriers are capable of promoting the expression of ALP in BMSCs, i.e. the multifunctional system exhibits good bone-promoting properties.
FIG. 4 shows the results of the present example, in which the expression of Vascular Endothelial Growth Factor (VEGF) gene was detected by PCR in cells cultured with PBLG microcarriers and polyamino acid multifunctional microcarriers for 7 days and 14 days, respectively (p < 0.05). The result shows that the polyamino acid multifunctional microcarrier can promote the expression of VEGF in BMSCs, namely the multifunctional system shows good vascularization promoting performance.
FIG. 5 shows the present example in which PBLG microcarriers and polyamino acid multifunctional microcarriers were placed on 1cm diameter filter paper, inverted and inoculated with Staphylococcus aureus (5X 10)5cfu/ml) at 37 ℃ for 24 hours, and the size of the zone of inhibition was measured. The result shows that the polyamino acid multifunctional microcarrier has obvious bacteriostatic ring generation, while the control group has no bacteriostatic ring; the antibacterial rate of the polyamino acid multifunctional microcarrier is realized by mixing 1ml of microcarrier and 1x108The OD value of the suspension is measured after the cfu/ml staphylococcus aureus suspension is co-cultured for 24 hours, and the result shows that the polyamino acid multifunctional microcarrier can obviously inhibit the proliferation of bacteria, and the bacteriostasis rate reaches 56%. Therefore, the polyamino acid multifunctional microcarrier can be proved to have good antibacterial performance.
FIG. 6 is a micro-CT image of rabbit alveolar bone defect repaired by polyamino acid multifunctional microcarrier for 4 weeks in this example. Wherein FIG. 6A is a blank control group and FIG. 6B is an experimental group after injection filling repair with the composite porous microcarrier. The result shows that a large amount of new bones exist in the polyamino acid multifunctional microcarrier group, while no new bones are generated in the control group, which indicates that the polyamino acid multifunctional microcarrier has good capability of promoting alveolar bone defect repair.
The present embodiment relates to a polyamino acid multifunctional microcarrier material for repairing alveolar bone defect and a preparation method thereof. In the method, after the surface of two nano particles of copper hydroxyphosphate (CP) and Hydroxyapatite (HA) is aminated and modified, gamma-benzyl-L-glutamic acid-N-carboxylic anhydride (BLG-NCA) is initiated to carry out ring-opening polymerization, and CP-g-PBLG and HA-g-PBLG which are chemically grafted are respectively obtained and are prepared with a micromolecule drug Icariin (ICA) according to a certain molar ratio through a double emulsion method to obtain the polyamino acid multifunctional microcarrier. In vitro cell experiments and in vivo animal experiments show that the polyamino acid multifunctional microcarrier prepared in the embodiment has good osteogenesis promoting, angiogenesis promoting and antibacterial effects, and the microcarrier prepared in the embodiment can promote the repair of alveolar bone defects.
Example two:
this embodiment is substantially the same as the first embodiment, and is characterized in that:
in this example, the CP-g-PBLG and HA-g-PBLG materials were prepared in the same manner as in example one.
In this embodiment, a method for preparing a multifunctional composite porous microcarrier material includes the following steps:
(1) dissolving 0.6mg of CP-g-PBLG, 150mg of HA-g-PBLG and 0.2mg of ICA into 10-30 mL of dichloromethane to obtain a first mixed liquid system;
(2) adding 5-10 mL of 1-10 wt% gelatin aqueous solution into the step (1), and emulsifying for 30-150 s by using an emulsifying machine to obtain a second mixed system;
(3) quickly adding the second mixed system prepared in the step (2) into 500-1000 mL of 0.1-1% polyvinyl alcohol (PVA) aqueous solution, and stirring at the rotating speed of 1000-2000 r/min until dichloromethane in the mixed system is completely volatilized;
(4) and (4) sieving the mixed system in the step (3) through a sieve with a specific size to obtain the polyamino acid multifunctional microcarrier with the size of 150-300 microns, placing the obtained microcarrier in a constant-temperature shaking table at 37 ℃, changing water every 30 minutes, repeatedly washing for multiple times until gelatin in the microcarrier is removed, freezing, drying and storing.
Experimental test analysis:
the present embodiment relates to a polyamino acid multifunctional microcarrier material for repairing alveolar bone defect and a preparation method thereof. In the method, two nano particles of copper hydroxyphosphate (CP) and Hydroxyapatite (HA) are usedPerforming surface amination modification to respectively obtain two kinds of CP-NH with a large number of amino groups on the surface2And HA-NH2A nanoparticle; respectively initiating the ring-opening polymerization of gamma-benzyl-L-glutamic acid-N-carboxylic anhydride (BLG-NCA) to respectively obtain two raw materials of CP-g-PBLG and HA-g-PBLG which are chemically grafted; then the raw materials and small molecular drug Icariin (ICA) are prepared into the polyamino acid multifunctional microcarrier by a double emulsion method according to a certain molar ratio. In vitro cell experiments and in vivo animal experiments show that the polyamino acid multifunctional microcarrier prepared in the embodiment has good osteogenesis promoting, angiogenesis promoting and antibacterial effects, and the microcarrier prepared in the embodiment can promote the repair of alveolar bone defects.
Example three:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the CP-g-PBLG and HA-g-PBLG materials were prepared in the same manner as in example one.
In this embodiment, a method for preparing a multifunctional poly-amino acid microcarrier material comprises the following steps:
(1) dissolving 0.9mg of CP-g-PBLG, 200mg of HA-g-PBLG and 0.3mg of ICA into 10-30 mL of dichloromethane to obtain a first mixed solution system;
(2) adding 5-10 mL of 1-10 wt% gelatin aqueous solution into the step (1), and emulsifying for 30-150 s by using an emulsifying machine to obtain a second mixed system;
(3) quickly adding the second mixed system prepared in the step (2) into 500-1000 mL of 0.1-1% polyvinyl alcohol (PVA) aqueous solution, and stirring at the rotating speed of 1000-2000 r/min until dichloromethane in the mixed system is completely volatilized;
(4) and (4) sieving the mixed system in the step (3) through a sieve with a specific size to obtain the polyamino acid multifunctional microcarrier with the size of 150-300 microns, placing the obtained microcarrier in a constant-temperature shaking table at 37 ℃, changing water every 30 minutes, repeatedly washing for multiple times until gelatin in the microcarrier is removed, freezing, drying and storing.
Experimental test analysis:
the present embodiment relates to a polyamino acid multifunctional microcarrier material for repairing alveolar bone defect and a preparation method thereof. In the method, two kinds of nano particles of copper hydroxyphosphate (CP) and Hydroxyapatite (HA) are subjected to surface amination modification to respectively obtain two kinds of CP-NH with a large number of amino groups on the surfaces2And HA-NH2A nanoparticle; respectively initiating the ring-opening polymerization of gamma-benzyl-L-glutamic acid-N-carboxylic anhydride (BLG-NCA) to respectively obtain two raw materials of CP-g-PBLG and HA-g-PBLG which are chemically grafted; then the raw materials and small molecular drug Icariin (ICA) are prepared into the polyamino acid multifunctional microcarrier by a double emulsion method according to a certain molar ratio. In vitro cell experiments and in vivo animal experiments show that the polyamino acid multifunctional microcarrier prepared in the embodiment has good osteogenesis promoting, angiogenesis promoting and antibacterial effects, and the microcarrier prepared in the embodiment can promote the repair of alveolar bone defects.
Example four:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the CP-g-PBLG and HA-g-PBLG materials were prepared in the same manner as in example one.
In this embodiment, a method for preparing a multifunctional poly-amino acid microcarrier material comprises the following steps:
(1) dissolving 1.2mg of CP-g-PBLG, 250mg of HA-g-PBLG and 0.4mg of ICA into 10-30 mL of dichloromethane to obtain a first mixed liquid system;
(2) adding 5-10 mL of 1-10 wt% gelatin aqueous solution into the step (1), and emulsifying for 30-150 s by using an emulsifying machine to obtain a second mixed system;
(3) quickly adding the second mixed system prepared in the step (2) into 500-1000 mL of 0.1-1% polyvinyl alcohol (PVA) aqueous solution, and stirring at the rotating speed of 1000-2000 r/min until dichloromethane in the mixed system is completely volatilized;
(4) and (4) sieving the mixed system in the step (3) through a sieve with a specific size to obtain the polyamino acid multifunctional microcarrier with the size of 150-300 microns, placing the obtained microcarrier in a constant-temperature shaking table at 37 ℃, changing water every 30 minutes, repeatedly washing for multiple times until gelatin in the microcarrier is removed, freezing, drying and storing.
Experimental test analysis:
the present embodiment relates to a polyamino acid multifunctional microcarrier material for repairing alveolar bone defect and a preparation method thereof. In the method, two kinds of nano particles of copper hydroxyphosphate (CP) and Hydroxyapatite (HA) are subjected to surface amination modification to respectively obtain two kinds of CP-NH with a large number of amino groups on the surfaces2And HA-NH2A nanoparticle; respectively initiating the ring-opening polymerization of gamma-benzyl-L-glutamic acid-N-carboxylic anhydride (BLG-NCA) to respectively obtain two raw materials of CP-g-PBLG and HA-g-PBLG which are chemically grafted; then the raw materials and small molecular drug Icariin (ICA) are prepared into the polyamino acid multifunctional microcarrier by a double emulsion method according to a certain molar ratio. In vitro cell experiments and in vivo animal experiments show that the polyamino acid multifunctional microcarrier prepared in the embodiment has good osteogenesis promoting, angiogenesis promoting and antibacterial effects, and the microcarrier prepared in the embodiment can promote the repair of alveolar bone defects.
Example five:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the CP-g-PBLG and HA-g-PBLG materials were prepared in the same manner as in example one.
In this embodiment, a method for preparing a multifunctional poly-amino acid microcarrier material comprises the following steps:
(1) dissolving 1.5mg of CP-g-PBLG, 300mg of HA-g-PBLG and 0.5mg of ICA into 10-30 mL of dichloromethane to obtain a first mixed liquid system;
(2) adding 5-10 mL of 1-10 wt% gelatin aqueous solution into the step (1), and emulsifying for 30-150 s by using an emulsifying machine to obtain a second mixed system;
(3) quickly adding the second mixed system prepared in the step (2) into 500-1000 mL of 0.1-1% polyvinyl alcohol (PVA) aqueous solution, and stirring at the rotating speed of 1000-2000 r/min until dichloromethane in the mixed system is completely volatilized;
(4) and (4) sieving the mixed system in the step (3) through a sieve with a specific size to obtain the polyamino acid multifunctional microcarrier with the size of 150-300 microns, placing the obtained microcarrier in a constant-temperature shaking table at 37 ℃, changing water every 30 minutes, repeatedly washing for multiple times until gelatin in the microcarrier is removed, freezing, drying and storing.
Experimental test analysis:
the present embodiment relates to a polyamino acid multifunctional microcarrier material for repairing alveolar bone defect and a preparation method thereof. In the method, two kinds of nano particles of copper hydroxyphosphate (CP) and Hydroxyapatite (HA) are subjected to surface amination modification to respectively obtain two kinds of CP-NH with a large number of amino groups on the surfaces2And HA-NH2A nanoparticle; respectively initiating the ring-opening polymerization of gamma-benzyl-L-glutamic acid-N-carboxylic anhydride (BLG-NCA) to respectively obtain two raw materials of CP-g-PBLG and HA-g-PBLG which are chemically grafted; then the raw materials and small molecular drug Icariin (ICA) are prepared into the polyamino acid multifunctional microcarrier by a double emulsion method according to a certain molar ratio. In vitro cell experiments and in vivo animal experiments show that the polyamino acid multifunctional microcarrier prepared in the embodiment has good osteogenesis promoting, angiogenesis promoting and antibacterial effects, and the microcarrier prepared in the embodiment can promote the repair of alveolar bone defects.
In conclusion, cell experiments, antibacterial experiments and animal experiments were performed on the polyamino acid multifunctional microcarrier obtained in the first example, and it was verified that the polyamino acid multifunctional microcarrier has good osteogenic property, vascularization promotion and antibacterial property, can accelerate the repair of alveolar bone defect of rabbit, and has good bone regeneration capability. The polyamino acid multifunctional microcarrier material for repairing alveolar bone defects in the embodiment of the invention is prepared by respectively carrying out surface amination modification on two nano particles of copper hydroxyphosphate (CP) and Hydroxyapatite (HA) through 3-Aminopropyltriethoxysilane (APS) in advance to respectively obtain two kinds of CP-NH with a large number of amino groups on the surfaces2And HA-NH2A nanoparticle; respectively initiating the ring-opening polymerization of gamma-benzyl-L-glutamic acid-N-carboxylic anhydride (BLG-NCA) to respectively obtain two raw materials of CP-g-PBLG and HA-g-PBLG which are chemically grafted; reaction strip at 0-5 DEG CPreparing three raw materials of CP-g-PBLG, HA-g-PBLG and ICA according to a certain molar ratio, and preparing a compound poly-amino acid multifunctional microcarrier material by using a double-emulsion method to serve as a filling material for repairing alveolar bone defects; wherein Cu in the microcarrier2+Can promote the expression of osteogenesis specific genes (ALP) and vascularization specific genes (VEGF) in BMSCs, and has obvious bacteriostatic effect on staphylococcus aureus, so that the whole system has obvious promotion effect on the repair of alveolar bone defects.
The embodiments of the present invention have been described with reference to the accompanying drawings, but the present invention is not limited to the embodiments, and various changes and modifications can be made according to the purpose of the invention, and any changes, modifications, substitutions, combinations or simplifications made according to the spirit and principle of the technical solution of the present invention shall be equivalent substitution patterns, so long as the object of the present invention is met, and the present invention shall fall within the protection scope of the present invention as long as the technical principle and inventive concept of the polyamino acid multifunctional microcarrier material for alveolar bone defect repair and the preparation method thereof of the present invention are not departed.

Claims (5)

1. A polyamino acid microcarrier for repairing alveolar bone defects is characterized in that the microcarrier is prepared from organic and inorganic hybridized polyamino acid macromolecules and icariin according to the weight ratio of 1: 1-1: 5 in a molar ratio; the structural formula of the organic-inorganic hybrid polyamino acid macromolecule is as follows:
Figure FDA0003055523240000011
a mixture according to a molar ratio of 1:1, wherein x is 2000-3000, y is 2500-3500, and R is: benzyl, methyl or ethyl; the structural formula of the icariin is respectively as follows:
Figure FDA0003055523240000012
2. the polyamino acid microcarrier for alveolar bone defect repair according to claim 1, wherein the polyamino acid macromolecules have a molecular weight distribution PDI of 1.25 to 1.32.
3. The polyamino acid microcarrier for alveolar bone defect repair according to claim 1, wherein the microcarrier has a size of 150 to 300 μm and a pore size and porosity ranging from 25 to 45 μm and 87.5% ± 3.6%, respectively.
4. A method for preparing the polyamino acid microcarrier for alveolar bone defect repair according to claim 1, 2 or 3, comprising the specific steps of:
a. ammoniation of hydroxyapatite HA and hydroxyl copper phosphate CP to obtain CP-NH2And HA-NH2Nanoparticles;
b. c, adding the CP-NH obtained in the step a2Dissolving amino acid and the mixed solution in a 1: 2-1: 10 molar ratio in a 1, 4-dioxane solution, stirring and reacting for 3-6 days at 25-50 ℃ in an inert atmosphere, settling by absolute ethyl alcohol, and performing post-treatment to obtain CP-g-polyamino acid;
c. subjecting the HA-NH obtained in the step a2Dissolving amino acid and the amino acid in a 1: 2-1: 10 molar ratio in a 1, 4-dioxane solution, stirring and reacting for 3-6 days at 25-50 ℃ in an inert atmosphere, settling by absolute ethyl alcohol, and performing post-treatment to obtain HA-g-polyamino acid;
d. dissolving the CP-g-polyamino acid obtained in the step b, the HA-g-polyamino acid obtained in the step c and icariin into dichloromethane according to a molar ratio of 1:1: 1-1: 1:5 to obtain a first mixed solution system;
e. and d, mixing 1-10 wt% of gelatin aqueous solution with the first mixed solution system obtained in the step d according to the weight ratio of gelatin: emulsifying the CP-g-polyamino acid at a molar ratio of 1: 1.2-1: 2.4 for 30-150 s to obtain a second mixed system;
f. and e, mixing 0.1-1% of polyvinyl alcohol PVA aqueous solution with the second mixed system obtained in the step e according to the weight ratio of polyvinyl alcohol: CP-g-polyamino acid 1: 0.1-1: mixing at a molar ratio of 0.5, and stirring at a rotating speed of 1000-2000 r/min until dichloromethane in a mixed system is completely volatilized;
g. and f, sieving the mixed system obtained in the step f through a sieve to obtain the polyamino acid microcarrier, then placing the obtained microcarrier in a constant-temperature shaking table at 37 ℃, changing water every 30 minutes, repeatedly washing for many times until gelatin in the microcarrier is removed completely, freezing, drying and storing to obtain the polyamino acid microcarrier for repairing the alveolar bone defect.
5. The method according to claim 1, 2 or 3, wherein the step a, the specific step five of the ammoniation, is:
a. adding 3-aminopropyltriethoxysilane into 75-100 wt% ethanol water solution to prepare a solution with the concentration of 0.03-0.06 moL/L;
b. respectively dissolving 10-20 mmol of hydroxyapatite HA and copper hydroxyphosphate CP in the solution a, stirring for 3-6 hours at 25-50 ℃, adjusting the pH value of the system to 9-12 by using ammonia water, continuing to react for 3-6 hours, washing, removing supernatant, and freeze-drying to obtain CP-NH2And HA-NH2Nanoparticles.
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