CN114836047A - Calcium phosphate oligomer-GelMA hydrogel composite system and preparation method thereof - Google Patents

Calcium phosphate oligomer-GelMA hydrogel composite system and preparation method thereof Download PDF

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CN114836047A
CN114836047A CN202210414368.7A CN202210414368A CN114836047A CN 114836047 A CN114836047 A CN 114836047A CN 202210414368 A CN202210414368 A CN 202210414368A CN 114836047 A CN114836047 A CN 114836047A
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gelma
solution
stirring
calcium phosphate
gelatin
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陈岑
黄波
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Zhejiang Sci Tech University ZSTU
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2389/00—Characterised by the use of proteins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/32—Phosphorus-containing compounds
    • C08K2003/321—Phosphates
    • C08K2003/325—Calcium, strontium or barium phosphate

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Abstract

The invention discloses a calcium phosphate oligomer-GelMA hydrogel composite system and a preparation method thereof, wherein the preparation method comprises the following steps: 1) preparing a gelatin solution dissolved in Phosphate Buffered Saline (PBS); 2) adding Methacrylic Anhydride (MA) into the gelatin solution obtained in the step 1), and stirring at 50 ℃ to crosslink the MA and the gelatin to prepare a methacrylate gelatin solution; 3) adding PBS to stop reaction, dialyzing, and lyophilizing to obtain methacrylate gelatin (GelMA) obtained in step 2)) A precursor; 4) preparation of CaCl 2 ·2H 2 Ethanol solution of O; 5) preparing an ethanol solution of phosphoric acid, and adding the ethanol solution into the solution obtained in the step 4); 6) centrifuging and washing to obtain calcium phosphate oligomer; 7) dissolving GelMA precursor in PBS, adding the Calcium Phosphate Oligomer (CPO) obtained in the step 4), adding a photoinitiator I2959, and performing ultraviolet irradiation for crosslinking to obtain GelMA-CPO hydrogel.

Description

Calcium phosphate oligomer-GelMA hydrogel composite system and preparation method thereof
Technical Field
The invention relates to the field of materials for repairing bone defects of critical dimensions, in particular to a preparation method of an inorganic-organic composite system of calcium phosphate oligomer-GelMA hydrogel.
Background
Titanium and its alloy have good mechanical properties, corrosion resistance and biocompatibility, can provide good mechanical support for critical dimension bone defect, and are widely applied to orthopedic implant materials. However, the titanium-based implant with a solid structure has poor mechanical compatibility, is easy to generate a stress shielding effect, causes the degradation and absorption of surrounding bone tissues, seriously influences the long-term service of the implant, and becomes a research hotspot of novel orthopedic materials along with the continuous popularization and maturation of a 3D printing technology.
The 3D printing porous titanium implant is a preferred material for repairing critical dimension bone defects, the porous titanium meeting the structural support requirement of a bearing part is optimally designed by adjusting a printing angle, a pore structure and parameters, but the porous structure is easy to cause bacterial adhesion and even formation of a biological membrane, so that related infection of the implant causes implantation failure, a bioactive coating is introduced to the surface of the implant material, and certain special biological functions (such as bone conduction/induction, antibiosis and protein/drug slow release) of the bioactive coating are given to the implant material, so that the implant material is a common modification strategy of a titanium-based material, but the surface coating method is mainly used for solid titanium materials, and is not suitable for porous titanium with a complex pore structure. And the infection risk of the implantation process, the healing period required by antibiosis and osteogenesis are different among patients, and how to regulate antibiosis and promote bone become a key according to the individual difference of the patients.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a novel organic-inorganic composite system as a carrier for modifying the pore structure of porous titanium, so as to achieve the purpose of high-efficiency osseointegration and have high use value.
The preparation method of the invention comprises the following steps:
(1) adding gelatin into PBS solution, stirring at 50 deg.C until all gelatin is dissolved;
(2) dripping methacrylic anhydride into the solution in the step (1), and stirring and reacting for 2 h at 50 ℃;
(3) adding PBS, and stirring for 10 min;
(4) pouring the reaction solution in the step (3) into a dialysis bag, changing water in the morning and at night, and continuously dialyzing for 6 days;
(5) freeze-drying the dialysate in the step (4) to obtain a GelMA precursor for later use;
(6) adding CaCl 2 ·2H 2 Adding absolute ethyl alcohol into the O, and stirring until the O is completely dissolved;
(7) adding phosphoric acid into absolute ethyl alcohol;
(8) adding the solution obtained in the step (7) into the solution obtained in the step (6), and reacting and stirring for 12 hours;
(9) centrifuging the solution of the step (8) at 8000 rpm/min for 10 min;
(10) washing the centrifugal precipitate obtained in the step (9) twice by using absolute ethyl alcohol, scattering the precipitate, and adding the absolute ethyl alcohol to prepare a CPO solution of 10 mg/mL for later use;
(11) dissolving the GelMA precursor in the step (5) into PBS to obtain GelMA solution;
(12) centrifuging the CPO solution obtained in the step (10) for 10min, dispersing the centrifugate, adding the dispersed centrifugate into the step (11), and stirring for 3 h at 40 ℃;
(13) adding 0.2 wt% of I2959 photoinitiator into the calcium phosphate oligomer-GelMA prepolymerization solution obtained in the step (12), stirring for 12 h at 40 ℃, and carrying out ultraviolet irradiation for 10 min;
(14) after being placed for a certain time at room temperature, the calcium phosphate oligomer-GelMA composite hydrogel can be successfully constructed.
The invention has the beneficial effects that:
the invention forms a homogeneous GelMA-CPO organic-inorganic compound in nano scale by the crosslinking reaction of CPOs dispersed in the photocuring GelMA precursor, and the compound can sustainably release Ca 2+ The porous titanium implant material has good capability of promoting the osteogenic differentiation of MSCs, and can be delivered into a void structure as an injectable material and then photocured into gel for modifying the porous titanium implant material.
Drawings
FIG. 1 is a stress-strain plot of GelMA-CPO for different organic/inorganic ratios;
FIG. 2 is a graph showing the release of Ca2+ from GelMA-CPO in physiological saline.
Detailed Description
In order to make the present invention more easily understood, and the technical solutions and advantages thereof more clearly understood, the present invention is further described in detail below with reference to the following embodiments and the accompanying drawings. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention, and that specific experimental procedures not mentioned in the following examples are generally conducted in accordance with routine experimentation.
Example 1
Adding 10 g of gelatin into 50 ml of PBS solution, stirring at 50 ℃ until the gelatin is completely dissolved, dropwise adding 8 ml of methacrylic anhydride, stirring at 50 ℃ for reacting for 2 hours, then adding 50 ml of PBS, stirring for 10min to terminate the reaction, pouring the reaction solution into a dialysis bag, changing water in the morning and at night, continuously dialyzing for 6 days, and freeze-drying to obtain a GelMA (methacrylic acid acylated gelatin) precursor for later use; adding CaCl 2 ·2H 2 Adding absolute ethyl alcohol into the solution O, stirring until the solution O is completely dissolved to obtain a solution A, adding phosphoric acid into the absolute ethyl alcohol to obtain a solution B, adding the solution B into the solution A, reacting and stirring for 12 hours, centrifuging at 8000 rpm/min for 10min, washing the obtained centrifugal precipitate with the absolute ethyl alcohol twice, centrifuging again, scattering the precipitate, adding the absolute ethyl alcohol to prepare a CPO (calcium phosphate oligomer) solution of 10 mg/mL for later use; dissolving GelMA precursor into PBS to prepare 10% w/v GelMA solution, centrifuging 20 ml of CPO solution for 10min, adding the centrifuge into 10% w/v GelMA solution, stirring for 3 h at 40 ℃, adding 0.2 wt% I2959 photoinitiator, stirring for 12 h at 40 ℃, and irradiating for 10min by ultraviolet to successfully prepare the CPOs-GelMA hydrogel.
Example 2
Adding 10 g of gelatin into 50 ml of PBS solution, stirring at 50 ℃ until the gelatin is completely dissolved, dropwise adding 8 ml of methacrylic anhydride, stirring at 50 ℃ for reacting for 2 hours, then adding 50 ml of PBS, stirring for 10min to terminate the reaction, pouring the reaction solution into a dialysis bag, changing water in the morning and at night, continuously dialyzing for 6 days, and freeze-drying to obtain a GelMA precursor for later use; adding CaCl 2 ·2H 2 Adding absolute ethyl alcohol into O, stirring until the absolute ethyl alcohol is completely dissolved to obtain a solution A, adding phosphoric acid into the absolute ethyl alcohol to obtain a solution B, adding the solution B into the solution A, reacting and stirring for 12 hours, centrifuging at 8000 rpm/min for 10min, washing the obtained centrifugal precipitate with the absolute ethyl alcohol twice, centrifuging again, scattering the precipitate, adding the absolute ethyl alcoholPreparing CPO solution of 10 mg/mL by using ethanol for later use; dissolving GelMA precursor into PBS to prepare 10% w/v GelMA solution, centrifuging 30 ml of CPO solution for 10min, adding the centrifuge into 10% w/v GelMA solution, stirring for 3 h at 40 ℃, adding 0.2 wt% I2959 photoinitiator, stirring for 12 h at 40 ℃, and irradiating for 10min by ultraviolet to successfully prepare the CPOs-GelMA hydrogel.
Example 3
Adding 10 g of gelatin into 50 ml of PBS solution, stirring at 50 ℃ until the gelatin is completely dissolved, dropwise adding 8 ml of methacrylic anhydride, stirring at 50 ℃ for reacting for 2 hours, then adding 50 ml of PBS, stirring for 10min to terminate the reaction, pouring the reaction solution into a dialysis bag, changing water in the morning and at night, continuously dialyzing for 6 days, and freeze-drying to obtain a GelMA precursor for later use; adding CaCl 2 ·2H 2 Adding absolute ethyl alcohol into the O, stirring until the absolute ethyl alcohol is completely dissolved to obtain a solution A, adding phosphoric acid into the absolute ethyl alcohol to obtain a solution B, adding the solution B into the solution A, reacting and stirring for 12 hours, centrifuging at 8000 rpm/min for 10min, washing the obtained centrifugal precipitate with the absolute ethyl alcohol twice, centrifuging again, scattering the precipitate, and adding the absolute ethyl alcohol to prepare a CPO solution of 10 mg/mL for later use; dissolving GelMA precursor into PBS to prepare 10% w/v GelMA solution, centrifuging 40 ml of CPO solution for 10min, adding the centrifuge into 10% w/v GelMA solution, stirring at 40 ℃ for 3 h, adding 0.2 wt% I2959 photoinitiator, stirring at 40 ℃ for 12 h, and performing ultraviolet irradiation for 10min to obtain the CPOs-GelMA hydrogel.
Example 4
Adding 10 g of gelatin into 50 ml of PBS solution, stirring at 50 ℃ until the gelatin is completely dissolved, dropwise adding 8 ml of methacrylic anhydride, stirring at 50 ℃ for reacting for 2 hours, then adding 50 ml of PBS, stirring for 10min to terminate the reaction, pouring the reaction solution into a dialysis bag, changing water in the morning and at night, continuously dialyzing for 6 days, and freeze-drying to obtain a GelMA precursor for later use; adding CaCl 2 ·2H 2 Adding absolute ethyl alcohol into the O, stirring until the absolute ethyl alcohol is completely dissolved to obtain a solution A, adding phosphoric acid into the absolute ethyl alcohol to obtain a solution B, adding the solution B into the solution A, reacting and stirring for 12 hours, centrifuging at 8000 rpm/min for 10min, washing the obtained centrifugal precipitate with the absolute ethyl alcohol twice, centrifuging again, scattering the precipitate, and adding the absolute ethyl alcohol to prepare a CPO solution of 10 mg/mL for later use; dissolving GelMA precursor in PBS to obtain 15% w/v GelMA solution, centrifuging 20 ml CPO solution for 10min, centrifugingAdding 10% w/v GelMA solution, stirring at 40 deg.C for 3 hr, adding 0.2 wt% I2959 photoinitiator, stirring at 40 deg.C for 12 hr, and ultraviolet irradiating for 10min to obtain CPOs-GelMA hydrogel.
Example 5
Adding 10 g of gelatin into 50 ml of PBS solution, stirring at 50 ℃ until the gelatin is completely dissolved, dropwise adding 8 ml of methacrylic anhydride, stirring at 50 ℃ for reacting for 2 hours, then adding 50 ml of PBS, stirring for 10min to terminate the reaction, pouring the reaction solution into a dialysis bag, changing water in the morning and at night, continuously dialyzing for 6 days, and freeze-drying to obtain a GelMA precursor for later use; adding CaCl 2 ·2H 2 Adding absolute ethyl alcohol into the O, stirring until the absolute ethyl alcohol is completely dissolved to obtain a solution A, adding phosphoric acid into the absolute ethyl alcohol to obtain a solution B, adding the solution B into the solution A, reacting and stirring for 12 hours, centrifuging at 8000 rpm/min for 10min, washing the obtained centrifugal precipitate with the absolute ethyl alcohol twice, centrifuging again, scattering the precipitate, and adding the absolute ethyl alcohol to prepare a CPO solution of 10 mg/mL for later use; dissolving the GelMA precursor into PBS to prepare 15% w/v GelMA solution, centrifuging 30 ml of CPO solution for 10min, adding the centrifuge into 10% w/v GelMA solution, stirring for 3 h at 40 ℃, adding 0.2 wt% I2959 photoinitiator, stirring for 12 h at 40 ℃, and irradiating by ultraviolet for 10min to successfully prepare the CPOs-GelMA hydrogel.
Example 6
Adding 10 g of gelatin into 50 ml of PBS solution, stirring at 50 ℃ until the gelatin is completely dissolved, dropwise adding 8 ml of methacrylic anhydride, stirring at 50 ℃ for reacting for 2 hours, then adding 50 ml of PBS, stirring for 10min to terminate the reaction, pouring the reaction solution into a dialysis bag, changing water in the morning and at night, continuously dialyzing for 6 days, and freeze-drying to obtain a GelMA precursor for later use; adding CaCl 2 ·2H 2 Adding absolute ethyl alcohol into the O, stirring until the absolute ethyl alcohol is completely dissolved to obtain a solution A, adding phosphoric acid into the absolute ethyl alcohol to obtain a solution B, adding the solution B into the solution A, reacting and stirring for 12 hours, centrifuging at 8000 rpm/min for 10min, washing the obtained centrifugal precipitate with the absolute ethyl alcohol twice, centrifuging again, scattering the precipitate, and adding the absolute ethyl alcohol to prepare a CPO solution of 10 mg/mL for later use; dissolving GelMA precursor into PBS to prepare 15% w/v GelMA solution, centrifuging 40 ml CPO solution for 10min, adding 10% w/v GelMA solution into the centrifuge, stirring at 40 ℃ for 3 h, adding 0.2 wt% I2959 photoinitiator, stirring at 40 ℃ for 12 h, and ultraviolet irradiating for 10min to successfully prepare CPOsGelMA hydrogel.

Claims (6)

1. A calcium phosphate oligomer-GelMA hydrogel composite system, comprising: the hydrogel composite system has good mechanical property and can continuously and slowly release Ca 2+ The Mesenchymal Stem Cells (MSCs) are degradable and have good osteogenic differentiation capacity.
2. The method for preparing a calcium phosphate oligomer-GelMA hydrogel composite system according to claim 1, comprising the steps of:
(1) adding gelatin into PBS solution, stirring at 50 deg.C until all gelatin is dissolved;
(2) dripping methacrylic anhydride into the solution in the step (1), and stirring and reacting for 2 h at 50 ℃;
(3) adding PBS, and stirring for 10 min;
(4) pouring the reaction solution in the step (3) into a dialysis bag, changing water in the morning and at night, and continuously dialyzing for 6 days;
(5) freeze-drying the dialysate in the step (4) to obtain a GelMA precursor for later use;
(6) adding CaCl 2 ·2H 2 Adding absolute ethyl alcohol into the O, and stirring until the O is completely dissolved;
(7) adding phosphoric acid into absolute ethyl alcohol;
(8) adding the solution obtained in the step (7) into the solution obtained in the step (6), and stirring for 12 hours;
(9) centrifuging the solution in the step (8) at 8000 rpm/min for 10 min;
(10) washing the centrifugal precipitate obtained in the step (9) with absolute ethyl alcohol twice;
(11) dissolving the GelMA precursor in the step (5) into PBS to obtain GelMA solution;
(12) dispersing the centrifugate obtained in the step (10), adding the dispersed centrifugate into the step (11), and stirring for 3 hours at 40 ℃;
(13) adding 0.2 wt% of I2959 photoinitiator into the calcium phosphate oligomer-GelMA prepolymerization solution obtained in the step (12), stirring for 12 h at 40 ℃, and carrying out ultraviolet irradiation for 10 min;
(14) after being placed for a certain time at room temperature, the calcium phosphate oligomer-GelMA composite hydrogel can be successfully constructed.
3. The method for preparing a calcium phosphate oligomer-GelMA hydrogel composite system according to claim 2, wherein the gelatin is type A gelatin derived from pig skin in each of the steps (1) to (14).
4. The method for preparing a calcium phosphate oligomer-GelMA hydrogel composite system according to claim 2, wherein the concentration of the methacrylic anhydride solution in the step (2) is 94% and the stirring speed is 300 rpm.
5. The method for preparing a calcium phosphate oligomer-GelMA hydrogel composite system according to claim 2, wherein in the step (4), the cut-off molecular weight of the dialysis bag is 12 kD.
6. The method for preparing a calcium phosphate oligomer-GelMA hydrogel composite system according to claim 2, wherein the GelMA solution is prepared at a concentration of 10% and 15% w/v in the step (11).
CN202210414368.7A 2022-04-20 2022-04-20 Calcium phosphate oligomer-GelMA hydrogel composite system and preparation method thereof Pending CN114836047A (en)

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CN115887780A (en) * 2022-09-30 2023-04-04 南充市中心医院 Oxygen sustained-release hydrogel and preparation method and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115570859A (en) * 2022-09-14 2023-01-06 浙江大学 Recyclable high-toughness composite hydrogel and preparation method and application thereof
CN115887780A (en) * 2022-09-30 2023-04-04 南充市中心医院 Oxygen sustained-release hydrogel and preparation method and application thereof

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