CN112158817B - Bone tissue repair material and preparation method and application thereof - Google Patents
Bone tissue repair material and preparation method and application thereof Download PDFInfo
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- CN112158817B CN112158817B CN202010962656.7A CN202010962656A CN112158817B CN 112158817 B CN112158817 B CN 112158817B CN 202010962656 A CN202010962656 A CN 202010962656A CN 112158817 B CN112158817 B CN 112158817B
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- C01B25/00—Phosphorus; Compounds thereof
- C01B25/16—Oxyacids of phosphorus; Salts thereof
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- C01B25/32—Phosphates of magnesium, calcium, strontium, or barium
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- 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
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses
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Abstract
The invention discloses a bone tissue repair material and a preparation method and application thereof, in a liquid phase system with the pH value of 10-12, soluble calcium salt and soluble phosphate or phosphoric acid with the molar ratio of Ca to P of 5:3 are subjected to in-situ coprecipitation reaction on a natural polymer A, and precipitates are dried and ground to form hydroxyapatite-natural polymer A nano composite particles; uniformly mixing the hydroxyapatite-natural polymer A nano composite particles with a solution of natural polymer B, placing the mixture in a mould for vacuum freeze drying, then soaking the mould in absolute ethyl alcohol, and drying the soaked mould to obtain the bone tissue repair material. The bone tissue repair material is loose and porous, hydroxyapatite is not easy to leak from the bone tissue repair material, the water absorption is good, the bone tissue repair material is soft after water absorption and has good elasticity, and the bone tissue repair material can be used for repairing bone defects common in the department of oral implantology, the department of oral repair, orthopedics and other surgeries; the preparation method is simple and convenient to operate, easy to automate, clean in reagents and low in cost.
Description
Technical Field
The invention relates to the field of bone tissue engineering materials, in particular to a bone tissue repair material and a preparation method and application thereof.
Background
At present, bone tissue substitute materials are hot spots in biomedical material research and application due to huge market demands. The endogenous bone tissue has good repairing effect, but the popularization and the application of the therapy are limited due to the shortage of the source of the substitute material; and the repair of the exogenous bone tissue has obvious immunological rejection reaction and may also introduce potential pathogen and other toxic side reaction. Therefore, the research and the preparation of the high-efficiency and safe artificial bionic bone tissue substitute material have great practical significance.
The natural bone tissue is a uniform and ordered biological complex composed of inorganic mineral (mainly nano-hydroxyapatite) and natural polymer (mainly collagen) which are arranged. Hydroxyapatite (HA for short) with chemical formula of Ca10(PO4)6(OH)2The chemical composition, crystal structure and physical and chemical properties of the bone collagen peptide are similar to those of inorganic components in normal bones of human, and the bone collagen peptide has good bone conductivity, biocompatibility and bioactivity. The above advantages make the hydroxyapatiteThe method has gained wide attention at home and abroad, and related researches and reports are in the tens of thousands. However, pure hydroxyapatite materials have inherent brittleness, low bending strength, extremely low toughness and extremely low degradation rate, and the application of the pure hydroxyapatite materials in bone grafting is greatly limited.
The ideal bionic bone matrix material should have good biological function, mechanical property, porosity, cell affinity, osteoinductivity and space-time matching property of material degradation disappearance and bone tissue regeneration. In order to overcome the inherent defects of hydroxyapatite, from the bionics perspective, nano hydroxyapatite is compounded with natural organic matter macromolecules to synthesize an inorganic-organic composite bionic structure, so that the biocompatibility and the mechanical property of the material are improved while the hydroxyapatite bone repairing function is maintained. Biomineralization is an effective technique for accomplishing this task, and is defined as: based on the bionic principle, biological macromolecules are used as templates, the growth of inorganic crystals is controlled through self-assembly, and a nano composite structure similar to natural bone tissues is prepared.
In the prior art, a Chinese patent 'nano hydroxyapatite/natural polymer composite material and a preparation method and application thereof' (publication No. CN101693774A) discloses a preparation method of a nano hydroxyapatite/natural polymer composite material, which is to put a natural polymer material into a mixed solution consisting of ethanol, water and urea, add a sodium dihydrogen phosphate solution and a calcium oxide solution, and then seal and react; however, the method focuses on the natural polymer material which is mineralized rapidly under mild conditions, the obtained main product is the mineralized chitosan porous sponge, and the nano hydroxyapatite covers the surface of the chitosan porous sponge and can fall off from the surface of the chitosan porous sponge when in use. Chinese patent 'mineralized silk protein material and preparation method thereof' (publication No. CN1241654A) discloses a mineralized silk protein material and preparation method thereof, wherein the organic component is degummed silk protein, and the mineral substance is calcium phosphate crystal, the specific method is that a solution containing calcium ion and a solution containing phosphate ion are added into a water-soluble degummed silk protein solution under the stirring state, the pH value of a reaction system is adjusted, the system is kept still after stirring for the same time, centrifugation and washing precipitation are repeated, and then the precipitation is dried in vacuum to obtain a product; the system has the problems of non-constant temperature, large capacity, pH deviation, most stable hydroxyapatite value and single product obtaining method. Meanwhile, in the using process, the risk that the fine mineralized silk protein particles are partially or completely separated from the bone grafting position is high.
Disclosure of Invention
In order to make up the defects in the prior art, the invention provides a bone tissue repair material and a preparation method thereof, the bone tissue repair material is a porous plastic block material, has good water absorption and water absorption softening, and hydroxyapatite particles are not easy to leak from the bone tissue repair material and can be used for repairing common bone defects of department of oral implantology, department of oral repair, orthopedics and other surgery; the preparation method is simple and convenient to operate, easy to automate, clean in reagents and low in cost.
The technical scheme provided by the invention is as follows:
a preparation method of a bone tissue repair material comprises the following steps:
(1) in a liquid phase system with the pH value of 10-12, carrying out in-situ coprecipitation reaction on soluble calcium salt and soluble phosphate or phosphoric acid with the molar ratio of Ca to P being 5:3 on a natural polymer A, and drying and grinding precipitates to form hydroxyapatite-natural polymer A nano composite particles;
(2) uniformly mixing hydroxyapatite-natural polymer A nano composite particles and natural polymer B in a liquid phase system, placing the mixture in a mould for vacuum freeze drying, and then soaking and drying the mixture by absolute ethyl alcohol to obtain a bone tissue repair material with a fixed shape;
the natural polymer A and the natural polymer B are the same or different.
In some embodiments, the hydroxyapatite content in the bone tissue repair material is 50wt% to 95 wt%.
In some embodiments, the in-situ co-precipitation reaction is carried out in a thermostatic water bath at 37-40 ℃. The reaction system is kept at a constant temperature of 37-40 ℃, so that the biological activity, nutritional ingredients and physical state of the natural polymer can be kept to the maximum extent, and the biological mineralization promotion effect can be exerted under appropriate conditions;
in some embodiments, the precipitate is soaked in deionized water and shaken, followed by centrifugation to remove the leachate, and the above steps are repeated multiple times.
In some embodiments, in step (1), the soluble calcium salt and the soluble phosphate salt are added to the liquid system in solid form, or the liquid phosphoric acid may be directly added, and the natural polymer a is added to the liquid system in solution form. The precursor salt of the hydroxyapatite is directly added in a solid form, so that the process of preparing the precursor salt solution is omitted, and the water resource is saved.
In some embodiments, natural polymer A or natural polymer B contains-OH, -NH-in its molecular chain2、-NH3-COOH.
In some embodiments, the natural polymer a or the natural polymer B is a charged natural polysaccharide or a natural protein, wherein the charged natural polysaccharide is at least one of chitin, chitosan oligosaccharide, alginic acid, cellulose, hyaluronic acid, pectin and derivatives thereof, and the charged natural protein is at least one of collagen, silk fibroin, sericin and derivatives thereof, preferably silk fibroin.
In some embodiments, the soluble calcium salt is calcium nitrate or calcium chloride; the soluble phosphate is at least one of potassium dihydrogen phosphate, potassium hydrogen phosphate, potassium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate and diammonium hydrogen phosphate. The soluble calcium salt is preferably calcium nitrate and the soluble phosphate salt is preferably potassium phosphate.
Preferably, in the step (1), the liquid phase system is adjusted to pH 10-12 by ammonia water.
In some embodiments, the collection of the product is varied, and the method for drying the precipitate includes vacuum freeze-drying technology and freshly used drying technology, wherein the hydroxyapatite-natural polymer a nanocomposite particles obtained by the drying technology have good particle size, micro-morphology and crystallinity.
In some embodiments, the precipitate is ground and then sieved to obtain hydroxyapatite-natural polymer a nanocomposite particles with different specifications.
In some embodiments, the hydroxyapatite-natural polymer a nanocomposite particles and the natural polymer B are uniformly mixed in a liquid phase system, and then placed in a mold for vacuum freeze drying, and then soaked in absolute ethyl alcohol and dried to obtain the bone tissue repair material with a fixed morphology.
The invention also provides a bone tissue repair material obtained by the preparation method, and the particle size of the hydroxyapatite-natural polymer A nano composite particles is 0.2-1.5 mm; the density of the bone tissue repair material is 0.45-0.50 g/cm3The porosity is 52-55%. The bone tissue repair material has a porous structure and good water absorption, the hydroxyapatite-natural polymer A nano composite particles are well combined with a matrix, the hydroxyapatite is not easy to leak from the bone tissue repair material, and the bone tissue repair material can be used for repairing and filling bone tissue defects of non-bearing parts such as oral cavities, limbs, jaw faces and the like and bone mass deficiency caused by excision of good and malignant tumors.
The principle of the preparation method of the bone tissue repair material provided by the invention is as follows:
the molecular chain of the natural polymer contains-OH, -NH and-NH2、-NH3Polar groups such as-COOH and the like react with calcium ions in the reaction to form a site matrix of hydroxyapatite crystallization, meanwhile, intermolecular electrostatic repulsion enables molecular chains of natural polymers to be unfolded, and calcium ions, phosphate ions and hydroxyl ions in a liquid phase system with the pH of 10-12 are further adsorbed, so that growth of hydroxyapatite crystals is promoted, the natural polymer template plays a role in regulating and controlling nucleation and growth of hydroxyapatite crystallization in the process, dispersion of in-situ synthesized nano hydroxyapatite particles in a composite organic matrix can be guaranteed, inorganic and organic compounding is realized, and a biomineralization-in-situ synthesized hydroxyapatite-natural polymer composite material with improved hardness, adjustable particle size and good appearance and crystallization form is formed; the prepared bone tissue repair material is processed into hydroxyapatite-natural polymer nano composite particles with different specifications; customizing different specifications and shapes (such as square, cylindrical or irregular shapes) according to the use requirement, and mixing the hydroxyapatite-natural polymer nano composite particles with the natural polymer solutionAfter mixing, the water is thoroughly removed by vacuum freeze drying, and the bone tissue repairing material with fixed appearance and porosity is obtained after the treatment with ethanol and drying.
Compared with the prior art, the technical scheme provided by the invention has the following advantages and beneficial effects:
the method is simple, easy to automate, quick and effective, and has mild reaction conditions, low energy consumption, low requirements on reaction equipment, and cheap and easily-obtained materials; the product can be made into structures with different shapes so as to meet the filling requirements of different wound surfaces or defects; the obtained product has certain cohesive force, the elasticity is increased after water absorption, the product is not easy to scatter after being implanted into a bone defect part, and hydroxyapatite particles can be effectively prevented from scattering or leaking.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
Fig. 1 is a scanning electron microscope image of a bone tissue repair material provided in example 1 of the present invention.
Fig. 2 is an XRD pattern of the bone tissue repair material provided in example 1 of the present invention.
Fig. 3 is a scanning electron microscope image of the bone tissue repair material provided in example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings and data in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The preparation method of the bone tissue repair material provided by the invention comprises the following steps:
(1) in a liquid phase system with the pH value of 10-12, carrying out in-situ coprecipitation reaction on soluble calcium salt and soluble phosphate (or phosphoric acid) with the molar ratio of Ca to P being 5:3 on a natural polymer A, and cleaning, drying, grinding and sieving precipitates to form hydroxyapatite-natural polymer A nano composite particles with different specifications;
(2) uniformly mixing hydroxyapatite-natural polymer A nano composite particles and natural polymer B in a liquid phase system, placing the mixture in a mould for vacuum freeze drying, and then soaking and drying the mixture by absolute ethyl alcohol to obtain a bone tissue repair material with a fixed shape;
wherein, the natural polymer A and the natural polymer B are the same or different.
The method specifically comprises the following steps:
(1) weighing natural polymer A, wherein the amount of the natural polymer A is 1.00-9.04 parts by weight, preferably 3.01-7.02 parts by weight, and more preferably 4.02-5.02 parts by weight; dissolving natural polymer A in 200 parts by volume of deionized water at 37-40 ℃ in a constant-temperature water bath, or diluting a high-concentration natural polymer A water solution at 37-40 ℃ in a constant-temperature water bath, wherein the concentration of the obtained natural polymer A water solution is 5-45 g/L, preferably 15-35 g/L, and more preferably 20.1-25.1 g/L.
(2) Putting the natural polymer A water solution into a constant-temperature water bath at 37-40 ℃, adding phosphate or phosphoric acid containing 5.7 parts by weight of phosphate radical under the stirring condition of the rotation speed of 1000-1500 rpm, wherein the phosphate is at least one selected from potassium dihydrogen phosphate, potassium hydrogen phosphate, potassium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate and diammonium hydrogen phosphate, preferably sodium dihydrogen phosphate or potassium phosphate, and more preferably potassium phosphate; after the phosphate is dissolved, adding calcium salt containing 4 parts by weight of calcium ions into the reaction system according to the molar ratio of Ca/P to 5:3, wherein the calcium salt is calcium nitrate or calcium chloride, and preferably calcium nitrate; after 5-10 minutes, adding enough ammonia water (NH) in one time3Concentration of 25-28 wt%), and ammonia water in an amount of 50-150 parts by volume, preferably 90-120 parts by volume, more preferably 100-110 parts by volumeIntegrating; and adjusting the stirring speed to 600-700 rpm, and stirring to fully react for 1-30 h, preferably 2-12 h, and more preferably 3-6 h.
(3) And stopping stirring after the reaction is finished, aging the reaction product in a reaction container at a constant temperature of 37-40 ℃ for 1-48 h, preferably 8-30 h, more preferably 12-24 h, adding ammonia water into the reaction system for multiple times at the same time interval during aging, and keeping the pH value of the system at 10-12, preferably 10.9-11.2.
(4) And after the aging is finished, removing the supernatant, transferring the precipitate into a centrifugal tube, and washing the centrifugal tube with deionized water until the washing liquid is neutral by using a pH meter.
(5) Drying the cleaned precipitate to obtain a hydroxyapatite-natural polymer A nano composite; preferably, the precipitate can be dried by two methods:
the method comprises the following steps: pouring out and spreading the precipitate on a glass watch glass, wherein the thickness of the precipitate is 0.1-2 cm, preferably 0.1-0.5 cm, more preferably 0.1-0.2 cm, drying the precipitate in an oven, wherein the temperature of the oven is set to 40 ℃, and the drying time is 24-72 hours, preferably 36-54 hours, more preferably 40-50 hours;
the second method comprises the following steps: pouring out and spreading the precipitate on a plastic surface dish, wherein the thickness of the precipitate is 0.1-2 cm, preferably 0.1-0.5 cm, more preferably 0.1-0.2 cm, pre-freezing the precipitate in a refrigerator at-20 ℃ overnight, and then transferring the precipitate into a vacuum freeze drying box for freeze drying, wherein the freeze drying time is 5-36 hours, preferably 8-18 hours, more preferably 10-12 hours.
(6) Taking out the dried hydroxyapatite-natural polymer A nano compound precipitate, primarily breaking, sequentially passing through a 10# sieve (aperture 2mm), a 18# sieve (aperture 1mm), a 35# sieve (aperture 0.5mm) and a 60# sieve (aperture 0.25mm) to distinguish three specifications of 0.25-0.5 mm, 0.5-1 mm and 1-2 mm, respectively encapsulating the hydroxyapatite-natural polymer A nano compound particles with the three specifications after each sieving, slightly grinding the bone powder particles with the specifications of more than 2mm and 1-2 mm in a mortar, continuously sieving, and circularly carrying out the operations of sieving for multiple times and grinding to obtain the hydroxyapatite-natural polymer A nano compound particles with the three specifications. The material of the sieve in the step is 304 stainless steel, the specification and the combination series of the sieve are not limited by the description, the specification of the sieve comprises all the specifications of 10-1000 meshes, and the combination of the sieves comprises any combination of all the specifications of 10-1000 meshes.
(7) Mixing the hydroxyapatite-natural polymer A nano composite particles with an aqueous solution of a natural polymer B, wherein the mass ratio of the hydroxyapatite-natural polymer A nano composite particles to the volume ratio of the aqueous solution of the natural polymer B is preferably 1g:1 mL-1 g:3 mL; preferably, the concentration of the solution of the natural polymer B is 1 to 10 weight percent; mixing the two materials, putting the mixture into customized moulds with different specifications and shapes, removing water by vacuum freeze drying, soaking the mixture in absolute ethyl alcohol for 24 hours, and drying the mixture to obtain the bone tissue repair material with fixed morphology and porosity.
The technical solutions of the present invention are further illustrated below by means of specific examples, which are intended to help the technical solutions of the present invention to be better understood, but these specific examples do not limit the scope of protection of the present invention in any way.
Example 1
(1) Weighing 75.3g of soluble silk fibroin, putting into 3L of purified water with the constant temperature of 40 ℃, and dissolving for 10min under the stirring of 150-1500 rpm to obtain a silk fibroin solution.
(2) 342.1g of trisodium phosphate dodecahydrate is weighed, the weighed trisodium phosphate dodecahydrate is put into the silk fibroin solution obtained in the step (1) at one time, the mixture is stirred at the speed of 150-1500 rpm until the solution is clear, 354.2g of calcium nitrate tetrahydrate is put into the mixture at one time, and the mixture is continuously stirred;
(3) after a large amount of milky white precipitate appeared in the reaction system, 1.5L of concentrated ammonia (NH) was added3The concentration is 25-28 wt%), the pH value of the system is ensured to be about 11, and the system is stirred for 3 hours to fully react.
(4) After the reaction is finished, the stirring is stopped, and the reaction product is aged for 20 hours in a reaction vessel at the constant temperature of 40 ℃.
(5) The aged reaction product was poured off.
(6) Firstly removing a reaction product solution, taking a centrifuge bottle, filling the reaction product into the centrifuge bottle, wherein the solution does not exceed 2/3 of the volume of the centrifuge bottle, the centrifugal speed is 5000rpm, the time is 7min, and the centrifugation is started. After centrifugation was completed, the supernatant was decanted.
(7) The precipitate was washed with purified water, centrifuged again, and washed repeatedly 6 times until the washing solution was neutral with a pH meter.
(8) Pouring the cleaned precipitate into a stainless steel tray, spreading hydroxyapatite with thickness not more than 2cm, and oven drying at 40 deg.C for 48 hr.
(9) Grinding the precipitate, sequentially passing through a 10-mesh sieve (aperture 2mm), a 18-mesh sieve (aperture 1mm), a 35-mesh sieve (aperture 0.5mm) and a 60-mesh sieve (aperture 0.25mm) to distinguish four sizes of particles of 1-2 mm, 0.5-1 mm, 0.25-0.5 mm and less than 0.25mm, and circularly grinding and sieving for multiple times until all the precipitate is completely ground into the four sizes of particles, wherein the precipitate is a hydroxyapatite-silk fibroin nano compound.
(10) Mixing the ground precipitate with a fibroin solution with the mass concentration of 5wt%, wherein the bath ratio is 1g: 2 mL; mixing, placing into a cylindrical mold with diameter of 2cm, freezing in a refrigerator at-20 deg.C, vacuum freeze drying for 48 hr to remove water, soaking in anhydrous ethanol for 24 hr, and oven drying at 40 deg.C to obtain bone tissue repairing material.
As shown in fig. 1, the bone tissue repair material prepared in example 1 was subjected to electron microscope scanning to find that: the silk fibroin forms a porous scaffold, and the hydroxyapatite is heavily surrounded, so that the hydroxyapatite is not easy to fall off or leak from the silk fibroin scaffold. The bone tissue repair material has a porosity of 55% and a density of 0.47g/cm3The aperture is 1-100 μm, and the particle size of hydroxyapatite particles wrapped in the bone tissue repair material is 0.2-1.5 mm. Fig. 2 is an XRD chart of the bone tissue repair material prepared in example 1, in which diffraction peaks around 26 °/32 °/34.1 °/39.7 °/46.8 °/49.5 °/53.2 ° are all characteristic peaks of hydroxyapatite crystals, representing crystallization of hydroxyapatite particles, confirming successful synthesis of hydroxyapatite crystals in the bone tissue repair material.
The water absorption experiment of the product shows that the weight of the bone tissue repair material after water absorption is 2.1 times of that of the dry bone tissue repair material, the dry compression strength is 35N, and the tensile strength is 10N; the wet compressive strength was 15N and the tensile strength was 5N. The result shows that the bone tissue repair material has strong water absorption and certain elasticity after absorbing water, and is a good bone tissue repair filling material.
Example 2
(1) Weighing 60.5g of soluble silk fibroin, putting into 3L of purified water with constant temperature of 37 ℃, and dissolving for 10min under stirring at 1000rpm to obtain a silk fibroin solution.
(2) 342.1g of monopotassium phosphate is added into the silk fibroin solution prepared in the step (2) at one time, the solution is stirred at the rotating speed of 500-1000 rpm until the solution is clear, 354.2g of anhydrous calcium chloride is added into the solution at one time, and the stirring reaction is continued.
(3) After a large amount of milky white precipitate appears in the reaction system, adding sodium hydroxide solution (with the concentration of 3M) in batches to ensure that the pH value of the system is about 11, and continuously stirring for 3 hours to ensure that the system is fully reacted.
(4) After the reaction is finished, the stirring is stopped, and the reaction product is aged for 16 hours in a reaction vessel at the constant temperature of 37 ℃.
(5) The aged reaction product was poured off.
(6) Firstly removing a reaction product solution, taking a centrifuge bottle, filling the reaction product into the centrifuge bottle, wherein the solution does not exceed 2/3 of the volume of the centrifuge bottle, the centrifugal speed is 5000rpm, the time is 7min, and the centrifugation is started. After centrifugation was completed, the supernatant was decanted.
(7) The precipitate was washed by starting, adding purified water to a centrifuge bottle, and then centrifuging, and washing was repeated 6 times in this manner until the washing solution was neutral by a pH meter.
(8) The precipitate was poured out and spread on a plastic petri dish with a thickness of 0.2cm, pre-frozen overnight in a refrigerator at-20 ℃ and then transferred to a vacuum freeze-drying oven for freeze-drying for 12 h.
(9) Grinding the precipitate, sequentially passing through a 10-mesh sieve (aperture 2mm), a 18-mesh sieve (aperture 1mm), a 35-mesh sieve (aperture 0.5mm) and a 60-mesh sieve (aperture 0.25mm) to distinguish four sizes of particles of 1-2 mm, 0.5-1 mm, 0.25-0.5 mm and less than 0.25mm, and circularly grinding and sieving for multiple times until all the precipitate is completely ground into the four sizes of particles, wherein the precipitate is a hydroxyapatite-silk fibroin nano compound.
(10) Mixing the ground precipitate with a fibroin solution with the mass concentration of 8 wt%, wherein the bath ratio is 1g: 2 mL; mixing, placing into a rectangular mold with width of 2cm, vacuum freeze drying at-40 deg.C for 12 hr to remove water, soaking in anhydrous ethanol for 24 hr, and oven drying at 60 deg.C to obtain bone tissue repairing material.
As shown in fig. 3, the bone tissue repair material prepared in example 2 was subjected to electron microscope scanning to find that: the silk fibroin forms a porous scaffold, and the hydroxyapatite is heavily surrounded, so that the hydroxyapatite is not easy to fall off or leak from the silk fibroin scaffold.
Product test data: the bone tissue repair material has a porosity of 54% and a density of 0.48g/cm3The aperture is 1-200 μm, and the particle diameter of hydroxyapatite particles wrapped in the bone tissue repair material is 0.2-1.5 mm.
The water absorption experiment of the product shows that the weight of the bone tissue repair material after water absorption is 2.1 times of that of the dry bone tissue repair material, the dry compression strength is 50N, and the tensile strength is 10N; the wet compressive strength was 25N and the tensile strength was 5N. The result shows that the bone tissue repair material has strong water absorption and becomes soft after absorbing water, thus being a good bone tissue repair filling material.
Example 3
The bone tissue repair material prepared in example 1 is polished to be matched with a bone defect part to be repaired, the bone tissue repair material absorbs water and becomes soft after being implanted into a body, the bone tissue repair material is completely matched with the repair part, silk fibroin starts to be rapidly degraded after about 2-3 weeks, and the proliferation rate of new bone at the bone defect part starts to be greatly increased after 2-3 weeks of bone implantation. In the early stage after bone grafting, silk fibroin provides a 'high-speed channel' for bone cell proliferation, and when the proliferation rate of bone cells in the later stage is increased, the degradation of silk fibroin can provide a large amount of amino acid and a growth space for cell growth.
Experiment results show that the hydroxyapatite-silk fibroin nanocomposite prepared in example 1 has excellent characteristics of inducing bone cell proliferation, silk fibroin fibers in a silk fibroin scaffold provide a large number of climbing bases for bone cell growth, and voids in the silk fibroin can provide sufficient proliferation space for bone cells, just like a criss-cross high-speed channel, so that the bone cell proliferation can be promoted to rapidly penetrate through the whole bone defect part.
The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. The preparation method of the bone tissue repair material is characterized by comprising the following steps:
(1) in a liquid phase system with the pH = 10-12, in-situ coprecipitation reaction is carried out on a soluble calcium salt and a soluble phosphate or phosphoric acid with the molar ratio of Ca to P being 5:3 on a natural polymer A, and precipitates are dried and ground to form hydroxyapatite-natural polymer A nano composite particles with the particle size of 10-1000 meshes;
(2) uniformly mixing hydroxyapatite-natural polymer A nano composite particles with a 1-10 wt% aqueous solution of natural polymer B, placing the mixture in a mold, performing vacuum freeze drying, soaking the mold in absolute ethyl alcohol, and drying the mold to obtain a bone tissue repair material with a fixed morphology; the ratio of the mass of the hydroxyapatite-natural polymer A nano composite particles to the volume of the water solution of the natural polymer B is 1g:1 mL-1 g:3 mL;
the natural polymer A is charged natural polysaccharide or natural protein, the charged natural polysaccharide is at least one of chitin, chitosan oligosaccharide, alginic acid, cellulose, hyaluronic acid, pectin and derivatives thereof, the charged natural protein is at least one of collagen, silk fibroin, sericin and derivatives thereof, and the natural polymer B is silk fibroin.
2. The method for producing a bone tissue repair material according to claim 1, characterized in that: the content of hydroxyapatite in the bone tissue repair material is 50-95 wt%.
3. The method for producing a bone tissue repair material according to claim 1, characterized in that: the in-situ co-precipitation reaction is carried out in a constant-temperature water bath at 37-40 ℃.
4. The method for producing a bone tissue repair material according to claim 1, characterized in that: in the step (1), soluble calcium salt and soluble phosphate are added into the liquid phase system in a solid form, and the natural polymer A is added into the liquid phase system in a solution form.
5. The method for producing a bone tissue repair material according to claim 1, characterized in that: the molecular chain of the natural polymer A contains-OH, -NH and-NH2、-NH3-COOH.
6. The method for producing a bone tissue repair material according to claim 1, characterized in that: the soluble calcium salt is calcium nitrate or calcium chloride; the soluble phosphate is at least one of potassium dihydrogen phosphate, potassium hydrogen phosphate, potassium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate and diammonium hydrogen phosphate.
7. The method for producing a bone tissue repair material according to claim 1, characterized in that: in the step (1), the pH of the liquid phase system is adjusted to 10-12 by ammonia water.
8. A bone tissue repair material characterized by: the bone tissue repair material is prepared by the preparation method of any one of claims 1 to 7, wherein the particle size of the hydroxyapatite is 0.2 to 1.5 mm;the density of the bone tissue repair material is 0.48g/cm3The porosity is 52-55%.
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| CN115054733B (en) * | 2022-07-05 | 2023-06-13 | 武汉纺织大学 | Preparation method and application of silk fibroin/hydroxyapatite composite micro-nano particle material |
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| CN105169475A (en) * | 2015-08-31 | 2015-12-23 | 中原工学院 | Preparation method of biomimetic bone material containing graphene, hydroxyapatite and fibroin |
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| GB0811542D0 (en) * | 2008-06-24 | 2008-07-30 | Knight David P | Bone repair material and a method for the preparation thereof |
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