CN114767927B

CN114767927B - Silicon/zinc ion doped biphasic calcium phosphate ceramic bracket and preparation method thereof

Info

Publication number: CN114767927B
Application number: CN202210342293.6A
Authority: CN
Inventors: 叶建东; 樊家佳; 陆特良
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2023-07-18
Anticipated expiration: 2042-04-02
Also published as: CN114767927A

Abstract

The invention discloses a silicon/zinc ion doped biphasic calcium phosphate ceramic bracket and a preparation method thereof, wherein the silicon ion doped hydroxyapatite powder and the zinc ion doped beta-tricalcium phosphate powder are mixed, a silicon/zinc ion doped biphasic calcium phosphate ceramic bracket blank is prepared through 3D printing, and then high-temperature sintering is carried out to obtain the silicon/zinc ion doped biphasic calcium phosphate ceramic bracket; on the basis, synthesizing a magnesium-doped calcium silicate precursor, preparing slurry, impregnating the slurry into a stent, coating the stent surface by permeation, repeatedly coating and drying, then performing heat treatment, forming a magnesium-doped calcium silicate surface layer on the stent surface, and finally obtaining the surface-modified silicon/zinc ion-doped biphasic calcium phosphate ceramic stent. The preparation method has the advantages of simple preparation process, adjustable doping amount and release amount of ions, synergism of various active ions to promote bone and vascularization and the like, and has important significance in expanding the clinical application of the calcium phosphate ceramic stent.

Description

Silicon/zinc ion doped biphasic calcium phosphate ceramic bracket and preparation method thereof

Technical Field

The invention relates to the technical field of bone injury repair medical materials, in particular to a silicon/zinc ion doped biphasic calcium phosphate ceramic bracket, a surface modified silicon/zinc ion doped biphasic calcium phosphate ceramic bracket and a preparation method thereof.

Background

The main inorganic component of the bone tissue is hydroxyapatite (HA; the Ca/P molar ratio of HA is 1.67), and the artificially synthesized hydroxyapatite HAs excellent biocompatibility, good bone conductivity and osseointegration, HAs higher mechanical property, but low biodegradability, is unfavorable for regeneration and repair of bone defect, and limits the wide application in the field of bone repair. Tricalcium phosphate (tricalcium phosphate, TCP; the Ca/P molar ratio of TCP is 1.5) HAs a chemical composition similar to that of bone mineral, HAs excellent biocompatibility, good bone conductivity and osseointegration, HAs high biodegradability, can realize regeneration and repair of bone defect, HAs been clinically standardized to be applied to repair of bone defect, but HAs mechanical properties inferior to those of HA ceramic. The biphase calcium phosphate (biphasic calcium phosphate, BCP) is formed by compounding HA and TCP, the degradability and the bioactivity can be regulated and controlled, and the mechanical property of the BCP ceramic is superior to that of the TCP ceramic. TCP has two crystal structures: α -TCP and β -TCP BCP is typically composed of HA and β -TCP because β -TCP HAs better chemical stability than α -TCP. BCP can be chemically synthesized directly or composited with synthesized HA and β -TCP. While BCP can combine the advantages of HA and beta-TCP, its lack of osteoinductive and angiogenic properties results in inadequate bone repair capacity (Boulter J M, pilet P, gauthier O, verron E. Biphsic calcium phosphate ceramics for bone reconstruction: A review of biological response [ J ]. Acta Biomaterialia,2017,53,1-12.).

Magnesium, zinc, silicon, strontium, etc. are microscale elements in inorganic minerals of natural bone that play an important role in biochemical reactions that are closely related to bone development and metabolism (Dorozhkin S V, epple M. Biological and medical significance of calcium phosphates [ J ]. Angewandte Chemie-International Edition,2002,41 (17): 3130-3146.). Zinc and strontium ion doping of calcium phosphate materials primarily contribute to bone, magnesium ion doping primarily contributes to hematopoiesis, and silicon ion doping simultaneously contributes to the combined action of bone and vascularization (Bose S, fielding G, tarafder S, et al Understanding of dopant-induced osteogenesis and angiogenesis in calcium phosphate ceramics J Trends in Biotechnology,2013,31 (10): 594-605.). The effect of zinc ion doping on bone is strong, silicon ion doping has good effect on bone and blood vessel formation, but the effect of unit ion doping is still not good enough, and active ions doped in the calcium phosphate ceramic matrix are released relatively slowly, so that early vascularization of the bracket is difficult to realize. The early vascularization is an important condition for repairing bone defects, and the blood vessel not only can bring oxygen and nutrient substances to surrounding cells and remove metabolic wastes, but also can convey cells involved in repairing to the bone defect position, so that the early vascularization is promoted to be more beneficial to accelerating the repairing process of the bone defects.

CN107441558A discloses a porous SiO for bone tissue engineering ₂ The composite support of biphasic calcium phosphate and the preparation method thereof, but the effect of promoting bones and vascularization of composite silicon oxide is inferior to that of silicon doping and silicon-zinc co-doping, and the preparation of the support by adopting polyurethane organic foam pore-forming is inferior to that of the support by 3D printing in terms of pore connectivity and mechanical properties. CN110613863a discloses a porous scaffold material for promoting vascularization based on silicon-doped hydroxyapatite, a preparation method and application thereof, calcium, phosphorus and silicon source solution are added into polyvinyl alcohol solution, and a small amount (2-5 wt.%) of silicon-doped hydroxyapatite is generated through chemical reaction to prepare the porous scaffold material for promoting vascularization of silicon-doped hydroxyapatite, namely vascularization is promoted by compounding a small amount of silicon-doped hydroxyapatite, however, the content of silicon-doped hydroxyapatite is small and the degradability is low, so that the ion release is very limited, the vascularization promoting effect is difficult to be remarkable, and the matrix material of the scaffold is polyvinyl alcohol, so that the osteogenic performance is lacking. CN107412855a discloses a 3D printing stent with a coating and a preparation method thereof, wherein the dopamine surface modified polybutylene succinate stent is prepared by impregnating a dopamine solution after 3D printing the polybutylene succinate stent, and the surface modified stent is coated with mesoporous magnesium silicate microspheres or copper-doped mesoporous magnesium silicate microspheres Mesoporous magnesium silicate microspheres or copper-doped mesoporous magnesium silicate microsphere coatings, however, the stent matrix is an artificially synthesized organic polymer, the bioactivity and the osteogenesis performance are lacked, and the mesoporous magnesium silicate microspheres or copper-doped mesoporous magnesium silicate microspheres are coated on the surface of a dopamine membrane, but the magnesium silicate microspheres and the dopamine membrane cannot be well combined, and especially the dopamine membrane and the microspheres are easy to fall off when the microspheres start to degrade. CN109793923B discloses a preparation method of a nano-structured calcium borosilicate biological coating, which comprises spraying calcium silicate powder on the surface of a titanium alloy material by plasma, then immersing in sodium borate solution to generate hydrothermal reaction, thus obtaining the calcium borosilicate biological coating, wherein the coating has the functions of promoting bone differentiation and anti-inflammatory function, but the coating is easy to crack and peel along with the extension of time in the application process due to the large difference between the thermal expansion coefficient and elastic modulus of the calcium borosilicate and the titanium alloy. For the regeneration and repair of bone injury, the calcium phosphate scaffold plays an important role in promoting bone and vascularization, and early vascularization is a key for realizing rapid bone formation, so that a preparation technology for improving early vascularization of the calcium phosphate scaffold based on a pro-vascularization active ion doped surface modification layer, which is simple and convenient in process and universal, is necessary to develop. At present, a silicon and zinc double-ion doped biphasic calcium phosphate ceramic bracket and a preparation method thereof are not reported.

Disclosure of Invention

The first aim of the invention is to overcome the defects and shortcomings of the prior art and provide a silicon/zinc ion doped biphasic calcium phosphate ceramic stent and a preparation method thereof.

The second object of the invention is to provide a surface modified silicon/zinc ion doped biphasic calcium phosphate ceramic stent and a preparation method thereof.

The first object of the invention is achieved by the following technical scheme: the preparation method of the silicon/zinc ion doped biphasic calcium phosphate ceramic bracket comprises the following steps:

1) Respectively dissolving a calcium source and a phosphorus source in deionized water to form a calcium source solution A1 and a phosphorus source solution A2, dissolving a silicon source in absolute ethyl alcohol to form a silicon source solution A3, uniformly mixing the silicon source solution A3 and the phosphorus source solution A2, dripping the silicon source solution A3 into the calcium source solution A1 to obtain a reaction solution A, adjusting the pH value of the reaction solution A, and sequentially stirring, carrying out hydrothermal treatment, centrifuging to obtain a precipitate, washing, centrifuging, drying and calcining to obtain silicon-doped hydroxyapatite powder; dissolving a calcium source and a zinc source in water to obtain a calcium/zinc source mixed solution B1, dissolving a phosphorus source in water to obtain a phosphorus source solution B2, dripping the phosphorus source solution B2 into the calcium/zinc source mixed solution B1 to obtain a reaction solution B, adjusting the pH value of the reaction solution B, and then sequentially stirring, ageing, centrifuging to obtain a precipitate, washing, centrifuging, drying and calcining to obtain zinc-doped beta-tricalcium phosphate powder;

2) Uniformly mixing the silicon-doped hydroxyapatite powder obtained in the step 1) with zinc-doped beta-calcium phosphate powder to obtain silicon/zinc ion-doped biphasic calcium phosphate mixed powder;

3) Fully and uniformly mixing the silicon/zinc ion doped biphasic calcium phosphate mixed powder obtained in the step 2) with a thickener, dripping a binder, fully stirring to prepare printing slurry, 3D printing and forming, and drying to obtain a silicon/zinc ion doped biphasic calcium phosphate ceramic bracket blank;

4) And 3) sintering the silicon/zinc ion doped biphasic calcium phosphate ceramic bracket blank obtained in the step 3) at high temperature to obtain the silicon/zinc ion doped biphasic calcium phosphate ceramic bracket.

Preferably, there is at least one of the following a-f:

a. the calcium source of the synthesized silicon doped hydroxyapatite powder is calcium nitrate, the phosphorus source is one or a combination of a plurality of diammonium hydrogen phosphate, monoammonium hydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate, and the silicon source is tetraethoxysilane;

b. the molecular formula of the silicon-doped hydroxyapatite powder is Ca ₁₀ (PO ₄ ) _6-x (SiO ₄ ) _x (OH) _2-x Wherein the Ca/(P+Si) molar ratio is 1.67 and the Si/(Si+P) molar ratio is 0.02 to 0.04;

c. the calcium source of the synthesized zinc doped beta-tricalcium phosphate powder is calcium nitrate, the zinc source is zinc nitrate, and the phosphorus source is one or a combination of a plurality of diammonium hydrogen phosphate, monoammonium phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate;

d. The (Zn+Ca)/P molar ratio of the zinc doped beta-tricalcium phosphate powder is 1.5, wherein the Zn/(Zn+Ca) molar ratio is 0.015-0.03;

e. the mass percentage of the mixed powder of the silicon doped hydroxyapatite powder and the zinc doped beta-calcium phosphate powder is 20-40:60-80; f. the thickener is methyl cellulose, and the binder is polyvinyl alcohol.

The inventor finds that silicon doping can destroy the stability of hydroxyapatite to enable the hydroxyapatite to be easier to decompose and produce alpha-TCP, and the alpha-TCP is degraded too quickly to provide a stable environment for the initial adhesion of stem cells, which is unfavorable for the subsequent proliferation and osteogenic differentiation of the stem cells, and meanwhile, the material degraded too quickly cannot provide mechanical support for bone defect tissues at the initial stage of implantation, and the degradation space has poor matching with new bone formation. The invention adopts a preparation method combining a chemical precipitation method with hydrothermal treatment, and further optimizes the technological parameter range, so that the problem of alpha-TCP generated by decomposing hydroxyapatite can be solved, and silicon doped hydroxyapatite with single phase is obtained. Zinc doping can improve the osteogenesis performance of the calcium phosphate material, but zinc doping reduces the degradation performance of the calcium phosphate material; the silicon doping can not only improve the bioactivity and the angiopoiesis performance of the calcium phosphate stent, but also accelerate the degradation speed of the calcium phosphate material. Therefore, the silicon and zinc double ion doping can lead the calcium phosphate material to have the degradation speed matched with the bone defect repair process, and simultaneously has excellent osteogenesis and vascularization performances. In addition, too high an ion doping amount causes cytotoxicity and reduces osteogenic/vasogenic activity; the amount of the dopant is too low, and the ability to promote bone and hematopoiesis is insufficient, so that the above-mentioned suitable amount range of the dopant is preferable.

The inventor finds that the two phases of the biphasic calcium phosphate have influence on the osteoinductive performance and the degradation performance of the material, if the content of the beta-tricalcium phosphate is too high, the degradation speed of the biphasic calcium phosphate is too high, and the material is unstable and is not beneficial to the formation of new bones; if the content of hydroxyapatite is too high, the degradation rate of biphasic calcium phosphate is low, which is unfavorable for the growth of new bone, so that the ratio of the two phases is preferably in a proper range.

The inventor finds that aiming at vascularization, the pore diameter of the bracket is too small to be beneficial to vascularization, the pore diameter reaches more than 300 mu m to be beneficial to vascularization, so that the pore diameter of the printed bracket exceeds 300 mu m after sintering when the pore diameter reaches 400 mu m, the pore diameter is further improved to be more beneficial to the vascularization of early pore channels of the surface modification of the magnesium-doped calcium silicate, but the pore diameter is too large to reduce the mechanical property of the bracket, and the pore diameter is not beneficial to the filling of osteoblasts into the pore channels, so that a proper pore diameter range is needed.

Preferably, in the step 1), the pH value of the reaction solution A for synthesizing the silicon doped hydroxyapatite powder is 10.5-11, the stirring speed is 400-600r/min, the stirring time is 40-60min, the hydrothermal treatment temperature is 140-180 ℃, the hydrothermal treatment time is 16-24h, the centrifugal speed of the solution after the reaction is 2000-4000r/min, the washing solution is deionized water, the stirring speed in the washing process is 400-600r/min, the stirring time is 10-20 min, the centrifugal speed of the solution after the washing is 4000-5000r/min, the drying temperature is 40-70 ℃, the drying time is 18-24h, the calcining temperature is 850-950 ℃ and the heat preservation time is 2-3h;

The pH value of the reaction solution B for synthesizing the zinc doped beta-tricalcium phosphate powder is 6.4-6.8, the stirring speed is 400-600r/min, the stirring time is 40-60min, the aging time is 12-24h, the centrifugal speed of the solution after reaction is 2000-4000r/min, the washing liquid is deionized water, the stirring speed in the washing process is 400-600r/min, the stirring time is 10-20min, the centrifugal speed of the solution after washing is 4000-5000r/min, the drying temperature is 40-70 ℃, the drying time is 18-24h, the calcining temperature is 850-950 ℃ and the heat preservation time is 2-3 h.

Preferably, in the step 4), the sintering temperature of the silicon/zinc ion doped biphasic calcium phosphate ceramic support blank is 1050-1150 ℃, the heating rate is 2-10 ℃/min, and the heat preservation time is 2-4h.

The invention provides a silicon/zinc ion doped biphasic calcium phosphate ceramic stent prepared by the method, which can be applied to preparing a bone injury repair medical material.

The second object of the invention is achieved by the following technical scheme: the method for preparing the surface modified silicon/zinc ion doped biphasic calcium phosphate ceramic bracket by using the silicon/zinc ion doped biphasic calcium phosphate ceramic bracket comprises the following steps:

dissolving a calcium source and a magnesium source in deionized water to obtain a calcium/magnesium source mixed solution C1, dissolving a silicon source in deionized water to obtain a silicon source solution C2, dropwise adding the calcium/magnesium source mixed solution C1 into the silicon source solution C2 to obtain a reaction solution C, and sequentially stirring, centrifuging, washing, centrifuging to obtain a precipitate, adding deionized water, adding a dispersing agent and stirring to obtain magnesium-doped calcium silicate precursor slurry;

Placing the silicon/zinc ion doped biphase calcium phosphate ceramic stent in magnesium doped calcium silicate precursor slurry for dipping, performing negative pressure lower surface infiltration coating, then drying, and performing repeated dipping infiltration coating to form a magnesium doped calcium silicate precursor surface layer, thereby obtaining the silicon/zinc ion doped biphase calcium phosphate ceramic stent with surface infiltration coating;

and carrying out heat treatment on the surface-permeation-coated doped biphasic calcium phosphate ceramic stent to obtain the surface-modified silicon/zinc ion-doped biphasic calcium phosphate ceramic stent.

Preferably, there is at least one of the following a, b:

a. the calcium source is calcium nitrate, the silicon source is sodium silicate, and the magnesium source is magnesium nitrate;

b. the Mg/(Mg+Ca) molar ratio of the magnesium-doped calcium silicate precursor slurry is 0.05-0.12.

Preferably, the stirring rate of the reaction solution C for preparing the magnesium-doped calcium silicate precursor slurry is 300-600r/min, the stirring time is 15-45min, the centrifugal rate is 2000-4000r/min, the stirring rate in the washing process is 300-600r/min, the stirring time is 5-10min, the centrifugal rate of the washed solution is 3000-4000 r/min, the washing solution and the slurry solution are deionized water, the dispersing agent is one or a combination of more of polyethylene glycol, polyacrylic acid and polymethacrylate ammonia, and the concentration of the dispersing agent is 2-6g/L;

The concentration of the magnesium-doped calcium silicate in the magnesium-doped calcium silicate precursor slurry is 0.4-0.6mol/L; the negative pressure range of the magnesium doped calcium silicate precursor slurry impregnated silicon/zinc ion doped biphasic calcium phosphate ceramic bracket is-0.05 to-0.25 MPa, the impregnation time is 10-30s, the times of impregnation and infiltration coating are 1-3, the drying temperature is 40-60 ℃, and the drying time is 30-60min; the final thickness of the surface layer of the magnesium doped calcium silicate precursor formed by dip-coating is 5-20 mu m.

Preferably, the heat treatment temperature of the surface-permeation-coated silicon/zinc ion-doped biphasic calcium phosphate ceramic bracket is 850-950 ℃, the heating rate is 5-10 ℃/min, and the heat preservation time is 1-3h.

The inventors found that the release of active ions from the doped biphasic calcium phosphate matrix was relatively slow and the promotion of early vascularization of the scaffold was not significant enough. The calcium silicate has faster ion release speed, can release silicon ions, and the magnesium ion doped calcium silicate can release silicon ions and magnesium ions simultaneously, so that the vascularization promoting performance is synergistically improved. Therefore, the magnesium-doped calcium silicate with higher ion release speed is used as the surface modification material of the silicon/zinc ion-doped biphasic calcium phosphate ceramic stent with lower ion release speed, which is beneficial to the release of active ions for promoting vascularization in the early stage of the stent and further promotes the initial vascularization of the stent. And the long-term slow release of the bracket matrix to the silicon and zinc ions is favorable for the later generation and growth of new bones. The regeneration and repair of bone defects can be accelerated and realized by proper time-series release of active ions by the surface layer and the bracket matrix.

The inventor finds that the ion doping amount of the magnesium doped calcium silicate has a certain influence on the early-stage mediated endothelial cell vascularization behavior and the coating stability of the material, and as the radius of magnesium ions is smaller than that of calcium ions, the excessive magnesium doping amount can damage the stability of the crystal structure of the calcium silicate, so that the release amount of magnesium ions in the early stage is excessive, and the concentration of magnesium ions in a cell culture solution is excessive, thereby inhibiting the endothelial cells from expressing vascularization related genes and even generating cytotoxicity; the above-mentioned proper amount range is preferable because the effect of promoting the expression of the gene related to angiogenesis is not remarkable when the amount of magnesium to be doped is too low.

The inventors have found that the conditions of the osmotic coating have a certain influence on the performance of the calcium phosphate stent. The negative pressure can enable the magnesium doped calcium silicate precursor slurry to permeate into pores on the surface of the calcium phosphate bracket, so that the surface coating layer and the matrix are combined more tightly, and the mechanical strength of the bracket is improved. If the negative pressure is too low, the slurry is not beneficial to permeate into the pores on the surface of the bracket; if the negative pressure is too high, the negative pressure permeates into pores on the surface of the bracket too deeply, and the degradation of the bracket matrix is affected. The thickness of the coated surface layer depends on the dipping time and the dipping times, and when the thickness of the surface layer is too low, active ions for promoting vascularization are released too little, and the sustained release time is short, so that the initial vascularization is not sufficiently stimulated; when the thickness of the surface layer is too high, the time required for degradation of the surface layer is prolonged, which is unfavorable for releasing zinc ions by the stent matrix, so that the generation and growth of subsequent new bones are limited, and the bone repair efficiency is reduced.

In addition, the heat treatment temperature has a certain influence on the stability and the biological activity of the magnesium-doped calcium silicate surface layer. If the heat treatment temperature is too low, the surface modification layer can not form chemical bonding with the bracket matrix, and the falling-off phenomenon is easy to occur; if the heat treatment temperature is too high, the modified layer is too compact, so that release of active ions for promoting vascularization is slowed down, and the vascularization performance of the stent is not improved.

The invention provides a surface modified silicon/zinc ion doped biphasic calcium phosphate ceramic bracket prepared by the method, which can be applied to preparing bone injury repair medical materials.

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

1. the silicon-doped hydroxyapatite and the zinc-doped beta-tricalcium phosphate are compounded to prepare the silicon-zinc ion-doped biphasic calcium phosphate ceramic bracket, wherein zinc doping has the effect of promoting bone and vascularization, and compared with an undoped biphasic calcium phosphate ceramic bracket, the silicon-doped biphasic calcium phosphate ceramic bracket has superior bone and vascularization promoting performance; the release amount of silicon and zinc active ions and the degradation performance of the bracket can be regulated and controlled by regulating and controlling the proportion of silicon-doped hydroxyapatite and zinc-doped beta-tricalcium phosphate.

2. Compared with a single-ion-doped biphasic calcium phosphate ceramic bracket, the silicon-zinc double-ion-doped biphasic calcium phosphate ceramic bracket prepared by the invention has the advantages that the silicon-zinc ion doping can play a role in synergy to improve the osteogenesis and angiogenesis capabilities of the material, the defect of single-ion doping can be made up, and the ion doping amount is easy to adjust, so that the ion release rate which is beneficial to promoting bone and angiogenesis can be adjusted.

3. According to the invention, the surface modified silicon/zinc ion doped biphasic calcium phosphate ceramic scaffold is prepared by further performing surface infiltration coating on the biphasic calcium phosphate ceramic scaffold, and because the dissolution rates of the scaffold base material and the surface infiltration coating material are different, mg and Si ions which have vascular promoting effects can be preferentially released through surface coating of the magnesium doped calcium silicate, and the cooperative vascularization promotion of Mg and Si ions can be beneficial to early promotion of scaffold pore channel vascularization, so that oxygen and nutrient substances are provided for the growth of subsequent new bones, metabolic wastes are eliminated, and the bone repair process is accelerated. Proper Mg and Si ion release rate and release period can be regulated and controlled by regulating and controlling proper magnesium ion doping amount, surface layer thickness and heat treatment temperature, thereby laying a foundation for early stage vascularization promotion.

4. The preparation method of the magnesium-doped calcium silicate surface modification layer of the bracket has simpler process, adopts a chemical precipitation method to synthesize the magnesium-doped calcium silicate precursor and prepare slurry, the precursor slurry has cohesiveness, adopts a dipping method under negative pressure to permeate and coat, can prepare the uniformly distributed bracket hole wall surface modification layer, can permeate into the surface holes of the bracket hole wall under proper negative pressure, and then obtains the crystalline magnesium-doped calcium silicate surface modification layer through heat treatment, so that the magnesium-doped calcium silicate surface modification layer is firmly combined with the bracket hole wall and is not easy to fall off; the combination of the magnesium-doped calcium silicate surface modification layer and the wall of the bracket hole can also improve the mechanical property of the bracket, improve the appearance and roughness of the bracket surface and be beneficial to cell adhesion and migration.

5. The invention can regulate the permeation quantity and the surface layer thickness of the magnesium-doped calcium silicate by controlling the negative pressure, the dipping time and the dipping times of the magnesium-doped calcium silicate precursor slurry dipping bracket.

Drawings

Fig. 1 is an X-ray diffraction pattern of comparative examples 1, 2 and example 1.

FIG. 2 is a graph showing the results of in vitro ion release test of example 1.

FIG. 3 is a graph showing the results of in vitro degradation tests of comparative example 1 and example 1.

FIG. 4 is a graph showing the results of the expression of osteoblast differentiation-related genes of mouse bone marrow mesenchymal stem cells on the surfaces of the samples of comparative example 1 and example 1.

FIG. 5 is a graph showing the results of the expression of vascular-related genes by human umbilical vein endothelial cells on the surfaces of the samples of comparative example 2 and example 1.

Fig. 6 is an X-ray diffraction pattern of the surface coating layers of examples 4 and 5.

FIG. 7 is a surface, cross-sectional electron microscopy and surface EDS elemental profile of the stent of example 5.

Fig. 8 is a graph of compressive strength for examples 1 and 5.

FIG. 9 is a graph showing the results of expression of the angiopoiesis-related gene by human umbilical vein endothelial cells cultured on the surface of examples 1, 4 and 5.

Detailed Description

The following examples are presented to further illustrate the practice of the invention, but are not intended to limit the practice and protection of the invention. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art. The reagents or apparatus used were not manufacturer-specific and were considered conventional products commercially available.

Comparative example 1

The comparative example uses undoped hydroxyapatite powder and beta-tricalcium phosphate powder with zinc doping amount of 2.5mol.% as raw materials, and the specific process steps comprise:

1) The method for synthesizing the undoped hydroxyapatite powder by using the chemical precipitation method and the hydrothermal method comprises the following steps: dissolving 118.075g of calcium nitrate in 1L of deionized water to obtain a calcium source solution, dissolving 39.618g of diammonium hydrogen phosphate in 1L of deionized water to obtain a phosphorus source solution, dropwise adding the phosphorus source solution into the calcium source solution to obtain a reaction solution, continuously stirring at a speed of 600r/min in the dropwise adding process, adding ammonia water to enable the pH value of the reaction solution to be 10.5, continuously stirring for 40min (at a stirring speed of 600 r/min) after the dropwise adding is finished, then filling the reaction solution into a high-pressure reaction kettle for hydrothermal treatment, wherein the hydrothermal temperature is 180 ℃, the hydrothermal time is 16h, centrifuging at a speed of 4000r/min to obtain a precipitate, repeatedly washing with deionized water to remove soluble salt, stirring at a stirring speed of 600r/min in the washing process, centrifuging at a speed of 5000r/min after washing, then drying in a 70 ℃ oven for 18h, calcining at a temperature of 950 ℃ in a muffle furnace, and preserving heat for 2h after primary grinding to obtain hydroxyapatite powder;

2) The method for synthesizing the zinc-doped beta-tricalcium phosphate powder by using the chemical precipitation method comprises the following steps: dissolving 207.222g of calcium nitrate and 6.694g of zinc nitrate in 1L of deionized water to obtain a calcium/zinc source mixed solution, dissolving 79.236g of diammonium phosphate in 1L of deionized water to obtain a phosphorus source solution, dripping the phosphorus source solution into the calcium/zinc source mixed solution to obtain a reaction solution, continuously stirring at a speed of 600r/min in the dripping process, simultaneously adding ammonia water to enable the pH value of the reaction solution to be 6.8, continuously stirring for 40min (at a stirring speed of 600 r/min) after the dripping is finished, aging at room temperature for 24h, centrifuging at a speed of 4000r/min to obtain a precipitate, repeatedly washing with deionized water to remove soluble salts, drying at a stirring speed of 600r/min for 10min in an oven at a stirring speed of 5000r/min after washing, calcining at 950 ℃ in a muffle furnace at a heating speed of 5 ℃/min after primary grinding, preserving heat for 2h, and obtaining zinc-doped beta-tricalcium phosphate powder after calcining;

3) Adding the undoped hydroxyapatite powder obtained in the step 1) and the beta-tricalcium phosphate powder with the zinc doping amount of 2.5mol.% obtained in the step 2) into a ball milling tank according to the mass percentage ratio of 40:60 to form mixed powder, performing ball milling on the mixed powder in a ratio of absolute ethyl alcohol to ball milling beads of 1:2:1 at a ball milling rate of 30Hz for 2h, and drying the mixed powder in a drying oven at 60 ℃ for 2h after ball milling to obtain zinc ion doped biphasic calcium phosphate powder;

4) Taking 5g of zinc ion doped biphasic calcium phosphate powder obtained in the step 3), adding 0.15 and g of methylcellulose, fully and uniformly mixing, continuously dripping 4.25g of polyvinyl alcohol solution with the concentration of 8wt.% into the mixture, uniformly stirring the mixture, filling the mixture into a 3D printing charging barrel, and obtaining a zinc ion doped biphasic calcium phosphate ceramic bracket blank through 3D printing;

5) And 4) sintering the biphase calcium phosphate ceramic bracket blank with zinc doping amount of 2.5mol.% obtained in the step 4) at a high temperature of 1150 ℃, wherein the heating rate is 2 ℃/min, and the heat preservation time is 2h, thus obtaining the zinc ion doped biphase calcium phosphate ceramic bracket.

Comparative example 2

The comparative example uses hydroxyapatite powder with silicon doping amount of 4mol.% and undoped beta-tricalcium phosphate powder as raw materials, and comprises the following specific process steps:

1) The method for synthesizing the silicon-doped hydroxyapatite powder by using the chemical precipitation method and the hydrothermal method comprises the following steps: dissolving 118.075g of calcium nitrate in 1L of deionized water to obtain a calcium source solution, dissolving 38.033g of diammonium hydrogen phosphate in 1L of deionized water to obtain a phosphorus source solution, uniformly mixing 2.72mL of tetraethyl orthosilicate with equal amount of absolute ethyl alcohol, adding the phosphorus source solution to obtain a phosphorus/silicon mixed solution, dripping the phosphorus/silicon mixed solution into the calcium source solution to obtain a reaction solution, continuously stirring at 600r/min in the dripping process, simultaneously adding ammonia water to enable the pH value of the reaction solution to be 10.5, continuously stirring for 40min (stirring speed is 600 r/min) after the dripping is finished, then filling the reaction solution into a high-pressure reaction kettle for hydrothermal treatment, wherein the hydrothermal temperature is 180 ℃, the hydrothermal time is 16h, centrifuging at 4000 r/min, removing soluble salt by repeated washing with deionized water, stirring at 600r/min, centrifuging at 5000r/min, drying at 70 ℃ for 18h in a baking oven at 950 ℃ after primary grinding, calcining at 5 ℃ for 2h, and preserving the temperature of hydroxyapatite powder;

2) The method for synthesizing undoped beta-tricalcium phosphate powder by using a chemical precipitation method comprises the following steps: dissolving 212.535g of calcium nitrate in 1L of deionized water to obtain a calcium source solution, dissolving 79.236g of diammonium hydrogen phosphate in 1L of deionized water to obtain a phosphorus source solution, dripping the phosphorus source solution into the calcium source solution to obtain a reaction solution, continuously stirring at a speed of 600r/min in the dripping process, simultaneously adding ammonia water to enable the pH value of the reaction solution to be 6.8, continuously stirring for 40min (stirring speed of 600 r/min) after the dripping is completed, aging at room temperature for 24h, centrifuging at a speed of 4000r/min to obtain a precipitate, repeatedly washing with deionized water to remove soluble salts, stirring at a stirring speed of 600r/min for 10min, centrifuging at a speed of 5000r/min, drying in an oven at 70 ℃ for 18h, calcining at a temperature of 950 ℃ in a muffle furnace after primary grinding, heating at a speed of 5 ℃/min, and preserving heat for 2h to obtain undoped beta-tricalcium phosphate powder after calcining;

3) Adding the hydroxyapatite powder with the silicon doping amount of 4mol.% obtained in the step 1) and the undoped beta-tricalcium phosphate powder obtained in the step 2) into a ball milling tank according to the mass percentage ratio of 40:60 to form mixed powder, ball milling the mixed powder with the mass ratio of absolute ethyl alcohol to ball milling beads of 1:2:1 at the ball milling rate of 30Hz for 2 hours, and drying the mixed powder in a baking oven at 60 ℃ for 2h after ball milling to obtain silicon ion doped biphasic calcium phosphate powder;

4) Taking 5g of the silicon ion doped biphasic calcium phosphate powder obtained in the step 3), adding 0.15 and g of methyl cellulose, fully and uniformly mixing, continuously dripping 4.25g of 8wt.% polyvinyl alcohol solution, manually stirring uniformly, loading into a 3D printing charging barrel, and performing 3D printing to obtain a silicon ion doped biphasic calcium phosphate ceramic bracket blank;

5) And (3) sintering the silicon ion doped biphasic calcium phosphate ceramic bracket blank obtained in the step (4) at a high temperature of 1150 ℃, wherein the heating rate is 2 ℃/min, and the heat preservation time is 2h, so as to obtain the silicon ion doped biphasic calcium phosphate ceramic bracket.

Example 1

The embodiment uses hydroxyapatite powder with silicon doping amount of 4mol.% and beta-tricalcium phosphate powder with zinc doping amount of 2.5 mol.% as raw materials, and the specific process steps comprise:

3) Adding the hydroxyapatite powder with the silicon doping amount of 4mol.% obtained in the step 2) and the beta-tricalcium phosphate powder with the zinc doping amount of 2.5mol.% obtained in the step 1) into a ball milling tank according to the mass percentage ratio of 40:60 to form mixed powder, performing ball milling according to the mass ratio of the mixed powder to absolute ethyl alcohol to ball milling beads of 1:2:1 at the ball milling rate of 30Hz for 2h, and drying in a baking oven at 60 ℃ for 2h after ball milling to obtain silicon/zinc ion doped biphasic calcium phosphate powder;

4) Taking 5g of the silicon/zinc ion doped biphasic calcium phosphate powder obtained in the step 3), adding 0.15 and g of methyl cellulose, fully and uniformly mixing, continuously dripping 4.25g of 8wt.% polyvinyl alcohol solution, manually stirring uniformly, loading into a 3D printing charging barrel, and performing 3D printing to obtain a silicon/zinc ion doped biphasic calcium phosphate ceramic bracket blank;

5) And (3) sintering the silicon/zinc ion doped biphasic calcium phosphate ceramic bracket blank obtained in the step (4) at a high temperature of 1150 ℃, wherein the heating rate is 2 ℃/min, and the heat preservation time is 2h, so as to obtain the silicon/zinc ion doped biphasic calcium phosphate ceramic bracket.

Fig. 1 is an X-ray diffraction pattern of the ceramic scaffolds of comparative example 1, comparative example 2 and example 1, and it can be seen from fig. 1 that the ceramic scaffolds of comparative example 1, comparative example 2 and example 1 each consisted of only β -tricalcium phosphate (JCPDF No. 090169) and hydroxyapatite (JCPDF No. 090432) without other impurity phases. The mass percentages of the hydroxyapatite and the beta-tricalcium phosphate of the comparative example 1, the comparative example 2 and the example 3 are maintained at about 40:60 after finishing calculation.

Fig. 2 is the inductively coupled plasma emission spectrometer (ICP) test results of example 1. Drying the ceramic brackets subjected to high-pressure sterilization, placing the ceramic brackets in a 48-well plate, setting 3 parallel samples in each group of ceramic brackets, adding a high-sugar basic culture medium according to 0.5ml of each group of ceramic brackets, changing culture medium liquid every other day, collecting soaking liquid of the ceramic brackets at time points of 1,3,7 and 14d, and detecting the concentration of Zn and Si elements in the soaking liquid by using ICP to obtain the ion release concentration at the corresponding time point. As shown by test data, the concentration of zinc ions in the culture medium is in the range of 0.3-0.4mg/L, the concentration of silicate ions is in the range of 3-4mg/L, and the combination of XRD test results can prove that silicate ions and zinc ions are successfully doped into hydroxyapatite and beta-tricalcium phosphate crystal lattices respectively.

FIG. 3 shows the results of in vitro degradation tests of comparative example 1 and example 1. Weighing the mass of the initial dry ceramic bracket, and marking the mass as m ₀ Transferring into 10mL centrifuge tube, adding acetic acid-sodium acetate soaking solution with pH of 4.5 at solid-liquid ratio of 0.02g/mL, placing 3 parallel samples each, incubating in constant temperature shaker at 37deg.C and 60r/min for 1, 2, 4 and 8 weeks, changing soaking solution weekly, and taking out ceramic bracket at corresponding time point, washing with deionized water, and bakingAnd (5) drying, weighing and recording the mass of the corresponding time point, marking as m, and substituting the mass into the ceramic bracket to calculate the weight loss rate of the ceramic bracket at the corresponding time point. Comparing comparative example 1 with example 1, it was found that the weight loss ratio of example 1 was consistently higher than that of comparative example 1, with example 1 having a weight loss ratio of 62.4% and comparative example 1 having a weight loss ratio of 46.5% at 8w, indicating that silicon incorporation can further improve the degradation performance of the biphasic calcium phosphate ceramic scaffold. FIG. 4 shows the expression of osteogenic differentiation-related genes by mouse bone marrow mesenchymal stem cells (mBMSCs) cultured on the ceramic scaffolds of comparative example 1 and example 1. Comparative example 1 and example 1 ceramic scaffolds were sterilized at high temperature and high pressure, then dried, placed in a 48-well plate, immersed in a complete medium for 6 hours, and then the surface of the scaffold was immersed for 6 hours according to a ratio of 4X 10 ⁴ cells/well were inoculated at a density of cells, replaced with osteoinductive fluid every other day, and cultured for 7d and 14d, followed by detection of the expression of osteogenic differentiation-related genes. Comparing comparative example 1 with example 1, it was found that stem cells cultured on example 1 had significantly higher expression of both early osteogenic differentiation related gene Alkaline phosphatase (ALP) and Collagen-I (Col-I) at the early stage of culture (day 7) than comparative example 1; the stem cells cultured on example 1 had higher expression of the late osteogenic differentiation related gene Bone Sialoprotein (BSP) in the late culture stage (day 14) than in comparative example 1. The results show that the promotion effect of the silicon/zinc double ion doping on the osteogenic differentiation of the stem cells is obviously superior to that of single zinc ion doping, and in addition, the previous research of the inventor discovers that the promotion effect of the silicon/zinc double ion doping on the osteogenic differentiation of the stem cells is obviously superior to that of single silicon ion doping. From this, it is known that the silicon/zinc ion doped biphasic calcium phosphate ceramic scaffold has better bone-promoting properties.

FIG. 5 shows the expression of angiogenesis-related genes by Human Umbilical Vein Endothelial Cells (HUVECs) cultured on the ceramic scaffolds of comparative example 2 and example 1. The materials of comparative example 2 and example 1 were sterilized at high temperature and high pressure, then dried, placed in a 48-well plate, immersed in a complete medium for 6 hours, and wetted, the surfaces of the materials were 4X 10 ⁴ cells/well density was inoculated, complete medium for endothelial cells was changed every other day, and cultures 3d and 5d were performed to examine the expression of the angiopoiesis-related gene. Comparison of comparative example 2 and example 1 shows that on day 3 eitherAlso on day 5, endothelial cells cultured on example 1 expressed significantly higher levels of three angiopoiesis-related genes, vascular Endothelial Growth Factor (VEGF), hypoxia inducible factor-1α (HIF-1α), endothelial nitric oxide synthase (eNOS), than comparative example 2. The result shows that the promoting effect of the silicon/zinc double ion doping on the endothelial cell vascularization differentiation is obviously superior to that of single silicon ion doping, and in addition, the inventor has found that the promoting effect of the silicon/zinc double ion doping on the endothelial cell vascularization differentiation is obviously superior to that of single zinc ion doping. From this, it can be seen that the silicon/zinc ion doped biphasic calcium phosphate ceramic stent has better vascular promoting performance.

Example 2

The embodiment uses hydroxyapatite powder with silicon doping amount of 4mol.% and beta-tricalcium phosphate powder with zinc doping amount of 1.5 mol.% as raw materials, and the specific process steps comprise:

1) The method for synthesizing the silicon-doped hydroxyapatite powder by using the chemical precipitation method and the hydrothermal method comprises the following steps: dissolving 118.075g of calcium nitrate in 1L of deionized water to obtain a calcium source solution, dissolving 38.033g of diammonium hydrogen phosphate in 1L of deionized water to obtain a phosphorus source solution, uniformly mixing 2.72mL of tetraethyl orthosilicate with equal amount of absolute ethyl alcohol, adding the phosphorus source solution to obtain a phosphorus/silicon mixed solution, then dropwise adding the phosphorus/silicon mixed solution into the calcium source solution to obtain a reaction solution, continuously stirring at 400r/min in the dropwise adding process, simultaneously adding ammonia water to enable the pH value of the reaction solution to be 11, continuously stirring for 60min (stirring speed is 400 r/min) after the dropwise adding, then filling the reaction solution into a high-pressure reaction kettle for hydrothermal treatment, wherein the hydrothermal temperature is 140 ℃, the hydrothermal time is 24h, centrifuging at 2000r/min, repeatedly washing with deionized water to remove soluble salts, stirring speed is 400r/min in the washing process, centrifuging at 4000r/min, then drying in a baking oven at 40 ℃ for 24h, calcining at 850 ℃ in a muffle furnace after primary grinding, heating up to obtain hydroxyapatite powder after the primary grinding, and preserving heat for 3 h;

2) The method for synthesizing the zinc-doped beta-tricalcium phosphate powder by using the chemical precipitation method comprises the following steps: dissolving 209.346g of calcium nitrate and 4.016g of zinc nitrate in 1L of deionized water to obtain a calcium/zinc source mixed solution, dissolving 79.236g of diammonium phosphate in 1L of deionized water to obtain a phosphorus source solution, dripping the phosphorus source solution into the calcium/zinc source mixed solution to obtain a reaction solution, continuously stirring at a speed of 400r/min in the dripping process, simultaneously adding ammonia water to enable the pH value of the reaction solution to be 6.4, continuously stirring for 60min (the stirring speed of 400 r/min) after the dripping is finished, aging at room temperature for 12h, centrifuging at a speed of 2000r/min to obtain a precipitate, repeatedly washing with deionized water to remove soluble salts, drying at a stirring speed of 600r/min for 20min in an oven at a speed of 4000r/min after washing, calcining at a temperature of 850 ℃ in a muffle furnace after primary grinding at a temperature of 5 ℃/min, preserving heat for 3h, and obtaining zinc-doped beta-tricalcium phosphate powder after calcination;

3) Adding the hydroxyapatite powder with the silicon doping amount of 4mol.% obtained in the step 1) and the beta-tricalcium phosphate powder with the zinc doping amount of 1.5mol.% obtained in the step 2) into a ball milling tank according to the mass percentage ratio of 20:80 to form mixed powder, performing ball milling according to the mass ratio of the mixed powder to absolute ethyl alcohol to ball milling beads of 1:2:1 at the ball milling rate of 30Hz for 2h, and drying in a baking oven at 60 ℃ for 2h after ball milling to obtain silicon/zinc ion doped biphasic calcium phosphate powder;

5) And (3) sintering the silicon/zinc ion doped biphase calcium phosphate ceramic bracket blank obtained in the step (4) at a high temperature of 1050 ℃, wherein the heating rate is 10 ℃/min, and the heat preservation time is 3 hours, so as to obtain the silicon/zinc ion doped biphase calcium phosphate ceramic bracket.

The phase analysis result shows that the ceramic bracket of the embodiment 2 consists of biphasic calcium phosphate without other impurity phases, and the mass percentages of the hydroxyapatite and the beta-tricalcium phosphate are maintained at about 20:80. From the ICP test results, zn ²⁺ Concentration in the MediumIs in the range of 0.2-0.28 mg/L; siO (SiO) ₄ ^4- The concentration of the ions in the culture medium is in the range of 3.2-4.5mg/L, and the combination of XRD test results shows that silicate ions and zinc ions are successfully doped into hydroxyapatite and beta-tricalcium phosphate crystal lattices respectively. The results of inoculating the mouse mesenchymal stem cells on the surfaces of the example 1 and the example 2 show that the mouse mesenchymal stem cells have slightly lower expression of the example 2 than the example 1 on proliferation, adhesion and osteogenic differentiation related genes (ALP, col-I, BSP), but have no significant difference, and the example 1 and the example 2 have good osteogenic activity; the results of the inoculation of human umbilical vein endothelial cells on the surfaces of example 1 and example 2 showed that there was no significant difference in proliferation, adhesion and expression of angiogenesis-related genes (VEGF, HIF-1α, eNOS) in both endothelial cells, indicating good angiogenic activity in both example 1 and example 2.

Example 3

The embodiment uses hydroxyapatite powder with silicon doping amount of 2mol.% and beta-tricalcium phosphate powder with zinc doping amount of 3mol.% as raw materials, and the specific process steps comprise:

1) The method for synthesizing the silicon-doped hydroxyapatite powder by using the chemical precipitation method and the hydrothermal method comprises the following steps: dissolving 118.075g of calcium nitrate in 1L of deionized water to obtain a calcium source solution, dissolving 38.826g of diammonium hydrogen phosphate in 1L of deionized water to obtain a phosphorus source solution, uniformly mixing 1.36mL of tetraethyl orthosilicate with equal amount of absolute ethyl alcohol, adding the phosphorus source solution to obtain a phosphorus/silicon mixed solution, dripping the phosphorus/silicon mixed solution into the calcium source solution to obtain a reaction solution, continuously stirring at a speed of 500r/min in the dripping process, simultaneously adding ammonia water to enable the pH value of the reaction solution to be 10.8, continuously stirring for 50min (the stirring speed is 500 r/min) after the dripping is finished, then filling the reaction solution into a high-pressure reaction kettle for hydrothermal treatment, the hydrothermal temperature is 160 ℃, the hydrothermal time is 20h, centrifuging at a speed of 3000 r/min, repeatedly washing with deionized water to remove soluble salt, the stirring speed of 500r/min in the washing process, centrifuging the solution at a speed of 4500r/min after the washing, then drying at a speed of 60 ℃ for 22h in a baking oven, calcining at a speed of 900 ℃ after primary grinding, heating up to obtain hydroxyapatite powder after the calcination, and preserving heat for 2.5 h;

2) The method for synthesizing the zinc-doped beta-tricalcium phosphate powder by using the chemical precipitation method comprises the following steps: dissolving 206.159g of calcium nitrate and 8.032g of zinc nitrate in 1L of deionized water to obtain a calcium/zinc source mixed solution, dissolving 79.236g of diammonium phosphate in 1L of deionized water to obtain a phosphorus source solution, dripping the phosphorus source solution into the calcium/zinc source mixed solution to obtain a reaction solution, continuously stirring at a speed of 500r/min in the dripping process, simultaneously adding ammonia water to enable the pH value of the reaction solution to be 6.6, continuously stirring for 50min (the stirring speed is 500 r/min) after the dripping is finished, aging at room temperature for 18h, centrifuging at a speed of 3000r/min to obtain a precipitate, repeatedly washing with deionized water to remove soluble salts, drying at a stirring speed of 500r/min for 15min in an oven at a speed of 4500r/min, calcining at 900 ℃ in a muffle furnace after primary grinding for 60 drying for 22h, maintaining the temperature at a speed of 5 ℃/min, and calcining to obtain zinc-doped beta-tricalcium phosphate powder;

3) Adding the hydroxyapatite powder with the silicon doping amount of 2mol.% obtained in the step 1) and the beta-tricalcium phosphate powder with the zinc doping amount of 3mol.% obtained in the step 2) into a ball milling tank according to the mass percentage ratio of 30:70 to form mixed powder, performing ball milling according to the mass ratio of the mixed powder to absolute ethyl alcohol to ball milling beads of 1:2:1, wherein the ball milling speed is 30Hz, the ball milling time is 2h, and drying the mixture in a baking oven at 60 ℃ for 2h after ball milling is finished to obtain silicon/zinc ion doped biphasic calcium phosphate powder;

5) And (3) sintering the silicon/zinc ion doped biphase calcium phosphate ceramic bracket blank obtained in the step (4) at a high temperature of 1100 ℃, wherein the heating rate is 5 ℃/min, and the heat preservation time is 3 hours, so as to obtain the silicon/zinc ion doped biphase calcium phosphate ceramic bracket.

The results of the phase analysis show that the ceramic of example 3The ceramic bracket is composed of biphasic calcium phosphate, has no other impurity phase, and the mass percentages of the hydroxyapatite and the beta-tricalcium phosphate are both maintained at about 30:70. From the ICP test results, zn ²⁺ The concentration in the culture medium is in the range of 0.36-0.52 mg/L; siO (SiO) ₄ ^4- The concentration of the ions in the culture medium is in the range of 1.2-2mg/L, and the combination of XRD test results shows that silicate ions and zinc ions are successfully doped into hydroxyapatite and beta-tricalcium phosphate crystal lattices respectively. The results of inoculating the mouse mesenchymal stem cells on the surfaces of the example 1 and the example 3 show that the mouse mesenchymal stem cells have no significant difference in proliferation, adhesion and expression of osteogenic differentiation related genes (ALP, col-I, BSP), which indicates that the example 1 and the example 3 have good osteogenic activity; the results of the inoculation of human umbilical vein endothelial cells on the surfaces of example 1 and example 3 show that both have good angiogenic activity on proliferation, adhesion and expression of angiogenesis-related genes (VEGF, HIF-1α, eNOS) in endothelial cells, example 3 being slightly lower than example 1, but not significantly different.

Example 4

The surface of the prepared calcium silicate slurry with 5mol.% magnesium doping amount is coated on the surface of the sample in the example 1 in a penetrating way, and the specific process steps comprise:

1) Dissolving 11.220g of calcium nitrate and 0.641g of magnesium nitrate in 100mL of deionized water to obtain a calcium/magnesium source mixed solution, dissolving 14.21g of sodium silicate in 100mL of deionized water to obtain a silicon source solution, dropwise adding the calcium/magnesium source mixed solution into the silicon source solution under stirring (600 r/min), continuously stirring at the speed of 600r/min for 45min after dropwise adding, centrifuging at the speed of 4000r/min to obtain a precipitate, repeatedly washing with deionized water to remove soluble salts, wherein the stirring speed of the washing process is 600r/min, the stirring time is 10min, centrifuging the washed solution at the speed of 4000r/min to obtain a magnesium-doped calcium silicate precursor reactant, adding 200mL of deionized water, adding 0.8g of polyethylene glycol dispersant, stirring at the speed of 400r/min for 30min to obtain 5mol.% magnesium-doped calcium silicate precursor slurry with the concentration of 0.6 mol/L;

2) Soaking the silicon/zinc co-doped calcium phosphate ceramic bracket of the embodiment 1 in the magnesium doped silicate slurry obtained in the step 1), coating the surface by permeation, wherein the permeation negative pressure is-0.20 MPa, the permeation time is 25s, then drying the silicon/zinc co-doped calcium phosphate ceramic bracket in a 50 ℃ oven for 40min, and repeating the soaking and drying for 2 times to form a magnesium doped calcium silicate surface layer with the thickness of 12 mu m, thereby obtaining the surface modified biphase calcium phosphate ceramic bracket;

3) And (3) placing the calcium phosphate ceramic bracket obtained in the step (2) in a muffle furnace, and performing heat treatment at 900 ℃ for 3 hours at a heating rate of 8 ℃/min to obtain the 5mol.% magnesium-doped calcium silicate surface modified silicon/zinc ion-doped biphasic calcium phosphate ceramic bracket.

Example 5

The surface of the sample prepared in the example 1 is coated with a calcium silicate slurry with 10mol.% magnesium doping, and the specific process steps comprise:

1) Dissolving 10.627g of calcium nitrate and 1.282g of magnesium nitrate in 100mL of deionized water to obtain a calcium/magnesium source mixed solution, dissolving 14.21g of sodium silicate in 100mL of deionized water to obtain a silicon source solution, dropwise adding the calcium/magnesium source mixed solution into the silicon source solution under stirring (600 r/min), continuously stirring at the speed of 600r/min for 45min after dropwise adding, centrifuging at the speed of 4000r/min to obtain a precipitate, repeatedly washing with deionized water to remove soluble salts, wherein the stirring speed of the washing process is 600r/min, the stirring time is 10min, centrifuging the washed solution at the speed of 4000r/min to obtain a magnesium-doped calcium silicate precursor reactant, adding 200mL of deionized water, adding 0.8g of polyethylene glycol dispersant, stirring at the speed of 400r/min for 30min to obtain uniform 10mol.% magnesium-doped calcium silicate precursor slurry with the concentration of 0.6 mol/L;

2) Soaking the silicon/zinc co-doped calcium phosphate ceramic bracket obtained in the embodiment 1) in the magnesium doped silicate slurry obtained in the step 1), coating by surface permeation, wherein the permeation negative pressure is-0.20 MPa, the permeation time is 25s, then drying for 40min in a 50 ℃ oven, and repeating soaking and drying for 2 times to form a magnesium doped calcium silicate surface layer with the thickness of 12 mu m, thereby obtaining the surface modified biphase calcium phosphate ceramic bracket;

3) And (3) placing the calcium phosphate ceramic bracket obtained in the step (2) in a muffle furnace, and performing heat treatment at 900 ℃ for 3 hours at a heating rate of 8 ℃/min to obtain the 10mol.% magnesium-doped calcium silicate surface modified silicon/zinc ion-doped biphasic calcium phosphate ceramic bracket.

Fig. 6 is an X-ray diffraction pattern of the surface coating of example 4 and example 5, showing by XRD pattern that the phase of the coating layer of the silicon/zinc ion doped biphasic calcium phosphate ceramic is calcium silicate, and as the amount of magnesium incorporation increases, the characteristic peak moves to a high angle, and it is seen that magnesium ions successfully enter calcium ion lattice sites.

Fig. 7 is a top coat characterization of example 5. From the figure, it can be seen (left upper panel is a surface-uncoated stent, and right upper panel is a surface-coated stent), that the surface-modified ceramic stent has macropores of about 400 μm. The coating thickness was about 12 μm (right panel below). As can be seen from the EDS element distribution diagram (lower middle diagram and lower left diagram are corresponding morphology diagrams), mg and Si elements are uniformly distributed on the surface of the ceramic bracket, which indicates that the magnesium-doped calcium silicate is successfully coated on the surface of the silicon/zinc ion-doped biphasic calcium phosphate ceramic bracket.

FIG. 8 shows the results of the compressive strength property tests of example 1 and example 5. The compressive strength of example 1 was 8.94MPa and the compressive strength of example 5 was 10.76MPa, indicating that the compressive strength of the doped calcium phosphate ceramic stent could be improved by surface infiltration coating of the magnesium doped calcium silicate.

FIG. 9 shows the expression of the genes related to angiogenesis by human umbilical vein endothelial cells after surface culture for 3d and 5d in example 1, example 4 and example 5. The materials of example 1, example 4 and example 5 were sterilized at high temperature and high pressure, then dried, placed in a 48-well plate, immersed in a complete medium for 6 hours, and the surface of the materials was 4X 10 ⁴ cells/well density was inoculated, complete medium for endothelial cells was changed every other day, and 3d and 5d cultures were used for detection of angiogenesis-related gene expression. From FIG. 9, it can be seen that examples 4 and 5 up-regulate the expression of three angiogenesis-related genes VEGF, HIF-1α and eNOS by endothelial cells on day 5, relative to example 1, wherein the up-regulation effect of example 5 is more remarkable. This demonstrates that the surface coating of magnesium-doped calcium silicate can further improve the vascularization promoting performance of the silicon/zinc ion-doped biphasic calcium phosphate ceramic stent, which is beneficial to Early vascularization of ceramic stents.

Example 6

The surface of the calcium silicate slurry with the magnesium doping amount of 12mol.% is prepared in the embodiment and is coated on the surface of the sample in the embodiment 2 in a penetrating way, and the specific process steps comprise:

1) Dissolving 10.390g of calcium nitrate and 1.538g of magnesium nitrate in 100mL of deionized water to obtain a calcium/magnesium source mixed solution, dissolving 14.21g of sodium silicate in 100mL of deionized water to obtain a silicon source solution, dropwise adding the calcium/magnesium source mixed solution into the silicon source solution under the condition of stirring (500 r/min), continuously stirring at the speed of 450 r/min for 30min after dropwise adding, centrifuging at the speed of 3000r/min to obtain a precipitate, repeatedly washing with deionized water to remove soluble salts, wherein the stirring speed of the washing process is 500r/min, the stirring time is 8min, centrifuging the washed solution at the speed of 4000r/min to obtain a magnesium-doped calcium silicate precursor reactant, adding 200mL of deionized water, adding 1.2g of polymethacrylate amine dispersant, stirring at the speed of 400r/min for 30min to obtain uniform and 0.4mol/L concentration of 12mol magnesium-doped calcium silicate precursor slurry;

2) Soaking the silicon/zinc co-doped calcium phosphate ceramic stent obtained in the embodiment 2 in the magnesium doped silicate slurry obtained in the step 1), coating the surface by permeation, wherein the permeation negative pressure is-0.25 MPa, the permeation time is 30s, then drying the ceramic stent in a 60 ℃ oven for 30min, and repeating the soaking and drying for 3 times to form a magnesium doped calcium silicate surface layer with the thickness of 20 mu m, thereby obtaining the surface modified biphase calcium phosphate ceramic stent;

3) And (3) placing the calcium phosphate ceramic bracket obtained in the step (2) in a muffle furnace, and performing heat treatment at 950 ℃ for 1h, wherein the heating rate is 10 ℃/min, so as to obtain the 12mol.% magnesium-doped calcium silicate surface modified silicon/zinc ion-doped biphasic calcium phosphate ceramic bracket.

The phase analysis result shows that the phase of the surface coating layer of the biphase calcium phosphate ceramic is calcium silicate. Scanning electron microscope observation shows that the calcium silicate doped coating uniformly coats the surface and the hole wall of the support, and EDS element distribution analysis also shows that magnesium element and silicon element are uniformly distributed on the surface of the biphase calcium phosphate ceramic support. These all illustrate the successful preparation of magnesium doped calcium silicate surface coated modified silicon/zinc ion doped biphasic calcium phosphate ceramic scaffolds. In comparison with example 2, example 6 can better promote proliferation and adhesion of human umbilical vein endothelial cells on the surface of a stent and expression of VEGF, HIF-1 alpha and eNOS genes. This demonstrates that a calcium silicate surface coated silicon/zinc ion doped biphasic calcium phosphate ceramic stent with a magnesium doping level of 12mol.% is more prone to angiogenesis, which is beneficial to achieving early vascularization of the stent.

Example 7

This example prepared a calcium silicate slurry surface having a magnesium doping level of 20mol.% was surface-osmotically coated onto the sample surface of example 2, by the following procedure:

1) Dissolving 9.446g of calcium nitrate and 2.564g of magnesium nitrate in 100mL of deionized water to obtain a calcium/magnesium source mixed solution, dissolving 14.21g of sodium silicate in 100mL of deionized water to obtain a silicon source solution, dropwise adding the calcium/magnesium source mixed solution into the silicon source solution under the condition of stirring (300 r/min), continuously stirring at the speed of 300r/min for 15min after dropwise adding, centrifuging at the speed of 2000r/min to obtain a precipitate, repeatedly washing with deionized water to remove soluble salts, wherein the stirring speed of the washing process is 300r/min, the stirring time is 5min, centrifuging the washed solution at the speed of 3000r/min to obtain a magnesium-doped calcium silicate precursor reactant, adding 200mL of deionized water, adding 1.0g of polyethylene glycol dispersant, stirring at the speed of 400r/min for 30min to obtain uniform 5mol.% magnesium-doped calcium silicate precursor slurry with the concentration of 0.4 mol/L;

2) Immersing the silicon/zinc ion doped calcium phosphate ceramic stent obtained in the embodiment 2 in the magnesium doped silicate slurry obtained in the step 1), coating by surface permeation, wherein the permeation negative pressure is-0.05 MPa, the permeation time is 10 s, and then drying in a baking oven at 40 ℃ for 60min to form a magnesium doped calcium silicate surface layer with the thickness of 5 mu m, thereby obtaining the surface modified biphasic calcium phosphate ceramic stent;

3) And (3) placing the calcium phosphate ceramic bracket obtained in the step (2) in a muffle furnace, and performing heat treatment at 850 ℃ for 1h, wherein the heating rate is 5 ℃/min, so as to obtain the 20mol.% magnesium-doped calcium silicate surface modified silicon/zinc ion-doped biphasic calcium phosphate ceramic bracket.

The phase analysis result shows that the phase of the surface coating layer of the biphase calcium phosphate ceramic is calcium silicate. Scanning electron microscope observation shows that the calcium silicate doped coating uniformly coats the surface and the hole wall of the support, and EDS element distribution analysis also shows that magnesium element and silicon element are uniformly distributed on the surface of the biphase calcium phosphate ceramic support. These all illustrate the successful preparation of magnesium doped calcium silicate surface coated modified silicon/zinc ion doped biphasic calcium phosphate ceramic scaffolds. The results of inoculating human umbilical vein endothelial cells on the surfaces of the materials of the examples 6 and 7 show that the example 6 can better promote the proliferation and adhesion of the human umbilical vein endothelial cells on the stent surface and the expression of VEGF, HIF-1 alpha and eNOS compared with the example 7.

The examples of the present invention are only examples for clearly illustrating the present invention, and are not limiting of the embodiments of the present invention. Other variations or modifications of the above examples may be made by those skilled in the art, and it is not necessary nor exhaustive of all embodiments. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention as set forth in the appended claims.

Claims

1. A method for preparing a surface modified silicon/zinc ion doped biphasic calcium phosphate ceramic scaffold by using a silicon/zinc ion doped biphasic calcium phosphate ceramic scaffold, which is characterized by comprising the following steps:

carrying out heat treatment on the silicon/zinc ion doped biphasic calcium phosphate ceramic stent with the surface being permeated and coated to obtain a surface modified silicon/zinc ion doped biphasic calcium phosphate ceramic stent;

the preparation method of the silicon/zinc ion doped biphasic calcium phosphate ceramic bracket comprises the following steps:

2. The method of claim 1, having at least one of the following a-f:

b. the Ca/(P+Si) molar ratio of the silicon-doped hydroxyapatite powder is 1.67, wherein the Si/(Si+P) molar ratio is 0.02-0.04;

e. the mass percentage of the mixed powder of the silicon doped hydroxyapatite powder and the zinc doped beta-calcium phosphate powder is 20-40:60-80;

f. the thickener is methyl cellulose, and the binder is polyvinyl alcohol.

3. The method according to claim 1, wherein in the step 1), the pH value of the reaction solution a of the synthetic silicon-doped hydroxyapatite powder is 10.5 to 11, the stirring rate is 400 to 600 r/min, the stirring time is 40 to 60 min, the hydrothermal treatment temperature is 140 to 180 ℃, the hydrothermal treatment time is 16 to 24 hours, the centrifugation rate of the solution after the reaction is 2000 to 4000 r/min, the washing solution is deionized water, the stirring rate of the washing process is 400 to 600 r/min, the stirring time is 10 to 20 min, the centrifugation rate of the solution after the washing is 4000 to 5000 r/min, the drying temperature is 40 to 70 ℃, the drying time is 18 to 24h, the calcining temperature is 850 to 950 ℃, and the heat preservation time is 2 to 3 h;

the pH value of the reaction solution B for synthesizing the zinc doped beta-tricalcium phosphate powder is 6.4-6.8, the stirring speed is 400-600 r/min, the stirring time is 40-60 min, the aging time is 12-24 h, the centrifugal speed of the solution after reaction is 2000-4000 r/min, the washing liquid is deionized water, the stirring speed in the washing process is 400-600 r/min, the stirring time is 10-20 min, the centrifugal speed of the solution after washing is 4000-5000 r/min, the drying temperature is 40-70 ℃, the drying time is 18-24 h, the calcining temperature is 850-950 ℃, and the heat preservation time is 2-3 h.

4. The method according to claim 1, wherein in step 4), the sintering temperature of the silicon/zinc ion doped biphasic calcium phosphate ceramic support body is 1050-1150 ℃, the heating rate is 2-10 ℃/min, and the holding time is 2-4 h.

5. The method of claim 1, having at least one of a, b:

a. the calcium source of the synthesized magnesium doped calcium silicate precursor slurry is calcium nitrate, the silicon source is sodium silicate, and the magnesium source is magnesium nitrate;

6. The method according to claim 1, wherein the stirring rate of the reaction solution C for preparing the magnesium-doped calcium silicate precursor slurry is 300-600 r/min, the stirring time is 15-45 min, the centrifugal rate is 2000-4000 r/min, the stirring rate in the washing process is 300-600 r/min, the stirring time is 5-10 min, the centrifugal rate of the washed solution is 3000-4000 r/min, the washing solution and the slurry solution are deionized water, the dispersing agent is one or a combination of polyethylene glycol, polyacrylic acid and polymethacrylate ammonia, and the concentration of the dispersing agent is 2-6 g/L;

the concentration of the magnesium-doped calcium silicate in the magnesium-doped calcium silicate precursor slurry is 0.4-0.6 mol/L; the negative pressure range of the magnesium doped calcium silicate precursor slurry impregnated silicon/zinc ion doped biphasic calcium phosphate ceramic bracket is-0.05 to-0.25 MPa, the impregnation time is 10-30 s, the times of impregnation and infiltration coating are 1-3, the drying temperature is 40-60 ℃, and the drying time is 30-60 min; the final thickness of the surface layer of the magnesium doped calcium silicate precursor formed by dip-coating is 5-20 mu m.

7. The method of claim 1, wherein the surface-infiltrated coated silicon/zinc ion doped dual phase calcium phosphate ceramic scaffold is heat treated at a temperature of 850-950 ℃, a ramp rate of 5-10 ℃/min, and a soak time of 1-3 h.

8. A surface-modified silicon/zinc ion-doped biphasic calcium phosphate ceramic scaffold obtainable by the process of any one of claims 1 to 7.