CN114767927A

CN114767927A - Silicon/zinc ion doped biphase calcium phosphate ceramic bracket and preparation method thereof

Info

Publication number: CN114767927A
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: 2022-07-22
Anticipated expiration: 2042-04-02
Also published as: CN114767927B

Abstract

The invention discloses a silicon/zinc ion doped biphase calcium phosphate ceramic bracket and a preparation method thereof, which comprises the steps of firstly mixing synthesized silicon ion doped hydroxyapatite powder with zinc ion doped beta-tricalcium phosphate powder, preparing a silicon/zinc ion doped biphase calcium phosphate ceramic bracket blank through 3D printing, and then sintering at high temperature to obtain the silicon/zinc ion doped biphase calcium phosphate ceramic bracket; on the basis, a magnesium-doped calcium silicate precursor is synthesized and prepared into slurry, the slurry is used for impregnating the stent and coating the surface of the stent in a penetrating way, the coating and the drying are repeated, then the heat treatment is carried out, a magnesium-doped calcium silicate surface layer is formed on the surface of the stent, and finally the surface-modified silicon/zinc ion-doped biphase calcium phosphate ceramic stent is obtained. The invention has the advantages of simple preparation process, adjustable ion doping amount and release amount, synergistic promotion of bone formation and vascularization by various active ions and the like, and has important significance for expanding the clinical application of the calcium phosphate ceramic stent.

Description

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

Technical Field

The invention relates to the technical field of medical materials for repairing bone injury, in particular to a silicon/zinc ion doped biphase calcium phosphate ceramic bracket, a surface modified silicon/zinc ion doped biphase 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), the artificially synthesized hydroxyapatite HAs excellent biocompatibility, good bone conductivity and bone integration, the HA ceramic HAs high mechanical property, but low biodegradability, is not favorable for the regeneration and repair of bone defects, and limits the wide application of the HA ceramic in the field of bone repair. The chemical composition of tricalcium phosphate (TCP; the Ca/P molar ratio of TCP is 1.5) is similar to that of bone mineral, and the tricalcium phosphate HAs excellent biocompatibility, good osteoconductivity and osseointegration, HAs high biodegradability, can realize the regeneration and repair of bone defects, is clinically and normatively applied to the bone defect repair, but the mechanical property of the TCP ceramic is inferior to that of HA ceramic. The Biphase 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 usually composed of HA and β -TCP because β -TCP HAs better chemical stability than α -TCP. BCP can be directly chemically synthesized, and can also be compounded by synthesized HA and beta-TCP. Although BCP can combine the advantages of HA and β -TCP, its lack of osteo-and angio-inductive properties results in an inadequate capacity for bone repair (Bouler J M, Pilet P, Gauthier O, Verron E.Biphasic calcium phosphate ceramics for bone retrieval: A review of biological response [ J ]. Acta biomaterials, 2017,53, 1-12.).

Magnesium, zinc, silicon, strontium, etc. are trace elements in inorganic minerals of natural bones, which play an important role in biochemical reactions closely related to bone development and metabolism (Dorozhkin S V, Epple M. Biological and medical signalling of calcium phosphates [ J ]. Angewandte Chemie-International Edition,2002,41(17): 3130-. Zinc and strontium ion doping of calcium phosphate materials primarily contributes to osteogenesis, magnesium ion doping primarily contributes to hematopoiesis, and silicon ion doping simultaneously contributes to osteogenesis and angiogenisis (Bose S, Fielding G, Tarafder S, et al. underpinning of human-induced osteogenesis and angiogenisis in calcium phosphate ceramics [ J ]. Trends in Biotechnology,2013,31(10): 594. 605.). The zinc ion doping has a strong effect of promoting bone formation, while the silicon ion doping has a good effect on promoting bone formation and blood vessel formation, but the unit ion doping effect is still not good enough, and the active ions doped in the calcium phosphate ceramic matrix are released relatively slowly, so that the early vascularization of the stent is difficult to realize. The early vascularization is an important condition for repairing the bone defect, and the blood vessel not only can bring oxygen and nutrient substances to peripheral cells and remove metabolic waste, but also can convey the cells participating in the repair to the bone defect part, so that the promotion of the early vascularization is more favorable for accelerating the repair process of the bone defect.

CN107441558A discloses a porous SiO used for bone tissue engineering₂The composite silicon oxide has the osteogenesis promoting and vascularization promoting effects which are not as good as the effects of silicon doping and silicon and zinc co-doping, and the stent prepared by adopting polyurethane organic foam pore-forming is inferior to the stent prepared by 3D printing in the aspects of pore connectivity and mechanical property. CN110613863A discloses a porous scaffold material for promoting vascularization based on silicon-doped hydroxyapatite, a preparation method and an application thereof, wherein a calcium, phosphorus and silicon source solution is added into a polyvinyl alcohol solution, a small amount (2-5 wt.%) of silicon-doped hydroxyapatite is generated through chemical reaction to prepare the porous scaffold material for promoting vascularization by compounding a small amount of silicon-doped hydroxyapatite,however, the content of the silicon-doped hydroxyapatite is low, the degradability is low, so that the ion release is limited, the effect of promoting vascularization is difficult to be obvious, and the matrix material of the scaffold is polyvinyl alcohol, so the osteogenic property is lacked. CN107412855A discloses a 3D printing support with a coating and a preparation method thereof, the dopamine surface modification poly (butylene succinate) support is prepared by dipping a dopamine solution after 3D printing the poly (butylene succinate) support, the surface modification support is coated with mesoporous magnesium silicate microspheres or copper-doped mesoporous magnesium silicate microspheres to obtain mesoporous magnesium silicate microspheres or copper-doped mesoporous magnesium silicate microsphere coating, however, the support matrix is an artificially synthesized organic polymer and lacks bioactivity and osteogenic performance, the surface of the dopamine membrane is coated with mesoporous magnesium silicate microspheres or copper-doped mesoporous magnesium silicate microspheres, but the magnesium silicate microspheres cannot be well combined with the dopamine membrane, and particularly, the dopamine membrane and the microspheres are easy to fall off when beginning to degrade. CN109793923B discloses a method for preparing a nanostructured boron-containing calcium silicate biological coating, which comprises plasma spraying calcium silicate powder on the surface of titanium alloy material, then soaking in sodium borate solution to generate hydrothermal reaction, thus obtaining the boron-containing calcium silicate biological coating, wherein the coating has the functions of promoting bone differentiation and anti-inflammatory, but because the thermal expansion coefficient and elastic modulus of boron-containing calcium silicate are greatly different from those of titanium alloy, the coating is easy to crack and peel along with the extension of time in the application process, the purpose of the patent is irrelevant to promoting vascularization, and the preparation method of the biological coating is not suitable for modifying calcium phosphate scaffold material with three-dimensional communicated porous structure, and can not construct modified layer on the inner pore wall of the scaffold material. For the regenerative repair of bone injury, the promotion of osteogenesis and vascularization of calcium phosphate scaffold is an important function, and the early vascularization is a key for realizing rapid osteogenesis, so that it is necessary to develop a preparation technology for improving the early vascularization of calcium phosphate scaffold based on the vasoactive ion-doped surface modification layer with simple and universal process. At present, no report is found on a silicon and zinc double-ion doped biphase calcium phosphate ceramic bracket and a preparation method thereof.

Disclosure of Invention

The first purpose of the invention is to overcome the defects of the prior art and provide a silicon/zinc ion doped biphase calcium phosphate ceramic bracket and a preparation method thereof.

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

The first purpose of the invention is realized by the following technical scheme: a preparation method of a silicon/zinc ion doped biphase 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 with the phosphorus source solution A2, then, dripping the mixture into the calcium source solution A1 to obtain a reaction solution A, adjusting the pH value of the reaction solution A, and then, 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, aging, 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 biphase calcium phosphate mixed powder;

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

4) and 3) sintering the silicon/zinc ion doped biphase calcium phosphate ceramic support blank obtained in the step 3) at a high temperature to obtain the silicon/zinc ion doped biphase calcium phosphate ceramic support.

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

a. the calcium source for synthesizing the silicon-doped hydroxyapatite powder is calcium nitrate, the phosphorus source is one or a combination of more of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate, and the silicon source is ethyl orthosilicate;

b. the molecular formula of the silicon-doped hydroxyapatite powder is Ca₁₀(PO₄)_6-x(SiO₄)_x(OH)_2-xWherein the molar ratio of Ca/(P + Si) is 1.67, and the molar ratio of Si/(Si + P) is 0.02-0.04;

c. the calcium source for synthesizing the 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 diammonium hydrogen phosphate, ammonium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate;

d. the molar ratio of (Zn + Ca)/P of the zinc-doped beta-tricalcium phosphate powder is 1.5, wherein the molar ratio of Zn/(Zn + Ca) 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 thickening agent 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 decomposed more easily to generate alpha-TCP, the alpha-TCP is degraded too fast to provide a stable environment for the initial adhesion of stem cells, the subsequent proliferation and osteogenic differentiation of the stem cells are not facilitated, meanwhile, the material degraded too fast cannot provide mechanical support for bone defect tissues in the initial implantation period, and the matching of the degradation space and the new osteogenic amount is poor. The invention adopts a preparation method combining a chemical precipitation method and a hydrothermal treatment, and further optimizes the range of the process parameters, so that the problem of alpha-TCP generated by the decomposition of the hydroxyapatite can be solved, and the silicon-doped hydroxyapatite with a single phase can be obtained. The zinc doping can improve the osteogenic property of the calcium phosphate material, but the zinc doping can reduce the degradation property of the calcium phosphate material; the silicon doping can not only improve the bioactivity and angiogenesis performance of the calcium phosphate bracket, but also accelerate the degradation speed of the calcium phosphate material. Therefore, the doping of silicon and zinc double ions can lead the calcium phosphate material to have the degradation speed matched with the bone defect repair process, and simultaneously have excellent osteogenesis and angiogenesis performance. In addition, too high an amount of ion doping may cause cytotoxicity and decrease osteogenesis/angiogenisis activity; the doping amount is preferably in the above-mentioned appropriate range because the ability to promote osteogenesis and hematopoiesis is insufficient due to too low doping amount.

The inventor finds that the two-phase proportion of the biphasic calcium phosphate has influence on the bone induction performance and the degradation performance of the material, and 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 not beneficial to the formation of new bones; if the content of the hydroxyapatite is too high, the degradation speed of the biphasic calcium phosphate is slow, which is not beneficial to the growth of new bones, so that the proportion of the two phases is preferably in a proper range.

The inventor finds that aiming at vascularization, the too small pore diameter of the stent is not beneficial to vascularization, and the pore diameter of the stent is more than 300 mu m, so that the pore diameter of the printed stent is more than 300 mu m after sintering when the pore diameter of the printed stent reaches 400 mu m, and the further improvement of the pore diameter is more beneficial to the subsequent magnesium-doped calcium silicate surface modification to promote the vascularization of early pore channels, but the too large pore diameter can reduce the mechanical property of the stent, and is not beneficial to the osteoblasts to fill the pore channels, so that a proper pore diameter range is required.

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 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 10-20min, 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 calcination temperature is 850-950 ℃, and the heat preservation time is 2-3 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 400-600r/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 solution 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 dual-phase calcium phosphate ceramic bracket blank is 1050-1150 ℃, the heating rate is 2-10 ℃/min, and the heat preservation time is 2-4 h.

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

The second purpose of the invention is realized by the following technical scheme: a method for preparing a surface modified silicon/zinc ion doped biphase calcium phosphate ceramic bracket by using the silicon/zinc ion doped biphase 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 then sequentially stirring, centrifuging, washing, centrifuging to obtain a precipitate, adding deionized water, adding a dispersing agent and stirring to obtain a magnesium-doped calcium silicate precursor slurry;

placing the silicon/zinc ion doped biphase calcium phosphate ceramic bracket in magnesium doped calcium silicate precursor slurry for dipping, performing negative pressure lower surface permeation coating, then drying, and performing repeated dipping permeation coating to form a magnesium doped calcium silicate precursor surface layer to obtain the silicon/zinc ion doped biphase calcium phosphate ceramic bracket with the surface permeation coating;

and carrying out heat treatment on the doped biphase calcium phosphate ceramic bracket with the surface permeated and coated to obtain the surface modified silicon/zinc ion doped biphase calcium phosphate ceramic bracket.

Preferably, at least one of the following a and 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 speed 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 speed is 2000-4000r/min, the stirring speed in the washing process is 300-600r/min, the stirring time is 5-10min, the centrifugal speed of the washed solution is 3000-4000r/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 for dipping the silicon/zinc ion-doped biphase calcium phosphate ceramic bracket is-0.05 to-0.25 MPa, the dipping time is 10 to 30s, the dipping, penetrating and coating times are 1 to 3, the drying temperature is 40 to 60 ℃, and the drying time is 30 to 60 min; the final thickness of the surface layer of the magnesium-doped calcium silicate precursor formed by dip-dip coating is 5-20 μm.

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

The inventors have found that the release of active ions from doped biphasic calcium phosphate matrices is relatively slow and the promotion of early vascularization of the scaffold is not significant enough. The calcium silicate ions release more rapidly and can release silicon ions, while the magnesium ion-doped calcium silicate can release silicon ions and magnesium ions simultaneously, and the vascularization promoting performance is synergistically improved. Therefore, the magnesium-doped calcium silicate with a high ion release speed is used as a surface modification material of the silicon/zinc ion-doped biphase calcium phosphate ceramic scaffold with a low ion release speed, so that the release of the active ions for promoting vascularization at the early stage of the scaffold is facilitated, and the early vascularization of the scaffold is further promoted. The long-term slow release of the silicon and zinc ions by the stent matrix is beneficial to the later generation and growth of new bones. The regenerative repair of bone defects can be accelerated and achieved by the properly timed release of active ions by the surface layer and the scaffold matrix.

The inventor finds that the ion doping amount of magnesium-doped calcium silicate has certain influence on the early-stage mediated endothelial cell angiogenesis behavior and the coating stability of the material, and because the radius of magnesium ions is smaller than that of calcium ions, the stability of a calcium silicate crystal structure is damaged due to overhigh magnesium doping amount, the early-stage release amount of magnesium ions is overlarge, and the concentration of magnesium ions in a cell culture solution is overhigh, so that the expression of endothelial cells on angiogenesis-related genes is inhibited, and even cytotoxicity is generated; on the other hand, the amount of magnesium doped is too low, and the effect of promoting the expression of the angiogenesis-related gene is not sufficiently significant, so that the above-mentioned appropriate range of the amount of magnesium doped is preferable.

The inventors have found that conditions of osmotic coating have an effect on the performance of calcium phosphate scaffolds. The negative pressure can enable the magnesium-doped calcium silicate precursor slurry to permeate into pores on the surface of the calcium phosphate support, so that the surface coating layer and the matrix are combined more tightly, and the mechanical strength of the support is improved. If the negative pressure is too low, the slurry is not favorable to penetrate into 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, so that the degradation of the bracket matrix is influenced. The thickness of the coated surface layer depends on the dipping time and the dipping times, and if the thickness of the surface layer is too low, active ions for promoting vascularization are too little and the sustained release time is short, so that the initial vascularization is not sufficiently stimulated; if the thickness of the surface layer is too high, the time required by the degradation of the surface layer is prolonged, the release of the zinc ions by the bracket matrix is not facilitated, the subsequent generation and growth of new bones are limited, and the bone repair efficiency is reduced.

In addition, the heat treatment temperature has some influence on the stability and bioactivity of the magnesium-doped calcium silicate surface layer. If the heat treatment temperature is too low, the surface modification layer cannot 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, the release of active ions for promoting vascularization is slowed down, and the vascularization performance of the stent is not favorably improved.

The invention provides a surface-modified silicon/zinc ion-doped biphase calcium phosphate ceramic bracket prepared by the method, which can be applied to preparation of medical materials for repairing bone injury.

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

1. according to the invention, the silicon and zinc ion doped biphase calcium phosphate ceramic scaffold is prepared by compounding the silicon doped hydroxyapatite and the zinc doped beta-tricalcium phosphate, wherein the zinc doping has an osteogenesis promoting effect, the silicon doping has osteogenesis and vascularization promoting effects, and compared with the undoped biphase calcium phosphate ceramic scaffold, the zinc ion doped biphase calcium phosphate ceramic scaffold has excellent osteogenesis and vascularization promoting properties; the release amount of active ions of silicon and zinc and the degradation performance of the stent can be adjusted and controlled by adjusting and controlling the proportion of the silicon-doped hydroxyapatite to the zinc-doped beta-tricalcium phosphate.

2. Compared with the single ion doped biphase calcium phosphate ceramic scaffold, the silicon and zinc ion doped biphase calcium phosphate ceramic scaffold prepared by the invention has the advantages that the silicon and zinc ion doping can play a synergistic role in improving the osteogenesis and angiogenization capacity of the material, the defect of single ion doping can be overcome, the ion doping amount is easy to adjust, and therefore, the ion release rate which is beneficial to promoting the osteogenesis and angiogenization can be adjusted.

3. According to the invention, the surface of the biphase calcium phosphate ceramic bracket is further coated with the magnesium-doped calcium silicate in a permeating manner to prepare the surface-modified silicon/zinc ion-doped biphase calcium phosphate ceramic bracket, because the dissolution rates of the bracket base material and the surface permeation coating material are different, Mg and Si ions which have the effect of promoting blood vessels can be preferentially released by coating the magnesium-doped calcium silicate on the surface, the synergistic vascularization promotion of Mg and Si ions is beneficial to early promotion of the vascularization of the bracket pore, oxygen and nutrient substances are provided for the growth of new bones, metabolic wastes are eliminated, and the bone repair process is accelerated. The release rate and the release period of Mg and Si ions can be properly regulated and controlled by regulating and controlling proper magnesium ion doping amount, surface layer thickness and heat treatment temperature, and a foundation is laid for early-stage vascularization promotion.

4. The process for preparing the magnesium-doped calcium silicate surface modification layer of the bracket is simple, a magnesium-doped calcium silicate precursor is synthesized by a chemical precipitation method and prepared into slurry, the precursor slurry has cohesiveness, the uniformly distributed bracket hole wall surface modification layer can be prepared by infiltration coating by a dipping method under negative pressure, the magnesium-doped calcium silicate slurry can infiltrate into surface pores of the bracket hole wall under proper negative pressure, and the crystallized magnesium-doped calcium silicate surface modification layer is obtained by 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 magnesium-doped calcium silicate surface modification layer is combined with the pore wall of the bracket, so that the mechanical property of the bracket can be improved, the appearance and roughness of the surface of the bracket can be improved, and cell adhesion and migration are facilitated.

5. The invention can regulate the infiltration capacity and the surface layer thickness of the magnesium-doped calcium silicate by controlling the negative pressure, the impregnation time and the impregnation frequency of the magnesium-doped calcium silicate precursor slurry impregnation support.

Drawings

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

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

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

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

FIG. 5 is a graph showing the results of expression of angiogenesis-related genes on the surface of human umbilical vein endothelial cells in 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 an EDS elemental map of the surface, a section electron micrograph, and a surface of the stent of example 5.

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

FIG. 9 is a graph showing the expression of angiogenesis-related genes cultured on the surface of human umbilical vein endothelial cells in examples 1, 4 and 5.

Detailed Description

The following examples are included to further illustrate the practice of the invention, but are not intended to limit the practice or protection of the invention. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.

Comparative example 1

The comparative example uses undoped hydroxyapatite powder and beta-tricalcium phosphate powder with the zinc doping amount of 2.5 mol.% 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: 118.075g of calcium nitrate is dissolved in 1L of deionized water to obtain a calcium source solution, 39.618g of diammonium hydrogen phosphate is dissolved in 1L of deionized water to obtain a phosphorus source solution, the phosphorus source solution is dropwise added into the calcium source solution to obtain a reaction solution, stirring is continuously carried out at a speed of 600r/min in the dropwise adding process, meanwhile, ammonia water is added to ensure that the pH value of the reaction solution is 10.5, stirring is continuously carried out for 40min (the stirring speed is 600r/min) after the dropwise adding is finished, then the reaction solution is put into a high-pressure reaction kettle for hydrothermal treatment, the hydrothermal temperature is 180 ℃, the hydrothermal time is 16h, then, precipitates are centrifugally taken at a speed of 4000r/min, deionized water is repeatedly used for washing to remove soluble salts, the stirring speed in the washing process is 600r/min, the stirring time is 10min, the washed solution is centrifuged at a speed of 5000r/min, then the washed solution is placed in a 70 ℃ oven for drying for 18h, primary grinding is carried out, and then calcination is carried out at 950 ℃ in a muffle furnace, the heating rate is 5 ℃/min, the temperature is kept for 2h, and hydroxyapatite powder is obtained after calcination;

2) the method for synthesizing the zinc-doped beta-tricalcium phosphate powder by using the chemical precipitation method comprises the following steps: 207.222g of calcium nitrate and 6.694g of zinc nitrate are dissolved in 1L of deionized water to obtain a calcium/zinc source mixed solution, 79.236g of diammonium phosphate is dissolved in 1L of deionized water to obtain a phosphorus source solution, the phosphorus source solution is dripped into the calcium/zinc source mixed solution to obtain a reaction solution, continuous stirring is carried out at the speed of 600r/min in the dripping process, ammonia water is added simultaneously to ensure that the pH value of the reaction solution is 6.8, the reaction solution is continuously stirred for 40min (the stirring speed is 600r/min) after dripping is finished, then room temperature aging is carried out for 24h, sediment is obtained by centrifugation at the speed of 4000r/min, deionized water is repeatedly used for washing to remove soluble salt, the stirring speed in the washing process is 600r/min, the stirring time is 10min, the washed solution is centrifuged at the speed of 5000r/min, then the washed solution is placed in a 70 ℃ oven for drying for 18h, the solution is calcined at the temperature of 950 ℃ in a muffle furnace after primary grinding, the heating rate is 5 ℃/min, the temperature is kept for 2h, and zinc-doped beta-tricalcium phosphate powder is obtained after calcination;

3) adding the undoped hydroxyapatite powder obtained in the step 1) and the beta-tricalcium phosphate powder with the zinc doping amount of 2.5 mol.% obtained in the step 2) into a ball milling tank according to the mass percentage of 40:60 to form mixed powder, carrying out ball milling on the mixed powder, namely absolute ethyl alcohol and ball milling beads according to the mass ratio of 1:2:1, wherein the ball milling speed is 30Hz, the ball milling time is 2 hours, and drying the ball milling powder in a 60 ℃ drying oven for 2 hours after the ball milling is finished to obtain zinc ion doped biphase calcium phosphate powder;

4) taking 5g of zinc ion doped biphase calcium phosphate powder obtained in the step 3), adding 0.15 g of methylcellulose, fully and uniformly mixing, continuously dropwise adding 4.25g of polyvinyl alcohol solution with the concentration of 8 wt.%, uniformly stirring, then loading into a 3D printing charging barrel, and obtaining a zinc ion doped biphase calcium phosphate ceramic support blank through 3D printing;

5) and (5) sintering the biphase calcium phosphate ceramic support blank with the zinc doping amount of 2.5 mol.% obtained in the step 4) at the high temperature of 1150 ℃, wherein the heating rate is 2 ℃/min, and the heat preservation time is 2h, so as to obtain the zinc ion doped biphase calcium phosphate ceramic support.

Comparative example 2

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

1) the method for synthesizing the silicon-doped hydroxyapatite powder by using a chemical precipitation method and a hydrothermal method comprises the following steps: 118.075g of calcium nitrate is dissolved in 1L of deionized water to obtain a calcium source solution, 38.033g of diammonium hydrogen phosphate is dissolved in 1L of deionized water to obtain a phosphorus source solution, 2.72mL of tetraethyl orthosilicate and equal amount of absolute ethyl alcohol are uniformly mixed and then added into the phosphorus source solution to obtain a phosphorus/silicon mixed solution, then the phosphorus/silicon mixed solution is dropwise added into the calcium source solution to obtain a reaction solution, the reaction solution is continuously stirred at the speed of 600r/min in the dropwise adding process, meanwhile, ammonia water is added to ensure that the pH value of the reaction solution is 10.5, the stirring speed is continuously kept for 40min (the stirring speed is 600r/min) after the dropwise adding is finished, then the reaction solution is put into a high-pressure reaction kettle for hydrothermal treatment, the hydrothermal temperature is 180 ℃, the hydrothermal time is 16h, then, the precipitate is centrifugally taken at the speed of 4000r/min, the deionized water is repeatedly washed to remove soluble salts, the stirring speed in the washing process is 600r/min, stirring for 10min, centrifuging the washed solution at the speed of 5000r/min, drying the solution in a 70 ℃ oven for 18h, primarily grinding the solution, calcining the solution in a muffle furnace at the temperature of 950 ℃, keeping the temperature for 2h at the temperature rise speed of 5 ℃/min, and calcining the calcined solution to obtain silicon-doped hydroxyapatite powder;

2) the method for synthesizing the undoped beta-tricalcium phosphate powder by using the chemical precipitation method comprises the following steps: 212.535g of calcium nitrate is dissolved in 1L of deionized water to obtain a calcium source solution, 79.236g of diammonium hydrogen phosphate is dissolved in 1L of deionized water to obtain a phosphorus source solution, the phosphorus source solution is dripped into the calcium source solution to obtain a reaction solution, the reaction solution is continuously stirred at the speed of 600r/min in the dripping process, ammonia water is added simultaneously to ensure that the pH value of the reaction solution is 6.8, the reaction solution is continuously stirred for 40min (the stirring speed is 600r/min) after the dripping is finished, then the reaction solution is aged for 24h at room temperature, precipitate is obtained by centrifugation at the speed of 4000r/min, soluble salts are removed by repeated washing with the deionized water, the stirring speed in the washing process is 600r/min, the stirring time is 10min, the washed solution is centrifuged at the speed of 5000r/min, then the washed solution is placed in a 70 ℃ oven for drying for 18h, the first time, the solution is calcined in a muffle furnace at the temperature of 950 ℃, the temperature rising speed is 5 ℃/min, the temperature is kept for 2h, calcining to obtain undoped beta-tricalcium phosphate powder;

3) adding the hydroxyapatite powder with the silicon doping amount of 4 mol.% 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 of 40:60 to form mixed powder, carrying out ball milling on the mixed powder according to the mass ratio of anhydrous ethanol to ball milling beads of 1:2:1, wherein the ball milling speed is 30Hz, the ball milling time is 2 hours, and drying the ball-milled powder in a 60 ℃ drying oven for 2 hours to obtain silicon ion doped biphase calcium phosphate powder;

4) taking 5g of the silicon ion-doped biphase calcium phosphate powder obtained in the step 3), adding 0.15 g of methylcellulose, fully and uniformly mixing, continuously dropwise adding 4.25g of polyvinyl alcohol solution with the concentration of 8 wt.%, manually stirring uniformly, then loading into a 3D printing charging barrel, and obtaining a silicon ion-doped biphase calcium phosphate ceramic support blank through 3D printing;

5) sintering the silicon ion doped biphase 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 biphase calcium phosphate ceramic bracket.

Example 1

In this embodiment, hydroxyapatite powder with a silicon doping amount of 4 mol.% and β -tricalcium phosphate powder with a zinc doping amount of 2.5 mol.% are used as raw materials, and the specific process steps include:

1) the method for synthesizing the silicon-doped hydroxyapatite powder by using a chemical precipitation method and a hydrothermal method comprises the following steps: 118.075g of calcium nitrate is dissolved in 1L of deionized water to obtain a calcium source solution, 38.033g of diammonium phosphate is dissolved in 1L of deionized water to obtain a phosphorus source solution, 2.72mL of tetraethyl orthosilicate and equal amount of absolute ethyl alcohol are uniformly mixed and then added into the phosphorus source solution to obtain a phosphorus/silicon mixed solution, the phosphorus/silicon mixed solution is dropwise added into the calcium source solution to obtain a reaction solution, the reaction solution is continuously stirred at a speed of 600r/min in the dropwise adding process, ammonia water is simultaneously added to ensure that the pH value of the reaction solution is 10.5, the stirring is continuously carried out for 40min (the stirring speed is 600r/min) after the dropwise adding is finished, then the reaction solution is put into a high-pressure reaction kettle for hydrothermal treatment, the hydrothermal temperature is 180 ℃, the hydrothermal time is 16h, then the precipitate is centrifugally taken at a speed of 4000r/min, the deionized water is repeatedly washed to remove soluble salts, the stirring speed is 600r/min in the washing process, stirring for 10min, centrifuging the washed solution at the speed of 5000r/min, drying the solution in a 70 ℃ oven for 18h, primarily grinding the solution, calcining the solution in a muffle furnace at the temperature of 950 ℃, keeping the temperature for 2h at the temperature rise speed of 5 ℃/min, and calcining the calcined solution to obtain silicon-doped hydroxyapatite powder;

2) the method for synthesizing the zinc-doped beta-tricalcium phosphate powder by using the chemical precipitation method comprises the following steps: 207.222g of calcium nitrate and 6.694g of zinc nitrate are dissolved in 1L of deionized water to obtain a calcium/zinc source mixed solution, 79.236g of diammonium hydrogen phosphate is dissolved in 1L of deionized water to obtain a phosphorus source solution, the phosphorus source solution is dripped into the calcium/zinc source mixed solution to obtain a reaction solution, continuous stirring is carried out at the speed of 600r/min in the dripping process, ammonia water is added simultaneously to ensure that the pH value of the reaction solution is 6.8, stirring is continued for 40min (the stirring speed is 600r/min) after dripping is finished, then aging is carried out for 24h at room temperature, precipitate is obtained by centrifugation at the speed of 4000r/min, deionized water is used for repeatedly washing to remove soluble salt, the stirring speed in the washing process is 600r/min, the stirring time is 10min, the washed solution is centrifuged at the speed of 5000r/min, then the washed solution is placed in a 70 ℃ oven for drying for 18h, primary grinding is carried out, then calcination is carried out at the temperature of 950 ℃ in a muffle furnace, the temperature rise rate is 5 ℃/min, the temperature is kept for 2h, and zinc-doped beta-tricalcium phosphate powder is obtained after calcination;

3) adding the hydroxyapatite powder with the silicon doping amount of 4 mol.% obtained in the step 2) and the beta-tricalcium phosphate powder with the zinc doping amount of 2.5 mol.% obtained in the step 1) into a ball milling tank according to the mass percentage of 40:60 to form mixed powder, carrying out ball milling on the mixed powder according to the mass ratio of the anhydrous ethanol to ball milling beads of 1:2:1, wherein the ball milling speed is 30Hz, the ball milling time is 2h, and drying in a 60 ℃ oven for 2h after the ball milling is finished to obtain silicon/zinc ion doped double-phase calcium phosphate powder;

4) taking 5g of the silicon/zinc ion doped biphase calcium phosphate powder obtained in the step 3), adding 0.15 g of methyl cellulose, fully and uniformly mixing, continuously dropwise adding 4.25g of polyvinyl alcohol solution with the concentration of 8 wt%, manually stirring uniformly, then loading into a 3D printing charging barrel, and obtaining a silicon/zinc ion doped biphase calcium phosphate ceramic support blank through 3D printing;

5) sintering the silicon/zinc ion doped biphase calcium phosphate ceramic support 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 biphase calcium phosphate ceramic support.

FIG. 1 is an X-ray diffraction pattern of the ceramic scaffolds of comparative example 1, comparative example 2 and example 1. from FIG. 1, it can be seen that the phases of the ceramic scaffolds of comparative example 1, comparative example 2 and example 1 are all composed of beta-tricalcium phosphate (JCPDF NO.090169) and hydroxyapatite (JCPDF NO.090432) only, and no other impurity phase. Through the calculation of refinement, the mass percentages of the hydroxyapatite and the beta-tricalcium phosphate in the comparative example 1, the comparative example 2 and the example 3 are all maintained at about 40: 60.

Fig. 2 shows the results of inductively coupled plasma emission spectrometer (ICP) testing in example 1. Drying the autoclaved ceramic supports, placing the ceramic supports in 48-hole plates, arranging 3 parallel samples in each group of ceramic supports, then adding a high-sugar basic culture medium according to 0.5ml of each hole, replacing culture medium liquid every other day, collecting soak solution of the ceramic supports at time points of 1,3,7 and 14d, and detecting the concentration of Zn and Si elements in the soak solution by using ICP (inductively coupled plasma), so as to obtain the ion release concentration at the corresponding time point. According to the 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 indicate that silicate ions and zinc ions are successfully doped into hydroxyapatite and beta-tricalcium phosphate lattices respectively.

Fig. 3 shows the results of the in vitro degradation tests of comparative example 1 and example 1. Weighing the mass of the initial dry ceramic support, and recording the mass as m₀And transferring the ceramic support to a 10mL centrifuge tube, adding acetic acid-sodium acetate soak solution with the pH value of 4.5 according to the solid-to-liquid ratio of 0.02g/mL, placing 3 parallel samples in each group, placing the samples in a constant-temperature shaking table at 37 ℃ and 60r/min for incubation for 1, 2,4 and 8 weeks, replacing the soak solution every week, taking out the ceramic support at a corresponding time point, washing the ceramic support with deionized water, drying, weighing and recording the mass of the corresponding time point, recording the mass as m, and substituting the mass into the corresponding time point to calculate the weight loss rate of the ceramic support 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, and that the weight loss ratio of example 1 was 62.4% and the weight loss ratio of comparative example 1 was 46.5% at 8w of soaking, which indicates 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. The ceramic stents of comparative example 1 and example 1 are dried after high temperature and high pressure sterilization, placed in a 48-hole plate, added with complete culture medium for soaking and wetting for 6h, and the surfaces of the stents are 4 multiplied by 10⁴cells are inoculated at the density of cells/hole, osteogenic inducing liquid is replaced every other day, and the expression condition of osteogenic differentiation related genes is detected after 7d and 14d of culture. Comparing comparative example 1 with example 1, it was found that the stem cells cultured in example 1 expressed the early osteogenic differentiation associated genes Alkaline phosphatase (ALP) and Collagen-I (Col-I) significantly higher than those of comparative example 1 at the early stage of culture (day 7); the stem cells cultured in example 1 expressed the late osteogenic differentiation associated gene Bone Sialoprotein (BSP) at the late stage of culture (day 14) more than that of 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 better than that of the single zinc ion doping, and in addition, the inventor finds that the promotion effect of the silicon/zinc double-ion doping on the osteogenic differentiation of the stem cells is obviously better than that of the single silicon ion doping. Therefore, the silicon/zinc ion doped biphasic calcium phosphate ceramic scaffold has better bone-promoting performance.

FIG. 5 is a drawing of cultivationExpression of angiogenesis-associated 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 oven-dried after autoclaving, placed in 48-well plates, soaked and wetted for 6h with complete medium at 4X 10 of the material surface⁴cells are inoculated at the density of cells/hole, the complete culture medium of endothelial cells is replaced every other day, and the cells are cultured for 3d and 5d to detect the expression condition of the angiogenesis-related genes. Comparison between comparative example 2 and example 1 revealed that the Endothelial cells cultured in example 1 expressed the three angiogenesis-related genes Vasculelelate growth factor (VEGF), Hypoxia indicle factor-1 alpha (HIF-1 alpha), and Endothelial oxide synthase (eNOS) significantly higher than those in comparative example 2 on either day 3 or day 5. The results show that the promotion effect of the silicon/zinc double-ion doping on the endothelial cell vascularization is obviously better than that of the single-silicon-ion doping, and in addition, the inventor finds that the promotion effect of the silicon/zinc double-ion doping on the endothelial cell vascularization is obviously better than that of the single-zinc-ion doping in earlier research. Therefore, the silicon/zinc ion doped biphasic calcium phosphate ceramic stent has better performance of promoting blood vessels.

Example 2

In this embodiment, hydroxyapatite powder with a silicon doping amount of 4 mol.% and β -tricalcium phosphate powder with a zinc doping amount of 1.5 mol.% are used as raw materials, and the specific process steps include:

1) the method for synthesizing the silicon-doped hydroxyapatite powder by using a chemical precipitation method and a hydrothermal method comprises the following steps: 118.075g of calcium nitrate is dissolved in 1L of deionized water to obtain a calcium source solution, 38.033g of diammonium hydrogen phosphate is dissolved in 1L of deionized water to obtain a phosphorus source solution, 2.72mL of tetraethyl orthosilicate and equal amount of absolute ethyl alcohol are uniformly mixed and then added into the phosphorus source solution to obtain a phosphorus/silicon mixed solution, the phosphorus/silicon mixed solution is dropwise added into the calcium source solution to obtain a reaction solution, the reaction solution is continuously stirred at the speed of 400r/min in the dropwise adding process, meanwhile, ammonia water is added to ensure that the pH value of the reaction solution is 11, the stirring speed is continuously kept for 60min (the stirring speed is 400r/min) after the dropwise adding is finished, then the reaction solution is put into a high-pressure reaction kettle for hydrothermal treatment, the hydrothermal temperature is 140 ℃, the hydrothermal time is 24h, then, precipitate is centrifugally taken at the speed of 2000r/min, the deionized water is repeatedly washed to remove soluble salts, the stirring speed in the washing process is 400r/min, stirring for 20min, centrifuging the washed solution at the speed of 4000r/min, then drying the solution in a 40 ℃ oven for 24h, carrying out primary grinding, calcining the solution in a muffle furnace at the temperature of 850 ℃ at the heating rate of 5 ℃/min, and keeping the temperature for 3h to obtain silicon-doped hydroxyapatite powder;

2) the method for synthesizing the zinc-doped beta-tricalcium phosphate powder by using the chemical precipitation method comprises the following steps: 209.346g of calcium nitrate and 4.016g of zinc nitrate are dissolved in 1L of deionized water to obtain a calcium/zinc source mixed solution, 79.236g of diammonium hydrogen phosphate is dissolved in 1L of deionized water to obtain a phosphorus source solution, the phosphorus source solution is dripped into the calcium/zinc source mixed solution to obtain a reaction solution, the stirring is continuously carried out at the speed of 400r/min in the dripping process, ammonia water is added simultaneously to ensure that the pH value of the reaction solution is 6.4, the stirring is continuously carried out for 60min (the stirring speed is 400r/min) after the dripping is finished, then the reaction solution is aged for 12h at room temperature, precipitate is obtained by centrifugation at the speed of 2000r/min, deionized water is repeatedly used for washing to remove soluble salt, the stirring speed in the washing process is 600r/min, the stirring time is 20min, the washed solution is centrifuged at the speed of 4000r/min, then the washed solution is placed in a 40 ℃ oven for drying for 24h, the primary grinding is carried out, then the calcination is carried out at the temperature of 850 ℃ in a muffle furnace, the temperature rise rate is 5 ℃/min, the temperature is kept for 3h, and zinc-doped beta-tricalcium phosphate powder is obtained after calcination;

3) adding the hydroxyapatite powder with the silicon doping amount of 4 mol.% obtained in the step 1) and the beta-tricalcium phosphate powder with the zinc doping amount of 1.5 mol.% obtained in the step 2) into a ball milling tank according to the mass percentage of 20:80 to form mixed powder, carrying out ball milling on the mixed powder according to the mass ratio of the anhydrous ethanol to ball milling beads of 1:2:1, wherein the ball milling speed is 30Hz, the ball milling time is 2h, and drying the ball milled powder in a 60 ℃ drying oven for 2h to obtain silicon/zinc ion doped biphase calcium phosphate powder;

5) sintering the silicon/zinc ion doped biphase calcium phosphate ceramic bracket blank obtained in the step 4) at 1050 ℃ at a heating rate of 10 ℃/min for 3h to obtain the silicon/zinc ion doped biphase calcium phosphate ceramic bracket.

The result of phase analysis shows that the ceramic bracket in the example 2 is composed of biphase calcium phosphate without other mixed phases, and the mass percentages of the hydroxyapatite and the beta-tricalcium phosphate are both maintained at about 20: 80. As can be seen from the ICP test results, Zn²⁺The concentration in the culture medium is in the range of 0.2-0.28 mg/L; SiO 2₄ ^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 incorporated into hydroxyapatite and beta-tricalcium phosphate crystal lattices respectively. The results of inoculating mouse mesenchymal stem cells on the surfaces of the example 1 and the example 2 show that the two are slightly lower than the example 1 in the expression of the genes (ALP, Col-I, BSP) related to the proliferation, adhesion and osteogenic differentiation of the mouse mesenchymal stem cells in the example 2, but have no significant difference, which indicates that the example 1 and the example 2 have good osteogenic activity; the results of inoculating human umbilical vein endothelial cells on the surfaces of example 1 and example 2 show that there is no significant difference in proliferation and adhesion of endothelial cells and expression of angiogenesis-related genes (VEGF, HIF-1. alpha., eNOS), indicating that example 1 and example 2 have good angiogenesis activity.

Example 3

In this embodiment, hydroxyapatite powder with a silicon doping amount of 2 mol.% and beta-tricalcium phosphate powder with a zinc doping amount of 3 mol.% are used as raw materials, and the specific process steps include:

1) the method for synthesizing the silicon-doped hydroxyapatite powder by using a chemical precipitation method and a hydrothermal method comprises the following steps: 118.075g of calcium nitrate is dissolved in 1L of deionized water to obtain a calcium source solution, 38.826g of diammonium phosphate is dissolved in 1L of deionized water to obtain a phosphorus source solution, 1.36mL of tetraethyl orthosilicate and equal amount of absolute ethyl alcohol are uniformly mixed and then added into the phosphorus source solution to obtain a phosphorus/silicon mixed solution, the phosphorus/silicon mixed solution is dropwise added into the calcium source solution to obtain a reaction solution, the reaction solution is continuously stirred at the speed of 500r/min in the dropwise adding process, meanwhile, ammonia water is added to ensure that the pH value of the reaction solution is 10.8, the stirring is continuously carried out for 50min (the stirring speed is 500r/min) after the dropwise adding is finished, then the reaction solution is put into a high-pressure reaction kettle for hydrothermal treatment, the hydrothermal temperature is 160 ℃, the hydrothermal time is 20h, then, precipitate is centrifugally taken at the speed of 3000r/min, the deionized water is repeatedly washed to remove soluble salts, the stirring speed in the washing process is 500r/min, stirring for 15min, centrifuging the washed solution at the speed of 4500r/min, drying in a 60 ℃ oven for 22h, primarily grinding, calcining at 900 ℃ in a muffle furnace at the temperature rise speed of 5 ℃/min, preserving heat for 2.5h, and calcining to obtain silicon-doped hydroxyapatite powder;

2) the method for synthesizing the zinc-doped beta-tricalcium phosphate powder by using the chemical precipitation method comprises the following steps: 206.159g of calcium nitrate and 8.032g of zinc nitrate are dissolved in 1L of deionized water to obtain a calcium/zinc source mixed solution, 79.236g of diammonium phosphate is dissolved in 1L of deionized water to obtain a phosphorus source solution, the phosphorus source solution is dripped into the calcium/zinc source mixed solution to obtain a reaction solution, continuous stirring is carried out at the speed of 500r/min in the dripping process, ammonia water is added simultaneously to ensure that the pH value of the reaction solution is 6.6, stirring is continued for 50min (the stirring speed is 500r/min) after dripping is finished, then aging is carried out for 18h at room temperature, precipitate is obtained by centrifugation at the speed of 3000r/min, deionized water is used for repeatedly washing to remove soluble salt, the stirring speed in the washing process is 500r/min, the stirring time is 15min, the washed solution is centrifuged at the speed of 4500r/min, then the washed solution is placed in a 60 oven for drying for 22h, primary grinding is carried out, then calcination is carried out in a muffle furnace at the temperature of 900 ℃, the temperature rising speed is 5 ℃/min, keeping the temperature for 2.5h, and calcining to obtain zinc-doped beta-tricalcium phosphate powder;

3) adding the hydroxyapatite powder with the silicon doping amount of 2 mol.% obtained in the step 1) and the beta-tricalcium phosphate powder with the zinc doping amount of 3 mol.% obtained in the step 2) into a ball milling tank according to the mass percentage of 30:70 to form mixed powder, carrying out ball milling on the mixed powder according to the mass ratio of anhydrous ethanol to ball milling beads of 1:2:1, wherein the ball milling speed is 30Hz, the ball milling time is 2h, and drying the ball milled powder in a 60 ℃ oven for 2h to obtain silicon/zinc ion doped double-phase calcium phosphate powder;

4) taking 5g of the silicon/zinc ion doped biphase calcium phosphate powder obtained in the step 3), adding 0.15 g of methylcellulose, fully and uniformly mixing, continuously dropwise adding 4.25g of polyvinyl alcohol solution with the concentration of 8 wt.%, manually stirring uniformly, then loading into a 3D printing charging barrel, and obtaining a silicon/zinc ion doped biphase calcium phosphate ceramic support blank through 3D printing;

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

The phase analysis result shows that the ceramic scaffold in the example 3 is composed of biphasic calcium phosphate without other impurity phases, and the mass percentages of the hydroxyapatite and the beta-tricalcium phosphate are both maintained at about 30: 70. From the ICP test results, Zn²⁺The concentration in the culture medium is in the range of 0.36-0.52 mg/L; SiO 2₄ ^4-The concentration of 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 examples 1 and 3 show that the mouse mesenchymal stem cells have no significant difference in the proliferation, adhesion and expression of the osteogenic differentiation related genes (ALP, Col-I, BSP) of the mouse mesenchymal stem cells, which indicates that the examples 1 and 3 have good osteogenic activity; the results of inoculating human umbilical vein endothelial cells on the surfaces of example 1 and example 3 show that the proliferation, adhesion and angiogenesis related genes (VEGF, HIF-1 alpha, eNOS) of endothelial cells of the two are slightly lower than those of example 1 in example 3, but have no significant difference, which indicates that the angiogenesis activities of example 1 and example 3 are good.

Example 4

In this example, calcium silicate slurry with 5 mol.% of magnesium doping amount is prepared and surface-permeated and coated on the surface of the sample in example 1, and the specific process steps include:

1) 11.220g of calcium nitrate and 0.641g of magnesium nitrate are dissolved in 100mL of deionized water to obtain a calcium/magnesium source mixed solution, 14.21g of sodium silicate is dissolved in 100mL of deionized water to obtain a silicon source solution, under the condition of stirring (600r/min), the calcium/magnesium source mixed solution is dripped into the silicon source solution, stirring at the speed of 600r/min for 45min after the dripping is finished, centrifuging at 4000r/min to obtain precipitate, repeatedly washing with deionized water to remove soluble salt, stirring at 600r/min for 10min, centrifuging the washed solution at 4000r/min to obtain 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 5 mol.% magnesium-doped calcium silicate precursor slurry with the concentration of 0.6 mol/L;

2) placing the silicon/zinc co-doped calcium phosphate ceramic stent in example 1) in the magnesium-doped silicate slurry obtained in step 1) for dipping, performing surface permeation coating, wherein the permeation negative pressure is-0.20 MPa, the permeation time is 25s, then placing the stent in an oven at 50 ℃ for drying for 40min, and performing dipping and drying for 2 times to form a magnesium-doped calcium silicate surface layer with the thickness of 12 mu m, thereby obtaining a surface-modified dual-phase calcium phosphate ceramic stent;

3) placing the calcium phosphate ceramic support obtained in the step 2) in a muffle furnace, and carrying out heat treatment at 900 ℃ for 3h, wherein the heating rate is 8 ℃/min, so as to obtain the 5 mol.% magnesium-doped calcium silicate surface-modified silicon/zinc ion-doped biphase calcium phosphate ceramic support.

Example 5

In this example, calcium silicate slurry with 10 mol.% magnesium doping amount is prepared and surface-permeated and coated on the surface of the sample of example 1, and the specific process steps include:

1) 10.627g of calcium nitrate and 1.282g of magnesium nitrate are dissolved in 100mL of deionized water to obtain a calcium/magnesium source mixed solution, 14.21g of sodium silicate is dissolved in 100mL of deionized water to obtain a silicon source solution, under the condition of stirring (600r/min), the calcium/magnesium source mixed solution is dripped into the silicon source solution, after the dripping is finished, stirring at the speed of 600r/min for 45min, centrifuging at 4000r/min to obtain precipitate, repeatedly washing with deionized water to remove soluble salt, stirring at 600r/min for 10min, centrifuging the washed solution at 4000r/min to obtain 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 10 mol.% magnesium-doped calcium silicate precursor slurry with the concentration of 0.6 mol/L;

2) placing the silicon/zinc co-doped calcium phosphate ceramic stent obtained in the example 1 in the magnesium-doped silicate slurry obtained in the step 1) for dipping, performing surface permeation coating, wherein the permeation negative pressure is-0.20 MPa, the permeation time is 25s, then placing the stent in an oven at 50 ℃ for drying for 40min, and performing dipping and drying for 2 times to form a magnesium-doped calcium silicate surface layer with the thickness of 12 mu m so as to obtain a surface-modified dual-phase calcium phosphate ceramic stent;

3) placing the calcium phosphate ceramic support obtained in the step 2) in a muffle furnace, and carrying out heat treatment at 900 ℃ for 3h, wherein the heating rate is 8 ℃/min, so as to obtain the 10 mol.% magnesium-doped calcium silicate surface-modified silicon/zinc ion-doped biphase calcium phosphate ceramic support.

Fig. 6 is an X-ray diffraction pattern of the surface coatings of examples 4 and 5, and XRD patterns show that the phase of the coating layer of the silicon/zinc ion-doped biphasic calcium phosphate ceramic is calcium silicate, and as the magnesium doping amount increases, the characteristic peak shifts to a high angle, and it can be seen that magnesium ions successfully enter the calcium ion lattice sites.

Figure 7 is a topcoat characterization of example 5. As can be seen (top left is a stent with an uncoated surface, while top right is a stent with a coated surface), the surface modified ceramic stent has macro pores of about 400 μm. The coating thickness was about 12 μm (lower right). It can be seen from the EDS element distribution diagram (the middle diagram at the bottom, and the left diagram at the bottom is the corresponding topography diagram), that the Mg and Si elements are uniformly distributed on the surface of the ceramic support, which indicates that the magnesium-doped calcium silicate is successfully coated on the surface of the silicon/zinc ion-doped biphase calcium phosphate ceramic support.

FIG. 8 shows the results of the compression strength test of examples 1 and 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 scaffold could be improved by surface permeation coating of magnesium-doped calcium silicate.

FIG. 9 shows the expression of the angiogenesis-related genes by human umbilical vein endothelial cells after surface culture for 3d and 5d in examples 1, 4 and 5. The materials of example 1, example 4 and example 5 were oven-dried after autoclaving, placed in 48-well plates, soaked in complete medium for 6h, and the material surface was 4X 10⁴cells are inoculated at the density of cells/hole, the complete culture medium of endothelial cells is replaced every other day, and the cells are cultured for 3d and 5d to detect the expression of the angiogenesis-related genes. As can be seen from FIG. 9, examples 4 and 5 were able to up-regulate the expression of three angiogenesis-related genes including VEGF, HIF-1. alpha. and eNOS by endothelial cells on day 5, compared to example 1, wherein the up-regulation effect of example 5 was more significant. The result shows that the surface coating of the magnesium-doped calcium silicate can further improve the vascularization promoting performance of the silicon/zinc ion-doped biphase calcium phosphate ceramic stent, and is beneficial to the early vascularization of the ceramic stent.

Example 6

In this example, calcium silicate slurry with 12 mol.% magnesium doping amount is prepared, and the slurry is surface-permeated and coated on the surface of the sample in example 2, and the specific process steps include:

1) 10.390g of calcium nitrate and 1.538g of magnesium nitrate are dissolved in 100mL of deionized water to obtain a calcium/magnesium source mixed solution, 14.21g of sodium silicate is dissolved in 100mL of deionized water to obtain a silicon source solution, under the condition of stirring (500r/min), the calcium/magnesium source mixed solution is dripped into the silicon source solution, after the dripping is finished, stirring at the speed of 450 r/min for 30min, centrifuging at 3000r/min to obtain precipitate, repeatedly washing with deionized water to remove soluble salt, stirring at 500r/min for 8min, centrifuging at 4000r/min to obtain magnesium-doped calcium silicate precursor reactant, adding 200mL deionized water, adding 1.2g ammonium polymethacrylate dispersant, stirring at the speed of 400r/min for 30min to obtain uniform 12 mol.% magnesium-doped calcium silicate precursor slurry with the concentration of 0.4 mol/L;

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

3) placing the calcium phosphate ceramic support obtained in the step 2) in a muffle furnace, and carrying out heat treatment at 950 ℃ for 1h at a heating rate of 10 ℃/min to obtain a 12 mol.% magnesium-doped calcium silicate surface-modified silicon/zinc ion-doped biphase calcium phosphate ceramic support.

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

Example 7

In this example, calcium silicate slurry with 20 mol.% of magnesium doping amount is prepared and surface-permeated and coated on the surface of the sample of example 2, and the specific process includes:

1) 9.446g of calcium nitrate and 2.564g of magnesium nitrate are dissolved in 100mL of deionized water to obtain a calcium/magnesium source mixed solution, 14.21g of sodium silicate is dissolved in 100mL of deionized water to obtain a silicon source solution, under the condition of stirring (300r/min), the calcium/magnesium source mixed solution is dripped into the silicon source solution, after the dripping is finished, stirring at the speed of 300r/min for 15min, centrifuging at 2000r/min to obtain precipitate, repeatedly washing with deionized water to remove soluble salt, stirring at 300r/min for 5min, centrifuging at 3000r/min to obtain magnesium-doped calcium silicate precursor reactant, adding 200mL deionized water, adding 1.0g polyethylene glycol dispersant, stirring at the speed of 400r/min for 30min to obtain uniform magnesium-doped calcium silicate precursor slurry with the concentration of 0.4mol/L and the concentration of 5mol percent;

2) placing the silicon/zinc ion doped calcium phosphate ceramic scaffold obtained in the example 2 into the magnesium doped silicate slurry obtained in the step 1) for dipping, performing surface permeation coating, wherein the permeation negative pressure is-0.05 MPa, the permeation time is 10 s, and then placing the scaffold into a drying oven at 40 ℃ for drying for 60min to form a magnesium doped calcium silicate surface layer with the thickness of 5 mu m, so as to obtain a surface modified dual-phase calcium phosphate ceramic scaffold;

3) and 3) placing the calcium phosphate ceramic support obtained in the step 2) in a muffle furnace, and carrying out heat treatment for 1h at 850 ℃ at a heating rate of 5 ℃/min to obtain the surface-modified silicon/zinc ion-doped biphase calcium phosphate ceramic support of 20 mol.% magnesium-doped calcium silicate.

The phase analysis result shows that the phase of the surface coating layer of the biphase calcium phosphate ceramic is calcium silicate. The observation of a scanning electron microscope shows that the magnesium-doped calcium silicate coating uniformly coats the surface and the pore walls of the bracket, and the EDS element distribution analysis also shows that magnesium and silicon are uniformly distributed on the surface of the biphase calcium phosphate ceramic bracket. These demonstrate 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 example 6 and example 7 show that, compared with example 7, example 6 can better promote the proliferation and adhesion of human umbilical vein endothelial cells on the surface of the stent and the expression of VEGF, HIF-1 alpha and eNOS.

The examples of the present invention are given for clarity of illustration only, and are not intended to limit the embodiments of the present invention. Other variants and modifications of the embodiments described above will be obvious to those skilled in the art, and it is not necessary or exhaustive for all embodiments to be considered. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A preparation method of a silicon/zinc ion doped biphase calcium phosphate ceramic bracket is characterized by comprising 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 with the phosphorus source solution A2, then, dripping the mixture into the calcium source solution A1 to obtain a reaction solution A, adjusting the pH value of the reaction solution A, and then, 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 the 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, aging, centrifuging to obtain a precipitate, washing, centrifuging, drying and calcining to obtain zinc-doped beta-tricalcium phosphate powder;

4) and (4) sintering the silicon/zinc ion doped biphase calcium phosphate ceramic support blank obtained in the step 3) at a high temperature to obtain the silicon/zinc ion doped biphase calcium phosphate ceramic support.

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

a. the calcium source for synthesizing the silicon-doped hydroxyapatite powder is calcium nitrate, the phosphorus source is one or a combination of diammonium phosphate, ammonium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate, and the silicon source is tetraethoxysilane;

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 thickening agent is methyl cellulose, and the binder is polyvinyl alcohol.

3. The preparation method as claimed in claim 1, wherein in step 1), the pH value of the reaction solution A for synthesizing the silicon-doped hydroxyapatite powder is 10.5-11, the stirring rate 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 rate of the solution after the reaction is 2000-4000r/min, the washing solution is deionized water, the stirring rate in the washing process is 400-600r/min, the stirring time is 10-20min, the centrifugal rate of the solution after the washing is 4000-5000r/min, the drying temperature is 40-70 ℃, the drying time is 18-24h, the calcination temperature is 850-950 ℃, and the heat preservation time is 2-3 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 400-600r/min, the stirring time is 40-60min, the aging time is 12-24h, the centrifugal speed of the solution after the 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 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 2-3 h.

4. The preparation method according to claim 1, wherein in the step 4), the sintering temperature of the silicon/zinc ion doped biphasic calcium phosphate ceramic stent blank is 1050-.

5. A method for preparing a surface-modified silicon/zinc ion-doped biphasic calcium phosphate ceramic scaffold according to claim 1, comprising the steps of:

soaking the silicon/zinc ion doped biphase calcium phosphate ceramic bracket in magnesium doped calcium silicate precursor slurry, performing negative pressure surface permeation coating, drying, and performing repeated soaking permeation coating to form a magnesium doped calcium silicate precursor surface layer to obtain a silicon/zinc ion doped biphase calcium phosphate ceramic bracket with the surface permeation coated;

and carrying out heat treatment on the silicon/zinc ion doped biphase calcium phosphate ceramic bracket with the surface permeated and coated to obtain the surface modified silicon/zinc ion doped biphase calcium phosphate ceramic bracket.

6. The method of claim 5, wherein at least one of the following a, b:

7. The method as claimed in claim 5, wherein 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 3000-4000r/min, the washing solution and the slurry solution are deionized water, the dispersant is one or more of polyethylene glycol, polyacrylic acid and polymethacrylate ammonia, and the concentration of the dispersant 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 for impregnating the silicon/zinc ion-doped biphase calcium phosphate ceramic bracket is-0.05 to-0.25 MPa, the impregnation time is 10 to 30s, the times of impregnation, penetration and coating are 1 to 3, the drying temperature is 40 to 60 ℃, and the drying time is 30 to 60 min; the final thickness of the surface layer of the magnesium-doped calcium silicate precursor formed by dip-dip coating is 5-20 μm.

8. The method as claimed in claim 5, wherein the heat treatment temperature of the surface-permeation coated silicon/zinc ion doped biphasic calcium phosphate ceramic stent is 850-950 ℃, the temperature rise rate is 5-10 ℃/min, and the heat preservation time is 1-3 h.

9. A silicon/zinc ion doped biphasic calcium phosphate ceramic scaffold prepared by the process of any one of claims 1-4.

10. Surface-modified silicon/zinc ion-doped biphasic calcium phosphate ceramic scaffolds prepared by the method of any one of claims 1-8.