CN102526797B - Preparation method of high-strength biological glass bone bracket with regular-hole distribution - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and in particular relates to a preparation method of a high-strength biological glass bone bracket with regular hole distribution. The preparation method of the glass bracket with bioactivity and complete degradability includes the following steps: preparing borate bioactive glass by melting method, pulverizing and sieving the glass block into glass powder of a certain size; mixing the glass powder with organic The liquid propylene glycol block polyether solution is prepared into a uniform slurry; through the computer three-dimensional printing process, the scaffold precursor (green body) is printed out under the designed program; after the green body is dried, it is sintered at high temperature, The resulting glass scaffold not only has excellent biological activity.

Description

A preparation method of high-strength bioglass bone scaffold with regular pore distribution

technical field

The invention belongs to the technical field of bioengineering, and in particular relates to a preparation method of a high-strength biological glass bone bracket with regular hole distribution.

Background technique

As a temporary structure for the growth of cells, the scaffold is a place that can induce cell differentiation and regenerate tissues and organs similar to the shape of the scaffold, and plays an important role in bone tissue engineering. The biggest feature of bone tissue is that the intercellular matrix has a large amount of calcium salt deposition, forming one of the hardest tissues in the human body. Structurally, hydroxyapatite can be regarded as a compressed material. For scaffold materials in bone tissue engineering, the primary requirement is the mechanical properties, that is, the mechanical properties of the scaffold must match those of the environmental tissue: low-strength scaffolds are not enough to support the daily activities of the human body. At present, the methods for preparing scaffolds mainly include organic foam impregnation method, pore-forming agent method, gas foaming method, and heat-induced phase separation method. For example, in the Chinese invention patent, CN 101050053 B, the bone scaffold prepared by organic foam impregnation method is introduced ,. These methods all try to use the material tissue itself to build a scaffold structure, and the strength of the scaffold obtained by these traditional methods is generally low, and the load-bearing capacity cannot match the real bone. On the other hand, the scaffold material requires that it must be able to promote tissue regeneration, and the ability of tissue regeneration will be affected by the interaction between the properties of the material surface and cells, and the substance conduction of the scaffold.

In recent years, a new type of bone tissue material has received more and more attention, which is borate bioglass. It has good biological activity, and there is a close ion exchange between the biological glass, soft tissue and bone, which leads to the formation of chemical bonds between the material interface and human bone tissue, and the surface can generate a biologically active hydroxyapatite layer , can be completely degraded with time. However, due to the strength problem of the scaffold obtained by the traditional preparation method, it is difficult to apply the bioglass scaffold as an implant material in the load-bearing part.

Based on the existing preparation of a glass bracket with controllable degradation performance (Chinese invention patent, CN 101050053 B and Chinese invention patent, CN 101125218 B), this patent application describes a bioglass with regular pore distribution The high-strength bone scaffold and its preparation method, compared with the previous process, the strength of the scaffold prepared by this patent is increased by an order of magnitude, and has the characteristics of regular distribution and connectivity of pores. In this patent, the composition of the borate bioglass support; the specific implementation method of the three-dimensional printing technology; the preparation method of the printing paste; the cell experiment and the animal experiment on the support are specified. The results show that the borate glass support does not devitrify during the sintering process, and still maintains the original glass phase state, and has good processing performance; due to the uniform pore distribution, the green body shrinks in all directions during sintering. Therefore, the pore structure will not be changed; compared with the traditional method, the compressive strength of the scaffold is greatly improved; in addition, it has been confirmed through cell experiments and animal experiments that the bone tissue engineering scaffold prepared by this method has good biocompatibility, degradability and stimulation Growth properties of bone cells.

Contents of the invention

The purpose of the present invention is to provide a method for preparing a high-strength bioglass bone scaffold with regular pore distribution, which solves the problem that the slurry cannot be adapted to rapid prototyping, and the bioactivity of the scaffold prepared by the traditional method is not high enough, or the compressive strength is not enough High disadvantage.

The preparation method of the high-strength bioglass bone scaffold with regular pore distribution proposed by the present invention uses three-dimensional printing technology to print (extrude) the special borate bioglass paste and accumulate it into a scaffold precursor (honeycomb green body), Finally, it is sintered into a high-strength bioglass scaffold with regular pore structure. This kind of scaffold can not only be gradually degraded in physiological simulated fluid, but also has good biocompatibility and biological activity, and has a stimulating effect on osteogenesis; it also has high mechanical properties, which is beneficial to segmental bone repair in clinical practice. potential applications. Specific steps are as follows:

(1) Preparation of glass powder

The bioglass used for the preparation of the scaffold is a borosilicate system glass with borate as the main body. The composition of the bioglass powder is: B ₂ O ₃ or P ₂ O ₅ as the main body of the glass network or SiO ₂ Calcium-containing glass, the total molar ratio is 30-90 mol%, and the outer body of the glass contains CaO, and also contains Na ₂ O, K ₂ O alkali metal oxides and MgO, SrO alkaline earth metal oxides or rare earth metal oxides; The total molar ratio of ion oxides outside the network accounts for 5-80mol% of the total molar weight of the glass composition, and the molar content of alkaline earth metal oxides accounts for 5-60 molar% of the total molar weight; according to the composition and proportion of the above glass, take The industrial raw materials of oxides, chlorides, carbonates, sulfates and phosphates corresponding to metal oxides are used as glass batch materials, mixed evenly, and the glass is melted at 1000-1400 ° C and kept for 0.5-8 hours; then quenched Get glass blocks. The obtained glass blocks are coarsely crushed by a horizontal ball mill, finely crushed by a planetary ball mill or finely crushed by a jet mill, and sieved to obtain glass powder with a final particle size of 0.05-50 μm;

(2) Preparation of propylene glycol block polyether organic blending solution

Propylene glycol block polyether is synthesized by nucleophilic substitution reaction of polyether (molecular weight 1800-2500) and 2,6-di-tert-butyl-p-cresol (purity greater than 98%). The molecular formula of the former is HO.(C ₂ H ₄ O)m.(C ₃ H ₆ O)nH, and the latter is C ₁₅ H ₂₄ O. Mix the two at a weight ratio of 1:1-2.5:1 to form a suspension, dissolve the suspension with 50wt% toluene solution, and place it in a three-necked flask. Platinum chloric acid (H ₂ PtCl ₆ 6H ₂ O) isopropanol solution (concentration: 0.01mol/L) is used as the catalyst, the amount of the catalyst is 50ug/g (catalyst weight/reactant weight), at 70-90 Stir vigorously at ℃, condense and reflux, and protect with nitrogen. After reacting for 5-8 hours, take out the reactants, remove monomers and low polymers through vacuum distillation, and obtain viscous propylene glycol block polyether. Dissolve the synthesized propylene glycol block polyether (g) in 50% ethanol (mL) at a ratio of 1:0.5-1:1.5 to prepare a solution to obtain the desired organic blend. The phase transition temperature of the organic blend liquid characterized by viscosity method is between 20-40°C. Because the slurry we need is in the form of a sol at low temperature, has a certain fluidity, and solidifies immediately above the phase transition temperature. The phase transition temperature in this experiment is around room temperature, which is beneficial to control the occurrence of phase transition, that is, the phase transition temperature is conducive to the synthesis of the stent blank.

(3) Preparation of glass slurry

Mix the obtained glass powder with the above-mentioned blending liquid in a certain proportion, and the mixing ratio (ie glass powder weight (g): blending liquid volume (ml)) is 1: 0.1-1: 0.5; after vigorous stirring 4- After 8 hours, store it in a water bath at 10°C-20°C to remove air bubbles and make a uniform glass slurry. Finally put it in the refrigerator to age for 4 hours before use.

(4) Preparation of honeycomb green body

First sieve the slurry with a 10-225μm sieve to remove large agglomerates, and pour the sieved slurry into the injection (jet) injector of the printer, and the slurry will pass through the injection port with a diameter of 0.2-2 The millimeter-adjustable injection (jet) injector is extruded from the injection port (nozzle) under the pressure of 0.01-0.05MPa, and the extrusion speed is 0.05-1.0mm/s to form a continuous linear slurry column. During the printing process, the nozzle and the extruded slurry are located in an oil bath (99% kerosene) at 40°C-70°C, so that the printed slurry can quickly solidify into a solid column, which not only ensures that the printed blank Regular in shape, it also immediately bears the weight of a solid column subsequently added to it. The extruded first layer of slurry is flatly spread on the Al ₂ O ₃ plate, and the subsequent slurry is accumulated layer by layer from bottom to top, and solidified into a honeycomb green body composed of solid columns. The green body preparation process is pre-designed by computer programs, and the speed of extruding slurry is controlled throughout the process to adapt to the solidification time of the solid column bonded by the slurry; the position of extrusion is controlled throughout the process to adjust the up and down, The three-dimensional pores of left, right and high and low constitute honeycomb blanks with different porosity and pore diameter.

(5) Sintering of honeycomb body

The stent body is first put into an oven at 10-90°C and kept warm for 12-48 hours to remove most of the kerosene. Then put it into a muffle furnace for degumming and sintering. The sintering conditions are: increase the temperature at 1°C/min to 400-550°C and keep it for 5-24 hours; increase the temperature at 1°C/min to 550-700°C. Keep warm for 1-5 hours.

The scaffold obtained by using the invention can be applied to segmental bone repair, can be used as a bone scaffold material in bone tissue engineering, and can be completely degraded to form new bone.

The glass support prepared by the method of the present invention has better biocompatibility and can adhere to osteoblasts; has better biological activity, and can be transformed into hydroxyapatite, an inorganic component of bone after the glass support is degraded; has excellent biodegradation Sex, completely degraded in vivo. The obtained glass bracket has regular pores, a porosity of 10-95%, a maximum pore size of 10-1000 μm, and a compressive strength of 30-70 MPa, which is higher than that of commonly obtained brackets prepared by traditional methods. And because of the characteristics of the 3D printing method itself, the slurry is extruded according to the pre-designed 3D shape software, and the structure and performance of the scaffold can be pre-designed to meet the needs of different parts of bone repair.

Description of drawings

Figure 1 shows the topography of the bracket in various directions: (a) perspective view, (b) top view, (c) side view.

Figure 2 is the determination of the phase transition temperature of propylene glycol block polyether.

Figure 3 is the compressive strength curve of the stent.

Fig. 4 is the phase XRD measurement of the glass powder and the prepared support.

Figure 5 is the XRD pattern of the scaffold sample soaked for 20 days.

Figure 6 is the FTIR spectrum of the stent sample soaked for 20 days.

Figure 7 shows the morphology of osteoblast MC3T3-E1 on the scaffold and the cell adhesion on it at 37°C. Among them: (a) scaffold morphology after soaking for 3 days, (b) cell adhesion after 5 days, (c) cell adhesion after 9 days.

Fig. 8 is the X-ray film of the segmental bone defect after the stent embedding experiment. (a) 4 weeks, (b) 8 weeks, (c) 12 weeks.

Detailed ways

The present invention is further illustrated below by way of examples.

Example 1: The preparation of a three-dimensional connected scaffold consists of the following five steps.

(1) Preparation of glass powder

Weigh and weigh 3.8185g anhydrous sodium carbonate, 9.9587g anhydrous potassium carbonate, 1.7502g basic magnesium carbonate, 19.8328g calcium carbonate, 7.9781g strontium carbonate, 9.7420g silicon dioxide, 41.2991g boric acid, 5.6206g dihydrogen phosphate sodium. After mixing evenly and grinding, the ingredients were placed in a platinum crucible and kept at 1200 °C for 30 min; then the obtained glass liquid was poured on a steel plate and quenched to obtain a glass block. The obtained glass blocks are coarsely crushed by a horizontal ball mill, finely crushed by a ball mill and sieved to obtain glass powder with a final particle size of 0.05-5 μm.

(2) Preparation of propylene glycol block polyether organic compound solution and determination of phase transition temperature

Take 50ml of toluene solution (50wt%) and place it in a three-necked flask. Then weigh 15 grams of polypropylene glycol ether (molecular weight: 2200), and weigh another 10 grams of 2,6-di-tert-butyl-p-cresol (purity greater than 98%). The two are fully mixed to form a uniform suspension, and added dropwise into a three-neck flask. Then take 1.25 mg of isopropanol solution (concentration: 0.01 mol/L) of platinum chloric acid (H ₂ PtCl ₆ ·6H ₂ O) as a catalyst, and add it dropwise into the three-necked flask. Stir vigorously at a temperature of 90°C, condense and reflux, and protect with nitrogen. After reacting for 5 hours, take out the reactants and distill under reduced pressure to obtain viscous propylene glycol block polyether.

Dissolve the viscous propylene glycol block polyether with 30 milliliters of 50% ethanol to obtain the desired organic blend. Viscosity method was used to characterize the sudden change point of viscosity of organic blending liquid at different temperatures, and the phase transition temperature was determined to be 27°C (see Figure 2).

(3) Preparation of glass slurry

Weigh 5g of the above-mentioned glass powder, add 2ml of organic blending liquid solution, place in a beaker and stir evenly, so that the glass powder is evenly dispersed in the blending liquid solution. Then put it in a 10°C water bath to remove air bubbles, make a uniform slurry, put it in a 4°C refrigerator, and refrigerate for 12 hours before use.

(4) Preparation of honeycomb green body

First, sieve the slurry with a 75 μm sieve to remove large agglomerates, and then put the sieved slurry into the needle tube of the 3D printer. During the printing process, the needle tube and the extruded slurry were located in an oil bath (99% kerosene) at 40 °C. Put the Al ₂ O ₃ plate in the oil bath, place the printing nozzle on the Al ₂ O ₃ plate, start the computer printing program, and extrude the honeycomb green body from the nozzle of the printer to accumulate on the Al ₂ O ₃ plate.

(5) Sintering of honeycomb body

Put the honeycomb body in an oven at 30°C for 24 hours to remove most of the kerosene, then put it in a muffle furnace for degumming and sintering. Hours; raise the temperature to 600°C at a rate of 1°C/min, keep it warm for 1 hour, and then turn off the power. After 24 hours, the furnace temperature drops to room temperature, and the honeycomb green body is sintered into a bracket.

Through the above steps, a high-strength bioglass scaffold with a regular pore structure was prepared (see Figures 1A, 1B and 1C).

Embodiment 2: the mensuration of the aperture of support, porosity and the mensuration of intensity

According to the method described in Example 1, a three-dimensional connected scaffold was prepared. Referring to the national standard GBT 5164-1985 "Determination of Porosity of Permeable Sintered Metal Materials", the porosity of the scaffold was measured, and the results showed that the porosity of the scaffold was 60%, and the maximum pore size was 400 μm, as shown in Figure 1B and C consistent with the SEM observations. The compressive strength of the bracket in Example 1 was measured by a compressive strength tester to be 30 MPa (see FIG. 3 ).

Embodiment 3: the mensuration of biological property:

According to the method described in Example 1, a three-dimensional connected scaffold was prepared. The bioactivity, biodegradability and biocompatibility tests were carried out on the prepared scaffold.

XRD analysis was carried out on the obtained scaffold and compared with glass powder (see Figure 4), which indicated that after a series of thermal processing, the obtained scaffold was still in the glass phase. Then the stent was soaked in a physiological simulation solution at a temperature of 37°C. After 20 days, the stent was taken out, and the reaction process and post-reaction products were characterized by XRD and FTIR (see Figures 5 and 6). The XRD spectrum of the product was shown as The spectrum of a typical hydroxyapatite crystal, and the FTIR spectrum of the product is shown as a typical infrared spectrum of hydroxyapatite. The results show that the borate glass bioactive scaffold sample can be converted into strontium-containing hydroxyapatite in the in vitro biomineralization reaction, and has good bioactivity and degradability.

In order to investigate the biocompatibility and cell adhesion of the prepared scaffolds and osteoblasts, the prepared scaffolds were placed in culture dishes filled with MC3T3-E1 osteoblast culture medium for 3, 5 and 9 days, and then removed. Scaffolds were fixed with glutaraldehyde, extracted with ethanol to remove water and freeze-dried. SEM showed that a large number of cells crawled on the scaffold (see Figure 7), indicating that the scaffold had good biocompatibility and cell adhesion.

Embodiment 4: the animal experiment of scaffold:

According to the method described in Example 1, a three-dimensional connected scaffold was prepared. Animal experiments were carried out on the prepared scaffolds.

A 15 mm segmental bone defect was made in the middle part of the bilateral radius of New Zealand white rabbits, and the scaffold prepared in Example 1 was embedded. Fig. 8 is an experimental X-ray film of the segmental bone defect. After 4, 8, and 12 weeks, the bone growth was observed by X-ray film. It can be seen that after 4 weeks of implantation, the scaffold material is directly combined with the bone; after 8 weeks, the density of the scaffold material decreases, and the two ends of the material are basically fused with the bone; After 12 weeks, the material was completely degraded, bone formation was obvious, and bone shaping was basically completed. It shows that the prepared scaffold has a good repairing effect on the bone defect of animals.

Claims (3)

1. a preparation method of a high-strength bioglass bone support with regular pore distribution, is characterized in that concrete steps are as follows: (1) Preparation of glass powder The bioglass used for the preparation of the scaffold is a borosilicate system glass with borate as the main body. The composition of the bioglass powder is: B ₂ O ₃ or P ₂ O ₅ as the main body of the glass network or SiO ₂ Calcium-containing glass, accounting for 30-90 mol% of the total glass composition, the outer body of the glass network contains CaO, and also contains Na ₂ O, K ₂ O alkali metal oxides, MgO, SrO alkaline earth metal oxides; The total molar proportion of bulk ion oxides accounts for 5-80 mol% of the total molar weight of the glass composition, and the molar content of alkaline earth metal oxides including CaO accounts for 5-60 mol% of the total molar weight; B ₂ O ₃ or P ₂ O ₅ is the total molar mass of the main body of the glass network or the calcium-containing glass containing _SiO2 and the ion oxide outside the network to meet 100%; according to the composition and proportion of the above-mentioned glass, the oxide and chloride corresponding to the metal oxide The industrial raw materials of carbonate, sulfate and phosphate are used as glass batch materials, mixed evenly, and the glass is melted at 1000-1400°C and kept for 0.5-8 hours; then quenched to obtain glass blocks; Type ball mill for coarse crushing, planetary ball mill for fine crushing or jet mill for fine crushing, and sieve to obtain glass powder with a final particle size of 0.05-50 μm; (2) Preparation of propylene glycol block polyether organic blending solution Mix polyether with a molecular weight of 1800-2500 and 2,6-di-tert-butyl-p-cresol at a weight ratio of 1:1-2.5:1 to form a suspension, and dissolve the suspension with 50wt% toluene solution , placed in a three-necked flask; use the isopropanol solution of platinum chloric acid as a catalyst, the amount of catalyst per gram of reactant is 50 μg, stir at a temperature of 70-90 ° C, condense and reflux, protect with nitrogen, and react for 5-8 After 1 hour, take out the reactant, remove the monomer and low polymer by distillation under reduced pressure, and obtain a viscous propylene glycol block polyether; dissolve the synthesized propylene glycol block polyether (g) in 50% ethanol (ml) Among them, the solution is prepared at a ratio of 1:0.5-1:1.5, and the desired organic blended liquid can be obtained; the phase transition temperature of the organic blended liquid characterized by the viscosity method is between 20-40 °C; (3) Preparation of glass slurry Mix the glass powder obtained in step (1) with the organic blending liquid obtained in step (2), the glass powder weight (grams): the blending liquid volume (ml) is 1: 0.1-1: 0.5; after stirring for 4-8 Store in a water bath at 10°C-20°C after 1 hour to remove air bubbles and make a uniform glass slurry; put it in the refrigerator for aging and set aside; (4) Preparation of honeycomb green body First, sieve the glass slurry obtained in step (3) with a 10-225 μm sieve to remove large agglomerates, and pour the sieved slurry into the syringe of the printer. The diameter of the injection port of the slurry is 0.2-2 The millimeter-adjustable syringe is extruded from the injection port under the pressure of 0.01-0.05MPa, and the extrusion speed is 0.05-1.0mm/s, forming a continuous linear slurry column; the nozzle and the extruded material during the printing process The slurry is located in an oil bath at 40°C-70°C. The first layer of slurry extruded is spread on the Al ₂ O ₃ plate, and the subsequent slurry accumulates layer by layer from bottom to top and solidifies into a solid column. The honeycomb blank body; the blank body preparation process is pre-designed by the computer program, and the speed of the extruded slurry is controlled throughout the process to adapt to the solidification time of the slurry bonded to the lower solid column; the position of the extrusion is controlled throughout the process to adjust. The three-dimensional pores of up and down, left and right, and high and low formed by solid columns form honeycomb blanks with different porosities and pore diameters; (5) Sintering of honeycomb body The stent body is first put into an oven at 10-90°C for 12-48 hours to remove most of the kerosene; then it is put into a muffle furnace for degumming and sintering. 550°C, keep warm for 5-24 hours; increase temperature at 1°C/min to 550-700°C, keep warm for 1-5 hours. 2. The method according to claim 1, characterized in that the obtained glass support has regular pores, a porosity of 10-95%, a maximum pore size of 10-1000 μm, and a compressive strength of 30-70 MPa. 3. The method according to claim 1, characterized in that the obtained scaffold can be applied to segmental bone repair, can be used as a bone scaffold material in bone tissue engineering, and can be completely degraded to form new bone.

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