CN109364304B - Method for preparing degradable uniform multifunctional biological bionic scaffold through 3D printing - Google Patents

Abstract

The invention provides a method for preparing an inorganic doped material for bone repair, which comprises the following steps: uniformly mixing polyester and a polycarboxyl compound in a solid phase manner, and adding the mixture into a double-screw extruder for mixing materials in a circulating manner, wherein the molar ratio of the polycarboxyl compound to the polyester is 0.1-20, the temperature of the materials is controlled to be 50-150 ℃, and the materials are continuously extruded for 5-200 min under the vacuum condition of 1-100 KPa, so that the polyester with the end modified by carboxylation is obtained; and adding inorganic bone inducing material particles into the double-screw extruder, wherein the addition amount of the inorganic bone inducing material is 1-30% of the weight of the polyester, controlling the temperature of the material at 50-150 ℃, and continuously extruding the material for 5-200 min under a vacuum condition to finally obtain the inorganic doped material for bone repair. The invention also provides a method for preparing the bone repair bionic scaffold by using the inorganic doped material through 3D printing. The method provided by the invention realizes uniform dispersion of inorganic materials in nanoscale under the solvent-free condition, has the advantages of simple process, low production cost, no generation of three wastes, environmental friendliness and suitability for industrial and continuous production.

Description

Method for preparing degradable uniform multifunctional biological bionic scaffold through 3D printing

Technical Field

The invention relates to the field of biomedical materials, in particular to a method for preparing a uniform bionic scaffold by 3D printing.

Background

Degradable biomaterials such as polylactic acid (PLA), polylactide glycolide (PLGA), etc. have good biocompatibility and exhibit great advantages in tissue or organ repair. In bone tissue repair, inorganic components such as Hydroxyapatite (HA), β -tricalcium phosphate (TCP), and the like are often introduced in order to accelerate bone regeneration. The common technical means is to directly carry out physical blending in the presence of a solvent or in the absence of the solvent, but inorganic materials cannot be uniformly dispersed in a high polymer material, so that the defects of low osteogenesis speed, low bone density, new bone deformity and the like are caused.

Inorganic doped biomedical material scaffolds can be prepared by a variety of methods. The most common method is natural drying or freeze drying after adding a porogen such as sodium chloride to the composite solution, sometimes with additional voids created by phase separation. The defects are that the connectivity of the hole is poor, the migration and differentiation of cells are influenced, and the in-vivo repair effect is further influenced. As an improved method, a scaffold with a definite open structure can be well obtained by using 3D printing and forming, such as solution low-temperature layer stack forming (e.g. using TissForm low-temperature rapid prototyping machine 3D printing bone repair material solution disclosed in patent document CN 102824657a, a three-dimensional hole structure which is communicated with each other can be formed).

Zhang Jian et al disclose a method for obtaining a uniform scaffold by an ion micelle pouring method in Advanced materials 2017,29, firstly, the end group of poly (lactic-co-glycolic acid) (PLGA) is subjected to carboxylation modification by succinic anhydride in the presence of a catalyst, and is self-assembled into a uniform and stable ion micelle precursor with nano-Hydroxyapatite (HA) in a 1, 4-dioxane solution, then a sodium chloride pore-forming agent is added, and the uniform material scaffold is obtained by freeze drying, so that a good in vivo bone defect repairing effect is shown, for example, the repairing speed is high, the bone density is high, a new bone rule is obtained, and the like.

However, the inorganic doped biomedical material has the end group only introduced with one carboxyl group, has low bonding strength with inorganic materials, uses organic chemical solvents, and has low preparation efficiency, difficult mass production and large environmental protection pressure; and the expense of the molding equipment is also a problem to be considered. There is therefore a need from an industrial point of view to find more economical, more efficient and more environmentally friendly alternatives.

The present invention aims to achieve uniform dispersion of inorganic materials in matrix polymerization and high-strength bonding with matrix materials in the form of chemical bonds, while it is desired to impart multi-functionalization to the stent.

Disclosure of Invention

The invention aims to: the method for preparing the uniform bionic scaffold is simple in process, free of organic solvent, free of pollution and low in cost, and not only can remarkably improve the performance of the bionic scaffold, but also can remarkably improve the industrial application value of the preparation method.

The above object of the present invention is achieved by the following technical solutions:

first, there is provided a method of preparing an inorganic doped material for bone repair, comprising: firstly, polyester with good biocompatibility and a polycarboxyl compound are subjected to esterification reaction under the solvent-free condition to obtain polyester with a carboxyl-modified tail end; and then calcium ions of the inorganic bone induction material and carboxyl of the modified polyester are crosslinked under the solvent-free condition to obtain the inorganic doped material with good dispersibility and high bonding strength of the inorganic material.

The method specifically comprises the following steps:

1) uniformly mixing biocompatible polyester and a polycarboxyl compound in a solid phase manner, and adding the mixture into a double-screw extruder for mixing materials in a circulating manner, wherein the molar ratio of the polycarboxyl compound to the polyester is 0.1-20, the temperature of the materials is controlled to be 50-150 ℃, and the materials are continuously extruded for 5-200 min under the vacuum condition of 1-100 KPa, so that the polyester with the end subjected to carboxylation modification is obtained;

2) after the reaction in the step 1) is finished, adding inorganic bone induction material particles into the double-screw extruder, wherein the addition amount of the inorganic bone induction material is 1-30% of the weight of the polyester added in the step 1), controlling the temperature of the material at 50-150 ℃, and continuously extruding the material for 5-200 min under a vacuum condition to finally obtain the inorganic doped material for bone repair.

Biocompatible polyesters useful in the methods of the present invention include, but are not limited to, any of the following polyesters: linear polyesters, examples include linear polylactic acid, linear polyglycolide lactide, linear polyglycolide, linear polycaprolactone, linear polylactide caprolactone, linear polyglycolide lactide caprolactone, linear polylactide polyethylene glycol, or linear polycaprolactone polyethylene glycol; or a branched polyester, examples including branched polylactic acid, branched polyglycolide lactide, branched polyglycolide, branched polycaprolactone, branched polylactide caprolactone, branched polyglycolide lactide caprolactone, branched polylactide polyethylene glycol, or branched polycaprolactone polyethylene glycol; branched polyesters are preferred according to the invention; most preferred is a branched polycaprolactone polyethylene glycol.

The carboxyl number n of the polycarboxyl compound which can be used in the method is more than or equal to 2, and the category of the polycarboxyl compound comprises any one of citric acid, tartaric acid, oxalic acid, succinic acid, glutaric acid, adipic acid, sebacic acid, succinic anhydride, glutaric anhydride, adipic anhydride, polymalic acid, polyaspartic acid or polymalic acid aspartic acid; preferably any one of succinic anhydride, polymalic acid, polyaspartic acid or polyaspartic acid; most preferred is polymalic acid or polyaspartic acid having a carboxyl group number of 10 to 20.

The polymalic acid, polyaspartic acid or polymalic acid aspartic acid is preferably prepared from D, L-malic acid, D-malic acid and L-aspartic acid which are monomers.

In the method of the present invention, a lubricant is preferably added along with the inorganic osteoinductive material particles in step 2) to promote the dispersibility of the inorganic material in the polyester material.

The lubricant which can be used in the method of the present invention includes but is not limited to any one or a mixture of more than two of stearic acid, glyceryl stearate, polyethylene glycol, polyester polyethylene glycol copolymer or acetyl tributyl citrate, wherein the weight of the lubricant is 0.3-50% of the weight of the polyester in the step 1), and preferably 0.3-20% of the weight of the polyester.

The inorganic osteoinductive material that can be used in the method of the invention may be selected from any one or a combination of two of hydroxyapatite and tricalcium phosphate; hydroxyapatite is preferred in the invention; nanoscale hydroxyapatite is most preferred.

In a more preferred embodiment of the present invention, the water content of the hydroxyapatite is controlled to be 0.1 to 10 wt%.

In the scheme of the invention, the molar ratio of the polycarboxyl compound to the polyester is preferably 0.1-15; more preferably 1.05.

In the embodiment of the present invention, the particle size of the inorganic osteoinductive material is preferably 20nm to 5 μm, and more preferably 20nm to 200 nm.

In the preferable scheme of the invention, the diameter of the screw of the double-screw extruder is 2-75 mm; the length-diameter ratio is more than or equal to 10, and more preferably 25-40; the rotating speed is 60-1000 rpm.

In a preferred embodiment of the present invention, the method for preparing an inorganic doped material for bone repair comprises the following steps:

1) uniformly mixing the branched polycaprolactone polyethylene glycol and succinic anhydride in a molar ratio of 1:1.05 in a solid phase manner, and then adding the mixture into a double-screw extruder for extruding and shearing, wherein the diameter of a screw of the double-screw extruder is 2-75 mm; the length-diameter ratio is 25-40, and the rotating speed is 60-1000 rpm; controlling the temperature of the material at 80-100 ℃, and continuously extruding the material for 5-20 min under the vacuum condition of 1-100 KPa to obtain branched polycaprolactone polyethylene glycol with the tail end modified by carboxylation;

2) after the reaction in the step 1) is finished, adding hydroxyapatite particles with the particle size of 20-200nm and stearic acid into the double-screw extruder, wherein the adding amount of the hydroxyapatite is 1-30% of the weight of the branched polycaprolactone polyethylene glycol added in the step 1), and the adding amount of the stearic acid is 0.3-20% of the weight of the branched polycaprolactone polyethylene glycol added in the step 1); and controlling the temperature of the material at 80-100 ℃, and continuously extruding the material for 5-20 min under a vacuum condition to finally obtain the inorganic doped material for bone repair.

In another preferred embodiment of the present invention, the method for preparing an inorganic doped material for bone repair comprises the steps of:

1) uniformly mixing the branched polycaprolactone and the polymalic acid in a molar ratio of 1:1.05 in a solid phase manner, and then adding the mixture into a double-screw extruder for extruding and shearing, wherein the diameter of a screw of the double-screw extruder is 2-75 mm; the length-diameter ratio is 25-40, and the rotating speed is 60-1000 rpm; controlling the temperature of the material at 80-120 ℃, and continuously extruding the material for 30-50 min under normal pressure to obtain the branched polycaprolactone with the tail end modified by carboxylation;

2) after the reaction in the step 1) is finished, adding hydroxyapatite particles with the particle size of 20-200nm into the double-screw extruder, wherein the adding amount of the hydroxyapatite is 10-15% of the weight of the branched polycaprolactone added in the step 1), controlling the temperature of the material at 80-120 ℃, and continuously extruding the material for 30-50 min under a vacuum condition to finally obtain the inorganic doped material for bone repair.

In another preferred embodiment of the present invention, the method for preparing an inorganic doped material for bone repair comprises the steps of:

1) uniformly mixing polyglycolide-lactide and polyaspartic acid in a molar ratio of 1:1.05 in a solid phase manner, and then adding the mixture into a double-screw extruder for extruding and shearing, wherein the diameter of a screw of the double-screw extruder is 2-75 mm; the length-diameter ratio is 25-40, and the rotating speed is 150-250 rpm; controlling the temperature of the material at 120-150 ℃, and continuously extruding the material for 15-20 min under the vacuum condition of 1-1.5 KPa to obtain polyglycolide lactide with a carboxyl modified tail end;

2) after the reaction in the step 1) is finished, adding hydroxyapatite particles with the particle size of 200nm and 100 meshes into the double-screw extruder, wherein the adding amount of the hydroxyapatite is 8-10% of the weight of the polyglycolide lactide added in the step 1), controlling the temperature of the material at 120-150 ℃, and continuously extruding the material for 5-10 min under a vacuum condition to finally obtain the inorganic doped material for bone repair.

On the basis, the invention further provides a method for preparing the bionic scaffold for bone repair by 3D printing, which comprises the following steps:

directly drawing the inorganic doped material for bone repair prepared by the method to prepare microfilaments with the diameter of 0.1-5 mm;

and secondly, the microfilament prepared in the step I is printed and formed through solid-phase 3D to obtain the bionic scaffold for bone repair.

In the scheme of the invention, the diameter of the microfilament in the step (i) is preferably 1.5-3 mm.

In the scheme of the invention, the 3D printer in the step (II) is in a conventional market specification, and the preferred precision is not lower than 0.02 mm.

Compared with the prior art, the method has the following beneficial effects:

1. the preparation process is simple and does not need any organic solvent. In the existing inorganic material doping technology, both polyester modification and 3D forming methods need to use organic solvents, the production process is complex, the production cost and the environmental protection pressure are extremely high, the obtained economic benefit is very limited, and the industrial production is not facilitated. The inventor finds out through experiments that a skillful method for promoting polyester and polyhydroxy compound to generate solid esterification by using mechanical force and further crosslinking the polyester and the polyhydroxy compound with an inorganic bone inducing material in a solid state is provided, the method can realize uniform dispersion of the inorganic material in a high polymer material, the process is very simple, more importantly, no solvent is needed, the obtained product can be used as a solid 3D printing material after being directly filamentized, compared with the liquid bone repairing 3D printing material in the prior art, the applicable printing resolution is obviously improved, and a vacuum treatment process in the printing and forming process can be omitted. The improvement in many aspects is significant to the industrial production of medical materials for bone repair and the like, the production cost of the inorganic doped biomedical bracket is obviously reduced, the environmental safety of the process is greatly improved, and the method has high industrial application value.

2. In the method, polyester and a polycarboxyl compound are subjected to esterification reaction in a solid state under the mechanical action provided by a double-screw extruder, such as extrusion force, shearing force and the like, a plurality of carboxyl functional groups are introduced at the tail end of the polyester, and inorganic materials are added for crosslinking after the esterification reaction is finished. Experiments show that compared with a blending reaction mode of adding polyester, a carboxyl modifier and an inorganic material at one time, the step reaction mode of the invention can enable the polycarboxyl of the polyester and the calcium ions of the inorganic material to be crosslinked more fully, not only can avoid the inorganic material from agglomerating and enable the inorganic material to be dispersed in the polyester very uniformly, but also can combine the inorganic material with the polyester material in a chemical bond form in high strength, thereby obtaining more excellent modification effect than the prior art.

3. After the scaffold is formed, the residual carboxyl groups which are not crosslinked on the surface of the material can be used for coupling active growth factors to further improve the biocompatibility of the material or for coupling hydroxyl-containing drugs for repair and treatment purposes.

4. In a preferred embodiment of the invention, the branched polyester is used, and the material processing temperature can also be significantly reduced. And continuous adjustment of processing temperature and mechanical properties of materials can be realized according to the number of the branched arms and the control of the molecular weight of the polyester. In a word, the method has the advantages of low production cost, no three wastes, environmental protection and suitability for industrial and continuous production.

Drawings

FIG. 1 is a four-arm polycaprolactone of example 1 of the present invention¹HNMR structure characterization map.

FIG. 2 shows a carboxylated modified four-arm polycaprolactone prepared in example 1 of the present invention¹HNMR structure characterization map.

FIG. 3 is an optical photograph of the physical appearance of the composite biological scaffold prepared by the method of example 2 of the present invention.

FIG. 4 is a low power electron micrograph of the composite bioscaffold prepared according to the method of example 2 of the present invention.

FIG. 5 is a high magnification electron micrograph of the composite bioscaffold prepared according to the method of example 2 of the present invention.

Detailed Description

The present invention will be further described with reference to specific examples, but it should not be construed that the subject matter of the present invention is limited to the examples.

All compounds and reagents used in the following examples are either available products or products that can be prepared by existing methods.

Example 1: preparation of four-arm polycaprolactone/hydroxyapatite composite material scaffold

(1) Preparation of polymalic acid

Putting 10g D L-malic acid into a 3-neck flask, introducing nitrogen for protection, controlling the reaction temperature at 130 ℃, carrying out electromagnetic stirring reaction for 4 hours, and measuring the molecular weight, wherein the Mn is 2420.

(2) Preparation of four-arm polycaprolactone/hydroxyapatite composite microfilament

Adding 8g of four-arm polycaprolactone (Mn 80,000) and 0.042g of polymalic acid prepared in step (1) into a silo of a twin-screw extruder (L/D20) with a screw diameter phi of 10mm, rotating at 200 rpm, and controlling the reaction temperature: naturally exhausting at 80 deg.c, extruding and shearing the material in a double screw extruder in circulating mode, and continuous reaction for 30 min. And then under the condition of not stopping the machine, adding 1g of nano-hydroxyapatite (the water content is 1.2%) into the feeding hole, continuously extruding and shearing the materials in the machine in a circulating manner, and continuously reacting for 30min to obtain the four-arm polycaprolactone/hydroxyapatite composite material.

Dissolving the four-arm polycaprolactone and the carboxylated modified four-arm polycaprolactone in CDCl₃To carry out¹And (3) HNMR characterization, wherein results are shown in figures 1 and 2, hydrogen nuclear magnetic spectra of the two are obviously different, and a peak near 7.2ppm shown in figure 2 is a characteristic peak of the fumaric acid end group of the polymalic acid, which indicates that the polymalic acid and polycaprolactone react to form a chemical bonding reaction.

(3) The four-arm polycaprolactone/hydroxyapatite composite material is directly extruded into filaments by a wire drawing machine, and the diameter of the filaments is controlled to be 1.75 mm.

(4) Preparation of four-arm polycaprolactone/hydroxyapatite composite material scaffold

By using a commercial FDM 3D printer, the temperature of the spray head is set to 80 ℃, the temperature of the hot bed is set to 25 ℃, and the printing speed is set to 30mm/s, so that a smooth and complete 3D porous support is finally obtained, and the overall appearance and microstructure of the support are shown in figures 3-5.

From the optical photograph of fig. 3, it can be seen that the entire structure of the stent prepared in this example is very complete; the clearer overall structure of the stent can be seen from the low power electron micrograph of fig. 4; from the high power electron micrograph of FIG. 5, it can be seen that the inorganic salts are uniformly distributed in the scaffold prepared in this example. In addition, the mechanical property detection shows that compared with the pure four-arm polycaprolactone stent, the compressive strength of the stent prepared by the embodiment is improved by at least 143.6% under the same deformation displacement.

Example 2: preparation of polyglycolide-lactide (PLGA)/hydroxyapatite composite material stent

(1) Preparation of polyaspartic acid

10g L-aspartic acid was put into a 3-neck flask, nitrogen was introduced for protection, the reaction temperature was controlled at 130 ℃, the reaction was carried out for 4 hours with electromagnetic stirring, and the molecular weight was determined to be 2600.

(2) Preparation of polyglycolide-lactide (PLGA)/hydroxyapatite composite filament

10g of PLGA (Mn 75,000) and 0.025g of polyaspartic acid were introduced into a silo of a 10 mm-screw extruder (L/D20) at a speed of 200 rpm, the reaction temperature being controlled: the reaction was carried out at 150 ℃ under a vacuum of 1.2KPa in a cyclic manner for 20 min. Then 1g of nano-hydroxyapatite (water content 1.2%) and 2g of lubricant PEG2000-PCL (Mn 1.2X 10) were added into the feed inlet⁴) And continuously reacting in the machine for 5min in a circulating manner, discharging and pelletizing.

And extruding the composite material into filaments by using a wire drawing machine, and controlling the diameter of the filaments to be 1.75 mm.

(3) Preparation of polyglycolide-lactide (PLGA)/hydroxyapatite composite scaffold

And (3) setting the temperature of a spray head to be 160 ℃, the temperature of a hot bed to be 45 ℃ and the printing speed to be 30mm/s by using a commercial FDM 3D printer, and finally obtaining the smooth and complete 3D porous composite material support.

Example 3: preparation of four-arm polycaprolactone/hydroxyapatite composite material bracket loaded with active growth factor RGD

Preparing a 0.5% RGD aqueous solution, adding NHS and EDI, and incubating the 3D scaffold obtained in example 1 in the solution for 24 h. And rinsing the composite material bracket with distilled water for three times to obtain the RGD modified composite material bracket.

Claims (12)

1. A method for preparing an inorganic doped material for bone repair specifically comprises the following steps:

1) uniformly mixing biocompatible branched polyester and a polycarboxyl compound in a solid phase, and adding the mixture into a double-screw extruder for mixing materials in a circulating mode, wherein the molar ratio of the polycarboxyl compound to the branched polyester is 0.1-20; controlling the temperature of the material at 50-150 ℃, and continuously extruding the material for 5-200 min under the vacuum condition of 1-100 KPa to obtain branched polyester with the tail end modified by carboxylation; the branched polyester is selected from branched polyglycolide lactide, branched polyglycolide, branched polycaprolactone, branched polylactide caprolactone, branched polylactide polyethylene glycol or branched polycaprolactone polyethylene glycol; the polycarboxyl compound is selected from polymalic acid or polyaspartic acid;

2) after the reaction in the step 1) is finished, adding inorganic bone inducing material particles and a lubricant into the double-screw extruder, wherein the adding amount of the inorganic bone inducing material is 1-30% of the weight of the branched polyester added in the step 1), the adding amount of the lubricant accounts for 0.3-20% of the weight of the branched polyester added in the step 1), and the inorganic bone inducing material is hydroxyapatite with the water content of 1.2-10 wt%; and controlling the temperature of the material at 50-150 ℃, and continuously extruding the material for 5-200 min under a vacuum condition to finally obtain the inorganic doped material for bone repair.

2. The method of claim 1, wherein: the branched polyester in the step 1) is branched polycaprolactone polyethylene glycol.

3. The method of claim 1, wherein: the polycarboxyl compound in the step 1) is polymalic acid or polyaspartic acid with the carboxyl number of 10-20.

4. The method of claim 1, wherein: the molar ratio of the polycarboxyl compound to the branched polyester in the step 1) is 0.1-15.

5. The method of claim 1, wherein: the molar ratio of the polycarboxyl compound to the branched polyester in the step 1) is 1.05.

6. The method of claim 1, wherein: the inorganic bone inducing material in the step 2) is nano-grade hydroxyapatite with the water content of 1.2 percent.

7. The method of claim 1, wherein the lubricant of step 2) is selected from one or more of stearic acid, glyceryl stearate, polyethylene glycol, polyester polyethylene glycol copolymer, and acetyl tributyl citrate.

8. The method of claim 1, wherein the twin screw extruder has a screw diameter of 2 to 75 mm; the length-diameter ratio is more than or equal to 10; the rotating speed is 60-1000 rpm.

9. The method of claim 1, wherein the twin screw extruder has a length to diameter ratio of 25 to 40.

10. A method of preparing an inorganic doped material for bone repair as claimed in claim 1 comprising the steps of:

1) uniformly mixing the branched polycaprolactone and the polymalic acid in a molar ratio of 1:1.05 in a solid phase manner, and then adding the mixture into a double-screw extruder for extruding and shearing, wherein the diameter of a screw of the double-screw extruder is 2-75 mm; the length-diameter ratio is 25-40, and the rotating speed is 60-1000 rpm; controlling the temperature of the material at 80-120 ℃, and continuously extruding the material for 30-50 min under normal pressure to obtain the branched polycaprolactone with the tail end modified by carboxylation;

2) after the reaction in the step 1) is finished, adding hydroxyapatite particles with the particle size of 20-200nm into the double-screw extruder, wherein the adding amount of the hydroxyapatite is 10-15% of the weight of the branched polycaprolactone added in the step 1), controlling the temperature of the material at 80-120 ℃, and continuously extruding the material for 30-50 min under a vacuum condition to finally obtain the inorganic doped material for bone repair.

11. A method for preparing a bionic scaffold for bone repair by 3D printing comprises the following steps:

directly drawing the inorganic doped material for bone repair prepared by the method of claim 1 into microfilaments with the diameter of 0.1-5 mm;

and secondly, the microfilament prepared in the step I is printed and formed through solid-phase 3D to obtain the bionic scaffold for bone repair.

12. The method of claim 11, wherein (i) said microwires have diameters of 1.5-3 mm.

Images