CN111214698B

CN111214698B - Composite bone repair material and preparation method thereof

Info

Publication number: CN111214698B
Application number: CN202010075510.0A
Authority: CN
Inventors: 杨杨; 牟亚男
Original assignee: Affiliated Hospital of Weifang Medical University
Current assignee: Affiliated Hospital of Weifang Medical University
Priority date: 2020-01-22
Filing date: 2020-01-22
Publication date: 2021-10-22
Anticipated expiration: 2040-01-22
Also published as: CN111214698A

Abstract

The invention relates to a filling bracket material containing a three-layer structure and suitable for bone repair and a preparation method thereof; the bottom end of the bone repair bracket has a hierarchical pore structure with high porosity, can slowly release bioactive factors and improve the inflammatory state of a bone damage part, has good bioactivity and bone induction capability, and is beneficial to enhancing osteogenesis and bone reconstruction; the upper end is a bone cement matrix barrier layer with low porosity, and the middle layer is an adhesive layer. The composite scaffold can effectively promote the repair and regeneration of bone tissues by utilizing the barrier effect of the upper layer and the bone-promoting effect of the lower layer, and the filling gel auxiliary material has good adhesion and filling effects, also has bone growth-promoting components and has a synergistic effect with a framework.

Description

Composite bone repair material and preparation method thereof

Technical Field

The invention belongs to the field of orthopedic materials, and particularly relates to a bioactive composite matrix orthopedic repair filling material containing a gradient pore structure and a preparation method thereof.

Background

Guided tissue regeneration using biomimetic bone is a common bone repair procedure in orthopedic clinical procedures. In oral clinics, tooth extraction and tooth implantation after tooth extraction are common operation operations. However, because of individual differences, alveolar bone defects or insufficient estimates of alveolar bone are common situations, and particularly when the thickness and bone density of alveolar bone are not ideal, the implant effect is seriously affected.

Therefore, the alveolar bone implant has the advantages that the alveolar bone is subjected to reparative growth and healing before oral implantation, the thickness and density of the alveolar bone are promoted, the defect of insufficient alveolar bone mass is overcome, and the alveolar bone implant has a good application prospect.

At present, in the prior art, a bone repair material is usually a degradable biological material loaded with active factors, but is different from bone repair of other parts, the oral environment is complex, in addition, teeth are frequently occluded, and alveolar bones bear pressure, so that the common load-type bone repair material is difficult to be suitable for pulling out tooth sockets, is poor in shape fitting degree with the alveolar bones, and is difficult to provide space and strength support for tissue growth of the alveolar bones. The gel filler has good filling effect, but has low adhesive strength, high exposure rate, easy washing and falling off, insufficient bionic property of the material and inconsistent degradability in vivo. Therefore, the preparation of the novel bone-imitating repair material is an effective method for solving the problem of insufficient bone quantity of the alveolar bone, but the prior art does not have a suitable repair material for the alveolar bone part.

The prior art currently used for bone repair can be listed as follows.

CN201611022649 provides a bone implant material, its preparation method and application. The preparation method of the bone implant material comprises the following steps: ultrasonically dispersing black phosphorus in a first solvent to obtain a dispersion liquid A; dissolving a high polymer material in a second solvent to obtain a solution B; mixing the dispersion liquid A and the solution B, and then carrying out ultrasonic treatment to obtain a uniformly dispersed BP @ high polymer material solution; and then injecting the mixture into a mold for molding or performing 3D printing molding, and volatilizing the solvent to prepare the bone implant material. The bone implant material has the function of promoting bone formation by photothermal degradation in physiological environment, and can be used as an implant of a bone defect filling part for clinical treatment of bone defects, but is difficult to apply to alveolar fossa parts.

CN201811228013 discloses a regenerated material with a tooth-like structure and a manufacturing method thereof, comprising three layers made of different materials, wherein the three layers are respectively an outermost layer, a middle layer and an innermost layer; the outermost layer is ellipsoidal and is coated outside the middle layer; the middle layer is distributed in the outermost layer in a gradient mode according to alveolar bone levels, and all levels of the middle layer can be tightly combined; the innermost layer is surrounded by the simulated dentin layer in the middle layer. Although this application can ensure the firmness of the implant, it only imitates the tooth structure, and lacks a porous structure, and cannot obtain a better function of promoting the growth of osteoblasts.

CN 201811648624 relates to an alveolar bone repair material and a personalized manufacturing method, the alveolar bone repair material is a high polymer material and is composed of an outer compact layer and an inner loose layer, the compact layer can prevent surrounding soft tissues from growing into a bone defect area to form a fibrous connective tissue, the loose layer at the bottom has a structure communicated with a spongy small hole, the hole structure is gradually enlarged from the outside to the inside, and the structure can adsorb and slowly release active substances to promote the growth of bone tissues. Although the material has better biocompatibility, and is not easy to fall off in the using process; however, the material is prepared by adopting a 3D printing technology, the manufacturing process is complicated, more importantly, the material needs to be customized according to individual differences of patients, the material cannot be applied to the industry in a large scale, and the application range and the application prospect of the material are limited. In addition, patent CN 105497981a discloses a method for molding alveolar bone based on three-dimensional printing technology, which directly prints porous implant by accurately measuring the volume of alveolar bone module, and also has the above-mentioned drawbacks.

CN201910002515 provides a porous calcium phosphate scaffold loaded microsphere composite material, a preparation method and applications thereof, which comprises a porous calcium phosphate scaffold, wherein polylactic acid-glycolic acid copolymer microspheres are loaded in pores of the porous calcium phosphate scaffold; the polylactic acid-glycolic acid copolymer microspheres coat one or more antituberculosis drugs. Although the porous calcium phosphate scaffold loaded microsphere composite material has the effects of bone filling reconstruction and bone induction osteogenesis, the loaded medicine is not suitable for the oral environment and is easy to dissipate under the action of cavity liquid.

In conclusion, the bone repair materials in the prior art are difficult to be effectively used in the alveolar socket part of the oral cavity, and the main problems are that:

(1) the gel type material has poor strength and is easy to fall off or wash out; (2) the bone cement filler is difficult to remove after being cured, and the growth space of the new bone tissue is severely limited; (3) the common degradable framework material has poor fitting degree and is difficult to fix in the oral alveolus, and particularly, the fixation is worse along with the degradation of the material; (4) although 3D printing has good fitting degree, the printing needs to be designed one by one aiming at individuation, a uniform preparation template is lacked, and the industrial application is not facilitated.

In addition, experiments prove that the scaffold type repairing material with the hierarchical porous structure is more beneficial to bone repair, because the scaffold has a three-dimensional porous structure and a larger specific surface area, a good growth space environment and active substances can be continuously provided for cell proliferation on the premise of slow degradation, and particularly, the scaffold type repairing material is beneficial to continuously supplying active ingredients.

However, at present, the bone repair material is mainly used in a closed bone defect environment, and is difficult to be used in an open complex environment of the oral cavity, and has various disadvantages: for example, the scaffold material is easily infiltrated by saliva, the slow-release bioactive factors are easily diluted and difficult to be enriched at the bone formation part, and simultaneously, other nutrient substances are difficult to be continuously provided for the required bone growth tissues, so that the bone proliferation is not facilitated and the growth of the bone tissues is promoted.

Therefore, there is a need for a prosthetic material capable of increasing the amount of alveolar bone, which can promote the growth and differentiation of osteoblasts and has excellent effects in terms of structure, strength, and fit of alveolar bone.

Disclosure of Invention

The invention aims to provide a bone repair filling matrix bracket material containing a three-layer structure and particularly suitable for alveolar bones and a preparation method thereof aiming at the defects of the prior art; the bottom end of the bone repair bracket has a hierarchical pore structure with high porosity, can slowly release bioactive factors and improve the inflammatory state of a bone damage part, has good bioactivity and bone induction capability, and is beneficial to enhancing osteogenesis and bone reconstruction; the upper end is a bone cement matrix barrier layer with low porosity, and the middle layer is an adhesive layer.

The composite scaffold provided by the invention can effectively promote the repair and regeneration of bone tissues by utilizing the barrier effect of the upper layer and the bone-promoting effect of the lower layer. Is particularly suitable for overcoming the defect of insufficient alveolar bone mass.

In a first aspect, the present invention provides a composite material, particularly suitable for alveolar bone repair, comprising a composite matrix scaffold and a filler gel adjuvant for adhering the matrix scaffold; the composite substrate support is composed of an upper-end substrate with low porosity, a lower-end substrate with high porosity and a middle bonding layer.

In a second aspect, the present invention provides a method for preparing the composite material for dental bone repair, which comprises the following specific steps.

(1) Preparing a porous bioactive lower-end substrate;

(2) preparing an upper-end matrix premixed slurry based on composite bone cement and a composite adhesive for bonding the upper-end matrix and the lower-end matrix;

(3) bonding the upper and lower substrates by using a composite adhesive to prepare a composite substrate support;

(4) preparing a filling gel auxiliary material capable of adhering to the matrix stent.

Wherein, the lower end substrate is prepared by mixing a bioactive particle suspension and a high molecular polymer solution.

The upper-end matrix premixing slurry is prepared by mixing PLGA microspheres/calcium phosphate-based composite bone cement powder and a curing liquid at room temperature.

The composite adhesive is prepared by mixing bioactive glass particles, a polymer skeleton pore-forming agent, a medical adhesive and a bacteriostatic agent in proportion.

Wherein, the sum of the height or the volume of the upper end matrix and the middle bonding layer accounts for 20-50% of the whole composite matrix bracket.

Wherein the preparation raw materials of the filling gel auxiliary material comprise: collagen powder containing hydroxyapatite/tricalcium phosphate, active polysaccharide (hyaluronic acid, carboxymethyl chitosan, chondroitin sulfate, sodium alginate), and povidone iodine powder.

Specifically, the technical scheme comprises the following specific steps.

Step (1): preparing a porous bioactive lower-end base material, comprising the following steps:

s1, preparing a bioactive particle suspension:

dispersing the composite bioactive particles comprising hydroxyapatite powder and bioactive glass powder by using a dioxane organic solvent to ensure that the solid content of a dispersion liquid is 5-15 wt%; and magnetically stirring for 10-15min, and uniformly mixing to uniformly disperse the bioactive particles in the organic solvent to obtain bioactive particle suspension.

Wherein the weight ratio of the hydroxyapatite powder to the bioactive glass powder is 2-3: 1; the particle size of the hydroxyapatite powder and the bioactive glass powder is less than 60 μm, and preferably 20-50 μm.

Wherein, the composition of the bioactive glass powder is 55% SiO₂-35％CaO-10％P₂O₅(molar ratio); commercial products of similar composition may also be purchased.

Preferably, the composite bioactive granules may further incorporate 5-50 wt% of a BMP powder, such as a recombinant human BMP powder, based on the mass of the hydroxyapatite powder.

S2, preparing a mixed high-molecular polymer solution:

mixing and compounding the poly epsilon-caprolactone-dioxane solution with the mass concentration of 0.1-0.2g/mL and the PLGA-methylene dichloride solution with the mass concentration of 0.2-0.5g/mL to ensure that the mass ratio of PLGA to poly epsilon-caprolactone in the mixed solution is 10:2-3 to obtain a PLGA/poly epsilon-caprolactone mixed high molecular polymer organic solution;

wherein the monomer ratio of polylactic acid and glycolic acid in PLGA is 50: 50.

S3, adding the bioactive particle suspension into the high molecular polymer organic solution in proportion, rapidly stirring and mixing, performing ultrasonic dispersion at room temperature for 1-2min after rapid stirring, pouring the mixed solution into a polytetrafluoroethylene mold with a certain shape at normal pressure, and filling the mold with the filling amount not more than 80% of the volume or height of the mold; pre-freezing the mold at-20 deg.C for 3-4 hr, further freezing at-60 deg.C to-80 deg.C or in liquid nitrogen, and vacuum freeze drying for 12-48 hr to remove organic solvent to obtain porous lower base material.

Wherein the mass ratio of the bioactive particles to the high molecular polymer in the mixed solution is 1: 2-3.

The polytetrafluoroethylene mold can be a cylinder or a cone suitable for filling the tooth socket, and is preferably in a tooth socket-like shape.

Wherein the modulus of filling in the mould is between 50% and 80%, preferably between 50 and 60%, of the volume or height of the mould.

Wherein the porosity of the prepared scaffold is about 50-85%.

Step (2): preparing the premixed slurry of the upper end matrix based on the composite bone cement and the composite adhesive for bonding the upper end matrix and the lower end matrix:

s1: preparation of upper-end matrix premix slurry based on composite bone cement

S1-1: preparing PLGA microsphere/calcium phosphate-based composite bone cement powder:

the calcium phosphate-based composite bone cement is formed by mixing PLGA microspheres and calcium phosphate-based composite bone cement with the particle size of less than 20 microns at room temperature, wherein the calcium phosphate-based composite bone cement comprises 80% of tricalcium phosphate (such as alpha-or beta-tricalcium phosphate), 15% of anhydrous calcium phosphate and 5% of hydroxyapatite by mass, and the PLGA microspheres account for 5-10% of the total mass of the powder.

The preparation method of the PLGA microspheres comprises the following steps:

adding a PLGA (polylactic acid and glycolic acid monomer ratio of 70:30 or 80:20) solution dissolved in an organic solvent into deionized water, and shearing and emulsifying; adding the emulsion into a 4-6% polyvinyl alcohol solution, continuing to shear at high speed, adding the solution into an isopropanol/polyvinyl alcohol mixed solution after the shearing is finished, stirring, and centrifuging to obtain the PLGA microspheres.

S1-2: preparing a curing liquid:

adding chitosan and collagen into 5-10 wt% polyvinyl alcohol aqueous solution to prepare a composite solution containing 1-3 wt% of chitosan and 0.1-0.2 wt% of collagen, and adding a proper amount of glacial acetic acid under the stirring condition to adjust the pH value to be fully dissolved so as to obtain a curing solution;

s1-3: mixing PLGA microspheres/calcium phosphate-based composite bone cement powder and curing liquid according to the proportion of 1g to 0.5-0.6mL at room temperature, and uniformly stirring to obtain slurry, namely the upper-end matrix premixed slurry.

Preferably, the PLGA microspheres/calcium phosphate-based composite bone cement powder and the curing liquid are mixed at room temperature according to the ratio of 1g to 0.6 mL.

Preferably, the composite bone cement powder is mixed with a setting fluid before use.

S2: preparation of composite adhesive for bonding upper and lower end substrate supports

The composite adhesive is prepared by mixing bioactive glass particles (55% SiO based on molar ratio)₂-35％CaO-10％P₂O₅The particle size is less than or equal to 50 microns, preferably less than or equal to 30 microns), and the polymer skeleton pore-forming agent, the medical adhesive and the bacteriostatic agent are mixed according to a proportion and stirred uniformly to obtain the skeleton adhesive.

The method comprises the following specific steps.

S2-1: adding bioactive glass particles into molten PLGA according to a mass ratio of 1:2-3, stirring and mixing at a melting temperature, fully stirring, cooling to room temperature, crushing and granulating, and controlling the particle size to be 50-150 mu m to obtain the embedded particles containing the bioactive glass particles.

Among them, the bioactive glass particles preferably have a particle size of 40 μm or less, more preferably 30 μm or less.

Wherein, preferably, the PLGA particle diameter is 50-100 μm, the number average molecular weight is 10000-100000g/mol, and LA/GA is 1.

Wherein, the melting temperature is preferably 180-.

S2-2: and (2) mixing 3-4 parts of the embedding particles, 4-6 parts of PLGA powder, 12-15 parts of alpha-n-butyl cyanoacrylate and 0.1-0.3 part of povidone iodine powder in parts by weight, stirring for 2-3 min, and uniformly mixing to obtain the composite adhesive.

Wherein, the povidone iodine contains 10 to 15 percent of effective iodine.

And (3): adhering the upper and lower substrates with a compound adhesive

S1: injecting a proper amount of composite adhesive into the mold in the step (1) to uniformly coat the upper surface of the cured lower-end base material in the mold;

s2: mixing the composite bone cement powder prepared in the step with a curing liquid to obtain an upper-end matrix premixed slurry; before the adhesive is solidified, injecting a proper amount of newly prepared upper-end matrix premixed slurry into a polytetrafluoroethylene mold to enable the polytetrafluoroethylene mold to uniformly cover an unset adhesive layer, standing at 37 ℃ for full solidification, then carrying out vacuum freeze drying treatment, further carrying out vacuum drying at 50-60 ℃ after freeze drying to constant weight, demolding, sampling, and carrying out irradiation sterilization to obtain the composite matrix support with the three-layer structure.

Wherein the sum of the height or volume of the upper matrix material and the middle bonding layer is not less than 20 percent of the whole matrix support, and is preferably 20 to 50 percent.

Wherein, the height or the volume of the bonding layer is not higher than that of the upper substrate, and preferably 10-80% of the height or the volume of the upper substrate.

Wherein, the compressive strength of the upper matrix of the repair material is not lower than 10MPa, and the porosity is lower than 60%.

Wherein, through the barrier effect on upper end base member and middle adhesive linkage, can also effectively avoid food waste or oral cavity bacterium to get into the alveolar pit depths, reduce inflammatory response.

Optionally, the composite matrix scaffold obtained can be soaked in an aqueous solution of bone growth inducing factor with a proper concentration, and then taken out and dried to enhance the bone inducing effect.

And (4): filling gel auxiliary material for preparing adhesive matrix support

The filling gel auxiliary material is a precursor of filling type hydrogel for adhering a matrix, and the preparation steps are as follows.

S1: preparation of collagen powder

Dispersing collagen in deionized water to prepare 1-3mg/mL dispersion, slightly heating and adjusting pH to 1-2 with hydrochloric acid to dissolve the collagen; adding hydroxyapatite and beta-tricalcium phosphate in the mass ratio of 1:1-2 into the dispersion liquid under the stirring condition, so that the total mass of the hydroxyapatite and the beta-tricalcium phosphate is 50-100% of the collagen; continuously stirring the obtained solution, dropwise adding sodium hydroxide to neutralize acid in the system until the system solution is nearly neutral (pH is 6.8-7.0), standing and precipitating for 6-12h, centrifugally separating and precipitating, washing with deionized water, drying and grinding to obtain collagen powder containing hydroxyapatite/tricalcium phosphate.

Wherein the collagen is type I collagen or type II collagen.

Wherein the particle size of the hydroxyapatite and the tricalcium phosphate is less than 50 μm; preferably less than 10 μm.

S2: preparation of Filler gel precursor Dry powder (adjuvant)

Weighing 60-70 parts of the collagen powder containing hydroxyapatite/tricalcium phosphate, 20-30 parts of hyaluronic acid, 5-15 parts of carboxymethyl chitosan powder, 5-10 parts of chondroitin sulfate, 2-3 parts of sodium alginate and 1-3 parts of povidone iodine powder according to parts by weight; and (3) uniformly mixing the materials to obtain the filling type hydrogel precursor auxiliary material.

Wherein the carboxymethyl degree of the carboxymethyl chitosan is not less than 80%, and the number average molecular weight is not less than 3 ten thousand.

Optionally, the filled gel precursor dry powder may also include bacteriostatic active components such as various antibiotics (e.g., vancomycin, bacteriostatic active polypeptides, etc.) or bone growth promoting components (e.g., bone morphogenic proteins).

The method of the invention further comprises the step of preparing the filled hydrogel by using the hydrogel precursor dry powder in use: at room temperature, adding distilled water accounting for 40-100% of the mass of the mixture into the precursor dry powder mixture, and uniformly stirring the mixture until a colloidal mixture with proper viscosity is formed, thus obtaining the filling type hydrogel product.

In a third aspect, the invention provides the use of the composite matrix scaffold described above for bone repair or promotion of bone growth, particularly alveolar bone.

In another aspect, the invention also includes a method of using the prepared composite material, comprising: preparing hydrogel liquid from the gel filling auxiliary materials by using distilled water, and injecting the hydrogel liquid into the alveolus; and then polishing and implanting the composite matrix support before the hydrogel is solidified, so that the hydrogel is fully adhered to the matrix support and infiltrates the matrix at the lower end of the support.

Alternatively, in addition to injecting the gel into the oral alveolar bone and then filling the composite matrix, the alveolar socket may be filled with a medium for infiltrating the hydrogel liquid or coating the filling gel auxiliary material.

For example, after a hydrogel liquid is soaked in a sterilized non-woven fabric or gauze or a filling gel auxiliary material is coated, a coated polished or non-polished substrate bracket is wound to fit a gap of an alveolar socket, and then the coated substrate bracket is implanted into the alveolar socket.

Wherein the total height of the composite matrix support is not less than 1mm, and preferably, the matrix height is equivalent to the depth of the dental socket.

Advantageous technical effects of the present invention include, but are not limited to, the following aspects.

1) Compared with the prior art, the alveolar bone filling type composite matrix material disclosed by the invention adopts biocompatible degradable materials capable of improving the osteogenesis of alveolar bone, and has the advantages of degradable implant materials, antibacterial substances and osteoinductive active substances; through the composite design of the upper and lower end matrixes, the porosity of the matrix presents the characteristic of gradient, the problem that the existing bone repair bracket is difficult to construct a multi-stage gradient pore structure is solved, and the technical effects of better bottom infiltration effect and obvious surface layer isolation effect are achieved.

Meanwhile, the three-section type matrix structure can ensure that the upper end of the matrix material implanted with the three-section type matrix structure has certain impact strength, can also ensure that the lower loose and brittle matrix with higher content of bioactive particles is not easy to break by external force or damage by washing, and the middle bonding layer has a barrier isolation effect with the oral environment besides a bonding effect, thereby not only improving the bone regeneration capacity, but also having certain capability of inhibiting germ infection.

2) The composite matrix material of the invention also comprises a filling gel, which has the function of filling the gap between the socket and the matrix, especially the deep part, besides the function of adhering the matrix. The bioactive particles, the active polysaccharide and the antibacterial ingredients contained in the gel fill the gap between the alveolar socket and the matrix, belong to soft filling, not only reduce potential inflammatory reaction, but also are in complete contact with wound tissues, thereby effectively promoting bone tissue generation and bone induction growth, shortening the healing time of alveolar bones and improving the quality of osteogenesis.

3) Importantly, the gel filler does not influence the growth space of bone tissues, effectively solves the problem of insufficient bone growth gaps caused by hard filling of 3D printing accurate implant materials, and simultaneously solves the problems that a pure gel filling material is low in firmness and easy to wash out or fall off from the dental alveolus of the oral cavity; the defect that the bone cement filled in the prior art cannot be taken out smoothly after being hardened is also overcome.

Meanwhile, the gel filler has the characteristic of convenient filling, is convenient to refill after degradation, and realizes the continuous stabilizing effect of the alveolar socket bone repair material.

4) Each component of the composite material of the invention contains biological active particles with good biocompatibility, such as HA, TCP, biological active glass, and the like; the bioactive particles are continuously released along with the gradual degradation of the high molecular polymer components, and most of the bioactive particles are not easy to diffuse to the oral cavity but are enriched at the alveolar socket part at the middle lower part of the matrix due to the barrier effect of the middle bonding layer, so that the loss of the dissipation of the bioactive particles is obviously reduced.

5) The composite matrix material prepared by the invention has high plasticity, can be molded or polished into various shapes such as sheets and bone nails, is beneficial to the growth of new bone tissues and the repair of bone defects because the composite matrix material contains bioactive materials with a hierarchical pore structure and polylactic acid-glycolic acid copolymer, and can also be applied to the fields of bone tissue repair of other parts, bone tissue engineering and the like.

6) The preparation method is simple in preparation process, the porous material is prepared by freeze drying, the method is simple and convenient, the process parameters are easy to control, and the quality is stable; compared with accurate materials such as 3D printing, the method does not need to perform independent modeling aiming at individuals, and because the method performs gap filling by means of gel or medium during use, individual difference does not need to be considered, the composite base material can be prepared in batches, the preparation period is short, the cost is low, and the method is suitable for large-scale industrial production.

Drawings

FIG. 1 section view of osteogenic zone induced by composite material

FIG. 2 is a scanning electron microscope image (partially enlarged) of the axial cutting sample of the lower substrate of the composite stent

Detailed Description

The present invention is described in detail below with reference to specific preparation examples and examples, but the use and purpose of these exemplary embodiments are merely to illustrate the present invention, and do not constitute any limitation to the actual scope of the present invention in any form.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

Preparation example 1

Preparation of porous bioactive lower matrix

S1 preparation of hydroxyapatite powder (4 g) with particle size of 10 μm and bioactive glass powder (2 g) (55% SiO)₂-35％CaO-10％P₂O₅) The composite particles are added into 65ml of 1, 4-dioxane solvent, and then are stirred by magnetic force for 12min and mixed evenly, so that the particles are evenly dispersed in the organic solvent, and the bioactive particle suspension is obtained.

S2, mixing and compounding 20mL of poly-epsilon-caprolactone-dioxane solution with the mass concentration of 0.1g/mL and 50mL of methylene dichloride solution of PLGA (the monomer ratio of polylactic acid to glycolic acid is 50:50) with the mass concentration of 0.2g/mL to obtain a PLGA/poly-epsilon-caprolactone mixed high molecular polymer organic solution;

s3, adding the bioactive particle suspension into the high molecular polymer organic solution, rapidly stirring for preliminary mixing, rapidly stirring and mixing, performing ultrasonic dispersion at room temperature for 1min, pouring a proper amount of mixed solution into a cylindrical polytetrafluoroethylene mold at normal pressure, and filling the mold with the filling modulus of 60% of the mold height; pre-freezing the mold at-20 ℃ for 4h, freezing at-80 ℃ overnight, and then carrying out vacuum freeze drying for 24h to remove the organic solvent, thus obtaining the porous lower-end substrate material.

And (4) retaining the prepared lower-end base material in a mold for later use in a subsequent process.

Preparation example 2

Preparation of Upper matrix premix slurry

S1: dissolving 10g of PLGA (molecular weight is 6-8 ten thousand, the monomer ratio of polylactic acid and glycolic acid is 70:30) in 50mL of dichloromethane, injecting 5mL of deionized water into the dichloromethane, shearing the mixture at high speed of 8500r/min for 1min, adding the mixture into 200mL of 5% polyvinyl alcohol solution, and continuing to shear the mixture at high speed for 1 min; then adding the mixture into a rotary evaporation bottle containing 1L of isopropanol/polyvinyl alcohol mixed aqueous solution, wherein the isopropanol content is 100ml, and the polyvinyl alcohol content is 12 g; removing the organic solvent by rotary evaporation at 35-45 ℃, standing and precipitating for 1h at room temperature, centrifugally separating and precipitating, fully washing with deionized water, and freeze-drying to obtain PLGA microspheres;

mixing 5g of PLGA microspheres and 55g of calcium phosphate-based composite bone cement with the particle size of 10-15 microns at room temperature, and drying to obtain PLGA microsphere/calcium phosphate-based composite bone cement powder;

wherein, the calcium phosphate based composite bone cement consists of 80 percent of beta-tricalcium phosphate, 15 percent of anhydrous calcium phosphate and 5 percent of hydroxyapatite according to the mass ratio.

S2: adding 2g of chitosan and 0.2g of collagen into 98g of 5 wt% polyvinyl alcohol aqueous solution to prepare a chitosan composite solution, and adding 2g of glacial acetic acid under the stirring condition to adjust the pH value to be fully dissolved to obtain a solidified solution;

s3: and mixing 10g of the prepared PLGA microsphere/calcium phosphate-based composite bone cement powder with 6ml of the curing liquid at room temperature, and uniformly stirring to obtain the upper-end matrix premixed slurry.

Preparation example 3

Preparation of composite adhesive for bonding upper and lower end substrate supports

S1: in a heated crucible, 2g bioactive glass particles (55% SiO based on molar ratio)₂-35％CaO-10％P₂O₅Particle size of 40 μm or less) to 5g of molten PLGA (particle size of 50 μm, LA/GA ═ 1), stirring and mixing at 200 ℃, then cooling to room temperature, pulverizing and granulating to make the particle size not more than 100 μm, to obtain the embedded particles containing bioactive glass particles.

S2: and (3) mixing 3g of the embedded particles, 4g of PLGA powder (LA/GA ═ 1), 13g of alpha-n-butyl cyanoacrylate and 150mg of povidone iodine (containing 12% of available iodine), and stirring for 3min until the mixture is uniform to obtain the composite adhesive.

The bonding strength of the adhesive and an aluminum plate after being cured for 15min is measured to be 5.85 +/-0.36 MPa according to GB/T7124-2008, and the adhesive has excellent bonding force.

Preparation example 4

Filling gel auxiliary material for preparing adhesive matrix support

S1: dispersing 2g type II collagen in deionized water to prepare 1mg/mL dispersion, slightly heating and adjusting pH with 1M hydrochloric acid to dissolve; 0.5g of hydroxyapatite and 1g of beta-tricalcium phosphate (particle size 20 μm) were added to the dispersion with stirring; continuously stirring the obtained solution, dropwise adding sodium hydroxide to neutralize acid in a system until the system solution is neutral, standing and precipitating for 10h, then centrifugally separating and precipitating, washing the obtained solid twice with deionized water, drying and grinding to obtain the collagen powder containing hydroxyapatite/tricalcium phosphate.

S2: weighing 3g of the collagen powder containing hydroxyapatite/tricalcium phosphate, 1.2g of hyaluronic acid, 550mg of carboxymethyl chitosan powder (the carboxymethyl degree is 90%), 320mg of chondroitin sulfate, 150mg of sodium alginate and 88mg of povidone-iodine powder, uniformly mixing the materials, and performing irradiation sterilization to obtain the filling type hydrogel precursor dry powder.

Preparation example 5

Preparation of filled hydrogels

To 2g of the hydrogel precursor dry powder mixture obtained in the above preparation example, 1.2mL of distilled water was added at room temperature, and the mixture was stirred uniformly to form a gel-like mixture, thereby obtaining an injectable filled hydrogel.

Embodiment mode 1

1) Bonding upper and lower substrates with a composite adhesive

Injecting a proper amount of the composite adhesive obtained in the preparation example into the mold in the preparation example 1 to uniformly coat the upper surface of the cured lower-end base material in the mold;

2) injecting the premixed slurry of the upper-end matrix obtained in the preparation example into a mold before the adhesive is solidified so as to enable the premixed slurry to uniformly cover the unset adhesive layer, standing at 37 ℃ for fully solidifying for 3h, then carrying out vacuum freeze-drying treatment for 12h, further carrying out vacuum drying at 50 ℃ after freeze-drying until the weight is constant, demolding, sampling, and carrying out irradiation sterilization to obtain the composite matrix support with the three-layer structure.

Wherein, the mold filling quality of the upper end matrix material is controlled to be 3 times of that of the composite adhesive; the modulus of the upper end matrix material and the middle bonding layer is 20% of the height of the mold, and the height of the obtained composite matrix accounts for 80% of the total height of the mold.

Wherein, the compressive strength of the upper matrix is 22MPa, and the porosity is about 30 percent.

And cutting and sampling the lower-layer matrix of the obtained repair material along the axial direction for characterization, carrying out surface observation by adopting a scanning electron microscope of Germany Zeiss company, and carrying out gold spraying treatment before the sample observation. Scanning electron microscope results (see fig. 2 and x 2000) show that the matrix has a porous interconnected structure, the pore walls are dense, active particle microspheres are distributed on the pore walls and the periphery of the pore walls to form a three-dimensional structure with hierarchical pores, the porosity is about 75%, and the pore diameters are mostly distributed in the range of 50-200 microns.

Embodiment mode 2

1) Bonding upper and lower substrates with a composite adhesive

2) injecting the premixed slurry of the upper-end matrix obtained in the preparation example into a mold before the adhesive is solidified so as to enable the premixed slurry to uniformly cover the unset adhesive layer, standing at 37 ℃ for fully solidifying for 3h, then carrying out vacuum freeze-drying treatment for 16h, further carrying out vacuum drying at 50 ℃ after freeze-drying until the weight is constant, demolding, sampling, and carrying out irradiation sterilization to obtain the composite matrix support with the three-layer structure.

Wherein, the mold filling quality of the upper end matrix material is controlled to be 4 times of that of the composite adhesive; the filling modulus of the upper end matrix material and the middle bonding layer is 30% of the height of the mold, and the height of the obtained composite matrix accounts for 90% of the total height of the mold.

Embodiment 3

1) Bonding upper and lower substrates with a composite adhesive

2) injecting the premixed slurry of the upper-end matrix obtained in the preparation example into a mold before the adhesive is solidified so as to enable the premixed slurry to uniformly cover the unset adhesive layer, standing at 37 ℃ for fully solidifying for 3h, then carrying out vacuum freeze-drying treatment for 24h, further carrying out vacuum drying at 60 ℃ after freeze-drying until the weight is constant, demolding, sampling, and carrying out irradiation sterilization to obtain the composite matrix support with the three-layer structure.

Wherein, the mold filling quality of the upper end matrix material is controlled to be 6 times of that of the composite adhesive; the modulus of the upper base material and the middle adhesive layer was 40% of the height of the mold, and the height of the resulting composite base was 100% of the total height of the mold (fully poured).

Embodiment 4

Polishing the composite substrate support with the three-layer structure prepared in the embodiment 1, and then putting the polished composite substrate support into a cylindrical polytetrafluoroethylene mold with a simulated dental socket shape and a diameter of 2cm, so that a certain gap (2-3mm) is formed between the composite substrate and the mold wall; a filling hydrogel liquid was prepared according to the method of preparation example 5, injected into a polytetrafluoroethylene mold, and the mold was placed in a water bath at 37 ℃ to observe the curing.

According to the observation result, the complete curing time at 37 ℃ is 3.5min, and the edge hole structure at the lower end of the matrix is filled with hydrogel; and the cured gel is completely adhered to the composite matrix support, so that the prepared hydrogel has a good wrapping effect on the matrix support.

Examples of effects

Animal bone effect test

Treating three adult healthy Hashima dogs with general anesthesia, stripping the gum of the 6 th teeth on the two sides of the lower jaw of each dog, removing the teeth by using dental forceps, and polishing the bottom area of the dental alveolus until the teeth are smooth so as to form a cylindrical defective hole; hemostasis after operation, intramuscular injection of penicillin and antiphlogosis for 3 days. After 3 days of tooth extraction, the left alveolar fossa at the tooth position is directly sutured without filling any material and is used as a control; the hydrogel obtained in preparation example 5 was injected and filled into the right socket, and then the composite base stent of embodiment 1 was ground to an appropriate shape and implanted into the socket to suture the gum. Two dogs were randomly selected to be sacrificed after 16 weeks, alveolar fossa was taken, fixed in 10% formalin for 2 days, dehydrated in gradient ethanol, embedded in PMMA, and the specimens were serially sectioned from the junction of the implant at the bottom of the alveolar fossa, stained with toluidine blue, and observed under 200-fold magnification. The results are shown in figure 1, and histology shows that after 16 weeks of implantation, newly formed bone regions are formed at the boundary edge of the implant matrix and the alveolar bottom, the implanted matrix material is combined with the alveolar bone bed in a bone manner, the edge of the matrix material is degraded and has formed new bone and communicated with the beam-shaped bone network and a mature Harvard system; the interface presents a alveolar osteogenic zone, and osteoblasts, osteocytes and capillaries are visible. And no osteogenic area appears at the corresponding position of the control side, but is replaced by fibrous connective tissue, no osteogenic tendency exists, and the phenomenon of slight alveolar atrophy appears.

The inflammation condition of the alveolar fossa of the dogs is observed during the test period, and no long-time continuous inflammation occurs in any of the three dogs; but the inflammation of the dental socket on the side of the implant disappears more quickly, the inflammation symptom basically disappears after the implant is implanted for 2 to 3 days, and the disappearance time of the symptom on the side of the control is 1 to 3 days more.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of a composite material for bone repair comprises a composite matrix support and a filling gel auxiliary material for adhering the matrix support, wherein the composite matrix support consists of an upper matrix, a lower matrix and a middle bonding layer;

the preparation method is characterized by comprising the following steps:

(1) preparing a porous lower end substrate;

the lower end substrate is prepared by mixing a bioactive particle suspension and a high molecular polymer solution;

(2) preparing an upper-end matrix premixed slurry based on the composite bone cement, and preparing a composite adhesive for bonding the upper-end matrix and the lower-end matrix;

wherein the upper-end matrix premixing slurry is prepared by mixing PLGA microspheres/calcium phosphate-based composite bone cement powder and a curing solution at room temperature;

the composite adhesive is prepared by mixing bioactive glass particles, a high-molecular skeleton pore-forming agent, a medical adhesive and a bacteriostatic agent in proportion;

(3) adhering the upper and lower end substrates by using a composite adhesive to prepare a composite substrate support;

wherein, the sum of the height or the volume of the upper end matrix and the middle bonding layer accounts for 20 to 50 percent of the whole composite matrix bracket;

(4) preparing a filling gel auxiliary material for adhering the matrix stent;

wherein the preparation raw materials of the filling gel auxiliary material comprise: collagen powder containing hydroxyapatite/tricalcium phosphate, active polysaccharide and povidone iodine powder;

wherein, the step (1) is specifically prepared as follows:

s1: preparing a bioactive particle suspension:

dispersing the composite bioactive particles comprising hydroxyapatite powder and bioactive glass powder by using a dioxane organic solvent to ensure that the solid content of a dispersion liquid is 5-15 wt%; magnetically stirring for 10-15min, and uniformly mixing to uniformly disperse the bioactive particles in the organic solvent to obtain bioactive particle suspension;

wherein the weight ratio of the hydroxyapatite powder to the bioactive glass powder is 2-3:1, and the molar composition of the bioactive glass powder is 55% of SiO 2-35% of CaO-10% of P2O 5;

s2: preparing a mixed high-molecular polymer solution:

mixing and compounding the poly-epsilon-caprolactone organic solution and the PLGA organic solution to ensure that the mass ratio of PLGA to poly-epsilon-caprolactone in the mixed solution is 10:2-3 to obtain a PLGA/poly-epsilon-caprolactone mixed high-molecular polymer organic solution;

s3: adding the bioactive particle suspension into the high molecular polymer organic solution in proportion, rapidly stirring and mixing, performing ultrasonic dispersion at room temperature for 1-2min after rapid stirring, pouring the mixed solution into a polytetrafluoroethylene mold with a certain shape under normal pressure, filling the mold, and performing vacuum freeze drying to remove the organic solvent to obtain a lower-end substrate material; wherein the mass ratio of the bioactive particles to the high molecular polymer in the mixed solution is 1: 2-3.

2. The method according to claim 1, wherein the specific steps for preparing the upper matrix premix slurry in the step (2) are as follows:

s1: preparing PLGA microsphere/calcium phosphate-based composite bone cement powder:

the calcium phosphate-based composite bone cement is formed by mixing PLGA microspheres and calcium phosphate-based composite bone cement at room temperature, wherein the calcium phosphate-based composite bone cement comprises tricalcium phosphate 80% by mass, anhydrous calcium phosphate 15% by mass and hydroxyapatite 5% by mass, and the using amount of the PLGA microspheres is 5-10% of the total mass of the powder;

s2: preparing a curing liquid:

adding chitosan and collagen into 5-10 wt% polyvinyl alcohol aqueous solution to prepare composite polyvinyl alcohol solution containing 1-3 wt% of chitosan and 0.1-0.2 wt% of collagen, and adding a proper amount of glacial acetic acid under the stirring condition to adjust the pH value to be fully dissolved so as to obtain solidifying solution;

s3: mixing PLGA microspheres/calcium phosphate-based composite bone cement powder and curing liquid according to a proportion at room temperature, and uniformly stirring to obtain upper-end matrix premixed slurry;

wherein the mixing ratio of the PLGA microspheres/calcium phosphate-based composite bone cement powder to the curing liquid is 1g to 0.5-0.6 mL.

3. The preparation method of claim 1, wherein the step of preparing the composite adhesive in the step (2) comprises the following steps:

s1: adding bioactive glass particles into molten PLGA according to a mass ratio of 1:2-3, stirring and mixing at a melting temperature, cooling to room temperature after fully stirring, and crushing and granulating to obtain embedded particles containing the bioactive glass particles;

wherein the particle size of the bioactive glass particles is less than or equal to 40 mu m, LA/GA in PLGA is 1, and the melting temperature is 180-200 ℃;

s2: and (2) mixing 3-4 parts of the embedding particles, 4-6 parts of PLGA powder, 12-15 parts of alpha-n-butyl cyanoacrylate and 0.1-0.3 part of povidone iodine powder in parts by weight, stirring for 2-3 min, and uniformly mixing to obtain the composite adhesive.

4. The method of claim 2, wherein the PLGA microspheres are prepared by the following steps:

adding deionized water into PLGA solution dissolved in an organic solvent, and shearing and emulsifying; adding the emulsion into a 4-6% polyvinyl alcohol solution, continuing to shear at high speed, adding the solution into an isopropanol/polyvinyl alcohol mixed solution after the shearing is finished, stirring, and centrifuging to obtain the PLGA microspheres.

5. The method according to claim 1, wherein the step (3) is specifically prepared by the following steps:

s1: injecting a proper amount of the prepared composite adhesive into the mold in the step (1) to uniformly coat the upper surface of the cured lower-end base material in the mold;

s2: before the adhesive is solidified, injecting the premixed slurry of the upper-end matrix prepared in the step into a polytetrafluoroethylene mold, uniformly covering the uncured adhesive layer with the premixed slurry, standing at 37 ℃ for full solidification, carrying out vacuum freeze drying treatment, further carrying out vacuum drying, demolding, sampling, and carrying out irradiation sterilization to obtain the composite matrix support with the three-layer structure;

wherein the height or volume of the bonding layer is 10-80% of the height or volume of the upper substrate.

6. The method according to claim 1, wherein the step (4) is specifically prepared by the following steps:

s1: dispersing collagen in deionized water to prepare 1-3mg/mL dispersion, slightly heating and adjusting pH with hydrochloric acid to dissolve the collagen, and adding hydroxyapatite and tricalcium phosphate into the dispersion under stirring to make the mass of the dispersion 50-100% of the collagen; continuously stirring the obtained solution, dropwise adding sodium hydroxide to make the system solution nearly neutral, standing and precipitating for 6-12h, centrifugally separating and precipitating, washing with deionized water, drying and grinding to obtain collagen powder containing hydroxyapatite/tricalcium phosphate;

s2: weighing 60-70 parts of the prepared collagen powder containing hydroxyapatite/tricalcium phosphate, 20-30 parts of hyaluronic acid, 5-15 parts of carboxymethyl chitosan powder, 5-10 parts of chondroitin sulfate, 2-3 parts of sodium alginate and 1-3 parts of povidone iodine powder according to parts by weight; and (3) uniformly mixing the materials to obtain the filling type hydrogel precursor auxiliary material.

7. A composite matrix scaffold made by the method of any one of claims 1-6.

8. A composite material for alveolar bone restoration, comprising a composite base scaffold prepared by the method of any one of claims 1 to 6 and a filling gel auxiliary material for adhering the base scaffold.