CN112587731B

CN112587731B - Composite stent and preparation method and application thereof

Info

Publication number: CN112587731B
Application number: CN202011400246.XA
Authority: CN
Inventors: 许为康; 王丽艳
Original assignee: GUANGDONG INSTITUTE OF MEDICAL INSTRUMENTS
Current assignee: Institute Of Health Medicine Guangdong Academy Of Sciences; Institute of Biological and Medical Engineering of Guangdong Academy of Sciences
Priority date: 2020-12-03
Filing date: 2020-12-03
Publication date: 2022-06-24
Anticipated expiration: 2040-12-03
Also published as: CN112587731A

Abstract

The invention discloses a composite stent and a preparation method and application thereof, the composite stent comprises a stent matrix, drug-loaded microspheres and alginate layers, wherein the stent matrix has a three-dimensional porous structure, the drug-loaded microspheres are fixed on the surface and in pore channels of the stent matrix, and the alginate layers are covered on the surfaces of the drug-loaded microspheres and the stent matrix; the drug-loaded microspheres comprise carrier microspheres and drugs loaded on the carrier microspheres, the drugs comprise bone repair drugs and/or growth factors, and the raw materials of the carrier microspheres comprise degradable polyester and mesoporous calcium silicate. The composite scaffold has stable structure, good drug slow release performance and osteogenic differentiation capacity, and can effectively promote the repair and reconstruction of bone tissues.

Description

Composite stent and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical engineering and biomedical materials, in particular to a composite stent and a preparation method and application thereof.

Background

With the continuous development of medical science, pharmacy, biology and other disciplines, new drugs for various diseases emerge endlessly, but how to make the drug release in vivo continuously and stably is still a problem. Whether the medicine is orally taken or injected intravenously, the change of the blood concentration can generate a peak-valley phenomenon, the medicine concentration is too high to cause larger toxic and side effects, and the treatment effect cannot be achieved if the medicine concentration is too low. Although the emerging bioactive macromolecular drugs are high in efficiency, the biological half-life period is short, and the drugs are easy to inactivate, so that the problem which troubles drug developers is also solved. The drug is embedded or adsorbed by adopting a proper biological material to form a drug controlled release system, and the drug controlled release system is implanted into a focus part of bone tissue for local administration, and the drug carrying system is called as a drug controlled release system, so that an effective solution way can be provided for the problems.

The polymer microsphere generally refers to a polymer aggregate with a diameter ranging from nanometer to micrometer and a spherical shape. The polymer microsphere attracts more and more interests of scientific workers due to designability and multiple functions, and is widely researched and applied in the field of tissue repair and regeneration. The materials currently used in microspheres are mainly classified into inorganic materials, natural polymer materials and synthetic polymer materials. The artificially synthesized degradable high molecular material can be used for designing the biological response characteristic by changing the chemical composition, the material structure, the surface property and the like of the raw materials. However, due to the shape, size, etc. of the microspheres themselves, the microspheres are not suitable for use alone in bone repair applications. Since the research of drug-loaded microsphere technology, the in vivo performance research cannot be separated from the composition with a gel system or a scaffold material, but the microsphere-containing composite scaffold prepared by the prior art generally has the problem of weak combination of drug-loaded microspheres and the scaffold material.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a composite stent and a preparation method and application thereof.

The invention provides a composite scaffold, which comprises a scaffold matrix, drug-loaded microspheres and a alginate layer, wherein the scaffold matrix is of a three-dimensional porous structure, the drug-loaded microspheres are fixed on the surface and in pore channels of the scaffold matrix, and the alginate layer is covered on the surfaces of the drug-loaded microspheres and the scaffold matrix; the drug-loaded microsphere comprises a carrier microsphere and a drug loaded on the carrier microsphere, wherein the drug comprises a bone repair drug and/or a growth factor, and the raw material of the carrier microsphere comprises degradable polyester and mesoporous calcium silicate.

The composite bracket provided by the embodiment of the invention at least has the following beneficial effects: the composite stent is formed by compounding a drug-loaded microsphere, a alginate layer and a stent matrix, wherein the mechanical property and the bone induction effect of the stent can be improved by adding the drug-loaded microsphere, and the drug-loaded microsphere also has good drug-loaded release performance; the alginate layers are covered on the surfaces of the drug-loaded microspheres and the stent matrix, so that the release rate of the drug can be further delayed, the drug-loaded microspheres can be prevented from falling off from the stent matrix, and the combination stability of the drug-loaded microspheres and the stent matrix is improved; the drug-loaded microspheres, the alginate layer and the stent matrix can effectively delay the release rate of the drug, have osteogenic differentiation capacity, can effectively promote the repair and reconstruction of bone tissues, and can overcome the defects that the drug-loaded microspheres are not suitable for bone repair alone due to the influence of the shape and the size of the drug-loaded microspheres, and the acellular matrix can only provide short-term release of the drug and cannot release the drug for a long time to continuously stimulate focus parts, so that the acellular matrix is difficult to treat defective tissues alone.

According to some embodiments of the invention, the degradable resin is selected from at least one of polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, poly 3-hydroxyalkanoate, poly (3-hydroxybutyrate), poly (3-hydroxybutyrate-co-3-hydroxyvalerate), polytrimethylene carbonate, polybutylene succinate; preferably, the molecular weight of the degradable polyester is 1.0-6.0 ten thousand daltons.

According to some embodiments of the invention, the mesoporous calcium silicate is prepared by a preparation method comprising the following steps: dissolving a template agent in water, sequentially adding a silicon source and a calcium source under the stirring state, stirring for reaction, carrying out solid-liquid separation to obtain a solid product, and then cleaning, drying and calcining the solid product to obtain the composite material. Wherein, the template agent can adopt hexadecyl trimethyl ammonium bromide and ammonium hydroxide, the silicon source can adopt ethyl orthosilicate, and the calcium source can adopt calcium nitrate tetrahydrate. The preparation method specifically comprises the following steps: dissolving 5-8 g of hexadecyl trimethyl ammonium bromide and 10-15 mL of ammonium hydroxide in 400-800 mL of deionized water, sequentially adding 28-35 mL of ethyl orthosilicate and 30-35 g of calcium nitrate tetrahydrate at intervals of 30min under the condition of stirring at 40 ℃, continuously stirring for 3h after completely adding, filtering and collecting a product, and washing with deionized water and ethanol for 3 times respectively; then, collecting the powder, drying the powder at 60 ℃ overnight, and calcining the powder at 500-700 ℃ for 2-4 h.

According to some embodiments of the invention, the drug is selected from at least one of bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), Vascular Endothelial Growth Factor (VEGF), alendronate sodium, naringin, resveratrol; further preferably, the drug loading rate of the drug-loaded microspheres is 40-80%, and the drug release period is 28-42 days.

According to some embodiments of the invention, the drug-loaded microspheres are prepared by an emulsion solvent evaporation method. Preferably, the drug-loaded microsphere is prepared by a preparation method comprising the following steps: mixing the medicine with mesoporous calcium silicate to obtain mixed powder; then dispersing the mixed powder into a degradable polyester solution to obtain a blending solution; and then dripping the blend into an aqueous solution of a surfactant, and then carrying out solid-liquid separation to prepare the drug-loaded microspheres. Wherein, the mass ratio of the medicine to the mesoporous calcium silicate can be controlled to be 1: (4-10); the degradable polyester solution can adopt a dichloromethane solution of degradable polyester; the surfactant can be polyvinyl alcohol, gelatin, methylcellulose, tween and the like, and the concentration of the aqueous solution of the surfactant is generally 10-30 mg/ml; after the drug-loaded microspheres are prepared, the drug-loaded microspheres with target particle sizes can be separated by a 270-1600-mesh stainless steel screen.

According to some embodiments of the invention, the scaffold matrix is selected from acellular matrices.

According to some embodiments of the invention, the acellular matrix is prepared by subjecting a bone tissue organ to an acellular treatment and then to supercritical carbon dioxide extraction; preferably, the acellular matrix is prepared by a preparation method comprising the following steps: removing cells of bone tissue organs by using a PBS (phosphate buffer solution) containing ethylenediaminetetraacetic acid, soaking the bone tissue organs by using a Tris buffer solution containing sodium dodecyl sulfate, washing the bone tissue organs by using the PBS buffer solution, and finally performing supercritical carbon dioxide extraction to obtain the bone tissue organs; further preferably, the extraction pressure of the supercritical carbon dioxide extraction is 15-20 MPa, the extraction temperature is 35-40 ℃, and the preferred extraction temperature is 37 ℃. Bone tissue organ can select for use at least one in cancellous bone, cortical bone, can specifically adopt the cancellous bone and the cortical bone of deriving from people, pig, dog, rabbit etc., if can adopt the inferior area bone trabecula of upper limbs cartilage of 2 ~ 16 weeks, and before carrying out the cell removal processing, can wash earlier, specifically can adopt high pressure water washing.

Specifically, when the acellular matrix is prepared, the bone tissue and organs can be acellular for 1-2 hours at room temperature by sequentially using PBS (phosphate buffer solution) containing 0.1-0.3% of ethylenediamine tetraacetic acid, soaking for 6-10 hours in 10mM Tris buffer solution containing 0.1-0.4% of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, performing supercritical carbon dioxide extraction, specifically setting the minimum pressure to be 6-8 MPa, the maximum pressure to be 15-20 MPa and the system temperature to be 35-40 ℃; specifically, the reaction solution is exposed for 10-15 min under the maximum pressure, the flow rate is 10kg/h, and then the reaction solution is reduced to the normal pressure within 5-10 min. By setting a minimum pressure, it is ensured that the carbon dioxide is always in a supercritical state during the treatment.

The acellular matrix is used as a scaffold matrix, has excellent biocompatibility, retains the biochemical components, biophysical structures and mechanical properties of natural bone ECM, and can be used as an induction template for providing various structural, mechanical and biological signals to guide the survival, proliferation and correct differentiation of MSCs.

In a second aspect of the present invention, there is provided a method for preparing any one of the composite scaffolds provided in the first aspect of the present invention, comprising the following steps:

s1, mixing the drug-loaded microspheres with the stent matrix to enable the drug-loaded microspheres to cover the surface of the stent matrix and to be filled in the pore channels of the stent matrix, and keeping the temperature at 37-55 ℃ until the drug-loaded microspheres are bonded on the stent matrix to prepare the stent containing the drug-loaded microspheres;

s2, soaking the stent containing the drug-loaded microspheres in an alginate solution, taking out and draining, and then soaking in an aqueous solution containing calcium ions and/or strontium ions to obtain the composite stent.

According to some embodiments of the invention, in step S1, the mass ratio of the drug-loaded microspheres to the scaffold matrix is 1: (3-10).

According to the preparation method, the drug-loaded microspheres are mixed with the stent matrix, so that the drug-loaded microspheres cover the surface of the stent matrix and are uniformly filled in the pore channels of the stent matrix, and then the drug-loaded microspheres are fixed on the stent matrix through a low-temperature fusion technology at 37-55 ℃, so that the combination mode of the drug-loaded microspheres and the stent matrix is simple, the requirement on equipment is low, and industrialization is easy to realize.

In a third aspect of the invention, there is provided a use of any one of the composite scaffolds provided by the first aspect of the invention in the preparation of a material for tissue repair and regeneration. The tissue repair and regeneration material can be a bone tissue repair and regeneration material.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a graph showing the results of the compressive strength test of composite scaffolds according to examples and comparative examples of the present invention;

FIG. 2 shows the results of in vitro drug release performance tests of the composite stents of examples 1-5 and comparative examples 2 and 4 of the present invention.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Preparation of (I) mesoporous calcium silicate

The mesoporous calcium silicate adopted in the preparation process of the composite scaffold is prepared by the following method:

dissolving 6.8g of hexadecyl trimethyl ammonium bromide and 12mL of ammonium hydroxide in 600mL of deionized water, sequentially adding 30mL of ethyl orthosilicate and 31.5g of calcium nitrate tetrahydrate at intervals of 30min under the stirring condition at 40 ℃, continuously stirring for 3h after the ethyl orthosilicate and the calcium nitrate tetrahydrate are completely added, filtering and collecting a product, and washing the product for 3 times by using deionized water and ethanol respectively; then, the collected powder was dried at 60 ℃ overnight and calcined at 600 ℃ for 2h to obtain mesoporous calcium silicate.

Preparation of composite scaffold

Example 1

A composite scaffold, the preparation method of which comprises the following steps:

s1, preparing an acellular matrix, comprising: taking the subchondral bone trabecula of the upper limb of the cow for 4 weeks, and cleaning the trabecula by using high-pressure water; at room temperature, sequentially carrying out decellularization for 1h by using PBS (phosphate buffer solution) containing 0.1% of ethylenediamine tetraacetic acid, soaking for 7h by using 10mM Tris buffer solution containing 0.25% of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, treating the cell-free matrix by using a supercritical carbon dioxide technology, setting the minimum pressure to be 8MPa, the maximum pressure to be 18MPa and the system temperature to be 37 ℃, specifically exposing the cell-free matrix for 10min under the maximum pressure with the flow rate of 10kg/h, and then reducing the temperature to normal pressure within 10min to prepare the acellular matrix with the three-dimensional porous structure as a scaffold matrix;

s2, preparing the drug-loaded microspheres, which comprises the following steps: mixing 10mg VEGF and 70mg mesoporous calcium silicate to obtain mixed powder of a medicament and the mesoporous calcium silicate, and then dispersing the mixed powder into 10ml of dichloromethane solution containing 1g of polycaprolactone (molecular weight: 2 ten thousand) to obtain medicament-carrying mesoporous calcium silicate/polycaprolactone blend liquid; preparing 400ml of aqueous solution containing 12g of polyvinyl alcohol 1788, slowly dripping the blended solution into the aqueous solution of polyvinyl alcohol 1788, continuously stirring at 400rpm for 20 hours, separating out the composite microspheres at the bottom of the container to prepare drug-loaded microspheres, and separating out the drug-loaded microspheres with target particle sizes by using 460 and 650-mesh stainless steel screens for later use;

s3, preparing the composite scaffold, which comprises the following steps: mixing 20mg of the drug-loaded microspheres prepared in the step S2 with 130mg of the stent matrix prepared in the step S1 to ensure that the drug-loaded microspheres cover the surface of the stent matrix and are filled in pore channels of the stent matrix, placing the stent matrix in an oven at 50 ℃, and keeping the temperature for 12 hours until the drug-loaded microspheres are firmly bonded on the stent matrix; and then soaking the stent containing the drug-loaded microspheres in 0.8% alginate aqueous solution for 10min, taking out the stent, draining, and soaking in 1mol/l calcium ion-containing aqueous solution to obtain the composite stent.

Example 2

s1, preparing an acellular matrix, comprising: taking the subchondral bone trabecula of the upper limb of the cow for 16 weeks, and cleaning the trabecula by using high-pressure water; then, sequentially carrying out cell removal for 2 hours by using PBS (phosphate buffer solution) containing 0.3 percent of ethylenediamine tetraacetic acid at room temperature, soaking for 10 hours by using 10mM Tris buffer solution containing 0.1 percent of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, treating the mixture by using a supercritical carbon dioxide technology, and setting the minimum pressure to be 7MPa, the maximum pressure to be 20MPa and the system temperature to be 37 ℃; specifically, the acellular matrix with the three-dimensional porous structure is prepared by exposing for 15min under the maximum pressure and reducing the flow rate to 10kg/h to normal pressure within 5min, and is used as a scaffold matrix;

s2, preparing the drug-loaded microspheres, which comprises the following steps: mixing 10mg of BMP-7 with 40mg of mesoporous calcium silicate to obtain mixed powder of a medicament and the mesoporous calcium silicate, and then dispersing the mixed powder into 10ml of dichloromethane solution containing 1g of polybutylene succinate (molecular weight: 5 ten thousand) to obtain medicament-loaded mesoporous calcium silicate/polybutylene succinate blending solution; preparing 500ml of aqueous solution containing 10g of polyvinyl alcohol 124, slowly dripping the blended solution into the aqueous solution of polyvinyl alcohol 124, continuously stirring at 500rpm for 15 hours, separating out the composite microspheres at the bottom of the container to prepare drug-loaded microspheres, and separating out the drug-loaded microspheres with target particle sizes by using 270-mesh and 460-mesh stainless steel screens for later use;

s3, preparing the composite scaffold, which comprises the following steps: mixing 20mg of the drug-loaded microspheres prepared in the step S2 with 60mg of the stent matrix prepared in the step S1 to ensure that the drug-loaded microspheres cover the surface of the stent matrix and are filled in pore channels of the stent matrix, placing the stent matrix in a 37 ℃ oven, and keeping the temperature for 16h until the drug-loaded microspheres are firmly bonded on the stent matrix; and then soaking the stent containing the drug-loaded microspheres in 0.2% alginate aqueous solution for 15min, taking out the stent, draining, and soaking in 3mol/l strontium ion-containing aqueous solution to obtain the composite stent.

Example 3

s1, preparing an acellular matrix, comprising: taking the subchondral bone trabecula of the upper limb of the cow for 8 weeks, and cleaning the trabecula by using high-pressure water; then at room temperature, sequentially carrying out cell removal for 1.5h by using PBS buffer solution containing 0.1% of ethylenediamine tetraacetic acid, soaking for 8h by using 10mM Tris buffer solution containing 0.2% of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, treating the cell-free matrix by using a supercritical carbon dioxide technology, setting the minimum pressure to be 8MPa, the maximum pressure to be 18MPa and the system temperature to be 37 ℃, specifically exposing the cell-free matrix for 10min under the maximum pressure with the flow rate of 10kg/h, and then reducing the temperature to normal pressure within 7min to prepare the acellular matrix with the three-dimensional porous structure as a scaffold matrix;

s2, preparing the drug-loaded microspheres, which comprises the following steps: mixing 10mg of BMP-2 with 50mg of mesoporous calcium silicate to obtain mixed powder of a drug and the mesoporous calcium silicate, and then dispersing the mixed powder into 10ml of dichloromethane solution containing 1g of polylactic acid (molecular weight: 1 ten thousand) to obtain drug-loaded mesoporous calcium silicate/polylactic acid blended solution; preparing 600ml of aqueous solution containing 18g of polyvinyl alcohol 124, slowly dripping the blended solution into the aqueous solution of polyvinyl alcohol 124, continuously stirring at 600rpm for 10 hours, separating out the composite microspheres at the bottom of the container to prepare drug-loaded microspheres, and separating out the drug-loaded microspheres with target particle sizes by using 650-mesh and 900-mesh stainless steel screens for later use;

s3, preparing the composite scaffold, which comprises the following steps: mixing 20mg of the drug-loaded microspheres prepared in the step S2 with 80mg of the stent matrix prepared in the step S1 to ensure that the drug-loaded microspheres cover the surface of the stent matrix and are filled in pore channels of the stent matrix, placing the stent matrix in a drying oven at 40 ℃, and keeping the temperature for 14h until the drug-loaded microspheres are firmly bonded on the stent matrix; and then soaking the stent containing the drug-loaded microspheres in 1% alginate aqueous solution for 10min, taking out the stent, draining, and soaking in 1.5mol/l strontium ion-containing aqueous solution to obtain the composite stent.

Example 4

s1, preparing an acellular matrix, comprising: taking the subchondral bone trabecula of the upper limb of the cow for 2 weeks, and cleaning the trabecula by using high-pressure water; then at room temperature, sequentially carrying out cell removal for 1h by using PBS buffer solution containing 0.3% of ethylenediamine tetraacetic acid, soaking for 6h by using 10mM Tris buffer solution containing 0.4% of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, processing the cell-free matrix by using a supercritical carbon dioxide technology, setting the minimum pressure to be 6MPa, the maximum pressure to be 16MPa, the system temperature to be 37 ℃, specifically exposing the cell-free matrix for 14min under the maximum pressure, and reducing the flow rate to be 10kg/h, and then reducing the pressure to normal pressure within 8min to prepare the acellular matrix with the three-dimensional porous structure as a scaffold matrix;

s2, preparing the drug-loaded microspheres, which comprises the following steps: mixing 10mg of alendronate sodium and 90mg of mesoporous calcium silicate to obtain mixed powder of a drug and the mesoporous calcium silicate, and then dispersing the mixed powder into 10ml of dichloromethane solution containing 1g of polylactic acid-glycolic acid copolymer (molecular weight: 3 ten thousand) to obtain drug-loaded mesoporous calcium silicate/polylactic acid-glycolic acid copolymer blended solution; preparing 200ml of aqueous solution containing 4g of polyvinyl alcohol 1799, slowly dripping the blended solution into the aqueous solution of polyvinyl alcohol 1799, continuously stirring for 12 hours at 300rpm, separating out the composite microspheres at the bottom of the container to prepare drug-carrying microspheres, and separating out the drug-carrying microspheres with target particle size by using 460 and 1600-mesh stainless steel screens for later use;

s3, preparing the composite scaffold, which comprises the following steps: mixing 20mg of the drug-loaded microspheres prepared in the step S2 with 180mg of the stent matrix prepared in the step S1 to ensure that the drug-loaded microspheres cover the surface of the stent matrix and are filled in pore channels of the stent matrix, placing the stent matrix in a 45 ℃ oven, and keeping the temperature for 12 hours until the drug-loaded microspheres are firmly bonded on the stent matrix; and then soaking the stent containing the drug-loaded microspheres in 0.5% alginate aqueous solution for 20min, taking out the stent, draining, and soaking in 2mol/l calcium ion-containing aqueous solution to obtain the composite stent.

Example 5

s1, preparing an acellular matrix, comprising: taking the subchondral bone trabecula of the upper limb of the cow for 6 weeks, and cleaning the trabecula by using high-pressure water; then at room temperature, sequentially carrying out cell removal for 2h by using PBS buffer solution containing 0.2% of ethylenediamine tetraacetic acid, soaking for 6h by using 10mM Tris buffer solution containing 0.3% of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, processing the cell-free matrix by using a supercritical carbon dioxide technology, setting the minimum pressure to be 6MPa, the maximum pressure to be 15MPa, the system temperature to be 37 ℃, specifically exposing the cell-free matrix for 12min under the maximum pressure, and reducing the flow rate to be 10kg/h, and then reducing the pressure to normal pressure within 6min to prepare the acellular matrix with the three-dimensional porous structure as a scaffold matrix;

s2, preparing the drug-loaded microspheres, which comprises the following steps: mixing 10mg of resveratrol and 100mg of mesoporous calcium silicate to obtain mixed powder of a medicament and the mesoporous calcium silicate, and then dispersing the mixed powder into 10ml of dichloromethane solution containing 1g of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (the molecular weight is 6 ten thousand) to obtain a medicament-loaded mesoporous calcium silicate/poly (3-hydroxybutyrate-co-3-hydroxyvalerate) blend solution; preparing 800ml of aqueous solution containing 6g of polyvinyl alcohol 1788, slowly dripping the blended solution into the aqueous solution of polyvinyl alcohol 1788, continuously stirring at 800rpm for 8 hours, separating out the composite microspheres at the bottom of the container to prepare drug-loaded microspheres, and separating out the drug-loaded microspheres with target particle sizes by using 650-mesh and 1600-mesh stainless steel screens for later use;

s3, preparing the composite scaffold, which comprises the following steps: mixing 20mg of the drug-loaded microspheres prepared in the step S2 with 200mg of the stent matrix prepared in the step S1 to ensure that the drug-loaded microspheres cover the surface of the stent matrix and are filled in pore channels of the stent matrix, placing the stent matrix in a 50 ℃ oven, and keeping the temperature for 10 hours until the drug-loaded microspheres are firmly bonded on the stent matrix; and then soaking the stent containing the drug-loaded microspheres in 0.6% alginate aqueous solution for 30min, taking out the stent, draining, and soaking in 2.5mol/l calcium ion-containing aqueous solution to obtain the composite stent.

Comparative example 1

A composite stent of this comparative example differs from example 4 in that: the microspheres are not loaded with drug. The preparation method specifically comprises the following steps:

s1, preparing the acellular matrix, including: taking the subchondral bone trabecula of the upper limb of the cow for 2 weeks, and cleaning the trabecula by using high-pressure water; then at room temperature, sequentially carrying out cell removal for 1h by using PBS buffer solution containing 0.3% of ethylenediamine tetraacetic acid, soaking for 6h by using 10mM Tris buffer solution containing 0.4% of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, processing the cell-free matrix by using a supercritical carbon dioxide technology, setting the minimum pressure to be 6MPa, the maximum pressure to be 16MPa, the system temperature to be 37 ℃, specifically exposing the cell-free matrix for 14min under the maximum pressure, and reducing the flow rate to be 10kg/h, and then reducing the pressure to normal pressure within 8min to prepare the acellular matrix with the three-dimensional porous structure as a scaffold matrix;

s2, preparing microspheres, including: dispersing 90mg of mesoporous calcium silicate in 10ml of dichloromethane solution containing 1g of polylactic acid-glycolic acid copolymer (molecular weight: 3 ten thousand) to obtain mesoporous calcium silicate/polylactic acid-glycolic acid copolymer blended solution; preparing 200ml of aqueous solution containing 4g of polyvinyl alcohol 1799, slowly dripping the blending solution into the aqueous solution of polyvinyl alcohol 1799, continuously stirring at 300rpm for 12h, separating out the composite microspheres at the bottom of the container to obtain microspheres, and separating out the microspheres with target particle size by using 460 and 1600-mesh stainless steel screens for later use;

s3, preparing the composite scaffold, which comprises the following steps: mixing 20mg of microspheres prepared in the step S2 with 180mg of stent matrix prepared in the step S1 to coat the drug-loaded microspheres on the surface of the stent matrix and fill the surface of the stent matrix in pore channels of the stent matrix, placing the stent matrix in a 45 ℃ oven, and keeping the temperature for 12 hours until the microspheres are firmly bonded on the stent matrix; and then soaking the bracket containing the microspheres in 0.5% alginate aqueous solution for 20min, taking out the bracket, draining, and soaking in 2mol/l calcium ion-containing aqueous solution to obtain the composite bracket.

Comparative example 2

A composite stent of this comparative example differs from example 4 in that: it contains no microsphere, and the medicine is directly compounded with acellular matrix. The preparation method specifically comprises the following steps:

s1, preparing an acellular matrix, comprising: taking the subchondral bone trabecula of the upper limb of the dairy cow for 2 weeks, and cleaning the trabecula by using high-pressure water; then at room temperature, sequentially carrying out cell removal for 1h by using PBS buffer solution containing 0.3% of ethylenediamine tetraacetic acid, soaking for 6h by using 10mM Tris buffer solution containing 0.4% of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, processing the cell-free matrix by using a supercritical carbon dioxide technology, setting the minimum pressure to be 6MPa, the maximum pressure to be 16MPa, the system temperature to be 37 ℃, specifically exposing the cell-free matrix for 14min under the maximum pressure, and reducing the flow rate to be 10kg/h, and then reducing the pressure to normal pressure within 8min to prepare the acellular matrix with the three-dimensional porous structure as a scaffold matrix;

s2, preparing the composite scaffold, which comprises the following steps: mixing 10mg of alendronate sodium and 180mg of the stent matrix prepared in the step S1, so that the drug is coated on the surface of the stent matrix and filled in the pore channel of the stent matrix, putting the stent matrix in a 45 ℃ oven, and keeping the temperature for 12 hours until the drug is firmly bonded on the stent matrix; and then soaking the stent containing the medicine in 0.5 percent alginate aqueous solution for 20min, taking out the stent, draining, and soaking in 2mol/l calcium ion-containing aqueous solution to obtain the composite stent.

Comparative example 3

A composite stent of this comparative example differs from example 4 in that: does not contain drug-loaded microspheres. The preparation method specifically comprises the following steps:

s1, preparing an acellular matrix, comprising: taking the subchondral bone trabecula of the upper limb of the dairy cow for 2 weeks, and cleaning the trabecula by using high-pressure water; then, sequentially carrying out cell removal for 1h by using PBS (phosphate buffer solution) containing 0.3 percent of ethylenediamine tetraacetic acid at room temperature, soaking for 6h by using 10mM Tris buffer solution containing 0.4 percent of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, processing the cell-free matrix by using a supercritical carbon dioxide technology, setting the minimum pressure to be 6MPa, the maximum pressure to be 16MPa, the system temperature to be 37 ℃, specifically exposing the cell-free matrix for 14min under the maximum pressure, and reducing the flow rate to be 10kg/h, and then reducing the pressure to normal pressure within 8min to prepare the acellular matrix with the three-dimensional porous structure as a scaffold matrix;

s2, preparing the composite scaffold, which comprises the following steps: putting 180mg of the bracket matrix prepared in the step S1 into a 45 ℃ oven, and preserving heat for 12 h; and then soaking the stent in 0.5% alginate aqueous solution for 20min, taking out the stent, draining, and soaking in 2mol/l calcium ion-containing aqueous solution to obtain the composite stent.

Comparative example 4

A composite stent of this comparative example differs from example 4 in that: it does not contain alginate. The preparation method specifically comprises the following steps:

s1, preparing an acellular matrix, comprising: taking the subchondral bone trabecula of the upper limb of the cow for 2 weeks, and cleaning the trabecula by using high-pressure water; then, sequentially carrying out cell removal for 1h by using PBS (phosphate buffer solution) containing 0.3 percent of ethylenediamine tetraacetic acid at room temperature, soaking for 6h by using 10mM Tris buffer solution containing 0.4 percent of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, processing the cell-free matrix by using a supercritical carbon dioxide technology, setting the minimum pressure to be 6MPa, the maximum pressure to be 16MPa, the system temperature to be 37 ℃, specifically exposing the cell-free matrix for 14min under the maximum pressure, and reducing the flow rate to be 10kg/h, and then reducing the pressure to normal pressure within 8min to prepare the acellular matrix with the three-dimensional porous structure as a scaffold matrix;

s3, preparing the composite scaffold, which comprises the following steps: and (3) mixing the drug-loaded microspheres prepared in the step S2 of 20mg with the stent matrix prepared in the step S1 of 180mg so that the drug-loaded microspheres cover the surface of the stent matrix and are filled in pore channels of the stent matrix, placing the stent matrix in a 45 ℃ oven, and keeping the temperature for 12 hours until the drug-loaded microspheres are firmly bonded on the stent matrix to prepare the composite stent.

Comparative example 5

A composite stent of this comparative example differs from example 4 in that: does not contain drug-loaded microspheres and alginate. The preparation method specifically comprises the following steps:

taking the subchondral bone trabecula of the upper limb of the cow for 2 weeks, and cleaning the trabecula by using high-pressure water; then at room temperature, sequentially carrying out cell removal for 1h by using PBS buffer solution containing 0.3% of ethylenediamine tetraacetic acid, soaking for 6h by using 10mM Tris buffer solution containing 0.4% of sodium dodecyl sulfate, and washing for multiple times by using PBS until no bubbles exist; then, the supercritical carbon dioxide is used for processing the mixture, the minimum pressure is set to be 6MPa, the maximum pressure is set to be 16MPa, the system temperature is 37 ℃, specifically, the mixture is exposed for 14min under the maximum pressure, the flow rate is 10kg/h, and then the mixture is reduced to the normal pressure within 8min to obtain the catalyst.

Comparative example 6

A composite stent of this comparative example differs from example 4 in that: the acellular matrix is not treated by a supercritical carbon dioxide extraction technology. The preparation method specifically comprises the following steps:

s1, preparing an acellular matrix, comprising: taking the subchondral bone trabecula of the upper limb of the cow for 2 weeks, and cleaning the trabecula by using high-pressure water; then at room temperature, sequentially carrying out acellular treatment for 1h by using PBS (phosphate buffer solution) containing 0.3% of ethylenediamine tetraacetic acid, soaking for 6h by using 10mM Tris buffer solution containing 0.4% of sodium dodecyl sulfate, washing for multiple times by using PBS until no bubbles exist, and preparing an acellular matrix with a three-dimensional porous structure as a scaffold matrix;

s2, preparing the drug-loaded microspheres, which comprises the following steps: mixing 10mg of alendronate sodium and 90mg of mesoporous calcium silicate to obtain mixed powder of a drug and the mesoporous calcium silicate, and then dispersing the mixed powder into 10ml of dichloromethane solution containing 1g of polylactic acid-glycolic acid copolymer (molecular weight: 3 ten thousand) to obtain drug-loaded mesoporous calcium silicate/polylactic acid-glycolic acid copolymer blended solution; preparing 200ml of aqueous solution containing 4g of polyvinyl alcohol 1799, slowly dripping the blended solution into the aqueous solution of polyvinyl alcohol 1799, continuously stirring at 300rpm for 12 hours, separating out the composite microspheres at the bottom of the container to prepare drug-loaded microspheres, and separating out the drug-loaded microspheres with target particle sizes by using 460 and 1600-mesh stainless steel screens for later use;

(III) Performance testing of composite scaffolds

The performance test of the composite supports of the examples 1-5 and the comparative examples 1-6 is respectively carried out by adopting the method, which comprises the following steps:

1. test for compressive Strength

And (3) testing the compressive strength and the compressive modulus of the composite support by adopting a universal material testing machine, wherein during testing, the descending speed of the probe is 5mm/min, and the specification of the composite support is as follows: the diameter was 10mm and the height was 20mm, and the results of the test are shown in FIG. 1. As can be seen from FIG. 1, after the scaffold matrix is compounded with the microspheres and the alginate, the compressive strength of the scaffold matrix is greatly improved, and the scaffold matrix is more suitable for repairing bone defects. In comparative example 6, the acellular matrix prepared by non-supercritical carbon dioxide extraction treatment is used for replacing the acellular matrix in example 4, substances such as fat and the like in the obtained acellular matrix are not beneficial to combination with the drug-loaded microspheres, the drug-loaded microspheres easily fall off from the scaffold, and the compressive strength of the prepared composite scaffold is lower than that of the composite scaffold of other examples.

2. In vitro cytotoxicity assessment

And (3) taking the prepared composite scaffold, evaluating according to the requirement of GB/T16886.5, and scoring according to 'qualitative grading of cytotoxicity of leach liquor'. The experimental results are shown in table 1 below.

Table 1 in vitro cytotoxicity scores of the composite scaffolds of the examples and comparative examples

As can be seen from Table 1 above, the composite scaffolds of the examples are not cytotoxic.

3. In vitro drug release Performance testing

The composite scaffolds of examples 1-5 and comparative examples 2 and 4 were evaluated for solute release in vitro by the following specific evaluation method: in vitro solute release experiments were performed at 37 ℃ and 60rmp in a constant temperature shaker. Specifically, 500mg of the stent was immersed in 200ml PBS (pH 7.4), test solutions were periodically collected and supplemented with an equal amount of PBS, and the solute contents of the collected test solutions were measured by High Performance Liquid Chromatography (HPLC); substituting the absorbance of the solute at a certain time point into a standard curve of the solute to obtain the actual release amount of the solute at the time point; and dividing the actual amount by the total amount of the loaded solute in the material to obtain the cumulative release amount of the solute at the time point. The composite scaffold was evaluated for solute release in vitro as described above, and the results are shown in fig. 2.

As can be seen from fig. 2, compared to the composite scaffold of example 4, the composite scaffold of comparative example 2 does not contain microspheres, the drug is directly compounded with the acellular matrix, and the composite scaffold lacks the sustained and controlled release performance for the drug and can only provide the transient release of the drug; comparative example 4 the composite stent does not contain alginate, and the composite stent has high burst release of the drug and short sustained-release period.

According to the invention, after the drug-loaded microspheres and the alginate are compounded to the acellular matrix, the good biocompatibility can be maintained, the compressive strength of the material can be greatly improved, and the material can be endowed with the sustained and controlled release function of the drug, so that the material is suitable for repairing and regenerating bone tissues and can be further applied to preparing tissue repairing and regenerating materials.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.

Claims

1. A composite scaffold is characterized by comprising a scaffold matrix, drug-loaded microspheres and a alginate layer, wherein the scaffold matrix is of a three-dimensional porous structure and is selected from a acellular matrix, and the acellular matrix is prepared by carrying out acellular treatment on bone tissues and organs and then carrying out supercritical carbon dioxide extraction; the drug-loaded microspheres are fixed on the surface and in the pore canal of the stent matrix, and the alginate layer is covered on the surfaces of the drug-loaded microspheres and the stent matrix; the drug-loaded microsphere comprises a carrier microsphere and a drug loaded on the carrier microsphere, wherein the drug comprises a bone repair drug and/or a growth factor, and the raw material of the carrier microsphere comprises degradable polyester and mesoporous calcium silicate.

2. The composite stent of claim 1, wherein the degradable polyester is selected from at least one of polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, poly-3-hydroxyalkanoate, poly (3-hydroxybutyrate-co-3-hydroxyvalerate), polytrimethylene carbonate, polybutylene succinate.

3. The composite stent of claim 2, wherein the degradable polyester has a molecular weight of 1.0 to 6.0 ten thousand daltons.

4. The composite scaffold according to claim 1, wherein the mesoporous calcium silicate is prepared by a preparation method comprising the steps of: dissolving a template agent in water, sequentially adding a silicon source and a calcium source under the stirring state, stirring for reaction, carrying out solid-liquid separation to obtain a solid product, and then cleaning, drying and calcining the solid product to obtain the composite material.

5. The composite stent of claim 1, wherein the drug is selected from at least one of bone morphogenetic protein-2, bone morphogenetic protein-7, vascular endothelial growth factor, alendronate sodium, naringin, and resveratrol.

6. The composite stent of claim 1, wherein the drug-loaded microspheres have a drug loading rate of 40-80%.

7. The composite stent of claim 1, wherein the drug-loaded microspheres are prepared by an emulsion solvent evaporation method.

8. The composite stent of claim 7, wherein the drug-loaded microspheres are prepared by a preparation method comprising the following steps: mixing the medicine with mesoporous calcium silicate to obtain mixed powder; then dispersing the mixed powder into a degradable polyester solution to obtain a blending solution; and then dripping the blend into an aqueous solution of a surfactant, and then carrying out solid-liquid separation to prepare the drug-loaded microspheres.

9. The composite scaffold according to claim 1, wherein the acellular matrix is prepared by a preparation method comprising the following steps: the bone tissue organ is decellularized by PBS buffer solution containing ethylene diamine tetraacetic acid, then is soaked by Tris buffer solution containing sodium dodecyl sulfate, is washed by the PBS buffer solution, and finally is extracted by supercritical carbon dioxide to obtain the bone tissue organ.

10. The composite bracket of claim 9, wherein the supercritical carbon dioxide extraction has an extraction pressure of 15 to 20MPa and an extraction temperature of 35 to 40 ℃.

11. The method of making a composite scaffold of any of claims 1 to 10, comprising the steps of:

s1, mixing the drug-loaded microspheres with a stent matrix to enable the drug-loaded microspheres to cover the surface of the stent matrix and fill in the pore channels of the stent matrix, and keeping the temperature at 37-55 ℃ until the drug-loaded microspheres are bonded on the stent matrix to obtain a stent containing the drug-loaded microspheres;

12. The method for preparing the composite scaffold according to claim 11, wherein in step S1, the mass ratio of the drug-loaded microspheres to the scaffold matrix is 1: (3-10).

13. Use of a composite scaffold according to any one of claims 1 to 10 in the preparation of a material for tissue repair and regeneration.