CN110665055A - Sericin/nano-hydroxyapatite tissue engineering bone graft and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of tissue engineering, and discloses a construction (preparation) method and application of a sericin/nano-hydroxyapatite tissue engineering bone graft containing bone morphogenetic protein-2. It includes growth factor, scaffold material and optional seed cells, and the seed cells adhere to the scaffold material to form a cell carrier complex with spatial structure and biological activity, and the scaffold material is composed of sericin. (SS) and nano-hydroxyapatite (nHAP) composite scaffold material, sericin encapsulates growth factor containing human recombinant bone morphogenetic protein-2, and bone marrow mesenchymal stem cells adhere to the composite scaffold material. The invention can adjust the pore size, degradation speed and biomechanical strength of the scaffold according to the ratio of SS and nHAP. The biomimetic artificial tissue engineering bone constructed in the present invention can be used as a bone graft for repairing large-segment bone defects, and it has been proved in animal experiments that it can repair large-segment bone defects well.

Description

Sericin/nano-hydroxyapatite tissue-engineered bone graft and its preparation method and application

technical field

The invention relates to a biodegradable active material for repairing bone defects, belonging to the technical field of tissue engineering, in particular to the construction of a bionic artificial bone and its application in orthopedics, and more particularly to sericin/nano-hydroxyapatite Stone tissue engineering bone graft and preparation method and application thereof.

Background technique

Bone tissue defect caused by trauma, tumor, congenital deformity, infection, pathology and other factors is one of the clinical difficulties, and bone grafting is the main method to solve this problem. Bone grafting is mainly divided into autologous bone grafting, allogeneic or allogeneic bone grafting. The disadvantages or limitations of these two methods mainly include insufficient donor source, damage to the donor site, complications after bone removal, and graft rejection. In recent years, with the development of tissue engineering disciplines, tissue engineering bone grafts constructed by tissue engineering principles and methods can improve the above drawbacks, and tissue engineering brings bright prospects for the repair of bone defects. There are four main aspects of bone tissue engineering research: scaffold materials, seed cells, cytokines, and clinical use.

Tissue engineered bone as a substitute for bone repair materials can avoid the defects of biologically derived repair materials. Combining a scaffold material with good biocompatibility and osteoconductivity and biodegradability in vivo with cytokines with strong osteoinductive activity can make the bone defect repair material have the dual properties of osteoconductivity and induction, and it can be used in rapid osteogenesis. At the same time, the implant material gradually degrades, which provides a new idea and method for the repair of clinical bone defects.

Bone marrow mesenchymal stem cells mainly exist in the bone marrow, and it has been confirmed that MSCs can differentiate into at least 9 kinds of mature cells, including osteoblasts and endothelial cells. The multidirectionality of differentiation suggests that MSCs may be used for cell therapy and tissue engineering artificial bone construction. Ideal seed cells, therefore, bone marrow MSCs become ideal seed cells for bone tissue engineering.

The safety and high-efficiency osteoinductive activity of bone morphogenetic proteins have been confirmed by more and more experiments. BMP-2 is considered to have the highest biological activity and is the most promising osteoinductive protein, which can induce in situ and ex situ Osteogenesis is considered to be the most promising osteoinductive substance. The US Food and Drug Administration officially approved rhBMP-2 in 2004 for the clinical treatment of long bone fractures. Dosing, so that the further research and application of BMP-2 is limited. Therefore, the ability of biomaterials to continuously and controllably release growth factors is of great significance for their effectiveness in clinical treatment.

Hydroxyapatite (HA) is the main inorganic mineral component of human and animal bones. People have done extensive research on HA. By improving the process technology, HA products with different shapes, porosity and degradation rates have been prepared. Commercially produced HA artificial bone has been certified by the US FDA and approved for clinical use. When the pore size of hydroxyapatite reaches the nanometer scale, it will show a series of unique properties. The nHAP composite material has better biological properties than the corresponding micrometer composite material. At the same time, optimizing the composition, structure and process of the material will make it possible to obtain mechanical properties. Bone repair material that better matches natural bone.

In recent years, in bone tissue engineering, research on nano-hydroxyapatite (nHAP) materials has developed rapidly in the world, and a complete scientific system is being formed. nHAP has inorganic components (calcium, phosphorus) similar to human bone tissue, and can prepare products with different shapes, porosity and degradation rates. Hydroxyapatite nanoparticles are introduced into non-hydrophilic biodegradable polyester matrix materials It is possible to obtain new bone repair materials that can be degraded, have better mechanical properties, and have superior osteoinductive properties.

However, a single artificial material is generally difficult to meet the requirements of extracellular scaffold materials for bone tissue engineering, and the refractory and brittle nature of nHAP restricts its clinical use.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the refractory and brittleness of nHAP in the existing extracellular scaffold materials for bone tissue engineering, as well as the in vivo content of biomaterials is very small, the half-life is short, and repeated administration is required. A tissue-engineered bone graft with sustained release of biomaterials and its preparation method and application.

Growth factors such as bone morphogenetic protein-2 are members of the β-transforming growth factor-β (TGF-β) superfamily and play an important role in embryogenesis and development, tissue and cell proliferation and differentiation, etc. The high-efficiency inducing osteogenic activity of BMP-2 has been confirmed by more and more experiments. The content of natural BMP-2 in the body is extremely small and the half-life is short, so it is difficult to maintain a sustained osteogenic effect in the body. Growth factors play an important role in the transfer of information between cells and the extracellular matrix. The controlled release of recombinant growth factors has become one of the important factors affecting whether biomaterials can be effectively used in tissue regeneration. Bone morphoprotein 2 (BMP-2) has been widely used in scientific research and clinical treatment of bone defects. BMP-2 used for clinical therapy should have the ability to control the sustained release to direct cell proliferation, osteogenic differentiation and bone formation. Therefore, the ability of biomaterials to continuously and controllably release growth factors is of great significance for their effectiveness in clinical treatment. Nanomaterials have significant advantages in delivering targeted drugs, regulating cell differentiation and promoting bone tissue regeneration. Relevant studies have shown that polymer nanoparticles composed of micelles and dendrimers and inorganic nanoparticles such as calcium phosphate and bioglass have been applied in tissue engineering such as muscles and bones as loading systems for bioactive drugs. However, a single artificial material is generally difficult to meet the requirements of extracellular scaffold materials for bone tissue engineering, and the refractory and brittle nature of nHAP restricts its clinical use.

Sericin (SS) is mainly derived from silk insects. Sericin is secreted by the middle silk glands of silkworms. It is a natural macromolecular viscous protein wrapped on the surface of silk fibroin. However, sericin has been in the process of degumming for a long time. It is discarded as waste, the degradation process requires a lot of oxygen, and the direct discharge of sericin causes environmental pollution. Through the study of pure sericin hydrogel with undestructed natural structure, it is found that sericin has the characteristics of good biocompatibility, adhesion, high porosity and maintenance of drug release. Therefore, the present invention uses sericin for the first time. Protein biomaterials are used as novel tissue engineering natural scaffolds and drug sustained release carriers.

In the present invention, sericin is compounded with nano-hydroxyapatite (the compounding ratio can be adjusted according to the biomechanical requirements of the bone repair site) to prepare a multi-porous scaffold material, which is used as a carrier to load and control release growth factors such as bone morphoprotein 2 (BMP-2), detected the loading and release of BMP-2, and studied its osteogenic induction. The biomimetic artificial bone was constructed in vitro. The experiments confirmed that the cells can release BMP-2 efficiently and continuously before and after compounding and promote the It proliferates and differentiates; in a preferred embodiment, the seed cells in the composite scaffold show good cell adhesion and growth under scanning electron microscopy, and the composite scaffold material can slowly and continuously release BMP-2 for up to 42 days. The bionic artificial bone implanted in the radial defect model constructed by the above method can repair the radial segmental bone defect very well. It was confirmed by experiments that the fracture was completely healed, the cortical bone was continuous, and the medullary cavity was recanalized at 12 weeks. The results of this study show that the ability to use biomaterials to continuously and controllably release growth factors is of great significance for their effectiveness in clinical treatment, which is mainly through the induction of osteogenesis by cartilage of bone, which is an effective method for the treatment of segmental bone defects. ideal method.

According to a first aspect of the present invention, the present invention provides a tissue engineered bone graft containing growth factors, comprising a composite scaffold material and a growth factor grown on the composite scaffold material, wherein the composite scaffold material comprises a cross-linking A complex of agent-crosslinked sericin and nano-hydroxyapatite.

According to a second aspect of the present invention, the present invention provides a method for preparing the tissue engineering bone graft of the present invention, the method comprising: preparing a solution containing growth factors and sericin, and then mixing with a cross-linking agent in the presence of a cross-linking agent. The suspension of nano-hydroxyapatite is mixed to obtain a mixed solution; the mixed solution is injected into the model for freezing and drying.

According to the third aspect of the present invention, there is provided a method for preparing a tissue engineering bone graft, wherein the nano-hydroxyapatite is prepared by a sol-flocculation method, the sericin solution is extracted by the LiBr method, and the cell scaffold carrier complex is prepared by using the LiBr method. After ultrasonic mixing, it is injected into the abrasive tool and then prepared by low temperature freeze-drying, including:

The preparation of composite artificial bone includes the following steps:

A. Preparation of nHAP artificial bone material powder

(1) chemically synthesize the aqueous solution of calcium nitrate and ammonium phosphate, add ammonia water, adjust the pH value of the solution to be 8-13, optionally add a dispersant, adjust the speed of the stirrer and the stirring time, make it precipitation completely, then pass washing, filtering;

(2) drying the precipitate at 80-120°C and sintering at 600-800°C for 2-3 hours to obtain nano-scale powder with a particle size of less than 100nm and similar to human bone tissue composition;

B. Preparation of sericin solution (different concentrations can be prepared as needed)

The 185Nd-s silkworm cocoons were cut into pieces, immersed in 6M LiBr aqueous solution, cleaved at 35°C for 24 hours, and then dialyzed the sericin solution with ultrapure water at room temperature for two days to obtain a sericin solution;

The synthesis steps of the sericin sustained-release solution loaded with rhBMP-2 are as follows:

Take a sericin solution with a sericin concentration of about 3% by weight, and mix the growth factor BMP-2 with the above 3% by weight sericin solution at a ratio of 1:50 (mass ratio) to prepare a concentration of 5μg/ml rhBMP-2 sericin solution;

C. Preparation of composite scaffolds with sericin/hydroxyapatite as a carrier for sustained release system

Mix the sericin solution loaded with rhBMP-2 into the nHAP mixture suspension, and then prepare the composite scaffold of slow-release rhBMP-2 by freeze-drying;

D. The composite scaffold is loaded with seed cells and prepared by negative pressure suction. The steps are as follows:

(1) The composite scaffold of rhBMP-2 was placed in a 6-well plate, pre-wetted with DMEM medium containing 10 wt% fetal bovine serum for 24 hours, and cultured in a 37°C, 5 vol% CO ₂ incubator for 24 hours,

(2) The bone marrow mesenchymal stem cells were evenly added to the above-mentioned pre-wetted scaffold, then placed in a vacuum negative pressure aspirator to maintain a negative pressure, placed in a 37°C incubator for 10 minutes, and then placed in a 37 After attaching in a 5% CO ₂ incubator for 2 hours, slowly add 1.5 ml of DMEM medium and continue to culture in a 37 ℃, 5 vol% CO ₂ incubator for 2-3 days and change the medium once.

According to the fourth aspect of the present invention, the present invention provides the tissue engineered bone graft of the present invention and the tissue engineered bone graft prepared by the preparation method of the present invention as bionic artificial bone in orthopaedics.

Through the research on pure sericin hydrogel whose natural structure is not destroyed, it is found that sericin has the characteristics of good biocompatibility, adhesion, high porosity and maintenance of drug release. As a new type of tissue engineering natural scaffold material and drug sustained-release carrier, the sericin biomaterial is used in orthopedics when the tissue engineering bone graft of the present invention is used as a bionic artificial bone, and the growth factor can be continuously and controllably released, thereby overcoming the problem of Inadequate direct application of BMP-2.

The growth factor of the present invention, such as rhBMP-2, uses sericin as a carrier to slow release to solve the problem

The problem of the release of rhBMP-2, so that the action time of rhBMP-2 can be maximized. The biomimetic artificial tissue engineered bone constructed in the present invention can be used for bone grafts for repairing large segmental bone defects, and has been used in animal experiments. It is proved that it can repair large segmental bone defects well, which makes the clinical application of sericin tissue engineered bone possible.

The present invention utilizes the biomaterial sericin as a carrier for sustained release to solve the problem of BMP-2 release, which is proved to be an effective means through experiments. Bone repair problems caused by defects and bone trauma.

Description of drawings

1 is a schematic structural diagram of a tissue engineered bone graft according to an embodiment of the present invention;

Fig. 2 is the embodiment of the present invention under the scanning electron microscope of nHAP powder artificial bone (200kv×100000);

Fig. 3 is the observation under the scanning electron microscope of artificial bone after compounding according to the embodiment of the present invention (5kv×300) (SS/HA mass ratio 1:1);

Fig. 4 is the observation under the scanning electron microscope of artificial bone after compounding according to the embodiment of the present invention (5kv×100) (SS/nHAP mass ratio 4:6);

Fig. 5 is the 12-week X-ray of group A in the embodiment of the present invention;

Description of reference numerals

1: tissue engineered bone graft;

2: sericin;

3: growth factor;

4: seed cell;

5: Nano-hydroxyapatite.

Detailed ways

The present invention will be described below by means of examples, and it should be noted that the enumerated examples should not be construed to limit the invention.

As shown in FIG. 1 , the present invention provides a tissue engineering bone graft 1 containing growth factors, comprising a composite scaffold material and a growth factor 3 grown on the composite scaffold material, and the composite scaffold material comprises a cross-linking Agent-crosslinked complexes of sericin 2 and nano-hydroxyapatite 5.

According to the bone graft of the present invention, wherein, as shown in FIG. 1 , the growth factor and the composite scaffold material constitute a cell carrier complex with a three-dimensional structure and biological activity, wherein the composite scaffold material is porous, and the pores The shape of the composite stent material is mainly circular, and the pores of the composite scaffold material communicate with each other so that the composite scaffold material forms a communication gap.

According to the bone graft of the present invention, in order to enable better sustained and controlled release of growth factors, it is preferred that the growth factors grow on sericin.

According to the bone graft of the present invention, preferably based on the total weight of the composite scaffold material, the content of sericin is 20-80% by weight, and the weight of nano-hydroxyapatite is 20-80% by weight.

According to the bone graft of the present invention, preferably the cross-linking agent is a mixture of horseradish peroxidase and H ₂ O ₂ , one or more of glutaraldehyde and genipin, since glutaraldehyde and Jing In vivo toxicity of _nipin , preferably the cross _- linking agent is a mixture of horseradish peroxidase and H2O2.

According to this aspect, the mass ratio of the horseradish peroxidase to H ₂ O ₂ has no special requirements, for example, 0.1-1:1.

According to the bone graft of the present invention, the weight ratio of sericin to cross-linking agent is preferably 0.01-100:1.

According to the bone graft of the present invention, preferably, the weight ratio of the growth factor to sericin is 0.01-1:1.

Constructing the bone graft of the present invention according to the aforementioned proportions not only enables better sustained and controlled release of growth factors, but also improves the refractory and brittleness of the scaffold material, increases the content of biomaterials in the body and prolongs its half-life.

According to a preferred embodiment of the present invention, preferably, the bone graft further comprises seed cells 4, and the seed cells 4 are adhered to the composite scaffold material, and more preferably, the seed cells are bone marrow mesenchymal stem cells.

According to the present invention, there is no special requirement for the type of the seed cells, which can be conventionally selected in the field. According to the present invention, preferably, the bone marrow mesenchymal stem cells are isolated, expanded and passaged in vitro into the third generation of bone marrow mesenchymal stem cells. mesenchymal stem cells.

According to the present invention, more preferably, the seed cell density is 1×10 ⁶ -5×10 ⁶ cells/ml.

According to a preferred embodiment of the present invention, the nano-hydroxyapatite has a pore diameter of 100-250 μm, a porosity of 90% or more, and the pores are connected pores.

According to a preferred embodiment of the present invention, the molecular weight of the sericin is 50 kDa to 250 kDa.

According to a preferred embodiment of the present invention, the sericin is a sericin with a natural structure extracted from silk fibroin-deficient mutant silkworm 185N-ds silkworm seeds.

According to a preferred embodiment of the present invention, the cross-linking agent is a mixture of glutaraldehyde, genipin, horseradish peroxidase and H ₂ O ₂ , preferably the cross-linking agent is horseradish A mixture of peroxidase and H2O2.

According to a preferred embodiment of the present invention, the growth factor can be conventionally selected in the field, for example, the growth factor is BMP2, preferably rhBMP2. The present invention has no special requirements for this, and will not be described in detail here.

According to the aforementioned composition and structure of the bone graft of the present invention, when it is used as a bionic artificial bone in orthopaedics, the growth factor can be continuously and controllably released. There is no special requirement for its preparation method, as long as it has the aforementioned composition and structure, the object of the present invention can be achieved.

According to a preferred embodiment of the present invention, the tissue-engineered bone graft of the present invention comprises a three-dimensional structure constructed from bone marrow mesenchymal stem cells, rhBMP-2-containing growth factor, nano-hydroxyapatite and sericin. Bioactive biomimetic artificial bone.

The construction method of the present invention can include:

Nano-hydroxyapatite (nHAP) was prepared by sol-flocculation method;

The sericin solution with undestructed extraction structure was prepared by low temperature LiBr method;

Bone marrow mesenchymal stem cells were isolated, cultured and expanded by density gradient centrifugation combined with adherence method;

The biomimetic artificial bone of cell scaffold carrier complex was constructed by negative pressure suction method.

According to a preferred embodiment of the present invention, there is provided a method for preparing the tissue engineering bone graft of the present invention, the method comprising: preparing a solution containing growth factors and sericin, and then combining with a cross-linking agent in the presence of a cross-linking agent. The suspension of nano-hydroxyapatite is mixed to obtain a mixed solution; the mixed solution is injected into the model for freezing and drying.

According to the method of the present invention, the sericin is preferably prepared by a low-temperature LiBr method, and more preferably includes the following steps: shredding silk cocoons, immersing them in an aqueous LiBr solution for cracking, and then purifying the cracked sericin solution. Concentrating or reducing the concentration of the sericin solution is carried out to obtain the sericin solution of the concentration.

According to a preferred embodiment of the present invention, the preferred cracking conditions include: the concentration of the LiBr aqueous solution is 4-12M, and/or the temperature is 25-40°C, and/or the time is 12-36h.

According to a preferred embodiment of the present invention, preferably the silkworm cocoon is a 185Nd-s silkworm cocoon.

In the present invention, preferably, the purification step includes: dialysis with ultrapure water at room temperature.

According to the present invention, preferably the nano-hydroxyapatite is prepared by a sol-flocculation method, and more preferably includes the following steps: under the condition of an alkaline aqueous solution, in the presence of a dispersant, calcium nitrate and ammonium phosphate are contacted and precipitated, and the obtained The precipitate is dried and sintered to obtain nano-hydroxyapatite with a particle size of less than 100 nm.

According to the present invention, the pH value of the solution is preferably adjusted to 8-13 by ammonia water, more preferably the drying conditions include a temperature of 80-120°C, and/or the sintering conditions include a temperature of 600-800°C, and/or a time of 2 -3 hours.

According to the present invention, the dispersing agent can be conventionally selected, and the present invention has no special requirements for this, and will not be described in detail here.

According to the present invention, the step of preparing a solution containing growth factors and sericin preferably comprises:

Mix the growth factor with the sericin solution, wherein the preferred dosage ratio of the growth factor to the sericin solution is 5-10 μg growth factor:ml sericin solution; more preferably the concentration of the sericin solution is 1-10 wt% .

According to the present invention, wherein, the preferred source of cross-linking agent is one or more of the mixture of horseradish peroxidase and hydrogen peroxide, glutaraldehyde and genipin; preferably the source of cross-linking agent is horseradish peroxidase The mixture of oxidase and hydrogen peroxide, more preferably the volume ratio of horseradish peroxidase to hydrogen peroxide is 1-10:1.

According to the present invention, the volume ratio of the sericin solution to the cross-linking agent is preferably 100:1-50.

According to the present invention, wherein, preferably, the method further comprises: performing negative pressure suction to load seed cells after freezing and drying: preferably comprising the following steps:

(1) pre-wetting and culturing the dry material to obtain a pre-wet scaffold;

(2) The seed cells are evenly added to the pre-wetted scaffold, and then placed in a vacuum negative pressure aspirator to maintain a negative pressure culture.

According to the present invention, both pre-wetting and vacuum suction can be carried out with reference to the steps of the prior art, and the present invention has no special requirements for this. For example, it can be carried out according to the following steps, but the present invention cannot only be applied to the following steps: ( 1) The composite scaffold of rhBMP-2 was placed in a 6-well plate, pre-wetted with DMEM medium containing 10 wt% fetal bovine serum for 24 hours, and cultured in a 37°C, 5 vol% CO ₂ incubator for 24 hours,

(2) The bone marrow mesenchymal stem cells were evenly added to the above-mentioned pre-wetted scaffold, then placed in a vacuum negative pressure aspirator to maintain a negative pressure, placed in a 37°C incubator for 10 minutes, and then placed in a 37 After attaching in a 5% CO ₂ incubator for 2 hours, slowly add 1.5 ml of DMEM medium and continue to culture in a 37 ℃, 5 vol% CO ₂ incubator for 2-3 days and change the medium once.

According to a preferred embodiment of the present invention, the present invention provides a method for preparing the tissue engineering bone graft of the present invention, wherein the nano-hydroxyapatite is prepared by a sol-flocculation method, and the sericin solution is prepared by adopting a sol-flocculation method. The LiBr method is used for extraction, and the cytoskeleton carrier complex is prepared by ultrasonic mixing, injection into the abrasive tool, and low temperature freeze-drying, including:

The preparation of composite artificial bone includes the following steps:

A. Preparation of nHAP artificial bone material powder

(1) chemically synthesize the aqueous solution of calcium nitrate and ammonium phosphate, add ammonia water, adjust the pH value of the solution to 8-13, add a dispersant, adjust the speed of the stirrer and the stirring time to make the precipitation complete, then wash, filter ;

(2) drying the precipitate at 80-120°C and sintering at 600-800°C for 2-3 hours to obtain nano-scale powder with a particle size of less than 100nm and similar to human bone tissue composition;

B. Preparation of sericin solution (different concentrations can be prepared as needed)

The 185Nd-s silkworm cocoons were cut into pieces, immersed in 6M LiBr aqueous solution, cleaved at 35°C for 24 hours, and then dialyzed the sericin solution with ultrapure water at room temperature for two days to obtain a sericin solution;

The synthesis steps of the sericin sustained-release solution loaded with rhBMP-2 are as follows:

Take a sericin solution with a sericin concentration of about 3% by weight, and mix the growth factor BMP-2 with the above 3% by weight sericin solution at a ratio of 1:50 (mass ratio) to prepare a concentration of 5μg/ml rhBMP-2 sericin solution;

C. Preparation of composite scaffolds with sericin/hydroxyapatite as a carrier for sustained release system

Mix the sericin solution loaded with rhBMP-2 into the nHAP mixture suspension, and then prepare the composite scaffold of slow-release rhBMP-2 by freeze-drying;

D. The composite scaffold is loaded with seed cells and prepared by negative pressure suction. The steps are as follows:

(1) The composite scaffold of rhBMP-2 was placed in a 6-well plate, pre-wetted with DMEM medium containing 10 wt% fetal bovine serum for 24 hours, and cultured in a 37°C, 5 vol% CO ₂ incubator for 24 hours,

(2) The bone marrow mesenchymal stem cells were evenly added to the above-mentioned pre-wetted scaffold, then placed in a vacuum negative pressure aspirator to maintain a negative pressure, placed in a 37°C incubator for 10 minutes, and then placed in a 37 After attaching in a 5% CO ₂ incubator for 2 hours, slowly add 1.5 ml of DMEM medium and continue to culture in a 37 ℃, 5 vol% CO ₂ incubator for 2-3 days and change the medium once.

The invention adopts the density gradient centrifugation combined with the adherence method to separate and culture the bone marrow mesenchymal stem cells, which greatly improves the success rate of separation.

The tissue engineering bone graft 1 prepared according to the aforementioned preparation method of the present invention includes a composite scaffold material 2 containing growth factor 3 and seed cells 4, and the seed cells 4 are adhered to the scaffold material 1 to form a three-dimensional structure. A cell carrier complex with spatial structure and biological activity, the scaffold material 2 is a composite scaffold of sericin/nano hydroxyapatite formed and composited, the composite scaffold contains growth factors, and bone marrow mesenchymal stem cells are adhered on it .

In the tissue engineering bone graft prepared according to the aforementioned preparation method of the present invention, the nano-hydroxyapatite (nHAP) is a porous active material with a pore diameter of 100-250 μm and a porosity of more than 90%, and the obtained Pores are connected pores.

For the tissue engineered bone graft prepared according to the aforementioned preparation method of the present invention, preferably, the sericin (sericin silk, SS) solution is sericin extracted from silk fibroin-deficient mutant silkworm 185N-ds variety.

The tissue engineering bone graft prepared according to the aforementioned preparation method of the present invention, through the above structure, the composite scaffold can slowly release the growth factor human recombinant bone morphogenetic protein-2 (rhBMP-2)

According to the aforementioned preparation method of the present invention, preferably, the seed cells are obtained from bone marrow, separated, expanded, and passaged in vitro to become the third generation of bone marrow mesenchymal stem cells, and the cell density is 1×10 ⁶ to 5×10 ⁶ /ml.

According to the present invention, preferably, the sericin solution is a mixed solution containing a certain concentration of rhBMP-2 growth factors.

According to the present invention, the configuration concentration of the rhBMP-2 is preferably 5 μg/ml.

According to the present invention, the cytokine bone morphogenetic protein-2 can be released slowly, continuously and efficiently.

According to the present invention, the sericin obtained by the LiBr method has good gel-forming properties in the presence of a cross-linking agent, and can form a sericin solution with an undamaged hydrogel structure.

The present invention provides the application of the tissue engineered bone graft of the present invention and the tissue engineered bone graft prepared by the preparation method of the present invention as biomimetic artificial bone in orthopedics.

The present invention is described in detail below by embodiment:

The embodiment of the present invention is constructed according to the following steps:

Preparation of nHAP artificial bone material powder

(1) chemically synthesize the aqueous solution of calcium nitrate and ammonium phosphate (the molar ratio of calcium nitrate and ammonium phosphate is 1:1), add ammonia water, adjust the pH value of the solution to 10, adjust the speed of the stirrer and the stirring time to make it The precipitation is complete, then washed and filtered;

(2) The precipitate was dried at 100°C and sintered at 700°C for 2 hours to obtain a nano-scale powder with a particle size of less than 100 nm and a similar composition to human bone tissue (observed under a scanning electron microscope (5kv×300), see Figure 2 );

Said sericin is constructed through the following steps:

1) Weigh silkworm cocoons (185 Nd-s) 600 mg and place in a reagent bottle;

2) add 24ml 6M LiBr solution;

3) 35℃ water bath for 24h;

4) 4000rpm×5min preliminary removal of insoluble substances;

5) Add 6ml Tris-HCl (1M pH 9.0);

6) Transfer the above solution into the pretreated dialysis bag (NWCO 3000Da);

7) The dialysis bag is placed in a reagent bottle containing ultrapure water;

8) Place on the stirrer and slowly stir the dialysis;

9) Change the water every 6h;

10) Dialysis for 48h;

11) Concentrate sericin solution with PEG 6000 aqueous solution until it reaches the required concentration;

The bionic artificial bone is constructed through the following steps:

(1) Take the above-mentioned sericin solution with a concentration of about 3 wt% (the concentration can be adjusted according to the biomechanical requirements of artificial bone preparation), in order to produce the sericin solution loaded with rhBMP-2, the growth factor is 1:50 ( mass ratio) of rhBMP-2 and the above-mentioned 3 wt % sericin solution (w/w) of SS were mixed to prepare a sericin solution with a concentration of 5 μg/ml rhBMP-2.

(2) Mix the nano-hydroxyapatite with the 3% sericin solution containing rhBMP-2 growth factor prepared above in a certain mass ratio (adjust the ratio of SS and nHAP according to the needs of applied biomechanics), and then put it into the Stir well in an ultrasonic oscillator, add HPR/H ₂ O ₂ (HRP 5mg/ml, H ₂ O 3/ _20,000 ) at a volume of 100:3:3 to make it evenly mixed. The mixed solution was injected into the model by injection, immediately placed in a -20°C refrigerator for 24 hours, and the prepared cylindrical or square scaffolds were then dried in a vacuum freeze dryer for 72 hours to obtain rhBMP-2-containing scaffolds. The sericin/hydroxyapatite composite scaffold material and the artificial bone material were sterilized by Co ⁶⁰ irradiation.

The artificial bone material is loaded with seed cells and prepared by negative pressure suction. The steps are as follows:

(1) The composite scaffold of rhBMP-2 was placed in a 6-well plate, pre-wetted with DMEM medium containing 10 wt% fetal bovine serum for 24 hours, and cultured in a 37°C, 5 vol% CO ₂ incubator for 24 hours,

(2) Bone marrow mesenchymal stem cells were evenly added to the above-mentioned pre-wetted scaffold (the mass ratio of SS/nHAP was 1:1 as the experimental sample, which can be adjusted according to the situation), and then placed in a vacuum negative pressure aspirator Medium suction to maintain negative pressure, put it in a 37°C incubator for 10 minutes, put it in a 37°C, 5% _CO2 incubator for 2 hours, and slowly add 1.5ml of DMEM culture medium to 37°C, 5% by volume. Continue the culture in a CO ₂ incubator for 2-3 d and change the medium once.

Figure 1 is a schematic structural diagram of a tissue engineered bone graft according to an embodiment of the present invention, and Figure 1 illustrates; a tissue engineered bone graft of the present invention includes a scaffold material, growth factors and seed cells, which constitute a three-dimensional spatial structure with porosity and biological Active cell carrier complex.

Figure 2 shows the pure nHAP powder artificial bone prepared in the embodiment of the present invention under a scanning electron microscope (200kv×100000). Figure 2 shows that the present invention prepares nano-hydroxyapatite particles with uniform size and good water dispersibility.

Fig. 3 is the observation under the scanning electron microscope (5kv×300) (SS/nHAP mass ratio 1:1) of the artificial bone after compounding according to the embodiment of the present invention. Fig. 3 shows that the sericin/hydroxyapatite composite scaffold has a good porous structure, The hydroxyapatite nanoparticles were uniformly distributed in the sericin scaffold.

Fig. 4 is the observation under the scanning electron microscope of the artificial bone after compounding according to the embodiment of the present invention ((5kv×100) (SS/nHAP mass ratio 4:6), Fig. 4 shows that the sericin/hydroxyapatite composite scaffold has a good porous structure , hydroxyapatite nanoparticles were uniformly distributed in the sericin scaffold, and the pores became smaller with the increase of nHA P content.

Figure 5 is a 12-week X-ray of Group A in Example 1 of the present invention. Figure 5 illustrates that at 12 weeks, the degradation of the implant material was completed, the medullary cavity was completely recanalized, the bone was completely reshaped, and the bone defect was repaired.

Application of bionic artificial bone in animal bone defect repair:

1. Animal experiment operation (New Zealand white rabbit)

1. Select ketamine hydrochloride injection 20mg/kg, inject anesthesia through the ear vein.

2. Routine depilation, disinfection, and surgical drape of the right forearm.

3. Make a 2cm longitudinal incision on the radial side of the forearm, expose the radial shaft, and cut the radius with a wire saw together with the periosteum at a distance of 2.5cm from the proximal end of the radius to form a 2cm bone defect animal model.

4. After rinsing the wound with normal saline, implanted respectively in group A: rhBMP2/SS/nHAp+BMSCs (bone graft of the present invention), group B: SS/nHAP+BMSC (prepared according to the method of the present invention, without Introduce sericin), group C: pure SS/HAP (prepared according to the method of the present invention, without introducing growth factor and bone marrow mesenchymal stem cells), blank control group is not filled with any material, and the wound is sutured according to the layers, group D: Blank control group.

5. Partial limbs are not fixed internally and externally, and wounds are not bandaged.

6. After awake from anesthesia, they were put into the cage for routine feeding, and 800,000 units of penicillin were injected intramuscularly for anti-inflammatory for three days after the operation.

Observation indicators and methods

1. General situation and gross specimens

The diet, activity and wound reaction of the rabbits were observed after operation, and the surface condition, osteogenesis and inflammatory reaction of the implanted materials were observed after 4 weeks, 8 weeks and 12 weeks. After the specimens were taken out, the connection of the bone defect and the growth of the bone end callus were observed.

2. X-ray inspection

The X-ray examinations of the test limbs were performed at 4, 8 and 12 weeks after the operation.

3. Biomechanical testing

Four animals were randomly selected and sacrificed at each time point of 4 weeks, 8 weeks and 12 weeks in each group, and the intact radial bone specimens on the surgical side were excised.

4. Scanning electron microscope observation

After the animals were sacrificed, the entire radial bone was taken out, and 2 specimens were randomly taken from the specimens of each material group in each period. The bone defect site and both ends of the junction with the material were cut with 0.5 cm, fixed with 3% glutaraldehyde, and a sharp knife was used to remove the middle. After dissection, dehydration, critical point drying, and gold spray coating, the compatibility of bone and material interface and the repair of bone defect were observed under scanning electron microscope (SEM).

result:

1. General situation and gross specimens

After the operation, the experimental animals had normal diet and activities, no wound infection, and the wound healed in the first stage about 1 week after the operation.

2. X-ray performance

Experimental group Group A: at 4 weeks, the material was partially degraded, the material was fused with bone tissue, and callus formed; at 8 weeks, the material was further degraded, and the contact boundary between bone and material was blurred; at 12 weeks, the material was degraded, and the medullary cavity was completely recanalized. Complete shaping and repair of bone defect; group B and group C had poor repair effect of bone defect; group D: bone defect was not repaired.

3. Mechanical analysis

Statistical analysis of the bending strength data measured in the three-point bending test of the specimens of the test groups in each period showed that the differences between groups A, B, and C within each group were 4 weeks < 8 weeks < 12 weeks, and the differences were statistically significant. (P < 0.05); compared between the groups at 4 weeks, 8 weeks and 12 weeks, there was a statistically significant difference between group A > group B > group C (P < 0.01); it indicated that the osteogenic ability of the materials in group A was better than that in group B The same material was implanted into the bone defect and its mechanical strength increased with time.

4. Scanning electron microscope observation

Group A: The material degraded at 4 weeks, and there was a gap between the material and normal bone, and callus filled the gap; 8 weeks: The material was further degraded, but the material was "fused" with normal bone, and a large number of new bone-like bones were produced between them. At 12 weeks, the material was completely degraded, the bone defect area was filled with new lamellar bone tissue, and the bone defect was completely repaired. Groups B and C showed poor material absorption and less new bone.

The preferred embodiments of the present invention have been described above in detail, however, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, a variety of simple modifications can be made to the technical solutions of the present invention, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention. All belong to the protection scope of the present invention.

Claims (10)

1. A tissue engineering bone graft containing growth factors, which comprises a composite scaffold material and the growth factors growing on the composite scaffold material, and is characterized in that the composite scaffold material comprises a compound of sericin and nano hydroxyapatite which are cross-linked by a cross-linking agent.

2. The bone graft of claim 1, wherein the growth factor and the composite scaffold material form a cell carrier composite having a three-dimensional structure and bioactivity, wherein the composite scaffold material is porous, the pores are mainly circular in shape, and the pores of the composite scaffold material are interconnected so that the composite scaffold material forms communicating voids; preferably said growth factor is encapsulated by said sericin; and/or

Based on the total weight of the composite scaffold material, the content of sericin is 20-80 wt%, the weight of nano hydroxyapatite is 20-80 wt%, and the weight ratio of sericin to a cross-linking agent is 0.01-100: 1; the weight ratio of the growth factor to the sericin is 0.01-1: 1;

preferably, the bone graft further comprises seed cells adhered to the composite scaffold material, preferably, the seed cells are bone marrow mesenchymal stem cells, and more preferably, the density of the seed cells is 1 x 10⁶-5×10⁶Per ml;

preferably, the mesenchymal stem cells are mesenchymal stem cells which are separated, amplified and passaged in vitro to generate the 3 rd generation.

3. The bone graft according to claim 1 or 2, wherein the nano-hydroxyapatite has a pore diameter of 100-250 μm and a porosity of 90% or more, and the pores are interconnected pores; and/or

The molecular weight of the sericin is 50kDa to 250 kDa; and/or

The sericin is extracted from silk fibroin deletion mutant silkworm 185N-ds silkworm eggs and has an undamaged natural structure; and/or

The cross-linking agent is horseradish peroxidase and H₂O₂One or more of glutaraldehyde and genipin; and/or

The growth factor is rhBMP 2.

4. A method of preparing the tissue engineered bone graft of any one of claims 1 to 3, comprising:

preparing a solution containing growth factors and sericin, and then mixing the solution with a suspension of nano hydroxyapatite in the presence of a cross-linking agent to obtain a mixed solution; and (4) injecting the mixed solution into a model for freezing and drying.

5. The method of claim 4, wherein,

the sericin is prepared by a low-temperature LiBr method, and preferably comprises the following steps:

shearing silkworm cocoons, soaking the silkworm cocoons in LiBr aqueous solution for cracking, then purifying cracked sericin solution, and optionally concentrating or concentrating the sericin solution to obtain sericin solution with the concentration;

preferred conditions for cleavage include: the LiBr aqueous solution has a concentration of 4-12M, a temperature of 25-40 ℃, and a time of 12-36h, wherein the silkworm cocoon is 185Nd-s silkworm cocoon;

preferably, the step of purifying comprises: dialyzing with ultrapure water at room temperature;

the nano hydroxyapatite is prepared by adopting a sol-flocculation method, and preferably comprises the following steps:

under the condition of alkaline aqueous solution, optionally in the presence of a dispersant, contacting calcium nitrate and ammonium phosphate for precipitation, drying the obtained precipitate, and sintering to obtain nano hydroxyapatite with the particle size of less than 100 nm;

the pH value of the solution is preferably adjusted to 8-13 by ammonia water, the drying conditions include a temperature of 80-120 ℃, the sintering conditions include a temperature of 600-800 ℃ and a time of 2-3 hours.

6. The method of claim 4 or 5, wherein the step of preparing a solution comprising growth factors and sericin comprises:

mixing the growth factor with the sericin solution, wherein the dosage ratio of the growth factor to the sericin solution is 5-10 mug growth factor: ml sericin solution; preferably, the concentration of the sericin solution is 1 to 10% by weight.

7. The method according to claim 4 or 5, wherein the cross-linking agent source is one or more of a mixture of horseradish peroxidase and hydrogen peroxide, glutaraldehyde and genipin; preferably, the cross-linking agent source is a mixture of horseradish peroxidase and hydrogen peroxide, and more preferably, the volume ratio of the dosage of the horseradish peroxidase to the dosage of the hydrogen peroxide is 1-10: 1; and/or

The volume ratio of the sericin solution to the cross-linking agent is 100: 1-50.

8. The method of claim 4 or 5, wherein the method further comprises: and (3) carrying out negative pressure suction on the loaded seed cells after freezing and drying: preferably comprising the steps of:

(1) carrying out prewetting culture on the dry material to obtain a prewetting bracket;

(2) uniformly adding the seed cells into the pre-wetting bracket, and then placing the bracket in a vacuum negative pressure aspirator to suck and maintain negative pressure culture.

9. A method for preparing the tissue engineering bone graft of any one of claims 1 to 3, wherein the nano-hydroxyapatite is prepared by a sol-flocculation method, a sericin solution is extracted by a LiBr method, and a cell scaffold carrier compound is prepared by mixing uniformly by ultrasound, injecting into a grinding tool and freeze-drying at a low temperature, and specifically comprises the following steps:

the preparation method of the composite artificial bone comprises the following steps:

A. preparation of nHAP artificial bone material powder

(1) Chemically synthesizing an aqueous solution of calcium nitrate and ammonium phosphate, adding ammonia water, adjusting the pH value of the solution to 8-13, optionally adding a dispersing agent, adjusting the speed and stirring time of a stirrer to completely precipitate, washing and filtering;

(2) drying the precipitate at 80-120 deg.C, sintering at 600-800 deg.C for 2-3 hr to obtain nanometer powder with particle size less than 100nm and similar to human bone tissue component;

B. preparation of sericin solution (different concentrations were prepared as required)

Shearing 185Nd-s silkworm cocoons, soaking in 6M LiBr aqueous solution, cracking for 24h at 35 ℃, and dialyzing sericin solution for two days at room temperature by using ultrapure water to obtain sericin protein solution;

the synthesis steps of the rhBMP-2 loaded sericin sustained-release solution are as follows:

taking a sericin solution with the sericin concentration of about 3 weight percent, mixing growth factor BMP-2 with the sericin solution with the weight percent of 3 according to a mass ratio of 1:50, and preparing the sericin solution with the rhBMP-2 concentration of 5 mu g/ml;

C. preparation of slow-release system composite scaffold material using sericin/hydroxyapatite as carrier

Mixing the sericin solution loaded with the rhBMP-2 into the nHAP mixture suspension, and then preparing the composite scaffold slowly releasing the rhBMP-2 by freeze drying;

D. the composite scaffold is loaded with seed cells and prepared by negative pressure suction, and the method comprises the following steps:

(1) placing the rhBMP-2 composite scaffold into a 6-well plate, pre-wetting with DMEM culture solution containing 10 wt% fetal calf serum for 24 hours at 37 ℃ and 5 vol% CO₂The culture is carried out in an incubator for 24 hours,

(2) uniformly adding bone marrow mesenchymal stem cells into the pre-wetted scaffold, placing in a vacuum negative pressure aspirator to aspirate and maintain negative pressure, placing in an incubator at 37 deg.C for 10 min, and placing in an incubator at 37 deg.C and 5% CO₂After 2 hours of attachment in the incubator, 1.5ml of DMEM medium was slowly added at 37 ℃ with 5 vol% CO₂The incubator continues to culture for 2-3 days and changes the culture solution once.

10. Use of the tissue engineered bone graft according to any one of claims 1 to 3 and the tissue engineered bone graft prepared by the preparation method according to any one of claims 4 to 9 as a biomimetic artificial bone in orthopedics.

Images