阅读说明：本技术 丝胶蛋白/纳米羟基磷灰石组织工程骨移植物及其制备方法和应用 (Sericin/nano-hydroxyapatite tissue engineering bone graft and preparation method and application thereof ) 是由 何春耒 张立兵 张业顺 于 2019-07-10 设计创作，主要内容包括：本发明属于组织工程学技术领域,公开了一种含骨形态发生蛋白-2的丝胶蛋白/纳米羟基磷灰石组织工程骨移植物的构建(制备)方法及其应用。包括生长因子、支架材料和可选的种子细胞,所述种子细胞粘附于所述支架材料上,构成了具有空间结构和生物活性的细胞载体复合物,所述的支架材料是由丝胶蛋白(SS)、纳米羟基磷灰石(nHAP)构成的复合支架材料,丝胶蛋白包裹了含有人重组骨形态发生蛋白-2生长因子,复合支架材料上粘附有骨髓间充质干细胞。本发明可根据SS、nHAP的比例来调节支架的孔径、降解速度及生物力学强度。本发明所述构建的仿生人工组织工程骨可以用于修复大段骨缺损的骨移植物,在动物实验中已经证明了能很好的修复大段骨缺损。(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. The composite scaffold material is a composite scaffold material consisting of sericin (SS) and nano hydroxyapatite (nHAP), the sericin wraps a growth factor containing human recombinant bone morphogenetic protein-2, and bone marrow mesenchymal stem cells are adhered on the composite scaffold material. The invention can adjust the aperture, degradation speed and biomechanical strength of the bracket according to the proportion of SS and nHAP. The bionic artificial tissue engineering bone constructed by the invention can be used for bone graft for repairing large bone defect, and animal experiments prove that the bionic artificial tissue engineering bone can well repair the large bone defect.)

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.

Technical Field

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

Background

Bone tissue defects caused by trauma, tumor, congenital malformation, infection, pathology and other factors are one of the clinical problems, and bone grafting is the main method for solving the problems. The bone grafting is mainly divided into autologous bone grafting, allogeneic or xenogeneic bone grafting. The drawbacks or limitations of these two approaches are mainly insufficient supply, supply area damage and complications after bone extraction, graft rejection, etc. With the development of the tissue engineering subject in recent years, the tissue engineering bone graft constructed by using the tissue engineering principle and method can improve the defects, and the tissue engineering brings good prospect for repairing bone defects. The bone tissue engineering research mainly has 4 aspects: scaffold material, seed cells, cytokines, clinical use.

The tissue engineered bone can be used as a substitute of bone repair materials to avoid the defects of biological source repair materials. The combination of the scaffold material which has good biocompatibility and bone conduction capability and is biodegradable in vivo and the cytokine with strong bone inducing activity can ensure that the bone defect repairing material has the dual characteristics of bone conduction and induction, and the implanted material is gradually degraded while bone formation is rapidly carried out, thereby providing a brand new thought and method for repairing clinical bone defects.

Bone marrow mesenchymal stem cells mainly exist in bone marrow, and it is proved that at least MSCs can be differentiated to more than 9 mature cells including osteoblasts and endothelial cells, and the differentiation polytropy suggests that the MSCs can become ideal seed cells for cell therapy and tissue engineering artificial bone construction, so that the MSCs become ideal seed cells for bone tissue engineering.

The safety and high-efficiency induced osteogenesis activity of bone morphogenetic protein are proved by more and more experiments, BMP-2 is considered to have the highest biological activity, is the most promising osteoinductive protein, can promote in-situ and ectopic osteogenesis, is considered to be the most promising osteoinductive substance, and the U.S. food and drug administration has formally approved rhBMP-2 for clinically treating long bone fracture in 2004, but due to the fact that BMP-2 has extremely tiny content in vivo and short half-life period, repeated administration is needed, further research and application of BMP-2 are limited. Thus, the ability of biological materials to sustain controlled release of growth factors is of great importance to their effectiveness in clinical treatment.

Hydroxyapatite (HA) is the main inorganic mineral component of human and animal bones, and HAs been widely studied, and HAs been able to prepare HA products of different shapes, porosities and degradation rates by improving process technology, and HAs been developed into commercially produced HA artificial bones, and HAs been approved by FDA in the united states for clinical application. When the aperture of the hydroxyapatite reaches the nanometer level, a series of unique performances are shown, and the nHAP composite material has better biological performance than a corresponding micron composite material; meanwhile, the composition, the structure and the process of the material are optimized, so that the bone repair material with mechanical property more matched with natural bone can be obtained.

In bone tissue engineering in recent years, research on nano hydroxyapatite (nHAP) materials science is rapidly developed internationally, and a complete scientific system is being formed. nHAP has inorganic components (calcium and phosphorus) similar to human bone tissues and can prepare products with different shapes, porosities and degradation rates, and hydroxyapatite nano particles are introduced into a non-hydrophilic biodegradable polyester matrix material, so that a novel bone repair material which can be degraded, has good mechanical properties and excellent bone induction performance can be obtained.

However, the single artificial material is generally difficult to meet the requirements of the extracellular scaffold material for bone tissue engineering, and the use of the nHAP in clinic is restricted by the degradability and brittleness of the nHAP.

Disclosure of Invention

The invention aims to overcome the defects of difficult degradability and brittleness of nHAP in the existing extracellular scaffold material for bone tissue engineering, extremely micro content of biological materials in vivo, short half-life period and need of repeated administration, and provides a tissue engineering bone graft capable of continuously releasing the biological materials, a preparation method and application thereof.

Growth factors such as bone morphogenetic protein-2 belong to one of the super family members of the beta-Transforming growth factor (TGF-beta), and play an important role in embryogenesis and development, proliferation and differentiation of tissues and cells, etc., and more experiments prove that the high-efficiency osteogenesis-inducing activity of BMP-2 has very small content in vivo and short half-life, and the sustained osteogenesis effect is difficult to maintain in vivo due to the fact that the natural BMP-2 has very small content in vivo. Growth factors play an important role in the transmission of information between cells and the extracellular matrix. Controlled release of recombinant growth factors is one of the important factors affecting whether biomaterials can be effectively applied for tissue regeneration. Bone morphogenetic protein 2(BMP-2) has been widely used in scientific research and clinical treatment of bone defects. BMP-2 for use in clinical treatment should have the ability of controlled sustained release to direct cell proliferation, osteogenic differentiation and bone formation. Thus, the ability of biological materials to sustain controlled release of growth factors is of great importance to their effectiveness in clinical treatment. The nano material has obvious advantages in the aspects of transferring targeted drugs, regulating cell differentiation and promoting bone tissue regeneration. Related researches show that polymer nanoparticles consisting of micelles and dendritic macromolecules and inorganic nanoparticles such as calcium phosphate and bioglass are applied to tissue engineering such as muscle and bone as a loading system of bioactive medicaments. However, the single artificial material is generally difficult to meet the requirements of the extracellular scaffold material for bone tissue engineering, and the use of the nHAP in clinic is restricted by the degradability and brittleness of the nHAP.

Sericin (SS) is mainly derived from silk-like insects, is secreted by the middle silk gland of silkworm and is a natural macromolecular viscous protein wrapped on the surface layer of silk fibroin, but sericin is discarded as waste in the degumming process for a long time, a large amount of oxygen is needed in the degradation process, and the direct discharge of sericin causes environmental pollution. According to the research on pure sericin hydrogel with an undamaged natural structure, the sericin is found to have the characteristics of good biocompatibility, adhesion, high porosity, drug release maintenance and the like, so that the sericin biomaterial is used as a novel tissue engineering natural scaffold material and a drug sustained-release carrier for the first time.

The invention compounds sericin and nano-hydroxyapatite (the compound proportion can be adjusted according to the requirements of the bone repair part on biomechanics) to prepare a compound porous scaffold material, takes the compound porous scaffold material as a carrier, loads and controls the release of growth factors such as bone morphogenetic protein 2(BMP-2), detects the loading and release conditions of the BMP-2, researches the osteogenic inductivity of the BMP-2, constructs a bionic artificial bone in vitro, and experiments prove that the BMP-2 can be efficiently and continuously released before and after cell compounding and promotes the proliferation and differentiation of the BMP-2; in a preferred embodiment, the seed cells are well attached and grow in the composite scaffold under the scanning electron microscope, and the composite scaffold material can slowly and continuously release BMP-2 for 42 days. The bionic artificial bone implanted radius defect model constructed by the method can well repair segmental radius bone defects. Experiments prove that the fracture is completely healed when the defect model is 12 weeks, the cortical bone is continuous, and the medullary cavity is communicated. The research result shows that the capability of sustained and controllable release of the growth factor by utilizing the biological material has important significance on the effectiveness of the biological material in clinical treatment, mainly induces osteogenesis in a chondromalacia mode, and is an ideal method for treating segmental bone defects.

According to a first aspect of the present invention, there is provided a tissue engineered bone graft containing a growth factor, comprising a composite scaffold material and a growth factor grown on the composite scaffold material, the composite scaffold material comprising a complex of sericin and nano-hydroxyapatite cross-linked by a cross-linking agent.

According to a second aspect of the invention, there is provided a method of preparing a tissue engineered bone graft according to the invention, the method 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.

According to a 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 LiBr method, the cell scaffold carrier complex is prepared by mixing the above components by ultrasound, injecting into a grinding tool, and lyophilizing at a low temperature, the method specifically comprising:

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 can be 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.

According to the fourth aspect of the invention, the invention provides the tissue engineering bone graft and the application of the tissue engineering bone graft prepared by the preparation method in orthopedics as a bionic artificial bone.

According to the invention, through research on pure sericin hydrogel with an undamaged natural structure, sericin is found to have the characteristics of good biocompatibility, adhesion, high porosity, drug release maintenance and the like, so that the sericin biomaterial is firstly adopted as a novel tissue engineering natural scaffold material and a drug slow release carrier, and when the tissue engineering bone graft is adopted as a bionic artificial bone to be applied in orthopedics, growth factors can be continuously and controllably released, thereby overcoming the defect of direct application of BMP-2.

The growth factor of the invention such as rhBMP-2 adopts sericin as a carrier for slow release to solve the problem

The constructed bionic artificial tissue engineering bone can be used for repairing bone graft of large bone defect, and animal experiments prove that the large bone defect can be well repaired, so that the clinical application of the sericin tissue engineering bone becomes possible.

The invention utilizes the sustained release of the biomaterial sericin as a carrier to solve the problem of BMP-2 release, is proved to be an effective means by experiments, is applied to the application of tissue engineering bone in treating bone defect, and is used for solving the bone repair problem caused by bone defect and bone trauma in clinic.

Drawings

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

FIG. 2 shows the nHAP powder artificial bone of the embodiment of the present invention under the scanning electron microscope (200 kv. times.100000);

FIG. 3 is a view of the artificial bone observed under a scanning electron microscope (5kv × 300) (SS/HA mass ratio 1:1) after compounding in the embodiment of the present invention;

FIG. 4 is the observation of the artificial bone of the present invention under scanning electron microscope after compounding (5kv X100) (SS/nHAP mass ratio 4: 6);

FIG. 5 shows a group A12 cycle X-ray according to example A of the present invention;

description of the reference numerals

1: a tissue engineered bone graft;

2: sericin;

3: a growth factor;

4: seed cells;

5: nano hydroxyapatite.

Detailed Description

The invention is described below by way of examples, it being noted that the examples given should not be construed as limiting the invention.

As shown in figure 1, the invention provides a tissue engineering bone graft 1 containing growth factors, which comprises a composite scaffold material and growth factors 3 growing on the composite scaffold material, wherein the composite scaffold material comprises a compound of sericin 2 and nano-hydroxyapatite 5 which are crosslinked by a crosslinking agent.

According to the bone graft of the invention, as shown in fig. 1, the growth factor and the composite scaffold material form a cell carrier composite with a three-dimensional space structure and bioactivity, wherein the composite scaffold material is porous, the shape of the pores is mainly circular, and the pores of the composite scaffold material are communicated with each other, so that the composite scaffold material forms a communicating gap.

In order to enable a better sustained and controlled release of the growth factor according to the bone graft of the present invention, it is preferred that the growth factor is grown on sericin.

The bone graft according to the invention preferably has a sericin content of 20-80 wt% and a nano-hydroxyapatite content of 20-80 wt% based on the total weight of the composite scaffold material.

According to the bone graft of the present invention, preferably, the cross-linking agent is horseradish peroxidase and H₂O₂Preferably, the cross-linking agent is horseradish peroxidase and H, due to the in vivo toxicity of glutaraldehyde and genipin₂O₂A mixture of (a).

According to this aspect, the horseradish peroxidase is conjugated to H₂O₂The mass ratio of (A) to (B) is not particularly limited, and is, for example, 0.1 to 1: 1.

The bone graft according to the invention preferably has a weight ratio of sericin to the crosslinking agent of 0.01 to 100: 1.

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

The bone graft is constructed according to the proportion, so that the growth factors can be better and continuously released in a controlled manner, the degradability and brittleness of the stent material can be improved, the content of the biological material in a body can be increased, and the half-life period of the biological material can be prolonged.

According to a preferred embodiment of the present invention, the bone graft further comprises seed cells 4, 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 invention, the category of the seed cell has no special requirement, and can be selected conventionally in the field, and according to the invention, the mesenchymal stem cell is preferably the mesenchymal stem cell which is separated and expanded and is subjected to in vitro passage to 3 rd generation.

According to the present invention, it is more preferable that the seed cell density is 1X 10⁶-5×10⁶Per ml。

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

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

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

According to a preferred embodiment of the invention, the cross-linking agent is glutaraldehyde, genipin, horseradish peroxidase and H₂O₂Preferably, the cross-linking agent is a mixture of horseradish peroxidase and H2O 2.

According to a preferred embodiment of the invention, the growth factor may be selected conventionally in the art, for example the growth factor is BMP2, preferably rhBMP 2. The present invention has no special requirement, and is not described in detail herein.

When the bone graft with the composition and the structure is used as a bionic artificial bone in orthopaedics, the growth factor can be continuously and controllably released. The preparation method has no special requirements, and the aim of the invention can be achieved as long as the composition and the structure are adopted.

According to the preferred embodiment of the invention, the tissue engineering bone graft comprises a bionic artificial bone which is constructed by bone marrow mesenchymal stem cells, rhBMP-2-containing growth factors, nano hydroxyapatite and sericin and has a three-dimensional structure and biological activity.

The construction method of the invention can comprise the following steps:

preparing nano hydroxyapatite (nHAP) by adopting a sol-flocculation method;

preparing a sericin solution with an undamaged extraction structure by adopting a low-temperature LiBr method;

separating, culturing and amplifying the mesenchymal stem cells by adopting a density gradient centrifugation method combined with a wall attaching method;

the negative pressure suction method is adopted to construct the cell scaffold carrier compound bionic artificial bone.

According to a preferred embodiment of the present invention, there is provided a method of preparing the tissue engineered bone graft according to the present invention, the method 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.

According to the method of the present invention, preferably, the sericin is prepared by a low-temperature LiBr method, and more preferably, the method 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 the sericin solution with the concentration.

According to a preferred embodiment of the invention, the conditions for preferred lysis comprise: the concentration of the LiBr aqueous solution is 4-12M, and/or the temperature is 25-40 ℃, and/or the time is 12-36 h.

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

In the present invention, it is preferable that the purification step comprises: dialysis was performed at room temperature with ultrapure water.

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

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

According to the invention, the dispersant may be chosen conventionally, for which the invention does not require special provisions, which will not be described in detail here.

According to the present invention, wherein the step of preferably preparing a solution containing 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 preferably 5-10 mug growth factor: ml sericin solution; more preferably, the concentration of the sericin solution is 1 to 10% by weight.

According to the invention, preferably, 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.

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

According to the present invention, wherein preferably 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.

According to the invention, both the prewetting and the vacuum suction can be carried out with reference to the prior art steps, for which the invention does not have particular requirements, but can be carried out, for example, according to the following steps, but it cannot be said that the invention is only applicable to 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.

According to a preferred embodiment of the present invention, the present invention provides a method for preparing the tissue engineering bone graft, wherein the nano-hydroxyapatite is prepared by a sol-flocculation method, the sericin solution is extracted by LiBr method, the cell scaffold carrier complex is prepared by mixing the mixture by ultrasound, injecting into a grinding tool and freeze-drying at low temperature, and the method 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, 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 can be 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% CO₂Culturing in an incubator24h，

(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.

The invention adopts a density gradient centrifugation combined adherence method to separate and culture the mesenchymal stem cells, thereby greatly improving the success rate of separation.

The tissue engineering bone graft 1 prepared according to the preparation method comprises a composite scaffold material 2 containing growth factors 3 and seed cells 4, wherein the seed cells 4 are adhered to the scaffold material 1 to form a cell carrier compound with a three-dimensional space structure and biological activity, the scaffold material 2 is a formed and compounded sericin/nano-hydroxyapatite composite scaffold, the composite scaffold contains the growth factors, and bone mesenchymal stem cells are adhered to the composite scaffold material.

According to the tissue engineering bone graft prepared by the preparation method, nano-hydroxyapatite (nHAP) is a porous active material with the pore diameter of 100-250 mu m and the porosity of more than 90%, and the obtained pores are communicated pores.

Preferably, the sericin solution of the tissue engineering bone graft prepared by the preparation method of the invention is sericin extracted from a silk fibroin deletion mutant silkworm 185N-ds variety.

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

According to the aforementioned preparation method of the present invention, preferably, the seed cells are mesenchymal stem cells obtained from bone marrow, isolated, expanded and passaged in vitro to passage 3, and the cell density is 1 × 10⁶～5×10⁶One per ml.

According to the invention, the sericin solution is preferably a mixed solution coated with rhBMP-2 growth factors with a certain concentration.

According to the present invention, it is preferable that the rhBMP-2 is prepared at a concentration of 5. mu.g/ml.

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

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

The invention provides the tissue engineering bone graft and application of the tissue engineering bone graft prepared by the preparation method in orthopedics as a bionic artificial bone.

The invention is illustrated in detail by the following examples:

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

preparation of nHAP artificial bone material powder

(1) Chemically synthesizing an aqueous solution of calcium nitrate and ammonium phosphate (the molar ratio of the calcium nitrate to the ammonium phosphate is 1:1), adding ammonia water, adjusting the pH value of the solution to 10, adjusting the speed and the stirring time of a stirrer to completely precipitate, washing and filtering;

(2) drying the precipitate at 100 deg.C, and sintering at 700 deg.C for 2 hr to obtain nanometer powder with particle diameter less than 100nm (observed under scanning electron microscope (5kv × 300), see fig. 2) similar to human bone tissue component;

the sericin is constructed by the following steps:

1) weighing 600mg of silkworm cocoon (185 Nd-s) and placing the silkworm cocoon in a reagent bottle;

2) adding 24ml of 6M LiBr solution;

3) water bath at 35 ℃ for 24 h;

4) primarily removing insoluble substances at 4000rpm multiplied by 5 min;

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

6) transferring the solution into a pretreated dialysis bag (NWCO 3000 Da);

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

8) placing on a stirrer, stirring slowly and dialyzing;

9) changing water every 6 h;

10) dialyzing for 48 h;

11) concentrating the sericin solution by using a PEG 6000 aqueous solution until the sericin solution reaches the required concentration;

the bionic artificial bone is constructed by the following steps:

(1) taking the prepared sericin solution with the concentration of about 3 wt% (the concentration can be adjusted according to the biomechanical requirements for preparing the artificial bone), mixing the rhBMP-2 with the growth factor of which the mass ratio is 1:50 (the mass ratio) with the sericin solution (w/w) with the weight of 3% of SS to prepare the sericin solution with the concentration of 5 mu g/ml of rhBMP-2 in order to produce the sericin solution loaded with the rhBMP-2.

(2) Mixing nano hydroxyapatite with the prepared rhBMP-2 growth factor-containing 3% sericin solution in a certain mass ratio (the ratio of SS to nHAP is adjusted according to the requirements of applied biomechanics), then placing the mixture into an ultrasonic oscillator for fully stirring, and adding HPR/H₂O₂(HRP5mg/ml，H₂O₂3 parts per million) are mixed uniformly according to the volume of 100:3: 3. Injecting the mixed solution into a model by injection, immediately freezing in a refrigerator at-20 deg.C for 24 hr to respectively prepare cylindrical or square scaffold material, and drying with a vacuum freeze drier for 72 hr to obtain sericin/hydroxyapatite composite scaffold material containing rhBMP-2, wherein the artificial bone material is Co⁶⁰And (5) performing irradiation sterilization for later use.

The artificial bone material loaded seed cells are prepared by negative pressure suction, and the steps are as follows:

(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 (with SS/nHAP mass ratio of 1:1 as the experimental sample, which can be adjusted according to the situation), placing in a vacuum negative pressure aspirator to maintain negative pressure, placing at 37 deg.CAfter keeping the temperature in an incubator for 10 minutes, the incubator is put into a room with the temperature of 37 ℃ and 5 percent 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.

FIG. 1 is a schematic structural diagram of a tissue engineered bone graft according to an embodiment of the present invention, and FIG. 1 illustrates the structure; the tissue engineering bone graft comprises a scaffold material, a growth factor and seed cells, and forms a three-dimensional space structure with porosity and a bioactive cell carrier compound.

Fig. 2 shows the pure nHAP powder prepared by the embodiment of the present invention under the scanning electron microscope (200kv × 100000), and fig. 2 shows that the present invention prepares nano hydroxyapatite particles with uniform size and good water dispersibility.

Fig. 3 is a view of the artificial bone observed under a scanning electron microscope after compounding (5kv × 300) (SS/nHAP mass ratio 1:1) in the embodiment of the present invention, and fig. 3 illustrates that the sericin/hydroxyapatite composite scaffold has a good porous structure, and hydroxyapatite nanoparticles are uniformly distributed in the sericin scaffold.

Fig. 4 is an observation of the artificial bone after compounding in the embodiment of the present invention under a scanning electron microscope ((5kv × 100) (SS/nHAP mass ratio 4: 6), and fig. 4 illustrates that the sericin/hydroxyapatite composite scaffold has a good porous structure, hydroxyapatite nanoparticles are uniformly distributed in the sericin scaffold, and pores become smaller as the nHAP content increases.

FIG. 5 is a 12-week line of group A in accordance with example of the present invention, and FIG. 5 illustrates that at 12 weeks the degradation of the implant material is complete, the medullary cavity is completely re-established, the bone is completely shaped, and the bone defect is repaired.

The application of the bionic artificial bone in the defect repair of animal bones comprises the following steps:

1. animal experiment operation (New Zealand white rabbit)

1. 20mg/kg of ketamine hydrochloride injection is selected and injected into the ear edge vein for anesthesia.

2. The right forearm is depilated conventionally, sterilized and covered with a towel for operation.

3. Taking the middle section of the radial side of the forearm to make a 2cm longitudinal incision, exposing the radial shaft, and sawing off the radius at a position 2.5cm away from the proximal end of the radius by using a wire saw together with a periosteum to prepare a 2cm bone defect animal model.

4. After the wounds are washed by the normal saline, the group A of rhBMP2/SS/nHAp + BMSCs (the bone graft of the invention), the group B of SS/nHAP + BMSC (prepared according to the method of the invention, sericin is not introduced), the group C of pure SS/HAP (prepared according to the method of the invention, growth factors and mesenchymal stem cells are not introduced), the blank control group is not filled with any material, and the wounds are sutured according to layers, and the group D of blank control group is implanted according to different groups.

5. The local limbs are not fixed internally and externally, and the wound is not bandaged.

6. After being anaesthetized and conscious, the animals are placed into a cage for regular feeding, and 80 ten thousand units of penicillin is injected into muscle for anti-inflammation every day three days after the operation.

Observation indicator and method

1. General condition and gross specimen

After operation, the rabbit is observed for diet, activity and wound reaction, and the surface condition, osteogenesis, inflammatory reaction and the like of the implant material are observed by drawing materials at 4 weeks, 8 weeks and 12 weeks. And taking out the specimen, and observing the bone defect connection condition and the bone end callus growth condition.

X-ray examination

X-ray examination of the test limb was performed at 4, 8, and 12 weeks after the operation.

3. Biomechanical testing

In each group, 4 animals were randomly selected at each time point of 4 weeks, 8 weeks, and 12 weeks, and then a full radius specimen on the operative side was cut out, and a three-point bending test was performed on a 858 miniBionix mechanical testing machine after periosteum and soft tissues were removed.

4. Observation by scanning electron microscope

Taking out the whole radius section after the animal is sacrificed, randomly taking out 2 specimens from the specimens of each period of each material group, cutting out 0.5cm of each of the two ends of the bone defect part and the joint part with the material, fixing by 3 percent glutaraldehyde, splitting from the middle by a sharp knife, dehydrating, drying at a critical point, spraying a gold coating film, and observing the compatibility condition of the bone and the material interface and the bone defect repairing condition under a Scanning Electron Microscope (SEM).

As a result:

1. general condition and gross specimen

The experimental animal after operation has normal diet and activity, no wound infection, wound healing for about 1 week after operation, wound suture falling automatically, normal limb activity, no limitation and no lameness.

X-ray representation

Experimental group a group: the material is partially degraded after 4 weeks, and the material is fused with bone tissues to form callus; the material is further degraded in 8 weeks, and the contact limit of the bone and the material is fuzzy; after 12 weeks, the material is degraded, the medullary cavity is completely communicated, the shaping is complete, and the bone defect is repaired; the bone defect repair effect of the B group and the C group is not good enough; group D: the bone defect is not repaired.

3. Mechanical analysis

The tested specimens of the test group in each period are subjected to a three-point bending test, and the statistical analysis of the measured bending strength data shows that: A. b, C comparing each group, wherein 4 weeks is less than 8 weeks and less than 12 weeks, the difference has statistical significance (P is less than 0.05); compared with groups at 4 weeks, 8 weeks and 12 weeks, the difference of the group A, the group B and the group C is statistically significant (P is less than 0.01); the osteogenesis capacity of the material in the group A is better than that of the material in the group B and the material in the group C, and the mechanical strength of the same material is enhanced along with the increase of the same material at the bone defect position along with the time.

4. Observation by scanning electron microscope

Group A: the material is degraded at 4 weeks, gaps are formed between the material and normal bone, and callus is filled in the gaps; 8 weeks: the material is further degraded, but the material is fused with normal bone, and a large amount of new bone-like tissues are generated; at 12 weeks, the material is completely degraded, the bone defect area is filled with the new plate-shaped bone tissue, and the bone defect is completely repaired. B. Group C showed poor material absorption and low bone mass.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.