阅读说明：本技术 一种透明质酸寡糖修饰的矿化胶原仿生骨修复材料 (Mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide ) 是由 陈宗刚 张秀丽 栗敏 顾国锋 郭忠武 于 2017-03-28 设计创作，主要内容包括：本发明涉及一种透明质酸寡糖修饰的矿化胶原仿生骨修复材料及其制备方法。透明质酸寡糖修饰的矿化胶原仿生骨修复材料,结构如下：胶原-羟基磷灰石复合材料中的胶原通过C-N键连接透明质酸寡糖,获得糖基化修饰的矿化胶原复合材料,透明质酸寡糖的分子量为776～5000Da。本发明首次利用希夫碱反应对胶原进行透明质酸寡糖修饰,可获得共价结合的糖基化胶原,并首次提出将糖基化胶原作为矿化模板用于骨支架设计中,除了发挥低分子量HA利于细胞迁移、增殖、分化及促创伤愈合的功能外,为体外构建血管化支架提供了新的材料基础及研究策略。(The invention relates to a mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide and a preparation method thereof. The mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide has the following structure: collagen in the collagen-hydroxyapatite composite material is connected with hyaluronic acid oligosaccharide through a C-N bond to obtain the glycosylation modified mineralized collagen composite material, and the molecular weight of the hyaluronic acid oligosaccharide is 776-5000 Da. The invention firstly utilizes Schiff base reaction to modify collagen with hyaluronic acid oligosaccharide to obtain covalently bound glycosylated collagen, and firstly proposes that the glycosylated collagen is used as a mineralization template in bone scaffold design, thereby not only exerting the functions of low molecular weight HA favorable for cell migration, proliferation, differentiation and wound healing promotion, but also providing a new material basis and a research strategy for in vitro construction of a vascularization scaffold.)

1. A mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide has the following structure:

collagen in the collagen-hydroxyapatite composite material is connected with hyaluronic acid oligosaccharide through a C-N bond to obtain the glycosylation modified mineralized collagen composite material, and the molecular weight of the hyaluronic acid oligosaccharide is 776-5000 Da.

2. The mineralized collagen-simulated bone repair material according to claim 1, wherein the hyaluronic acid oligosaccharide-modified collagen has a sugar content of 0.909% to 5.266%;

preferably, the collagen is type I collagen, and the molecular weight of the collagen is 8-12 KD.

3. The mineralized collagen-simulated bone repair material according to claim 1, prepared by the following steps:

(1) mixing collagen, hyaluronic acid and NaBH₃CN is added into the reaction system, stirred and reacted for 1 to 3 days at the temperature of 36 to 38 ℃ in the dark, then an acetic acid solution is added for dilution, ultrafiltration and centrifugation are carried out, and after precipitation and drying, the glycosylated collagen is prepared;

the reaction system is a mixed solution of one of Hexafluoroisopropanol (HFP) or dimethyl amide (DMF) solvent and sodium bicarbonate, the molar concentration of the sodium bicarbonate is 0.05-0.15M, and the volume ratio of the hexafluoroisopropanol or dimethyl amide to the sodium bicarbonate is 3 (1-3);

the mass concentration of the collagen added into the reaction system is 18-22 g/L, the mass concentration of the hyaluronic acid added into the reaction system is 4-6 g/L, and NaBH is added₃The mass concentration of CN added into the reaction system is 6 g/L-18 g/L;

(2) adding the glycosylated collagen prepared in the step (1) into hydrochloric acid, stirring and dissolving to prepare a glycosylated collagen solution with the concentration of 0.5-0.7 mg/ml, and then adding CaCl₂Uniformly mixing the solution, standing for 8-15 min, and then stirring and adding NaH₂PO₄Adjusting the pH of the solution to 7, standing for 2-24 h at 25-38 ℃, performing solid-liquid separation, washing a precipitate, and drying to obtain the glycosylated collagen mineralized composite;

the CaCl is₂The addition amount of the solution is that 0.023-0.0913 mol of CaCl is added to each gram of collagen in the step (1)₂；CaCl₂With NaH₂PO₄The molar ratio of (1.5-1.8): 1;

(3) dissolving a forming agent in hexafluoroisopropanol to prepare a solution with the mass concentration of 2-4%, and then adding the glycosylated collagen mineralized composite material prepared in the step (2), wherein the mass ratio of the glycosylated collagen mineralized composite material to the forming is (1-3): 1, uniformly mixing, and performing electrostatic spinning to prepare a mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide;

the forming agent is collagen, polylactic acid or glycosylated collagen prepared in the step (1).

4. The mineralized collagen-mimicked bone repair material according to claim 3, wherein in step (1), the molar concentration of sodium bicarbonate is 0.1M, the volume ratio of hexafluoroisopropanol or dimethylamide solvent to sodium bicarbonate is 3: 2; the collagen is type I collagen, the mass concentration of the collagen added into the reaction system is 20g/L, the mass concentration of the hyaluronic acid added into the reaction system is 5g/L, and NaBH is added₃The mass concentration of CN added into the reaction system is 6 g/L;

preferably, in the step (1), the reaction is stirred for 1 day at 37 ℃ under the condition of keeping out of the light;

preferably, in the step (1), the mass concentration of the acetic acid solution is 5%, and the dilution volume multiple is 8 times;

preferably, in the step (1), the ultrafiltration tube for ultrafiltration and centrifugation is 30k, and the centrifugation conditions are as follows: 4000g/min, 25 min/time and 3-8 times of ultrafiltration;

preferably, in the step (1), the drying is vacuum freeze drying.

5. The mineralized collagen-simulated bone repair material according to claim 3, wherein in step (2), the concentration of hydrochloric acid is 0.01M;

preferably, in the step (2), the concentration of the glycosylated collagen solution is 0.6 mg/ml.

6. The mineralized collagen-simulated bone repair material according to claim 3, wherein in the step (2), the pH regulator is NaOH solution with a concentration of 0.1-0.5M;

preferably, in the step (2), standing is carried out for 22-24 hours at 37 ℃; preferably, the cleaning process is repeated centrifugal cleaning of deionized water for 3 times, wherein the centrifugal cleaning is carried out for 8min at 8000-10000 r/min;

preferably, in the step (2), the drying is vacuum freeze drying.

7. The mineralized collagen-simulated bone repair material according to claim 3, wherein in the step (3), the final electro-spinning solution has a mass concentration of 8%;

preferably, the forming agent is glycosylated collagen;

preferably, in the step (3), the uniform mixing is that after magnetic stirring and mixing are carried out for 5-10 min, ultrasonic treatment is carried out for 20min at 400-500W, and stirring is continued for 24-48 h; preferably, the spinning voltage is 15-20 kv, the speed is 0.6-1.0 mL/h, the receiving distance is 8-15 cm, and the receiving device is an aluminum foil or a cover glass with the diameter of 14mm placed on the aluminum foil.

8. A biocompatibility detection method of a mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide is characterized by comprising the following steps:

a. carrying out fixed cross-linking treatment and drying on the mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide to be detected to prepare a pretreatment material;

b. sterilizing the pretreatment material, soaking for 2.5-3.5 h by using an ethanol solution with the mass concentration of 70%, then transferring into sterilized PBS (phosphate buffer saline) for buffer soaking for 2h, taking out and repeatedly soaking for 3-4 times, and then soaking for 0.5h by using a 1640 culture medium or an alpha-MEM culture medium to prepare a pretreated sample;

c. co-culturing endothelial cell PIEC or precursor osteoblast MC3T3-E1 with the pretreated sample obtained in step c at a cell density of 10³～10⁴Per cm²，37℃、5％CO₂Culturing under the conditions, detecting the proliferation of cells at 1d, 3d, 5d and 7d after culturing, observing the adhesion and growth forms of the scaffolds after culturing the cells at 3d and 5d by using a Scanning Electron Microscope (SEM), detecting the expression of ALP of the alkaline phosphatase of MC3T3-E1 during culturing, and evaluating the biocompatibility according to the results.

9. The method for detecting biocompatibility according to claim 8, wherein in the step a, the fixing and crosslinking treatment is carried out by steam treatment with 25% glutaraldehyde for 24-36 h or soaking in a 95% ethanol system containing EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) for 20-24 h; preferably, the drying is performed by standing in a vacuum drying oven for 3-5 days.

10. The method according to claim 8, wherein in the step b, the culture medium is changed every 2 to 3 days during the culture.

Technical Field

The invention relates to a mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide and a preparation method thereof, belonging to the technical field of biomedical materials.

Background

Bone defects caused by trauma, tumors, infection, pathological factors and the like are quite common in clinic, and become one of the difficult problems which puzzle the healthy life of human beings. Bone grafting has become the largest volume graft second only to blood transfusion and has a tendency to increase year by year. At present, the bone repair materials for clinical application comprise autogenous bone, allogeneic bone, xenogeneic bone, artificial bone and the like, but have certain problems and can not completely solve the huge clinical requirements on the bone materials. Despite limited sources and secondary trauma issues, autologous bone remains the best clinically effective bone implant material today and has been the "gold standard" for the treatment of bone defects. Therefore, by simulating the components and the structure of natural bone, scientists at home and abroad prepare bionic composite bone materials of different forms of collagen-hydroxyapatite by an in vitro artificial synthesis method, and the bionic composite bone materials are applied to clinic. However, with the intensive research and the follow-up of clinical application, it is found that these materials have the disadvantages of insufficient bioactivity or insufficient bone repair effect compared with autologous bone, and it is difficult to obtain ideal repair effect. In addition, the bone is a highly vascularized tissue, and the current research on tissue engineering bone mostly neglects the revascularization of the graft, for large bone defects, the range of nutrition and oxygen permeation of peripheral tissues is limited, and the formation of the endogenous blood vessels of the implanted bone material is slow or difficult to vascularize, which finally results in the remarkable reduction of the osteogenic activity of the transplanted bone and even the necrosis of central cells of the tissue. Therefore, the problems of insufficient biological activity, vascularization and the like of the traditional bone scaffold material still remain the key point of tissue engineering bone research.

Hyaluronic Acid (HA) is a natural extracellular matrix, HAs good biocompatibility and physicochemical properties, can affect proliferation, migration and differentiation of cells, and particularly HAs biological activities of promoting vascularization, wound repair and immunoregulation of oligosaccharide oHA. At present, the binding of hyaluronic acid to proteins such as collagen for the construction of tissue materials has been studied, but the use of hyaluronic acid having a large molecular weight, which is an oligosaccharide of at least 5kD, has been focused. This is mainly because oHA below 5kD is difficult and expensive to produce and the lower the molecular weight, the greater the difficulty in designing the material.

Chinese patent document CN105903081A (application No. 201510926063.4) discloses a preparation method of a novel double-layer proteoglycan-based repair material, wherein hyaluronic acid and type I collagen (Col I) are mainly combined through charge and hydrogen bond effects, and then a three-dimensional reticular composite material is obtained through freeze drying and thermal crosslinking treatment, and the polysaccharide and collagen composite material is mainly applied to the repair of soft tissues.

The basic material which is more important in the tissue engineering bone is collagen/hydroxyapatite composite material, the preparation process and the components of the material are continuously improved, the prior mechanical mixing of calcium phosphate and collagen is gradually developed into the directional arrangement of inorganic particles on polymer matrixes such as collagen and the like to realize the effective compounding of inorganic components and organic components, and in addition, the defect of poor mechanical property of the collagen is improved by introducing other components. Chinese patent document CN101590293A (application No. 200910149959.0) discloses a method for preparing HA (herein referred to as hydroxyapatite)/collagen/chitosan interpenetrating polymer network scaffold, which comprises dispersing hydroxyapatite sol prepared by using PVP as a template in a collagen and chitosan blend solution, performing post-treatment such as pressure reduction and degassing after two-step cross-linking to obtain a composite scaffold, wherein although inorganic particles in the scaffold are well dispersed in a collagen/chitosan matrix, the overall operation process is more, and natural bone is a hierarchical structure formed by using collagen as a template through regulation of non-collagen or interaction between collagen and a mineral phase to realize nucleation and mineralization of calcium phosphate and based on a self-assembly process of collagen, the multi-level ordered structure formed from micro-level to macro-level of the bone is very important for the performance and function of the bone, and the separately prepared hydroxyapatite is only cross-linked and combined on the polymer matrix in the document, microstructural biomimetics of bone was not achieved.

Disclosure of Invention

Aiming at the huge demand of the current clinical bone transplantation and the defects of the existing bone repair material, the invention overcomes the defects of the prior art and provides a mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide and a preparation method thereof.

The technical scheme of the invention is as follows:

a mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide has the following structure:

collagen in the collagen-hydroxyapatite composite material is connected with hyaluronic acid oligosaccharide through a C-N bond to obtain the glycosylation modified mineralized collagen composite material, and the molecular weight of the hyaluronic acid oligosaccharide is 776-5000 Da.

According to the invention, the preferable sugar-carrying mass percentage of the hyaluronic acid oligosaccharide modified collagen is 0.909% -5.266%.

According to the invention, the collagen is preferably type I collagen, and the molecular weight of the collagen is 8-12 KD.

The structure assembly process of the mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide is as follows:

self-assembling collagen molecules modified by hyaluronic acid oligosaccharide to form collagen microfibrils; regulating the calcium-phosphorus salt to be arranged along the axial orientation of the microfibrils by glycosylated collagen molecules, and further assembling to form mineralized collagen fibers; the mineralized collagen fibers are further assembled to form glycosylation mineralized collagen fiber bundles in an orientation arrangement (as shown in figure 2); the mineralized materials are assembled into a nanofiber bionic bone repair material (shown in figure 1) through electrostatic spinning.

A preparation method of a mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide comprises the following steps:

(1) mixing collagen and gelatinHyaluronic acid and NaBH₃CN is added into the reaction system, stirred and reacted for 1 to 3 days at the temperature of 36 to 38 ℃ in the dark, then an acetic acid solution is added for dilution, ultrafiltration and centrifugation are carried out, and after precipitation and drying, the glycosylated collagen is prepared;

the reaction system is a mixed solution of one of Hexafluoroisopropanol (HFP) or dimethyl amide (DMF) solvent and sodium bicarbonate, the molar concentration of the sodium bicarbonate is 0.05-0.15M, and the volume ratio of the hexafluoroisopropanol or dimethyl amide to the sodium bicarbonate is 3 (1-3);

the mass concentration of the collagen added into the reaction system is 18-22 g/L, the mass concentration of the hyaluronic acid added into the reaction system is 4-6 g/L, and NaBH is added₃The mass concentration of CN added into the reaction system is 6 g/L-18 g/L;

(2) adding the glycosylated collagen prepared in the step (1) into hydrochloric acid, stirring and dissolving to prepare a glycosylated collagen solution with the concentration of 0.5-0.7 mg/ml, and then adding CaCl₂Uniformly mixing the solution, standing for 8-15 min, and then stirring and adding NaH₂PO₄Adjusting the pH of the solution to 7, standing for 2-24 h at 25-38 ℃, performing solid-liquid separation, washing a precipitate, and drying to obtain the glycosylated collagen mineralized composite;

the CaCl is₂The addition amount of the solution is that 0.023-0.0913 mol of CaCl is added to each gram of collagen in the step (1)₂；CaCl₂With NaH₂PO₄The molar ratio of (1.5-1.8): 1;

(3) dissolving a forming agent in hexafluoroisopropanol to prepare a solution with the mass concentration of 2-4%, and then adding the glycosylated collagen mineralized composite material prepared in the step (2), wherein the mass ratio of the glycosylated collagen mineralized composite material to the forming is (1-3): 1, uniformly mixing, and performing electrostatic spinning to form porous nanofiber, thereby preparing the mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide;

the forming agent is collagen, polylactic acid or glycosylated collagen prepared in the step (1);

preferably, in the step (1), the molar concentration of the sodium bicarbonate is 0.1M, and the volume ratio of the hexafluoroisopropanol or the dimethylamide solvent to the sodium bicarbonate is3: 2; the collagen is type I collagen, the mass concentration of the collagen added into the reaction system is 20g/L, the mass concentration of the hyaluronic acid added into the reaction system is 5g/L, and NaBH is added₃The mass concentration of CN added into the reaction system is 6 g/L.

Preferably, in step (1), the reaction is stirred for 1 day at 37 ℃ in the absence of light.

According to the invention, in the step (1), the mass concentration of the acetic acid solution is 5%, and the dilution volume is 8 times.

Preferably, in step (1), the ultrafiltration tube for ultrafiltration centrifugation is 30k, and the centrifugation conditions are as follows: 4000g/min, 25 min/time and 3-8 times of ultrafiltration.

According to the present invention, in the step (1), the drying is vacuum freeze drying.

According to the present invention, in the step (2), the concentration of hydrochloric acid is preferably 0.01M.

Preferably, in the step (2), the concentration of the glycosylated collagen solution is 0.6 mg/ml.

According to the invention, in the step (2), the pH regulator is NaOH solution with the concentration of 0.1-0.5M. In the process of pH adjustment, when the pH is close to or reaches 6, the solution begins to be turbid, the change fluctuation is large when the pH is about 6.2, the reaction is still carried out at the moment, the stirring is continued, the NaOH solution is dripped until the pH of the system is 7, and then the stirring is continued for 2 hours.

Preferably, in the step (2), standing is carried out for 22-24 hours at 37 ℃; preferably, the cleaning process is repeated centrifugal cleaning for 3 times by using deionized water, and the centrifugal cleaning is carried out for 8min at 8000-10000 r/min.

According to the present invention, in the step (2), the drying is vacuum freeze drying.

According to the invention, in the step (3), the mass concentration of the final electrospinning solution is 8%; preferably, the forming agent is glycosylated collagen.

According to the invention, in the step (3), the uniform mixing is performed by adopting magnetic stirring and mixing for 5-10 min, then carrying out ultrasonic treatment at 400-500W for 20min, and continuing stirring for 24-48 h; preferably, the spinning voltage is 15-20 kv, the speed is 0.6-1.0 mL/h, the receiving distance is 8-15 cm, and the receiving device is an aluminum foil or a cover glass with the diameter of 14mm placed on the aluminum foil;

a biocompatibility detection method of a mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide comprises the following steps:

a. carrying out fixed cross-linking treatment and drying on the mineralized collagen bionic bone repair material modified by hyaluronic acid oligosaccharide to be detected to prepare a pretreatment material;

b. sterilizing the pretreatment material, soaking for 2.5-3.5 h by using an ethanol solution with the mass concentration of 70%, then transferring into sterilized PBS (phosphate buffer saline) for buffer soaking for 2h, taking out and repeatedly soaking for 3-4 times, and then soaking for 0.5h by using a 1640 culture medium or an alpha-MEM culture medium to prepare a pretreated sample;

c. co-culturing endothelial cell PIEC or precursor osteoblast MC3T3-E1 with the pretreated sample obtained in step c at a cell density of 10³～10⁴Per cm²，37℃、5％CO₂Culturing under the conditions, detecting the proliferation of cells at 1d, 3d, 5d and 7d after culturing, observing the adhesion and growth forms of the scaffolds after culturing the cells at 3d and 5d by using a Scanning Electron Microscope (SEM), detecting the expression of ALP of the alkaline phosphatase of MC3T3-E1 during culturing, and evaluating the biocompatibility according to the results.

The biocompatibility evaluation described above can be evaluated by methods that are conventional in the art. For example, endothelial cell PIEC can be referred to a related method in "collagen-chitosan nanofiber biomimetic extracellular matrix prepared by electrospinning" (Chen Zong-gang; Donghua university, 2007.); MC3T3-E1 cells can be referenced to the methods related in biocompatibility of modified poly (D, L-lactic acid) with MC3T3-E1 cells (Zhengdanfang; Chongqing university, 2008.).

Preferably, in the step a, the fixing and crosslinking treatment is carried out by steam treatment with 25% glutaraldehyde for 24-36 h or soaking in a 95% ethanol system containing EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) for 20-24 h; preferably, the drying is performed by standing in a vacuum drying oven for 3-5 days.

Preferably, in the step c, the culture medium is changed every 2 to 3 days during the culture process.

The 1640 medium and the α -MEM medium are commercially available media which are conventional in the art. Endothelial cells PIEC and precursor osteoblasts MC3T3-E1 are commercially available cells routinely used in the art.

Advantageous effects

1. According to the invention, the Schiff base reaction is utilized for carrying out hyaluronic acid oligosaccharide modification on collagen for the first time, so that the covalently bound glycosylated collagen can be obtained, and besides the functions of low molecular weight HA favorable for cell migration, proliferation and differentiation, the covalently bound glycosylated collagen also HAs potential influences on the aspects of collagen space configuration and activity, molecular recognition, cell communication, signal transduction and the like; compared with the prior art that the crosslinking of micromolecule oligosaccharide and collagen is realized by utilizing a physical mode, the glycosylated collagen prepared by the invention has remarkable advantages; in addition, the method is simple and low in cost by using the low-molecular-weight HA to modify the collagen, and the collagen can be used for a template for calcium phosphate deposition in a subsequent mineralization reaction, so that the collagen HAs potential application in the research of repair and regeneration of hard tissues;

2. the invention utilizes hyaluronic acid oligosaccharide fragment capable of promoting vascularization to modify collagen bionic extracellular matrix, and the prepared material can endow the material with anticoagulation and vascularization promoting functions while improving the biocompatibility of the material, so that the stent can survive for a long time after being transplanted into a body, thereby providing a new material foundation and a research mode for constructing the vascularization promoting stent of vascularization tissue engineering bone and other tissues;

3. the scaffold designed by the invention is based on the bionics principle and the self-assembly technology, realizes the nucleation and mineralization of calcium phosphate by using glycosylated collagen as a template for the first time, has the components and the microstructure closer to natural bone tissues, is expected to promote angiogenesis in new bones and improve the osteogenesis effect, and has the characteristic of biological activity compared with the composite scaffold which is obtained by dispersing the existing independently prepared hydroxyapatite sol in the collagen/chitosan blended solution to obtain the oriented arrangement of inorganic particles in a polymer matrix;

4. the invention utilizes the electrostatic spinning technology to form the bracket material, which is different from the simple mechanical mixing of inorganic powder and high molecular material in the prior art, the preparation of the electrospinning system is that the mineralization and nucleation of calcium and phosphorus salt on collagen are realized through biomineralization to obtain the mineralized composite material, so that the inorganic/organic two phases can form a tight bond and have a certain orientation relation, and then a certain amount of polymers such as collagen or polylactic acid and the like and the freeze-dried mineralized composite powder are introduced to form a uniform mixing system for electrospinning, so that the nano porous fiber of the mineralized material is obtained, is more close to the form of natural extracellular matrix, is beneficial to the adhesion and growth of cells, and good pores are easier to be connected with the inward migration of cells and the diffusion of nutrient substances; the method for pre-precipitating the polymer to obtain the composite material and then optimizing the conditions for electrospinning can effectively improve the problems of poor inorganic particle dispersibility or phase separation in a mechanical mixing system.

Drawings

FIG. 1 is an infrared spectrum of collagen Col, collagen/hydroxyapatite Col/HAP, glycosylated collagen/hydroxyapatite Col/oHAs/HAP;

FIG. 2 is a TEM morphology of collagen/oHAs/hydroxyapatite material synthesized by reaction standing for 24 h;

FIG. 3 is an SEM morphology of a Col/HAP-Col-3-1 blend fiber scaffold;

FIG. 4A is an SEM image of endothelial cells when cultured in Col/HAP-Col-3-1 for 3 days;

FIG. 4B is an SEM image of endothelial cells when cultured in Col/HAP-PLA-3-1 for 3 days;

FIG. 5 shows the results of the proliferation tendency of endothelial cells on various mineralized collagen fiber scaffolds;

FIG. 6A is an SEM image of endothelial cells cultured on Col/HAP-Col-3-2 for 5 days;

FIG. 6B is an SEM image of endothelial cells cultured on Col/oHAs/HAP-Col-3-2 for 5 days;

FIG. 6C is an SEM image of endothelial cells cultured on Col/HA/HAP-Col-3-2 for 5 days;

FIG. 7 is a bar graph showing the results of ALP detection of MC3T3-E1 on Col/HAP-Col-3-2, Col/oHAs/HAP-3-2, Col/HA/HAP-Col-3-2 scaffolds;

FIG. 8 is a bar graph showing the results of the proliferation tendency of MC3T3-E1 on Col/HAP-Col-3-2, Col/oHAs/HAP-Col/oHAs-3-2, Col/HA/HAP-Col/HA-3-2 fibrous scaffolds;

FIG. 9 is an SEM topography of MC3T3-E1 on Col/oHAs/HAP-Col/oHAs-3-2 fibrous scaffolds.

Detailed Description

The technical solutions of the present invention are further described below with reference to the following embodiments and the drawings of the specification, but the scope of the present invention is not limited to these embodiments.

Source of raw materials

Sodium hyaluronate raw material HA was purchased from Huaxi furuida biomedical limited, molecular weight 5 kD;

the collagen is type I collagen with a molecular weight of 10kD and is purchased from Doctorle biotechnology limited;