CN110947031A - A kind of bone tissue engineering scaffold material with high biological activity and its preparation method and application - Google Patents
A kind of bone tissue engineering scaffold material with high biological activity and its preparation method and application Download PDFInfo
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- CN110947031A CN110947031A CN201910913778.4A CN201910913778A CN110947031A CN 110947031 A CN110947031 A CN 110947031A CN 201910913778 A CN201910913778 A CN 201910913778A CN 110947031 A CN110947031 A CN 110947031A
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- engineering scaffold
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Abstract
本发明公开了一种具有高生物活性的骨组织工程支架材料及其制备方法和应用。将纳米鱼骨和经过甲基丙烯酸酐改性的明胶(GelMA)，在光引发剂的存在下进行光聚合反应形成复合水凝胶，所述复合水凝胶即为骨组织工程支架材料；所述纳米鱼骨的含量为0.5％～90％。本发明的复合水凝胶型骨组织工程支架材料不仅具有良好的骨传导性以及骨诱导性，在体内可降解，而且具备三维多孔结构和良好的力学性能，能够在短时间内黏附较高数量及质量的细胞，并且能够促进细胞生长、增殖、迁移和分化，有利于新生组织的结构重塑和构建，达到快速治疗骨缺损的目的满足临床需要。本发明产品安全性好，性能可靠，操作简便，重复性好，易于产业化。
The invention discloses a bone tissue engineering scaffold material with high biological activity and a preparation method and application thereof. The nano-fish bone and the gelatin (GelMA) modified by methacrylic anhydride are photopolymerized in the presence of a photoinitiator to form a composite hydrogel, and the composite hydrogel is a bone tissue engineering scaffold material; The content of the nano fish bone is 0.5% to 90%. The composite hydrogel type bone tissue engineering scaffold material of the present invention not only has good osteoconductivity and osteoinductivity, and is degradable in vivo, but also has a three-dimensional porous structure and good mechanical properties, and can adhere to a relatively high amount in a short time. It can promote the growth, proliferation, migration and differentiation of cells, which is conducive to the structural remodeling and construction of new tissues, and achieves the purpose of rapid treatment of bone defects to meet clinical needs. The product of the invention has good safety, reliable performance, simple operation, good repeatability and easy industrialization.
Description
Technical Field
The invention belongs to the technical field of tissue engineering. More particularly, relates to a bone tissue engineering scaffold material with high bioactivity, a preparation method and application thereof.
Background
The bone defect is difficult to repair by self, which causes great pain to the patient. Bone defect repair is a troublesome problem which troubles clinical treatment, the number of patients is large worldwide, and the incidence of bone defects is on a growing trend along with the aging of society. The clinical bone defect repair mainly adopts autologous bone graft, allogeneic bone graft and artificial bone prosthesis. Autologous cartilage transplantation has greatly limited the clinical application of this method due to the limited source of cartilage tissue. The bone regeneration and self-repair capability is extremely limited, while the existing artificial bone prosthesis has the problems of insufficient bone repair effect, poor biological safety, strong immune stimulation, material shortage or complex treatment, high cost in manufacturing and use, poor operability and the like. Therefore, no perfect method for completely solving the repair problem of damaged tissues and organs exists clinically so far.
With the rapid development of bone tissue engineering in recent years, the selection and preparation of scaffold materials become hot spots for research, and the ideal scaffold material for bone tissue engineering should satisfy the following points: 1. the biocompatibility is high: besides meeting the general requirements of biological materials such as no toxicity, no aberration and the like, the degradation product also has the advantages of no toxic action on cells, no inflammatory reaction, contribution to adhesion and proliferation of seed cells and promotion on growth and differentiation of the cells; 2. the biodegradability is suitable for: the scaffold material has a degradation function, and the growth rate of bone tissue cells is adapted to the degradation rate; 3. has a suitable three-dimensional porous structure: the scaffold material has a three-dimensional structure, corresponding porosity and high specific surface area, can provide an optimal microenvironment for adhesion, proliferation, growth and function exertion of osteoblasts, and can also provide a space and a scaffold for formation of new bone tissues; 4. plasticity and suitable mechanical strength: the scaffold material has good plasticity and proper mechanical strength, and can support the new tissue until the new tissue has proper mechanical properties; 5. good scaffold-cell interface: the material should provide a good scaffold-cell interface, facilitating cell adhesion. The hydrogel is a three-dimensional cross-linked hydrophilic polymer material, can absorb and retain certain water, but is insoluble in water, has good biocompatibility, and is one of the hot spots of current material science research. The application fields of the current hydrogel comprise biology, medicine, chemical industry, food and the like. The hydrogel structure and the extracellular matrix are similar to each other, so that necessary three-dimensional space and mechanical support are provided for the reconstruction of cells and tissues, and the function of simulating the extracellular matrix is achieved; has good histocompatibility, proper pore size and porosity, and is beneficial to the proliferation and adhesion of chondrocytes, the infiltration of nutrient substances and the discharge of cell metabolites; has good biological activity and is widely used for the research of bone defect regeneration.
Methacrylic anhydride modified gelatin (GelMA) is the product of reacting gelatin with methacrylic anhydride. The GelMA hydrogel is a polymer derived from gelatin and methacrylic acid, has stable chemical properties, proper expansibility, good biocompatibility, good flexibility and the like, and is widely used in the fields of tissue engineering such as wound repair, artificial tissue, bone repair and the like. However, the GelMA hydrogel alone has the disadvantages of poor mechanical properties, especially insufficient elastic modulus at the bone repair level to support the required mechanical strength, and the GelMA hydrogel alone does not have corresponding biological functionality, such as antibacterial property, tissue regeneration promotion, bone regeneration, etc., and various materials are usually required to be added so as to achieve the corresponding functions better. It should be noted that the preparation process, molecular weight, concentration and other conditions of different GelMA hydrogels have an effect on the degradation and mechanical properties. Many composite hydrogel materials promoting relevant performances are developed at present, but the main problems are difficult to realize clinically and the corresponding effects are poor.
The main components of natural bone are collagen fiber and hydroxyapatite nanocrystal, and since the self bone tissue has limited material, the granular bone is mainly derived from animal bone tissue (bovine bone, pig bone, coral bone, fish bone, etc.) or hydroxyapatite, calcium silicate, calcium phosphate, calcium carbonate, etc. with similar components. However, the particle bone has the inherent defect of insufficient space stability (loose structure and easy dispersion by blood or flushing fluid) in the application process (Sunzhi, Xiaoyumei, Van hong Song, Zhang xing, influence of hydroxyapatite on the preparation and performance of calcium alginate hydrogel [ J ] the report on inorganic materials, 2010,25(10): 1087-. The research direction has thus focused on the study of osteoinductive hydrogels, which are expected to impart better steric stability to the particulate bone by means of hydrogel loading. The inventor subject group previously disclosed a composite hydrogel based on black phosphorus and gelatin and application thereof in bone tissue engineering in chinese patent document CN 108653809a, wherein black phosphorus and gelatin modified by double bond are used as bone tissue engineering scaffold materials, and the composition and proportion of black phosphorus and gelatin modified by double bond are adjusted to ensure that the hydrogel scaffold materials have good mechanical properties, mechanical properties and biological properties. The development of the tissue engineering scaffold with high biological activity and high cell affinity is the main development direction of the biological materials in the future.
The prior art is mostly limited to simple loading of hydrogel and granular bone, and has poor compatibility of an organic phase and a granular bone interface; insufficient osteoinductive properties; the adhesion rate of bone cells in various scaffolds is poor; the proliferation effect of the adhered bone cells is poor; slow speed of cell growth, etc.
Disclosure of Invention
The primary object of the present invention is to overcome the above-mentioned drawbacks and disadvantages of the prior art and to provide a method for preparing a scaffold material for bone tissue engineering with high bioactivity. The bone tissue engineering scaffold material not only has good osteoconductivity and osteoinductivity, is degradable in vivo, but also has suitable appearance, three-dimensional porous structure and good mechanical property, can provide calcium, phosphorus, magnesium and other ions for bone tissues, can adhere cells with higher quantity and quality in a short time, can promote cell growth, proliferation, migration and differentiation, is beneficial to structural remodeling and construction of new tissues, and achieves the aim of quickly treating bone defects to meet clinical requirements.
The invention also aims to provide the bone tissue engineering scaffold material with high bioactivity, which is prepared by the method.
The invention further aims to provide the application of the bone tissue engineering scaffold material in serving as or preparing a bone graft.
It is still another object of the present invention to provide a bone graft comprising the above bone tissue engineering scaffold material.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a bone tissue engineering scaffold material with high biological activity comprises the steps of carrying out photopolymerization on nano fishbone and gelatin (GelMA) modified by methacrylic anhydride in the presence of a photoinitiator to form composite hydrogel, wherein the composite hydrogel is the bone tissue engineering scaffold material; the content of the nano fishbone is 0.5-90%.
The nanometer fishbone is a main solid byproduct of the fish slices and the minced fillet, has wide sources, and contains rich components such as protein, lipid and the like besides mineral elements such as calcium, magnesium, phosphorus and the like; in addition, the bone regeneration water-soluble powder has better water solubility, water retention and suspension stability, and can provide corresponding nutrients for bone regeneration. Gelatin has good biocompatibility and is often used to mimic the extracellular matrix. The invention takes nano fishbone as a main material, and prepares the methacrylic acid derivative polymer by gelatin, so that the methacrylic acid derivative polymer and the nano fishbone are solidified and crosslinked through photopolymerization to generate the composite hydrogel type bone tissue engineering scaffold material, the bone tissue engineering scaffold material not only has good bone conductivity and bone inductivity, but also has a three-dimensional porous structure and good mechanical property, can adhere cells with higher quantity and quality in a short time, can promote the growth, proliferation, migration and differentiation of the cells, is beneficial to the structural remodeling and construction of new tissues, and achieves the aim of quickly treating bone defects to meet the clinical requirements.
The invention can further adjust the physical and chemical properties, mechanical properties and biological properties of the scaffold material by adjusting the composition and proportion of the nano fishbone and the methacrylic anhydride modified gelatin, and better induce the growth, proliferation, migration and differentiation of cells, thereby realizing the accurate and controllable biological function.
In a preferred embodiment of the invention, the content of the nano fishbone is 1-30% based on the osteoconductivity, osteoinductivity, mechanical strength and biodegradability of the bone tissue engineering scaffold material; preferably 3% -10%; more preferably 5% to 7%.
The method utilizes nano fishbone and methacrylic anhydride modified gelatin to synthesize the composite hydrogel under a controlled condition, and the storage modulus of the composite hydrogel is 1000-100000 Pa.
In a preferred embodiment of the present invention, the particle size of the nano fishbone is 1-200 nm; preferably 10 to 100 nm.
In a preferred embodiment of the invention, the nano fishbone is prepared by the following method:
s11, cleaning the fishbone, adding water to immerse the fishbone, cooking and softening the fishbone, rinsing until a washing liquid is clear, and draining;
s12, mixing the raw materials in a material-liquid ratio of 1: 1-10, adding ice water, and crushing at 2000-4000 r/min to obtain fishbone powder or fishbone paste with the particle size of less than 105 mu m;
s13, mixing the raw materials in a material-liquid ratio of 1: 1-10 adding a water solution of triton to the fishbone powder or fishbone paste, stirring at room temperature for 24-48 h, changing the solution every 8-10 h, and rinsing until the washing solution is foamless to obtain acellular bone paste;
s14, adding water with the solid content of 1% -10%, uniformly mixing, and then carrying out ball milling for 1-12 h.
In the preferred embodiment of the present invention, in step S11, the ratio of the feed to the liquid (the ratio of the mass of the fishbone to the volume of the water) is 1: 3 to 5.
In a preferred embodiment of the present invention, in step S11, the washing solution is rinsed repeatedly with hot water of 60-80 ℃ until the washing solution is clear.
In a preferred embodiment of the present invention, the cooking softening conditions are as follows: steaming at 100-150 deg.C for 30-120 min.
In a preferred embodiment of the invention, the concentration of the triton aqueous solution is 0.1-10%; preferably 0.3 to 5%.
In the preferred embodiment of the present invention, in step S14, the ball milling conditions are as follows: the stirring speed is 1000-5000 r/min, the ball milling filling rate is 60-85%, and the diameter of the grinding ball is 0.5-1 mm.
In a preferred embodiment of the present invention, the methacrylic anhydride modified gelatin preferably has a molecular weight of 150 to 1200000, more preferably 250 to 100000, and most preferably 250 to 80000, from the viewpoint of biocompatibility, porosity, plasticity, and mechanical strength.
In a preferred embodiment of the invention, the methacrylic anhydride modified gelatin is prepared by the following method: reacting 7-18% of gelatin-PBS solution with methacrylic anhydride at 45-60 ℃ for 5-20 h, dialyzing and drying a reaction product to obtain a white and soft spongy product, namely the photo-crosslinkable gelatin modified by the methacrylic anhydride.
In a preferred embodiment of the invention, the gelatin preferably has a molecular weight of from 1 to 30 ten thousand, more preferably from 2 to 5 ten thousand.
In a preferred embodiment of the invention, the methacrylic anhydride modified gelatin has a degree of methacrylation of at least 10%, preferably 20% to 80%. Experiments show that when the degree of the methacrylic acid esterification is higher, the photocrosslinking performance of the product is improved, the strength of the composite hydrogel is favorably improved, and the prepared composite hydrogel is more favorable for cell adhesion, growth, proliferation and migration under different conditions.
As used herein, the term "degree of methacrylation" refers to the amount of carboxylic acid groups present in a methacrylic acid-derivatized polymer, which carboxylic acid groups are capable of reacting to produce methacrylate groups.
In a preferred embodiment of the invention, the photoinitiator is one or more of I2959, benzophenone, benzoin butyl ether, isopropyl thioxanthone, diphenylethanone or methyl benzoylformate.
In a preferred embodiment of the present invention, the addition amount of the photoinitiator is 0.05% to 5%.
In a preferred embodiment of the present invention, the photopolymerization conditions are as follows: and ultraviolet irradiation is carried out for 10-300 s.
The invention also provides a culture method of the fibroblast, which comprises the following specific steps: the bone tissue engineering scaffold material is used as a culture carrier, and fibroblasts are suspended and inoculated on the surface of the bone tissue engineering scaffold material.
The preparation method of the fibroblast suspension comprises the following steps: culturing in a culture medium until the cell fusion is 80-90%, digesting, centrifuging, and resuspending to obtain cell suspension.
In a preferred embodiment of the invention, the culture medium comprises the following components: penicillin of 90-110 mu g/mL, streptomycin of 90-110 mu g/mL, 8% -12% fetal bovine serum, and the balance of L-DMEM and/or DMEM/F12.
In a preferred embodiment of the invention, the digestion is passaged with 0.2% to 0.3% trypsin containing 0.1% to 0.15% EDTA.
In the preferred embodiment of the invention, when the fibroblasts are cultured on the surface of the composite hydrogel, the sterilized composite hydrogel is paved in a 6-well plate, a fibroblast suspension is inoculated, after incubation for 10 min-2 h, a proper amount of culture medium is added and placed at 37 ℃ and 5% CO2Culturing in an incubator.
The bone tissue engineering scaffold material with high bioactivity prepared by the method and the application thereof in serving as or preparing bone graft are also within the protection scope of the invention.
The invention also provides a bone graft comprising: the bone tissue engineering scaffold material with high bioactivity; and cells seeded on the bone tissue engineering scaffold material.
The composite hydrogel type bone tissue engineering scaffold material prepared by the invention not only has the shape of the traditional hydrogel product and the water absorption and plasticity of the traditional composite hydrogel, but also has the following beneficial effects:
1. the invention takes the nanometer fishbone as the main material, and the natural high-biocompatibility material can provide a suitable microenvironment for the growth, proliferation and differentiation of cells, so as to ensure that a local environment enriched with a large number of cells is formed around the implant, and the material has good osteoconductivity and osteoinductivity, and can quickly express the proliferation activity after the cells are attached and spread on the surface of the material, thereby being beneficial to the structural remodeling and construction of new tissues, and achieving the purpose of quickly treating bone defects and meeting the clinical requirements.
2. The invention obtains the gelatin modified by methacrylic anhydride by modifying the gelatin with excellent biocompatibility, and the gelatin is compounded with the nano fishbone, thereby not only forming stable colloid under lower solid content, but also being beneficial to improving the strength of the product, and further preparing the bone tissue engineering scaffold material with good mechanical property and biological property and high biological activity.
3. The composite hydrogel type bone tissue engineering scaffold material prepared by the invention can realize the rapid preparation of bone tissue engineering scaffold materials with different shapes and thicknesses by regulating and controlling the proportion of the nano fishbone and the methacrylic anhydride modified gelatin.
4. The composite hydrogel type bone tissue engineering scaffold material prepared by the invention is of a three-dimensional porous three-dimensional structure, has a large communicated pore structure which can enable cells to smoothly enter the interior of the scaffold, and also has a small pore structure which can enable the cells to adhere, migrate and exchange nutrition; meanwhile, the compound has good biodegradability, biocompatibility and extremely low cytotoxicity, and is suitable for cell adhesion, growth, proliferation and migration; in addition, the material has good mechanical property and can bear the pressure generated in physiological activities.
5. The composite hydrogel type bone tissue engineering scaffold material prepared by the invention has better bone repair capability in an animal skull defect model, and can be beneficial to bone regeneration; and the existence of the nano fishbone not only improves the adhesion rate of cells, but also ensures that bone cells which survive per se can be used as seed cells to participate in osteogenesis, thereby being more beneficial to bone regeneration, and showing that the composite hydrogel type bone tissue engineering scaffold material can effectively promote the generation of new bone and the repair of bone defect.
Drawings
Fig. 1 shows the potential change of the Nano Fishbone (NFB) at different milling times.
FIG. 2 is a particle size distribution of Nanometer Fishbone (NFB) after ball milling for 8 h.
Fig. 3 is the composite hydrogel type bone tissue engineering scaffold material packaged by the invention.
FIG. 4 is a scanning electron microscope image of the Nano Fishbone (NFB) -gelatin (GelMA) composite hydrogel type bone tissue engineering scaffold material modified by methacrylic anhydride, wherein a (10% GelMA/0% NFB), b (GelMA/1% NFB), c (GelMA/3% NFB), and d (GelMA/5% NFB).
FIG. 5 shows the rheological properties of the composite hydrogel type bone tissue engineering scaffold material of the present invention.
FIG. 6 shows the living and dead staining of the composite hydrogel bone tissue engineering scaffold material surface inoculated with cells in culture, wherein a (control, blank control), b (10% GelMA/0% NFB), c (GelMA/1% NFB), d (GelMA/3% NFB), e (GelMA/5% NFB), f (GelMA/7% NFB), and g (GelMA/10% NFB).
FIG. 7 shows the cell proliferation results of the composite hydrogel bone tissue engineering scaffold material of the present invention.
FIG. 8 is a nuclear magnetic image of methacrylic anhydride modified gelatin (GelMA).
FIG. 9 shows the bone repair situation after one month of the implantation of the composite hydrogel type bone tissue engineering scaffold material of the present invention into rat skull defect model, wherein a (control, blank control), b (10% GelMA/0% NFB), c (GelMA/1% NFB), d (GelMA/3% NFB), e (GelMA/5% NFB).
Detailed Description
The invention is further described below with reference to the figures and the specific examples. The following examples are preferred embodiments of the present invention, but are not intended to limit the scope of the present invention in any manner. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1
1. Preparing the nano fishbone:
(1) weighing the backbone of the grass carp with meat, and mixing the components in a ratio of 1: 3 (fishbone mass: water volume) adding tap water, and stewing for 60min at 121 ℃ in a back-pressure high-temperature stewing pan (ZM-100 Guangzhou Daizhou Dayuanjian packaging equipment Co., Ltd.); repeatedly rinsing with 80 deg.C hot water to retain white solid, and removing the suspended oil until the lotion is clear;
(2) crushing the white solid by a conditioner, wherein the material-liquid ratio is 1: 1 adding ice water, micronizing and crushing with bone paste machine (MKCA6-2 Japan MASUKO) at 2000r/min until the grinding disc gap is reduced to 0.3mm to obtain bone paste;
(3) according to the material-liquid ratio of 1: 10, adding 0.3 percent triton aqueous solution, stirring for 24 hours at room temperature, changing the solution once every 8 hours, and then rinsing with a large amount of deionized water until the washing solution is foamless to obtain acellular bone paste;
(4) measuring the water content in the decellularized bone paste, adding distilled water with the solid content of 5%, uniformly mixing, pouring into a wet high-energy ball mill (MINI ZETA 03E Germany Chinesh instruments manufacturing Co., Ltd.) for ball milling, stirring at the rotating speed of 3000r/min, the ball milling filling rate of 85%, the diameter of a milling ball of 0.5mm, obtaining decellularized nano bone liquid after ball milling for 6h, and freeze-drying and storing to obtain the nano bone with the particle size of 10-100 nm.
Example 2
1. Preparing the nano fishbone:
(1) weighing the backbone of the grass carp with meat, and mixing the components in a ratio of 1: 5 (fishbone mass: water volume) adding tap water, and stewing at 150 ℃ for 30min in a counter-pressure high-temperature stewing pan (ZM-100 Guangzhou Daizhou Dazhuan packaging equipment Co., Ltd.); repeatedly rinsing with 60 deg.C hot water to retain white solid, and removing the suspended oil until the lotion is clear;
(2) crushing the white solid by a conditioner, wherein the material-liquid ratio is 1: 10 adding ice water, micronizing and crushing with bone paste machine (MKCA6-2 Japan MASUKO) at 4000r/min to obtain bone paste with a grinding disc gap of 0.3 mm;
(3) according to the material-liquid ratio of 1: 1, adding 0.1% of water solution of triton, stirring at room temperature for 48h, changing the solution every 10h, and then rinsing with a large amount of deionized water until the washing solution is foamless to obtain acellular bone paste;
(4) measuring the water content in the decellularized bone paste, adding distilled water with the solid content of 10%, uniformly mixing, pouring into a wet-process high-energy ball mill (MINI ZETA 03E Germany Steady instruments manufacturing Co., Ltd.) for ball milling, stirring at the rotating speed of 5000r/min, ball milling with the filling rate of 60%, grinding ball diameter of 1mm, obtaining decellularized nano bone liquid after ball milling for 12h, and freeze-drying and storing to obtain the nano bone with the particle size of 10-100 nm.
Example 3
1. Preparing the nano fishbone:
(1) weighing the backbone of the grass carp with meat, and mixing the components in a ratio of 1: 3 (fishbone mass: water volume) adding tap water, and stewing for 120min at 100 ℃ in a counter-pressure high-temperature stewing pan (ZM-100 Guangzhou Daizhou Dayuanjian packaging equipment Co., Ltd.); repeatedly rinsing with 70 deg.C hot water to retain white solid, and removing the suspended oil until the lotion is clear;
(2) crushing the white solid by a conditioner, wherein the material-liquid ratio is 1: adding ice water, micronizing and crushing with bone paste machine (MKCA6-2 Japan MASUKO) at 4000r/min until the grinding disc gap is reduced to 0.3mm to obtain bone paste;
(3) according to the material-liquid ratio of 1: 10, adding 10% of water solution of triton, stirring at room temperature for 48h, changing the solution once every 10h, and then rinsing with a large amount of deionized water until the washing solution is foamless to obtain acellular bone paste;
(4) measuring the water content in the decellularized bone paste, adding distilled water with the solid content of 1%, uniformly mixing, pouring into a wet high-energy ball mill (MINI ZETA 03E Germany Chinesh instruments manufacturing Co., Ltd.) for ball milling, stirring at the rotating speed of 1000r/min, ball milling with the filling rate of 70%, grinding ball diameter of 0.8mm, obtaining decellularized nano bone liquid after ball milling for 1h, and freeze-drying and storing to obtain the nano bone with the particle size of 1-200 nm.
Example 4
1. A nanometer fishbone-GelMA composite hydrogel type bone tissue engineering scaffold material is prepared by the following steps:
(1) nano fishbones were prepared according to the method of example 1, 2 or 3:
(2) synthesizing Gel-MA material: type A gelatin (Sigma-Aladdin) is dissolved in PBS buffer solution at 50 ℃ in a water bath to prepare a gelatin solution with the concentration of 15% (w/v); slowly dropwise adding methacrylic anhydride at the speed of 0.7mL/min, and controlling the ratio of the methacrylic anhydride to the gelatin to be 7.5: 1 (w/v); stirring in a water bath at 50 ℃ for 12 hours, and adding 4 times volume of PBS buffer solution to stop reaction; putting the obtained milky white liquid into a dialysis bag (8-14KD), dialyzing in distilled water for 7d, freeze-drying to obtain white and soft spongy product, i.e. Gel-MA material, and storing in a low-temperature drying place; wherein, the degree of methacrylic esterification of the gelatin is 70 percent;
(3) preparing a pre-polymerization solution: dissolving sterilized Gel-MA with sterile pure water, mixing the obtained solution with the nano fishbone obtained in the step (1), controlling the content of the nano fishbone to be 50%, adding 0.1% (w/w) of photoinitiator I2959 dissolved in DMSO, and uniformly mixing to obtain a pre-polymerization solution;
(4) preparation of composite hydrogel: and removing bubbles from the obtained prepolymerization solution, placing the prepolymerization solution in a mould, and irradiating by ultraviolet for 50s to obtain the nano fishbone-GelMA composite hydrogel type bone tissue engineering scaffold material.
Example 5
The nano fishbone of the example 4 and the Gel-MA solution which is filtered, sterilized, dissolved and sterilized by pure water are mixed, the content of the nano fishbone is controlled to be 0.5%, 1%, 3%, 5%, 7%, 10%, 30% and 90%, and the rest is the same as the example 4, so that the composite hydrogel type bone tissue engineering scaffold material with different nano fishbone contents is obtained. The bone tissue engineering scaffold material has osteoconductivity and osteoinductivity, has a three-dimensional porous structure and certain mechanical properties, can promote cell growth, proliferation, migration and differentiation, and is beneficial to effectively promoting the generation of new bone and repairing bone defects.
The product performance with the nano fishbone content of 1%, 3%, 5%, 7% and 10% is shown in example 6.
Example 6 Performance testing
The following performance tests were performed on the nano fishbones prepared in examples 1 to 3 and the partially composite hydrogel bone tissue engineering scaffold material prepared in example 5 (the content of the nano fishbones is 1%, 3%, 5%, 7%, 10%, respectively):
(1) as can be seen from FIG. 1, the potential change was the largest at 8h of ball milling, while the ball milling time continued to be longer and the potential change decreased.
(2) As shown in FIG. 2, the particle size distribution of the nano fishbone is 40-100 nm when the ball milling is carried out for 8 hours.
(3) FIG. 3 shows that when the content of the nano fishbone is 0.5% -90%, the nano fishbone and Gel-MA can be successfully prepared into the composite hydrogel type bone tissue engineering scaffold material.
(4) As can be seen from fig. 4, the nano fishbone and Gel-MA can form a three-dimensional porous composite hydrogel after being illuminated, and have a large communicated pore structure which can enable cells to smoothly enter the interior of the scaffold, and a small pore structure which can enable the cells to adhere, migrate and exchange nutrients, so that the nano fishbone-MA hydrogel is suitable for cell adhesion, growth, proliferation and migration; moreover, the overall porosity is reduced along with the increase of the proportion of the nano fishbone, the pore size is in a trend of increasing along with the increase of the proportion of the nano fishbone, and the composite hydrogel with a pore structure suitable for cell growth can be prepared by adjusting the proper proportion.
(5) As can be seen from FIG. 5, the composite hydrogel formed by the nano fishbone and Gel-MA after illumination has a controllable storage modulus, and the overall strength of the composite hydrogel tends to increase with the proportion of the nano fishbone. The composite hydrogel has the storage modulus of 1000-100000 Pa, has good mechanical property and can bear the pressure generated in physiological activities.
(6) FIG. 6 shows the proliferation results of cells in the composite hydrogel leaching solution in 1, 3 and 5 days. As can be seen from FIG. 6, the composite hydrogel formed by the nano fishbone and Gel-MA in different proportions by the cells is beneficial to the proliferation of the cells, and the proliferation rate is increased along with the increase of the content of the nano fishbone, and the proliferation rate of the added nano fishbone is better than that of the pure Gel-MA hydrogel, which indicates that the addition of the nano fishbone is beneficial to the proliferation and growth of the cells, the existence of the nano fishbone not only improves the adhesion rate of the cells, but also the bone cells which survive per se can be used as seed cells to participate in osteogenesis.
(7) FIG. 7 shows the result of staining 5 days after the cells were seeded on the surface of the nano fishbone composite hydrogel. As can be seen from FIG. 7, the composite hydrogel formed by the nanometer fishbone and Gel-MA with different proportions of the cells can grow well, and after the cells are cultured for 5 days under the same conditions, the cell density tends to increase along with the increase of the proportion of the nanometer fishbone, which indicates that the addition of the nanometer fishbone is beneficial to cell culture and can effectively promote cell adhesion, growth, proliferation and migration.
(8) Fig. 8 is a nuclear magnetic diagram of methacrylic anhydride-modified gelatin, and it can be seen from fig. 8 that gelatin was successfully modified.
(9) FIG. 9 shows the results of one month after the implantation of the nano-fish bone composite hydrogel into the rat skull defect model.
Constructing a skull defect model: taking a rat (250-300 g), carrying out intraperitoneal injection anesthesia by 10% chloral hydrate, after general anesthesia succeeds, removing hairs by using a shaver, wiping surface skin by using alcohol, then sequentially cutting the skin and subcutaneous tissues at the midline of the head by using a scalpel, stripping periosteum, exposing the skull, and then drilling two symmetrical 5mm bone defect models at two sides of the skull by using bone drills.
Skull repair surgery: implanting the nano fishbone composite hydrogel at the bone defect part, suturing the skin, and wiping the sutured part by iodophor to clean the sutured part; observing the physiological status of the patient at regular time after operation; one month post-surgery rats were sacrificed with anesthesia; and taking out the skull, cleaning the skull with normal saline, fixing with 4% paraformaldehyde, observing the obtained bone tissue sample, and judging the effect of the composite hydrogel on the skull repair.
As can be seen from fig. 9, the number of new bones is significantly increased, which can rapidly promote bone repair, and the bone repair effect is more significantly increased with the increase of the content of the nano fishbone, which indicates that the addition of the nano fishbone can promote bone regeneration, and that the bone cells survived by the nano fishbone can also be used as seed cells to participate in osteogenesis, which is more beneficial to bone regeneration, indicating that the composite hydrogel type bone tissue engineering scaffold material of the present invention can effectively promote the generation of new bones and the repair of bone defects.
2. In addition, a large number of experiments prove that the gelatin with higher methacrylic acid esterification degree (the methacrylic acid esterification degree is 20-80%) can improve the photocrosslinking performance of the product, is favorable for improving the strength of the composite hydrogel and enables the composite hydrogel to be more favorable for cell growth under different conditions.
The results show that the composite hydrogel type bone tissue engineering scaffold material prepared by the invention has good biocompatibility and high safety, simultaneously has good mechanical property and functionality, is suitable for bone cell growth, can be used as a bone scaffold material to provide good support and nutrition for the growth and differentiation of bone cells, can support the growth and osteogenic differentiation of cells, can effectively promote the generation of new bones and the repair of bone defects, and achieves the aim of quickly treating bone defects to meet the clinical requirements.
Example 7 cell culture
The bone tissue engineering scaffold material of the above embodiment is used as a culture carrier, and fibroblasts are suspended and surface-inoculated on the bone tissue engineering scaffold material.
(1) Inoculating fibroblasts into a culture bottle, culturing until the coverage of the cells reaches 80-90%, digesting and passaging the cells by using 0.25% trypsin containing 0.125% EDTA, and preparing a cell suspension; wherein the culture medium comprises the following components: 100 mu g/mL penicillin, 100 mu g/mL streptomycin, 10% fetal bovine serum and the balance of L-DMEM and/or DMEM/F12;
2) inoculating fibroblast on the surface of the bone tissue engineering scaffold material for culture: spreading the sterilized composite membrane in a 6-hole plate, inoculating the purified fibroblast suspension, incubating for 10-60 min, adding appropriate amount of culture medium, and standing at 37 deg.C and 5% CO2Culturing in an incubator.
The observation of an electron microscope shows that the cells are well attached to the surface of the bone tissue engineering scaffold material, and the cells gradually cover the surface of the material along with the prolonging of the culture time, so that the state of the scaffold is good, the scaffold has no obvious toxicity to the cells, has good cell compatibility, and obviously enhances the effects of attracting, inducing and regulating the growth of the cells.
Comparative example
The preparation process is otherwise identical to that of example 1, with the only difference that: the gelatin used was not modified but 15% gelatin was used directly.
As a result, compared with the gelatin modified by methacrylic anhydride, the gelatin which is 15% directly used cannot be gelatinized under the ultraviolet irradiation condition, is in a solid state only under the condition of lower than 10 ℃, is difficult to be used as a bone repair material, and has poor overall result.
Those skilled in the art will appreciate that, in the embodiments of the methods of the present invention, the sequence numbers of the steps are not used to limit the sequence of the steps, and it is within the scope of the present invention for those skilled in the art to change the sequence of the steps without inventive work. The examples described herein are intended to aid the reader in understanding the practice of the invention and it is to be understood that the scope of the invention is not limited to such specific statements and examples. Those skilled in the art can make various other specific changes and combinations based on the teachings of the present invention without departing from the spirit of the invention, and these changes and combinations are within the scope of the invention.
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