CN112156227A

CN112156227A - Composition and preparation of bone filling material, and preparation method and application thereof

Info

Publication number: CN112156227A
Application number: CN202010646853.8A
Authority: CN
Inventors: 李倩; 杨春; 赵继志; 祖岩; 穆月
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-12-08
Filing date: 2016-12-05
Publication date: 2021-01-01
Also published as: CN106729972A

Abstract

The invention belongs to the technical field of medical biomaterials, and particularly relates to a composition and a preparation of a bone filling material, and a preparation method and application thereof. The bone filling material composition comprises bone meal, gelatin and a cross-linking agent, and has the advantages of induction, elasticity and good plasticity. The bone filling material is prepared by freeze-drying the solution prepared from the bone filling material composition. A method of preparing a bone filler material comprising mixing the bone filler material composition or preparation with a pharmaceutically acceptable solvent. The bone filling material has wide raw material sources and simple preparation, and can provide the bone filling material which can be filled in time during clinical application. The bone filling material can be widely used in the scientific research field or as a medical material.

Description

Composition and preparation of bone filling material, and preparation method and application thereof

The present application is a divisional application of an invention patent application having an application date of 2016, 12 and 05, and an application number of 201611102957.2, entitled "composition and preparation of bone filling material, and a method for producing the same and an application thereof".

Technical Field

The invention belongs to the technical field of medical biomaterials, and particularly relates to a composition and a preparation of a bone filling material, and a preparation method and application thereof.

Background

The human bone repair technology has been developed for a long time, and is mainly characterized by making great improvements on the biocompatibility of materials, from original wicker to metal and then to medical polymers, and the bone repair materials have already entered the stage of bionic materials. However, no matter what kind of material, the clinical use means is not improved much, and the granular or strip-shaped materials which are not easy to shape are still basically used, so that the technical requirements on doctors in clinical use are high, and particularly, the operation is very inconvenient for bone cracks or unhealed bone seams. Bone wounds due to trauma, tumors, pathological disease or other forms of trauma and in dental bone implants, the shape of the bone defect is often very irregular or the wound site is deep. The filler commonly used in the bone grafting is regular in shape fixation (or is scattered into particles and is not shaped), and is not easy to shape randomly, so that dead spaces are remained at the defect part in the filling process, the bone forming effect is influenced, and the healing time is prolonged. In order to improve the bone grafting effect, if the bone filling material can be shaped by adopting the modes of film coating, finger shaping, injection by an injector and the like, a better repairing effect can be obtained inevitably, but higher requirements are provided for the shaping property of the bone filling material.

Chinese patent document CN102755668 discloses a plastic medical bone paste, which is a paste-like composite material mainly composed of polysaccharide or protein glue solution or organic binder and bone powder. Due to the characteristics of softness and easiness in molding, the bone filler in the form of the bone paste can still meet the molding requirements of most bone grafting operations except for the fact that a certain defect exists in the filling of fine bone gaps, but the following problems still exist in the application of bone filling materials.

For bone meal, for example, Bio-Os bone meal is generally extracted from bovine bone, and all organic components are thoroughly removed from bovine cancellous bone through technical processing, while fine bone trabecular structures and internal voids are preserved, so that a scaffold is provided for osteoblast growth, blood clot stabilization and blood vessel regeneration are ensured, and the Bio-Os bone meal is a natural, non-antigenic and osteoconductive graft material and is widely applied clinically at present. The chemical components of Bio-Os bone powder are close to the inorganic structure of human bone (low crystal natural apatite), and the macroscopic and microscopic physical structures are very similar to human cancellous bone. But has the following defects in clinical use and osteogenesis inducing capability: (1) the elastic modulus of the bone meal particles is about 1GPa and is far higher than the hardness (about 0.1MPa) of the accepted elastic substrate capable of inducing the osteogenic differentiation of the stem cells; (2) simple inorganic bone materials cannot promote osteogenesis under the induction condition of chemical or biological factors; (3) in those cases where the granular bone powder is used, the granular bone powder also has the disadvantages of displacement, migration, difficulty in forming and difficulty in placement, which causes inconvenience in clinical operation and difficulty in ensuring the bone grafting effect.

At present, the bone filling materials commonly used in clinic comprise Bio-Os bone powder and Bio-Os collagen, which means that the bone filling materials consist of 90% of the Bio-Os and 10% of the collagen, are loose porous structures and are in a three-dimensional rectangular block shape (3mm by 5mm by 7 mm). The elastic modulus of the material is reduced (about 1MPa) compared with Bio-Os bone meal, but is still higher than the ideal range for inducing stem cell bone differentiation, and the material is expensive and brings economic burden to patients.

In summary, the existing bone filling materials are difficult to solve the following problems simultaneously:

(1) bone filling materials (allogenic bones or artificially synthesized bone materials) which are commonly used clinically have too high hardness and are not suitable for the osteogenic differentiation of mesenchymal stem cells and the maintenance of the functions of the bone cells;

(2) the material only provides physical support for the defect part and lacks the induction function of growth factors;

(3) the material has poor plasticity, can not meet the defect transplantation operation required by large area or special shape, and is not suitable for the individual requirement of clinical bone defect treatment.

In addition, commercial products of partial bone filling materials are expensive and difficult to be widely used.

Disclosure of Invention

In view of the above, the present invention provides a bone filling material composition, which comprises bone meal, gelatin and a cross-linking agent.

The invention also provides a bone filling material preparation, which is a freeze-dried product obtained by freeze-drying a solution prepared from the bone filling material composition.

The invention also provides a method for preparing the bone filling material preparation, which comprises the step of carrying out freeze drying treatment on the solution prepared from the bone filling material composition.

The present invention further provides a method of preparing a bone filler material comprising mixing the bone filler material composition or bone filler material preparation with a pharmaceutically acceptable solvent.

In addition, the invention also provides application of the bone filling material prepared by the method in the scientific research field and application of the bone filling material as a medical material.

The bone filling material composition can be used for preparing a bone filling material with proper elastic modulus, and can further promote bone differentiation of bone marrow mesenchymal stem cells and osteoblast function due to proper hardness of the bone filling material. The bone filling material composition subjected to freeze drying forms a bone powder scaffold structure with a large number of gaps, is more elastic and tough, is easy to shape, and has a more induction effect. It can be used in the form of powder, fluid, or semisolid, and has good moldability. Therefore, the bone filling material has the characteristics of elasticity, induction function and good plasticity. The method has wide raw material sources and simple preparation, can provide the bone filling material which can be filled in time during clinical application, and can adapt to the shaping of various bone repair situations.

Drawings

FIG. 1 is an appearance diagram of a bone meal-gelatin film prepared by the experimental group 4 of example 1;

FIG. 2 is an external view of the product after the first prefreezing in the process of preparing the gelatin/genipin-bone powder scaffold of example 2;

FIG. 3 is an appearance of the product soaked in genipin aqueous solution after the first freeze-drying in the process of preparing gelatin/genipin-bone powder scaffold of example 2;

FIG. 4 is a graph showing the appearance of the product after a second freeze-drying in an aqueous genipin solution during the preparation of the gelatin/genipin-bone powder scaffold of example 2;

FIG. 5 is an environmental scanning electron micrograph of the gelatin/genipin-bone powder scaffold prepared in example 2;

FIG. 6 is a representation of BMSCs in a form X100 under an inverted microscope;

FIG. 7 is a diagram showing the expression of the level of the protein of the osteogenic marker in immunoblotting;

FIG. 8 is a graph showing mRNA level expression of the osteogenic marker in Real-time fluorescent quantitative PCR assay;

FIG. 9 shows the distribution of the osteoblast markers detected by immunofluorescence staining, which is the result of cell staining of Col-I osteoblast differentiation marker protein markers under a confocal laser microscope;

FIG. 10 shows the distribution of the markers detected by immunofluorescence staining, the results of cell staining by OCN markers of osteogenic differentiation under confocal laser microscopy;

FIG. 11 shows the distribution of the osteoblast markers detected by immunofluorescence staining-the result of cell staining of OPN osteoblast differentiation marker protein markers under a confocal laser microscope;

FIG. 12 is a graph showing the statistical results of the distribution of the markers for bone formation detected by immunofluorescence staining;

FIG. 13 is a visual observation (upper) and an observation (lower) under an inverted microscope of cell staining in a dish for measuring alkaline phosphatase activity;

FIG. 14 is a graph showing the statistical results of the alkaline phosphatase (ALP) activity assay.

Detailed Description

In the present invention, unless otherwise specified, all operations are carried out under room temperature and normal pressure conditions, and all "%" are mass percentages.

The bone filling material composition comprises bone meal, gelatin and a cross-linking agent.

The gelatin used in the invention is one of osteogenesis induction factors as degradable biological macromolecules, and can effectively promote the regeneration of bone around a bone grafting area; gelatin also reduces production costs compared to collagen. Finished bone powder is used as a raw material, the chemical characteristics of the material are kept, and the porous scaffold structure of the material is reconstructed by mixing the bone powder with gelatin gel. The ratio of gelatin and bone powder can adjust the elastic modulus of the material, can prepare bone filling materials with different elastic moduli, promotes the osteogenesis effect after bone transplantation by utilizing the induction action of a physical environment, and is suitable for the osteogenic differentiation of mesenchymal stem cells and the maintenance of the function of osteocytes. The gelatin has the effect of bonding solids, forms semisolid after cross-linking, is easy to shape after being added with bone meal particles, is not limited by the shape and size of bone defect, and can be prepared into personalized materials according to the needs.

We know that the number and function of mesenchymal stem cell-derived osteoblasts are critical in promoting new bone formation. In recent years, studies have found that extracellular matrix (ECM) elasticity, i.e., the elasticity of materials that cells perceive, can influence the process of inducing differentiation of stem cells into osteoblasts. We have found that the osteogenic differentiation of stem cells is about 10⁵Pa matrix obviously predominates, and the over-soft or over-hard extracellular matrix is unfavorable for the osteogenic differentiation and osteoblast function maintenance of the stem cells cultured in vitro. The elastic modulus of the clinically common bone meal particles is about 1GPa, and the clinically simple use of the materials may not achieve the optimal bone-promoting effect in the aspect of physical environment. That is, the conventional bone filling material (including most bone cement) has very high hardness, and studies have shown that an excessively hard substrate (i.e., a substrate formed of the bone filling material) is disadvantageous in stem cell osteogenic differentiation, and a material having suitable hardness can promote bone cell viability and osteogenesis. The bone filling material composition can control the hardness of the material by adjusting the component proportion, and provides a good physical environment for the differentiation of stem cells to osteoblasts.

The bone filling material composition of the present invention may further include a pharmaceutically acceptable solvent. Particularly when packaged as a product, the components may be packaged separately, or some or all of the components may be mixed and packaged.

Collagen is the main component of extracellular matrix, is a structural protein which is most abundant in human body, and accounts for more than 30% of the total amount of human body protein. The collagen has the characteristics of wide source, easy obtainment, good biological safety, low immunogenicity and the like, can be degraded by organisms in vivo, can be absorbed by organisms, has controllable degradation rate, is beneficial to the adhesion, proliferation and differentiation of stem cells, and is beneficial to the maintenance of phenotype. However, collagen has limited applications due to its high degradation rate, low mechanical strength of the prepared scaffold, and high cost of obtaining high purity collagen. Gelatin is a degradation product of collagen, a natural polypeptide polymer. In use, the gelatin has the advantages of being capable of being modified, convenient to process and mold and the like. The gelatin is similar to collagen, and has the advantages of strong bioactivity, and promoting cell growth and proliferation. The gelatin water solution can form heat reversible gel at a temperature below 35 deg.C, and is in gel state at a temperature below 35 deg.C, and is in solution at a temperature above 35 deg.C. The thermal and mechanical stability of gelatin hydrogels is not high and is generally improved by chemical crosslinking.

The gelatin used in the invention has the effect of induction of biological factors, and simultaneously reduces the cost compared with the type I collagen. More unexpectedly, the matching use of the gelatin and the bone powder can also adjust the elastic modulus of the bone filling material, promote the osteogenesis effect after bone transplantation and simultaneously facilitate the shaping.

In a preferred embodiment, the bone meal is a hydroxyapatite-based bone substitute material; Bio-Os bone meal is preferred. The Bio-os bone meal mainly contains Hydroxyapatite (HA), and HA is a main inorganic component of human and animal bones, HAs good biocompatibility, high hardness, good bearing property, osteoinductivity and can be firmly combined with bone tissues. However, HA HAs poor fatigue resistance, high brittleness and low strength, and HAs limited direct application in biomaterials. The bone meal and the gelatin are used cooperatively, so that the problem is avoided.

The bone powder takes the size of microscopic particles and the shape of the required bone filling material as standards, and the finished product bone powder can be ground to different degrees. In a preferred embodiment, the particle size of the bone meal is between 10um and 200 um.

In a preferred embodiment, the crosslinking agent is one or more of genipin, glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC HCl), and the like; preferably genipin.

Genipin (genipin) is an iridoid compound obtained by extracting geniposide from gardenia and hydrolyzing the geniposide by beta-glucosidase, can be cross-linked with protein, collagen, gelatin, chitosan and the like to prepare biological materials, is an excellent natural biological cross-linking agent, and has far lower toxicity than traditional chemical cross-linking agents such as formaldehyde, glutaraldehyde and the like. Studies have shown that genipin is less cytotoxic than 1/10, glutaraldehyde⁴While the ability to promote cell proliferation is 5X 10 of glutaraldehyde³And (4) doubling.

The dosage relationship of the bone meal and the gelatin is as follows: the greater the proportion of bone meal, the greater the concentration of gelatin, and the harder the material, for the same cross-linker concentration. In a preferred embodiment, the mass ratio of gelatin to bone meal is 0.05-0.4:1, more preferably 0.15-0.4: 1.

The amount of the crosslinking agent is limited to the amount that will be understood by those skilled in the art to achieve crosslinking. In a preferred embodiment, the cross-linking agent is used in an amount of 0.05 to 0.5%, preferably 0.2%, based on the total weight of the bone filling material.

The invention also provides a bone filling material preparation, which is a freeze-dried product obtained by freeze-drying a solution prepared from the bone filling material composition. The prepared product is prepared by a freeze drying process, forms a bone meal scaffold structure with a large number of gaps, is more elastic and tough, and is easy to shape.

The present invention also provides a method for preparing a bone filling material preparation, which comprises freeze-drying a solution prepared from the bone filling material composition.

In a preferred embodiment, the freeze-drying process is carried out in two steps:

firstly, preparing a gelatin solution, mixing the gelatin solution with bone meal to form a mixture of the gelatin solution and the bone meal, and freeze-drying the mixture of the gelatin solution and the bone meal to obtain a gelatin-bone meal scaffold;

and secondly, preparing a cross-linking agent solution, mixing the cross-linking agent solution with the gelatin-bone meal scaffold to form a mixture of the cross-linking agent solution and the gelatin-bone meal scaffold, and freeze-drying the mixture of the cross-linking agent solution and the gelatin-bone meal scaffold to obtain the gelatin/cross-linking agent-bone meal scaffold, thus obtaining the bone filling material preparation.

In the above step, a solvent commonly used for preparing a solution is a solvent conventionally used for a freeze-drying process, and is typically physiological saline.

The gelatin/cross-linking agent-bone powder scaffold prepared by the freeze-drying method utilizes the chemical properties and biological characteristics of materials, and the formed three-dimensional scaffold structure can simulate the structure of trabecula cancellous bone in a body, so that a good space structure is provided for vascularization of a bone defect area and attachment of mesenchymal stem cells after transplantation. Furthermore, the mechanical properties of the material can be improved and controlled by mixing with Bio-os bone meal. The freeze-drying method used here prepares the porous scaffold by using the principle that a deep-frozen solvent sublimes under vacuum. By adopting secondary freeze-drying operation, a more uniform and large-amount gap bone meal scaffold can be obtained, so as to be beneficial to bone formation; moreover, the cross-linked structure thus obtained is more elastic and tough and is easy to shape.

In addition, when the three-dimensional scaffold is prepared by lyophilization, the size of the pores can also be changed by changing the concentration of the mixture. In a more preferred embodiment, the gelatin solution is 5-10% by mass when freeze-dried in the first step. In another more preferred embodiment, the mass fraction of the crosslinker solution at the second freeze-drying stage is between 0.3% and 1%, preferably 0.3%.

The present invention also provides a method for preparing a bone filling material, comprising: mixing the bone filler composition or the bone filler preparation with a pharmaceutically acceptable solvent.

The medically acceptable solvent is, for example, blood, physiological saline, or the like. The amount of solvent is limited to form a semi-solid bone filler material that facilitates the clinical bone grafting procedure. The semi-solid bone filling material can be used for shaping various bone filling situations. Particularly in clinical application, the bone powder, the gelatin and the cross-linking agent can be mixed and then directly applied to a bone transplantation area of a patient, and body fluid, blood and the like of the patient react with the mixture to realize bone filling.

In a preferred embodiment, the bone filling material obtained has a modulus of elasticity in the range of 0.05-0.8MPa, more preferably 0.05-0.3MPa, most preferably 0.1-0.2 MPa.

The invention also includes embodiments formed after any combination of the bone filler composition and bone filler preparations and methods of making the same.

The bone filling material prepared by the method can be widely used in the scientific research field, such as animal model establishment, experimental analysis and the like, as an experimental material.

The bone filling material prepared by the method can be widely used in the medical field as a medical material, for example, a bone or tooth repairing material and the like.

The present invention will be further described below with reference to examples and the accompanying drawings, in which experimental methods not specifying specific conditions were carried out according to conventional conditions.

Example 1

(1) The finished Bio-Os bone meal (from GeistlichPharma AG) was ground under sterile conditions to a powder and the particles of the ground powder were observed under an environmental scanning electron microscope to have a particle size of about 10-200 um. The particles are properly and uniformly mixed to prepare the bracket material with uniform elasticity and hardness, and gaps with proper size are formed to facilitate the growth of cells and the bone formation.

(2) Preparing gelatin water solution with concentration of 5% and 10%, stirring in water bath at 50 deg.C to promote dissolution, and recovering to room temperature for use; 5% glutaraldehyde solution was prepared.

(3) Bone meal-gelatin films with different proportions are respectively prepared according to the dosage of the table 1 and the following steps.

The method comprises the following operation steps:

a. mixing the ground bone powder with gelatin solution and glutaraldehyde solution rapidly, covering the surface of the mixed colloid with another glass sheet, and pressurizing to form the mixed colloid into a complete and uniform film with a thickness of about 1 mm.

b. Standing at room temperature for 10 min, placing the bone meal-gelatin film covered with the glass sheet into pure water, carefully removing the glass sheet to ensure the integrity of the film, and carefully avoiding long-time exposure in the air to prevent gelatin water loss from causing film deformation.

c. Soaking in 1% glycine solution after suction filtration and repeatedly eluting for several times, at least 24 hours, to neutralize free glutaraldehyde.

d. And (5) washing the membrane for 3 times by using a PBS solution, and irradiating for 30-40min by using ultraviolet rays to finish the preparation of the material.

The elastic modulus of the prepared bone meal-gelatin film is shown in table 1, and the appearance of the bone meal-gelatin film prepared in experimental group 4 is shown in fig. 1.

TABLE 1 preparation of bone meal-gelatin film and elastic modulus results

Remarking: the elastic modulus is measured by measuring the substrate material through an atomic force microscope (the test method is specifically shown in the experimental example part), and the measurement method is widely accepted and applied to biomechanical elasticity measurement experiments.

As can be seen from table 1, when the concentration of the gelatin solution is 5%, we gradually increase the mass ratio of gelatin in the composition to adjust the elastic modulus of the composition, and at the same concentration of the gelatin solution, the increase of the mass ratio of gelatin accompanied by the increase of the solvent (water) can make the elastic modulus of the composition decrease as the mass ratio of gelatin increases, and at this time, the key substance for adjusting the elastic modulus of the composition is the solvent-water; however, the addition of a biologically active substance was the initial and advantageous of the present invention, so we tried to increase the concentration of gelatin solution, found that at 10% gelatin solution concentration a smaller range of elasticity change was obtained compared to 5% gelatin solution concentration, and that at a solution-to-bone meal mass ratio of 4:1, the elastic modulus value obtained meets the ideal hardness range of the osteogenic differentiation of stem cells known at present. In addition, when the material is prepared, we find that a 10% gelatin solution has good semi-solid property, so that the bone meal-gelatin film has good plasticity in preparation, and the obtained bone meal-gelatin film has good toughness and is easy to store, as shown in figure 1.

Example 2

(1) The finished Bio-Os powder (from GeistlichPharma AG) was ground under sterile conditions to a powder, and the particle size of the ground powder was observed under an environmental scanning electron microscope to be about 10-200 um. The particle size is suitable for uniform mixing to prepare the bracket material with uniform elasticity and hardness, so as to be beneficial to cell growth and osteogenesis.

(2) Preparing 10% gelatin water solution, stirring in 50 deg.C water bath to promote dissolution, and returning to room temperature for use; stock solutions of 1% genipin in water were prepared (genipin is available from shanghai frontier biotechnology limited).

(3) Gelatin/genipin-bone powder scaffolds were prepared according to the following procedure, according to the amounts used in table 2. The elastic modulus of the prepared gelatin/genipin-bone powder scaffold is shown in table 2.

The method comprises the following operation steps:

a. operating on ice, slowly solidifying gelatin water solution at low temperature, stirring continuously during solidification to make bone powder uniformly distributed in gel, stabilizing for about 5min, immediately placing at-20 deg.C, pre-freezing overnight. The obtained gelatin-bone powder is shown in FIG. 2.

b. And taking the sample out of a refrigerator at the temperature of-20 ℃, putting the sample into a vacuum freeze dryer, and freeze-drying for 24 hours.

c. The freeze-dried sample is in a sponge shape, soaked in prepared genipin aqueous solution with the mass fraction of 0.3%, kept at the constant temperature of 37 ℃ for 24 hours, and then the gel is dark blue due to the cross-linking reaction, as shown in figure 3.

d. The pre-freezing and freeze-drying processes are performed again to obtain the gelatin/genipin-bone powder scaffold which is a dark blue porous structure as shown in figure 4.

TABLE 2 preparation of gelatin/genipin-bone powder scaffolds and elastic modulus results

(4) Observation by electron microscope

The obtained gelatin/genipin-bone powder scaffold has uniform pore distribution, and pore size of 20-100 μm, as shown in FIG. 5. In the figure, a and b show the surface topography of the material, with magnifications of 500X and 2000X, respectively, and the arrows indicate the bone meal particles and the signs indicate the pore structure of the gelatin scaffold. The surface of the material is in a uniform porous structure, and the bone meal particles are uniformly distributed in the gelatin porous structure.

From the above experiments and results, it can be known that a scaffold structure with uniformly distributed pores and a suitable size can be obtained by using a freeze-drying method, wherein the size of the pores is 20-100 micrometers, so that a good spatial structure is provided for the attachment and spreading of stem cells in a material scaffold; the ground bone powder is uniformly embedded in the scaffold structure, so that bioactive substances playing a role in the scaffold are well exposed, and the effect of the bioactive substances on inducing stem cell osteogenic differentiation is played; the elastic modulus of the obtained composition is controllable by mixing the gelatin and the Bio-oss bone powder, and an ideal hardness range suitable for stem cell bone differentiation is achieved; the shape and size of the obtained composition can be adjusted in the steps, and the space requirements of different defects can be met. Therefore, the method skillfully integrates the factors influencing stem cell osteogenesis, such as spatial structure, scaffold elasticity, biological induction and the like, and furthest ensures the osteogenesis effect after bone grafting.

Experimental example osteogenic differentiation Induction experiment

(1) Culture of rat bone marrow mesenchymal stem cells

Rat bone marrow mesenchymal stem cells (BMSCs) can be stably passaged, spread cells are fusiform under an inverted phase contrast microscope (as shown in figure 6), the density of the spread cells reaches 90% in about three days, passage is carried out at a ratio of 1:2, and 3-6 generation cells are taken for osteogenic differentiation induction experiment observation.

(2) Preparation of the substrate Material

The experimental substrates were prepared as follows using the crosslinker ratios in table 3:

a. an elastic template material, Polydimethylsiloxane (PDMS) precursor a agent (basic component) was taken into a centrifuge tube, and the mass thereof was weighed.

b. Adding the agent B (cross-linking agent) according to the mass ratio of 10:1, 20:1, 35:1, 60:1 and 80:1 respectively, fully and uniformly mixing, carrying out centrifugation for 5 minutes at 5000rpm after balancing, and removing bubbles generated in the colloid due to stirring.

c. And pouring the mixed PDMS into a culture dish, horizontally standing for 1h, and keeping the mixed PDMS horizontal after the colloid is completely spread at the bottom of the dish, and baking the mixed PDMS in a blast drying oven at 70 ℃ for 24 h.

d. After being placed in a clean bench and irradiated by ultraviolet light for 2 hours, the gel surface was covered with a 0.2mg/mL layer of type I collagen solution (type I collagen is purchased from Sigma Ltd.) and incubated overnight.

e. Before inoculating the cells, washing the gel for 3 times by PBS, and irradiating for 30-40min by ultraviolet rays until the cells are inoculated.

(3) Hardness measurement of base Material

The elasticity of the substrate material was measured using an Atomic Force Microscope (AFM), and the instrument used in this experiment was AFM (NT-MDT, NTEGRA). In the experiment, a rectangular pyramid needle point is adopted, an AFM probe is positioned on the surface of a substrate, measurement is carried out in an AFM force curve mode, and a Hertz-Steiner model is adopted to fit the obtained curve to obtain a Young modulus value. The mechanical determination method is used for researching the mechanical properties of materials, AFM force-distance curve measurement systems are used for measuring the surface force curves of the materials at the same load rate, at least 10 points are measured on each material with hardness, and at least 10 force curves are obtained. The measured values are shown in Table 3, and it is understood that the elastic hardness of the substrate decreases with the decrease of the crosslinking agent, showing a difference of order of magnitude.

TABLE 3 base material ratios and hardness measurements

(4) Immunoblot detection (Western blot) of osteogenic marker protein level expression

Rat bone marrow mesenchymal stem cells are respectively cultured on the extracellular matrix with the 5 different hardnesses and a blank culture dish, total protein of the cells after 7 days of inoculation is extracted, an immunoblotting experiment is carried out by using specific antibodies of Col-I, OPN and BMP2, and GAPDH is used as an internal reference.

The results are shown in FIG. 7, where A is the immunoblot banding results and B, C, D is the data statistics for A results. The results of this experiment were from 8 independent experiments, and the statistical values were normalized after averaging and standard deviation for each group, and values for each group were compared to 1:10 groups, "-" indicating P < 0.05.

(5) Real-time fluorescent quantitative PCR detection of mRNA level expression of osteogenic marker

Rat bone marrow mesenchymal stem cells are respectively cultured on the 5 extracellular matrixes with different hardness and a blank culture dish, total RNA of the cells inoculated for 7 days is extracted and collected, the total RNA is reversely transcribed into cDNA, primers of promoter regions of Col-I, OPN and BMP2 are used for real-time fluorescence quantitative PCR to detect the amount of the enriched DNA fragments, and GAPDH is used as an internal reference.

The results are shown in FIG. 8, wherein A, B, C, D is the data statistics of the real-time quantitative PCR results versus the CT values obtained from the experiment. The results of this experiment were from 4 independent experiments, and the statistical values were normalized after averaging and standard deviation for each group, and the values for each group were compared to 1:10 groups, "+" indicates P <0.05 and "+" indicates P < 0.01.

(6) Immunofluorescence staining detection of distribution state of osteogenic markers

After 6 groups of cells were cultured on the above different substrates for 7 days, the cells were collected, stained with antibodies against Col-I, OCN, OPN, FITC, rhodamine and DAPI, and observed with a confocal laser microscope (20X oil lens).

The results are shown in FIGS. 9-12. FIGS. 9-11 show the results of cell staining under a confocal laser microscope; randomly selecting 5 fields, calculating the fluorescence intensity of cells, and counting the total cell amount to be more than 50, wherein the statistical result is shown in a D picture. The results of this experiment were from 3 independent experiments, and the statistical values were normalized after averaging and standard deviation for each group, and the values for each group were compared to 1:10 groups, as shown in fig. 12, "+" indicates P <0.05, and "+" indicates P < 0.01. Green: Col-I and OPN, red: OCN, blue: indicating the cell nucleus.

(7) Alkaline phosphatase (ALP) Activity assay

After 6 groups of cells were cultured on different substrates for 7 days, the alkaline phosphatase activity was detected by staining, and then the cells were observed by naked eye and under the mirror.

The results are shown in FIGS. 13-14. FIG. 13 shows the visual observation (upper) and the observation under an inverted microscope of the staining of cells in a culture dish (lower); the staining density was counted by randomly selecting 5 fields under the mirror, comparing the values of each group with 1:10 groups (high elastic modulus), and the statistical results are shown in FIG. 14, where "+" indicates P < 0.05.

And (4) conclusion: from the above experiments, it was demonstrated that rat mesenchymal stem cells (BMSC) exhibited approximately the same differentiation between bone and bone under the induction of Polydimethylsiloxane (PDMS) elastic substrate, and the peak of differentiation occurred in the range of 0.354 ± 0.04MPa of elastic modulus of substrate compared to the induction of harder substrate using different detection methods, and the result demonstrated that bone filling material with high elastic modulus such as Bio-os bone powder (elastic modulus about 1GPa) may not provide the most ideal physical environment for differentiation of stem cells to osteogenic bone, while the present invention uses the mixing of bone powder with high elastic modulus and gelatin with low elastic modulus to reduce the elastic modulus of the material, and through the trial, a specific material preparation process was achieved, simulating a structure suitable for growth and differentiation of stem cells. Most importantly, under the theoretical guidance of the experiment, the optimal gelatin-bone powder ratio and elastic modulus values close to the experimental conclusion are obtained. Under the guidance of the numerical value, the bone filling material with proper hardness/elasticity, bone guiding and bone inducing functions and good plasticity can be prepared by the bone filling material composition.

Finally, the above embodiments are only used to illustrate the technical solution of the present invention and are not limited. Modifications and equivalents of the present invention may be made by those skilled in the art without departing from the spirit and scope of the present invention, and are intended to be included within the scope of the appended claims.

Claims

1. A bone filling material composition is characterized by comprising bone meal, gelatin and a cross-linking agent.

2. The bone filling material composition according to claim 1, wherein the bone powder is a bone substitute material containing hydroxyapatite as a main component; Bio-Os bone meal is preferred.

3. The bone filling material composition of claim 1 or 2, wherein the cross-linking agent is one or more of genipin, glutaraldehyde, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC HCl); preferably genipin.

4. The bone filling material composition according to any one of claims 1 to 3,

the mass ratio of the gelatin to the bone meal is 0.05-0.4:1, preferably 0.15-0.4: 1; and/or

The amount of the crosslinking agent is an amount that can achieve the purpose of crosslinking, and is preferably 0.05 to 0.5%, preferably 0.2%, of the total weight of the bone filling material.

5. A bone filling material preparation, which is a freeze-dried product obtained by freeze-drying a solution prepared from the bone filling material composition according to any one of claims 1 to 4.

6. A method for preparing a bone filler material preparation, comprising freeze-drying a solution prepared from the bone filler material composition according to any one of claims 1 to 4.

7. The method according to claim 6, wherein the freeze-drying process is carried out in two steps:

8. The method of claim 7,

during the first step of freeze drying, the mass fraction of the gelatin solution is 5-10%; and/or

In the second step of freeze drying, the mass fraction of the cross-linking agent solution is 0.3-1%, preferably 0.3%.

9. A method of preparing a bone filler material, comprising:

mixing the bone filling material composition of any one of claims 1 to 4 with a pharmaceutically acceptable solvent; or

Mixing a bone filling material preparation prepared according to any one of claims 6 to 8 with a pharmaceutically acceptable solvent.

10. Use of the bone filling material prepared by the method according to claim 9 in scientific research and as a medical material.