CN112370571A

CN112370571A - Bone tissue engineering scaffold material for treating jaw and face bone defect and synthesis method thereof

Info

Publication number: CN112370571A
Application number: CN202011350944.3A
Authority: CN
Inventors: 马鹏飞; 林淑贤; 吴珺华
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2020-11-26
Filing date: 2020-11-26
Publication date: 2021-02-19

Abstract

The invention relates to a bone tissue engineering scaffold material for treating jaw face bone defect and a synthesis method thereof, wherein the scaffold material is a gelatin-chitosan-based scaffold material which comprises gelatin, chitosan, polyvinyl alcohol and nano-hydroxyapatite according to the mass ratio of (1.5-2.5) to (3-6) to (1-7). The synthesis method comprises the following steps: (1) taking gelatin and water to form gelatin solution; (2) taking chitosan, water and acetic acid solution to form a chitosan suspension; (3) taking polyvinyl alcohol and water to form polyvinyl alcohol solution; (4) mixing the gelatin solution, the chitosan suspension and the polyvinyl alcohol solution, adding nano hydroxyapatite, stirring, and cooling to room temperature to obtain a material precursor solution; (5) freezing the material precursor solution, freeze-drying, post-crosslinking and freeze-drying repeatedly. Compared with the prior art, the scaffold material has good osteogenesis promoting capacity, excellent mechanical strength, good biocompatibility, low toxicity and small side effect, and provides a new material choice for repairing the maxillofacial bone defect.

Description

Bone tissue engineering scaffold material for treating jaw and face bone defect and synthesis method thereof

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a bone tissue engineering scaffold material for treating maxillofacial bone defects and a synthesis method thereof.

Background

The oral and maxillofacial regions have important physiological functions such as chewing, swallowing, speech and the like, and the jaw bone is an important bone scaffold for supporting the morphology and functions of the maxillofacial region. Bone defects caused by inflammation such as maxillofacial trauma, tumor, periodontal disease and other anatomical factors not only affect the appearance of a patient, but also have adverse effects on the mental health of the patient. Realizes quick and good repair of the defects of the maxillofacial bone, and has positive significance for the subsequent development of other treatments and healthy life of patients.

Compared with other parts of a body, the bone of the jaw face has special anatomical physiological appearance, has the characteristics of complex structure, more cavities and the like, the bone defect of the part is complex, and the requirement of large-scale industrial production is difficult to meet. At present, the maxillofacial bone tissue defect repair method comprises the technologies of autologous bone transplantation, allogeneic bone transplantation, distraction osteogenesis, prosthesis repair, vascularized skeletal muscle flap transplantation and the like, wherein the autologous bone transplantation is the gold standard for bone defect repair treatment, but the treatment means can bring additional donor area trauma. The other methods have defects, such as immunological rejection, long time consumption, and unsatisfactory postoperative treatment effect.

Since birth, tissue engineering provides wide application prospect for repairing maxillofacial bone defects. The bone tissue engineering means that by combining the medical principle and the engineering technology, seed cells obtained after in vitro culture and amplification are inoculated on a scaffold material which has good biocompatibility and can be absorbed and degraded, and induction factors are used for inducing the proliferation of cells and the growth of new blood vessels, so that the aim of gradually repairing bone defects is fulfilled. Compared with the traditional method, the regeneration and repair technology based on the biological material has the advantages of small wound, no immunogenicity, accurate forming and the like.

The currently commonly used bone tissue engineering scaffolds include natural hydrogel scaffolds and artificial synthetic gel scaffolds. The natural hydrogel scaffold is derived from extracts of animals and plants, has the characteristics of good biocompatibility, low toxicity and low economic cost, and is widely used in the field of biological medicines. The natural hydrogels commonly used at present include the following: (1) collagen and gelatin scaffolds; (2) a hyaluronic acid hydrogel scaffold; (3) an alginate hydrogel scaffold; (4) a chitosan hydrogel scaffold. However, the natural hydrogel often has the problems of insufficient mechanical strength, excessively fast degradation rate and the like, and the effect is often not good when the natural hydrogel is used alone. The artificially synthesized hydrogel, such as polylactic acid, racemic polylactic acid, polylactic-co-glycolic acid, polycaprolactone, polyvinyl alcohol and the like, can effectively improve the mechanical properties of the material, but a large amount of initiator needs to be added in the preparation process of the materials, so that the toxicity of the materials is increased, the degradation speed of the materials is slow, the materials cannot adapt to the osteogenesis process in a living body, and the final bone defect repair effect is influenced.

Meanwhile, a pure hydrogel scaffold material does not have osteogenesis inducing activity, and a corresponding active factor is required to be applied. Most of the growth factors are soluble polypeptides with small molecular weight, can regulate and control the proliferation, movement, contraction, differentiation and tissue reconstruction of target cells through paracrine, autocrine, endocrine and other ways, and have very important functions in the bone development and repair process. The most important growth factors are transforming growth factor-beta, bone morphogenetic protein BMP, fibroblast growth factor, platelet derived growth factor PDEF and insulin-like growth factor IGF. However, since these exogenous osteogenic growth factors have a short half-life and are rapidly diluted and metabolized by local use, they are very expensive to prepare because they are repeatedly used in large doses, and thus, even though exogenous recombinant growth factors can promote bone regeneration to some extent, their use is limited due to their high cost and short half-life in vivo.

Therefore, it is necessary to construct a composite bone tissue engineering scaffold material which has good biocompatibility, good mechanical strength, degradation rate matched with the in vivo osteogenesis process and continuous and stable osteogenesis inducing activity.

Patent CN102406963A discloses a multi-component bone tissue engineering scaffold material and a preparation method thereof. The multi-component bone tissue engineering scaffold material consists of chitosan, gelatin, pectin and nano hydroxyapatite in percentage by mass. The preparation method comprises the following steps: selecting chitosan and pectin, and preparing hydroxyapatite/chitosan-pectin compound by codeposition technology, wherein the particle diameter of the hydroxyapatite is 10-100 nm; dispersing nHCP into chitosan-gelatin solution, cross-linking with glutaraldehyde, and freeze-drying to obtain the multi-component porous scaffold material for bone tissue engineering. According to the stent material prepared by the invention, nHCP is uniformly dispersed in the stent and is not easy to agglomerate; the pore diameter of the stent material is 100-300 mu m, and the porosity is more than 80 percent. This patent suffers from the following disadvantages: 1. in the composition, pectin is adopted as one of the components of the bracket, the structure and the composition of the pectin are unstable due to the change of plant types, storage time and processing technology, the preparation of the bracket material with continuous stability and high efficiency cannot be realized, and the pectin produced by using only citrus peel and apple peel as production raw materials can meet the requirements of commercial production at present due to the immature pectin production technology. In contrast, polyvinyl alcohol, as an artificially synthesized high molecular polymer, has a definite structure and properties, a mature production process, a wide source and a low cost, and the low-viscosity polyvinyl alcohol adopted in the invention has definite and stable physicochemical properties. 2. Functionally, the addition of pectin does not significantly improve the mechanical properties of the scaffold material. The polyvinyl alcohol can effectively enhance the compressive strength of the stent material, and the degradation rate of the stent can be controlled by adding the polyvinyl alcohol, so that the stent is more adaptive to the in-vivo osteogenesis process.

Disclosure of Invention

The invention aims to provide a bone tissue engineering scaffold material for treating maxillofacial bone defects and a synthesis method thereof, wherein the scaffold material has good osteogenesis promoting capability and excellent mechanical strength, and meanwhile, the scaffold material has good biocompatibility, low toxicity and small side effect, and provides a new material selection for maxillofacial bone defect repair.

The purpose of the invention is realized by the following technical scheme:

a bone tissue engineering scaffold material for treating jaw face bone defects is a gelatin-chitosan-based scaffold material and comprises gelatin (gelatin), chitosan (cs), polyvinyl alcohol (PVA) and nano-hydroxyapatite (nHA), wherein the mass ratio of gelatin, chitosan, polyvinyl alcohol and nano-hydroxyapatite is (1.5-2.5): (3-6): 1-7). The materials of the present invention mimic natural bone tissue in composition and structure.

The gelatin and the chitosan form a porous three-dimensional matrix, the polyvinyl alcohol is fused in the matrix in a physical penetration mode to form a uniform net-shaped structure, and the nano-hydroxyapatite is distributed in the net-shaped structure and on the surface of the net-shaped structure.

The mass ratio of the gelatin to the chitosan to the polyvinyl alcohol to the nano-hydroxyapatite is preferably 2:2:3 (1-7), and preferably 2:2:3: 1.

The stent material also comprises acetic acid and paraformaldehyde.

The gelatin has the gel strength of more than or equal to 250bloom g, the deacetylation degree of chitosan is more than or equal to 95%, the viscosity of chitosan is 100-200 mpa · s, and the relative molecular mass of a polyvinyl alcohol polymer is 25000-35000.

The porosity of the support material is 70-85%, preferably 71-76.37%, preferably 76.37%, the pore diameter is 100-200 um, the particle size of nano hydroxyapatite is 10-100nm, the compression strength is 1.16-1.71 MPa, the elastic modulus is 1.70-3.73 MPa, the swelling ratio is 4.05-6.33, the pH value of the material is maintained at 6.8-7.2 after the material is soaked in a phosphate buffer solution for 7 days, and the degradation rate of the material after the material is soaked in the phosphate buffer solution for four weeks is less than 50%.

A method for synthesizing the bone tissue engineering scaffold material, which comprises the following steps:

(1) mixing gelatin and water, heating, and dissolving to obtain gelatin solution as yellowish viscous liquid;

(2) mixing chitosan, water and acetic acid solution, and dissolving to obtain chitosan suspension in the form of yellowish transparent viscous liquid, wherein acetic acid is used as cosolvent;

(3) heating polyvinyl alcohol and water for dissolving to form polyvinyl alcohol solution which is colorless transparent viscous liquid;

(4) mixing and heating a gelatin solution, a chitosan suspension and a polyvinyl alcohol solution for dissolving, adding nano hydroxyapatite, stirring, and cooling to room temperature to obtain a material precursor solution;

(5) freezing and freeze-drying the material precursor solution, adding a paraformaldehyde solution (paraformaldehyde is a volatile cross-linking agent) for cross-linking, and repeatedly freeze-drying to obtain the bone tissue engineering scaffold material. Acetic acid and paraformaldehyde cannot be completely volatilized by freeze-drying, so that the prepared scaffold material also contains partial acetic acid and paraformaldehyde, but the two substances cannot affect other substances in use and can be slowly degraded, the pH of the scaffold material can be maintained at a neutral level, and the leaching liquor of the material has no obvious toxicity to cells.

In the step (1), the heating temperature is 50-65 ℃, the gelatin solution is placed at the temperature of less than or equal to 4 ℃ for standby, and the stability of the structure and the property of the effective components in the gelatin solution can be maintained by low-temperature storage.

In the step (1), the mass-to-volume ratio of the gelatin to the water is 1g/10 ml.

In the step (2), the chitosan suspension is mixed and dissolved at room temperature, the chitosan suspension is placed at the temperature of less than or equal to 4 ℃ for standby, and the stability of the structure and the property of the effective components in the chitosan suspension can be maintained by low-temperature storage.

In the step (2), the mass volume ratio of the chitosan to the acetic acid solution is 1g/20ml, and the volume fraction of the acetic acid solution is 2%.

In the step (3), the heating temperature is 90-95 ℃, the polyvinyl alcohol solution is placed at the temperature of less than or equal to 4 ℃ for standby, and the stability of the structure and the properties of the effective components in the polyvinyl alcohol solution can be maintained by low-temperature storage.

In the step (3), the mass-to-volume ratio of the polyvinyl alcohol to the water is 6g/100 ml.

In the step (4), the heating temperature is 50-65 ℃, magnetic stirring is adopted for stirring, and the stirring time is 1.5-2.5 h, preferably 2 h.

In the step (5), the freezing temperature is-90 to-70 ℃, the freezing time is preferably-80 ℃, the freezing time is 7.5 to 8.5 hours, the freezing time is preferably 8 hours, the freeze-drying time is 11 to 13 hours, the freeze-drying time is preferably 12 hours, the cross-linking time is 1 to 2 hours, in order to keep the shape of the material, the freezing and then freeze-drying are carried out, and the two steps are separately carried out.

In the step (5), the mass-to-volume ratio of the paraformaldehyde solution is 4g/100 ml.

It is known that natural bone matrix mainly includes collagen, inorganic salts, bioactive factors, and the like. Wherein the collagen is a scaffold formed by bone tissue; the inorganic salt is mainly distributed in the organic matter of the bone in the form of hydroxyapatite crystals and is an important substance for mineralization and deposition of bone tissues; although the content of the active factors is small, the active factors are involved in regulating the biological behaviors of cells related to bone tissues. The three major components play an important role in the formation and mineralization processes of bone tissues, and are synergistic and have no effect. If the repair material for artificial bone regeneration can simulate three main components in bone matrix and load stem cells with osteogenic activity, the interaction of the components is expected to effectively enhance the osteogenic induction activity of the material, facilitate the mineralization of bone tissues and finally realize the regeneration and repair of bone defects.

Gelatin is a macromolecular polypeptide polymer obtained by partial hydrolysis of natural collagen, is used as a macromolecular hydrophilic colloid, has thermal reversibility, has the temperature higher than 37 ℃, is cooled to form gelatin, is implanted into a body for 1 to 2 weeks and starts to degrade, and has too high degradation speed and low mechanical strength, thereby influencing the application of the gelatin in bone tissue biology. The chitosan is a partial deacetylation product of chitin, 1000-3000 acetylglucosamine residues are connected with each other through p1, 4 glucoside chains to form a polymer, the chitosan has positive ion property, macromolecules with negative charges can be attracted to be gathered in the scaffold, growth factors can be loaded, the chitosan is non-water-soluble, and degradation is started after the chitosan is implanted in vivo for 3 months. The polyvinyl alcohol (PVA, medical grade) is a safe polymer organic matter, has no toxicity to human bodies, no side effect, good biocompatibility, certain water solubility and certain degradability (degraded in 1.5-2.5 months after being implanted into human bodies), can obviously improve the mechanical properties of the scaffold material, increases the compression strength and the elastic modulus of the material, and can adjust the degradation rate of the scaffold material to be matched with the repair process of bone defects by controlling the added amount of the polyvinyl alcohol. The nano hydroxyapatite is used as a main inorganic component in natural bone tissues, can obviously improve the mechanical property of the composite material, and simultaneously, calcium and phosphorus ions slowly released by the nano hydroxyapatite can promote the differentiation and the function of effective osteoblasts, thereby being beneficial to the mineralization of the bone tissues.

Finally, the support material prepared by the method has the appearance of a light white porous support, the porosity is 70-80%, the compression strength reaches 1.16-1.71 Mpa, and the elastic modulus reaches 1.70-3.73 Mpa.

Compared with the prior art, the beneficial effects of the invention are embodied in the following aspects:

(1) the scaffold material is a gelatin-chitosan-based scaffold material, has good biocompatibility, and can effectively improve the mechanical strength of the composite material after polyvinyl alcohol is added into gelatin and chitosan.

(2) The nano-hydroxyapatite adopted in the material is an inorganic component in natural bone tissues, and can stably and continuously release calcium and phosphorus ions and promote osteogenic differentiation of bone marrow stem cells.

(3) The material can balance various factors such as mechanical strength, porosity, degradation rate and the like, and ensure that the scaffold material can be matched with the osteogenesis process in a living body.

(4) The preparation method is simple.

Drawings

FIG. 1 is a schematic structural diagram of a bone tissue engineering scaffold material;

FIG. 2 is a schematic representation of a scaffold material for bone tissue engineering prepared in example 1;

FIG. 3 is a scanning electron microscope image of the scaffolds prepared in example 1;

FIG. 4 is a scanning electron microscope image of nano-hydroxyapatite in the scaffold material prepared in example 1;

FIG. 5 is a microscopic view of the bone tissue engineering scaffold material prepared in example 1;

FIG. 6 is a graph showing the results of inducing osteogenic directional differentiation of rat bone marrow mesenchymal stem cells by the scaffold material for bone tissue engineering prepared in example 1;

FIG. 7 shows the results of inducing osteogenesis of rat bone marrow mesenchymal stem cells by using the scaffold material for bone tissue engineering prepared in example 1;

FIG. 8 shows that the scaffold material for bone tissue engineering prepared in example 1 is applied to the repair of rat skull defect model.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

A bone tissue engineering scaffold material for treating jaw face bone defects is a gelatin-chitosan-based scaffold material and comprises gelatin, chitosan, polyvinyl alcohol and nano-hydroxyapatite, wherein the mass ratio of the gelatin to the chitosan to the polyvinyl alcohol to the nano-hydroxyapatite is (1.5-2.5) to (3-6) to (1-7), the scaffold material further comprises acetic acid and paraformaldehyde, the gel strength of the gelatin is not less than 250bloom g, the deacetylation degree of the chitosan is not less than 95%, the viscosity of the chitosan is 100-200 mpa · s, and the relative molecular mass of a polymer of the polyvinyl alcohol is 25000-35000.

A method for synthesizing the bone tissue engineering scaffold material comprises the following steps:

(1) mixing gelatin and water, heating at 50-65 ℃ to dissolve the gelatin to form a gelatin solution, and placing the gelatin solution at a temperature of less than or equal to 4 ℃ for later use, wherein the mass-to-volume ratio of the gelatin to the water is 1g/10 ml;

(2) mixing chitosan, water and an acetic acid solution at room temperature for dissolving to form a chitosan suspension, placing the chitosan suspension at a temperature of less than or equal to 4 ℃ for later use, wherein the mass-volume ratio of the chitosan to the acetic acid solution is 1g/20ml, and the volume fraction of the acetic acid solution is 2%;

(3) heating polyvinyl alcohol and water at 90-95 ℃ for dissolving to form a polyvinyl alcohol solution, placing the polyvinyl alcohol solution at a temperature of less than or equal to 4 ℃ for later use, wherein the mass volume ratio of the polyvinyl alcohol to the water is 6g/100 ml;

(4) mixing a gelatin solution, a chitosan suspension and a polyvinyl alcohol solution, heating at 50-65 ℃ for dissolving, adding nano hydroxyapatite, magnetically stirring for 1.5-2.5 h, and cooling to room temperature to obtain a material precursor solution;

(5) freezing the material precursor solution at-90 to-70 ℃ for 7.5 to 8.5 hours, then freeze-drying for 11 to 13 hours, adding a paraformaldehyde solution with the mass-to-volume ratio of 4g/100ml for crosslinking for 1 to 2 hours, and then repeatedly freeze-drying to obtain the bone tissue engineering scaffold material.

Example 1

A bone tissue engineering scaffold material for treating maxillofacial bone defect is shown in figure 1, and comprises gelatin (gel), chitosan (cs), polyvinyl alcohol (pva) and nano-hydroxyapatite (nHA). The material simulates natural bone tissues in components and structure, the mass ratio (w/w) of the effective components is 2:2:3:1 (gelatin: chitosan: polyvinyl alcohol: nano-hydroxyapatite), and after the volatilizable cross-linking agent is added, a porous composite scaffold structure capable of effectively promoting bone defect repair is finally formed.

The bone tissue engineering scaffold material for treating the maxillofacial bone defect comprises the following steps:

(1) preparing a gelatin solution: weighing a certain amount of gelatin (gel) (the gelatin has a gel strength of 250bloom g), adding a proper amount of distilled water, ensuring that the mass-to-volume ratio (w/v) is 1g/10ml, putting the gelatin into a 65 ℃ water bath kettle to be dissolved into light yellow viscous liquid, and then putting the gelatin into a 4 ℃ refrigerator for later use;

(2) preparing a chitosan solution: weighing a certain amount of chitosan (cs) (the deacetylation degree of the chitosan is 95%, the viscosity of the chitosan is 200mpa · s), adding a proper amount of distilled water, slowly dropping an acetic acid solution at room temperature to assist dissolution, ensuring that the mass volume ratio (w/v) is 1g/20ml, stirring to form a suspension, fully dissolving the suspension to form a light yellow transparent viscous liquid, standing overnight, and placing the light yellow transparent viscous liquid into a refrigerator at 4 ℃ for later use;

(3) preparing a polyvinyl alcohol solution: weighing a certain amount of polyvinyl alcohol (pva) (the relative molecular mass of a polymer of the polyvinyl alcohol is 31000), adding a proper amount of distilled water, ensuring that the mass-volume ratio is 6g/100ml, putting the polyvinyl alcohol into a water bath kettle at 95 ℃, repeatedly shaking and dissolving to form colorless transparent viscous liquid, and putting the liquid into a refrigerator at 4 ℃ for later use;

(4) mixing a gelatin solution, a chitosan solution and a polyvinyl alcohol solution to enable the mass ratio of the effective components of the three to be 2:2:3, placing the three into a 65 ℃ water bath kettle for dissolving, adding nano hydroxyapatite (nHA) after fully dissolving, and enabling the mass ratio of the four to be gel: cs: pva: magnetically stirring for 2h and then cooling to room temperature, wherein nHA is 2:2:3: 1;

(5) freezing the mixed solution at-80 ℃ for 8h, putting the frozen mixed solution into a freeze dryer for freeze drying for 12h to obtain a porous scaffold material, adding 4% paraformaldehyde solution for crosslinking for 2h, repeating the freeze drying process once, and finally obtaining the bone tissue engineering scaffold material, wherein the microstructure schematic diagram of the material is shown in figure 1, gelatin, chitosan and polyvinyl alcohol form a porous three-dimensional scaffold structure with certain mechanical strength and proper degradation rate (the microstructure electron microscope diagram of a specific structure can be shown in figures 3 and 4), hydroxyapatite is used as an active factor for promoting osteogenesis and is uniformly distributed in the matrix, and the mechanical property of the scaffold material is further enhanced. The physical size of the material is shown in figure 2, the scale bar is 1cm, the microscopic images of the material are shown in figures 3, 4 and 5, the porosity of the material is very high, 76.37%, the pore diameter is 200um, and the particle size of the nano-hydroxyapatite is 10nm (shown in figure 4). The compressive strength of the material is 1.18Mpa, the elastic modulus is 2.11Mpa and the swelling ratio is 5.82 measured by a universal mechanical tester, the PH of the material is kept at about 6.9 after the material is soaked in 5ml of phosphate buffer solution for 7d, and the residual mass of the stent material is 65.27% of the original mass after the stent material is soaked in the phosphate buffer solution for 4 weeks. When in use, the material can be directly placed in a bone defect area of a rat skull, as shown in figure 8, and can be fixed by suturing through surrounding connective tissues or fixed by compressing through mucosal suture.

The bone tissue engineering scaffold material prepared in this example was used to induce osteogenic directional differentiation of rat bone marrow mesenchymal stem cells, and as a result, the MB-HA 12.5% (12.5% represents the percentage of hydroxyapatite in total scaffold material, the same applies below) in this example shows that fig. 6 shows the expression level of the osteogenesis related gene Runx2 (the same applies below) on the ordinate, and the expression level of Runx2 of MB-HA 12.5% is 0.009, which shows that the expression level of the osteogenesis related gene Runx2 in this example is significantly enhanced compared with other groups, and fig. 7 shows a concrete graph of the differentiation result.

Example 2

A bone tissue engineering scaffold material for treating maxillofacial bone defects is prepared by a synthesis method which comprises the following steps of: cs: pva: the obtained material was recorded as MB-HA 50% except that nHA was 2:2:3:7, and the obtained material was tested by universal mechanics to obtain a material having a compressive strength of 1.71Mpa, an elastic modulus of 3.73Mpa, a swelling ratio of 4.05, a porosity of 71%, a pore size of 100um, and a particle size of nano-hydroxyapatite of 10 to 100nm, and MB-HA 50% obtained in this example was used to induce osteogenic direction differentiation of rat mesenchymal stem cells, and as a result, as shown in fig. 6, the expression level of Runx2 of MB-HA 50% was 0.0046.

Example 3

A bone tissue engineering scaffold material for treating jaw face bone defects is prepared by the same synthesis method as in example 1 except that cross-linking time is 1h, the obtained material is subjected to universal mechanical test to obtain the material with the compression strength of 1.16Mpa, the elastic modulus of 1.70Mpa and the swelling ratio of 6.33, the pH value of the material is kept at about 6.8 after the material is soaked in 5ml of phosphate buffer solution for 7 days, the porosity of the material is 73%, the pore diameter of the material is 150um, and the particle size of nano-hydroxyapatite is 10-100 nm.

Example 4

A bone tissue engineering scaffold material for treating maxillofacial bone defects is synthesized by the following steps:

(1) preparing a gelatin solution: weighing a certain amount of gelatin (gel) (the gel strength of the gelatin is 300bloomg), adding a proper amount of distilled water, ensuring that the mass-to-volume ratio (w/v) is 1g/10ml, putting the gelatin into a50 ℃ water bath kettle to be dissolved into light yellow viscous liquid, and then putting the gelatin into a 2 ℃ refrigerator for later use;

(2) preparing a chitosan solution: weighing a certain amount of chitosan (cs) (the deacetylation degree of the chitosan is 97%, the viscosity of the chitosan is 200mpa · s), adding a proper amount of distilled water, slowly dropping an acetic acid solution at room temperature to assist dissolution, ensuring that the mass volume ratio (w/v) is 1g/20ml, stirring to form a suspension, fully dissolving the suspension to form a light yellow transparent viscous liquid, standing overnight, and placing the light yellow transparent viscous liquid into a refrigerator at 2 ℃ for later use;

(3) preparing a polyvinyl alcohol solution: weighing a certain amount of polyvinyl alcohol (pva) (the relative molecular mass of a polymer of the polyvinyl alcohol is 35000), adding a proper amount of distilled water, ensuring that the mass-volume ratio is 6g/100ml, putting into a 90 ℃ water bath kettle, repeatedly shaking for dissolution to form colorless transparent viscous liquid, and putting into a 2 ℃ refrigerator for later use;

(4) mixing a gelatin solution, a chitosan solution and a polyvinyl alcohol solution to enable the mass ratio of active ingredients of the three to be 1.5:1.5:4, putting the three into a 65 ℃ water bath kettle for dissolving, adding nano hydroxyapatite (nHA) after fully dissolving, and enabling the mass ratio of the four to be gel: cs: pva: performing magnetic stirring for 2.5h and then cooling to room temperature, wherein nHA is 1.5:1.5:4: 1;

(5) freezing the mixed solution at-70 ℃ for 8.5h, putting the frozen mixed solution into a freeze dryer for freeze drying for 11h to obtain a porous scaffold material, adding 4% paraformaldehyde solution for crosslinking for 1.5h, and repeating the freeze drying process once to finally obtain the bone tissue engineering scaffold material. The obtained material has excellent compression strength and elastic modulus, and the PH of the material is kept at about 6.9 after the material is soaked in phosphate buffer solution for 7 days.

Example 5

(1) preparing a gelatin solution: weighing a certain amount of gelatin (gel) (the gel strength of the gelatin is 350bloomg), adding a proper amount of distilled water, ensuring that the mass-to-volume ratio (w/v) is 1g/10ml, putting the gelatin into a water bath kettle at 60 ℃ to dissolve the gelatin into light yellow viscous liquid, and putting the gelatin into a refrigerator at 2 ℃ for later use;

(2) preparing a chitosan solution: weighing a certain amount of chitosan (cs) (the deacetylation degree of the chitosan is 95%, the viscosity of the chitosan is 300mpa · s), adding a proper amount of distilled water, slowly dropping an acetic acid solution at room temperature to assist dissolution, ensuring that the mass volume ratio (w/v) is 1g/20ml, stirring to form a suspension, fully dissolving the suspension to form a light yellow transparent viscous liquid, standing overnight, and placing the light yellow transparent viscous liquid into a refrigerator at 2 ℃ for later use;

(3) preparing a polyvinyl alcohol solution: weighing a certain amount of polyvinyl alcohol (pva) (the relative molecular mass of a polymer of the polyvinyl alcohol is 25000), adding a proper amount of distilled water, ensuring that the mass-volume ratio is 6g/100ml, putting the polyvinyl alcohol into a water bath kettle at 93 ℃, repeatedly shaking and dissolving to form colorless transparent viscous liquid, and putting the liquid into a refrigerator at 2 ℃ for later use;

(4) mixing a gelatin solution, a chitosan solution and a polyvinyl alcohol solution to ensure that the mass ratio of the effective components of the three is 2.5:2.5:6, putting the three into a water bath kettle at 60 ℃ for dissolving, adding nano hydroxyapatite (nHA) after fully dissolving, and ensuring that the mass ratio of the four is gel: cs: pva: stirring for 1.5h under magnetic force, and cooling to room temperature;

(5) freezing the mixed solution at-90 ℃ for 7.5h, putting the frozen mixed solution into a freeze dryer for freeze drying for 13h to obtain a porous scaffold material, adding 4% paraformaldehyde solution for crosslinking for 1.5h, and repeating the freeze drying process once to finally obtain the bone tissue engineering scaffold material. The obtained material has excellent compression strength and elastic modulus, and the PH of the material is kept at about 7.2 after the material is soaked in phosphate buffer solution for 7 days.

Comparative example 1

When a commercially available osteogenic induction medium (cygen, HUXMA-90021) was used to induce osteogenic directional differentiation of rat bone marrow mesenchymal stem cells, which was designated as control, the result is shown in FIG. 6, the Runx2 expression level of control was 0.0026, and FIG. 7 is a concrete graph of the differentiation result, and it can be seen that 12.5% of MB-HA produced distinct calcified nodules, while no distinct nodule production was observed in the control group.

Comparative example 2

A bone tissue engineering scaffold material for treating maxillofacial bone defects is prepared by a synthesis method which comprises the following steps of: cs: pva: the obtained material was designated as MB except that nHA was 2:2:3:0, and MB prepared in this example was used to induce osteogenic differentiation of rat bone marrow mesenchymal stem cells, as shown in fig. 6, the expression level of Runx2 of MB was 0.0026, and fig. 7 is a concrete graph of differentiation results, and it can be seen that MB-HA 12.5% produced a distinct calcified nodule, while no distinct nodule was produced in the MB group.

Comparative example 3

A bone tissue engineering scaffold material for treating maxillofacial bone defects is prepared by a synthesis method which comprises the following steps of: cs: pva: the same procedure as in example 1 was repeated except that nHA was 2:2:3:0.0529, the obtained material was designated as MB-HA 0.75%, and 0.75% of MB-HA prepared in this example was used to induce osteogenic differentiation of rat bone marrow mesenchymal stem cells, and as a result, as shown in fig. 6, the expression level of Runx2 of MB-HA 0.75% was 0.0026.

Comparative example 4

A bone tissue engineering scaffold material for treating maxillofacial bone defects is prepared by a synthesis method which comprises the following steps of: cs: pva: the same procedure as in example 1 was repeated except that nHA was 2:2:3:0.2165, and the obtained material was designated as MB-HA 3%, and MB-HA 3% obtained in this example was used to induce osteogenic differentiation of rat bone marrow mesenchymal stem cells, and as a result, as shown in fig. 6, the expression level of Runx 2% in MB-HA 3% was 0.0026.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. The bone tissue engineering scaffold material for treating the jaw face bone defect is characterized in that the scaffold material is a gelatin-chitosan-based scaffold material and comprises gelatin, chitosan, polyvinyl alcohol and nano-hydroxyapatite, wherein the mass ratio of the gelatin to the chitosan to the polyvinyl alcohol to the nano-hydroxyapatite is (1.5-2.5) to (3-6) to (1-7).

2. The bone tissue engineering scaffold material for treating the maxillofacial bone defect according to claim 1, wherein the mass ratio of gelatin, chitosan, polyvinyl alcohol and nano-hydroxyapatite is 2:2:3 (1-7).

3. The scaffold material for bone tissue engineering for the treatment of maxillofacial bone defects according to claim 1, wherein said gelatin has a gel strength of 250bloom g or more, chitosan has a deacetylation degree of 95% or more, chitosan has a viscosity of 100 to 200 mpa-s, and polyvinyl alcohol has a relative molecular mass of 25000 to 35000.

4. The bone tissue engineering scaffold material for treating maxillofacial bone defects according to claim 1, wherein the scaffold material has a porosity of 70-85%, a pore size of 100-200 um, and a particle size of nano-hydroxyapatite of 10-100 nm.

5. A method for synthesizing the scaffold material for bone tissue engineering according to any one of claims 1 to 4, wherein the method for synthesizing comprises the following steps:

(1) mixing gelatin and water, heating and dissolving to form gelatin solution;

(2) mixing chitosan, water and acetic acid solution for dissolving to form a chitosan suspension;

(3) heating polyvinyl alcohol and water for dissolving to form polyvinyl alcohol solution;

(5) and freezing and freeze-drying the material precursor solution, adding a paraformaldehyde solution for crosslinking, and repeatedly freeze-drying to obtain the bone tissue engineering scaffold material.

6. The method for synthesizing the bone tissue engineering scaffold material according to claim 5, wherein in the step (1), the heating temperature is 50-65 ℃, and the gelatin solution is placed at the temperature of less than or equal to 4 ℃ for standby.

7. The method for synthesizing scaffold material for bone tissue engineering as claimed in claim 5, wherein in step (2), the chitosan suspension is mixed and dissolved at room temperature, and the chitosan suspension is kept at a temperature of 4 ℃ or lower for further use.

8. The method for synthesizing the bone tissue engineering scaffold material according to claim 5, wherein in the step (3), the heating temperature is 90-95 ℃, and the polyvinyl alcohol solution is placed at a temperature of less than or equal to 4 ℃ for standby.

9. The method for synthesizing the bone tissue engineering scaffold material according to claim 5, wherein in the step (4), the heating temperature is 50-65 ℃, the stirring is performed by magnetic stirring, and the stirring time is 1.5-2.5 hours.

10. The method for synthesizing the bone tissue engineering scaffold material according to claim 5, wherein in the step (5), the freezing temperature is-90 to-70 ℃, the freezing time is 7.5 to 8.5 hours, the freeze-drying time is 11 to 13 hours, and the crosslinking time is 1 to 2 hours.