阅读说明：本技术 一种仿生复合骨支架及其制备方法 (Bionic composite bone scaffold and preparation method thereof ) 是由 王建南 裔洪根 殷音 于 2020-08-06 设计创作，主要内容包括：本发明提供了一种仿生复合骨支架的制备方法,包括以下步骤：A)将蚕丝置于温水中进行预处理,再置于65～95℃的水中处理,干燥,得到初处理的蚕丝；B)将所述初处理的蚕丝在中性盐溶液中处理,将得到的丝素丝胶蛋白复合溶解液再纯化,得到丝素丝胶蛋白复合溶液；C)将所述丝素丝胶蛋白复合溶液浓缩后冷冻干燥,将得到的初始骨架置于小分子一元醇中处理后冻干,得到仿生复合骨支架。本申请采用温和脱胶、一步溶解法制备了丝素丝胶蛋白复合溶液,利用该种丝素丝胶蛋白复合溶液制备得到的仿生复合骨支架具有高强度和高模量高韧性,解决了骨再生修复的天然生物聚合物组织工程支架力学性能不足的卡脖子技术难题。(The invention provides a preparation method of a bionic composite bone scaffold, which comprises the following steps: A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk; B) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin sericin composite dissolved solution to obtain a silk fibroin sericin composite solution; C) and concentrating the silk fibroin and sericin composite solution, freeze-drying, treating the obtained initial skeleton in micromolecule monohydric alcohol, and freeze-drying to obtain the bionic composite bone scaffold. The silk fibroin sericin composite solution is prepared by adopting a mild degumming and one-step dissolving method, the bionic composite bone scaffold prepared from the silk fibroin sericin composite solution has high strength, high modulus and high toughness, and the technical problem of neck clamping which is insufficient in mechanical property of a natural biopolymer tissue engineering scaffold for bone regeneration and repair is solved.)

1. A preparation method of a bionic composite bone scaffold comprises the following steps:

A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;

B) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin sericin composite dissolved solution to obtain a silk fibroin sericin composite solution;

C) and concentrating the silk fibroin and sericin composite solution, freeze-drying, treating the obtained initial skeleton in micromolecule monohydric alcohol, and freeze-drying to obtain the bionic composite bone scaffold.

2. The method according to claim 1, wherein the temperature of the warm water is 35 to 60 ℃ and the time of the pretreatment is 1 to 10 min.

3. The preparation method according to claim 1, wherein in the step A), the treatment time in the water at 65-95 ℃ is 1-8 h.

4. The method according to claim 1, wherein in step a), the ratio of silk to warm water is 1 g: (40-60) ml, wherein the ratio of the pretreated silk to water is 1 g: (40-60) ml.

5. The method according to claim 1, wherein the step a) further comprises, after the pretreatment: taking out the pretreated silk, washing in water at 25-30 ℃, and dehydrating;

the method also comprises the following steps of: washing the treated silk in water at 25-30 ℃ and then dehydrating; the drying is carried out by hot air at 40-60 ℃.

6. The preparation method according to claim 1, wherein the neutral salt solution is 8-10M lithium bromide aqueous solution, and the ratio of the primary treated silk to the lithium bromide aqueous solution is 1 g: (20-30) ml.

7. The method according to claim 1, wherein the temperature of the treatment in step B) is 50 to 80 ℃.

8. The preparation method according to claim 1, wherein the purification method is specifically:

and (3) filling the obtained silk fibroin sericin composite dissolved solution into a dialysis bag with the molecular weight cutoff of 10-50 kDa, and placing the dialysis bag in deionized water for continuous dialysis for 1-3 days.

9. The preparation method according to claim 1, wherein in the step C), the small molecule monohydric alcohol is an ethanol solution with a concentration of 70-80%, and the treatment time is 10-60 min; the freezing temperature of the freeze drying is-10 to-80 ℃, the time is 12 to 36 hours, and the drying time is 24 to 72 hours.

10. A bionic composite bone scaffold is prepared from a silk fibroin sericin composite solution, and the preparation method of the silk fibroin sericin composite solution comprises the following steps:

A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;

B) and (3) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin and sericin composite dissolved solution to obtain the silk fibroin and sericin composite solution.

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a bionic composite bone scaffold and a preparation method thereof.

Background

Bones or teeth are the most rigid connective tissues in the human body, and in the case of bones, with the aging of the world population and the increasing number of accidents, the patients with bone diseases face increasing pressure on clinical medical treatment related to bones, particularly bone repair and bone transplantation. As early as the 19 th century, bone graft surgery began to repair a wide range of bone defects. At present, the common bone repair methods include traditional autologous bone transplantation, allogeneic bone transplantation and artificial bone transplantation. Although the treatment effect of the autologous bone transplantation repair is optimal, the autologous bone transplantation repair has serious limitations on factors such as supply area damage, insufficient supply and complications. Allograft bone transplantation faces the problems of immunological rejection, disease transmission and the like. Tissue engineering was proposed in the 90 s of the 20 th century, and then research on scaffold materials for bone tissue engineering was rapidly developed. The bioceramic material has hydroxyapatite with the same important components of organism skeleton and teeth, has excellent bioactivity and biomechanical strength, but has high brittleness and poor toughness, and is not beneficial to remodeling of new bone. The synthesized organic polymer material has the advantages of wide sources, controllable physicochemical properties, excellent processing and forming properties and the like, such as polyester, polyanhydride, polyorthoester, polyphosphazene, polylactic acid, polyglycolic acid and copolymers thereof and other synthetic polymers, but the material lacks biological activity, has poor cell compatibility, and does not degrade or degrade products to influence the tissue microenvironment.

Natural organic polymers, such as collagen, gelatin, alginate, hyaluronic acid, chitosan, fibroin, etc., are widely favored for the study of tissue engineering materials because of their excellent biological activity. In contrast to other natural polymers, especially protein polymers: the silk is composed of fibroin fibers (the weight ratio is 70-80%) and sericin outside the silk fibers (the weight ratio is 20-30%), the composition is unique, the protein purity is very high, the preparation method can be simple and mild, small damage to fibroin macromolecules can be controlled, and the silk fibroin composite material is not like collagen and other natural macromolecules which are complex in purification, small in molecular weight, scattered in distribution, easy to change and the like. The silk fibroin macromolecule is connected by 6 heavy chains with 390kDa and 6 light chains with 26kDa in disulfide bonds and is combined with another glycoprotein/P25 with 25kDa in a hydrophobic interaction manner, wherein the heavy chains have a sequence structure basis forming a compact-structure and high-performance material. The molecular weight of sericin is also larger, more than 9 sericins exist, relating to Chuanminglan points out the outer, middle and inner three layers of sericin from the histological angle, and the small loose meter suggests that the outer sericin has 4 types from the outside and the inner, namely sericin I, sericin II, sericin III and sericin IV, and the solubility property from the outside and the inner is lower and lower, and the beta conformation is increased.

Silk fibroin and sericin have excellent cell, blood and tissue compatibility and controllable molding, and have been widely studied in tissue engineering applications such as skin, blood vessels, bones and the like as a scaffold material for tissue engineering. The regenerated silk fibroin solution is obtained by dissolving strong alkali weak acid salt or enzyme degumming salt, and the methods cause the damage of silk fibroin macromolecules; the regenerated sericin solution is mainly from silkworm cocoons, and the two solutions are generally prepared by independent separation. For example, CN02138129.1 discloses a preparation method of a silk fibroin and sericin composite tissue engineering scaffold, wherein a composite material is prepared by mixing pre-prepared regenerated silk fibroin and regenerated sericin in proportion, a degumming method of silk fibroin adopts a strong base and weak acid salt solvent, and sericin extracts the outer layer of silk sericin coated on cocoon silk. For example, CN201510353363.8 discloses a scaffold material for bone tissue engineering containing silk fibroin and sericin and a preparation method thereof, wherein regenerated silk fibroin and regenerated sericin are obtained respectively step by step and then mixed to prepare the scaffold material, wherein the preparation of silk fibroin does not disclose specific process conditions, and sericin is obtained from silkworm cocoon and treated with organic solvents such as alcohol.

However, the regenerated silk (silk fibroin, sericin) protein material of the above research is also poor in mechanical properties, and difficult to meet the requirements of replacement of hard tissues such as bones, teeth and the like, and the key point of the problem is that the existing degumming method and extraction method have large damage to macromolecular structures of silk fibroin and sericin, and the polymerization degree (molecular weight) of macromolecules is an extremely important intrinsic factor for determining the performance of macromolecules.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a preparation method of a bionic composite framework, and the bionic composite framework has high strength and modulus.

In view of this, the application provides a method for preparing a bionic composite bone scaffold, comprising the following steps:

A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;

B) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin sericin composite dissolved solution to obtain a silk fibroin sericin composite solution;

C) and concentrating the silk fibroin and sericin composite solution, freeze-drying, treating the obtained initial skeleton in micromolecule monohydric alcohol, and freeze-drying to obtain the bionic composite bone scaffold.

Preferably, the temperature of the warm water is 35-60 ℃, and the pretreatment time is 1-10 min.

Preferably, in the step A), the treatment time in the water at 65-95 ℃ is 1-8 h.

Preferably, in the step a), the ratio of the silk to the warm water is 1 g: (40-60) ml, wherein the ratio of the pretreated silk to water is 1 g: (40-60) ml.

Preferably, step a) further comprises, after the pretreatment: taking out the pretreated silk, washing in water at 25-30 ℃, and dehydrating;

the method also comprises the following steps of: washing the treated silk in water at 25-30 ℃ and then dehydrating; the drying is carried out by hot air at 40-60 ℃.

Preferably, the neutral salt solution is 8-10M of lithium bromide aqueous solution, and the ratio of the primarily treated silk to the lithium bromide aqueous solution is 1 g: (20-30) ml.

Preferably, in the step B), the treatment temperature is 50-80 ℃.

Preferably, the purification method specifically comprises:

and (3) filling the obtained silk fibroin sericin composite dissolved solution into a dialysis bag with the molecular weight cutoff of 10-50 kDa, and placing the dialysis bag in deionized water for continuous dialysis for 1-3 days.

Preferably, in the step C), the micromolecular monohydric alcohol is an ethanol solution with the concentration of 70-80%, and the treatment time is 10-60 min; the freezing temperature of the freeze drying is-10 to-80 ℃, the time is 12 to 36 hours, and the drying time is 24 to 72 hours.

The application also provides a bionic composite bone scaffold which is prepared from the silk fibroin sericin composite solution, and the preparation method of the silk fibroin sericin composite solution comprises the following steps:

A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;

B) and (3) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin and sericin composite dissolved solution to obtain the silk fibroin and sericin composite solution.

The application provides a preparation method of a bionic composite skeleton, which adopts mild degumming and a one-step dissolving method to prepare a silk fibroin sericin composite solution, and adopts pretreatment in warm water and subsequent treatment in water at a specific temperature to remove part or all of sericin on the surface of silk, wherein the existence of sericin increases strong hydrogen bond combination and network structure among molecular chains of a composite membrane, thereby improving the strength and modulus of the composite skeleton, or adopts degumming in water at a specific temperature and protection of a fibroin protein macromolecular structure in the subsequent degumming process of fibroin fiber, thereby improving the strength of the composite skeleton due to high molecular weight of regenerated fibroin protein; the sericin of the silk fibroin outer layer is different from the inside and the outside of the coating layer, the type, the structure and the performance of the sericin are different, the high-strength and high-toughness silk fibroin composite three-dimensional scaffold with different compositions and controllable mechanical property is obtained by controlling the degumming degree, the technical problem of neck clamping that the mechanical property of the natural biopolymer tissue engineering three-dimensional scaffold for bone regeneration and repair is insufficient is solved, and the composite scaffold has the osteogenesis activity of rapidly inducing hydroxyapatite deposition in situ.

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.

Aiming at the problem that the strength and the modulus of a silk fibroin sericin composite framework are influenced by serious damage of silk fibroin macromolecules or respective preparation of a silk fibroin sericin composite solution in the prior art, the application provides a bionic composite framework, the composite framework is prepared from the silk fibroin sericin composite solution, in the preparation process of the silk fibroin sericin bionic composite framework, the silk fibroin sericin composite solution with high molecular weight and high viscosity is prepared by adopting a deionized water mild degumming and one-step dissolving method, the method can avoid the damage of the silk fibroin macromolecules, and finally the silk fibroin in the prepared silk fibroin sericin composite solution has high viscosity and high molecular weight, so that the silk fibroin bionic composite framework prepared from the silk fibroin composite solution has better compression modulus and strength. Specifically, the embodiment of the invention discloses a preparation method of a bionic composite bone scaffold, which comprises the following steps:

A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;

B) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin sericin composite dissolved solution to obtain a silk fibroin sericin composite solution;

C) and concentrating the silk fibroin and sericin composite solution, freeze-drying, treating the obtained initial skeleton in micromolecule monohydric alcohol, and freeze-drying to obtain the bionic composite bone scaffold.

In the preparation process of the silk fibroin bionic composite framework, the silk is firstly pretreated in warm water, then is treated in water at 65-95 ℃ and is dried to obtain the primarily treated silk; in the process, sericin of the silk is completely or partially dissolved, the fibroin is still in a fiber state, if undissolved sericin exists, the undissolved sericin is wrapped on the outer surface of the fibroin fiber, and the dissolved sericin exists in water and is in a solution state. The temperature of the warm water is 35-60 ℃, namely the silk can be pretreated in the warm water at 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃, and the ratio of the silk to the warm water is 1 g: (40-60) ml, more specifically, the ratio of the silk to the warm water is 1 g: 42ml, 1 g: 45ml, 1 g: 48ml, 1 g: 52ml, 1 g: 55ml, 1 g: 58 ml. The pretreatment is carried out in the constant-temperature warm water for 1-10 min, and the pretreatment is carried out in stirring all the time so as to ensure that the silk can be preliminarily and completely immersed in the water. And after the pretreatment is finished, taking out the pretreated silk, washing the silk in deionized water at 25-30 ℃ and dehydrating the silk. The dehydrated silk is treated in water at 65-95 deg.C, more specifically, the dehydrated silk is treated in water at 68 deg.C, 70 deg.C, 72 deg.C, 75 deg.C, 78 deg.C, 80 deg.C, 82 deg.C, 84 deg.C, 87 deg.C, 90 deg.C or 93 deg.C. This application silk is at the mode of aquatic preliminary treatment again in water treatment earlier, can dissolve sericin in the undamaged while of protection fibroin macromolecular structure. The ratio of the pretreated silk to water is 1 g: (40-60) ml, more specifically, the ratio of the pretreated silk to water is 1 g: 42ml, 1 g: 45ml, 1 g: 48ml, 1 g: 50ml, 1 g: 52ml, 1 g: 55ml, 1 g: 58ml or 1 g: 60 ml. The degumming rate of the silk sericin can be adjusted according to different temperatures and time of water so as to realize the preparation of silk sericin composite solutions with different viscosities and different molecular weights; in the application, the time for treating the silk in the water is preferably 1-8 hours, so that the silk is partially or completely degummed; more specifically, the time for the treatment in water is 1.5h, 2h, 2.5h, 3h, 3.2h, 3.6h, 4.2h, 4.5h, 5.0h, 5.5h, 6.0h, 6.5h, 6.8h, 7.0h or 7.5 h. After treatment, the treated silk is washed in water at 25-30 ℃, dehydrated and dried; the drying is carried out by hot air at 40-60 ℃. The silk is subjected to the treatment to obtain the primary treated silk, the silk still presents a fiber state, the protection of the silk fibroin macromolecules is realized, and the viscosity of the silk fibroin sericin is ensured.

According to the invention, the silk after primary treatment is treated in a neutral salt solution and purified, and then the silk fibroin sericin composite solution is obtained. In the process, the primary-treated fibrous silk is dissolved to obtain the silk fibroin sericin composite solution, wherein the inner core of the silk is silk fibroin fibers, and the periphery of the silk fibroin fibers is wrapped with sericin with different contents. Neutral salt solution specifically is selected from the lithium bromide aqueous solution in this application, and its concentration is 8 ~ 10M, the silk of preliminary treatment with the proportion of lithium bromide aqueous solution is 1 g: (20-30) ml, more specifically, the proportion of the primary treated silk rain to the lithium bromide aqueous solution is 1 g: 22ml, 1 g: 24ml, 1 g: 25ml, 1 g: 27ml or 1 g: 29 ml. The treatment temperature is specifically 50-80 ℃, and more specifically, the treatment temperature is specifically 52 ℃, 57 ℃, 62 ℃, 65 ℃, 67 ℃, 68 ℃, 72 ℃, 75 ℃ or 78 ℃.

After the treatment, purifying the obtained silk fibroin sericin dissolving solution to remove lithium bromide and small molecular weight silk fibroin; the purification method specifically comprises the steps of filling the obtained silk fibroin and sericin composite dissolved solution into a dialysis bag with the molecular weight cutoff of 10-50 kDa, and placing the dialysis bag in deionized water for continuous dialysis for 1-3 days.

The method comprises the steps of preparing a silk fibroin and sericin composite solution, namely preparing a skeleton from the silk fibroin and sericin composite solution, concentrating the silk fibroin and sericin composite solution to a certain degree, removing bubbles, pre-freezing at-20 to-80 ℃ for 12 to 48 hours, then freeze-drying for 24 to 72 hours, treating the obtained initial composite skeleton in a micromolecule monohydric alcohol solution for 10 to 60 minutes to ensure that the obtained composite skeleton is insoluble in water and tissue fluid, finally soaking in water, air-drying at constant temperature and humidity, and freeze-drying to obtain the silk fibroin and sericin bionic composite skeleton; more specifically, the time of the treatment is 15min, 20min, 22min, 26min, 32min, 36min, 47min, 46min, 52min, 54min, 56min or 58 min.

The silk fibroin and sericin composite solution is prepared by adopting a mild degumming and one-step dissolving method, and the traditional method that silk fibroin and sericin are respectively prepared, respectively dissolved and then mixed for preparing a composite material is broken through. Importantly, the invention provides a preparation technology of the high-performance regenerated silk fibroin protein composite material based on the fundamental factor that the molecular weight of the high polymer influences the performance of the polymer. In the preparation process of the silk fibroin solution by the mild degumming one-step dissolution method, a hot water degumming method is adopted, so that the silk fibroin macromolecular chain (especially a heavy chain) cannot be damaged in the degumming process, the silk fibroin molecular weight in the composite material is concentrated in a high molecular weight region above 150kDa, and the reported silk fibroin prepared by the conventional method or the material prepared by mixing silk fibroin and sericin presents continuous distribution, mainly distributed below 85kDa to 15kDa or even smaller, so that the silk fibroin provided by the invention has the advantages of large molecular weight and small molecular weight dispersion degree, and the strength and the modulus of a composite three-dimensional framework are improved. The existence of sericin increases the strong hydrogen bond bonding between the molecular chains of the composite framework and also improves the strength and modulus of the composite framework. The sericin in the outer layer of the silk fibroin fiber is different from the outer layer to the inner layer along with the coating layer, and the type, structure and performance (especially mechanical property) of sericin are also different. According to the invention, the high-strength high-toughness silk fibroin composite three-dimensional scaffold with different compositions and controllable mechanical properties is obtained by controlling the degumming degree, the technical problem of neck clamping that the mechanical properties of the natural biopolymer tissue engineering three-dimensional scaffold for bone regeneration and repair are insufficient is solved, and the composite scaffold has osteogenesis activity for rapidly inducing hydroxyapatite deposition in situ.

Meanwhile, the application also provides a bionic composite bone scaffold which is prepared from the silk fibroin sericin composite solution, and the preparation method of the silk fibroin sericin composite solution comprises the following steps:

A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;

B) and (3) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin and sericin composite dissolved solution to obtain the silk fibroin and sericin composite solution.

The step a) and the step B) are processes for preparing the silk fibroin and sericin composite solution, and are not described herein again. The silk fibroin bionic composite bone scaffold is prepared from a silk fibroin sericin composite solution.

For further understanding of the present invention, the following examples are provided to illustrate the preparation method of the silk fibroin composite scaffold provided by the present invention, and the scope of the present invention is not limited by the following examples.