CN114681682B

CN114681682B - Biological scaffold, preparation material and preparation method thereof

Info

Publication number: CN114681682B
Application number: CN202210208002.4A
Authority: CN
Inventors: 宋柏杨; 程跃
Original assignee: Ningbo First Hospital
Current assignee: Ningbo First Hospital
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2023-04-21
Anticipated expiration: 2042-03-03
Also published as: CN114681682A

Abstract

The invention provides a biological stent, a preparation material and a preparation method thereof, wherein the biological stent comprises a ureteral stent, an intestinal canal stent, an esophagus stent, a urethra stent or a vascular stent, and the like, silk fibroin and gelatin protein are used as basic materials, and the biological stent with compact surface and internal porous structure is prepared through the steps of freeze-drying, compression and cross-linking, so that the biological stent is beneficial to the adhesion and proliferation of epithelial cells and the circulation of 3D growing cells such as smooth muscle and nutrient substances, the preparation process is simple, and the prepared biological stent can meet the clinical use requirements.

Description

Biological scaffold, preparation material and preparation method thereof

Technical Field

The invention relates to the field of biomedical materials, in particular to a biological stent, a preparation material and a preparation method thereof.

Background

The majority of the materials for manufacturing the biological scaffold at present are high polymer materials, such as polylactic acid, polyglycolic acid, polycaprolactone, chitosan, gelatin and the like, and the natural high polymer materials are medical materials which are used by human beings earliest, so that the biological scaffold has good biocompatibility, almost all the degradation products are degradable and nontoxic, the periodic taking out of the biological scaffold in secondary operation is avoided, and the pain of patients is relieved. Particularly, in the field of tissue engineering, the studies of scaffolds of gelatin-based materials and scaffolds of fibroin-based materials have been very extensive in recent years, because gelatin is a protein obtained by partially hydrolyzing collagen in connective or epidermal tissues of animals, has many excellent physical and chemical properties, has good biocompatibility and degradability, and fibroin is a natural polymer material derived from the nature, has excellent mechanical properties, controllable biodegradability, and is easy to process and form, and particularly, has biocompatibility equivalent to collagen as a raw material of an ideal regenerative medical scaffold.

It is known that many hollow organs in the human body, such as blood vessels, ureters, urethra, esophagus, and intestine, can be divided into three layers, i.e., an inner epithelial layer, an intermediate smooth muscle layer, and an outer connective tissue layer, respectively, histologically. Epithelial cells are usually barrier, tight connection between cells and invasion of cell membrane specific proteins to tissues such as urine and blood can be realized, and the diameter of human epithelial cells is usually larger than 10 mu m, so that the diameter of the layer of scaffold is required to be small and compact, thereby avoiding leakage of cells into the scaffold, and being unfavorable for epithelialization. Smooth muscle cells in the middle layer are arranged in 3D cells, so that a large number of through holes are needed in the scaffold, the smooth muscle cells are convenient to shuttle, and the porous structure is also beneficial to conveying nutrient substances such as extracellular matrixes. At present, the epithelial layer and the smooth muscle layer scaffold can be manufactured by a layering manufacturing method respectively, but the operation is complex, and the adhesion problem of the two-layer scaffold is a main obstacle, so that the application of layering manufacturing is limited. Therefore, there is an urgent need for a simple method of manufacturing such layered scaffolds to facilitate the development of tissue engineering regeneration.

Disclosure of Invention

The invention has the advantages that the biological scaffold which takes the silk fibroin and the gelatin protein as main materials is prepared by the freeze-drying-compression-crosslinking process, the process is simple, and the mechanical property and the medical property of the prepared biological scaffold can meet the clinical requirements.

Another advantage of the present invention is to provide a biological scaffold, and a preparation material and a preparation method thereof, wherein the biological scaffold is layered, the surface of the scaffold is compact, adhesion and proliferation of epithelial cells are facilitated, the interior of the scaffold is porous, the pore size formed is large, shuttling of 3D grown cells such as smooth muscle is facilitated, and the porous structure facilitates circulation of nutrients.

The invention also has the advantages of providing a biological scaffold, a preparation material and a preparation method thereof, wherein gelatin and silk fibroin forming the scaffold are natural proteins, the biological compatibility is good, degradation products are nontoxic, RGD sequences are arranged on the surfaces, and the cell recognition and the adhesion growth are facilitated.

Another advantage of the present invention is to provide a biological scaffold, and a preparation material and a preparation method thereof, by changing the ratio of silk fibroin and gelatin, and the crosslinking time and concentration of a crosslinking agent, the mechanical properties and pore size of the biological scaffold can be changed to construct biological scaffolds with different requirements.

The invention also has the advantages of providing a biological stent, a preparation material and a preparation method thereof, wherein the porous structure and the active groups on the surface of the biological stent can adsorb and graft various active substances, thereby increasing the stent function, expanding the application range of the biological stent and better meeting the clinical demands.

Another advantage of the present invention is to provide a biological stent, a preparation material and a preparation method thereof, wherein the degradation time of the biological stent can be controlled by controlling the preparation process, and the requirements can be better satisfied.

The invention also provides a biological stent, a preparation material and a preparation method thereof, and the preparation material and the preparation method for the biological stent can be applied to the preparation of biological stents of various tissues and organs, and the stents with different shapes can be manufactured by using the molds with different sizes and shapes only by changing the relative proportion and the molds so as to meet the requirements of different tissues and organs.

According to one aspect of the present invention, there is provided a method for preparing a biological stent, comprising the steps of:

(A) Preparing a silk fibroin solution and a gelatin solution;

(B) Lyophilizing, mixing silk fibroin solution and gelatin solution in step (A) uniformly, and lyophilizing to obtain biological scaffold preform;

(C) Compressing the biological stent preform in step (B);

(D) Crosslinking, namely placing the compressed biological stent preform in the step (C) into ethanol solution for preliminary crosslinking, and then placing the biological stent preform into glutaraldehyde solution for further crosslinking; and

(E) Removing the aldehyde groups remaining in step (D).

Wherein the concentration of the silk fibroin solution prepared in the step (A) is 2% -10%, and the concentration of the gelatin solution is 1% -5%.

Wherein said step (D) comprises the steps of:

(D1) Placing the compressed biological stent in 95% ethanol solution for 2h for primary crosslinking; and (D2) placing the preliminarily crosslinked stent in a 1% glutaraldehyde solution for 24 hours to further crosslink.

In the step (E), the crosslinked stent of the step (D2) is rinsed with a large amount of deionized water to remove residual aldehyde groups.

In the step (a), the preparation of the silk fibroin solution includes the steps of:

(A1) Extraction of silk fibroin: cutting cocoon shell into small pieces, boiling in 0.5% sodium carbonate solution for three times each for 30min, removing silk fibroin fiber surface sericin, cleaning, and drying to obtain silk fibroin fiber;

(A2) Dissolving the silk fibroin fibers dried in the step (A1) until the solute molar ratio is CaCl ₂ :C ₂ H ₅ OH:H ₂ Stirring the three-element solution with O=1:2:8 until the three-element solution is completely dissolved, and centrifuging or filtering to remove impurities to obtain a silk fibroin solution;

(A3) Freeze-drying the silk fibroin solution obtained in the step (A2) to obtain silk fibroin, and preserving for later use; and

(A4) Dissolving the silk fibroin in the step (A3) in water to obtain silk fibroin solution with required concentration.

According to another aspect of the invention, the silk fibroin solution and gelatin solution prepared in step (a) are in a ratio of 1:1.

Wherein the concentration of the silk fibroin solution is 2% -10% and the concentration of the gelatin solution is 1% -5%.

The crosslinking agents used for the primary crosslinking and the further crosslinking in the step (D) are respectively 95% ethanol and 1% glutaraldehyde solution.

The preparation method of the biological scaffold of the invention further comprises a step (F): the ureteral stent, the intestinal canal stent, the esophagus stent, the urethra stent or the vascular stent which are clinically required are prepared through the ureteral stent mold, the intestinal canal stent mold, the esophagus stent mold, the urethra stent mold or the vascular stent mold.

The invention also provides a preparation material of the biological scaffold, which comprises the following components: silk fibroin and gelatin proteins in a 1:1 ratio.

Wherein the preparation material of the biological scaffold is prepared by the following steps:

(A) Preparing a silk fibroin solution and a gelatin solution;

(C) Compressing the biological stent preform in step (B);

(E) Removing the aldehyde groups remaining in step (D).

Wherein said step (D) comprises the steps of:

The preparation material of the biological stent is used for preparing ureteral stents, urethral stents, esophageal stents, intestinal stents or vascular stents.

The invention also provides a biological stent, comprising a ureteral stent, an intestinal stent, an esophageal stent, a urethral stent or a vascular stent, wherein the biological stent is prepared by the following method:

(A) Preparing a silk fibroin solution and a gelatin solution;

(C) Compressing the biological stent preform in step (B);

(D) Crosslinking, namely placing the compressed biological stent preform in the step (C) into ethanol solution for preliminary crosslinking, and then placing the biological stent preform into glutaraldehyde solution for further crosslinking;

(E) Removing aldehyde groups remaining in step (D); and

(F) The ureteral stent, the intestinal canal stent, the esophagus stent, the urethra stent, the stomach tube stent or the vascular stent which are clinically needed are prepared by a ureteral stent mold, an intestinal canal stent mold, an esophagus stent mold, a urethra stent or a vascular stent mold.

Drawings

Fig. 1 is a schematic diagram of a preparation method of a biological scaffold according to an embodiment of the invention.

Fig. 2 is a schematic view of a tubular biological stent according to one embodiment of the present invention.

FIG. 3 is a graphical representation of tensile property test results after varying the ratio of silk fibroin to gelatin in accordance with some embodiments of the present invention.

FIG. 4 is an electron micrograph of a biological stent prepared in example I at 5% gelatin and silk fibroin concentrations.

FIG. 5 is an electron micrograph of a biological stent made without the compression step in the thirteenth embodiment.

Detailed Description

The following description is presented to enable one of ordinary skill in the art to make and use the invention. The preferred embodiments in the following description are by way of example only and other obvious variations will occur to those skilled in the art. The basic principles of the invention defined in the following description may be applied to other embodiments, variations, modifications, equivalents, and other technical solutions without departing from the spirit and scope of the invention.

Many hollow organs in the human body, such as blood vessels, ureters, urethra, esophagus, intestine, etc., can be divided into three layers histologically, an inner epithelial layer, an intermediate smooth muscle layer, and an outer connective tissue layer, respectively. The epithelial cells usually have a barrier function, the tight connection between cells and the invasion of cell membrane specific proteins can be caused by urine, blood and other substances into tissues, and the diameter of human epithelial cells is usually larger than 10 mu m, so that the diameter of the layer of scaffold is required to be small and compact, thereby avoiding leakage of cells into the scaffold and being unfavorable for epithelialization. Smooth muscle cells in the middle layer are arranged in 3D cells, so that a large number of through holes are needed in the scaffold, the smooth muscle cells are convenient to shuttle, and the porous structure is also beneficial to conveying nutrient substances such as extracellular matrixes.

The present invention produces such a bioscaffold by employing a simple lyophilization-compression-crosslinking procedure. The basic materials adopted are gelatin and silk fibroin, and the surface of the gelatin modified material is favorable for the adhesion and phenotype of urothelial cells, but the gelatin is poor in mechanical property and is unfavorable for the support and suture of the stent. The silk fibroin is taken from silk and is a common polymer material, and after crosslinking, the mechanical property of the bracket can be enhanced. The two materials are natural materials, the biocompatibility is good, the RGD sequence is formed on the surface, the RGD sequence is composed of arginine, glycine and aspartic acid, exists in various extracellular matrixes, can be specifically combined with 11 integrins, can effectively promote the adhesion of cells to biological materials, can competitively inhibit the combination of various adhesion proteins including fibrin and platelets, and can inhibit the combination of platelets and fibrin, so that the recognition and adhesion of cells are facilitated. In addition, the material itself can be degraded, and degradation products are nontoxic and harmless, thereby being beneficial to realizing in-situ regeneration of tissues.

The ureteral stent is a hollow catheter with a plurality of side holes, has flexibility and elasticity, one end is positioned at the kidney, the other end is positioned at the bladder, has the functions of supporting, expanding and internally draining the ureter, and is used for various urological surgeries such as kidneys, ureters, bladders and the like, so as to ensure smooth and thorough urine, relieve ureteral obstruction, reduce hydronephrosis and protect renal functions. Thus, ureteral stents are one of the commonly used devices for urology. Since the gelatin used in the present invention contributes to the adhesion and phenotype of urothelial cells, a ureteral stent can be manufactured using the materials and manufacturing methods of the present invention.

In addition, the mechanical property and the pore diameter condition of the scaffold can be changed by changing the proportion of the silk fibroin and the gelatin and the crosslinking time and the concentration of the crosslinking agent so as to construct biological scaffolds with different requirements.

In detail, according to one aspect of the present invention, there is provided a method for preparing a biological scaffold, first, a silk fibroin solution and a gelatin solution having a certain concentration are prepared separately, then the silk fibroin solution and the gelatin solution are mixed, and freeze-drying treatment is performed, and then the scaffold is compressed. The compressed scaffold makes the silk fibroin insoluble in water and can enhance the strength of the silk fibroin after the first treatment. The compressed stent is further crosslinked after the second treatment, so that the strength of the stent is enhanced. The biological stent prepared by the method has compact surface, loose interior and proper physical properties, and is used as a ureteral stent or a biological stent at other positions.

The biological stent and the preparation materials and the preparation methods thereof according to the present invention will be further described with reference to specific examples.

Embodiment one:

referring to fig. 1, the preparation method of the biological scaffold of the present invention comprises the following steps:

(A) Preparing 5% silk fibroin solution and 5% gelatin solution;

(B) Freeze-drying, mixing 5% silk fibroin solution and 5% gelatin solution uniformly, and freeze-drying to obtain a biological scaffold preform;

(C) Compressing the biological stent preform in step (B);

(D) Crosslinking, namely placing the compressed biological stent prefabricated product in the step (C) into a 95% ethanol solution for 2 hours for preliminary crosslinking, and then placing the biological stent prefabricated product into a 1% glutaraldehyde solution for 24 hours for further crosslinking;

and

(E) Rinsing with a large amount of deionized water to remove residual aldehyde groups to obtain the biological scaffold.

Further, the biological stent can be used for manufacturing organ stents with different requirements through corresponding molds. For example, ureteral stents of clinically desirable shapes can be made by molding the ureteral stent; the intestinal canal bracket can be manufactured through a die of the intestinal canal bracket; stents required by other hollow organs such as vascular stents and the like, such as urethral stents, esophageal stents and the like, can be manufactured through the die of the vascular stents and clinical requirements. Referring to fig. 1 and 2, the lyophilized biological stent preform may be compressed into a tube shape by an axial mold or may be directly compressed into a flat shape. In fig. 2 is schematically shown a tubular ureteral stent, the thickness dimension T of which is below 1mm and the diameter D of which may be about 2 to 3 mm.

In said step (a), the preparation of the silk fibroin solution and the gelatin solution comprises the steps of:

(A1) Extraction of silk fibroin: cutting cocoon shell into small pieces, boiling in 0.5% sodium carbonate solution for three times each for 30min to remove silk fibroin fiber surface sericin, washing with distilled water, and drying in an incubator to obtain silk fibroin fiber;

(A2) Dissolving the dried silk fibroin fibers into a ternary solution, wherein the solute molar ratio CaCl in the solution ₂ :C ₂ H ₅ OH:H ₂ O=1:2:8, placing the weighed components in a beaker, slowly stirring until the components are completely dissolved, and clarifying the solution for later use;

(A3) Water bath at constant temperature of 80 ℃ is carried out, and impurities are removed, thus obtaining silk fibroin solution;

(A4) Freeze-drying the silk fibroin solution to obtain silk fibroin, and preserving for later use;

(A5) Dissolving silk fibroin in water to obtain 5% silk fibroin solution; and

(A6) The purchased gelatin protein was dissolved in water to make a 5% gelatin solution.

Wherein in the step (A3), impurities are removed by filtration or centrifugation.

The experimental materials used in the invention are derived from:

gelatin protein: sigma-Aldrich, CAS:9000-70-8

95% alcohol: chinese medicine

Glutaraldehyde: aladine, CAS:111-30-8

The electron microscope image of the biological stent manufactured by the embodiment is shown as figure 4 of the accompanying drawings, and the surface of the biological stent is compact, the section of the biological stent is porous, so that the requirements of the stent materials required by the hollow organs of the human body are met. Namely, the surface of the stent material is compact, the adhesion and proliferation of epithelial cells are facilitated, the inside is porous, the cell shuttling of smooth muscle and other 3D growth is facilitated, the transportation of nutrient substances is facilitated, and the clinical use requirements can be met.

Embodiment two:

the preparation method of the biological scaffold of the embodiment comprises the following steps:

(A) Preparing 8% silk fibroin solution and 5% gelatin solution; the method comprises the steps of carrying out a first treatment on the surface of the

(B) Lyophilizing, mixing 8% silk fibroin solution and 5% gelatin solution in step (A), and lyophilizing to obtain biological scaffold preform;

(C) Compressing the biological stent preform in step (B);

and

(E) The biological scaffold is prepared by washing with a large amount of deionized water to remove residual aldehyde groups.

Further, the biological stent can be used for manufacturing organ stents with different requirements through corresponding molds. For example, ureteral stents of clinically desirable shapes can be made by molding the ureteral stent; the intestinal canal bracket can be manufactured through a die of the intestinal canal bracket; the stent required by other hollow organs such as vascular stents and the like, such as gastric tube stents, urethral stents, esophageal stents and the like, can be manufactured through the die of the vascular stents and clinical requirements.

In said step (A), the preparation of silk fibroin solution and gelatin solution can be referred to in example 1.

In this embodiment, the rigidity of the bracket is increased compared with that of the first embodiment, so that the clinical use requirement can be met.

Embodiment III:

(A) Preparing 10% silk fibroin solution and 5% gelatin solution;

(B) Lyophilizing, mixing silk fibroin solution 10% and gelatin solution 5% in step (A), and lyophilizing to obtain biological scaffold preform;

(C) Compressing the biological stent preform in step (B);

and

Further, the biological stent is used for manufacturing organ stents with different requirements through corresponding molds. For example, ureteral stents of clinically desirable shapes can be made by molding the ureteral stent; the intestinal canal bracket can be manufactured through a die of the intestinal canal bracket; stents of other hollow organs such as vascular stents, e.g. urethral stents, esophageal stents, etc. can be manufactured through the die of vascular stents and clinical requirements.

In this embodiment, the rigidity of the bracket is increased compared with the first embodiment and the second embodiment, so that the clinical use requirement can be met.

Embodiment four:

(A) Preparing a 15% silk fibroin solution and a 5% gelatin solution;

(B) Lyophilizing, mixing silk fibroin solution 15% and gelatin solution 5% in step (A), and lyophilizing to obtain biological scaffold preform;

(C) Compressing the biological stent preform in step (B);

and

In this embodiment, the rigidity of the stent is increased compared with the first, second and third embodiments, but the stent is brittle and is easy to break, and cannot meet clinical requirements.

Fifth embodiment:

(A) Preparing a 2% silk fibroin solution and a 5% gelatin solution;

(B) Lyophilizing, mixing silk fibroin solution 2% and gelatin solution 5% in step (A), and lyophilizing to obtain biological scaffold preform;

(C) Compressing the biological stent preform in step (B);

and

Further, the biological stent is used for manufacturing organ stents with different requirements through corresponding molds. For example, ureteral stents of clinically desirable shapes can be made by molding the ureteral stent; the intestinal canal bracket can be manufactured through a die of the intestinal canal bracket; stents of other hollow organs such as vascular stents and the like, such as gastric tube stents, urethral stents, esophageal stents and the like, can be manufactured through the die of the vascular stents and clinical requirements.

In this embodiment, the rigidity of the bracket is reduced compared with the four embodiments, and the clinical use requirement can still be satisfied.

Example six:

(A) Preparing 5% silk fibroin solution and 7.5% gelatin solution;

(B) Lyophilizing, mixing 5% silk fibroin solution and 7.5% gelatin solution in step (A), and lyophilizing to obtain biological scaffold preform;

(C) And (3) compressing, wherein the biological stent prefabricated product in the step (B) is hard and cannot be compressed into a flake shape.

(D) Crosslinking, namely placing the compressed biological stent prefabricated product in the step (C) into a 95% ethanol solution for 2 hours for preliminary crosslinking, and then placing the biological stent prefabricated product into a 1% glutaraldehyde solution for 24 hours for further crosslinking; and

(F) The biological scaffold is prepared by washing with a large amount of deionized water to remove residual aldehyde groups.

The biological stent in this embodiment cannot be compressed to obtain a structure with compact surface and loose interior, so that clinical use requirements cannot be met.

Embodiment seven:

(A) Preparing 5% silk fibroin solution and 10% gelatin solution;

(B) Lyophilizing, mixing 5% silk fibroin solution and 10% gelatin solution in step (A), and lyophilizing to obtain biological scaffold preform;

Example eight:

(A) Preparing 5% silk fibroin solution and 15% gelatin solution;

(B) Lyophilizing, mixing 5% silk fibroin solution and 2% gelatin solution in step (A), and lyophilizing to obtain biological scaffold preform;

(C) Compressing, namely, the biological stent prefabricated product in the step (B) is hard and cannot be manually compressed into a flake shape;

The biological stent obtained by the embodiment cannot be compressed to obtain a structure with compact surface and loose inside, so that clinical requirements cannot be met.

Example nine:

(A) Preparing 5% silk fibroin solution and 2% gelatin solution;

(B) Lyophilizing, mixing 5% silk fibroin solution and 1% gelatin solution in step (A), and lyophilizing to obtain biological scaffold preform;

(C) Compressing the biological stent preform in step (B);

and

The biological stent obtained by the embodiment has rigidity and toughness which can meet clinical requirements.

Example ten:

(A) Preparing 5% silk fibroin solution and 5% gelatin solution;

(B) Lyophilizing, mixing 5% silk fibroin solution and 5% gelatin solution in step (A), and lyophilizing to obtain biological scaffold preform;

(C) Compressing the biological stent preform in step (B);

(D) Crosslinking, namely placing the compressed biological stent prefabricated product in the step (C) into 80% ethanol solution for 2 hours for preliminary crosslinking, and then placing the biological stent prefabricated product into 1% glutaraldehyde solution for 24 hours for further crosslinking;

and

(E) Biological stents, such as gastric tube stents, urethral stents, esophageal stents, and the like, are made by rinsing with a large amount of deionized water to remove residual aldehyde groups.

Further, the biological stent is used for manufacturing organ stents with different requirements through corresponding molds. For example, ureteral stents of clinically desirable shapes can be made by molding the ureteral stent; the intestinal canal bracket can be manufactured through a die of the intestinal canal bracket; the stent of other hollow organs such as the vascular stent can be manufactured through the mold of the vascular stent and clinical requirements.

The ureteral stent prepared in the embodiment has poor mechanical properties, is easy to collapse, cannot play a role in supporting hollow organs such as a ureter, and the like, or has short supporting time, and cannot meet clinical use requirements.

Example eleven:

(A) Preparing 5% silk fibroin solution and 5% gelatin solution;

(C) Compressing the biological stent preform in step (B);

(D) Crosslinking, namely placing the compressed biological stent prefabricated product in the step (C) into a 95% ethanol solution for 1h for preliminary crosslinking, and then placing into a 1% glutaraldehyde solution for 18h for further crosslinking;

and

Embodiment twelve:

(A) Preparing 5% silk fibroin solution and 5% gelatin solution;

(C) Compressing the biological stent preform in step (B);

(D) Crosslinking, namely placing the compressed biological stent in the step (C) into a 95% ethanol solution for 2 hours to perform primary crosslinking, and then placing the biological stent into a 1% glutaraldehyde solution for 30 hours to perform further crosslinking;

and

The ureteral stent manufactured in the embodiment can meet clinical use requirements.

Embodiment thirteen:

(A) Preparing 5% silk fibroin solution and 5% gelatin solution;

(C) Crosslinking, namely placing the biological stent preform in the step (B) into a 95% ethanol solution for 2 hours to perform primary crosslinking, and then placing into a 1% glutaraldehyde solution for 24 hours to perform further crosslinking; and

(D) The biological scaffold is prepared by washing with a large amount of deionized water to remove residual aldehyde groups.

Further, the method further comprises a step (F): and (3) manufacturing organ scaffolds with different requirements through corresponding moulds by the biological scaffolds in the step (E). For example, ureteral stents of clinically desirable shapes can be made by molding the ureteral stent; the intestinal canal bracket can be manufactured through a die of the intestinal canal bracket; stents of other hollow organs such as vascular stents, e.g. urethral stents, esophageal stents, etc. can be manufactured through the die of vascular stents and clinical requirements.

In this example, the lyophilized biological scaffold was directly crosslinked without compression, and the experimental results showed that the surface of the biological scaffold prepared in this example formed a porous structure, as shown in fig. 5. Obviously, without the compression step, the surface of the stent formed after the compression step has a compact structure compared with the embodiment after the compression step, which indicates that the biological stent prepared by the compression step has compact surface and porous interior.

Further, fig. 3 is a drawing of a scaffold with different ratios of silk fibroin and gelatin, A, B, C, D and E represent examples 4, 3, 1, 7, 8, respectively, with the ratios of silk fibroin and gelatin being 3:1, 2:1, 1:1, 1:2, 1:3, respectively.

By way of the above examples, it was found that the rigidity of the scaffold increased after increasing the proportion of silk fibroin, which is associated with single sided beta-sheet of silk fibroin, and that the toughness of the scaffold increased after increasing gelatin. Therefore, through adjusting the proportion of silk fibroin and gelatin, the scaffold with compact surface and porous interior can be finally obtained, which is beneficial to the adhesion and proliferation of epithelial cells, the shuttle of 3D cytotoxin such as smooth muscle and the circulation of nutrient substances, and the scaffold with different shapes is manufactured through moulds with various sizes and shapes to meet the requirements of different tissues and organs, so as to be beneficial to the development of tissue engineering regeneration.

Further, according to the above embodiment, it has been found that the concentration of silk fibroin in the biological scaffold material is usually 2% -10%, and the scaffold structure with larger pore size can be formed after freeze-drying, which is beneficial to compression treatment. However, when the gelatin protein concentration is increased, manual compression is often difficult, so that the gelatin concentration needs to be adjusted accordingly to find a proper concentration. Experiments show that the gelatin protein with the concentration of 10% is hard after freeze-drying and cannot be compressed into a flake shape; gelatin protein at 7.5% concentration is still hard after lyophilization and cannot be compressed into flakes; gelatin proteins less than or equal to 5% can be manually compressed into flakes.

In the crosslinking step of the invention, 95% of alcohol can induce silk fibroin to be insoluble in water, whereas silk fibroin in a stent can be soluble in water to cause stent collapse; thus, the preliminary crosslinking uses 95% alcohol as a crosslinking agent.

The invention shows that the compression can form a compact structure on the surface of the bracket, otherwise, a porous structure is formed.

Glutaraldehyde can react with amino groups in the material to perform chemical crosslinking, so that on one hand, the mechanical property of the material is improved, and on the other hand, the degradation time of the material is prolonged, so that the prepared biological scaffold can better meet clinical use requirements. Thus glutaraldehyde may also be replaced by other aldehyde crosslinkers.

It will be appreciated by those skilled in the art that by varying the crosslinking time and concentration of the crosslinking agent, the mechanical properties and pore size of the bioscaffold can be varied to meet the needs of different tissue organs.

In addition, the surface of the biological scaffold prepared by the invention contains active groups, such as-COOH, and can adsorb and graft various active substances, so that the performance of the scaffold is enhanced, and the clinical application is better satisfied.

It will be appreciated by persons skilled in the art that the embodiments of the invention described above and shown in the drawings are by way of example only and are not limiting. The objects of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been shown and described in the examples and embodiments of the invention may be modified or practiced without departing from the principles described.

Claims

1. A method for preparing a biological stent, comprising the steps of:

(A) Preparing a silk fibroin solution and a gelatin solution, wherein the concentration range of the silk fibroin solution is 2% -10%, and the concentration range of the gelatin solution is 1% -5%;

(C) Compressing, namely compressing the biological stent prefabricated product in the step (B) to obtain the biological stent prefabricated product with compact surface and loose interior; and

(D) And (3) crosslinking, namely placing the compressed biological stent in the step (C) in a 95% ethanol solution for 2 hours for preliminary crosslinking, and then placing the biological stent in a 1% glutaraldehyde solution for 24 hours for further crosslinking to obtain the biological stent with enhanced strength.

2. The method for preparing a biological stent according to claim 1, wherein the step (D) comprises the steps of:

preliminary cross-linking, inducing silk fibroin beta-sheet to enhance mechanical properties of the scaffold, while reducing solubility of silk fibroin in water; and

further crosslinking, the amino groups in the biological stent preform and the crosslinking agent undergo Schiff base reaction to perform chemical crosslinking.

3. The method for preparing a biological stent according to claim 1, wherein in the step (D), the crosslinked stent is rinsed with deionized water to remove residual aldehyde groups.

4. The method for preparing a bioscaffold according to claim 1, wherein the ratio of silk fibroin solution and gelatin solution prepared in step (a) is 1:1.

5. The method for producing a bioscaffold according to any one of claims 1-4, wherein in the compressing step: the ureteral stent, the intestinal canal stent, the esophagus stent, the urethra stent or the vascular stent which are clinically required are prepared through the ureteral stent mold, the intestinal canal stent mold, the esophagus stent mold, the urethra stent mold or the vascular stent mold.

6. The bioscaffold according to any one of claims 1-4, wherein in the compressing step: the freeze-dried biological stent preform can be selectively compressed and formed into a tubular shape or a flat shape.

7. A biological stent comprising a ureteral stent, an intestinal stent, an esophageal stent, a urethral stent, or a vascular stent, wherein the biological stent is prepared by:

(D) And (3) crosslinking, namely placing the compressed biological stent prefabricated product in the step (C) into a 95% ethanol solution for 2 hours for preliminary crosslinking, and then placing the biological stent prefabricated product into a 1% glutaraldehyde solution for 24 hours for further crosslinking to obtain the biological stent with enhanced strength.

8. The bioscaffold of claim 7, wherein the bioscaffold is tubular or flat.

9. The bioscaffold of claim 7, wherein the ratio of gelatin solution to silk fibroin solution in the method is 1:1.

10. The bioscaffold of claim 7, wherein in the method, the crosslinked scaffold is rinsed with deionized water to remove residual aldehyde groups.