CN117982738A - Biological scaffold, biological scaffold preform and method for preparing same - Google Patents
Biological scaffold, biological scaffold preform and method for preparing same Download PDFInfo
- Publication number
- CN117982738A CN117982738A CN202410137600.6A CN202410137600A CN117982738A CN 117982738 A CN117982738 A CN 117982738A CN 202410137600 A CN202410137600 A CN 202410137600A CN 117982738 A CN117982738 A CN 117982738A
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- China
- Prior art keywords
- polylactic acid
- gelatin
- stent
- polycaprolactone copolymer
- scaffold
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- Pending
Links
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- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
- A61F2002/045—Stomach, intestines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
- A61F2002/047—Urethrae
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
- A61F2002/048—Ureters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/82—Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2002/823—Stents, different from stent-grafts, adapted to cover an aneurysm
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- Health & Medical Sciences (AREA)
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- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
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- Oral & Maxillofacial Surgery (AREA)
- Cardiology (AREA)
- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
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Abstract
The invention provides a biological scaffold, a biological scaffold preform and a preparation method thereof, wherein the biological scaffold is prepared by the following method: (A) Preparing a homogeneous polymer solution of polylactic acid/polylactic acid-polycaprolactone copolymer (PLLA/PLCL); (B) pouring the above polymer solution into a preheated mold; (C) freeze-drying to obtain a PP stent; and (D) grafting gelatin, and fixing the gelatin to the PP bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP-gel bracket with high micropores and high hydrophilicity. The biological scaffold has higher hydrophilicity and good biocompatibility, is favorable for tissue regeneration, and can be applied to clinical treatment of various organ diseases.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to a biological stent for tubular tissues, a biological stent prefabricated product and a preparation method thereof.
Background
Any ureteral diseases, such as stones, tumors, deformities and other internal factors or external factors such as injury, infection, inflammation and the like, can cause ureteral obstruction, cause hydronephrosis or damage to renal functions, and seriously affect the physical and mental health of patients. Particularly when there is a long-term abnormality or lesion in the ureter, it is a great challenge for urologists, and at present, such as kidney transplantation, ureterostomy, ureterohal replacement and ureterostomy, have not been successfully performed all the time, and problems such as serious complications, anastomotic stricture, chronic renal failure, and donor tissue collection are faced. In summary, current clinical treatments for long-segment ureteral defects also face a variety of challenges.
With the development of technology, ureteral tissue engineering has achieved a stand as a potential alternative to autograft. Various other natural polymers and synthetic polymers such as collagen, silk fibroin, gelatin, polylactic acid, polycaprolactone and the like are commonly used for ureteral stents, but on the one hand, due to the formation of fistulae and stenosis caused by the inertness of the stent, the ureteral repair effect is not ideal. On the other hand, ureteral repair is a complex process involving the reconstruction of the urothelium layer, smooth muscle layer, etc., which serves as a barrier to protect underlying tissue from toxic components in the urine, which can lead to ureteral fibrosis, constriction, and stenosis. While a different scaffold may affect the adhesion and movement of urothelial cells.
Thus, for the fields of urology and tissue engineering, research into more suitable stents is of great importance for the treatment of ureteral diseases.
Disclosure of Invention
An object of the present invention is to provide a biological scaffold, a biological scaffold preform and a preparation method thereof, wherein the biological scaffold firstly obtains a biological scaffold preform-PP scaffold through polylactic acid and polylactic acid-polycaprolactone copolymer, and then grafts gelatin to obtain a PP-gel scaffold, and the PP-gel scaffold has high toughness and good biocompatibility, and has great clinical application potential.
It is another object of the present invention to provide a bioscaffold, a bioscaffold preform, and a method for preparing the same, which are prepared by using a thermal phase separation method and an ammonolysis GA crosslinking method, and which are simple to operate.
Another object of the present invention is to provide a biological stent, a biological stent preform and a method for preparing the same, wherein the prepared biological stent has a high hydrophilicity, is helpful for adhesion and proliferation of urinary epithelial cells, and is suitable for being applied to clinical treatment of ureter long segment defects as an ideal stent.
Another object of the present invention is to provide a biological stent, a biological stent preform, and a method for manufacturing the same, which have a strong biocompatibility, can be manufactured through different molds, are suitable for various organ applications, and can be applied to human body for treating related organ diseases.
It is another object of the present invention to provide a biological stent, a biological stent preform, and a method for preparing the same, in which the biological stent prepared by the thermal phase separation method has a desired porosity and pore size on the surface and inside, and is very suitable for clinical application as a biological stent.
It is another object of the present invention to provide a biological stent, a preform of the biological stent, and a method for preparing the same, which have a porous and dense surface, which is advantageous for epithelialization while preventing cells from entering the stent.
It is another object of the present invention to provide a biological scaffold, a biological scaffold preform, and a method of preparing the same, which has high porosity and macropores, such physical structure allowing cell migration and nutrient circulation, facilitating promotion of tissue regeneration.
Another object of the present invention is to provide a bio-scaffold, a bio-scaffold preform and a method for preparing the same, which have optimal porosity and pore size, not only facilitate nutrition circulation, but also have anti-fibrosis effect, so that the bio-scaffold can be built in ureter, can remarkably improve healing of urinary epithelium and delay renal function decrease, and is suitable for clinical application.
According to one aspect of the present invention, there is provided a method for preparing a biological scaffold, comprising the steps of:
(A) Preparation of organisms from polylactic acid/polylactic acid-polycaprolactone copolymer (PLLA/PLCL)
A support preform PP support; and
(B) And (3) grafting gelatin, and fixing the gelatin to the PP bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP-gel bracket.
Wherein in step (a) PLLA: the mass ratio of PLCL ranges from 1:9 to 9:1.
Wherein the PP scaffold is prepared by the steps of:
(A1) Polymer for preparing homogeneous polylactic acid/polylactic acid-polycaprolactone copolymer (PLLA/PLCL)
A solution;
(A2) Pouring the polymer solution into a preheated mold;
(A3) Phase separation and freeze-drying to obtain a PP stent;
In step (A1), PLLA and PLCL are dissolved in 1, 4-dioxane at 65 ℃ according to the mass ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 respectively to form a homogeneous polymer solution, and in step (A3), the polymer solution and a mold are placed in-80 ℃ for 24 hours and then freeze-dried for 72 hours to obtain the PP stent.
In the step (A1), PLLA and PLCL are dissolved in 1, 4-dioxane at a predetermined mass ratio at 65 ℃ to form a homogeneous polymer solution, and in the step (A3), the polymer solution and the mold are placed in-80 ℃ for 24 hours and then lyophilized for 72 hours to obtain the PP stent.
The step (B) comprises the following steps:
removing impurities by soaking and drying;
preparing an aldehyde bracket by ammonolysis and glutaraldehyde crosslinking; and
Grafting gelatin to obtain the PP-gel scaffold.
Wherein said step (B) comprises the steps of:
(B1) Soaking the PP bracket in 95% alcohol for 1h, drying in a vacuum oven, further soaking in 0.06g/ml 1.6-hexamethylenediamine/isopropanol solution for 10min, washing with a large amount of water to remove free hexamethylenediamine, and further obtaining the bracket with impurities removed;
(B2) Soaking an ammoniated PP bracket in 1.0wt% GA aqueous solution for 3 hours at room temperature, and then washing with a large amount of water to remove free GA to obtain an aldehyde bracket;
(B3) The aldehyde scaffold was incubated in a 1.0wt% gelatin/PBS solution for 24h at room temperature, and then the ungrafted gelatin was washed off with water to obtain a PP-gel scaffold.
In said step (A3), the PP stent is manufactured as a tubular stent or a circular stent by means of a matched mold.
Wherein the mass ratio of PLLA to PLCL in step (A1) is 4:6.
The PP-gel stent is used for preparing a ureteral stent, an intestinal canal stent, an esophageal stent, a urethral stent, a gastric tube stent or a vascular stent which are clinically required through a ureteral stent mold, an intestinal canal stent mold, an esophageal stent mold, a gastric tube stent mold or a vascular stent mold.
According to another aspect of the present invention, there is also provided 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 homogeneous polymer solution of polylactic acid/polylactic acid-polycaprolactone copolymer (PLLA/PLCL) at a concentration of 10% (w/v);
(B) Pouring the polymer solution into a preheated mold;
(C) Phase separation and freeze-drying to obtain a PP stent; and
(D) And (3) grafting gelatin, and fixing the gelatin to the PP bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP-gel bracket with high micropores and high hydrophilicity.
The invention also provides a biological stent, wherein the biological stent comprises a ureteral stent, an intestinal stent, an esophageal stent, a urethral stent or a vascular stent, and is characterized in that the biological stent comprises the following materials: polylactic acid, polylactic acid-polycaprolactone copolymer, and gelatin.
The invention also provides a biological stent prefabricated product, wherein the biological stent prefabricated product is used for preparing a ureteral stent, an intestinal stent, an esophageal stent, a urethral stent or a vascular stent which are clinically required by adopting a ureteral stent mould, an intestinal stent mould, an esophageal stent mould, a urethral stent mould or a vascular stent mould.
Wherein the biological scaffold prefabricated product is prepared by the following method:
(A1) Polymer for preparing homogeneous polylactic acid/polylactic acid-polycaprolactone copolymer (PLLA/PLCL)
A solution;
(A2) Pouring the polymer solution into a preheated mold; and
(A3) Phase separation and freeze drying to obtain the PP stent.
Drawings
FIG. 1 is a schematic illustration of an ammonolysis crosslinking reaction process of a PP-gel stent according to an embodiment of the invention.
Fig. 2 is an electron microscopy image of a PP46 stent prepared according to a fourth embodiment of the present invention.
Fig. 3 is an electron microscope (SEM) image analysis of nine PP stents prepared in accordance with nine embodiments of the present invention.
Fig. 4 is a graph of pore, pore size and porosity analysis of PP scaffolds of different specific gravities according to various embodiments of the present invention.
FIG. 5 is a graph showing the results of contact angle image analysis of PP46 and PP46-gel scaffolds prepared according to the fourth embodiment of the invention.
FIG. 6 shows stress-strain curves of PP46 and PP46-gel according to a fourth embodiment of the invention.
FIG. 7 shows the total reflection IR spectrum of PP46 and PP46-gel with gelatin powder according to a fourth embodiment of the invention.
FIG. 8 is an image showing the proliferation results of epithelial cells according to at least one embodiment of the present invention and a comparative embodiment.
Fig. 9 shows the results of a subcutaneous embedding animal experiment according to at least one embodiment of the present invention and a comparative embodiment.
Fig. 10 shows images after stent graft according to at least one embodiment of the present invention and a comparative embodiment.
FIG. 11 shows the results of an epithelialization phenotype of urothelial cells in accordance with at least one embodiment of the present invention and a comparative embodiment.
FIG. 12 is a graph showing the results of protein expression studies according to at least one embodiment of the present invention and a comparative 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.
Gelatin is a natural protein, a partial hydrolysate of collagen, containing arginine-glycine-aspartic acid (RGD) peptide sequences associated with integrins, and is commonly used in the field of tissue engineering. However, since gelatin has low mechanical properties, it is difficult to reconstruct ureters by surgery, and thus various synthetic polymers must be grafted. Polylactic acid (PLLA) is a commonly used polymer that can be made into a nanofiber porous scaffold by Thermal Induced Phase Separation (TIPS), however, PLLA is rigid and brittle and when applied to ureteral stents, it is difficult to perform ureteral remodeling. Therefore, we combine PLLA with polylactic acid-polycaprolactone copolymer (PLCL) with good elasticity. However, since synthetic polyesters are poorly hydrophobic and cell-adhesive, they do not properly modulate cell phenotype, and therefore we choose to graft natural gelatin to increase the hydrophilicity and cell-adhesion of the scaffold.
In the invention, polylactic acid/polylactic acid-polycaprolactone copolymer (PLLA/PLCL) mixed chemical modification gelatin materials with different proportions are used, a tubular stent with a highly microporous topological structure is created by adopting a thermally induced phase separation method, namely, the polylactic acid/polylactic acid-polycaprolactone copolymer (PLLA/PLCL) stent, namely, a PP stent is prepared by adopting a thermally induced phase separation method (TIPS), then the influence of gelatin (gel) modified synthetic materials on long-segment ureter abnormality is studied, and the conduction of biological signals of channels which are accompanied by the application of the gelatin synthetic materials as biological stents is explored, so that a novel stent material and a manufacturing method are provided for the ureter stent. Therefore, the stent of the invention can be used for ureter tissue engineering and can be applied to different organs as various small-diameter tubular tissues in the future.
The invention adopts hexamethylenediamine ammonolysis and glutaraldehyde crosslinking to graft gelatin into polymeric ester. FIG. 1 is an experimental process for preparing a PP46-gel scaffold by ammonolysis of GA. Specifically, the ester bond in the material is opened by amino groups, the surface of the scaffold is subjected to excessive GA hydroformylation, and finally, the amino groups on the gelatin react with the surface of the scaffold to crosslink.
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.
Materials and reagents:
polylactic acid (PLLA) (mw=20 kDa), purchased from the Ningbo materials technology and engineering institute;
Polylactic acid-polycaprolactone copolymer (PLCL) (50:50, mw=30 kDa), purchased from bioengineering limited in napa, inc;
1, 4-dioxane, hexamethylenediamine (HMD) and Glutaraldehyde (GA), purchased from Aba Ding Shiji company (Shanghai, china);
Gelatin, available from Sigma-Aldrich Inc. (Misu, USA).
Embodiment one:
(A) PLLA and PLCL in a mass ratio of 1:9 were dissolved in 1, 4-dioxane at 65℃to form a homogeneous polymer solution at a concentration of 10% (w/v);
(B) Pouring into a preheated mold;
(C) Phase separation, namely placing the polymer solution and the die in a temperature of minus 80 ℃ for 24 hours, and then freeze-drying for 72 hours to obtain a biological scaffold preform-PP 19 scaffold;
(D) And (3) grafting gelatin, and fixing the gelatin to the PP19 bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP19-gel bracket.
In the step (C), the PP19 stent may be a tubular stent or a circular stent, wherein the circular stent is fabricated in a steel mold (diameter 60mm, height 1 mm). In addition, in the step (C), the biological scaffold preform is prepared by performing phase separation by means of thermally induced phase separation.
In the step (D), the step (D) includes the steps of:
(D1) Removing impurities: soaking the PP19 bracket in 95% alcohol for 1h, drying in a vacuum oven, further soaking in 0.06g/ml 1.6-hexamethylenediamine/isopropanol solution for 10min, and washing with a large amount of water to remove free hexamethylenediamine;
(D2) An hydroformylation scaffold: soaking the PP19 bracket in 1.0wt% GA aqueous solution for 3 hours at room temperature, and then washing with a large amount of water to remove free GA;
(D3) Grafted gelatin: the above-mentioned aldehyde scaffold was incubated in a gelatin/PBS solution of 1.0wt% for 24 hours at room temperature, and then ungrafted gelatin was washed off with water to obtain a PP19-gel scaffold.
Wherein the step (D1) is to remove impurities in the step (C) before grafting gelatin.
In said step (A), a 10% w/v homogeneous polymer solution is formed.
Wherein the PP19 scaffold prepared in the steps (A), (B) and (C) is used as a biological scaffold preform for subsequent gelatin grafting.
Further comprising a step (E) of manufacturing the PP19-gel scaffold into a biological scaffold by adopting a corresponding mould for clinical application.
Embodiment two:
(A) PLLA and PLCL in a mass ratio of 2:8 were dissolved in 1, 4-dioxane at 65℃to form a homogeneous polymer solution at a concentration of 10% (w/v);
(B) Pouring into a preheated mold;
(C) Phase separation, namely placing the polymer solution and a die in a temperature of minus 80 ℃ for 24 hours, and then freeze-drying for 72 hours to obtain a PP28 bracket;
(D) And (3) grafting gelatin, and fixing the gelatin to the PP28 bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP28-gel bracket.
In the step (C), the PP28 stent may be a tubular stent or a circular stent, wherein the circular stent is fabricated in a steel mold (diameter 60mm, height 1 mm). In addition, in the step (C), the biological scaffold preform is prepared by performing phase separation by means of thermally induced phase separation.
In the step (D), the step (D) includes the steps of: (D1) removing impurities; (D2) an hydroformylation scaffold; and (D3) grafting gelatin. The specific operations of steps (D1) - (D3) are described with reference to example one.
In said step (A), a 10% w/v homogeneous polymer solution is formed.
Wherein the PP28 scaffold prepared in the steps (A) (B) (C) is used as a biological scaffold preform for subsequent gelatin grafting.
Further comprising a step (E) of manufacturing the PP28-gel scaffold into a biological scaffold by adopting a corresponding mould for clinical application.
Embodiment III:
(A) PLLA and PLCL in a mass ratio of 3:7 were dissolved in 1, 4-dioxane at 65℃to form a homogeneous polymer solution at a concentration of 10% (w/v);
(B) Pouring into a preheated mold;
(C) Phase separation, namely placing the polymer solution and a die in a temperature of minus 80 ℃ for 24 hours, and then freeze-drying for 72 hours to obtain a PP37 bracket;
(D) And (3) grafting gelatin, and fixing the gelatin to the PP37 bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP37-gel bracket.
In the step (C), the PP37 stent may be a tubular stent or a circular stent, wherein the circular stent is fabricated in a steel mold (diameter 60mm, height 1 mm).
In the step (D), the step (D) includes the steps of: (D1) removing impurities; (D2) an hydroformylation scaffold; and (D3) grafting gelatin. The specific operations of steps (D1) - (D3) are described with reference to example one.
In said step (A), a 10% w/v homogeneous polymer solution is formed.
Wherein the PP37 scaffold prepared in the steps (A) (B) (C) is used as a biological scaffold preform for subsequent gelatin grafting.
In addition, in the step (C), the biological scaffold preform is prepared by performing phase separation by means of thermally induced phase separation.
Further comprising a step (E) of manufacturing the PP37-gel scaffold into a biological scaffold by adopting a corresponding mould for clinical application.
Embodiment four:
(A) Dissolving PLLA and PLCL in a mass ratio of 4:6 in 1, 4-dioxane at 65 ℃ to form a homogeneous polymer solution;
(B) Pouring into a preheated mold;
(C) Phase separation, namely placing the polymer solution and a die in a temperature of minus 80 ℃ for 24 hours, and then freeze-drying for 72 hours to obtain a PP46 bracket;
(D) And (3) grafting gelatin, and fixing the gelatin to the PP46 bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP46-gel bracket.
In the step (C), the PP46 stent may be a tubular stent or a circular stent, wherein the circular stent is fabricated in a steel mold (diameter 60mm, height 1 mm). In addition, in the step (C), the biological scaffold preform is prepared by performing phase separation by means of thermally induced phase separation.
In the step (D), the step (D) includes the steps of: (D1) removing impurities; (D2) an hydroformylation scaffold; and (D3) grafting gelatin. The specific operations of steps (D1) - (D3) are described with reference to example one.
In said step (A), a 10% w/v homogeneous polymer solution is formed.
Wherein the PP46 scaffold prepared in the steps (A), (B) and (C) is used as a biological scaffold preform for subsequent gelatin grafting.
Further comprising a step (E) of manufacturing the PP46-gel scaffold into a biological scaffold by adopting a corresponding mould for clinical application.
Fifth embodiment:
(A) PLLA and PLCL in a mass ratio of 5:5 were dissolved in 1, 4-dioxane at 65℃to form a homogeneous polymer solution at a concentration of 10% (w/v);
(B) Pouring into a preheated mold;
(C) Phase separation, namely placing the polymer solution and a die in a temperature of minus 80 ℃ for 24 hours, and then freeze-drying for 72 hours to obtain a PP55 bracket;
(D) And (3) grafting gelatin, and fixing the gelatin to the PP55 bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP55-gel bracket.
In the step (C), the PP55 stent may be a tubular stent or a circular stent, wherein the circular stent is fabricated in a steel mold (diameter 60mm, height 1 mm). In addition, in the step (C), the biological scaffold preform is prepared by performing phase separation by means of thermally induced phase separation.
In the step (D), the step (D) includes the steps of: (D1) removing impurities; (D2) an hydroformylation scaffold; and (D3) grafting gelatin. The specific operations of steps (D1) - (D3) are described with reference to example one.
In said step (A), a 10% w/v homogeneous polymer solution is formed.
Wherein the PP55 scaffold prepared in the steps (A), (B) and (C) is used as a biological scaffold preform for subsequent gelatin grafting.
Further comprising a step (E) of manufacturing the PP55-gel scaffold into a biological scaffold by adopting a corresponding mould for clinical application.
Example six:
(A) PLLA and PLCL in a mass ratio of 6:4 were dissolved in 1, 4-dioxane at 65℃to form a homogeneous polymer solution at a concentration of 10% (w/v);
(B) Pouring into a preheated mold;
(C) Phase separation, namely placing the polymer solution and a die in a temperature of minus 80 ℃ for 24 hours, and then freeze-drying for 72 hours to obtain a PP64 bracket;
(D) And (3) grafting gelatin, and fixing the gelatin to the PP64 bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP64-gel bracket.
In the step (C), the PP64 stent may be a tubular stent or a circular stent, wherein the circular stent is fabricated in a steel mold (diameter 60mm, height 1 mm). In addition, in the step (C), the biological scaffold preform is prepared by performing phase separation by means of thermally induced phase separation.
In the step (D), the step (D) includes the steps of: (D1) removing impurities; (D2) an hydroformylation scaffold; and (D3) grafting gelatin. The specific operations of steps (D1) - (D3) are described with reference to example one.
In said step (A), a 10% w/v homogeneous polymer solution is formed.
Wherein the PP64 scaffold prepared in the steps (A), (B) and (C) is used as a biological scaffold preform for subsequent gelatin grafting.
Further comprising a step (E) of manufacturing the PP64-gel stent into a biological stent by adopting a corresponding mould for clinical application.
Embodiment seven:
(A) PLLA and PLCL in a mass ratio of 7:3 were dissolved in 1, 4-dioxane at 65℃to form a homogeneous polymer solution at a concentration of 10% (w/v);
(B) Pouring into a preheated mold;
(C) Phase separation, namely placing the polymer solution and a die in a temperature of minus 80 ℃ for 24 hours, and then freeze-drying for 72 hours to obtain a PP73 bracket;
(D) And (3) grafting gelatin, and fixing the gelatin to the PP73 bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP73-gel bracket.
In the step (C), the PP73 stent may be a tubular stent or a circular stent, wherein the circular stent is fabricated in a steel mold (diameter 60mm, height 1 mm). In addition, in the step (C), the biological scaffold preform is prepared by performing phase separation by means of thermally induced phase separation.
In the step (D), the step (D) includes the steps of: (D1) removing impurities; (D2) an hydroformylation scaffold; and (D3) grafting gelatin. The specific operations of steps (D1) - (D3) are described with reference to example one.
In said step (A), a 10% w/v homogeneous polymer solution is formed.
Wherein the PP73 scaffold prepared in the steps (A), (B) and (C) is used as a biological scaffold preform for subsequent gelatin grafting.
Further comprising a step (E) of manufacturing the PP73-gel scaffold into a biological scaffold by adopting a corresponding mould for clinical application.
Example eight:
(A) PLLA and PLCL in a mass ratio of 8:2 were dissolved in 1, 4-dioxane at 65℃to form a homogeneous polymer solution at a concentration of 10% (w/v);
(B) Pouring into a preheated mold;
(C) Phase separation, namely placing the polymer solution and a die in a temperature of minus 80 ℃ for 24 hours, and then freeze-drying for 72 hours to obtain a PP82 bracket;
(D) And (3) grafting gelatin, and fixing the gelatin to the PP82 bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP82-gel bracket.
In the step (C), the PP82 stent may be a tubular stent or a circular stent, wherein the circular stent is fabricated in a steel mold (diameter 60mm, height 1 mm). In addition, in the step (C), the biological scaffold preform is prepared by performing phase separation by means of thermally induced phase separation.
In the step (D), the step (D) includes the steps of: (D1) removing impurities; (D2) an hydroformylation scaffold; and (D3) grafting gelatin. The specific operations of steps (D1) - (D3) are described with reference to example one.
In said step (A), a 10% w/v homogeneous polymer solution is formed.
Wherein the PP82 scaffold prepared in the steps (A) (B) (C) is used as a biological scaffold preform for subsequent gelatin grafting.
Further comprising a step (E) of manufacturing the PP82-gel scaffold into a biological scaffold by adopting a corresponding mould for clinical application.
Example nine:
(A) PLLA and PLCL in a mass ratio of 9:1 were dissolved in 1, 4-dioxane at 65℃to form a homogeneous polymer solution at a concentration of 10% (w/v);
(B) Pouring into a preheated mold;
(C) Phase separation, namely placing the polymer solution and a die in a temperature of minus 80 ℃ for 24 hours, and then freeze-drying for 72 hours to obtain a PP91 bracket;
(D) And (3) grafting gelatin, and fixing the gelatin to the PP91 bracket by adopting an ammonolysis and Glutaraldehyde (GA) crosslinking method to obtain the PP91-gel bracket.
In the step (C), the PP91 stent may be a tubular stent or a circular stent, wherein the circular stent is fabricated in a steel mold (diameter 60mm, height 1 mm). In addition, in the step (C), the biological scaffold preform is prepared by performing phase separation by means of thermally induced phase separation.
In the step (D), the step (D) includes the steps of: (D1) removing impurities; (D2) an hydroformylation scaffold; and (D3) grafting gelatin. The specific operations of steps (D1) - (D3) are described with reference to example one.
In said step (A), a 10% w/v homogeneous polymer solution is formed.
Wherein the PP91 scaffold prepared in the steps (A), (B) and (C) is used as a biological scaffold preform for subsequent gelatin grafting.
Further comprising a step (E) of manufacturing the PP91-gel stent into a biological stent by adopting a corresponding mould for clinical application.
It will be appreciated by those skilled in the art that by the above method of the present invention, each of the embodiments may be augmented with a step (E): the gelatin grafted scaffold is manufactured into a biological scaffold which can be applied to clinic by adopting a corresponding mould. It will be appreciated that different organ scaffolds may be manufactured using different molds and thus applied to different organ treatments. For example, the PP-gel stent is manufactured into a clinically required ureteral stent, an intestinal stent, an esophageal stent, a urethral stent or a vascular stent by a ureteral stent mold, an intestinal stent mold, an esophageal stent mold, a urethral stent mold or a vascular stent mold.
The characteristics of the biological scaffold preform and the gelatin grafted biological scaffold prepared in the above examples were tested.
1. Porosity measurement: the scaffold porosity was measured using ethanol displacement, since ethanol can enter pores, but does not shrink or expand the scaffold.
2. And (3) observing the morphology of the bracket: the morphology of the scaffolds was observed by scanning electron microscopy (SEM: TM-3000, hitachi).
3. Determination of pore size and wall thickness: the pore size and wall thickness of the stent were measured using image-j software, with at least 50 positions measured per image.
4. Measurement of contact angle: the hydrophilicity of the gel film before and after coating was measured with a contact angle meter (datphysicsoca, germany), the samples were dried under vacuum at 30 ℃ before measurement, deionized water was measured by software 10 seconds after the nanofiber scaffold surface, at least three independent measurements were made for each sample and averaged.
5. Biocompatibility studies: permanent human urine-bearing cells were used for culture in F12K medium (Gibco) containing 10% fetal bovine serum (FBS, gibco), 100U/ml penicillin and 100g/ml streptomycin (Gibco) in a humidified environment of 5% carbon dioxide at 37℃and changed every two days. In the biocompatibility experiment, HUCs is placed on a PP and PP-gel stent, the proliferation condition of cells on the stent is detected by using a cell counting kit-8 (CCK-8), and the distribution and the morphology of the cells on the stent are observed by using a laser confocal microscope (CLSM, SP8, leica, germany) and a Scanning Electron Microscope (SEM).
6. Cell activity and epithelialization assays: cell cycle was detected with a flow cytometer, epidermal growth factor was detected with an EGF ELISA kit, and epithelialization level was assessed by quantitative PCR.
7. In vivo evaluation of tissue regeneration effect: the experiments were performed subcutaneously by selecting 12 male SD rats, and dividing them into two groups, four weeks and eight weeks. The stent is cut into round slices, sterilized with 75% alcohol for 12 hours and then implanted. Four and eight weeks later, scaffolds were removed and tested by H & E, masson, immunohistochemistry (IHC), immunofluorescence (IF) staining.
8. And (3) detecting biocompatibility of the stent: the CD68 test scaffolds were biocompatible and divided into three groups of 18 adult new zealand male rabbits for 8 weeks and 12 weeks, PP group, PP-gel group and autologous ureter graft group, respectively. After anesthetizing rabbits with pentobarbital (30 mg/kg), an incision is made in the middle of the abdomen, the left ureter is found along the paracolonic sulcus, the left ureter defect (about 4 cm) is surgically excised, and after excision, the graft is placed at the ureter defect and sutured under an operation microscope. The ureteral stent is placed in the ureter, and the stent is taken out from the bladder incision two weeks after operation. Histological observations were made 8 weeks and 12 weeks after surgery.
9. Detection of epithelial cells: epithelial cells were examined with AE1/AE3 and the function after ureteral reconstruction of each group was analyzed using CT and CT urography (CTU).
The experimental results of the above examples are shown in the following figures: FIG. 2 shows SEM analysis of PP46 and pore size and wall thickness measurements, where a1 and a2 are cross-sectional pore sizes, b1 and b2 are cross-sectional wall thicknesses, and c1 and c2 are indicated pore sizes; FIG. 3 shows SEM analysis and pore diameter and wall thickness measurement results of PP19 to PP91 and PP19-gel to PP91-gel prepared in the above nine examples, wherein A1-A9 in FIG. 3 are SEM analysis and cross-sectional pore diameter measurement structures of the biological stent preform PP19-PP91, and A1-A9 are SEM analysis and cross-sectional wall thickness measurement results of the PP19-gel-PP 91-gel. FIG. 4 shows the results of the porosity analysis of the preform for a biological stent (PP stent) of the above example. FIG. 5 is a graph showing contact angle images and contact angle analysis of PP46 and PP46-gel scaffolds. FIG. 6 shows the stress curves of PP46 and PP 46-gel. FIG. 7 shows the total reflection IR spectra of PP46 and PP46-gel with gelatin powder. FIG. 8 shows the results of epithelial proliferation. Fig. 9 shows the results of subcutaneous embedding animal experiments. Fig. 10 shows an image after stent implantation. FIG. 11 shows the results of urothelial cell epithelialization detection. FIG. 12 shows the results of protein expression studies.
The invention is mainly researched by PP46 and PP46-gel, and other proportions of biological scaffolds and biological scaffold prefabricated products are verified and analyzed by adopting the same mode.
As can be seen from figures 2 to 4, the internal pore structure of the PP46 stent is good, the average pore diameter of the section is 32.48 +/-6.51 mu m (shown as a1-a2 in figure 2), the wall thickness of the section is 613.80 +/-175.03 nm (shown as b1-b2 in figure 2), the surface of the PP46 stent is porous, and the average pore diameter is 3.74+/-1.02 mu m (shown as c1-c2 in figure 2).
Of all the scaffold ratios (PP 19-PP 91) of the above examples, the internal pore size structure of PP46 is best seen by electron microscopy, which is 75.48 ±3.90% (as shown in fig. 4), so that the PP46 scaffold material has high porosity and macropores, such physical structure allows cell migration and nutrient circulation, which is advantageous for promoting tissue regeneration.
As shown in fig. 5, the contact angle of the PP46 scaffold was 98.50±2.93°, and the contact angle of the PP46-gel scaffold was 48.86 ±5.20°, which indicates that the fixation of gelatin on the scaffold significantly improved the hydrophilicity of the scaffold, resulting in a PP-gel scaffold with higher hydrophilicity.
As shown in FIG. 6, the stress-strain curves of the PP46 and the PP46-gel brackets show that the tensile strength of the PP46-gel brackets is reduced (0.78+ -0.02 Mpa vs 0.72+ -0.03 Mpa, p < 0.01) due to the ammonolysis effect, but the final strain is unchanged (84.62 + -3.09 Mpa vs 84.88+ -3.22 Mpa, p > 0.05), which shows that the toughness of the brackets is the same as the toughness of the brackets before, and meanwhile, the toughness of the PP46 and the PP46-gel brackets is enhanced compared with that of polylactic acid, so that the brackets are more beneficial to being applied to tissue engineering.
The PP46 stent provided by the invention has optimal porosity and pore diameter, is beneficial to nutrition circulation, and has an anti-fibrosis effect. In addition, since the stent surface cools faster than the stent interior, a relatively dense pore structure can be formed on the stent surface, facilitating epithelialization while preventing cells from entering the stent.
The total reflection infrared spectra of PP46 and PP46-gel with gelatin powder are shown in fig. 7, where the gelatinized scaffold has two characteristic absorption peaks of gelatin, whereas the PP46 scaffold does not, and the results indicate that gelatin was successfully grafted onto the PP46 scaffold.
Repair of the urothelium is critical to ureter regeneration, and therefore, whether urothelium cells can grow on stents is an important issue. Hus cultured on PP46 and PP46-gel scaffolds proliferated for seven days with tissue-cultured polystyrene (TCPS) as positive control.
Over time hus on both scaffolds continued to grow and proliferate, indicating that the scaffolds could support hus proliferation without toxic effects. On day seven, the PP46-gel scaffold had more living cells than the PP46 scaffold, indicating that the scaffold was more favorable for proliferation of hus after gelatin implantation, as shown by A in FIG. 8.
Cells were grown on scaffolds by DAPI staining of cytoskeleton and confocal microscopy for three and seven days, as shown in B in fig. 8, with cytoskeletal disturbance occurring within three days for cells on both scaffolds, and increased in number on both scaffolds, but the cytoskeleton on PP46 scaffold was still chaotic, while the cell morphology on PP46-gel scaffold was good, with more actin bundles expressed on the scaffold surface.
As seen from SEM analysis of C in FIG. 8, hus grew on both scaffold surfaces and covered some of the pores on the scaffold surface, and on day seven, cells were observed to cover more surface voids, especially on the PP46-gel scaffold.
As shown in D in FIG. 8, EGF is an important factor for promoting epithelialization, and can guide G0 phase cells to return to G1 phase and carry out cell cycle proliferation, and after seven days of culture, the GEF level of the PP46-gel group is obviously higher than that of the PP46 group, and 818.39 +/-68.96 pg/ml is reached.
As shown in E and F in fig. 8, the flow cytometer was used to detect the urothelial cell cycle distribution, and the cell proliferation index was calculated as: proliferation index (%) = (s+g2/M)/(g0/g1+s+g2/M) x100%. Proliferation index increased from (50.16.+ -. 2.25)% in the PP46 group to (56.04.+ -. 5.06)% in the PP46-gel group.
Animal experiment: the PP46 and PP46-gel scaffolds were implanted subcutaneously in SD rats for 4 and 8 weeks, respectively, as shown in a in fig. 9, the degree of vascularization of the scaffold surface increased with time, and the degree of vascularization of the PP46-gel scaffold surface was higher compared to the two scaffolds in the same period, and the vascularization of the outer layer of the scaffold was critical for ureteral regeneration. From HE and Masson staining, cells only migrate to the surface of the scaffold, secrete collagen, and facilitate cell adhesion and proliferation, and in gelatin-transplanted scaffolds, cells accumulate within the scaffold and secrete collagen. In addition, after 8 weeks, gelatin scaffolds were significantly degraded and fibroblasts passed through the scaffolds.
Inflammation is reflected primarily in the early stages and is mostly expressed on the stent surface, as shown by B in fig. 9, and semi-quantitative analysis shows that at 8 weeks, the inflammatory expression of the stent tissue cross section is significantly reduced. Clinically, iatrogenic ureteral injuries are the most common cause, especially in some typical surgical ureteroscopy procedures. The experimental result shows that at 4 weeks, positive signals are generated on the surface and the inside of the two scaffolds, and the PP46 is higher than the PP46-gel scaffold, which indicates that after gelatin grafting, the biocompatibility of the scaffold is improved. As shown in C in fig. 9, after 8 weeks, the in-stent positive model significantly decreased, with no significant difference in the positive area ratio.
Incomplete ureteral regeneration can cause ureteral dilation and hydronephrosis, thereby disrupting renal function. As shown in fig. 10, the naked eye and CT/CTU photographs 12 weeks after the autograft group showed some degree of ureteral stenosis, but the PP46-gel group had a lower degree of stenosis than the PP46 group. The PP46 group showed a significant ureter, a significant expansion of the renal pelvis, and hydronephrosis, and the left renal pelvis was not developed, suggesting serious damage to renal function, and in the PP46-gel group, the degree of ureteral expansion was reduced and reversible, as shown by a in fig. 10.
Hydronephrosis was observed by CT cross-section and the effect of the scaffold on renal function was assessed by Cr and BUN. The reduced hydronephrosis, cr, BUN following PP46-gel implantation, indicated that the PP-gel group significantly reduced kidney function impairment, as shown by B-D in FIG. 10.
Histological analysis was performed 8 weeks and 12 weeks after surgery, and the analysis results are shown as E in fig. 10. At 8 weeks, inflammatory cells in the PP46 group infiltrate more than in the PP46-gel group, and disappeared at 12 weeks. The Masson staining results showed that the PP46-gel scaffold degraded after two months, whereas the PP46 scaffold did not, and in addition, the normal collagen mechanism replaced the scaffold site in the PP46-gel group, whereas the PP 46-group exhibited fibrosis due to collagen hyperproliferation, i.e., scar formation. The regeneration of urothelium was observed by AE1/AE immunohistochemical staining, and after 8 weeks in the PP46 group, a thinner urothelium layer appeared on the surface, whereas the number of cells increased with time in the PP46-gel group increased. Thus, the gelatin grafted stent promotes the epithelialization of the urinary tract, remarkably reduces the expansion degree of the ureter and hydronephrosis, and protects the renal function.
Three-dimensional ureteral engineering is a better strategy for treating ureteral stenotic disorders, where one of the main purposes is epithelialization of the material surface. The present experiment detects the expression of functional proteins related to urothelial cells on two scaffolds by immunofluorescence, and finds that the cells on the PP46-gel scaffold are more, and the urothelial cells maintain an epithelial phenotype, as shown by A in FIG. 11. ZO-1 and UPKIII are urothelial proteins, closely related to the action of urinary epithelial obstruction, and RT-qPCR detects the transcriptional level of cell-related genes, and the results show that UPK, ZO-1 and other membrane proteins are significantly increased, as shown by B and C in FIG. 11.
These results indicate that gelatin grafted scaffolds have the ability to promote urinary tract epithelium, including cell proliferation and epithelial function-related gene expression, and furthermore, integrin α6, β4, K-RAS and N-RAS genes are also activated, demonstrating that activation of FAK/RAS-related pathways further promotes cell proliferation.
FIG. 12 is a protein expression study, wherein A in FIG. 12 shows the expression of several pathway proteins detected by Western blotting. Consistent with the commercial tenant RT-qPCR results, expression of p-FAK, integrin α6, β4, ras, p-RAF and p-ERK proteins was significantly increased in gelatin-modified scaffold cells, as shown by B in fig. 12. Thus, the combined effect of the PP46-gel scaffold on urothelium in vitro behavior and in vivo urolithiation can be developed, as shown at C in FIG. 12.
It is known that integrin alpha 6/beta 4 perceives the RGD sequence on the surface of the grafted gelatin scaffold, phosphorylates FAK and activates downstream MAPK/ERK pathway, and in addition, its internal porous structure also provides more EGF to promote growth and proliferation of epithelial cells. The urinary tract epithelialization realizes the formation of tight connection between cells and AUM, thereby blocking the influence of urine on submucosa. Therefore, the PP porous scaffold grafted with gelatin has the effect of promoting ureter regeneration.
It will be appreciated by those skilled in the art that changing the ratio of PLL to PLCL can change the pore diameter and wall thickness of the PP stent, or can adjust the ratio of PLL to PLCL to other ratio, so as to synthesize more PP stents, and then study the performance of the PP stents to adapt to the stent requirements of different organs and satisfy more clinical applications.
In the invention, the performance of the PP stent and the PP-gel stent serving as ureteral stents can be studied, and the same or similar experimental methods can be adopted to study the performance of the PP stent and the PP-gel stent serving as biological stents of other organs so as to increase the clinical application range of the PP stent and the PP-gel stent.
According to the research results, the PP biological stent can be internally arranged in a ureter, can remarkably improve healing of an epithelial layer of the urinary tract and delay renal function reduction, and the stent grafted with gelatin can stimulate integrin alpha 6/beta 4 on a cell membrane of the epithelial cell of the urinary tract, and then is phosphorylated under the action of focal adhesion kinase, so that the formation of the epithelium is promoted through a signal channel of mitogen activated protein kinase/extracellular signal regulation kinase, and the biomimetic stent is constructed by combining nano morphology and biochemistry, so that ureter regeneration is stimulated together.
In a word, the invention grafts gelatin to polyester by using hexamethylenediamine ammonolysis and glutaraldehyde crosslinking method, and the obtained various tubular porous PP-gel scaffolds, especially the PP46-gel scaffolds, have higher hydrophilicity and good biocompatibility, can promote cell adhesion and proliferation, are beneficial to ureter epithelialization and ureter regeneration, and have protection effect on ureter and renal function, so the PP46-gel scaffold can be used as an ideal scaffold for constructing long-segment ureter abnormality or defect. In addition, with the development of tissue engineering, the PP-gel scaffold can also be used as a scaffold for other tissues and further applied to the treatment of various organ diseases.
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 (15)
1. A method for preparing a gelatin modified polylactic acid/polylactic acid-polycaprolactone copolymer, which is characterized by comprising the following steps:
(A) Dissolving polylactic acid/polylactic acid-polycaprolactone copolymer with a predetermined proportion to form a homogeneous polymer solution;
(B) Carrying out phase separation and freeze-drying on the polymer solution; and
(C) And (5) grafting gelatin.
2. The method for preparing a gelatin-modified polylactic acid/polylactic acid-polycaprolactone copolymer according to claim 1, wherein the step (B) comprises the steps of: (B1) Pouring the polymer solution in step (a) into a preheated mold; (B2) phase separation is carried out by adopting a thermally induced phase separation mode; and (B3) placing the polymer solution and the die in a preset temperature, and freeze-drying to obtain the polylactic acid/polylactic acid-polycaprolactone copolymer product.
3. The method for preparing a gelatin-modified polylactic acid/polylactic acid-polycaprolactone copolymer according to claim 2, wherein in the step (C), gelatin is immobilized to the polylactic acid/polylactic acid-polycaprolactone copolymer by ammonolysis and Glutaraldehyde (GA) crosslinking to obtain a gelatin-modified polylactic acid/polylactic acid-polycaprolactone copolymer product.
4. The method for producing a gelatin-modified polylactic acid/polylactic acid-polycaprolactone copolymer according to claim 3, wherein in step (B3), the polymer solution and the mold are placed in-80 ℃ for 24 hours, and then lyophilized for 72 hours, to obtain a polylactic acid/polylactic acid-polycaprolactone copolymer product.
5. The method for producing a gelatin-modified polylactic acid/polylactic acid-polycaprolactone copolymer according to any one of claims 1 to 4, wherein polylactic acid and polylactic acid-polycaprolactone copolymer are each dissolved in 1, 4-dioxane at a mass ratio of a predetermined ratio in step (a) to form a homogeneous polymer solution having a concentration of 10% (w/v).
6. The method for preparing a gelatin-modified polylactic acid/polylactic acid-polycaprolactone copolymer according to claim 5, wherein the step (C) comprises the steps of: (C1) The polylactic acid/polylactic acid-polycaprolactone copolymer product is soaked in 95% alcohol for 1h, then dried in a vacuum oven, further soaked in 0.06g/ml of 1, 6-hexamethylenediamine/isopropanol solution for 10min, and then washed with a large amount of water to remove free hexamethylenediamine, thus obtaining the impurity-removed bracket; (C2) Soaking the ammonolysis polylactic acid/polylactic acid-polycaprolactone copolymer in 1.0wt% GA aqueous solution for 3 hours at room temperature, and then washing with a large amount of water to remove free GA to obtain an aldehyde bracket; (C3) The aldehyde bracket is cultured for 24 hours in a gelatin/PBS solution with the weight percent of 1.0 at room temperature, and then ungrafted gelatin is washed off by water, so as to obtain the polylactic acid/polylactic acid-polycaprolactone copolymer modified by gelatin.
7. The method for producing a gelatin-modified polylactic acid/polylactic acid-polycaprolactone copolymer according to claim 6, wherein the mass ratio of polylactic acid to polylactic acid-polycaprolactone copolymer in step (a) is 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1.
8. A gelatin modified polylactic acid/polylactic acid-polycaprolactone copolymer comprising:
a polymer formed by polylactic acid and polylactic acid-polycaprolactone copolymer, wherein the polymer is provided with at least one ester group and is suitable for decomposing an external group; and
Gelatin grafted onto a product formed by polylactic acid and a polylactic acid-polycaprolactone copolymer under preset conditions, wherein the mass ratio of the polylactic acid to the polylactic acid-polycaprolactone ranges from 1:9 to 9:1, a plurality of pores are formed after the gelatin is grafted, and the pores are distributed in a polymer main body and are distributed more densely than inside the surface of the main body.
9. The gelatin-modified polylactic acid-polycaprolactone copolymer according to claim 8, wherein the mass ratio of polylactic acid to polylactic acid-polycaprolactone is 4:6.
10. The gelatin-modified polylactic acid/polylactic acid-polycaprolactone copolymer according to claim 8 or 9, prepared by the method of claims 1 to 7.
11. The application of the gelatin modified polylactic acid-polycaprolactone copolymer is characterized in that the gelatin modified polylactic acid-polycaprolactone copolymer is suitable for being applied to human tissue organs as a biological scaffold.
12. Use of the gelatin-modified polylactic acid-polycaprolactone copolymer according to claim 11, wherein the gelatin-modified polylactic acid-polycaprolactone copolymer (PP-gel) is used to produce a clinically desirable ureteral stent, intestinal stent, esophageal stent, urethral stent or vascular stent by means of a ureteral stent mould, an intestinal stent mould, an esophageal stent mould, a urethral stent mould or a vascular stent mould.
13. Use of the gelatin-modified polylactic acid-polycaprolactone copolymer according to claim 12, wherein the mass ratio of polylactic acid to polylactic acid-polycaprolactone ranges from 1:9 to 9:1.
14. Use of a gelatin-modified polylactic acid-polycaprolactone copolymer according to any of claims 11 to 13, wherein the biological scaffold is a ureteral scaffold and the ureteral scaffold is formed with a plurality of pores, the pores being arranged denser than the inside at the surface of the scaffold body to facilitate epithelial layer growth.
15. Use of a gelatin-modified polylactic acid-polycaprolactone copolymer according to claim 14, wherein the bioscaffold is prepared by the method of any one of claims 1 to 7.
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