CN115414535A - Preparation method and application of exosome-modified tea polyphenol-enhanced tissue repair scaffold - Google Patents
Preparation method and application of exosome-modified tea polyphenol-enhanced tissue repair scaffold Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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Abstract
The invention provides a preparation method and application of an exosome-modified tea polyphenol-enhanced tissue repair scaffold. The preparation method comprises the following steps: step 1, dissolving a polymer matrix material in an aqueous solution, adding a photoinitiator I2959, and then processing and molding the obtained slurry by using a 3D printing technology to obtain a porous repair support; step 2, soaking the repair scaffold in a polylysine solution for 12-24 hours to obtain the repair scaffold with a grafted adhesion layer; step 3, carrying out surface grafting modification on the repair support with the grafted adhesion layer by using tea polyphenol to obtain a tea polyphenol reinforced repair support; and 4, taking the stem cell exosome suspension, and soaking the tea polyphenol reinforced scaffold in the stem cell exosome suspension for 12-24 hours to obtain the exosome modified tea polyphenol reinforced repair scaffold. The invention has the advantages of good biological safety, no introduction of exogenous cells and excellent processing performance.
Description
Technical Field
The invention relates to a preparation method and application of an exosome-modified tea polyphenol-enhanced tissue repair scaffold.
Background
The penis is a differentiated organ at the tail end of a human body, and once the penis is damaged, the penis cannot be repaired by itself, so that the natural fertility and other normal physiological functions are influenced. The corpus cavernosum tissue is an important component of the penis, and the healthy and intact corpus cavernosum of the penis is a prerequisite for maintaining the normal erection function and natural reproduction of the penis. Various congenital male external genitalia dysplasia, penis cancer, penis defect caused by perineum trauma, vascular erectile dysfunction and the like all need to be subjected to extensive penis reconstruction surgery to repair the structural and functional defects of the penis.
The current clinical treatment scheme aiming at the cavernous defect is limited. Although there are some successful cases of human allogeneic penis transplantation, penis transplantation is still in the starting stage, there are many ethical disputes, and the limited sources of the cavernous body grafts of penis, the potential immunogenicity and the like become the biggest bottleneck of the success of the operation. Tissue engineered cavernous stents are the most desirable biomaterials for use in penile reconstructive surgery.
The rapid development of stem cell technology enables exosome to be easily extracted from one or more of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells and myogenic stem cells. Because it contains no genetic component and has lower immunogenicity, it can avoid immunological rejection reaction, so that it has excellent biological safety. The outer portion of the exosome is coated with phospholipid bilayers, and the content of the exosome can be protected from degradation, so that degradation failure of certain active ingredients is prevented. In addition, the exosome also has the biological functions of promoting angiogenesis and injury repair.
The rapid development of the 3D printing technology and the maturity of the stem cell exosome extraction technology provide a better solution direction for the sponge to build a proper repair material. Based on the above, the application provides a preparation method of an exosome-modified tea polyphenol enhanced tissue repair scaffold and an application of the exosome-modified tea polyphenol enhanced tissue repair scaffold in preparation of products related to cavernous defect repair.
Disclosure of Invention
The invention aims to provide a preparation method and application of an exosome-modified tea polyphenol-enhanced tissue repair scaffold, which have the advantages of good biological safety, no introduction of exogenous cells and excellent processing performance.
In order to achieve the above object, a first aspect of the present invention provides a method for preparing an exosome-modified tea polyphenol-enhanced tissue repair scaffold, comprising the steps of:
step 1: dissolving a high polymer matrix material in a water solution according to a predetermined ratio, adding a photoinitiator I2959, and processing and molding the obtained slurry by using a 3D printing technology to obtain a porous repair scaffold;
step 2: soaking the repair stent in a polylysine solution for 12-24 hours to obtain the repair stent with a grafted adhesion layer;
wherein the temperature of the lysine solution is kept between 4 and 8 ℃;
and step 3: carrying out surface grafting modification on the repair bracket with the grafted adhesion layer by using tea polyphenol to obtain a tea polyphenol reinforced repair bracket;
and 4, step 4: taking the stem cell exosome suspension, and soaking the tea polyphenol enhanced repair scaffold in the stem cell exosome suspension for 12-24 hours to obtain the exosome modified tea polyphenol enhanced repair scaffold.
According to an embodiment of the present invention, the polymeric matrix material comprises at least one of a methacrylated gelatin, a methacrylated polysaccharide, and a methacrylated polylactic acid.
According to a specific embodiment of the present invention, the stem cell exosome comprises at least one of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, myogenic stem cells.
According to an embodiment of the present invention, the lysine solution in step 2 has a mass fraction of 2%.
According to a specific embodiment of the invention, the mass fraction of the stem cell exosome suspension in the step 4 is 0.5-3%.
The second aspect of the invention is to provide an application of the tissue repair scaffold prepared by the preparation method of the exosome-modified tea polyphenol-enhanced tissue repair scaffold in preparation of products related to cavernous defect repair.
The invention has the following beneficial effects:
the exosome-modified tea polyphenol-enhanced tissue repair scaffold prepared by the invention has the advantages of good biological safety, no introduction of exogenous cells and excellent processing performance, can realize personalized customization according to the requirements of different patients, and can be used for repairing the cavernous body injury of the penis and reconstructing the reproductive function caused by trauma, operation and the like.
In the invention, the porous repairing bracket is subjected to exosome-modified tea polyphenol enhancement, so that the porosity and the mechanical property can be improved, the improvement of the porosity is favorable for better attaching bioactive factor components to the tissue repairing bracket, and the optimization of the mechanical property is favorable for adapting to the change of biomechanics.
The present invention will be described in further detail with reference to the accompanying drawings.
Drawings
FIG. 1 is a microscopic view of a repair scaffold obtained in a comparative example of the present invention;
FIG. 2 is a microscopic view of the exosome-modified tea polyphenol-enhanced tissue repair scaffold obtained in example 1 of the present invention;
FIG. 3 is an electron microscope image of the exosome-modified tea polyphenol-enhanced tissue repair scaffold obtained in example 1 of the present invention.
Detailed Description
In order that the above objects, features and advantages of the present invention can be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as specifically described herein and, therefore, the scope of the present invention is not limited by the specific embodiments disclosed below.
In view of the complex tissue structure of the corpus cavernosum, the traditional penile repair scheme usually realizes appearance repair through skin flap transplantation or realizes erectile function repair through prosthesis implantation, but does not recover the autonomous erectile function of the patient; the invention realizes the reconstruction of the cavernous body structure through the modification of exosome and the enhancement of tea polyphenol, recovers the erection function of the penis to a certain degree and brings good prognosis effect for patients.
Example 1
The embodiment provides a preparation method of an exosome-modified tea polyphenol enhanced tissue repair scaffold, which comprises the following steps:
step 1: dissolving a high polymer matrix material in an aqueous solution according to a predetermined proportion, adding a photoinitiator I2959, and fully and uniformly dissolving to obtain a slurry; then processing and molding the slurry by using a 3D printing technology to obtain a porous hydrogel repairing support;
wherein the polymer matrix material comprises at least one of methyl propylene gelatin, methyl propylene polysaccharide and methyl propylene polylactic acid;
step 2: soaking a repair stent in a polylysine solution with the mass fraction of 2% for 12-24 hours, for example, for 16 hours, so as to obtain the repair stent with a grafted adhesion layer;
wherein the lysine solution is maintained at a temperature of 4 ℃ to 8 ℃, e.g., 6 ℃;
and step 3: carrying out surface grafting modification on the repair bracket with the grafted adhesion layer by using tea polyphenol to obtain a tea polyphenol reinforced repair bracket;
and (3) the tea polyphenol has a polyhydroxy structure, the repair support with the grafted adhesion layer obtained in the step (2) is soaked in a tea polyphenol solution with the weight ratio of 2%, and the surface grafting modification of the porous repair support by the tea polyphenol can be realized through the electrostatic adsorption effect.
Wherein the tea polyphenols can be extracted and separated from tea by solvent extraction, ion precipitation, column separation, etc.
And 4, step 4: taking a stem cell exosome suspension with the mass fraction of 3%, and soaking the tea polyphenol enhanced scaffold in the stem cell exosome suspension for 12-24 hours, for example, for 16 hours to obtain the exosome modified tea polyphenol enhanced repair scaffold.
In the invention, the exosome-modified tea polyphenol enhanced repair scaffold comprises the following components in percentage by mass:
in a specific embodiment, the exosome-modified tea polyphenol enhanced repair scaffold may comprise the following components in percentage by mass:
comparative example
The comparative example differs from example 1 in that: the following steps are performed after step 1: and (3) soaking the porous repairing scaffold obtained in the step (1) in an epsilon-polylysine solution and a heparin sodium solution in sequence, repeating for many times, and combining by utilizing the action of ionic charges to realize layer-by-layer self-assembly to obtain the repairing scaffold with a multilayer polylysine and heparin surface modification structure. Step 4 was then performed directly to obtain an exosome-modified repair scaffold.
As can be seen from the microscopic view shown in FIG. 1, the porosity of the repair stent obtained in the comparative example is low, between 30% and 35%, and the microstructure required by the penis cannot be achieved; meanwhile, the repair stent only increases the strain of 20 to 25 percent of the length of the repair stent under the action of the external stress of 0.05 to 0.1Mpa, and the repair stent can be fractured after the external stress of 0.1Mpa is exceeded; therefore, the mechanical strength of the prosthetic stent cannot be well matched with the mechanical properties required for penile erection.
As can be seen from the microscopic image shown in fig. 2 and the electron microscope image shown in fig. 3, the porosity of the exosome-modified tea polyphenol-enhanced repair scaffold prepared in the embodiment is obviously improved, the porosity can reach 50% -60%, and the improvement of the porosity is beneficial to better attaching bioactive factor components to the tissue repair scaffold and better exerting the biological function of the tissue repair scaffold; the mechanical property of the exosome-modified tea polyphenol-enhanced repair scaffold prepared by the embodiment is obviously improved, strain exceeding 100% -110% of the length of the exosome-modified tea polyphenol-enhanced repair scaffold can be generated under the action of 0.4-0.5Mpa of external stress, the optimization of the mechanical property is more beneficial to adapting to the change of biomechanics, and the repair of damage of a damaged cavernous body and the recovery of reproductive function are facilitated.
In order to further verify the effect of the exosome-modified tea polyphenol enhanced repair scaffold prepared in the embodiment, the inventor adopts experimental white rabbits for test and verification, and the specific process is as follows:
selecting a mild New Zealand white rabbit, anesthetizing the rabbit with 3% pentobarbital sodium according to the dosage of 1ml/kg, preparing skin conventionally, disinfecting, paving, winding the root of the rabbit cavernous body with a rubber ring, clamping the rubber ring with an artery clamp, making a 5mm longitudinal incision on the right penis cavernous body, cutting open the white membrane, exposing the cavernous body, removing part of cavernous body tissues, and quickly transplanting the exosome-modified tea polyphenol-enhanced cavernous body repair scaffold to the defect part of the penis cavernous body and suturing.
The same cavernous body injury model was established, without the placement of the repair material, with direct suturing of the tunica albuginea, fascia, and skin, as a control group.
After 1 month of operation, the male rabbit and the normal female rabbit after the cavernous body repair are in the same cage, and the dates of the same cage and the new-born rabbit are recorded; and 2 months after the operation, the blood vessel repair condition of the cavernous body of the penis of the rabbit is detected by adopting MRI and blood vessel Doppler ultrasound, and 4 months after the operation, the recovery condition of the erectile function is detected by adopting ICP and the like.
As a result: after the experimental group male rabbits and the normal female rabbits are in the same cage for 2 months, the newly born rabbits are born, and after the control group male rabbits and the normal female rabbits are in the same cage for 2 months, the rabbits are not born, which shows that the exosome-modified cavernous body repairing bracket can promote the damage repair of the cavernous body and can recover the normal mating function of the rabbits to realize normal reproduction.
In conclusion, the exosome-modified tea polyphenol-enhanced repair scaffold prepared in the embodiment has customizability, and the cavernous body defect repair model is obtained before 3D printing, so that the repair scaffold matched with the cavernous body defect repair model is manufactured for the cavernous body defect repair model, the application in preparation of products related to cavernous body defect repair is realized, and finally, the repair and function regeneration of cavernous body defects are realized.
Although the present invention has been described with reference to the preferred embodiments, it is not intended to limit the scope of the invention. It will be appreciated by those skilled in the art that changes may be made without departing from the scope of the invention, and it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
Claims (6)
1. A preparation method of an exosome-modified tea polyphenol enhanced tissue repair scaffold comprises the following steps:
step 1: dissolving a high polymer matrix material in a water solution according to a predetermined ratio, adding a photoinitiator I2959, and processing and molding the obtained slurry by using a 3D printing technology to obtain a porous repair scaffold;
step 2: taking a polylysine solution, and soaking the repair stent in the polylysine solution for 12-24 hours to obtain the repair stent with the grafted adhesion layer;
wherein the temperature of the lysine solution is kept between 4 and 8 ℃;
and step 3: carrying out surface grafting modification on the repair bracket with the grafted adhesion layer by using tea polyphenol to obtain a tea polyphenol reinforced repair bracket;
and 4, step 4: taking the stem cell exosome suspension, and soaking the tea polyphenol enhanced repair scaffold in the stem cell exosome suspension for 12-24 hours to obtain the exosome modified tea polyphenol enhanced repair scaffold.
2. The method of preparing an exosome-modified tea polyphenol-enhanced tissue repair scaffold according to claim 1, wherein the polymeric matrix material comprises at least one of a methacrylated gelatin, a methacrylated polysaccharide, a methacrylated polylactic acid.
3. The method of preparing an exosome-modified tea polyphenol-enhanced tissue repair scaffold according to claim 1, wherein the stem cell exosome comprises at least one of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, myogenic stem cells.
4. The method of preparing an exosome-modified tea polyphenol-enhanced tissue repair scaffold according to claim 1, wherein the mass fraction of the lysine solution in step 2 is 2%.
5. The method for preparing an exosome-modified tea polyphenol-enhanced tissue repair scaffold according to claim 1, wherein the mass fraction of the stem cell exosome suspension in the step 4 is 0.5-3%.
6. Use of the tissue repair scaffold prepared by the method for preparing an exosome-modified tea polyphenol-enhanced tissue repair scaffold according to any one of claims 1 to 5 in the preparation of a preparation related to cavernous defect repair.
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| CN115957378A (en) * | 2023-02-08 | 2023-04-14 | 上海交通大学医学院附属瑞金医院 | Bone repair composition and preparation method and application thereof |
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| CN110680960A (en) * | 2018-07-04 | 2020-01-14 | 苏州纳晶医药技术有限公司 | Controllable gradient degradation spiral tectorial membrane stent, preparation method and application thereof |
| CN114601968A (en) * | 2022-03-30 | 2022-06-10 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Repair method for promoting rapid vascularization of damaged cavernous body |
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