CN110721344A - Injectable biological gel for promoting myocardial repair and preparation method thereof - Google Patents
Injectable biological gel for promoting myocardial repair and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an injectable biological gel for promoting myocardial repair, which comprises omentum acellular matrix solution, chitosan derivative solution and 1, 4-butanediol diglycidyl ether solution.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to an injectable biological gel for promoting myocardial repair and a preparation method thereof.
Background
Myocardial infarction is a common type of ischemic heart disease, myocardial cells in a central area of a lesion die due to oxygen deficiency within minutes after acute attack of coronary artery occlusion, myocardial cells remaining at an ischemic edge are gradually apoptotic due to insufficient blood supply and oxygen supply, an infarcted area is gradually enlarged, the wall thickness of the infarcted area becomes thin, a left ventricle is enlarged, the diastolic end pressure of the left ventricle is increased, the cardiac function is continuously reduced, and finally congestive heart failure is caused. As a world population, China is large, and along with the aging of social population, the life threat of myocardial infarction to the public in China is increasingly severe.
The biological material transplanted into the damaged cardiac muscle has certain effect on controlling the microenvironment of the myocardial infarction area and improving the cardiac function. For example, Kofidis et al demonstrate that injection of Matrigel gel into infarcted myocardium can restore damaged myocardium without distorting the geometry of the heart. Christman et al injected fibrin glue into a rat myocardial infarction model and showed that it had protective effects on the ventricular wall and cardiac function after myocardial infarction.
In recent years, the emergence of organ and tissue decellularization strategies has opened up new approaches for regenerative medicine research. The acellular matrix scaffold material is beneficial to the adhesion, proliferation, differentiation and other biological functions of cells because the acellular matrix scaffold material reserves the natural components and three-dimensional structures of the extracellular matrix, such as type I collagen, type III collagen, type IV collagen, fibronectin, laminin, glycosaminoglycan (GAGs) and the like; furthermore, because of the relative conservation of extracellular matrix components among different species, acellular matrix scaffold materials have little or no immunological rejection, and, therefore, acellular matrices have incomparable advantages over other biomaterials in tissue repair and regeneration. At present, researchers have successfully obtained acellular matrix materials of various tissue and organ sources through acellular technology. The greater omentum is one of the peritoneum, a layer of mucous membrane present in the abdominal cavity of higher animals, and a membranous tissue formed by connective tissue. The greater omentum is the peritoneum that connects the greater curvature of the stomach to the transverse colon. The omentum majus is rich in extremely strong elasticity and a highly vascularized structure, and has wide application prospect in the fields of tissue engineering and regenerative medicine due to the advantages. However, there are no reports on the use of a retinal acellular matrix to create an injectable biogel for promoting myocardial repair. On the other hand, chitosan has obvious antioxidation and important function for eliminating a large amount of active oxygen free radicals in ischemic myocardium. At present, no report related to the establishment of injectable biological gel for promoting myocardial repair by compounding chitosan and omentum acellular matrix exists.
Disclosure of Invention
The present application aims to overcome the above problems or at least partially solve or alleviate the above problems, and the present invention discloses an injectable bio-gel prepared by the method for preparing an injectable bio-gel, which can effectively promote the repair of damaged cardiac muscle after myocardial infarction, reduce fibrosis, and significantly improve cardiac function.
According to a first aspect of the present application, there is provided an injectable biogel for promoting myocardial repair, characterised in that: the injectable biogel comprises a omentum acellular matrix solution, a chitosan derivative solution and a 1, 4-butanediol diglycidyl ether solution.
Compared with the prior art, the technical scheme disclosed by the invention has the advantages that on one hand, the omentum acellular matrix contains various cytokines which are beneficial to tissue repair and regeneration, and on the other hand, the chitosan derivative can effectively remove active oxygen free radicals at the myocardial infarction part, so that the injectable biogel disclosed by the invention can effectively promote the repair of damaged cardiac muscle after myocardial infarction, reduce fibrosis and obviously improve cardiac function.
According to a second aspect of the present application, there is provided a method of preparing an injectable biogel for promoting myocardial repair, characterized by the steps of:
soaking omentum majus in buffer solution containing sodium dodecyl sulfate and DNA enzyme for decellularization, and then digesting the decellularized omentum tissue by combining combined digestive enzyme solution and a digestive bottle with a rotor to obtain a omentum decellularized matrix;
mixing a chitosan derivative aqueous solution and a beta-sodium glycerophosphate aqueous solution according to the volume ratio of 1 (0.1-0.2) to obtain a chitosan derivative solution;
mixing omentum acellular matrix solution, chitosan derivative solution and 1, 4-butanediol diglycidyl ether according to the volume ratio of 1: (0.1-0.5) and (0.01-0.05) to obtain the injectable biological gel for promoting myocardial repair.
Compared with the prior art, the preparation method of the injectable biological gel for promoting myocardial repair provided by the invention has the same beneficial effect as that of the injectable biological gel for promoting myocardial repair in the technical scheme, and the detailed description is omitted here.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. Some specific embodiments of the present application will be described in detail hereinafter by way of illustration and not limitation with reference to the accompanying drawings. The same reference numbers in the drawings identify the same or similar elements or components. Those skilled in the art will appreciate that the drawings are not necessarily drawn to scale. In the drawings:
FIG. 1 is a flow chart of a method of preparing an injectable biogel for promoting myocardial repair;
FIG. 2 is an appearance of an injectable biogel for promoting myocardial repair;
FIG. 3 is a photograph of echocardiography (horizontal short axis section of mitral valve) of heart function of rats under microscope for experimental group and control group;
FIG. 4 is a photograph showing the degree of fibrosis in the myocardial infarction region observed under a microscope by Masson trichrome staining of specimens of the experimental group and the control group.
Detailed Description
In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The following further describes a method for preparing an injectable bio-gel for promoting myocardial repair provided by the present application.
In the present application, the abbreviations have the following meanings:
SDS (sodium dodecyl sulfate): sodium lauryl sulfate;
PBS: phosphate buffer;
DNA enzyme: deoxyribonuclease;
mrFP: a red fluorescent protein;
min: and (3) minutes.
The invention discloses an injectable biological gel for promoting myocardial repair, which comprises three solutions of omentum acellular matrix, chitosan and 1, 4-butanediol diglycidyl ether, or consists of three solutions of the omentum acellular matrix, chitosan derivative and 1, 4-butanediol diglycidyl ether. Wherein the omentum acellular matrix is omentum acellular matrix solution with the concentration of 10-20 mg/mL; the chitosan derivative solution is a chitosan solution with the chitosan derivative content of 1-3% mass concentration, which is obtained by mixing chitosan and a beta-sodium Glycerophosphate (GP) water solution according to the proportion of 1 (0.1-0.2), and the 1, 4-butanediol diglycidyl ether solution is a solution with the concentration of 0.5-1.0% mass concentration, which is prepared by PBS.
It should be noted that the chitosan derivative may be carboxylated chitosan, and may also be other types of chitosan compounds, such as carboxymethyl chitosan, chitosan quaternary ammonium salt, etc., which is not limited in this respect.
The injectable biogel for promoting myocardial repair is prepared by the following preparation method of the injectable biogel for promoting myocardial repair.
The invention discloses an injectable biological gel for promoting myocardial repair, which has the advantages that on one hand, because omentum acellular matrix contains a plurality of cytokines beneficial to tissue repair and regeneration, and on the other hand, chitosan derivatives can effectively remove active oxygen free radicals at myocardial infarction positions, the injectable biological gel disclosed by the invention can effectively promote the repair of damaged myocardium after myocardial infarction, reduce fibrosis and obviously improve cardiac function, has simple operation process and mild implementation conditions, and has wide application prospect in the treatment of ischemic heart diseases.
According to one aspect of the present invention, there is also provided a method of preparing an injectable biogel for promoting myocardial repair; the method comprises the following steps:
step S01, soaking the omentum majus in a buffer solution containing sodium dodecyl sulfate and DNA enzyme for decellularization, and then digesting the decellularized omentum tissue by combining the combined digestive enzyme solution with a digestive bottle with a rotor to obtain a omentum decellularized matrix;
step S02, mixing the chitosan derivative aqueous solution and the beta-sodium glycerophosphate aqueous solution according to the volume ratio of 1 (0.1-0.2) to obtain a chitosan derivative solution;
step S03, mixing the omentum acellular matrix solution, the chitosan derivative solution and 1, 4-butanediol diglycidyl ether according to the volume ratio of 1: (0.1-0.5) and (0.01-0.05) to obtain the injectable biological gel for promoting myocardial repair.
The preparation of the omentum decellularized matrix comprises the step of soaking omentum majus in a buffer solution containing sodium dodecyl sulfate and DNase to perform decellularization treatment.
Optionally, a shaking operation is further performed in the step of performing the cell removal treatment.
In this embodiment, the preparation of the omentum decellularized matrix further comprises a step of defatting treatment. In an exemplary embodiment, the step of the above-described degreasing treatment is performed before the step of the decellularization treatment. In certain preferred embodiments, the step of degreasing uses a volume ratio of 1: 1: 0.2-1 of mixed solution of methanol, chloroform and diethyl ether, preferably, the volume ratio of the methanol, the chloroform and the diethyl ether can also be 1: 1: 0.4-0.7, preferably, the volume ratio of the methanol, the chloroform and the diethyl ether can also be 1: 1: 0.5.
in certain embodiments, the step of decellularizing is performed in a gradient comprising at least a first gradient and a second gradient.
In certain preferred embodiments, in the first gradient, the concentration of sodium dodecyl sulfate is 1.5-3.5% by weight, the concentration of DNase is 3500-4500U/L, and the pH is 7.2-7.4; preferably, in the first gradient, the weight ratio of the concentration of the sodium dodecyl sulfate is 1.5-2.5%, and the concentration of the DNase is 3600-4400U/L; preferably, in the first gradient, the weight ratio of the concentration of the sodium dodecyl sulfate is 1.8-2.2%, and the concentration of the DNase is 3800-; preferably, in the first gradient, the concentration of sodium dodecyl sulfate is 2.0% by weight and the concentration of DNase is 4000U/L.
In certain preferred embodiments, in the second gradient, the concentration of sodium dodecyl sulfate is 0.6 to 1.5% by weight, the concentration of DNase is 2500-; preferably, in the second gradient, the weight ratio of the concentration of the sodium dodecyl sulfate is 0.7-1.3%, and the concentration of the DNase is 2600-3400U/L; preferably, in the second gradient, the weight ratio of the concentration of the sodium dodecyl sulfate is 0.8-1.2%, and the concentration of the DNase is 2800 and 3200U/L; preferably, in the second gradient, the concentration of sodium dodecyl sulfate is 1.0% by weight and the concentration of DNase is 3000U/L.
In certain preferred embodiments, the gradient further comprises a third gradient, wherein in the third gradient, the concentration of sodium dodecyl sulfate is 0.1 to 0.6% by weight, the concentration of DNase is 1500-; preferably, in the third gradient, the weight ratio of the concentration of the sodium dodecyl sulfate is 0.2-0.6%, and the concentration of the DNase is 1600-2400U/L; preferably, in the third gradient, the weight ratio of the concentration of the sodium dodecyl sulfate is 0.4-0.6%, and the concentration of the DNase is 1800-2200U/L; preferably, in the third gradient, the concentration of sodium dodecyl sulfate is 0.5% by weight and the concentration of DNase is 2000U/L.
According to another aspect of the present invention, there is provided a method for preparing an injectable biogel for promoting myocardial repair, characterized by the following specific operating steps:
1. preparation of degreasing solution
Methanol, chloroform and diethyl ether are mixed in a volume ratio of 1: 1: mixing at a ratio of 0.2-1;
2. preparation of cell-removing liquid
1) Cell removal liquid I: weighing sodium dodecyl sulfate and DNase, dissolving in PBS, wherein the weight ratio of the concentration of the sodium dodecyl sulfate in the PBS is 1.5-3.5%, the concentration of the DNase in a phosphate buffer solution is 3500-4500U/L, the pH is 7.2-7.4, and filtering for sterilization;
2) and (3) cell removal liquid II: weighing sodium dodecyl sulfate and DNase, dissolving in PBS, wherein the weight ratio of the concentration of the sodium dodecyl sulfate in the PBS is 0.6-1.5%, the concentration of the DNase in the PBS is 2500-3500U/L, the pH is 7.2-7.4, and filtering for sterilization;
3) and (3) removing cell sap III: weighing sodium dodecyl sulfate and DNase, dissolving in PBS, wherein the weight ratio of the concentration of the sodium dodecyl sulfate in the PBS is 0.1-0.6%, the concentration of the DNase in the PBS is 1500-;
3. preparation of omentum decellularized matrix digestive juice
Mixing pancreatin, collagenase and protease dispaseII in a mass ratio of 2: 2: 1-2, wherein the concentrations of pancreatin, collagenase and protease dispaseII in the PBS solution are all 0.05-0.25%, and after complete dissolution, the pH is regulated to 7.2-7.4 to obtain the omentum acellular matrix digestive juice.
4. Omentum majus decellularization
1) Cleaning fresh omentum majus tissue in PBS, and treating the cleaned tissue by shaking and soaking in a degreasing solution, wherein the degreasing solution soaking time is 18-30h, and the shaking frequency is 150-;
2) cleaning the fat-removed omentum majus tissue in PBS, and treating the omentum majus tissue in a cell removal solution I by shaking and soaking for 4-12h at the shaking frequency of 100-;
3) cleaning the omentum majus tissue treated by the cell removal liquid I in PBS, and treating the omentum majus tissue in the cell removal liquid II by shaking and soaking for 4-12h at the shaking frequency of 100-;
4) cleaning the omentum majus tissue treated by the cell removal liquid II in PBS, and treating the omentum majus tissue by shaking and soaking the omentum majus tissue in the cell removal liquid III, wherein the soaking time is 4-12h, and the shaking frequency is 100-;
5) the omentum decellularized matrix was obtained by washing with PBS.
5. Preparation of omentum acellular matrix
Adding the cleaned and cut omentum acellular matrix into a digestion bottle containing the digestion solution, adding 10-20 g of omentum acellular matrix into every 100ml of the digestion solution, covering the membrane, putting the membrane on a magnetic stirrer for stirring at the rotating speed of 60-100rpm, digesting for 30-45min in an incubator with 5% CO2 at 37 ℃ and under the rotating speed of 60-100rpm (the digestion time is properly adjusted according to the concentration of the digestive enzyme), and then stopping the digestion reaction by using serum, wherein the volume ratio of the serum to the digestion solution is 1-2: 10. collecting digestive juice to obtain liquid omentum acellular matrix biogel, and adjusting the concentration of the omentum acellular matrix to 10-20 mg/mL.
6. Preparation of injectable biogel for promoting myocardial repair
Mixing the omentum acellular matrix, chitosan and 1, 4-butanediol diglycidyl ether solution according to the weight ratio of 1: (0.1-0.5): (0.01-0.05) by volume.
In certain embodiments, the fresh omentum majus tissue is derived from a mammal such as a pig, cow, sheep, or the like.
In the present application, macroreticular tissue is commercially available, for example from slaughter houses.
The following will explain details of the present invention by specific examples.
The formulation of the reagents required for the decellularization of the omentum majus and their identification used in the following examples:
PBS: weighing 8g NaCl, 0.2g KCl, 3.491g Na2HPO4·12H2O，0.2g KH2PO4Dissolving in 1L ultrapure water, adjusting pH to 7.2-7.4, sterilizing with high pressure steam at 121 deg.C for 20min, and storing at 4 deg.C.
2) Degreasing solution: 400mL of methanol, 400mL of chloroform and 200mL of diethyl ether were mixed well.
3) Cell removal liquid I: weighing 20g SDS and dissolving in PBS, adding DNase and fixing in 1L to make the DNase concentration reach 4000U/L, adjusting pH to 7.2-7.4, filtering and sterilizing, and storing at normal temperature.
4) And (3) cell removal liquid II: weighing 10g SDS and dissolving in PBS, adding DNase and fixing in 1L to make the DNase concentration reach 3000U/L, adjusting pH to 7.2-7.4, filtering and sterilizing, and storing at normal temperature.
5) And (3) removing cell sap III: weighing 5g SDS and dissolving in PBS, adding DNase and fixing in 1L to make the DNase concentration reach 2000U/L, adjusting pH to 7.2-7.4, filtering and sterilizing, and storing at normal temperature.
6) Omentum decellularized matrix digestive juice: dissolving pancreatin 0.05 g, collagenase 0.1g and Dispase II 0.1g in 100ml PBS solution, adjusting pH value to 7.2-7.4, and filtering and sterilizing through a 0.22 mu m microporous membrane. It is prepared before use and is used for digesting the omentum acellular matrix.
7) A digestion bottle: a200 mL glass vial having a cap and a magnetic stirrer rotor (diameter. times. length: 6. times.20 mm) was placed inside the vial.
Step 1 decellularization of omentum
Fresh porcine peritoneal macroreticular tissue (purchased from a slaughterhouse) was washed 3 times in PBS. And (3) shaking and soaking the cleaned tissue in 2L of degreasing solution for treatment for 24h, wherein the shaking frequency is 200 r/min. Then, the fat-removed omentum majus tissue is washed in PBS for 3 times, and is treated for 8 hours by shaking and soaking in 1L of cell removal liquid I, and the shaking frequency is 150 r/min. Subsequently, the omentum majus tissue treated by the cell removal fluid I is washed in PBS for 3 times, and is soaked in 1L of cell removal fluid II for treatment for 8 hours with the oscillation frequency of 150 r/min. Subsequently, the omentum majus tissue treated by the cell removal fluid II is washed in PBS for 3 times, and is soaked in 1L of cell removal fluid III in a shaking way for 8 hours, and the shaking frequency is 150 r/min. Finally, the omentum majus tissue treated by the cell removal fluid III is washed by PBS and stored at 4 ℃.
Step 2 preparation of omentum acellular matrix
Adding 10g of the cleaned and cut omentum acellular matrix into a digestion bottle containing the digestion solution, then adding 100mL of the digestion solution, covering the digestion solution, putting the digestion bottle on a magnetic stirrer, stirring the digestion solution at the rotating speed of 100rpm, putting the magnetic stirrer and the digestion bottle together into an incubator at 37 ℃, continuously digesting for 30 to 45min, and then adding 2mL of serum to stop the digestion reaction, wherein the volume ratio of the serum to the digestion solution is 0.1 to 0.2: 10. collecting digestive juice to obtain liquid omentum acellular matrix biogel. The concentration of the omentum acellular matrix was adjusted to 20 mg/mL.
Step 3 preparation of injectable biogel for promoting myocardial repair
Mixing a chitosan water solution and a beta-sodium Glycerophosphate (GP) water solution according to a ratio of 1:0.2 to obtain a chitosan solution; preparing 1.0% 1, 4-butanediol diglycidyl ether (BDDE) solution by using PBS solution; mixing the 20mg/mL omentum acellular matrix solution, the chitosan solution and 1, 4-butanediol diglycidyl ether according to the weight ratio of 1: the mixture is mixed evenly according to the volume ratio of 0.5: 0.05.
Step 4 preparation of rat myocardial infarction model
SD rats were anesthetized with pentobarbital sodium and connected to a small animal ventilator. Thoracotomy was performed, the pericardium was cut open, and the heart was exposed. And (3) sewing and binding the lower edge of the left auricle by using a 0-7 number silk thread at a position of 2 mm. After ligation, the left ventricular wall becomes pale and the ventricular wall motion is weakened. The ST segment of I, II lead is obviously raised in the electrocardiographic monitoring, which shows that the preparation of the coronary infarction animal model is successful.
And 5: transplantation of injectable biogel for promoting myocardial repair and cardiac function detection
The SD rats were randomly divided into an injectable bio-gel injection experimental group and a physiological saline injection control group. After 14 days of myocardial infarction, the rat was again thoracically opened for injection transplantation. The chest was opened to expose the heart and multiple injections were made over the macroscopic infarcted area and surrounding healthy myocardium for a total injection of 100 μ L. Cardiac ultrasound examination was performed on the rats of each experimental group 4 weeks after the transplantation, as shown in fig. 3, and the results showed that the cardiac function of the rats of the experimental group injected with the bio-gel was significantly improved compared to the control group injected with the physiological saline (fig. 2). Subsequently, the rats were sacrificed by intraperitoneal injection of pentobarbital sodium (200mg/kg), the hearts were removed by rapid thoracotomy, embedded with OCT, and frozen in frozen sections (5 μm) after 20s of liquid nitrogen snap-freezing. As shown in fig. 4, all the specimens of the experimental group and the control group were stained with collagen fibers (Masson trichrome stain), and the degree of fibrosis in the myocardial infarction region was observed under a microscope, and it was revealed that the degree of fibrosis of the heart of the rats in the experimental group injected with biogel was significantly improved as compared with the control group with physiological saline.
According to the technical scheme disclosed by the invention, on one hand, because the omentum decellularized matrix contains various cytokines which are beneficial to tissue repair and regeneration, and on the other hand, the chitosan derivative can effectively remove active oxygen free radicals at the myocardial infarction part, the injectable biogel disclosed by the invention can effectively promote the repair of damaged cardiac muscle after myocardial infarction, reduce fibrosis and obviously improve the cardiac function, and the injectable biogel has the advantages of simple operation process, mild implementation conditions and wide application prospect in the treatment of ischemic cardiac diseases.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. An injectable biogel for promoting myocardial repair, characterized by: the injectable biogel comprises a omentum acellular matrix solution, a chitosan derivative solution and a 1, 4-butanediol diglycidyl ether solution.
2. The injectable biogel for promoting myocardial repair according to claim 1, which is characterized in that: the volume ratio of the omentum acellular matrix solution to the chitosan derivative solution to the 1, 4-butanediol diglycidyl ether is 1: (0.1-0.5):(0.01-0.05).
3. The injectable biogel for promoting myocardial repair according to claim 1 or 2, which is characterized in that: the concentration of the omentum acellular matrix solution is 10-20 mg/mL; the mass concentration of the chitosan derivative solution is 1-3%; the mass concentration of the 1, 4-butanediol diglycidyl ether solution is 0.5-1.0%.
4. The injectable biogel for promoting myocardial repair according to claim 3, which is characterized in that: the chitosan derivative solution is obtained by mixing a chitosan derivative and a beta-sodium glycerophosphate aqueous solution according to the volume ratio of 1 (0.1-0.2), and the 1, 4-butanediol diglycidyl ether solution is prepared by using a phosphate buffer solution.
5. A method for preparing an injectable biogel for promoting myocardial repair, comprising the steps of:
soaking omentum majus in buffer solution containing sodium dodecyl sulfate and DNA enzyme for decellularization, and then digesting the decellularized omentum tissue by combining combined digestive enzyme solution and a digestive bottle with a rotor to obtain a omentum decellularized matrix;
mixing a chitosan derivative aqueous solution and a beta-sodium glycerophosphate aqueous solution according to the volume ratio of 1 (0.1-0.2) to obtain a chitosan derivative solution;
mixing omentum acellular matrix solution, chitosan derivative solution and 1, 4-butanediol diglycidyl ether according to the volume ratio of 1: (0.1-0.5) and (0.01-0.05) to obtain the injectable biological gel for promoting myocardial repair.
6. The method of preparing an injectable biogel for promoting myocardial repair according to claim 5, wherein: the step of obtaining a omentum decellularized matrix further comprises a step of defatting treatment, wherein the step of defatting treatment is performed before the step of decellularizing the omentum majus.
7. The method of preparing an injectable biogel for promoting myocardial repair according to claim 6, wherein: the step of degreasing treatment is carried out by using methanol, chloroform and diethyl ether in a volume ratio of 1: 1: treating with 0.2-1 mixed solution.
8. The method of preparing an injectable biogel for promoting myocardial repair according to claim 5, wherein: the step of soaking the omentum majus for decellularization is performed by a graded treatment.
9. The method of preparing an injectable biogel for promoting myocardial repair according to claim 8, wherein: the gradient comprises a first gradient, a second gradient and a third gradient;
in the first gradient, treatment is carried out by using the weight ratio of the concentration of sodium dodecyl sulfate to 1.5-3.5% and the concentration of DNA enzyme to 3500-4500U/L;
in the second gradient, treatment is carried out by using the weight ratio of the concentration of the sodium dodecyl sulfate to the concentration of 0.6-1.5 percent, the concentration of the DNA enzyme to 2500-3500U/L and the pH value to 7.2-7.4;
in the third gradient, treatment was carried out using a weight ratio of the concentration of sodium dodecyl sulfate of 0.1 to 0.6% and the concentration of DNase of 1500-.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111714698A (en) * | 2020-05-18 | 2020-09-29 | 北京奥泰康医药技术开发有限公司 | Biological gel composition containing cells, preparation method and application thereof |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101288779A (en) * | 2007-04-18 | 2008-10-22 | 中国人民解放军军事医学科学院基础医学研究所 | Syringeability cardiac muscle tissue engineering products based on thermo-sensitive chitosan hydrogel |
| CN101574514A (en) * | 2008-05-06 | 2009-11-11 | 中国人民解放军军事医学科学院基础医学研究所 | Product for treating myocardial infarction based on chitosan aquagel and growth factor |
| WO2009089110A3 (en) * | 2008-01-04 | 2010-03-18 | Boston Scientific Scimed, Inc. | Methods for repair and regeneration of bone marrow |
| CN102038977A (en) * | 2010-11-29 | 2011-05-04 | 中国人民解放军军事医学科学院基础医学研究所 | Tissue engineered cardiac muscle using OPF (Oligo(Poly(Ethylene Glycol) Fumarate) hydrogel as a carrier and preparation method thereof |
| CN103937014A (en) * | 2014-04-10 | 2014-07-23 | 北京爱美客生物科技有限公司 | Chitosan double-network quick response-type injectable hydrogel and preparation method thereof |
| CN104826165A (en) * | 2015-04-21 | 2015-08-12 | 北京帝康医药投资管理有限公司 | Preparation method of acellular bionet and acellular bionet prepared by preparation method |
| CN104888276A (en) * | 2015-05-05 | 2015-09-09 | 北京帝康医药投资管理有限公司 | Cardiac muscle tissue engineering product and its preparation method and use |
| EP3205349A1 (en) * | 2006-10-02 | 2017-08-16 | Orthocell Limited | A method of producing native components, such as growth factors or extracellular matrix proteins, through cell culturing of tissue samples for tissue repair |
| CN107224617A (en) * | 2017-05-24 | 2017-10-03 | 中国人民解放军第三军医大学 | A kind of hydrogel using spleen cell epimatrix as raw material and preparation method thereof |
| CN107708675A (en) * | 2015-04-17 | 2018-02-16 | 罗查尔工业有限责任公司 | The composition and kit of pseudoplastic behavior microgel matrix |
| US10265439B2 (en) * | 2015-09-03 | 2019-04-23 | Arizona Board Of Regents On Behalf Of Arizona State University | Injectable cell-laden biohybrid hydrogels for cardiac regeneration and related applications |
-
2019
- 2019-11-21 CN CN201911145252.2A patent/CN110721344B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3205349A1 (en) * | 2006-10-02 | 2017-08-16 | Orthocell Limited | A method of producing native components, such as growth factors or extracellular matrix proteins, through cell culturing of tissue samples for tissue repair |
| CN101288779A (en) * | 2007-04-18 | 2008-10-22 | 中国人民解放军军事医学科学院基础医学研究所 | Syringeability cardiac muscle tissue engineering products based on thermo-sensitive chitosan hydrogel |
| WO2009089110A3 (en) * | 2008-01-04 | 2010-03-18 | Boston Scientific Scimed, Inc. | Methods for repair and regeneration of bone marrow |
| CN101574514A (en) * | 2008-05-06 | 2009-11-11 | 中国人民解放军军事医学科学院基础医学研究所 | Product for treating myocardial infarction based on chitosan aquagel and growth factor |
| CN102038977A (en) * | 2010-11-29 | 2011-05-04 | 中国人民解放军军事医学科学院基础医学研究所 | Tissue engineered cardiac muscle using OPF (Oligo(Poly(Ethylene Glycol) Fumarate) hydrogel as a carrier and preparation method thereof |
| CN103937014A (en) * | 2014-04-10 | 2014-07-23 | 北京爱美客生物科技有限公司 | Chitosan double-network quick response-type injectable hydrogel and preparation method thereof |
| CN107708675A (en) * | 2015-04-17 | 2018-02-16 | 罗查尔工业有限责任公司 | The composition and kit of pseudoplastic behavior microgel matrix |
| CN104826165A (en) * | 2015-04-21 | 2015-08-12 | 北京帝康医药投资管理有限公司 | Preparation method of acellular bionet and acellular bionet prepared by preparation method |
| CN104888276A (en) * | 2015-05-05 | 2015-09-09 | 北京帝康医药投资管理有限公司 | Cardiac muscle tissue engineering product and its preparation method and use |
| US10265439B2 (en) * | 2015-09-03 | 2019-04-23 | Arizona Board Of Regents On Behalf Of Arizona State University | Injectable cell-laden biohybrid hydrogels for cardiac regeneration and related applications |
| CN107224617A (en) * | 2017-05-24 | 2017-10-03 | 中国人民解放军第三军医大学 | A kind of hydrogel using spleen cell epimatrix as raw material and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| SONYA B.等: "Safety and efficacy of an injectable extracellular matrix hydrogel for treating myocardial infarction", 《SCIENCE TRANSLATIONAL MEDCINE》 * |
| 姚日生: "《药用高分子材料》", 30 April 2008, 北京：化学工业出版社 * |
| 梁岳龙: "新型生物可降解支架材料的生物学特性研究和支架法联合大网膜修复胆道损伤的动物实验研究", 《中国优秀博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111714698A (en) * | 2020-05-18 | 2020-09-29 | 北京奥泰康医药技术开发有限公司 | Biological gel composition containing cells, preparation method and application thereof |
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