Controllable degradation bionic hydrogel and application thereof

Abstract

The invention discloses a biomimetic hydrogel capable of being controllably degraded and an application of the biomimetic hydrogel. Belongs to the technical field of medical materials. The controllable degradation of the hydrogel is successfully realized by precisely regulating and controlling the composition proportion of the low-viscosity sodium alginate, the high-viscosity sodium alginate and the oxidized high-viscosity sodium alginate in the mixed sodium alginate. The biomimetic artificial organ is constructed by mixing the material with an acellular matrix, gelatin and PBS, so that controllable degradation of the organ is realized, and the problems of cell loss and function impairment caused by the fact that the artificial organ loses structural support due to too fast degradation of the material are effectively avoided; the problems of inflammation and foreign body reaction caused by too slow degradation of the material are also avoided; the structural stability and the function durability of the artificial organ are improved. In addition, the bionic hydrogel which is subjected to bionic design and research and development is suitable for survival of organ cells, the condition of liver injury of mice with hepatic fibrosis is relieved, the lifetime is prolonged, and the hydrogel has artificial liver transplantation treatment potential.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and more particularly to a controllably degradable bionic hydrogel and application thereof. Background Art

[0002] Sodium Alginate is a natural polysaccharide extracted from brown algae. Due to its good biocompatibility, gelling and functionality, it is widely used in many fields such as food industry, medicine and biomedicine, cosmetics and personal care, industrial field, and agricultural environmental protection field. In the fields of medicine and biomedicine, sodium alginate can be used as a carrier material for drug sustained-release systems. By controlling the degradation rate of sodium alginate gel, the drug can be released at different rates. It can be used to construct artificial organs or tissue engineering scaffolds to provide a growth environment for cells. It can be used for cell encapsulation to protect them from immune rejection. However, the degradation rate of sodium alginate is affected by many factors (such as environmental pH, the presence of enzymes, etc.). This uncontrollability may affect its application effect in drug sustained-release systems, artificial organ construction or tissue engineering.

[0003] Especially in the process of artificial organ construction, due to the uncontrollable degradation of hydrogels, the following technical problems often exist: (1) Uncontrollable material degradation: After transplantation, artificial organs often lose their structural support due to rapid material degradation, resulting in the loss of organ cells and impaired function; (2) Slow material degradation: If the material remains in the body for a long time, it may cause chronic inflammation and foreign body reaction, and even interfere with the normal organ function of the host; (3) Limited functional duration: Since the survival and functional expression of cells in the artificial matrix are limited by the characteristics of the material, most artificial organs find it difficult to achieve long-term biological functions.

[0004] Therefore, how to provide a controllably degradable bionic hydrogel and apply it to the construction of artificial organs to improve the stability of the artificial organ structure and the durability of its function is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides a controllable degradable bionic hydrogel and application thereof. By precisely regulating the composition ratio of low-viscosity sodium alginate, high-viscosity sodium alginate and oxidized high-viscosity sodium alginate in the mixed sodium alginate, the controllable degradation of the hydrogel is successfully achieved. Applying it to the construction of artificial organs can significantly improve the structural stability and functional durability of the artificial organs, solve the problem of poor therapeutic effect after artificial organ transplantation, prolong the survival period, and provide technical support for the treatment of various organ diseases.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A controllably degradable bionic hydrogel, comprising mixed sodium alginate; the mixed sodium alginate comprises high-viscosity sodium alginate, low-viscosity sodium alginate and oxidized high-viscosity sodium alginate; The oxidized high-viscosity sodium alginate is prepared by the following method: An oxidant is added to a high-viscosity sodium alginate solution, and the solution is oxidized for 6-12 hours under low temperature, sealed and light-proof conditions. Then, ethylene glycol is added to neutralize excess oxidant, and the solution is precipitated and dried to prepare oxidized high-viscosity sodium alginate.

[0007] Another object of the present invention is to provide: application of the above-mentioned controllably degradable biomimetic hydrogel, wherein the application is in any of the following directions: (1) Application in the preparation of biological scaffold materials; (2) Application in the preparation of drug sustained-release carriers; (3) Application in the preparation of cell encapsulation materials.

[0008] Another object of the present invention is to provide: a controllably degradable biomimetic hydrogel for preparing artificial organs, comprising mixed sodium alginate, decellularized matrix, PBS and gelatin; The mixed sodium alginate includes high-viscosity sodium alginate, low-viscosity sodium alginate and oxidized high-viscosity sodium alginate; The oxidized high-viscosity sodium alginate is prepared by the following method: An oxidant is added to a high-viscosity sodium alginate solution, and the solution is oxidized for 6-12 hours under low temperature, sealed and light-proof conditions. Then, ethylene glycol is added to neutralize excess oxidant, and the solution is precipitated and dried to prepare oxidized high-viscosity sodium alginate.

[0009] Preferably, the concentration of the decellularized matrix is ​​4-8% w / v; the concentration of the gelatin is 8-16% w / v; the concentration of the mixed sodium alginate is 4-5% w / v; and the volume ratio of the decellularized matrix, PBS, gelatin and mixed sodium alginate is 1:1:2:1.

[0010] Preferably, the viscosity of the high-viscosity sodium alginate is in the range of 15-25 cp; and the viscosity of the low-viscosity sodium alginate is in the range of 4-12 cp.

[0011] Preferably, when the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 0-20%, the mass percentage of high-viscosity sodium alginate is 60-100%, and the mass percentage of oxidized high-viscosity sodium alginate is 0-20%, the degradation time of the artificial organ is ≥45d.

[0012] When the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 20-40%, the mass percentage of high-viscosity sodium alginate is 20-60%, and the mass percentage of oxidized high-viscosity sodium alginate is 20-40%, the degradation time of the artificial organ is 15-45 days.

[0013] When the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 40-50%, the mass percentage of high-viscosity sodium alginate is 0-20%, and the mass percentage of oxidized high-viscosity sodium alginate is 40-50%, the degradation time of the artificial organ is 5-15 days.

[0014] Another object of the present invention is to provide: application of the controllably degradable bionic hydrogel for preparing artificial organs in preparing controllably degradable artificial organs.

[0015] Another object of the present invention is to provide: a controllably degradable artificial organ, wherein the artificial organ is prepared by using the controllably degradable bionic hydrogel for preparing the artificial organ.

[0016] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention provides a controllable bionic hydrogel and its application. By precisely controlling the composition ratio of low-viscosity sodium alginate, high-viscosity sodium alginate and oxidized high-viscosity sodium alginate in the mixed sodium alginate, the controllable degradation of the hydrogel is successfully achieved. The hydrogel is applied to the construction of artificial organs and mixed with decellularized matrix, gelatin and PBS to construct bionic artificial organs, achieving controllable degradation of the organs. The problem of cell loss and functional impairment caused by the loss of structural support of artificial organs due to excessive material degradation can be effectively avoided; the problem of inflammation and foreign body reaction caused by excessively slow material degradation can also be avoided; the stability of the artificial organ structure and the durability of its function are significantly improved. In addition, the present invention also carries out bionic design, and the bionic hydrogel used to construct artificial organs is more suitable for the survival of organ cells, provides a suitable environment for their growth, and significantly improves the therapeutic effect of the functional sustainability of artificial organs.

[0018] (2) The present invention applies the above-mentioned bionic hydrogel to the process of constructing an artificial liver, and verifies its effect of reducing liver damage and prolonging survival in a mouse model of liver fibrosis, indicating that it has the potential for artificial liver transplantation therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 In vitro degradation rate of artificial liver with different components.

[0021] Figure 2 Figure 3: The degradation of artificial livers with different components in vivo.

[0022] Figure 3 For: In vitro functional characterization of the bionic artificial liver (glycogen staining).

[0023] Figure 4 This is the survival curve of mice with liver fibrosis treated with bionic artificial liver.

[0024] Figure 5 For: Body weight changes in mice with liver fibrosis treated with bionic artificial liver.

[0025] Figure 6 Figure 3: Changes in the number of hepatocytes in different groups at different culture times. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] A controllable degradable biomimetic hydrogel, including mixed sodium alginate, is prepared by the following method: (1) Preparation of oxidized high-viscosity sodium alginate: Add 10g of high-viscosity sodium alginate (15-25cp, sigma, item number 180947) to 900ml of water, stir magnetically for about 8h, wait for the sodium alginate to completely dissolve, and place in a refrigerator for 2h to lower its temperature to 4°C. Dissolve 10.7g of potassium periodate in 100ml of water, quickly mix with the dissolved sodium alginate solution, oxidize for 6-12h under low temperature, sealed, and light-proof conditions, and then add 3ml of ethylene glycol to neutralize the excess oxidant (potassium periodate) to terminate the oxidation reaction. Add this reaction solution to an ethanol-salt mixture (500ml ethanol-1g sodium chloride) in a volume ratio of 1:1 to precipitate the oxidized alginate. After standing for 10min, pour off the supernatant, take out the precipitate and put it into a freeze dryer for drying. Repeat the purification process twice to obtain a dry oxidized high-viscosity sodium alginate solid; (2) High viscosity sodium alginate (15-25 cp, sigma, catalog number 180947), low viscosity sodium alginate (4-12 cp, sigma, catalog number A1112) and the oxidized high viscosity sodium alginate solid dried in step (1) are mixed in different mass percentages to prepare a mixed sodium alginate; then the mixed sodium alginate is dissolved in PBS at a ratio of 4% w / v, sterilized, and stored at 4° C. to prepare a controllably degradable biomimetic hydrogel, which is heated to 37° C. to melt before use.

[0029] Example 2

[0030] A controllable degradable biomimetic hydrogel for preparing an artificial liver, comprising a liver decellularized matrix, PBS, gelatin and mixed sodium alginate, and the specific preparation method is as follows: (1) Preparation of liver decellularized matrix: Liver tissues of 6-8 week old C57 mice were surgically isolated and perfused with PBS to remove blood stains on the surface. The liver tissues were then incubated in PBS containing 1% sodium dodecyl sulfate (SDS) and 1% Triton X-100 for 72 hours for decellularization. The decellularized liver tissues were freeze-dried, and the freeze-dried powder was dissolved in PBS containing 3% acetic acid (Merck Millipore, USA) and 1 mg / ml pepsin (Sigma Aldrich, USA) and shaken at 37°C for 72 hours. Finally, NaOH was used to adjust the pH of the dissolved liver decellularized matrix to neutral, and the corresponding liver decellularized matrix concentration here was 8% w / v; (2) Preparation of gelatin solution: Dissolve gelatin powder in PBS at a ratio of 16% (w / v), sterilize and store at 4°C. Heat the liquid to 37°C until it melts before use. (3) Preparation of mixed sodium alginate: Same as steps (1) and (2) of Example 1; high viscosity sodium alginate (15-25 cp, sigma, Catalog No. 180947), low viscosity sodium alginate (4-12 cp, sigma, Catalog No. A1112) and oxidized high viscosity sodium alginate were mixed in different mass percentages, and the final concentration of the mixed sodium alginate was 4% w / v; (4) Take 1 ml of the liver decellularized matrix prepared in step (1), 1 ml of PBS, 2 ml of the gelatin prepared in step (2), and 1 ml of the mixed sodium alginate prepared in step (3), and mix them to prepare a controllably degradable biomimetic hydrogel for preparing an artificial liver.

[0031] Example 3

[0032] Study on the degradation of bioartificial livers with different components (1) Construction of artificial livers with different components: According to the preparation method of Example 2, prepare different proportions of high-viscosity sodium alginate, low-viscosity sodium alginate and oxidized high-viscosity sodium alginate for the preparation of controllable biomimetic hydrogels for artificial livers, and explore the effect of the proportions of different types of sodium alginate on the degradation performance of hydrogels. Mix the prepared hydrogel with liver cells and refrigerate at 4°C for 15 minutes, then select a suitable extrusion nozzle diameter, adjust the printing nozzle temperature to 10-15°C, and adjust the printing platform temperature to 5-10°C. Use 1-2mm 3 / s extrusion speed to print the controllably degradable biomimetic hydrogel containing cells in a 6 cm sterile culture dish; (2) Study on the in vitro degradation of artificial livers with different components The artificial livers were divided into multiple groups according to their components, and two artificial livers were printed in each group with the same requirements. The printed livers were placed in a 3% CaCl 2 The solution was rapidly cross-linked. After cross-linking was completed, one artificial liver was taken from each group for freeze-drying and the original weight (weight before culture) was weighed. The other one was added with liver cell culture medium and cultured at 37°C. After 14 days of culture, the culture medium was sucked away, the remaining hydrogel was freeze-dried, and the remaining weight was weighed. The gel degradation before and after culture was calculated by dry weight ratio = remaining weight / original weight. The experimental results are shown in Figure 1 As shown ( Figure 1 The horizontal axis is the proportion of oxidized high-viscosity sodium alginate, and the vertical axis is the proportion of low-viscosity sodium alginate. The proportion of high-viscosity sodium alginate makes up 100%).

[0033] Result analysis: Figure 1 It can be seen that the degradation rate of artificial liver is different when the ingredients are different, and the degradation rate of artificial liver is proportional to the proportion of low-viscosity sodium alginate and oxidized high-viscosity alginate.

[0034] (3) Study on the in vivo degradation of artificial livers with different components

[0035] An easily degradable artificial liver (40% oxidized high-viscosity sodium alginate + 40% low-viscosity sodium alginate + 20% high-viscosity sodium alginate) and a difficult-to-degrade artificial liver (20% oxidized high-viscosity sodium alginate + 20% low-viscosity sodium alginate + 60% high-viscosity sodium alginate) were prepared, and 0.01 mg / ml of cy7 fluorescent dye was added during gel preparation. In order to prevent the slow degradation of the gel from affecting the function of the native liver, the easily degradable artificial liver was transplanted in situ in the liver. In order to prevent the gel from degrading too quickly and losing the compensatory function of the liver, the difficult-to-degrade artificial liver was transplanted to the abdominal mesentery. After different periods of time, the degradation of the transplanted artificial liver was observed using a small animal living imaging device. The experimental results are shown as follows. Figure 2 shown.

[0036] The results were analyzed by Figure 2 It can be seen that the easily degradable artificial liver can be rapidly degraded in about 7 days when transplanted in situ in the liver, and will not affect the growth and function of the native liver; the non-degradable artificial liver can still exist 21 days after transplantation in the abdominal cavity, and can continue to maintain the morphology of the artificial liver and exert the compensatory function of the liver.

[0037] Example 4

[0038] Liver function tests in vivo transplantation

[0039] On the basis of Example 3, a biodegradable artificial liver (40% oxidized high-viscosity sodium alginate + 40% low-viscosity sodium alginate + 20% high-viscosity sodium alginate) was selected for in situ transplantation and cultured in vitro for 2 days. Glycogen staining was performed to observe the glycogen secretion and storage. The experimental results are as follows: Figure 3 shown.

[0040] Result analysis: Figure 3 It can be seen that the artificial liver showed obvious glycogen accumulation after in vitro culture, which means that the artificial liver has acquired mature liver function during in vitro culture.

[0041] Example 5

[0042] Detection of the survival and body weight of mice transplanted in vivo

[0043] Six-week-old C57 mice were intraperitoneally injected with carbon tetrachloride to establish a liver fibrosis mouse model. The controllably degradable in vivo transplanted artificial liver (40% oxidized high-viscosity sodium alginate + 40% low-viscosity sodium alginate + 20% high-viscosity sodium alginate) was transplanted into the liver fibrosis mouse model (liver in situ), and a blank control group and a sham operation group were set up for comparison. The survival time and weight changes of the mice in the control group and the experimental group were recorded. The experimental results are shown in the figure. Figure 4 and Figure 5 shown.

[0044] Result analysis: Figure 4 and Figure 5 It can be seen that the survival time of mice with liver fibrosis that underwent orthotopic liver transplantation was prolonged, and there was no significant change in body weight, indicating that artificial liver can effectively alleviate the damage caused by liver fibrosis after orthotopic liver transplantation and provide liver compensatory function.

[0045] Example 6

[0046] Comparison of biomimetic hydrogel cell culture

[0047] Controllable degradable biomimetic hydrogels containing decellularized matrix (the concentration of decellularized matrix is ​​8% w / v; the concentration of gelatin is 16% w / v; the concentration of mixed sodium alginate is 4% w / v; the volume ratio of decellularized matrix, PBS, gelatin and mixed sodium alginate is 1:1:2:1) and gelatin-sodium alginate hydrogels without decellularized matrix (the concentration of gelatin is 16% w / v; the concentration of mixed sodium alginate is 4% w / v; the volume ratio of decellularized matrix, PBS, gelatin and mixed sodium alginate is 0:1:2:1) were prepared respectively. The two were mixed with the same number of hepatocytes, and the changes in the number of hepatocytes were observed after culturing for different time periods. The results are as follows: Figure 6 shown.

[0048] Result analysis: Figure 6 It can be seen that after adding decellularized matrix, the proliferation rate of liver cells is higher than that without adding decellularized matrix, which indicates that the bionic hydrogel with added decellularized matrix is ​​more suitable for cell survival and proliferation.

[0049] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0050] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A controllable degradable biomimetic hydrogel, characterized in that: Including mixed sodium alginate; the mixed sodium alginate includes high-viscosity sodium alginate, low-viscosity sodium alginate and oxidized high-viscosity sodium alginate; The oxidized high-viscosity sodium alginate is prepared by the following method: An oxidant is added to a high-viscosity sodium alginate solution, and the solution is oxidized for 6-12 hours under low temperature, sealed and light-proof conditions. Then, ethylene glycol is added to neutralize excess oxidant, and the solution is precipitated and dried to prepare oxidized high-viscosity sodium alginate.

2. The use of the controllably degradable biomimetic hydrogel according to claim 1, characterized in that: The application is any of the following: (1) Application in the preparation of biological scaffold materials; (2) Application in the preparation of drug sustained-release carriers; (3) Application in the preparation of cell encapsulation materials.

3. A controllably degradable biomimetic hydrogel for preparing artificial organs, characterized in that: Including mixed sodium alginate; also including decellularized matrix, PBS and gelatin; The mixed sodium alginate includes high-viscosity sodium alginate, low-viscosity sodium alginate and oxidized high-viscosity sodium alginate; The oxidized high viscosity sodium alginate is prepared by the following method: An oxidant is added to a high-viscosity sodium alginate solution, and the solution is oxidized for 6-12 hours under low temperature, sealed and light-proof conditions. Then, ethylene glycol is added to neutralize excess oxidant, and the solution is precipitated and dried to prepare oxidized high-viscosity sodium alginate.

4. The controllably degradable biomimetic hydrogel for preparing artificial organs according to claim 3, characterized in that: The concentration of the decellularized matrix is ​​4-8% w / v; the concentration of the gelatin is 8-16% w / v; the concentration of the mixed sodium alginate is 4-5% w / v; and the volume ratio of the decellularized matrix, PBS, gelatin and mixed sodium alginate is 1:1:2:

1.

5. The controllably degradable biomimetic hydrogel for preparing artificial organs according to claim 4, characterized in that: The viscosity of the high-viscosity sodium alginate is in the range of 15-25 cp; the viscosity of the low-viscosity sodium alginate is in the range of 4-12 cp.

6. The controllably degradable biomimetic hydrogel for preparing artificial organs according to claim 3, characterized in that: When the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 0-20%, the mass percentage of high-viscosity sodium alginate is 60-100%, and the mass percentage of oxidized high-viscosity sodium alginate is 0-20%, the degradation time of the artificial organ is ≥45d; When the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 20-40%, the mass percentage of high-viscosity sodium alginate is 20-60%, and the mass percentage of oxidized high-viscosity sodium alginate is 20-40%, the degradation time of the artificial organ is 15-45 days; When the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 40-50%, the mass percentage of high-viscosity sodium alginate is 0-20%, and the mass percentage of oxidized high-viscosity sodium alginate is 40-50%, the degradation time of the artificial organ is 5-15 days.

7. Use of the bionic hydrogel according to any one of claims 3 to 6 in preparing controllable degradable artificial organs.

8. A controllable degradable artificial organ, characterized in that: The artificial organ is prepared by using the bionic hydrogel described in any one of claims 3-6.

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