Bionic hydrogel capable of performing artificial organ orthotopic transplantation and application

Abstract

The invention discloses bionic hydrogel capable of being used for artificial organ orthotopic transplantation and application of the bionic hydrogel. Belongs to the technical field of medical materials. The bionic hydrogel comprises an adhesion hemostasis layer and a cell carrying layer, and the adhesion hemostasis layer is formed by polyvinyl alcohol (PVA) and boric acid and can rapidly stop bleeding and provide stable support for artificial organs; the cell carrying layer is composed of an acellular matrix, a phosphate buffer solution, gelatin and sodium alginate, can simulate the cell microenvironment of natural organs, and supports functional culture of organ parenchymal cells. According to the design, a dynamic cross-linked network is formed through chemical boric acid ester bonds, and excellent hemostatic performance, tissue adhesion, self-healing capacity and shape adaptability are achieved; meanwhile, mature organ function phenotypes are shown in vitro and can be quickly integrated with native organs, so that functions are exerted, and technical support is provided for human organ transplantation.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and more particularly to a bionic hydrogel capable of in-situ transplantation of artificial organs and its application. Background Art

[0002] Due to the malfunction of organs caused by diseases, injuries and aging of human tissues and organs, there is a huge gap in tissue and organ replacement therapy clinically. A bioartificial organ is an organ made of artificial materials that partially or completely replaces the functions of natural human organs. In recent years, artificial organs have played an increasingly important role in saving critically ill patients and gaining time for organ transplantation. Currently, many artificial organs have been implanted into our bodies: liver, lung, heart, gonad, larynx, cornea and upper limb, etc., and the initial replacement of many human "parts" has been achieved. Therefore, the research on artificial organs has always been a hot topic in the research of regenerative medicine and has great clinical application prospects.

[0003] In recent years, hydrogel materials have attracted much attention in the construction of artificial organs due to their excellent tissue compatibility, plasticity and potential for biofunctionalization. However, existing hydrogel-based artificial organs mostly rely on external equipment to assist functions and cannot completely replace the functions of diseased organs. In addition, these technologies usually have problems such as poor biocompatibility, limited function recovery and complex operations during transplantation, which limit their clinical application effects. Moreover, in the treatment of patients with severe organ failure, partial or total resection of organs is often required. Traditional in-situ transplanted artificial organs have no hemostatic function and often cannot adapt to large-scale in-situ transplantation. They can only be attached to the outside of the organ capsule in the form of patches, which affects the function of the transplanted organ. Commercially available bioadhesives or hemostatic patches have single functions and often cannot support the long-term survival of transplanted cells.

[0004] Therefore, how to provide a hydrogel-based material and an in-situ transplantation method that can be transplanted in-situ and effectively maintain the function of the transplanted organ is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0005] In view of this, the present invention provides a bionic hydrogel capable of in-situ transplantation of artificial organs and its application. Through the rapid integration of the adhesion hemostasis layer and the cell-loading layer, not only the transplantation process is accelerated, but also seamless fusion with the native organ can be achieved. Taking liver transplantation as an example, the recovery effect of liver function is significantly improved and the survival period of liver failure model mice is prolonged.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A bionic hydrogel capable of in-situ transplantation of artificial organs, comprising an adhesion hemostasis layer and a cell-loading layer;

[0008] The adhesion hemostatic layer includes polyvinyl alcohol and boric acid with a volume ratio of 0.5 - 1.0:0.5 - 1.0;

[0009] The cell - loaded layer includes decellularized matrix, phosphate - buffered saline, gelatin, and sodium alginate with a volume ratio of 0.8 - 1.0:0.8 - 1.0:1.5 - 2.0:0.8 - 1.0.

[0010] Preferably, the concentration of the polyvinyl alcohol is 4 - 8% w / v; the concentration of the boric acid is 1 - 2% w / v; the concentration of the decellularized matrix is 2 - 6% w / v; the concentration of the gelatin is 4 - 8% w / v; the concentration of the sodium alginate is 1 - 3% w / v.

[0011] Preferably, the artificial organs include the heart, lungs, liver, kidneys, pancreas, small intestine, and cornea; correspondingly, the decellularized matrix corresponds to the decellularized matrix of each of the above - mentioned organs.

[0012] Another object of the present invention is to provide the application of the above - mentioned bionic hydrogel in the construction and in - situ transplantation of artificial organs for non - disease diagnosis and treatment.

[0013] Another object of the present invention is to provide a method for in - situ transplantation of artificial organs for non - disease diagnosis and treatment. Using the above - mentioned bionic hydrogel, in - situ transplantation of artificial organs is carried out, which specifically includes the following steps:

[0014] (1) Mix the cells of the organ to be transplanted with the cell - loaded layer, and then print the printed body of the transplanted organ;

[0015] (2) Place the printed organ printed body in a calcium chloride solution for cross - linking reaction. After the reaction ends, place it in a culture medium for culture to prepare an artificial organ;

[0016] (3) Coat the adhesion hemostatic layer on the surface of the wound of the native organ. After the bleeding of the native organ stops, attach the artificial organ obtained in step (2) to the adhesion hemostatic layer to complete the in - situ transplantation of the artificial organ.

[0017] Preferably, the printing temperature of the printed body of the transplanted organ in step (1) is 25 - 37°C; the cross - linking reaction time in step (2) is 2 - 4 min.

[0018] Preferably, the culture temperature in step (2) is 37°C, and the culture time is 2 - 5 d.

[0019] Another object of the present invention is to provide the application of the above - mentioned method in the in - situ transplantation of artificial organs for non - disease diagnosis and treatment.

[0020] Through the above - mentioned technical solutions, compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention provides a bionic hydrogel for in-situ transplantation of artificial organs, which includes two parts: an adhesion and hemostasis layer and a cell-loading layer. The adhesion and hemostasis layer is formed by polyvinyl alcohol (PVA) and boric acid, which can rapidly stop bleeding and provide stable support for artificial organs. This design forms a dynamic cross-linked network through chemical borate bonds, achieving excellent tissue adhesion, self-healing ability, and shape adaptability. The cell-loading layer is composed of decellularized matrix, phosphate buffer solution, gelatin, and sodium alginate, simulating the cell microenvironment of natural organs and supporting the functional culture of organ parenchymal cells. At the same time, the requirements of hemostasis and in-situ transplantation are realized through the dynamic cross-linking process between the gelatin in the cell-loading layer and the adhesion and hemostasis layer.

[0022] (2) This system can combine advanced bioprinting and cross-linking technologies to prepare artificial organs with ideal shapes as needed;

[0023] (3) The transplantation method is simple and efficient: through the rapid integration of the adhesion and hemostasis layer and the cell-loading layer, it not only accelerates the transplantation process but also seamlessly integrates with the native organ, thereby restoring partial organ function. Animal experiments further verified its significant effects in organ function recovery and prolonging the survival period of mice in the organ failure model. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0025] Figure 1 It is: the hemostatic function of the hemostasis layer in Example 3.

[0026] Figure 2 It is: the rapid adhesion function of the hemostasis layer and the cell-loading layer in Example 3.

[0027] Figure 3 It is: the verification of the rapid adhesion function of the hemostasis layer and the cell-loading layer by test tube experiment in Example 3.

[0028] Figure 4 It is: the scanning electron micrograph of the in-situ transplanted artificial liver in Example 3.

[0029] Figure 5 It is: the in-vitro liver function test (ICG uptake / release) of the in-situ transplanted artificial liver in Example 3.

[0030] Figure 6 It is: the detection of CYP450 enzyme activity in vitro of the in-situ transplanted artificial liver in Example 3.

[0031] Figure 7 It is: The survival curve of mice with liver failure treated by orthotopic transplantation of artificial liver in Example 3.

[0032] Figure 8 It is: HE staining image 14 days after transplantation of orthotopic transplantation of artificial liver in Example 3.

[0033] Figure 9 It is: The hemostatic function of the hemostatic layer in Example 4.

[0034] Figure 10 It is: HE staining image 14 days after transplantation of orthotopic transplantation of artificial liver in Example 4. Detailed implementation manners

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] Example 1

[0037] A bionic hydrogel capable of orthotopic liver transplantation, comprising an adhesion hemostatic layer and a cell-loaded layer;

[0038] The adhesion hemostatic layer includes: 1 ml of PVA (8% w / v) and 1 ml of boric acid (2% w / v);

[0039] The cell-loaded layer includes: 1 ml of liver acellular matrix (6% w / v), 1 ml of phosphate buffer, 2 ml of gelatin (8% w / v), and 1 ml of sodium alginate (3% w / v);

[0040] The specific preparation method is as follows:

[0041] Preparation of the adhesion hemostatic layer and the cell-loaded layer:

[0042] Preparation of the adhesion hemostatic layer: Dissolve PVA and boric acid in phosphate buffer at concentrations of 8% w / v and 2% w / v respectively to obtain PVA solution and boric acid solution respectively. After sterilization and aliquoting, store at 4 °C. Before use, heat the liquid to 37 °C until it melts. When in use, mix the PVA solution and the boric acid solution in a volume ratio of 1 ml:1 ml to obtain the adhesion hemostatic layer;

[0043] Preparation of the cell-laden layer: The liver tissues of 6-8-week-old C57 mice were surgically isolated, and after perfusion, the blood stains on their surfaces were removed using phosphate buffer solution; then the liver tissues were incubated in phosphate buffer solution 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 phosphate buffer solution containing 5% acetic acid (Merck Millipore, USA) and 1 mg / ml pepsin (Sigma Aldrich, USA), and shaken at 37 °C for 48 hours. Finally, the pH of the dissolved liver decellularized matrix was adjusted to neutral using NaOH to prepare the liver decellularized matrix;

[0044] The gelatin powder and sodium alginate powder were dissolved in phosphate buffer solution at ratios of 8% w / v and 3% w / v respectively to prepare gelatin solution and sodium alginate solution. After sterilization and aliquoting, they were stored at 4 °C. Before use, the liquid was heated to 37 °C until it melted;

[0045] Then, 1 ml of liver decellularized matrix (6% w / v), 1 ml of phosphate buffer solution, 2 ml of gelatin (8% w / v), and 1 ml of sodium alginate (3% w / v) were mixed to prepare the cell-laden layer.

[0046] Example 2

[0047] A bionic hydrogel capable of in-situ liver transplantation, comprising an adhesion and hemostasis layer and a cell-laden layer;

[0048] The adhesion and hemostasis layer comprises: 0.5 ml of PVA (4% w / v) and 0.5 ml of boric acid (1% w / v);

[0049] The cell-laden layer comprises: 0.8 ml of liver decellularized matrix (2% w / v), 0.8 ml of phosphate buffer solution, 1.5 ml of gelatin (4% w / v), and 0.8 ml of sodium alginate (1% w / v);

[0050] The specific preparation method is as follows:

[0051] Preparation of the adhesion and hemostasis layer and the cell-laden layer:

[0052] Preparation of the adhesion and hemostasis layer: PVA and boric acid were dissolved in phosphate buffer solution at concentrations of 4% w / v and 1% w / v respectively to prepare PVA solution and boric acid solution. After sterilization and aliquoting, they were stored at 4 °C. Before use, the liquid was heated to 37 °C until it melted. When in use, the PVA solution and boric acid solution were mixed at a volume ratio of 0.5 ml:0.5 ml to prepare the adhesion and hemostasis layer;

[0053] Preparation of cell-laden layer: The liver tissues of 6-8-week-old C57 mice were surgically isolated, and after perfusion, the blood stains on their surfaces were removed using phosphate buffer solution; then the liver tissues were incubated in phosphate buffer solution 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 phosphate buffer solution containing 5% acetic acid (Merck Millipore, USA) and 1 mg / ml pepsin (Sigma Aldrich, USA), shaken at 37 °C for 48 hours, and finally the pH of the dissolved liver decellularized matrix was adjusted to neutral with NaOH to prepare the liver decellularized matrix;

[0054] Gelatin powder and sodium alginate powder were dissolved in phosphate buffer solution at ratios of 4% w / v and 1% w / v respectively to prepare gelatin solution and sodium alginate solution, which were sterilized, aliquoted, and stored at 4 °C. Before use, the liquid was heated to 37 °C until it melted;

[0055] Then, 0.8 ml of liver decellularized matrix (2% w / v), 0.8 ml of phosphate buffer solution, 1.5 ml of gelatin (4% w / v), and 0.8 ml of sodium alginate (1% w / v) were mixed to prepare the cell-laden layer.

[0056] Example 3

[0057] A method for orthotopic transplantation of an artificial organ for non-disease diagnosis and treatment specifically includes the following steps:

[0058] (1) Addition of primary hepatocytes: The cultured primary hepatocytes were mixed in the cell-laden layer prepared in Example 1 at a concentration of 5×10 6 cells / ml;

[0059] (2) Cultivation of orthotopic transplantation type artificial liver

[0060] The cell-laden layer gel prepared in step (1) was placed in a refrigerator at 4 °C for 10 minutes, and at 25 °C - 37 °C, an artificial extrusion or 3D printing method was used to prepare a biological liver with an ideal shape or size. The printed liver body was placed in 3% CaCl2 solution to rapidly crosslink it, and then it was cultured in hepatocyte medium (DMEM + 10% fetal bovine serum) at 37 °C for 2 - 5 days;

[0061] (3) Characterization of the transplantation performance of orthotopic transplantation type artificial liver

[0062] The hemostatic layer prepared in Example 1 was applied to the liver wound, and then the cell-laden layer was placed above the adhesion layer to observe the hemostasis of the native liver of the mouse ( Figure 1)。The liver tissue was cut open, and the hemostatic layer and the adhesion layer were applied to both sides respectively to observe the adhesion ability of the two to the liver tissue after combination. Figure 2 );The hemostatic layer and the adhesion layer were applied to two test tubes respectively, and the two were joined together to further observe their adhesion ability. Figure 3 )。

[0063] Result analysis: As Figure 1 shown, after transplantation, the bleeding at the liver wound disappeared; as Figure 2 shown by the treatment with the adhesion layer, the cut liver re-polymerized together, proving its excellent tissue adhesion; the following test tube adhesion experiment further demonstrated its adhesion performance. Figure 3 )。

[0064] (4) Liver function detection of the in-situ transplantation type artificial liver

[0065] Observe the microstructure of the in-situ transplantation type artificial liver under a scanning electron microscope. Figure 4 )。The in-situ transplantation type artificial liver was cultured in a culture medium supplemented with progesterone, pregnenolone-16α-carbonitrile, and 8-bromo-cAMP for 48 hours. Subsequently, the artificial liver was co-incubated with a medium containing ICG (1 mg / ml) at 37 °C for 1 hour, washed three times with phosphate buffer, and changed to a hepatocyte culture medium. The ICG uptake / release was observed under a microscope. Figure 5 )。

[0066] Result analysis: As Figure 4 shown, the scanning electron microscope results showed that there were a large number of hepatocytes in the in-situ transplantation type artificial liver, arranged in clusters, which played an important role in supporting the liver function.

[0067] Result analysis: As Figure 5 shown, the artificial liver could normally take up ICG and release ICG (the green disappeared) 6 hours after changing to a normal culture medium.

[0068] Inducers such as 3-methylcholanthrene (cyp1a2, cyp3a13), ethanol (cyp2e1), and rifampicin (cyp3a11) were co-incubated with the artificial liver at 37 °C for 72 hours respectively to detect their CYP enzyme activities. Figure 6 )。

[0069] Result analysis: As Figure 6 shown, after induction by the inducers, the artificial liver highly expressed the cyp450 metabolic enzyme gene, which was similar to that of primary hepatocytes, indicating that the artificial liver had good liver metabolic function.

[0070] (5) Transplant the in-situ transplanted artificial liver to the liver position of Fah- / - mice. After 1 week, discontinue NTBC treatment for Fah-deficient mice, and record the survival periods of the control group and experimental group mice ( Figure 7 ), and take the transplanted mouse livers for HE staining ( Figure 8 ).

[0071] Result analysis: As Figure 7 shown, the artificial liver transplantation can extend the survival period of liver failure mice, which proves its good liver compensatory function in vivo;

[0072] Figure 8 shows the HE results of the artificial liver after being transplanted in situ to the native liver, indicating that it has a mature liver morphology and structure after transplantation.

[0073] Example 4

[0074] A method for in-situ transplantation of an artificial organ for non-disease diagnosis and treatment specifically includes the following steps:

[0075] (1) Addition of primary hepatocytes: Mix the cultured primary hepatocytes at a concentration of 5×10 6 cells / ml into the cell-loaded layer prepared in Example 2;

[0076] (2) Cultivation of the in-situ transplanted artificial liver

[0077] Place the cell-loaded layer gel in step (1) in a 4°C refrigerator for 10 minutes. At 25°C - 37°C, use artificial extrusion or 3D printing to prepare a biological liver with an ideal shape or size. Place the printed liver body in a 3% CaCl2 solution to quickly crosslink it, and then use a hepatocyte culture medium (DMEM + 10% fetal bovine serum) to culture it at 37°C for 2 - 5 days;

[0078] (3) Characterization of the transplantation performance of the in-situ transplanted artificial liver

[0079] Apply the hemostatic layer prepared in Example 2 to the liver wound, and then place the cell-loaded layer above the adhesion layer to observe the hemostasis of the native mouse liver ( Figure 9 ).

[0080] Result analysis: As Figure 9 shown, after transplantation, the bleeding at the liver wound disappeared.

[0081] (4) Transplant the in-situ transplanted artificial liver to the liver position of Fah- / - mice. After 1 week, discontinue NTBC treatment for Fah-deficient mice, and take the transplanted mouse livers for HE staining ( Figure 10 ).

[0082] Result analysis:Figure 10 The HE results of the artificial liver after transplantation in situ into the native liver are shown, indicating that it has a mature liver morphology and structure after transplantation.

[0083] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other.

[0084] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can 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 will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A bionic hydrogel capable of performing in-situ transplantation of artificial organs, characterized in that, It includes an adhesion hemostatic layer and a cell-carrying layer; The adhesion hemostatic layer includes polyvinyl alcohol and boric acid with a volume ratio of 0.5 - 1.0:0.5 - 1.0; The cell-carrying layer includes acellular matrix, phosphate buffer solution, gelatin and sodium alginate with a volume ratio of 0.8 - 1.0:0.8 - 1.0:1.5 - 2.0:0.8 - 1.

0.

2. The bionic hydrogel capable of in-situ transplantation of artificial organs according to claim 1, wherein The concentration of the polyvinyl alcohol is 4 - 8% w / v; the concentration of the boric acid is 1 - 2% w / v; the concentration of the acellular matrix is 2 - 6% w / v; the concentration of the gelatin is 4 - 8% w / v; the concentration of the sodium alginate is 1 - 3% w / v.

3. The bionic hydrogel capable of in-situ transplantation of artificial organs according to claim 1, wherein The artificial organs include heart, lung, liver, kidney, pancreas, small intestine and cornea; correspondingly, the acellular matrix corresponds to the acellular matrix of each of the above organs.

4. Use of the biomimetic hydrogel according to any one of claims 1 - 3 in the construction and in-situ transplantation of artificial organs for non-disease diagnosis and treatment.

5. A method for in-situ transplantation of an artificial organ for non-disease diagnosis and treatment, characterized in that, Using the biomimetic hydrogel according to any one of claims 1 - 3 for in-situ transplantation of artificial organs, specifically including the following steps: (1) Mix the cells of the organ to be transplanted in the cell-carrying layer, and then print the printed body of the transplanted organ; (2) Place the printed organ printed body in a calcium chloride solution for crosslinking reaction, and after the reaction is completed, place it in a culture medium for culture to prepare an artificial organ; (3) Coat the adhesion hemostatic layer on the surface of the wound of the native organ. After the bleeding of the native organ stops, attach the artificial organ obtained in step (2) to the adhesion hemostatic layer to complete the in-situ transplantation of the artificial organ.

6. The method for in-situ transplantation of an artificial organ for non-disease diagnosis and treatment according to claim 5, characterized in that, The printing temperature of the printed body of the transplanted organ in step (1) is 25 - 37°C; the crosslinking reaction time in step (2) is 2 - 4 min.

7. The method for in-situ transplantation of an artificial organ for non-disease diagnosis and treatment according to claim 5, characterized in that, The culture temperature in step (2) is 37°C, and the culture time is 2 - 5 d.

8. Use of the method according to any one of claims 5 - 7 in the in-situ transplantation of artificial organs for non-disease diagnosis and treatment.

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