CN102718928B - Hydrogel for intelligent desorption of cell sheet layer and application of hydrogel - Google Patents

Abstract

The invention relates to a cell sheet intelligent desorption hydrogel and application thereof. The hydrogel is formed by dissolving functional monomer M1, chemical or physical cross-linking agent M2 and water-soluble biopolymer M3 in a solvent M4 and adopting a redox initiation system for free radical polymerization. The M1, M2, M3 And the mass percentage of M4 is M1: 5-10%, M2: 2-10%, M3: 1-15%, M4: 75-92%. The hydrogel has excellent temperature or pH value response characteristics, as well as biocompatibility, and can effectively promote the rapid proliferation and detachment of cells. The cell sheet formed after detachment retains the complete extracellular matrix, which avoids the damage to the biological function of the cells caused by the enzymatic hydrolysis method and the damage to the cells caused by the degradation components of the scaffold material. The intelligently desorbed cell sheet of the hydrogel of the invention can be used for the treatment and repair of skin, corneal epithelium and periodontal ligament related tissues and organs.

Description

A kind of cell sheet intelligent desorption hydrogel and its application

technical field

The invention relates to the fields of biomedical engineering and intelligent polymer materials, in particular to a cell sheet intelligent desorption hydrogel and its application.

Background technique

For a long time, the shortage of donors and transplant rejection have been two major obstacles to the development of organ transplantation. The currently used natural materials and synthetic scaffold materials have poor clinical efficacy due to problems such as low strength and inflammation caused by degradation. Cell sheet technology is a new technology for harvesting seed cells in tissue engineering. It can be successfully transplanted without a scaffold, and avoids the problems of immunogenicity, inflammatory response, and optical interference caused by traditional scaffold materials after transplantation.

Cell sheet medicine is a technology that takes out the patient's own cells and cultures them in a petri dish, and then transplants the proliferated sheets to the affected area to promote the regeneration of the affected organs. When the cell sheet is attached to the organ whose function cannot be restored due to serious illness or injury, substances called cytokines equivalent to growth hormone will be secreted from the cell sheet to promote telangiectasia, damaged Cells will regenerate. Since it is the patient's own cells, it cannot be rejected. The possibility of curing patients with diabetes, periodontal disease, lung air leak, liver dysfunction and joint dysfunction can be greatly improved through cell sheet technology. Chinese patent CN101896600A discloses a patterned cell sheet and its production method. The method comprises the steps of: providing a continuous sheet of cells disposed on a substrate comprising shape-transforming properties and/or modifiable surface properties; exposing the continuous sheet of cells to a release agent in a patterned manner washing the cell sheet after exposure to the releasing agent to remove cells that have been affected by the releasing agent, and repopulating with a second cell type remaining after the cells affected by the releasing agent have been removed; down the gap.

Polyisopropylacrylamide (polyN-isopropylacrylamide, PNIPAAm) has temperature-sensitive properties, and its volume phase transition temperature is ~32°C. Yamato et al. (Tissue Eng., 2001, 7: 473.) grafted PNIPAAm onto the surface of the cell culture plate and inoculated the cells at 37°C. Since PNIPAAm is hydrophobic, the cells can adhere, stretch and proliferate; when When the temperature is lowered to below 32°C, PNIPAAm is hydrophilic, the volume expands, the cells are separated from the surface of the culture plate, and the overall cell population is detached in a sheet form while maintaining the connection between cells, thereby obtaining a cell sheet that retains cell activity . Harvesting the cell sheet by changing the temperature avoids the damage to the biological function of the cells caused by the enzyme digestion method, and can directly adhere to the tissue without suturing, and at the same time avoids the damage to the cells caused by the degradation components of the scaffold material.

Hydrogel is a multi-component system composed of a polymer with a three-dimensional cross-linked network structure and a medium constructed by hydrophilic polymer chains. It has good biomedical materials such as controlled drug release, soft tissue scaffold construction, and cell encapsulation. Application prospect. As a scaffold for filling defects, a carrier for controlled release of bioactive molecules, and a transfer system for tissues/cells, hydrogel materials are developing in the direction of intelligence. Temperature-sensitive hydrogel is a polymer hydrogel whose volume changes with the change of environmental temperature; pH-sensitive hydrogel is a polymer hydrogel whose volume changes with the change of environmental pH value. This kind of gel macromolecular network There are groups (carboxyl, sulfonic acid or amino) that can dissociate into ions, which can take or release protons according to changes in the pH value of the environment. Utilizing the soft-wet and sensitive properties of temperature or pH responsive hydrogels, cell sheet detachment can be achieved by changing the temperature or pH value. Pure synthetic hydrogels have poor cytocompatibility, and the introduction of natural biocompatible monomers such as collagen, chitosan, and sericin into synthetic hydrogels can promote cell proliferation and detachment.

From the above analysis, it can be seen that there is no report on the combination of temperature-sensitive or pH-sensitive hydrogel, biocompatible polymer and cell sheet desorption technology. The present invention comprehensively considers the temperature or pH value sensitivity and soft-wet characteristics of the hydrogel, and the good cell compatibility of the biocompatible polymer, uses deionized water as a solvent, and adopts free radical polymerization to obtain a hydrogel that can pass through Adjust the temperature or pH value to realize the hydrogel of automatic detachment of cell sheets, and can be applied to the treatment and repair of tissues and organs such as skin, corneal epithelium and periodontal ligament.

Contents of the invention

The technical problem to be solved by the present invention is: in order to effectively promote cell adhesion, avoid damage to cell activity and function caused by traditional enzymatic hydrolysis, and at the same time reduce complications related to traditional tissue engineering, such as inflammatory reactions, etc., to provide A cell sheet intelligent desorption hydrogel for biomedical engineering, the hydrogel not only has good biocompatibility, can promote rapid cell proliferation, but also has excellent temperature or pH value response performance, only by changing the temperature Or the pH value of the gel can quickly change between hydrophilicity and hydrophobicity, so as to realize the intelligent regulation of cell proliferation and desorption.

A cell sheet intelligent desorption hydrogel of the present invention is formed by dissolving functional monomer M1, chemical or physical cross-linking agent M2 and water-soluble biopolymer M3 in solvent M4 and performing free radical polymerization using a redox initiation system Into, the mass percent of M1, M2, M3 and M4 is M1: 8-15%, M2: 1-10%, M3: 1-15%, M4: 60-90%, after changing the temperature or pH value Cells detach from the hydrogel surface to form a sheet structure.

The M1 is temperature-sensitive or pH-sensitive, and is N-isopropylacrylamide or N, N'-bisethylacrylamide or bisethoxyacrylamide or methacrylic acid N, N-dimethyl Aminoethyl ester or one of acrylic acid or methacrylic acid or itaconic acid, and blends thereof.

The M2 is a chemical cross-linking agent or a physical cross-linking agent, which is N, N'-methylenebisacrylamide or ethylene glycol dimethacrylate or piperamide or glutaraldehyde or hectorite or One of magnesium saponite or sauconite or lithium magnesium silicate, and mixtures thereof.

The M3 is a water-soluble biopolymer, which is starch or sodium carboxymethylcellulose or sodium alginate or chitosan or sodium humate or gelatin or guar gum or polyvinylpyrrolidone or β-cyclodextrin or silk A type of gelatin, and its mixture.

Described M4 is deionized water.

The hydrogel detachment cells are mouse fibroblasts or corneal cells or skeletal muscle cells or bone marrow mesenchymal stem cells or skin fibroblasts or human umbilical vein endothelial cells or rat islet cells or periodontal ligament stem cells kind of.

When the temperature of the hydrogel is 3-15° C. or the pH value is 2-5, the proliferating cells are rapidly detached from the surface of the hydrogel to form a cell sheet.

The cell sheet after the intelligent desorption of the hydrogel is implanted into the recipient site, and a three-dimensional structure tissue is constructed through the superimposition of homotype or heterotype cell sheets.

The application of the hydrogel intelligently desorbed cell sheet in the treatment and repair of skin, corneal epithelium and periodontal ligament tissue or organs.

The principle of the present invention is: temperature-sensitive or pH value responsive hydrogel has good temperature and pH value sensitivity, and when the ambient temperature is higher or lower than the phase transition temperature or critical pH value, the gel will undergo hydrophilic and hydrophobic change, and its volume shrinks or swells. At high temperature or low pH value, due to the dominant role of hydrophobic groups or the protonation of acid radical ions, the gel macroscopically shows volume shrinkage, and because the hydrogel contains biocompatible polymers, cells adhere and spread rapidly on the surface of the gel. 1. After the proliferation reaches a certain amount, the ambient temperature drops below the phase transition temperature or the ambient pH value rises above the critical pH value. At this time, the volume of the gel expands, and the cells attached to the surface of the gel detach automatically, thereby obtaining Highly active cell sheets can rapidly proliferate when transferred to skin and other tissue defects.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The hydrogel that can be used for the intelligent desorption of cell sheets in the present invention has simple components, is easy to operate, has excellent temperature or pH value response characteristics, and biocompatibility, and can effectively promote the rapid proliferation and desorption of cells.

(2) The cytocompatibility and cell proliferation or desorption rate of the hydrogel can be regulated by monomer feed ratio, cross-linking degree and reaction temperature, etc.

(3) The cell sheet detachment is simple and easy, and the automatic detachment of the cells can be realized only by controlling the temperature or pH value, thereby avoiding the damage to the cell activity and function caused by the traditional enzymatic hydrolysis method.

(4) The obtained cell sheet retains a complete extracellular matrix, and no scaffold material is required. The three-dimensional tissue of the whole cell is obtained by stacking the same or different cell sheets, which overcomes the disadvantages of degradation of traditional tissue engineering scaffolds. It has a good application prospect in the fields of week and skin tissue regeneration.

Detailed ways

The present invention will be described in further detail below in conjunction with specific examples. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention, and any other changes, modifications, substitutions, combinations, and simplifications that do not violate the spirit and principles of the present invention should be the same as All effective replacement methods are included within the protection scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

A smart detachment hydrogel for cell sheets, composed of N-isopropylacrylamide, N,N'-methylenebisacrylamide and sodium carboxymethylcellulose dissolved in deionized water The methylethylenediamine initiation system is formed by radical polymerization, and the mass percentage of the isopropylacrylamide, N, N'-methylenebisacrylamide, carboxymethylcellulose sodium and deionized water is 10% %, 2%, 8%, 80%, reduce the hydrogel environment temperature to 5°C, and the dermal fibroblasts proliferating for 7 days are desorbed from the surface of the hydrogel to form a sheet structure. The desorbed skin fibroblast sheet can be used for the treatment and repair of burnt skin epidermis.

Example 2

A kind of cell sheet intelligent desorption hydrogel, which is formed by free radical polymerization by dissolving N-isopropylacrylamide, hectorite and sodium alginate in deionized water and using ammonium persulfate/tetramethylethylenediamine initiation system into, the mass percent of the isopropylacrylamide, hectorite, sodium alginate and deionized water is 11%, 1%, 3%, 85%, and the ambient temperature of the hydrogel is reduced to 4°C, and the proliferation is 7 Day corneal cells detach from the hydrogel surface to form a sheet structure. The detached corneal cell sheet can be used for the treatment and repair of the cornea.

Example 3

A cell sheet intelligent desorption hydrogel, composed of N,N-dimethylaminoethyl methacrylate, ethylene glycol dimethacrylate and sodium humate dissolved in deionized water using ammonium persulfate/tetramethyl The base ethylenediamine initiation system carries out radical polymerization, and the mass percent of described N,N-dimethylaminoethyl methacrylate, ethylene glycol dimethacrylate, sodium humate and deionized water is 8 %, 3%, 4%, 85%, lower the ambient temperature of the hydrogel to 3°C, and the bone marrow mesenchymal stem cells proliferated for 7 days detach from the surface of the hydrogel to form a sheet structure. The desorbed bone marrow mesenchymal stem cell sheet can be used for the treatment and repair of scleroderma.

Example 4

A cell sheet intelligent desorption hydrogel, composed of N,N'-diethylacrylamide, ethylene glycol dimethacrylate and polyvinylpyrrolidone dissolved in deionized water using ammonium persulfate/tetramethylethyl ether Diamine initiation system is formed by free radical polymerization, and the mass percent of N, N'-diethylacrylamide, ethylene glycol dimethacrylate, polyvinylpyrrolidone and deionized water is 10%, 4% , 6%, 80%, reduce the temperature of the hydrogel environment to 4°C, and the human umbilical vein endothelial cells proliferating for 7 days are desorbed from the surface of the hydrogel to form a sheet structure. The desorbed human umbilical vein endothelial cell sheet can be used for the treatment and repair of myocardial tissue.

Example 5

A cell sheet intelligent desorption hydrogel, composed of a blend of isopropylacrylamide and acrylic acid (3:1 mass ratio), a mixture of N,N'-methylenebisacrylamide/hectorite Dissolve sericin in deionized water and use ammonium persulfate/tetramethylethylenediamine to initiate free radical polymerization. The blend of isopropylacrylamide and acrylic acid, N,N'- The mass percentages of methylbisacrylamide, sericin and deionized water were 12%, 6%, 12%, and 80%. The surface of the glue is desorbed to form a lamellar structure. The desorbed periodontal ligament stem cell sheet can be used for the treatment and repair of periodontitis.

Example 6

A kind of cell sheet intelligent desorption hydrogel, which is composed of a blend of N, N-dimethylaminoethyl methacrylate and methacrylic acid (mass ratio is 4:1), N, N'-methylene Bisacrylamide and chitosan are dissolved in deionized water and formed by radical polymerization using ammonium persulfate/tetramethylethylenediamine initiation system. The N,N-dimethylaminoethyl methacrylate and methyl The mass percentage of the blend of acrylic acid, N,N'-methylenebisacrylamide, chitosan and deionized water is 14%, 8%, 3%, 75%, and the ambient temperature of the hydrogel is reduced to 3°C , dermal fibroblasts proliferated for 7 days were detached from the surface of the hydrogel to form a sheet structure. The desorbed skin fibroblast sheet can be used for the treatment and repair of burnt skin epidermis.

Claims (1)

1. cell sheets intelligence desorption hydrogel, it is characterized in that, described hydrogel is by methacrylic acid N, the blend of N-dimethylaminoethyl and methacrylic acid, N, N '-methylene-bisacrylamide and chitosan are dissolved in deionized water, adopting ammonium persulphate and Tetramethyl Ethylene Diamine initiator system to carry out radical polymerization forms, described methacrylic acid N, the blend of N-dimethylaminoethyl and methacrylic acid, N, N,-methylene-bisacrylamide, the mass percent of chitosan and deionized water is 14%, 8%, 3% and 75%, methacrylic acid N, the mass ratio of N-dimethylaminoethyl and methacrylic acid is 4: 1,

Described hydrogel envrionment temperature is 3 ℃, breeds the skin fiber archeocyte of 7 days from hydrogel surface desorption, forms laminated structure;

Described hydrogel desorption cell is skin fiber archeocyte.