CN116496511A - Macroporous hydrogel with rapid degradation of enzyme response and preparation method and application thereof - Google Patents
Macroporous hydrogel with rapid degradation of enzyme response and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a macroporous hydrogel capable of being rapidly degraded in enzyme response, a preparation method and application thereof. The macroporous hydrogel with the rapid degradation of enzyme response can be widely applied to cell culture, proliferation, migration, differentiation and collection, is particularly suitable for the expansion of mesenchymal stem cells, and the mesenchymal stem cells after the expansion obtained through the degradation of the hydrogel mediated by the tobacco etch virus protease are mesenchymal stem cell spheres with high stem property maintenance, function enhancement and high extracellular matrix retention.
Description
Technical Field
The invention relates to the field of new materials, in particular to a macroporous hydrogel with rapid degradation of enzyme response, a preparation method and application thereof.
Background
The mesenchymal stem cells have the excellent characteristics of wide cell sources, high differentiation potential, high growth speed and the like, and are widely applied to tissue engineering, stem cell treatment, drug screening and individuation disease models. These applications often require a sufficient number and quality of mesenchymal stem cells to obtain the desired effect. The commonly used two-dimensional expansion methods often result in mesenchymal stem cells losing stem properties and changing phenotypes, thus resulting in undesirable downstream application results. In contrast, mesenchymal stem cells exhibit more robust and versatile features in three-dimensional culture. Three-dimensional culture can be roughly classified into stentless three-dimensional culture and three-dimensional culture based on hydrogel scaffolds.
Common methods for the stentless three-dimensional culture of mesenchymal stem cells include hanging drop, low adhesion surface, magnetic suspension, rotating bottle, rotating wall container, etc. In these methods, mesenchymal stem cells are aggregated into mesenchymal stem cell pellets through cell-cell interactions. The mesenchymal stem cell spheres have various advantages over single mesenchymal stem cell suspensions, and are characterized by the benefits of increased dryness, increased activity, and enhanced paracrine immune regulation and nutrition (anti-apoptosis, angiogenesis, anti-fibrosis and anti-inflammatory). However, one of the most significant disadvantages of these methods is that these cells lose anoikis due to the lack of initial extracellular matrix binding, resulting in a portion of the cells losing activity during culture.
The mesenchymal stem cells in the body are surrounded by their secreted extracellular matrix niche, such a niche structure can maintain the stem, multipotential and survival of the mesenchymal stem cells and enable the mesenchymal stem cells to respond effectively to differentiation stimuli. The extracellular matrix is thus considered to have a very important role in the three-dimensional culture of stem cells. Based on this, hydrogel scaffolds mimicking the extracellular matrix have been widely used in tissue engineering. For example, the short peptide arginine-glycine-aspartic acid (hereinafter abbreviated as RGD) is a cell adhesion short peptide derived from extracellular matrix proteins, and the covalent attachment of RGD in a hydrogel network provides an initial binding site for anchoring cells to a hydrogel scaffold, so that the problem of anoikis caused by incapacity of cells to adhere to artificially synthesized hydrogels is well solved. These applications illustrate that extracellular matrix plays a major role in hydrogel manufacture. However, the natural extracellular matrix niche is a complex network of proteins and polysaccharides that act as a "harbor" for cell signaling molecules (e.g., growth factors and matrix fragments) that permeate it, so that these molecules can be released back into the cell to regulate the function and activity of the cell. The formation of such natural extracellular matrix niches requires post-secretion assembly of extracellular matrix proteins. This also directly determines its irreplaceability and is an important factor limiting the use of artificial extracellular matrix in tissue engineering.
Mesenchymal stem cells secrete extracellular matrix upon expansion within the hydrogel scaffold, remodelling the hydrogel network for further proliferation and migration. Thus, proliferation of mesenchymal stem cells and proliferation of extracellular matrix inside the hydrogel are two simultaneous events. However, two major drawbacks of long-term expansion of scaffold-based mesenchymal stem cells are: the limited proliferation space makes macro-expansion difficult and lacks adequate methods to ensure that high quality mesenchymal stem cells are harvested. In fact, it is also difficult to achieve this important factor in preserving the natural extracellular matrix secreted during cell proliferation during the harvesting process of expanding mesenchymal stem cells. These reasons have all greatly affected the downstream use of mesenchymal stem cells, so that the results of these uses cannot reach the expected level. For this reason, new materials combining the advantages of both stentless and hydrogel stent-based technologies have been named innovative and powerful tools for tissue engineering, with urgent need for breakthrough to solve the key problems.
Thus, with respect to the limitations in proliferation space and harvesting methods, applicants wish to address the problem of proliferation space limitation by utilizing macroporous hydrogels in combination with the advantages of stent-free and stent-based culture; meanwhile, the introduction of tobacco etch virus protease and its substrate sequence mediates rapid degradation of the hydrogel to achieve rapid release of cells inside the hydrogel. Therefore, the two problems of the two classical pain points in the technical field of three-dimensional expansion of mesenchymal stem cells are solved.
Disclosure of Invention
Aiming at the two problems existing in the three-dimensional mesenchymal stem cell amplification technology, the invention provides an innovative scheme, and designs the macroporous hydrogel which can respond to degradation under mild and rapid conditions by utilizing the advantages of large space of the macroporous hydrogel and the property of efficient enzyme digestion activity of tobacco etch virus protease.
In order to solve the problems, the invention adopts the following scheme: the macroporous hydrogel is characterized in that a macroporous structure space-containing three-dimensional network which is formed by copolymerization of hydrophilic monomer molecules and enzyme response crosslinking molecules and can be rapidly degraded by tobacco etch virus protease.
Further, the enzyme-responsive rapidly-degrading macroporous hydrogel is characterized in that the hydrophilic monomer molecule is acrylamide; the enzyme response crosslinking molecule is prepared by adding four-arm polyethylene glycol-maleimido and cysteine-containing tobacco etch virus protease response short peptide
Furthermore, the enzyme response quick degradation macroporous hydrogel is characterized in that the enzyme response crosslinking molecule also comprises cell adhesion short peptide, namely the enzyme response crosslinking molecule is prepared from quadrifilar polyethylene glycol-maleimide, cysteine-containing tobacco etch virus protease response short peptide and cell adhesion short peptide through addition reaction.
Further, the enzyme response rapid degradation macroporous hydrogel is characterized in that the cysteine-containing tobacco etch virus protease response short peptide is a short peptide with an acrylic acid connected at the tail end, and the sequence of the short peptide is glutamic acid-asparagine-leucine-tyrosine-phenylalanine-glutamine-serine-cysteine (ENLYFQSC).
Further, the enzyme-responsive rapidly degrading macroporous hydrogel is characterized in that the cell adhesion oligopeptide monomer is a specific oligopeptide with sulfhydryl groups, and the sequence of the specific oligopeptide is cysteine-arginine-glycine-aspartic acid (CRGD).
The preparation method of the macroporous hydrogel with the rapid degradation of enzyme response is characterized by comprising the following steps:
the four-arm polyethylene glycol-maleamide and cysteine-containing tobacco etch virus protease response short peptide are mixed according to the mole ratio of 1.5:10, mixing the mixture in DMSO solvent in proportion, reacting at room temperature, removing unreacted reactants through dialysis, and freeze-drying liquid obtained through dialysis to obtain enzyme response crosslinking molecules;
the acrylamide is weighed according to the amount of the macroporous hydrogel prepared as required;
adding the prepared enzyme response crosslinking molecules and acrylamide into a mixed solvent of glucan and polyethylene glycol, adding a photoinitiator, performing oscillation to form liquid-liquid phase separation, and then inducing the acrylamide dissolved in the polyethylene glycol phase to polymerize under the ultraviolet condition, wherein the enzyme response crosslinking molecules participate in crosslinking of a network to obtain hydrogel (the acrylamide is pre-dissolved in the polyethylene glycol phase, and when two phases are mixed for ten seconds, only a very small part of acrylamide can diffuse into the glucan, and although the small part of acrylamide is polymerized under the initiator, a crosslinked network cannot be formed);
soaking the hydrogel in water for swelling, wherein in the swelling process, polyethylene glycol solid (polyethylene glycol solid is a hydrogel network and comprises polyacrylamide and cross-linked molecules) is swelled by absorbing water, and simultaneously dextran and polyethylene glycol liquid phases diffuse towards the outside of the hydrogel, and after the dextran and polyethylene glycol liquid phases are completely replaced by water, the large pore diameter inside the hydrogel is formed, so that the macroporous hydrogel is obtained.
Furthermore, the preparation method of the macroporous hydrogel with the rapid degradation of the enzyme response is characterized in that cell adhesion short peptide is added into the DMSO solvent in the first step, and the cell adhesion short peptide participates in the addition reaction of the quadrifilar polyethylene glycol-maleimide and the cysteine-containing tobacco etch virus protease response short peptide.
The application of enzyme-responsive fast-degrading macroporous hydrogel is characterized in that the enzyme-responsive fast-degrading macroporous hydrogel is applied to aspects of cell culture, proliferation, migration, differentiation and collection.
The application of the macroporous hydrogel with the rapid enzyme response degradation is characterized in that the macroporous hydrogel with the rapid enzyme response degradation is applied to the fields of biological medicine, tissue engineering and medical cosmetology.
Tetraarm polyethylene glycol-maleamide (molecular weight 20000), cysteine-containing tobacco etch virus protease response short peptide monomer and cell adhesion short peptide were mixed in a molar ratio of 1.5:10:1 (the ratio is 1:4 in theory as long as the excess of the cysteine-containing tobacco etch virus protease response short peptide is satisfied), and the reaction is carried out at room temperature, then the dialysis is carried out on the synthesized product by using a dialysis bag with a cut-off molecular weight of 3.5kDa, the dialysis is carried out in deionized water, unreacted reactants are removed, and the liquid obtained by dialysis is freeze-dried, thus obtaining the enzyme response cross-linked molecules. The cysteine-containing tobacco etch virus protease response short peptide monomer is a short peptide with the end connected with acrylic acid, and the sequence of the short peptide is glutamic acid-asparagine-leucine-tyrosine-phenylalanine-glutamine-serine-cysteine (ENLYFQSC), and the short peptide is prepared by using a solid phase synthesis method; the cell adhesion oligopeptide monomer is a specific oligopeptide with sulfhydryl group, and the sequence of the specific oligopeptide is cysteine-arginine-glycine-aspartic acid (CRGD), and the specific oligopeptide is prepared by using a solid phase synthesis method.
The hydrogel has a macroporous structure inside, and is formed by liquid-liquid phase separation based on dextran and polyethylene glycol. 200mg/ml dextran (molecular weight 40000) and 140mg/ml polyethylene glycol (molecular weight 20000) in a volume ratio of 1:1, after mixing and shaking, liquid-liquid phase separation is formed, wherein the dextran is uniformly dispersed in the polyethylene glycol continuous phase in the form of liquid drops. The hydrophilic monomer molecule is acrylamide, and the gel forming is to induce the polymerization of the acrylamide dissolved in a polyethylene glycol phase under the ultraviolet condition by a photoinitiator, and the crosslinking point participates in the crosslinking of a network.
The invention combines the advantages of a bracket-free and a hydrogel bracket-based mesenchymal stem cell amplification strategy, and invents a macroporous hydrogel capable of being enzymatically degraded so as to realize the efficient amplification and the nondestructive harvest of the mesenchymal stem cells. The mesenchymal stem cells obtained by the method after amplification are harvested in the form of cell spheres, and the outer layers of the cell spheres are shells rich in natural extracellular matrix. The mesenchymal stem cell pellet has the advantages of high maintenance of the stem property, improved differentiation potential and obviously improved relevant important functions. The invention has great promotion effect on breaking through the double classical problems of quantity and quality existing in the technical field of stem cell expansion. And has potential application value in the aspects of biological medicine, tissue engineering, medical cosmetology and the like.
The invention has the following technical effects: 1. compared with the traditional three-dimensional culture hydrogel, the average diameter of a large number of large holes uniformly distributed in the macroporous hydrogel is controllable, can reach hundred micrometers, provides sufficient space for proliferation of mesenchymal stem cells, and can realize 27 times of proliferation of the mesenchymal stem cells in a 14-day culture period. And, cell binding sequence RGD of covalent anchor in the macroporous hydrogel network, have effectively avoided the anoikis of mesenchymal stem cells, show extremely high cell survival rate.
2. Compared with the traditional three-dimensional culture method, the macroporous hydrogel effectively combines the advantages of the bracket-free three-dimensional culture and the bracket-based three-dimensional culture, and is characterized in that: first, cells aggregate into cell spheres in the macroporous cavity; second, the cell sphere is anchored to the hydrogel scaffold by RGD, while allowing the cell to migrate along the scaffold inside the hydrogel.
3. In contrast to traditional post-expansion hydrogel-based stem cell harvesting methods, the harvesting of cells in the present invention is based on tobacco etch virus protease-mediated degradation of hydrogels. Due to the high cleavage efficiency of tobacco etch virus protease and mild reaction conditions, the stem cells of the invention are harvested rapidly without loss of activity.
4. Compared with the traditional harvesting method based on trypsin, the protease based on the tobacco etch virus is an orthogonal protease, has high specificity in enzyme digestion reaction, and the extracellular matrix secreted in the stem cell proliferation process is highly reserved in the hydrogel degradation process.
5. Compared with the traditional mesenchymal stem cell pellet, the mesenchymal stem cells harvested in the invention are the stem cell pellets with high stem maintenance, high differentiation potential, enhanced function and high extracellular matrix retention.
6. The pore size of the macroporous hydrogel can be regulated and controlled by regulating the concentration ratio of polyethylene glycol and dextran in liquid-liquid phase separation, the pore size in the hydrogel is in direct proportion to the ratio of dextran to polyethylene glycol solution, and the more the dextran, the larger the pore size. The method comprises the steps of carrying out a first treatment on the surface of the The mechanical property of the hydrogel can be regulated and controlled by adjusting the concentration of the crosslinking points, and the higher the mass ratio of crosslinking molecules in the gel forming mixed solution is, the higher the modulus of the obtained hydrogel is.
7. The enzyme response quick degradation macroporous hydrogel of the invention has breakthrough application value in the aspects of cell culture, proliferation, migration, differentiation, collection and the like. And has great potential application value in the aspects of biological medicine, tissue engineering, medical cosmetology and the like.
Drawings
FIG. 1 is a schematic diagram of a macroporous hydrogel with rapid degradation of the enzyme response.
FIG. 2 is a schematic representation of the preparation of a macroporous hydrogel with rapid degradation of the enzyme response.
FIG. 3 is a structural characterization of a macroporous hydrogel that rapidly degrades in response to an enzyme.
FIG. 4 is a degradation characterization of an enzyme response to a rapidly degrading macroporous hydrogel.
FIG. 5 effect of tobacco etch virus protease on mesenchymal stem cell activity.
FIG. 6 shows a 14-day proliferation curve of mesenchymal stem cells.
Figure 7 mesenchymal stem cells maintained stem properties after 14 days of proliferation and in situ differentiation potential inside hydrogels.
FIG. 8 shows a schematic representation of the harvesting of mesenchymal stem cell spheres and characterization of the dryness.
FIG. 9 characterization of important performance gene expression associated with mesenchymal stem cell spheres.
FIG. 10 effect of concentration ratio of polyethylene glycol and dextran on pore size of macroporous hydrogel in liquid-liquid phase separation.
FIG. 11 effect of concentration of cross-linking points on mechanical properties of hydrogels.
Description of the embodiments
The invention is described in further detail below with reference to the accompanying drawings.
A macroporous hydrogel with enzyme response and rapid degradation is characterized in that a macroporous structure space-containing three-dimensional network which is formed by copolymerization of hydrophilic monomer molecules and enzyme response crosslinking molecules and can be rapidly degraded by tobacco etch virus protease; the enzyme response cross-linking molecule is a polypeptide-containing synthetic multi-arm polymer responded by tobacco etch virus protease, and can be catalyzed and cut off by the tobacco etch virus protease enzyme, as shown in figure 1.
The enzyme response crosslinking molecule is characterized by comprising the following preparation method: tetraarm polyethylene glycol-maleamide (molecular weight 20000), cysteine-containing tobacco etch virus protease response short peptide monomer and cell adhesion short peptide were mixed in a molar ratio of 1.5:10:1 in DMSO solvent, and dialyzing the synthesized product by using a dialysis bag with a cut-off molecular weight of 3.5kDa, dialyzing in deionized water, removing unreacted reactants, and freeze-drying the liquid obtained by dialysis to obtain the enzyme response cross-linked molecules. The cysteine-containing tobacco etch virus protease response short peptide monomer is a short peptide with the end connected with acrylic acid, and the sequence of the short peptide is glutamic acid-asparagine-leucine-tyrosine-phenylalanine-glutamine-serine-cysteine (ENLYFQSC), and the short peptide is prepared by using a solid phase synthesis method; the cell adhesion oligopeptide monomer is a specific oligopeptide with sulfhydryl group, and the sequence of the specific oligopeptide is cysteine-arginine-glycine-aspartic acid (CRGD), and the specific oligopeptide is prepared by using a solid phase synthesis method.
A macroporous hydrogel with fast degradation of enzyme response, which is characterized in that a macroporous structure exists in the hydrogel and is formed by liquid-liquid phase separation based on dextran and polyethylene glycol. Liquid-liquid phase separation formation conditions characterized in that 200mg/ml dextran (molecular weight 40000) and 140mg/ml polyethylene glycol (molecular weight 20000) are present in a volume ratio of 1:1, after mixing and shaking, liquid-liquid phase separation is formed, wherein the dextran is uniformly dispersed in the polyethylene glycol continuous phase in the form of liquid drops.
The macroporous hydrogel is characterized in that hydrophilic monomer molecules are acrylamide, gel formation is performed by inducing acrylamide dissolved in a polyethylene glycol phase to polymerize through a photoinitiator under an ultraviolet condition, and crosslinking points (enzyme response crosslinking molecules) participate in crosslinking of a network.
The preparation process of the macroporous hydrogel with the rapid degradation of the enzyme response is shown in fig. 2: polyethylene glycol solution, dextran solution and photoinitiator solution are mixed according to the volume ratio of 10:10:1, oscillating to form liquid-liquid phase separation, transferring the formed liquid-liquid phase separation mixture to a silica gel mold, and demolding after 5 minutes of irradiation of an ultraviolet lamp to obtain the hydrogel. The hydrogel swells by soaking in water. During the swelling process, the polyethylene glycol solid phase absorbs water and swells, and the dextran liquid diffuses towards the outside of the hydrogel. After the dextran is completely replaced by water, the large pore size inside the hydrogel is formed.
The following examples are set forth to demonstrate the various properties of the present invention.
Example 1 characterization of the enzyme response degrading macroporous hydrogel of the invention the internal macroporous structure was characterized.
In the invention, the enzyme response degradation macroporous hydrogel internal macroporous structure is characterized by an optical microscope and a scanning electron microscope. As shown in fig. 3, the macroporous hydrogel can observe a closely adjacent macroporous structure under an optical microscope, and is a spherical cavity structure; the same structure can be observed as a result of the electron scanning microscope imaging. Through statistics, the distribution of the pore sizes of the macropores is normal, and the distribution is shown that most of the pore diameters are concentrated in a range of 100-500 mu m, and the average pore diameter is 318 mu m.
Example 2 enzyme-responsive degradation of macroporous hydrogels of the present invention rapidly degraded in tobacco etch virus protease solutions.
As shown in FIG. 4, the hydrogel having a volume of 500. Mu.l after swelling was transferred to 2ml of a solution of tobacco etch virus protease having a concentration of 300. Mu.g/ml, and slowly shaken at a uniform speed at room temperature, the hydrogel was degraded to 50% after 30 minutes, degraded to 10% after one hour, and complete degradation was achieved within 2 hours. In addition, the tobacco etch virus protease has strong specificity and high enzyme activity, and is an orthogonal enzyme for extracellular matrix integrins, so that the cell microenvironment is not damaged. In addition, the effect of tobacco etch virus protease on cell activity was minimal, as shown in FIG. 5, with tobacco etch virus protease concentrations up to 1mg/ml having minimal effect on cell activity. Therefore, the hydrogel with mild degradation conditions and rapid degradation has important advantages in accordance with rapid cell harvest from the hydrogel.
Example 3 application of the enzyme-responsive degradable macroporous hydrogels of the present invention to expansion of mesenchymal stem cells.
In the invention, an enzyme-responsive degradable macroporous hydrogel is applied to the expansion of mesenchymal stem cells. In particular, cells are seeded into the cavities inside the hydrogel by injection, as shown in fig. 6 a. Mesenchymal stem cells aggregate into cytoballs in the cavity firstly through cell-cell interaction, and are anchored to the hydrogel scaffold through RGD sites exposed on the inner wall of the cavity secondly. As shown in fig. 6b, in the subsequent proliferation phase, the proliferation of mesenchymal stem cells is manifested in two aspects: expansion of the cell sphere caused by cell proliferation inside the cell sphere; and cells outside the cell sphere migrate and proliferate along the hydrogel scaffold. We quantified the number of mesenchymal stem cells on days 1, 4, 7, 10, 14, respectively, during the proliferation of mesenchymal stem, and as a result, as shown in fig. 6c, the mesenchymal stem cells showed a steady proliferation of the whole in the hydrogel over time, with a proliferation rate of up to 27 times over 14 days. Furthermore, we assessed the viable and dead state of cells within the hydrogels on day 1 as well as day 14 by a viable and dead cell staining technique. As a result, as shown in FIG. 6d, the cells were in a good state of survival in the interior of the hydrogel. The results show that the enzyme response of the enzyme is high in biological affinity for degrading the macroporous hydrogel and has satisfactory amplification effect on mesenchymal stem cells.
Example 4 enzymatic response degrading macroporous hydrogels of the present invention favored mesenchymal stem cell maintenance.
The enzyme response degradation macroporous hydrogel is applied to the expansion of mesenchymal stem cells, and is favorable for the maintenance of the stem property of the mesenchymal stem cells. This example provides an assessment of the maintenance of stem properties of mesenchymal stem cells expanded by enzymatic degradation of macroporous hydrogels of the present invention. Specifically, after the mesenchymal stem cells are amplified for 14 days, the hydrogel and the whole cells are subjected to fluorescence co-calibration of OCT4 and SOX2 in the mesenchymal stem cells by an immunofluorescence technology. We classified the cells within the hydrogel according to different proliferation morphologies: the cell pellet growth zone and the cell migration proliferation zone were evaluated for the stem properties of cells in both zones. As shown in fig. 7a, cells in both regions showed high levels of OCT4 and SOX2 expression, indicating that the stem properties of mesenchymal stem cells remained intact during expansion. Further, to verify the differentiation potential of mesenchymal stem cells after expansion, we performed in situ induced differentiation. Specifically, after mesenchymal stem cells are amplified for 14 days, osteoblast differentiation and adipogenic differentiation induction factors are respectively added into a culture medium, and after 14 days and 7 days of induction, immunofluorescence calibration is carried out on the whole hydrogel. For osteogenic differentiation, we performed fluorescent co-targeting of Osteocalcin (OCN) and collagen (Col 1A) on cells; for adipogenic differentiation, we performed fluorescent labeling of cells with lipid-droplet coat protein (Perilipin). The results are shown in fig. 7b, where cells in both regions exhibit high differentiation potential for osteogenesis as well as adipogenesis. Thus, the enzyme-responsive degradable macroporous hydrogels of the present invention facilitate the maintenance of stem properties of mesenchymal stem cells during expansion.
Example 5 enzymatic response degradation of macroporous hydrogels of the present invention achieved non-destructive harvesting of mesenchymal stem cells after expansion.
The enzyme response degradation macroporous hydrogel can be rapidly degraded by tobacco etch virus protease to release the amplified mesenchymal stem cells, so that the harvesting of the cells is greatly facilitated. First, as illustrated in fig. 8a, mesenchymal stem cells were harvested in the form of cytoballs. As shown in fig. 8b, after the hydrogel is degraded, the mesenchymal stem cell pellet is released and suspended in the culture solution. Next, the mesenchymal stem cell pellet has a good re-adherence function as shown in fig. 8 c. To verify the differentiation potential of the harvested mesenchymal stem cells, we performed differentiation induction of osteogenesis and adipogenesis, respectively. The results are shown in fig. 8d, in which mesenchymal stem cells obtained by hydrogel degradation in the present invention exhibit high osteogenic and adipogenic differentiation potential. Again, we compared the potential of conventional two-way expansion of the resulting mesenchymal stem cells in these respects. As shown in fig. 8e, the mesenchymal stem cells obtained by degrading the macroporous hydrogel through the enzyme response in the present invention are significantly stronger in adipogenic and osteogenic differentiation potential than those obtained by the conventional method. Finally, an important factor in the present invention, which is embodied in the non-destructive harvesting and expansion of mesenchymal stem cells, is that the extracellular matrix is well preserved and tightly packed in the form of a shell layer on the outer layer of the cytosphere, as shown in fig. 8 f. Compared with the traditional method for damaging the extracellular matrix based on trypsin harvesting, the tobacco etch virus protease utilized in the invention only degrades the hydrogel and does not damage the extracellular matrix, thereby realizing the nondestructive harvesting of the double layers of the cell and the extracellular matrix. The important factor of intact extracellular matrix will largely ensure the enhancement of the versatile function of mesenchymal stem cells in downstream applications.
Example 6 enzymatic response degradation of macroporous hydrogels of the present invention achieved enhancement of mesenchymal stem cell stem properties and multifaceted performance.
In order to evaluate the stem properties of mesenchymal stem cell spheres obtained by enzymatic response degradation of perforated hydrogels in the present invention and other relevant important clinical functions, we compared the expression levels of mesenchymal stem cells before and after amplification at genes related to these functions by PCR array technology. As a result, as shown in FIG. 9a, mesenchymal stem cell spheres obtained by degrading perforated hydrogel in response to an enzyme in the present invention were greatly changed in gene expression level as compared with that before amplification. By gene functional analysis, we found that changes in these important genes, especially up-regulated gene expression, corresponded to the following important functions of mesenchymal stem cells: the overall elevation of dry function, cartilage developmental function, skeletal system developmental function, inflammatory response function, angiogenic function, and immune response function is summarized in fig. 9b.
Example 6 the pore size and the mechanical properties of the macroporous hydrogels of the present invention were freely adjustable.
The pore size of the macroporous hydrogel can be regulated and controlled by regulating the concentration ratio of polyethylene glycol and dextran in liquid-liquid phase separation, the pore size in the hydrogel is in direct proportion to the ratio of dextran to polyethylene glycol solution, and the more the dextran, the larger the pore size is, as shown in figure 10. The mechanical properties of the hydrogel can be regulated and controlled by adjusting the concentration of the crosslinking points, and the higher the mass ratio of crosslinking molecules in the gel forming mixed solution is, the higher the modulus of the obtained hydrogel is, as shown in fig. 11.
Claims (9)
1. The macroporous hydrogel is characterized by being formed by copolymerization of hydrophilic monomer molecules and enzyme response crosslinking molecules, and containing a macroporous structure space three-dimensional network.
2. The enzyme responsive rapidly degrading macroporous hydrogel of claim 1, wherein the hydrophilic monomer molecule is acrylamide; the enzyme response crosslinking molecule is prepared by adding a short peptide of the protease response of the cysteine-containing tobacco etch virus of quadrifilar polyethylene glycol-maleamide.
3. The enzyme-responsive rapidly degrading macroporous hydrogel according to claim 2, wherein the enzyme-responsive cross-linking molecule further comprises a cell adhesion oligopeptide, wherein the enzyme-responsive cross-linking molecule is prepared from a four-arm polyethylene glycol-maleimido, a cysteine-containing tobacco etch virus protease-responsive oligopeptide and a cell adhesion oligopeptide by an addition reaction.
4. The enzyme-responsive rapidly degrading macroporous hydrogel according to claim 2, wherein the cysteine-containing tobacco etch virus protease responsive oligopeptide is an acrylic acid terminated oligopeptide having the sequence glutamic acid-asparagine-leucine-tyrosine-phenylalanine-glutamine-serine-cysteine (ENLYFQSC).
5. The enzyme-responsive rapidly degrading macroporous hydrogel according to claim 3, wherein the cell-adhering oligopeptide monomer is a thiol-bearing specific oligopeptide having the sequence cysteine-arginine-glycine-aspartic acid (CRGD).
6. The preparation method of the macroporous hydrogel with the rapid degradation of enzyme response is characterized by comprising the following steps:
the four-arm polyethylene glycol-maleamide and cysteine-containing tobacco etch virus protease response short peptide are mixed according to the mole ratio of 1.5:10, mixing the mixture in DMSO solvent in proportion, reacting at room temperature, removing unreacted reactants through dialysis, and freeze-drying liquid obtained through dialysis to obtain enzyme response crosslinking molecules;
the acrylamide is weighed according to the amount of the macroporous hydrogel prepared as required;
adding the prepared enzyme response crosslinking molecules and acrylamide into a mixed solvent of glucan and polyethylene glycol, adding a photoinitiator, performing oscillation to form liquid-liquid phase separation, and then inducing the acrylamide dissolved in the polyethylene glycol phase to polymerize under the ultraviolet condition, wherein the enzyme response crosslinking molecules participate in crosslinking of a network to obtain hydrogel;
soaking the hydrogel in water for swelling, absorbing water by the polyethylene glycol solid phase for swelling, diffusing the dextran and the polyethylene glycol liquid phase to the outside of the hydrogel, and forming a large pore diameter inside the hydrogel after the dextran and the polyethylene glycol liquid phase are completely replaced by water to obtain the macroporous hydrogel.
7. The method for preparing the enzyme-responsive fast-degrading macroporous hydrogel according to claim 6, wherein a cell adhesion oligopeptide is added into the DMSO solvent in the first step, and the cell adhesion oligopeptide participates in the addition reaction of the quadrifilar polyethylene glycol-maleimide and the cysteine-containing tobacco etch virus protease-responsive oligopeptide.
8. Use of the enzyme-responsive fast degrading macroporous hydrogel according to claim 1, wherein the enzyme-responsive fast degrading macroporous hydrogel is used in cell culture, proliferation, migration, differentiation and collection.
9. Use of the enzyme-responsive rapidly degrading macroporous hydrogel according to claim 1, wherein the enzyme-responsive rapidly degrading macroporous hydrogel is used in biomedical, tissue engineering and medical cosmetic fields.
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