Cellulose cross-linked porcine small intestinal submucosa hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to cellulose cross-linked porcine small intestinal submucosa hydrogel as well as a preparation method and application thereof. The cellulose cross-linked pig small intestine submucosa hydrogel comprises a pig small intestine submucosa hydrogel and dialdehyde cellulose cross-linked with the pig small intestine submucosa hydrogel, the crosslinking is that the primary amino group of the porcine small intestinal submucosa hydrogel and the aldehyde group of the dialdehyde cellulose form an imine bond. The cellulose cross-linked porcine small intestinal submucosa hydrogel has excellent mechanical properties and biocompatibility, and can be better used for promoting stem cell osteogenic differentiation and preparing refined organoid structures.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a cellulose-crosslinked porcine small intestinal submucosa hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] Organoids are cell aggregates derived from stem cells or progenitor cells, and are miniature models based on an in vitro 3D cell culture system that can reflect the functions of some organs in vivo. Compared with traditional two-dimensional cell culture, organoids have the advantages of a more physiological cell composition and behavior, a more stable genome, being more suitable for biological transfection and high-throughput screening, etc. The most fundamental technology for constructing organoids is the in vitro 3D culture of cells. The microenvironment (culture matrix) in which stem cells are located plays a crucial role in their growth and differentiation. Cells undergo a series of biological processes under the regulation of the microenvironment and are gradually induced to differentiate into specific three-dimensional structures. Therefore, mimicking the in vivo environment to allow cells to grow, develop, and differentiate under conditions close to the physiological environment is the key to precisely constructing an organoid model.

[0003] The extracellular matrix (ECM) is a dynamic three-dimensional network composed of macromolecules synthesized and secreted by cells into the extracellular space, distributed on the cell surface or between cells, and its main components are polysaccharides and proteins or proteoglycans. The ECM wraps cells, not only providing mechanical support and physical strength for cells, but also participating in the regulation of signal transduction, cell adhesion, spreading, differentiation and other behaviors. Constructing ECM in vitro to simulate the in vivo cell microenvironment can make the growth and differentiation of cells closer to the in vivo real situation, and then replicate the complex spatial morphology of tissues, showing the interactions and spatial position morphologies between cells and between cells and their surrounding matrix.

[0004] In recent years, researchers have developed a variety of ECM materials for organoid culture, including natural decellularized ECM and synthetic ECM-mimicking materials. Decellularized ECM contains more active substances such as cytokines than synthetic ECM-mimicking materials and is currently the preferred material for in vitro cell culture and organoid manufacturing. Among them, the gel-like matrix glue formed by the basement membrane of porcine small intestinal submucosa (SIS) and the matrix glue (Matrigel) purified from Engelbreth-Holm-Swarm mouse sarcoma are the most widely used. Compared with Matrigel, SIS has a lower production cost, higher biological activity, can also be modified and remodeled by host cells, and shows higher phenotypic protein expression. However, the mechanical strength of the porcine small intestinal submucosa hydrogel is poor, which limits its popularization in practical applications. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a cellulose-crosslinked porcine small intestinal submucosa hydrogel and its preparation method and application. The cellulose-crosslinked porcine small intestinal submucosa hydrogel has excellent mechanical properties and biocompatibility, and can be better used to promote stem cell osteogenic differentiation and prepare refined organoid structures.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a cellulose-crosslinked porcine small intestinal submucosa hydrogel, including a porcine small intestinal submucosa hydrogel and dialdehyde cellulose crosslinked with the porcine small intestinal submucosa hydrogel; the crosslinking is that the primary amino group of the porcine small intestinal submucosa hydrogel and the aldehyde group of the dialdehyde cellulose form an imine bond.

[0008] Preferably, the dialdehyde cellulose is 2,3-dialdehyde cellulose.

[0009] Preferably, the molar ratio of the primary amino group of the porcine small intestinal submucosa in the porcine small intestinal submucosa hydrogel to the aldehyde group of the dialdehyde cellulose is (10-20):1.

[0010] Preferably, the content of the primary amino group of the porcine small intestinal submucosa in the porcine small intestinal submucosa hydrogel is 18-20 mmol / g; the content of the aldehyde group in the dialdehyde cellulose is 6-8 mmol / g.

[0011] The present invention also provides a preparation method of the cellulose-crosslinked porcine small intestinal submucosa hydrogel described in the above technical solution, including the following steps:

[0012] Mix the porcine small intestinal submucosa buffer solution and the dialdehyde cellulose buffer solution, adjust the pH value of the obtained mixed solution to 7.0-7.4, and then carry out a Schiff base reaction to obtain the cellulose-crosslinked porcine small intestinal submucosa hydrogel. The dialdehyde cellulose buffer solution is a phosphate buffer solution of dialdehyde cellulose.

[0013] Preferably, the concentration of the porcine small intestinal submucosa in the mixed solution is 1-3% (w / V).

[0014] Preferably, the temperature of the Schiff base reaction is 25-37 °C; the time of the Schiff base reaction is 15-30 min.

[0015] Preferably, the preparation method of the porcine small intestinal submucosa buffer solution includes the following steps: sequentially inactivate viruses, degrease, decellularize and dry the porcine small intestinal submucosa to obtain a porcine small intestinal submucosa acellular matrix;

[0016] The acellular matrix of porcine small intestinal submucosa is subjected to protease enzymolysis. After the obtained enzymolysis solution is freeze-dried, it is successively crushed and sieved to obtain freeze-dried powder of porcine small intestinal submucosa;

[0017] The freeze-dried powder of porcine small intestinal submucosa is dissolved in a mixed solution of acetic acid and phosphate buffered saline to obtain a buffered solution of porcine small intestinal submucosa.

[0018] The present invention also provides the application of the cellulose-crosslinked porcine small intestinal submucosa hydrogel described in the above technical solution or the cellulose-crosslinked porcine small intestinal submucosa hydrogel prepared by the preparation method described in the above technical solution in the preparation of biomaterials.

[0019] Preferably, the biomaterial includes one or several of products for promoting osteogenic differentiation of mesenchymal stem cells, products for three-dimensional culture of mesenchymal stem cells, products for organoid culture, and products for joint filling.

[0020] The present invention provides a cellulose-crosslinked porcine small intestinal submucosa hydrogel, which includes a porcine small intestinal submucosa hydrogel and dialdehyde cellulose crosslinked with the porcine small intestinal submucosa hydrogel; the crosslinking is that the primary amino group of the porcine small intestinal submucosa hydrogel and the aldehyde group of the dialdehyde cellulose form an imine bond. In the present invention, dialdehyde cellulose is a natural polymer composed of glucose units, and its molecular structure contains abundant free hydroxyl groups, which can participate in intermolecular interactions and form hydrogen bonds, endowing dialdehyde cellulose with excellent mechanical properties; the porcine small intestinal submucosa hydrogel contains abundant collagen, sulfated glycosaminoglycan components and a large number of unbound amino groups. Dialdehyde cellulose can form hydrogen bonds with the free amino groups inside the porcine small intestinal submucosa hydrogel, and can also undergo a Schiff base reaction with the free amino groups (primary amino groups) to generate imine bonds to form a crosslinked network. Therefore, adding dialdehyde cellulose to the porcine small intestinal submucosa hydrogel to re-crosslink it into a gel makes the internal structure of the porcine small intestinal submucosa hydrogel more dense, increases the mechanical strength of the porcine small intestinal submucosa hydrogel, and further promotes the differentiation of cells towards the osteogenic direction; the present invention prepares hydrogels with different hardnesses by adding different amounts of dialdehyde cellulose to meet the different matrix hardnesses required by different cells and improve the disadvantage of single mechanical properties. Moreover, both the porcine small intestinal submucosa and dialdehyde cellulose have good biocompatibility. The crosslinking of the two not only improves the hardness of the porcine small intestinal submucosa hydrogel, but also has low cytotoxicity, and can be better applied to promote the osteogenic differentiation of stem cells. Different from synthetic ECM-mimicking materials, natural acellular ECM (porcine small intestinal submucosa hydrogel) contains more active substances such as cytokines than synthetic ECM-mimicking materials, and is the preferred material for current in vitro cell culture and organoid fabrication. The cellulose-crosslinked porcine small intestinal submucosa hydrogel makes the organoid structure more refined, its functionality is improved, and thus the development of complex organoid 3D culture is promoted.

[0021] The present invention also provides a preparation method of the above-mentioned cellulose-crosslinked porcine small intestinal submucosa hydrogel. The raw materials of this method are cheap and easily available, meeting the requirements of green economy. Description of the Drawings

[0022] Figure 1 Fourier transform infrared spectra of the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3, the porcine small intestinal submucosa hydrogel (SIS) of Comparative Example 1, and 2,3-dialdehyde cellulose (DAC) of Comparative Example 2;

[0023] Figure 2 Frequency sweep diagrams of the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3;

[0024] Figure 3Compressive strength diagrams of the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3;

[0025] Figure 4 Compressive modulus diagrams of the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3;

[0026] Figure 5 Degradation result diagrams of the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3 and the porcine small intestinal submucosa hydrogel (SIS) of Comparative Example 1;

[0027] Figure 6 Biocompatibility test result diagrams of the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3, the porcine small intestinal submucosa hydrogel (SIS) of Comparative Example 1, and the blank control group (Ctrl) of human umbilical cord mesenchymal stem cells (UMSC);

[0028] Figure 7 Biocompatibility test result diagrams of the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3, the porcine small intestinal submucosa hydrogel (SIS) of Comparative Example 1, and the blank control group (Ctrl) of L929 mouse fibroblasts (L929);

[0029] Figure 8 Cell adhesion diagrams of the gels (SIS-DAC) with different mechanical properties in Application Examples 1 to 3, the SIS gel of Comparative Application Example 1, and the blank control group (ctrl) of Comparative Application Example 2;

[0030] Figure 9 Result diagrams of the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3 and the porcine small intestinal submucosa hydrogel (SIS) of Comparative Example 1 in promoting osteogenic differentiation of stem cells. Detailed implementation manners

[0031] The present invention provides a cellulose-crosslinked porcine small intestinal submucosa hydrogel, comprising a porcine small intestinal submucosa hydrogel and dialdehyde cellulose crosslinked with the porcine small intestinal submucosa hydrogel; the crosslinking is that the primary amino group of the porcine small intestinal submucosa hydrogel and the aldehyde group of the dialdehyde cellulose form an imine bond.

[0032] Unless otherwise specified, the present invention has no special requirements for the sources of the raw materials used, and commercially available products well-known to those skilled in the art can be used.

[0033] As an implementation manner, the dialdehyde cellulose is 2,3-dialdehyde cellulose.

[0034] As an implementation manner, the preparation method of the 2,3-dialdehyde cellulose comprises the following steps:

[0035] Mix the aqueous suspension of cellulose and an oxidizing agent, carry out a selective oxidation reaction under dark conditions, add ethylene glycol to continue the reaction to terminate the reaction, and after dialysis of the obtained reaction product, dry it to obtain the 2,3-dialdehyde cellulose.

[0036] As an embodiment, the concentration of the aqueous suspension of cellulose is 10 - 15 mg / mL, and in specific examples it is 10 - 12 mg / mL; the preparation method of the aqueous suspension of cellulose is to suspend cellulose in water; the water is deionized water; the oxidizing agent is sodium periodate; the mass ratio of cellulose to the oxidizing agent is 1:(2 - 4), and in specific examples it is 1:(3.5 - 4); the selective oxidation reaction is carried out under the conditions of water bath and stirring; the temperature of the water bath is 25 - 40 °C, and in specific examples it is 30 - 35 °C; the stirring rate is 800 - 1000 rpm, and in specific examples it is 900 - 1000 rpm; the time of the selective oxidation reaction is 2 - 4 h, and in specific examples it is 3 - 4 h; the mass ratio of ethylene glycol to the oxidizing agent is 1 - 1.5:1, and in specific examples it is 1.1 - 1.25:1; the time of the continued reaction is 1 - 1.5 h, and in specific examples it is 1 h; the dialysis is carried out with a dialysis bag having a cut-off molecular weight of 3000 - 5000, and in specific examples it is 3000 - 4000; the dialysis is carried out with stirring in a dialysis solution at room temperature; the stirring rate is 500 - 800 rpm, and in specific examples it is 500 - 600 rpm; the time of the dialysis is 48 - 72 h, and in specific examples it is 60 - 72 h; the dialysis solution is deionized water; the dialysis solution is changed every 12 h during the dialysis process; the drying is freeze-drying; the temperature of the freeze-drying is -40 - 80 °C, and in specific examples it is -60 - 80 °C; the time of the freeze-drying is 48 - 72 h, and in specific examples it is 48 - 60 h.

[0037] In the present invention, the adjacent dihydroxy groups on cellulose are oxidized to adjacent dialdehyde groups by an oxidizing agent. When the oxidizing agent is sodium periodate, the reaction mechanism is as follows: sodium periodate is used to obtain an aldehyde group substituent by selectively oxidizing the hydroxyl groups on the 2,3 positions of the sugar ring of cellulose, and its reaction formula is as follows:

[0038]

[0039] As an embodiment, the molar ratio of the primary amino groups of porcine small intestinal submucosa to the aldehyde groups of dialdehyde cellulose in the porcine small intestinal submucosa hydrogel is (10 - 20):1, and in specific examples it is 10:1, 15:1 or 20:1.

[0040] As an implementation method, the determination method of the primary amino group content of the porcine small intestinal submucosa in the porcine small intestinal submucosa hydrogel is the acid-base titration method. The specific process is as follows: At room temperature, dissolve 5 mg of the porcine small intestinal submucosa hydrogel in 10 mL of 0.1 mol / L hydrochloric acid solution, then add calibrated 0.1 mol / L sodium hydroxide solution for titration, and continuously monitor with a pH meter. When the pH value approaches 2, record the pH value and the volume of the added NaOH solution until the pH value approaches 3. Record a total of 5 - 6 groups of data and substitute them into Formula 1 and Formula 2 to obtain V e1 , in the presence of two drops of phenolphthalein indicator, add more 0.1 mol / L sodium hydroxide solution until the phenolphthalein turns red, and record the volume as V e2 , and calculate the amino group content according to the formula.

[0041]

[0042] F(V) = V e1 -kV Formula 2

[0043]

[0044] where F(V) is the A. Johansson function expression, V0 is the volume of HCl (mL), V is the volume of the NaOH standard solution (mL), N is the concentration of the NaOH standard solution (mol / L), [H + is the concentration of H + , [OH - is the concentration of OH - , m is the mass of the porcine small intestinal submucosa hydrogel (SIS) (g), and k is the slope.

[0045] As an implementation method, the content of the primary amino group of the porcine small intestinal submucosa in the porcine small intestinal submucosa hydrogel is 18 - 20 mmol / g, and in specific examples, it is 18.5 - 19.8 mmol / g.

[0046] As an implementation method, the determination method of the aldehyde group content in the dialdehyde cellulose is the hydroxylamine hydrochloride titration method. The specific process is as follows: Take accurately weighed dialdehyde cellulose and disperse it in water, heat and dissolve it at 60 °C to obtain a dialdehyde cellulose buffer solution. Add a certain amount of accurately weighed hydroxylamine hydrochloride to dissolve in the dialdehyde cellulose buffer solution, then add methyl orange solution and stir to mix evenly. Stir at room temperature for 3 h to obtain a pink mixed solution. Titrate the pink mixed solution with a calibrated 0.1 mol / L NaOH solution and record the volume of the added NaOH solution until the solution changes from orange to yellow; each sample is tested 3 times and the average value is taken.

[0047] The aldehyde group content ([-CHO], mmol / g) in dialdehyde cellulose is calculated by the formula [-CHO] = ΔV × cNaOH / mDAC, where ΔV is the volume (mL) of NaOH solution consumed at the titration end point, cNaOH is the concentration (mol / L) of the NaOH solution, and mDAC is the weight (g) of the weighed dialdehyde cellulose.

[0048] As an implementation manner, the aldehyde group content in the aldehyde cellulose is 6 - 8 mmol / g, and in specific embodiments, it is 6.5 - 7.5 mmol / g.

[0049] Cellulose is a natural polymer composed of glucose, which is widely distributed in nature and has advantages such as good biocompatibility, renewability, and biodegradability. Cellulose itself belongs to polysaccharides, so it has extremely high affinity with the ECM rich in glycosaminoglycans. In addition, most cellulose is a high - molecular linear structure, and the molecular chain structure contains a large number of free hydroxyl groups, which can attract each other and form hydrogen bonds, having excellent mechanical properties and modifiability. Based on the fact that the porcine small intestinal submucosa hydrogel contains rich collagen, sulfated glycosaminoglycan components, and a large number of unbound amino groups, a certain amount of polymer containing aldehyde groups can form hydrogen bonds with the free amino groups inside the SIS, and can also react with the free amino groups to form Schiff base reactions to generate enamine bonds, thus forming a cross - linked network. Therefore, adding a certain amount of aldehyde - modified cellulose to the porcine small intestinal submucosa hydrogel to re - cross - link and form a gel makes the internal structure of the cellulose - cross - linked porcine small intestinal submucosa hydrogel more dense, increases the mechanical strength of the cellulose - cross - linked porcine small intestinal submucosa hydrogel, improves the hardness of the cellulose - cross - linked porcine small intestinal submucosa hydrogel by increasing the content of dialdehyde cellulose, and both the porcine small intestinal submucosa and cellulose have good biocompatibility and low cytotoxicity, which can further promote the differentiation of cells towards the osteogenic direction, laying a theoretical foundation for the development of organoid technology and significantly improving the application potential of organoids.

[0050] The present invention also provides a preparation method of the above - mentioned cellulose - cross - linked porcine small intestinal submucosa hydrogel, which includes the following steps:

[0051] Mix the porcine small intestinal submucosa buffer solution and the dialdehyde cellulose buffer solution, adjust the pH value of the obtained mixed solution to 7.0 - 7.4, and then carry out the Schiff base reaction to obtain the cellulose - cross - linked porcine small intestinal submucosa hydrogel. The dialdehyde cellulose buffer solution is a phosphate buffer solution of dialdehyde cellulose.

[0052] As an implementation manner, the preparation method of the dialdehyde cellulose buffer solution is as follows: disperse dialdehyde cellulose in a phosphate buffered saline solution, heat and dissolve it to obtain a dialdehyde cellulose buffer solution; the concentration of the phosphate buffered saline solution is 0.01 mol / L; the temperature for heating and dissolving is 60-100 °C, and in specific embodiments it is 70-80 °C; the concentration of dialdehyde cellulose in the dialdehyde cellulose buffer solution is 5.6-7.5 mg / mL, and in specific embodiments it is 6-7 mg / mL. The present invention does not have special limitations on the heating and dissolving time, as long as it can be completely dissolved. The present invention uses a phosphate buffer solution to ensure physiological conditions and is used for subsequent induction of gel self-assembly.

[0053] As an implementation manner, the preparation method of the porcine small intestinal submucosa buffer solution includes the following steps:

[0054] Inactivate viruses, degrease, decellularize and dry porcine small intestinal submucosa in sequence to obtain a decellularized matrix of porcine small intestinal submucosa;

[0055] Perform protease enzymolysis on the decellularized matrix of porcine small intestinal submucosa. After the obtained enzymolysis solution is freeze-dried, it is crushed and sieved in sequence to obtain a freeze-dried powder of porcine small intestinal submucosa;

[0056] Dissolve the freeze-dried powder of porcine small intestinal submucosa in a mixed solution of acetic acid and phosphate buffered saline solution to obtain a porcine small intestinal submucosa buffer solution.

[0057] As an implementation manner, the preparation method of the porcine small intestinal submucosa is as follows: perform pretreatment on porcine small intestine to obtain porcine small intestinal submucosa; the pretreatment is: scrape off the mucosal layer, intestinal muscular layer and serosa layer of the longitudinally opened porcine small intestine segment, and then wash it to obtain porcine small intestinal submucosa; the preparation method of the porcine small intestine segment is: take 100 cm long from 10 cm distal to the duodenum of fresh porcine small intestine and cut it transversely every 10 cm; the scraping method is: place the longitudinally opened porcine small intestine segment on a gauze moistened with physiological saline, wrap the surgical knife handle with gauze with the mucosal layer facing up and scrape off the mucosal layer, and rinse with physiological saline while scraping. When the surface is a milky white translucent basement membrane-like tissue, turn the porcine small intestine over and scrape off the intestinal muscular layer and serosa layer in the same way, and rinse while scraping; the reagent used for washing is physiological saline.

[0058] As an implementation manner, the virus inactivation is ultraviolet lamp irradiation; the time of ultraviolet lamp irradiation is 30-60 min, and in specific embodiments it is 40-50 min; the present invention achieves the purpose of virus inactivation through ultraviolet lamp irradiation.

[0059] As an implementation manner, the degreasing is as follows: soaking the porcine small intestinal submucosa after virus inactivation in a sodium deoxycholate solution, taking it out and then washing; the mass concentration of the sodium deoxycholate solution is 3-5%, specifically 4% in a specific embodiment; the soaking is carried out at room temperature; the soaking time is 3-4 h, specifically 4 h in a specific embodiment; the washing is soaking and washing with ultrapure water; the washing is carried out at room temperature; the washing time is 24-36 h, specifically 24-30 h in a specific embodiment. The present invention has no special limitation on the dosage of the sodium deoxycholate solution, and it is only necessary to completely immerse the porcine small intestinal submucosa after virus inactivation.

[0060] As an implementation manner, the decellularization is as follows: soaking the degreased porcine small intestinal submucosa in a salt solution of deoxyribonuclease I (DNase-I), taking it out and then washing; the enzyme activity of the deoxyribonuclease I is 2000-3000 kU, specifically 2000-2500 kU in a specific embodiment; the concentration of deoxyribonuclease I in the salt solution of deoxyribonuclease I is 0.1-1 mg / mL, specifically 0.2-0.5 mg / mL in a specific embodiment; the salt in the salt solution of deoxyribonuclease I is NaCl; the concentration of the salt in the salt solution of deoxyribonuclease I is 1-2 mol / L, specifically 1 mol / L in a specific embodiment; the soaking is carried out at room temperature; the soaking time is 2-4 h, specifically 3 h in a specific embodiment; the washing is soaking and washing with ultrapure water; the washing is carried out at room temperature; the washing time is 36-48 h, specifically 36 h in a specific embodiment. The present invention has no special limitation on the dosage of the salt solution of deoxyribonuclease I, and it is only necessary to completely immerse the degreased porcine small intestinal submucosa. The present invention removes the residual DNA enzyme by washing.

[0061] As an implementation manner, the drying is freeze-drying; the temperature of the freeze-drying is -40 to -80 °C, specifically -60 to -80 °C in a specific embodiment; the time of the freeze-drying is 48-72 h, specifically 48-60 h in a specific embodiment.

[0062] As an implementation manner, the protease is pepsin; the protease enzymolysis is as follows: after crushing the acellular matrix of porcine small intestinal submucosa, adding a mixed solution of pepsin and acetic acid, and stirring at room temperature for digestion to obtain an enzymolysis solution; the mass ratio of the pepsin to the acellular matrix of porcine small intestinal submucosa is 1:10 - 15, and in specific embodiments, it is 1:10 - 12; the volume concentration of the acetic acid is 0.5 - 1% (V / V), and in specific embodiments, it is 0.5 - 0.8% (V / V); the mass ratio of the acetic acid to the acellular matrix of porcine small intestinal submucosa is 10 - 15:1, and in specific embodiments, it is 10 - 12:1; the rotation speed of the stirring is 500 - 800 rpm, and in specific embodiments, it is 500 - 600 rpm; the digestion time is 48 - 72 h, and in specific embodiments, it is 48 - 60 h.

[0063] As an implementation manner, the temperature of the freeze-drying is -40 - 80 °C, and in specific embodiments, it is -60 - 80 °C; the time of the freeze-drying is 48 - 72 h, and in specific embodiments, it is 48 - 60 h; the crushing is grinding; the sieving is carried out with an 80 - 100 mesh sieve, and in specific embodiments, it is an 80 - 90 mesh sieve.

[0064] As an implementation manner, the concentration of acetic acid in the mixed solution of acetic acid and phosphate buffered saline (PBS) is 0.5 - 1% (V / V), and in specific embodiments, it is 0.5% (V / V); the concentration of the phosphate buffered saline is 0.01 mol / L.

[0065] As an implementation manner, the preparation method of the porcine small intestinal submucosa hydrogel comprises the following steps: after adjusting the pH value of the porcine small intestinal submucosa buffer solution to 7.0 - 7.4, carrying out incubation to obtain the porcine small intestinal submucosa hydrogel.

[0066] As an implementation manner, the pH value adjustment is 7.0 - 7.2; the reagent used for pH value adjustment is sodium hydroxide; the incubation temperature is 30 - 37 °C, and in specific embodiments, it is 37 °C; the incubation time is 15 - 30 min, and in specific embodiments, it is 20 - 30 min.

[0067] As an implementation manner, the mixing is carried out at room temperature; the mixing is vortex mixing; the concentration of the porcine small intestinal submucosa in the mixed solution is 1 - 3% (w / V), and in specific embodiments, it is 2% (w / V); the pH value of the mixed solution is adjusted to 7.0 - 7.2; the reagent used for pH value adjustment is sodium hydroxide; the temperature of the Schiff base reaction is 25 - 37 °C, and in specific embodiments, it is 37 °C; the time of the Schiff base reaction is 15 - 30 min, and in specific embodiments, it is 20 - 30 min.

[0068] In the present invention, dialdehyde cellulose can form hydrogen bonds with free amino groups inside the porcine small intestinal submucosa hydrogel, and can also react with free amino groups to form imine bonds through Schiff base reaction, thereby forming a cross-linked network to form a gel.

[0069] The present invention also provides the use of the cellulose-crosslinked porcine small intestinal submucosa hydrogel described in the above technical solution or the cellulose-crosslinked porcine small intestinal submucosa hydrogel prepared by the preparation method described in the above technical solution in the preparation of biomaterials.

[0070] As an embodiment, the biomaterial includes one or several of products for promoting osteogenic differentiation of mesenchymal stem cells, products for three-dimensional culture of mesenchymal stem cells, products for organoid culture, and products for joint filling. In a specific embodiment, it is a product for promoting osteogenic differentiation of mesenchymal stem cells.

[0071] As an embodiment, the preparation method of the product for promoting osteogenic differentiation of mesenchymal stem cells includes the following steps: spreading the cellulose-crosslinked porcine small intestinal submucosa hydrogel on the bottom of a well plate, sterilizing after gel formation, washing and soaking overnight with phosphate buffered saline (PBS) to make the gel reach physiological pH value, then soaking in complete stem cell medium to replace the absorbed phosphate buffered saline in the gel. Using well-grown passage 5-8 mesenchymal stem cells, after trypsin digestion, inoculating on the gel, and moving the well plate to a cell culture incubator for culture.

[0072] As an embodiment, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells (UMSC); each well in the well plate contains 100 μL of the cellulose-crosslinked porcine small intestinal submucosa hydrogel; the sterilization treatment is ultraviolet irradiation; the time of ultraviolet irradiation is 8-12 h, specifically 10-12 h in the specific embodiment; the number of overnight washing and soaking times is 1-3 times, specifically 2 times in the specific embodiment; the physiological pH value is 7.0-7.4, specifically 7.2-7.4 in the specific embodiment; the time of soaking again in complete stem cell medium is 10-20 min, specifically 15 min in the specific embodiment; 1×10 4 passage 5-8 mesenchymal stem cells are inoculated on each gel, the culture temperature is 37 °C; the culture is carried out under the condition of 5% (V / V) CO2 concentration.

[0073] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention, but they cannot be understood as limiting the protection scope of the present invention.

[0074] Example 1

[0075] The preparation of the cellulose-crosslinked porcine small intestinal submucosa hydrogel (SIS-DAC) is as follows:

[0076] (1) Preparation of porcine small intestinal submucosa buffer solution:

[0077] 1) Preparation of acellular matrix of porcine small intestinal submucosa: The naturally sourced porcine small intestinal submucosa was successively subjected to pretreatment, virus inactivation, degreasing, decellularization, and drying to obtain the acellular matrix of porcine small intestinal submucosa;

[0078] The pretreatment was as follows: Take fresh porcine small intestine, starting from 10 cm distal to the duodenum, take 100 cm in length, cross-section it every 10 cm, longitudinally cut open the small intestine, place the serosa layer on a gauze moistened with physiological saline, wrap the surgical knife handle with gauze with the mucosal layer facing up, scrape the mucosal layer on the surface of the small intestine, rinse it with physiological saline while scraping, turn the small intestine over when the surface is a milky white semi-transparent basement membrane-like tissue; Scrape the muscular layer and serosa layer of the small intestine in the same way, rinse while scraping, and after scraping all clean, place the SIS in physiological saline for cleaning;

[0079] Virus inactivation: The above-mentioned washed small intestinal submucosa was irradiated with an ultraviolet lamp for 30 min to achieve the purpose of virus inactivation;

[0080] Degreasing: The above-mentioned virus-inactivated SIS was placed in 100 mL of a solution containing 4 wt% sodium deoxycholate and soaked at room temperature for 4 h, then taken out and soaked and washed with ultrapure water at room temperature for 24 h;

[0081] Decellularization: The above-mentioned degreased SIS was placed in 100 mL of a 1 mol / L NaCl solution containing 20 mg of 2000 kU deoxyribonuclease I (DNase-I) and soaked at room temperature for 3 h, then taken out and soaked and washed with ultrapure water at room temperature for 36 h to remove the residual DNA enzyme;

[0082] Drying: The above-mentioned decellularized SIS was programmed to cool to -80 °C and freeze-dried for 48 h;

[0083] 2) Enzymolysis with pepsin solution: After crushing the acellular matrix of porcine small intestinal submucosa (SIS), add pepsin (the mass ratio of pepsin to SIS is 1:10) and 0.5% (V / V) acetic acid solution (the mass ratio of 0.5% (V / V) acetic acid to SIS is 10:1), stir at room temperature for 48 h for digestion to obtain porcine small intestinal submucosa buffer solution;

[0084] The above-mentioned porcine small intestinal submucosa buffer solution was freeze-dried at -80 °C for 48 h, then ground and passed through an 80-mesh sieve to obtain porcine small intestinal submucosa lyophilized powder;

[0085] Dissolve the freeze-dried powder of porcine small intestinal submucosa in a mixed solution of acetic acid with a concentration of 0.5% (V / V) and PBS with a concentration of 0.01 mol / L to obtain a porcine small intestinal submucosa buffer solution;

[0086] (2) Prepare a 2,3-dialdehyde cellulose buffer solution: Suspend 1 g of cellulose in 100 mL of deionized water, and react with sodium periodate under dark conditions in a 40 °C water bath with stirring at 1000 rpm for 4 h. The mass ratio of cellulose to sodium periodate is 1:4. Add 4 mL of ethylene glycol to the reaction system and continue to stir and react at 1000 rpm for 1 h to terminate the reaction. Dialyze the reaction product with a dialysis bag with a molecular cut-off of 3000 in deionized water with stirring at 500 rpm at room temperature for 72 h. Replace the dialysis solution every 12 h, and then freeze-dry at -80 °C for 48 h to obtain 2,3-dialdehyde cellulose;

[0087] Disperse the obtained 2,3-dialdehyde cellulose in a 0.01 mol / L phosphate buffered saline (PBS solution), and heat and dissolve it at 60 °C to obtain a 2,3-dialdehyde cellulose buffer solution with a concentration of 6 mg / mL;

[0088] (3) Prepare a cellulose-crosslinked porcine small intestinal submucosa hydrogel (SIS-DAC)

[0089] Mix the above-mentioned porcine small intestinal submucosa buffer solution and 6 mg / mL 2,3-dialdehyde cellulose buffer solution evenly at room temperature. The molar ratio of the primary amino group of porcine small intestinal submucosa to the aldehyde group of 2,3-dialdehyde cellulose is 10:1. The concentration of porcine small intestinal submucosa in the obtained mixed solution is 2% (w / V). Then adjust the pH value of the obtained mixed solution to 7.0 with NaOH, and incubate at 37 °C for 30 min for Schiff base reaction to obtain a cellulose-crosslinked porcine small intestinal submucosa hydrogel (SIS-DAC = 10:1). The content of the primary amino group of porcine small intestinal submucosa in the porcine small intestinal submucosa hydrogel is 19.8 mmol / g; the content of the aldehyde group in the dialdehyde cellulose is 7.5 mmol / g.

[0090] Example 2

[0091] The difference from Example 1 is that the molar ratio of the primary amino group of porcine small intestinal submucosa to the aldehyde group of 2,3-dialdehyde cellulose is 15:1, and the rest is the same as Example 1, to obtain a cellulose-crosslinked porcine small intestinal submucosa hydrogel (SIS-DAC = 15:1). The content of the primary amino group of porcine small intestinal submucosa in the porcine small intestinal submucosa hydrogel is 19.8 mmol / g; the content of the aldehyde group in the dialdehyde cellulose is 7.5 mmol / g.

[0092] Example 3

[0093] The difference from Example 1 is that the molar ratio of the primary amino group of porcine small intestine submucosa to the aldehyde group of 2,3-dialdehyde cellulose is 20:1, and the rest is the same as in Example 1, to obtain a cellulose-crosslinked porcine small intestine submucosa hydrogel (SIS-DAC = 20:1). The content of the primary amino group of porcine small intestine submucosa in the porcine small intestine submucosa hydrogel is 19.8 mmol / g; the content of the aldehyde group in the dialdehyde cellulose is 7.5 mmol / g.

[0094] Comparative Example 1

[0095] The difference from Example 1 is that the pH value of the porcine small intestine submucosa buffer solution in Example 1 was adjusted to 7.0 with sodium hydroxide and incubated at 37 °C for 30 min to obtain a porcine small intestine submucosa hydrogel (SIS), and the rest is the same as in Example 1.

[0096] Comparative Example 2

[0097] The difference from Example 1 is that no porcine small intestine submucosa hydrogel was added, and the gel only contained 2,3-dialdehyde cellulose (DAC), and the rest is the same as in Example 1.

[0098] Application Example 1

[0099] Application of the cellulose-crosslinked porcine small intestine submucosa hydrogel. The cellulose-crosslinked porcine small intestine submucosa hydrogel can be used to promote the osteogenic differentiation of mesenchymal stem cells. The specific steps are as follows:

[0100] The mixed solution of the porcine small intestine submucosa buffer solution and the 2,3-dialdehyde cellulose buffer solution in Example 1 was spread at 100 μL per well on the bottom of a 48-well plate, the pH value was adjusted to 7.0, incubated at 37 °C for 30 min to form a gel, then sterilized by ultraviolet irradiation for 12 h, and the obtained gel was washed and soaked twice overnight with PBS solution to make the gel reach the physiological pH value (pH = 7.4). Subsequently, it was soaked in the complete stem cell medium for 15 min to replace the PBS solution absorbed in the gel. Using well-grown P5-8 generation UMSC cells, after trypsin digestion, 1×10 4 cells were inoculated on each gel, and the plate was transferred to a cell culture incubator at 37 °C and 5% (V / V) CO2 concentration for culture. After 24 h, a fluorescence microscope was used to take pictures and observe the cell adhesion on the surface of different gels.

[0101] Application Example 2

[0102] The difference from Example 1 is that the mixed solution of the porcine small intestine submucosa buffer solution and the 2,3-dialdehyde cellulose buffer solution in Example 1 was replaced with the mixed solution of the porcine small intestine submucosa buffer solution and the 2,3-dialdehyde cellulose buffer solution in Example 2, and the rest is the same as in Example 1.

[0103] Application Example 3

[0104] The difference from Example 1 is that the mixed solution of porcine small intestinal submucosa buffer solution and 2,3-dialdehyde cellulose buffer solution in Example 1 is replaced with the mixed solution of porcine small intestinal submucosa buffer solution and 2,3-dialdehyde cellulose buffer solution in Example 3, and the rest is the same as Example 1.

[0105] Comparative Application Example 1

[0106] The difference from Example 1 is that the mixed solution of porcine small intestinal submucosa buffer solution and 2,3-dialdehyde cellulose buffer solution in Example 1 is replaced with porcine small intestinal submucosa buffer solution in Comparative Example 1, and the rest is the same as Example 1.

[0107] Comparative Application Example 2

[0108] The difference from Example 1 is that UMSC cells are directly plated on the bottom of a 48-well plate as a blank control group (ctrl).

[0109] Performance Test

[0110] (1) After freeze-drying the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3, the porcine small intestinal submucosa hydrogel (SIS) in Comparative Example 1, and 2,3-dialdehyde cellulose (DAC) in Comparative Example 2 at -80 °C for 48 h, Fourier transform infrared spectroscopy (FTIR) tests were performed in the range of 400 - 4000 cm -1 with a resolution of 4 cm -1 . The FTIR results are as Figure 1 shown.

[0111] As can be seen from Figure 1 , in all gels, the aldehyde characteristic peak of dialdehyde cellulose originally at 1725 cm -1 disappeared, proving that the reaction to form imine was complete and the gel was successfully prepared. The characteristic peak of the imine bond at 1635 cm -1 could not be distinguished due to interference from the amide peak on SIS.

[0112] (2) At a constant strain amplitude of 4%, frequency scans were performed on the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3 in the oscillation frequency range of 0.01 to 100 rad / s. The results are as Figure 2 shown, where G' is the elastic modulus and G'' is the viscous modulus.

[0113] As can be seen from Figure 2 , the SIS-DAC gel prepared by the present invention shows an increase in mechanical strength with the increase in the content of dialdehyde cellulose.

[0114] (3) The compressive strength of the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3 was measured using a 50 N sensor at a speed of 0.1 mm / min. The results are as follows Figure 3 shown. A strain interval of 10% to 20% was selected to fit a straight line, and the slope was obtained, which is the compressive modulus. The results are as follows Figure 4 shown.

[0115] From Figure 3 and Figure 4 it can be seen that for the SIS-DAC gels prepared in the present invention, as the content of dialdehyde cellulose increases, the compressive modulus of the gels increases.

[0116] (4) After freeze-drying the cellulose-crosslinked porcine small intestinal submucosa hydrogels prepared in Examples 1 to 3 and the porcine small intestinal submucosa hydrogel (SIS) of Comparative Example 1 at -80 °C for 48 h, the original weight after drying was weighed. Then they were soaked in PBS solution and placed in a 37 °C constant temperature incubator to simulate the environment in a cell culture incubator. The gels were taken out at fixed time points (1 day, 3 days, 5 days, 7 days, 14 days, 21 days), the PBS residues on the gel surface were washed, and after freeze-drying at -80 °C for 48 h, the mass was weighed to calculate the remaining rate of the gels after degradation for a certain number of days. The results are as follows Figure 5 shown.

[0117] From Figure 5 it can be seen that the remaining rate of the SIS-DAC gels prepared in the present invention reaches about 50% after 21 days of degradation, and they can be preferably used for subsequent cell culture.

[0118] (5) L929 mouse fibroblasts (L929) and human umbilical cord mesenchymal stem cells (UMSC) were seeded at 6000 cells per well in a 96-well plate, and 200 μL of DMEM and αMEM media containing 10 wt% fetal bovine serum and 1 wt% penicillin / streptomycin were added. After culturing in a 37 °C, 5% (V / V) CO2 incubator for 24 h, 200 μL of the extracts of the SIS-DAC gels prepared in Examples 1 to 3 and the SIS gel prepared in Comparative Example 1 were added respectively, and the sample without adding the extract was used as the blank control group (Ctrl). The plates were incubated in a 37 °C, 5% CO2 incubator. After 24 h or 72 h, the medium was removed, 100 μL of thiazolyl blue (MTT) (diluted and filtered with the medium, final concentration 0.5 mg / mL) was added to each well, and the cells were incubated at 37 °C in the dark for 4 h. The MTT was discarded, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and the plates were shaken on a shaker at 37 °C for 15 min. Then the absorbance of each well at a wavelength of 490 nm was measured using a microplate reader to compare the cytotoxicity of each sample. The results are as follows Figure 6 and Figure 7 shown.

[0119] From Figure 6 and Figure 7 It can be seen that the SIS-DAC gel prepared by the present invention has low cytotoxicity.

[0120] (6) Human umbilical cord mesenchymal stem cells (UMSCs) were selected for cell adhesion and osteogenic differentiation experiments to test whether the gels (SIS-DAC) with different mechanical properties in Application Examples 1-3, the SIS gel in Comparative Application Example 1, and the blank control group (ctrl) in Comparative Application Example 2 have the ability to induce osteogenic differentiation of cells. The results are as Figure 8 shown.

[0121] From Figure 8 It can be seen that with the increase in the content of dialdehyde cellulose in the SIS-DAC gel of the present invention, the mechanical properties are improved, the hardness of the SIS-DAC gel is enhanced, and the stretching ability of UMSCs is improved, laying a foundation for promoting the osteogenic differentiation of human umbilical cord mesenchymal stem cells.

[0122] (7) 1 mL of the SIS-DAC gels prepared in Examples 1-3 and the SIS gel prepared in Comparative Example 1 were respectively injected into 6-well plates. After gel formation, they were sterilized by ultraviolet irradiation for 12 h, washed and soaked in PBS solution overnight twice to make the gels reach physiological pH value (pH = 7.4), and then soaked in complete stem cell medium for 15 min to replace the PBS solution absorbed in the gels. Using well-grown UMSC cells of P5-8 generations, after trypsin digestion, 5×10 4 cells were inoculated on each gel. The plates were transferred to a cell culture incubator at 37 °C and 5% (V / V) CO2 concentration for culture, and only complete medium was used for culture, which was changed every 3 days. After 14 days of induction, total RNA of cells in each group was extracted by Trizol method, and cDNA was reverse transcribed using a cDNA synthesis kit. Thereafter, real-time quantitative polymerase chain reaction (PCR) was performed using a qPCR SYBR Green MasterMix kit and each positive and negative primer template, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. There were three parallel wells in each group, and the relative expression levels of osteogenic differentiated stem cells on each gel were calculated. The results are as Figure 9 shown.

[0123] From Figure 9 It can be seen that with the increase in hardness of the SIS-DAC gel of the present invention, the relative expression level of the osteogenic marker Runx2 is increased, and when the ratio of amino group to aldehyde group in SIS and DAC is 10:1, the relative expression level of the osteogenic marker is the highest, indicating the most significant differentiation towards the osteogenic direction.

[0124] Although the above embodiments have described the present invention in detail, they are only a part rather than all of the embodiments of the present invention. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A cellulose-crosslinked porcine small intestinal submucosa hydrogel, characterized in that, It includes a porcine small intestinal submucosa hydrogel and dialdehyde cellulose crosslinked with the porcine small intestinal submucosa hydrogel; the crosslinking is that the primary amino group of the porcine small intestinal submucosa hydrogel and the aldehyde group of the dialdehyde cellulose form an imine bond.

2. The cross-linked cellulose porcine small intestinal submucosa hydrogel according to claim 1, wherein The dialdehyde cellulose is 2,3-dialdehyde cellulose.

3. The cross-linked cellulose porcine small intestinal submucosa hydrogel according to claim 1 or 2, wherein The molar ratio of the primary amino group of the porcine small intestinal submucosa in the porcine small intestinal submucosa hydrogel to the aldehyde group of the dialdehyde cellulose is (10 - 20):

1.

4. The cross-linked cellulose porcine small intestinal submucosa hydrogel according to claim 1 or 2, characterized in that, The content of the primary amino group of the porcine small intestinal submucosa in the porcine small intestinal submucosa hydrogel is 18 - 20 mmol / g; the content of the aldehyde group in the dialdehyde cellulose is 6 - 8 mmol / g.

5. The preparation method of the cellulose-crosslinked porcine small intestinal submucosa hydrogel according to any one of claims 1 to 4, characterized in that, It includes the following steps: Mix the porcine small intestinal submucosa buffer solution and the dialdehyde cellulose buffer solution, and after adjusting the pH value of the obtained mixed solution to 7.0 - 7.4, carry out a Schiff base reaction to obtain the cellulose-crosslinked porcine small intestinal submucosa hydrogel. The dialdehyde cellulose buffer solution is a phosphate buffer solution of dialdehyde cellulose.

6. The preparation method according to claim 5, characterized in that, The concentration of the porcine small intestinal submucosa in the mixed solution is 1 - 3% (w / V).

7. The preparation method according to claim 5, characterized in that, The temperature of the Schiff base reaction is 25 - 37 °C; the time of the Schiff base reaction is 15 - 30 min.

8. The preparation method according to claim 5, characterized in that, The preparation method of the porcine small intestinal submucosa buffer solution includes the following steps: successively inactivate viruses, degrease, decellularize, and dry the porcine small intestinal submucosa to obtain a porcine small intestinal submucosa acellular matrix; Carry out protease enzymolysis on the porcine small intestinal submucosa acellular matrix, freeze-dry the obtained enzymolysis solution, and then successively carry out pulverization and sieving to obtain a porcine small intestinal submucosa freeze-dried powder; Dissolve the porcine small intestinal submucosa freeze-dried powder in a mixed solution of acetic acid and phosphate buffered saline solution to obtain a porcine small intestinal submucosa buffer solution.

9. Use of the cellulose-crosslinked porcine small intestinal submucosa hydrogel according to any one of claims 1 - 4 or the cellulose-crosslinked porcine small intestinal submucosa hydrogel prepared by the preparation method according to any one of claims 5 - 8 in the preparation of a biomaterial.

10. The application according to claim 9, characterized in that, The biomaterial includes one or more of products for promoting osteogenic differentiation of mesenchymal stem cells, products for three-dimensional culture of mesenchymal stem cells, products for organoid culture, and products for joint filling.

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