PREPARATION METHOD OF 4D CHITOSAN-BASED THERMOSENSITIVE HYDROGEL

Abstract

Medical material production and preparation, and a preparation method of a 4D chitosan-based thermosensitive hydrogel. First, chitosan is dissolved in acetic acid solution; a chitosan-based thermosensitive hydrogel is printed by a 4D bioprinter and lyophilized after solvent extraction, to obtain lyophilized chitosan; subsequently, aqueous -sodium glycerophosphate solution is prepared with ultrapure water and -sodium glycerophosphate, and then aqueous carboxymethyl chitosan solution is prepared with ultrapure water and aqueous -sodium glycerophosphate solution are charged into and mixed well with aqueous carboxymethyl chitosan solution to prepare a mixture; finally, the lyophilized chitosan is crosslinked with the mixture to obtain the 4D chitosan-based thermosensitive hydrogel. With scientific and reliable principles thereof, the present invention solves a problem that conventional thermosensitive hydrogels have uneven pore sizes, and improves the entrapment efficiency and ability of limbal stem cells.

Claims

1. A preparation method of a 4D chitosan-based thermosensitive hydrogel, comprising the following steps: (1) at room temperature, weighing chitosan, dissolving the chitosan in acetic acid solution, stirring until the chitosan has completely dissolved, to obtain a chitosan solution; using a 4D bioprinter to print the chitosan solution into a chitosan-based thermosensitive hydrogel with a pore size of 50 to 100 m according to preset print parameters, and shaping the chitosan-based thermosensitive hydrogel as desired; lyophilizing after solvent extraction, to obtain lyophilized chitosan; (2) charging ultrapure water and -sodium glycerophosphate into a water bath kettle at 60 to 70 C.; after dissolution of -sodium glycerophosphate, naturally cooling to room temperature to obtain an aqueous -sodium glycerophosphate solution; (3) preparing an aqueous carboxymethyl chitosan solution with ultrapure water at room temperature, dripping the aqueous -sodium glycerophosphate solution obtained in step (2) dropwise into the aqueous carboxymethyl chitosan solution, and mixing well to obtain a mixture; and (4) crosslinking the lyophilized chitosan obtained in step (1) with the mixture obtained in step (3) for 1 to 2 min, to obtain the 4D chitosan-based thermosensitive hydrogel of uniform pore size.

2. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 1, wherein a molar concentration of the acetic acid solution in step (1) is 0.2 Mol/L, and a concentration of the chitosan solution ranges from 2.2 wt. % to 6.7 wt. %.

3. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 1, wherein a concentration of the aqueous -sodium glycerophosphate solution ranges from 6 wt. % to 8 wt. %.

4. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 1, wherein a concentration of the aqueous carboxymethyl chitosan solution prepared in step (3) ranges from 2.2 wt. % to 6.7 wt. %.

5. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 1, wherein concentrations of the chitosan, the carboxymethyl chitosan, and the -sodium glycerophosphate in the 4D chitosan-based thermosensitive hydrogel prepared in step (4) are 10 wt. % to 30 wt. %, 10 wt. % to 30 wt. %, and 60 wt. % to 80 wt. %, respectively.

6. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claims 1, wherein the 4D chitosan-based thermosensitive hydrogel may be preserved in a sterile environment at 4 to 15 C. for 6 to 12 months.

7. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 1, wherein after entrapping limbal stem cells, the 4D chitosan-based thermosensitive hydrogel is applied on the surface of an alkali burned cornea and releases limbal stem cells to repair and treat a wound.

8. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 2, wherein the 4D chitosan-based thermosensitive hydrogel may be preserved in a sterile environment at 4 to 15 C. for 6 to 12 months.

9. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 3, wherein the 4D chitosan-based thermosensitive hydrogel may be preserved in a sterile environment at 4 to 15 C. for 6 to 12 months.

10. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 4, wherein the 4D chitosan-based thermosensitive hydrogel may be preserved in a sterile environment at 4 to 15 C. for 6 to 12 months.

11. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 5, wherein the 4D chitosan-based thermosensitive hydrogel may be preserved in a sterile environment at 4 to 15 C. for 6 to 12 months.

12. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 2, wherein after entrapping limbal stem cells, the 4D chitosan-based thermosensitive hydrogel is applied on the surface of an alkali burned cornea and releases limbal stem cells to repair and treat a wound.

13. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 3, wherein after entrapping limbal stem cells, the 4D chitosan-based thermosensitive hydrogel is applied on the surface of an alkali burned cornea and releases limbal stem cells to repair and treat a wound.

14. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 4, wherein after entrapping limbal stem cells, the 4D chitosan-based thermosensitive hydrogel is applied on the surface of an alkali burned cornea and releases limbal stem cells to repair and treat a wound.

15. The preparation method of the 4D chitosan-based thermosensitive hydrogel according to claim 5, wherein after entrapping limbal stem cells, the 4D chitosan-based thermosensitive hydrogel is applied on the surface of an alkali burned cornea and releases limbal stem cells to repair and treat a wound.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0015] FIG. 1 illustrates the structure of chitosan described in step (1) of Example 1 of the present invention.

[0016] FIG. 2 illustrates the structure of a 4D chitosan-based thermosensitive hydrogel described in step (4) of Example 1 of the present invention.

[0017] FIG. 3 illustrates the state of a 4D chitosan-based thermosensitive hydrogel entrapping limbal stem cells at 40-fold magnification described in Example 2 of the present invention.

[0018] FIG. 4 illustrates the state of the 4D chitosan-based thermosensitive hydrogel entrapping limbal stem cells at 10-fold magnification described in Example 2 of the present invention.

[0019] FIG. 5 illustrates the comparison of efficacy of conventional thermosensitive hydrogel-entrapped limbal stem cells versus 4D chitosan-based thermosensitive hydrogel-entrapped limbal stem cells in the treatment of rabbits with corneal alkali burns.

DETAILED DESCRIPTION

[0020] The present invention is described in detail below with reference to the accompanying drawings and specific examples.

Example 1

[0021] A preparation method of a 4D chitosan-based thermosensitive hydrogel as provided by the example included the following steps:

[0022] at room temperature, weighing chitosan, dissolving the chitosan in 0.2 Mol/L acetic acid solution, stirring for 12 h until the chitosan had completely dissolved, to obtain a 4.4 wt. % chitosan solution; printing the chitosan solution into round chitosan-based thermosensitive hydrogel with a pore size of 50 to 100 m according to preset print parameters (hole interval 600 m, layer height 100 m, bed temperature 25 C., printing speed 15 mm/s, nozzle diameter 0.3 mm, and output 0.1 g/min) by means of a UN-4DBI-C01 4D bioprinter; lyophilizing after solvent extraction, to obtain lyophilized chitosan, as shown in FIG. 1;

[0023] charging ultrapure water and -sodium glycerophosphate into a water bath kettle at 65 C.; after dissolution of -sodium glycerophosphate, naturally cooling to room temperature to obtain a 7 wt. % aqueous -sodium glycerophosphate solution;

[0024] preparing a 4.4 wt. % aqueous carboxymethyl chitosan solution with ultrapure water at room temperature, dripping the aqueous -sodium glycerophosphate solution obtained in step (2) dropwise into the aqueous carboxymethyl chitosan solution, and mixing well to obtain a mixture; and

[0025] crosslinking the lyophilized chitosan obtained in step (1) with the mixture obtained in step (3) for 1 to 2 min, to obtain a 4D chitosan-based thermosensitive hydrogel of uniform pore size as shown in FIG. 2, where concentrations of the chitosan, the carboxymethyl chitosan, and the -sodium glycerophosphate in the 4D chitosan-based thermosensitive hydrogel were 18 wt. %, 18 wt. %, and 64 wt. %, respectively.

Example 2

[0026] This example relates to a process of the 4D chitosan-based thermosensitive hydrogel prepared in Example 1 entrapping limbal stem cells:

[0027] The 4D chitosan-based thermosensitive hydrogel was placed and fully soaked in a Petri dish to obtain a 4D chitosan-based thermosensitive hydrogel to be entrapped.

[0028] Limbal stem cells were separated, cultured, and charged into the 4D chitosan-based thermosensitive hydrogel to be entrapped as obtained in step (1) for further culture.

[0029] After 24 h culture, the state of the 4D chitosan-based thermosensitive hydrogel entrapped limbal stem cells were observed under a microscope at 40- and 10-fold magnification, respectively.

Example 3

[0030] This example relates to a comparative test of efficacy of 4D chitosan-based thermosensitive hydrogel-entrapped limbal stem cells versus conventional thermosensitive hydrogel-entrapped limbal stem cells in the treatment of rabbits with corneal alkali burns.

[0031] Corneas of living rabbits were subject to alkali burns to obtain animal models of alkali burns.

[0032] The animal models of alkali burns obtained in step (1) were treated with conventional thermosensitive hydrogel-entrapped limbal stem cells, to obtain models of treatment with conventional thermosensitive hydrogel.

[0033] The animal models of alkali burns obtained in step (1) were treated with 4D chitosan-based thermosensitive hydrogel-entrapped limbal stem cells prepared in Example 1, to obtain models of treatment with 4D chitosan-based thermosensitive hydrogel.

[0034] Efficacy of both models was observed 28 days after treatment, respectively. As shown in FIG. 5, after alkali burns procedure, corneal wound areas (white areas in the figure) of each group decrease gradually over time; moreover, 4D chitosan-based thermosensitive hydrogel treatment group has significantly better repairing effect than both conventional thermosensitive hydrogel group and non-repairing group.