Preparation method of acellular cartilage photosensitive hydrogel microspheres

Abstract

The invention relates to a preparation method of decellularized cartilage photosensitive hydrogel microspheres. The preparation method comprises the following steps: preparing a decellularized cartilage matrix, preparing decellularized cartilage photosensitive hydrogel, and preparing the decellularized cartilage photosensitive hydrogel microspheres. The cartilage tissue is treated by adopting physical crushing, a chemical reagent and an enzymatic method, the prepared acellular cartilage matrix can retain bioactive components in the cartilage matrix to the greatest extent, and a good microenvironment is provided for growth and differentiation of cartilage cells. Besides, the decellularized cartilage hydrogel microspheres are prepared by adopting a micro-fluidic method, the operation is relatively simple, complicated equipment and conditions are not needed, and the prepared microspheres are excellent in performance, uniform in particle size distribution, good in mechanical property, complete in bioactive component retention, good in biocompatibility and free of obvious immune and inflammatory reactions, and can be used for preparing the decellularized cartilage hydrogel microspheres. And in combination with drug release, remarkable optimization of cartilage repair is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical materials, and particularly relates to a preparation method of decellularized cartilage photosensitive hydrogel microspheres. BACKGROUND

[0002] Cartilage tissue has poor self-repairing ability due to its own characteristics such as no blood vessel, nerve and lymph distribution. Traditional cartilage repair methods such as microfracture surgery and cartilage transplantation have many problems. Although microfracture surgery can stimulate the release of bone marrow stem cells from subchondral bone, the repair tissue formed is mostly fibrocartilage, and its biomechanical properties are far inferior to normal hyaline cartilage. Cartilage transplantation faces challenges such as limited donor sources, immune rejection and difficulty in integrating the transplanted cartilage. Hydrogel, as a material with good biocompatibility and adjustable physical and chemical properties, has been widely used in cartilage tissue engineering.

[0003] However, traditional hydrogels often lack biological activity and cannot well simulate the microenvironment of natural cartilage, limiting their effectiveness in cartilage repair. Decellularized cartilage matrix retains the biological active components of natural cartilage, such as collagen and glycosaminoglycans, which can provide a suitable growth microenvironment for cells and promote the adhesion, proliferation and differentiation of chondrocytes. Combining decellularized cartilage matrix with hydrogel to prepare decellularized cartilage hydrogel microspheres is expected to solve the problems of traditional cartilage repair methods and hydrogel materials and provide a more effective strategy for cartilage repair. However, current methods for preparing decellularized cartilage hydrogel microspheres have complex preparation processes and unstable microsphere performance, so it is of great practical significance to develop a method for efficiently preparing decellularized cartilage hydrogel microspheres with excellent performance. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a preparation method of decellularized cartilage photosensitive hydrogel microspheres. The method not only removes the cell components in the cartilage tissue that cause immune reactions and retains important cartilage matrix structures, but also makes the decellularized cartilage into photosensitive hydrogel and further prepares it into injectable microsphere form, combining practicality and controllability.

[0005] The present application provides a preparation method of decellularized cartilage photosensitive hydrogel microspheres, comprising the following steps: (1) Take fresh pig cartilage tissue, remove impurities; cut the pig cartilage tissue into thin slices, rinse with a phosphate buffered saline (PBS) solution to remove blood and impurities on the surface, and obtain cartilage pieces; perform decellularization treatment on the cartilage pieces by using a chemical-enzyme combined method to remove cell membranes and intracellular components; then perform ultrasonic cleaning to obtain a decellularized cartilage matrix; freeze-dry and grind the decellularized cartilage matrix to obtain a decellularized cartilage matrix powder; (2) Digest the decellularized cartilage matrix powder, then dialyze and freeze-dry to obtain a decellularized cartilage hydrogel freeze-dried product; add a photosensitizer and a photoinitiator in sequence after dissolving the freeze-dried product to obtain a decellularized cartilage photosensitive hydrogel solution; (3) Use the decellularized cartilage photosensitive hydrogel solution as a dispersed phase to form decellularized cartilage photosensitive hydrogel droplets by using a microfluidic device, and then perform ultraviolet radiation to obtain decellularized cartilage photosensitive hydrogel microspheres.

[0006] Preferably, the decellularization in step (1) is specifically: first, immerse the cartilage pieces in a solution containing trypsin and EDTA, and stir at 37°C for 96 hours; then immerse the cartilage pieces in a solution containing Triton X-100 and Tris, and stir at room temperature for 24 hours.

[0007] Preferably, the digestion in step (2) is specifically: immerse the decellularized cartilage matrix powder in a solution containing pepsin and hydrochloric acid at a ratio of 10-20 mg / mL, and stir at 37°C for 72 hours.

[0008] Preferably, after the digestion in step (2), sodium chloride is added for precipitation.

[0009] Preferably, the photosensitizer in step (2) includes one or more of methacrylate (MA), dimethyl acrylate-terminated polyethylene glycol (MeAc-PEG-MeAc), and methylene bisacrylamide (MBAA); the amount added is 1-2 mL / g.

[0010] Preferably, the photoinitiator in step (2) includes one or more of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2,2-dimethoxy-2-diphenylacetophenone (DMPA), and phenyl bis(2,4,6-trimethylbenzoyl) phosphine oxide (PBPO); the amount added is 1-2 mg / mL.

[0011] Preferably, in step (3), mineral oil containing Span 80 is used as a continuous phase.

[0012] Preferably, the ultraviolet light radiation power in the step (3) is 100-200 W; the time is 5-10 min.

[0013] Advantages The application adopts physical crushing, chemical reagent and enzymatic method to treat cartilage tissue, and the prepared decellularized cartilage matrix can maximize the retention of biological active ingredients (such as collagen, glycosaminoglycan, etc.) in the cartilage matrix, and provides a good microenvironment for the growth and differentiation of chondrocytes. In addition, the application adopts microfluidic method to prepare decellularized cartilage hydrogel microspheres, which is relatively simple to operate, does not require complex equipment and conditions, and the prepared microspheres have excellent performance, uniform particle size distribution, good mechanical properties, complete retention of biological active ingredients, good biocompatibility, do not cause obvious immune and inflammatory reactions, can also be combined with drug release, and realize the significant optimization of cartilage repair. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Schematic diagram for preparing decellularized cartilage matrix of example 1.

[0015] Figure 2 Schematic diagram of staining results of decellularized cartilage matrix prepared in example 1 and normal cartilage.

[0016] Figure 3 Schematic diagram of component quantification results of decellularized cartilage matrix prepared in example 1 and normal cartilage (n≥3).

[0017] Figure 4 Scanning electron microscope graph of decellularized cartilage matrix prepared in example 1 and normal cartilage.

[0018] Figure 5 Microfluidic injection pump (left) and chip (right) for example 3.

[0019] Figure 6 Decellularized cartilage photosensitive hydrogel droplet prepared in example 3.

[0020] Figure 7 Light microscope graph of decellularized cartilage photosensitive hydrogel microspheres prepared in example 3.

[0021] Figure 8 Scanning electron microscope graph of decellularized cartilage photosensitive hydrogel microspheres prepared in example 3. DETAILED DESCRIPTION

[0022] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0023] Example 1 Preparation of decellularized cartilage matrix: (1) Take fresh pork rib cartilage and remove surrounding soft tissue, periosteum and other impurities; cut the pork cartilage tissue into thin slices and rinse with PBS buffer 3 times, 5 minutes each time, to remove surface blood and impurities, and obtain cartilage slices (about 2 mm thick).

[0024] (2) Soak the cartilage slices in a solution containing 2.5 g / L trypsin + 0.5 mM EDTA, stir at 37°C for 96 hours, and change the solution every 24 hours.

[0025] (3) Transfer the cartilage slices to a 1% Triton X-100 + 1% Tris solution and stir at room temperature for 24 hours to remove the cell membrane and intracellular components.

[0026] (4) Wash with PBS buffer by sonication 3 times (10 minutes each time), stir for 3 days, freeze dry and grind into powder.

[0027] Experimental results: ①For example Figure 1 As shown, decellularized cartilage matrix was prepared by treating cartilage tissue using physical pulverization, chemical reagents, and enzymatic methods.

[0028] ② The effectiveness of the decellularization method was verified through histological staining, immunofluorescence staining, and biochemical analysis: such as... Figure 2 DAPI and H&E staining revealed the presence of cell nuclei in native cartilage tissue, while nuclei were absent in decellularized cartilage matrix. Collagen II, a major ECM component in cartilage, remained highly expressed in the decellularized cartilage matrix after decellularization.

[0029] ③ Biochemical analysis of normal cartilage and decellularized cartilage matrix: such as Figure 3 The results showed that the DNA content retained in the decellularized cartilage matrix was 1.22%, far lower than that in natural tissue. Furthermore, the decellularized cartilage matrix still retained 65.92% glycosaminoglycans and 81.68% type II collagen.

[0030] ④ The observation results of scanning electron microscopy are as follows Figure 4 As shown, the surface of natural cartilage tissue is relatively smooth and lacks obvious pores. Meanwhile, a certain amount of porosity is present on the surface of the decellularized cartilage matrix.

[0031] The above results show that the decellularization method of the present application has a significant effect, and can retain the main ECM components and remove almost all cells.

[0032] Example 2 Preparation of decellularized cartilage photosensitive hydrogel: (1) Cartilage powder digestion: 10 g of cartilage powder was soaked in a solution containing 1 mg / mL pepsin + 0.01 M hydrochloric acid, and stirred at 37°C for 72 hours. Pepsin digestion can decompose non-collagen proteins in the decellularized cartilage matrix.

[0033] (2) Purification and dialysis: 15% NaCl solution (volume is 1 / 3 of the remaining digestion solution) was added, centrifuged at 4°C, 3000 rpm for 15 minutes, and the precipitate was taken. After dissolving the precipitate in deionized water, it was loaded into a dialysis bag (molecular weight cutoff 8 kDa), and dialyzed at 4°C for 48 hours, with water changed every 8 hours.

[0034] (3) Photosensitive modification: The freeze-dried powder was dissolved in 0.5 M acetic acid solution at 10 mg / mL, and the pH was adjusted to 8-9 with 5 M NaOH under dark conditions at 4°C. 1.5 mL / g of methacrylate (MA) was added at a rate of 0.5 ml / min, and stirred for 3 days. MA modification introduces photo-crosslinking sites, and the freeze-dried powder is obtained after dialysis for 96 hours, adjustment of pH to 7, and freeze-drying.

[0035] (4) Solution preparation: The freeze-dried powder was dissolved in PBS at 20 mg / mL, and after ultrasonic dissolution, 1.5 mg / mL lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP) photoinitiator was added, and the solution was shaken in the dark for 30 seconds. LAP as a photoinitiator can initiate free radical polymerization under ultraviolet light.

[0036] Example 3 Preparation of decellularized cartilage photosensitive hydrogel microspheres: (1) PDMS chip was used, containing a dispersed phase inlet, a continuous phase inlet, and a microsphere collection outlet (Figure 1) Figure 5 Right).

[0037] (2) Injection pump 1 was connected to the dispersed phase (decellularized cartilage photosensitive hydrogel solution) at a flow rate of 10 μL / min; injection pump 2 was connected to the continuous phase (mineral oil containing 5% Span 80) at a flow rate of 100 μL / min (Figure 2) Figure 5 Left).

[0038] (3) Droplets were formed under the action of shear force in the chip channel, and flowed into -20°C anhydrous ethanol (Figure 3) Figure 6 ).

[0039] (4) Irradiate with a 150W UV lamp for 5 minutes, discard the ethanol after gelation, remove the mineral oil with acetone, wash with PBS 3 times, and collect the microspheres by centrifugation.

[0040] Experimental results: ①For example Figure 7 As shown, under a bright-field microscope, the hydrogel microspheres are regularly spherical and exhibit good monodispersity. They have a uniform texture, smooth boundaries, and an average diameter of 148.8 ± 82.21 µm.

[0041] ② The morphology of the hydrogel microspheres was observed using scanning electron microscopy (SEM). For example... Figure 8 As shown, the surface of the microspheres exhibits a porous structure with an average pore size of 4.21 ± 2.22 µm, which is beneficial for drug loading / sustained release and cell adhesion.

Claims

1. A method for preparing a decellularized cartilage photo-sensitive hydrogel microsphere, characterized in that, It comprises the following steps: (1) Take fresh pig cartilage tissue, remove impurities; cut the pig cartilage tissue into thin slices, rinse with phosphate buffered saline solution to remove blood and impurities on the surface, and obtain cartilage pieces; the cartilage pieces are subjected to decellularization treatment by chemical-enzyme combined method to remove cell membranes and intracellular components; then ultrasonic cleaning is carried out to obtain decellularized cartilage matrix; the decellularized cartilage matrix is freeze-dried and ground to obtain decellularized cartilage matrix powder; (2) The above decellularized cartilage matrix powder is digested, then dialyzed and freeze-dried to obtain a decellularized cartilage hydrogel freeze-dried; after the freeze-dried is dissolved, a photosensitizer and a photoinitiator are added in sequence to obtain a decellularized cartilage photosensitive hydrogel solution; (3) The above decellularized cartilage photosensitive hydrogel solution is used as the dispersed phase to form decellularized cartilage photosensitive hydrogel droplets by using a microfluidic device, and then subjected to ultraviolet radiation to obtain decellularized cartilage photosensitive hydrogel microspheres.

2. The production method according to claim 1, characterized by, The decellularization in step (1) is specifically: first immerse the cartilage pieces in a solution containing trypsin and EDTA, and stir at 37℃ for 96 hours; then immerse the cartilage pieces in a solution containing triton X-100 and tris-hydroxymethyl aminomethane, and stir at room temperature for 24 hours.

3. The production method according to claim 1, characterized by, The digestion in step (2) is specifically: immerse the decellularized cartilage matrix powder in a solution containing pepsin and concentrated hydrochloric acid at a ratio of 10-20 mg / mL, and stir at 37℃ for 72 hours.

4. The method of claim 1, wherein, After digestion in step (2), sodium chloride is added for precipitation.

5. The production method according to claim 1, characterized by, The photosensitizer in step (2) includes one or more of methacrylate, dimethyl acrylate-terminated polyethylene glycol, and methylene bisacrylamide; the amount added is 1-2 mL / g.

6. The production method according to claim 1, characterized by, The photoinitiator in step (2) includes one or more of lithium phenyl-2,4,6-trimethylbenzoylphosphinate, 2,2-dimethoxy-2-diphenyl ketone, and phenyl bis(2,4,6-trimethylbenzoyl) phosphine oxide; the amount added is 1-2 mg / mL.

7. The preparation method according to claim 1, characterized in that, In step (3), mineral oil containing Span 80 is used as the continuous phase.

8. The production method according to claim 1, characterized by, The ultraviolet radiation power in step (3) is 100-200 W; the time is 5-10 min.

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