Preparation method of meniscus extracellular matrix hydrogel

Abstract

The invention provides a preparation method of meniscus extracellular matrix hydrogel, which comprises the following steps: cutting meniscus into blocks, circularly freezing and thawing, and treating with a decellularized solution to obtain a decellularized meniscus; freeze-drying and crushing the decellularized meniscus, and digesting the decellularized meniscus with digestive juice to form pregel; and then adjusting the pH value to 7.0-7.5, so as to prepare the meniscus extracellular matrix hydrogel. Nanofiber structures exist in micropores of the prepared acellular matrix hydrogel, adhesion sites are provided for cells, stable adhesion and proliferation of the cells are facilitated, and tissue repair is well promoted.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a method for preparing meniscus extracellular matrix hydrogel. Background Art

[0002] The meniscus is an important structure in the knee joint, and meniscus injury is also the most common knee injury, with an average annual incidence of 0.66%. The meniscus has many important biomechanical functions, including transmitting loads, absorbing shock, stabilizing joints, and lubricating and nourishing articular cartilage. In view of the important functions of the meniscus, it should be preserved and repaired as much as possible in the treatment after its injury. However, the meniscus tissue is basically avascular, and its dense extracellular matrix limits the mobility and proliferation potential of relatively few cells. Therefore, the ability of the meniscus to repair itself is very limited.

[0003] The material of the invention retains many inherent components and bioactive factors of the meniscus and has broad application prospects in clinical treatment as a new type of meniscus injury repair material. Summary of the invention

[0004] The present invention provides a method for preparing a meniscus extracellular matrix hydrogel, the preparation method comprising: A) Cut the meniscus into pieces, freeze-thaw them repeatedly, soak them in 1% hydrogen peroxide for 1-2 hours, wash them with PBS solution, and freeze them in deionized water; B) Soak in 0.4%-0.8% trypsin and incubate at 37°C for 2-3 minutes to terminate digestion; C) The sample prepared in step B) was placed in PBS solution, 5%-8% Triton X-100 solution, 5%-8% sodium deoxycholate solution, 2%-4% Triton X-100 solution and PBS solution in sequence, 4% of each solution. o C constant temperature shaking for 24-36h, and each solution was washed with PBS after treatment to prepare the decellularized meniscus; D) The decellularized meniscus was freeze-dried, crushed, and digested with a digestion solution. The reaction viscous solution was then neutralized with an aqueous NaOH solution and 10×PBS and placed at 37° C. to prepare a meniscus extracellular matrix hydrogel.

[0005] The present invention first carries out decellularized extracellular matrix treatment, and the cell can be more fully lysed by the method of cyclic freeze-thaw. After being treated in 1% hydrogen peroxide, the permeability of the cell membrane can be effectively destroyed. Afterwards, the sample is placed in PBS solution, Triton X-100 solution, sodium deoxycholate solution, Triton X-100 solution and PBS solution for treatment, cell debris and cell nuclear components are removed, and the porosity of the tissue is increased. The decellularized extracellular matrix immunogenic component clearance rate obtained by the above method is high, and a large amount of collagen and GAG are retained, with good biological activity. Further, there is a nanofiber structure in the prepared hydrogel micropores, which provides adhesion sites for cells, is conducive to stable cell adhesion and proliferation, and has a better promoting effect on tissue repair.

[0006] In some embodiments, in step A), the cyclic freeze-thaw process includes: after cutting the meniscus into pieces, placing them in deionized water, freezing, and thawing; and then placing them in a 10% NaCl solution, freezing, and thawing.

[0007] In some embodiments, in step A), the cyclic freeze-thaw process includes: after cutting the meniscus, placing it in deionized water, freezing it at -80°C for 1-2 hours, and then thawing it at 37°C; then placing it in 10% NaCl solution, freezing it at -80°C for 1-2 hours, and then thawing it at 37°C.

[0008] In some embodiments, in step B), the digestion is stopped using BSA.

[0009] In some embodiments, in step B), the concentration of trypsin is 5%.

[0010] In some embodiments, in step C), the sample prepared in step B) is sequentially placed in a PBS solution, a 5% Triton X-100 solution, a 6% sodium deoxycholate solution, a 3% Triton X-100 solution, and a PBS solution, with 4% of each solution. o The cells were shaken at constant temperature at C for 24 h, and washed with PBS after being treated with each solution to prepare the decellularized meniscus.

[0011] In some embodiments, in step D), the digestive fluid is an aqueous solution of HCl containing pepsin.

[0012] In some embodiments, in step D), the mass ratio of the pepsin to the decellularized meniscus is 1:5 to 20. Preferably, the mass ratio is 1:10.

[0013] In some embodiments, in step D), the decellularized meniscus is freeze-dried, crushed, placed in a 0.01 M HCL aqueous solution containing pepsin, stirred at room temperature to form a fluid mucus solution, and the reactive viscous solution is neutralized with an aqueous NaOH solution and 10×PBS, and placed at 37° C. for several minutes to prepare an extracellular matrix hydrogel.

[0014] Beneficial effects: 1. The components of the decellularized matrix of the present invention are basically close to those of natural tissues and can provide good biological functions.

[0015] 2. The molding conditions of the decellularized matrix gel preparation method of the present invention are normal human physiological conditions, which are suitable for in vivo application and can be directly injected for use, or can be implanted in vivo for use after in vitro molding.

[0016] 3. In the process of preparing the decellularized matrix, the sample is sequentially placed in PBS solution, Triton X-100 solution, sodium deoxycholate solution, Triton X-100 solution and PBS solution for treatment, so as to effectively remove the immunogenic components and increase the porosity of the tissue. The prepared extracellular matrix hydrogel can spontaneously form a nanofiber network structure, provide adhesion sites for cells, facilitate stable cell adhesion and proliferation, and has a good promoting effect on tissue repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The histological analysis results of the inner, middle and outer regions of the meniscus extracellular matrix in the embodiment of the present invention are shown in FIG. Figure 1 a in Figure 1 b in Figure 1 c in the figure represent the tissue hematoxylin-eosin (H&E) staining, toluidine blue staining, and Sirius red staining of the inner, middle, and outer regions of the meniscus extracellular matrix, respectively); Figure 2 Quantitative analysis of the components of the inner, middle and outer regions of the meniscus in the embodiment of the present invention ( Figure 2 a in Figure 2 b in the figure respectively represents the distribution diagram of collagen and GAG content in the decellularized extracellular matrix); Figure 3 Electron microscopic images of hydrogels in the inner, middle, and outer regions of the meniscus extracellular matrix ( Figure 3 a in Figure 3 (b) represents scanning electron micrographs of the inner, middle and outer regions of the natural meniscus and the meniscus extracellular matrix hydrogel, respectively. DETAILED DESCRIPTION

[0018] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0019] Unless otherwise specified, the reagents or consumables used in the present invention are commercially available.

[0020] Unless otherwise specified, % in this article refers to mass volume percentage.

[0021] Test Materials: TritonX-100, sodium deoxycholate, hematoxylin and eosin (H&E) staining kit, toluidine blue staining kit, Sirius red staining kit and FITC-phalloidin: Beijing Solarbio Technology Co., Ltd. Pepsin: Shanghai Yuanye Biotechnology Co., Ltd. Hydroxyproline detection kit and glycosaminoglycan detection kit: Jiangsu Jingmei Biotechnology Co., Ltd. Penicillin-streptomycin solution (double antibody), phosphate buffer, recombinant trypsin digestion solution (without phenol red): Wuhan Punosai Life Science Technology Co., Ltd. 4% paraformaldehyde fixative: Shanghai Biyuntian Biotechnology Co., Ltd.

[0022] Example 1 Preparation of extracellular matrix hydrogels inside, in and outside the meniscus 1.1 Preparation of extracellular matrix inside, inside and outside the meniscus The meniscus was obtained from fresh pig knee joints, and the tissue was divided into inner, middle and outer regions using a surgical blade. The innermost region was the inner region, with a length of 2-3 mm, the outermost region was the outer region, with a length of 4-6 mm, and the region between the inner and outer regions was the middle region, with a length of 7-9 mm. The meniscus tissues in the inner, middle and outer regions were cut into small pieces and decellularized as follows: (1) Place the cut meniscus in deionized water, freeze at -80°C for 2 h, then thaw in a 37°C water bath; place in 10% NaCl solution, freeze at -80°C for 2 h, then thaw in a 37°C water bath, and rinse with PBS; (2) Soak in 1% hydrogen peroxide for 1 h, rinse with PBS, place in deionized water, and freeze at -20°C for 48 h; (3) Soak in 0.5% (m / v) trypsin solution, digest at 37°C for 2.5 min, and add BSA to terminate digestion; (4) Placed in PBS solution, 5% TritonX-100 solution, 6% sodium deoxycholate solution, 3% TritonX-100 solution and PBS solution in sequence, each solution was oscillated at 4°C for 24 h at a speed of 200 rpm. After treatment, each solution was washed with deionized water at 4 o The cells were washed at 400 °C and freeze-dried to prepare the acellular meniscus.

[0023] 1.2 Preparation of extracellular matrix hydrogels inside, in, and outside the meniscus (1) Cut the decellularized meniscus into pieces, freeze-dry for 48-72 hours, and then further mechanically powderize; (2) adding the fine powder at 15 mg / mL into a pepsin / 0.01 M HCl solution, with the mass ratio of pepsin / decellularized meniscus fine powder being 1:10, and stirring the mixed system at room temperature until no visible powder is formed, thereby forming a flowable viscous solution; (3) The pH (to 7.0-7.5) and osmotic pressure of the reaction viscous solution were adjusted with NaOH aqueous solution and 10×PBS, and the solution was placed at 37°C for several minutes to prepare the meniscus extracellular matrix hydrogel. The material is thermosensitive and can change from a solution state to a gel state at 37°C.

[0024] Example 2 Preparation of extracellular matrix hydrogels inside, in and outside the meniscus 1.1 Preparation of extracellular matrix inside, inside and outside the meniscus The meniscus was obtained from fresh pig knee joints, and the tissue was divided into inner, middle and outer regions using a surgical blade. The innermost region was the inner region, with a length of 2-3 mm, the outermost region was the outer region, with a length of 4-6 mm, and the region between the inner and outer regions was the middle region, with a length of 7-9 mm. The meniscus tissues in the inner, middle and outer regions were cut into small pieces and decellularized as follows: (1) Place the cut meniscus in deionized water, freeze at -80°C for 1.5 h, then thaw in a 37°C water bath; place in 10% NaCl solution, freeze at -80°C for 1.5 h, then thaw in a 37°C water bath, and rinse with PBS; (2) Soak in 1% hydrogen peroxide for 1 h, rinse with PBS, place in deionized water, and freeze at -20°C for 48 h; (3) Soak in 0.5% trypsin solution, digest at 37°C for 3 min, and add BSA to terminate digestion; (4) Placed in PBS solution, 5% TritonX-100 solution, 6% sodium deoxycholate solution, 3% TritonX-100 solution and PBS solution in sequence, each solution was oscillated at 4°C for 24 h at a speed of 200 rpm. After treatment, each solution was washed with deionized water at 4 o The cells were washed at 400 °C and freeze-dried to prepare the acellular meniscus.

[0025] 1.2 Preparation of extracellular matrix hydrogels inside, in, and outside the meniscus (1) Cut the decellularized meniscus into pieces, freeze-dry for 48-72 hours, and then further mechanically powderize; (2) adding the fine powder at 15 mg / mL into a pepsin / 0.01 M HCl solution, with the mass ratio of pepsin / decellularized meniscus fine powder being 1:10, and stirring the mixed system at room temperature until no visible powder is formed, thereby forming a flowable viscous solution; (3) The viscous solution was neutralized with NaOH aqueous solution and 10×PBS, and then placed at 37°C for several minutes to prepare the meniscus extracellular matrix hydrogel. The material is thermosensitive and can change from a solution state to a gel state at 37°C.

[0026] Comparative Example 1 The other steps are the same as in Example 1, except for the preparation of the extracellular matrix inside, in and outside the meniscus, and the preparation process is as follows: (1) Place the cut meniscus in deionized water, freeze at -80℃ for 2 h, and then thaw in a 37℃ water bath; place in 10% NaCl solution, freeze at -80℃ for 2 h, and then thaw in a 37℃ water bath, rinse with PBS; then repeat the above freeze-thaw cycle.

[0027] (2) Soak in 0.5% trypsin solution and digest at 37°C for 20 min. Add BSA to terminate the digestion and rinse with deionized water at 4 o The cells were washed at 400 °C and freeze-dried to prepare the acellular meniscus.

[0028] Advantages of the embodiment over the comparative example: the glycosaminoglycan components in the decellular meniscus prepared according to the comparative example are more damaged, while the glycosaminoglycan components in the decellular meniscus prepared according to the embodiment are better preserved.

[0029] Comparative Example 2 The other steps are the same as in Example 1, except for the preparation of the extracellular matrix inside, in and outside the meniscus, and the preparation process is as follows: (1) Soak the cut meniscus for 1 hour until the meniscus turns white, and then rinse with PBS three times; (2) Soak in 1% hydrogen peroxide for 1 h and rinse with PBS; (3) Add deionized water and freeze and thaw at -20°C for 48 h (freeze and thaw four times, freezing for 11 h and thawing for 1 h each time), and discard the deionized water; (4) Place the samples in PBS solution, 5% TritonX-100 solution, 6% sodium deoxycholate solution, 3% TritonX-100 solution and PBS solution in sequence, and oscillate at 4°C for 24 h at a speed of 200 rpm. After each solution treatment, wash the samples three times with PBS at 4°C and freeze-dry to prepare the decellularized meniscus.

[0030] Advantages of the embodiment over the comparative example: the decellularized meniscus prepared according to the comparative example still has residual cells inside, which poses a risk of inducing an immune response, while the decellularized meniscus prepared according to the embodiment completely removes immunogenic substances and has good safety and reliability.

[0031] Test Example 1 Characterization and Performance Testing of Meniscus Extracellular Matrix Hydrogel 1.1. Histological analysis of the decellularization effect inside, middle and outside the meniscus Take an appropriate amount of decellularized matrix from the inner, middle and outer parts of the meniscus and natural porcine meniscus tissue, and fix them with 4% paraformaldehyde to keep the cell structure intact. Then, embed the tissue samples in paraffin, and use a paraffin slicer to cut 5 μm thick slices from the embedded tissue. Finally, perform H&E staining, toluidine blue staining, and Sirius red staining to observe the decellularization effect of the inner, middle and outer parts of the meniscus. The results showed that the H&E staining results of the natural porcine meniscus, such as Figure 1 As shown in a, the meniscus cells are distributed in round or oval shapes, with a large number of blue nuclei and rich extracellular matrix. After decellularization, no blue nuclei were found in the inner, middle and outer regions of the meniscus, and the cell pits were vacuolated, indicating that the immunogenic components such as cells have been completely removed. The results of toluidine blue staining are shown in Figure 1. Figure 1 As shown in b, after decellularization, the glycosaminoglycan components (blue areas) in the inner, middle and outer regions of the meniscus are well preserved, which is similar to the staining results of the natural meniscus. Figure 1 As shown in c, after decellularization, the collagen (red area) in the inner, middle and outer regions of the meniscus is well preserved, the collagen is continuous, and the staining results are basically indistinguishable from those of the natural meniscus.

[0032] 1.2. Quantitative analysis of decellularized components in the inner, middle, and outer parts of the meniscus Take appropriate amounts of the inner, middle and outer decellularized matrices of the meniscus and natural porcine meniscus to determine the content of collagen (COL) and glycosaminoglycan (GAG). The measurements were performed according to the experimental instructions of the porcine hydroxyproline kit and the glycosaminoglycan kit. Figure 2 As shown in the figure, the content of collagen and GAG is lower than that of normal extracellular matrix, which is the loss caused by the decellularization process. However, some collagen and GAG are still retained in dECM, which is very beneficial for the subsequent preparation of hydrogels with intrinsic bioactivity.

[0033] 1.3. Morphology of meniscus extracellular matrix hydrogel The microstructures of the inner, middle and outer regions of the natural meniscus and the meniscus extracellular matrix hydrogel prepared in the embodiment were observed by scanning electron microscopy. Figure 3As shown, the results show that the fibers inside the natural meniscus show a regular radial orientation. The inner, middle and outer regions of the meniscus extracellular matrix hydrogel all show a porous structure after freeze-drying, which is conducive to cell growth, diffusion of nutrients and waste removal. In addition, the presence of nanofiber structures in the micropores provides adhesion sites for cells, which is conducive to stable cell adhesion and proliferation.

[0034] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of the present invention. These improvements and substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a meniscus extracellular matrix hydrogel, characterized in that: The preparation method comprises: A) The meniscus was cut into pieces, and after a freeze-thaw cycle, it was immersed in 1% hydrogen peroxide for 1-2 hours, rinsed with PBS solution, and frozen in deionized water; B) Soak in 0.4%-0.8% trypsin and incubate at 37°C for 2-3 minutes to terminate digestion; C) placing the sample prepared in step B) in PBS solution, 5%-8% Triton X-100 solution, 5%-8% sodium deoxycholate solution, 2%-4% Triton X-100 solution and PBS solution in sequence, each solution is shaken at 4°C for 24-36 hours, and each solution is washed with PBS after treatment to prepare acellular meniscus; D) The decellularized meniscus was freeze-dried, crushed, and digested with a digestion solution. The reaction viscous solution was then neutralized with an aqueous NaOH solution and 10×PBS and placed at 37° C. to prepare a meniscus extracellular matrix hydrogel.

2. The method for preparing the meniscus extracellular matrix hydrogel according to claim 1, characterized in that: In step A), the freeze-thaw cycle process includes: after cutting the meniscus into pieces, placing them in deionized water, freezing them, and thawing them; and then placing them in a 10% NaCl solution, freezing them, and thawing them.

3. The method for preparing the meniscus extracellular matrix hydrogel according to claim 1 or 2, characterized in that: In step A), the freeze-thaw cycle process includes: after cutting the meniscus, placing it in deionized water, freezing it at -80°C for 1-2 hours, and then thawing it at 37°C; then placing it in a 10% NaCl solution, freezing it at -80°C for 1-2 hours, and then thawing it at 37°C.

4. The method for preparing the meniscus extracellular matrix hydrogel according to claim 1, characterized in that: In step B), the digestion is stopped with BSA.

5. The method for preparing the meniscus extracellular matrix hydrogel according to claim 1, characterized in that: In step B), the concentration of trypsin is 5%.

6. The method for preparing the meniscus extracellular matrix hydrogel according to claim 1, characterized in that: In step C), the sample prepared in step B) is placed in PBS solution, 5% Triton X-100 solution, 6% sodium deoxycholate solution, 3% Triton X-100 solution and PBS solution in sequence, each solution is shaken at a constant temperature of 4°C for 24 hours, and each solution is washed with PBS after treatment to prepare a decellularized meniscus.

7. The method for preparing the meniscus extracellular matrix hydrogel according to claim 1, characterized in that: In step D), the digestive fluid is an aqueous solution of HCl containing pepsin.

8. The method for preparing the meniscus extracellular matrix hydrogel according to claim 1, characterized in that: In step D), the mass ratio of the pepsin to the decellularized meniscus is 1:5-20.

9. The method for preparing the meniscus extracellular matrix hydrogel according to claim 1 or 8, characterized in that: The mass ratio of the pepsin to the decellularized meniscus is 1:10.

Images