A thermosensitive hydrogel of porcine testicular acellular extracellular matrix and its preparation method

Abstract

The present invention discloses a thermosensitive hydrogel of porcine testicular acellular extracellular matrix and a preparation method thereof, including: using freeze-thaw cycles to disrupt cell membranes, and performing decellularization treatment with a mild detergent TritonX-100. While effectively removing testicular cells, the biological composition and structural scaffold of the testis are highly retained. This hydrogel can rapidly solidify and form after being injected into the testis, adapt to the complex environment of the testis, recruit testicular interstitial stem cells at the same time, regulate their differentiation into testicular interstitial cells, and then secrete testosterone. In addition, the hydrogel can also degrade under the action of in vivo enzymes, release cytokines, and reduce the inflammatory reaction of testicular interstitium. The hydrogel also has high biological safety and low immunogenicity.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedical materials, tissue engineering and regenerative medicine, and particularly to a porcine testicular acellular extracellular matrix thermosensitive hydrogel and a preparation method thereof. Background Art

[0002] Currently, the biomedical materials for treating testosterone deficiency in clinical practice are still limited to exogenous testosterone supplementation, including transdermal gels and transdermal patches. These biomedical materials can penetrate through the skin into the blood or act on the testis through the scrotum to play a role, thereby rapidly increasing the body's testosterone level and further improving male reproductive ability, and having a certain therapeutic effect on mild testosterone deficiency. However, for individuals who need long-term drug administration to maintain the body's testosterone level, exogenous testosterone supplementation not only fails to meet the need for long-term testosterone normalization, but also produces serious side effects, disrupting the endocrine regulation of the hypothalamus-pituitary-testis axis and gradually reducing the ability to produce endogenous testosterone. Transdermal biomedical materials also have adverse effects such as low delivery efficiency, skin infection, and contact transmission to others.

[0003] Acellular extracellular matrix hydrogel is a natural biomaterial derived from animal tissues. After a series of decellularization treatments, the obtained material has advantages such as high biosafety and low immunogenicity. Testicular acellular extracellular matrix hydrogel has biological cues specific to the testicular microenvironment and can have a good regulatory effect on the improvement of the testicular microenvironment. At the same time, acellular extracellular matrix hydrogel is beneficial to cell adhesion and proliferation and also has a positive promoting effect on stem cell differentiation. Based on the characteristics that collagen can self-assemble thermosensitively, the present invention designs a unique decellularization treatment and digestion process for testicular tissue to obtain testicular acellular extracellular matrix hydrogel, which can be directly implanted into the testis by in-situ injection and solidify into a shape by changing the temperature, recruiting Leydig stem cells in the testis to adhere and proliferate and differentiate into Leydig cells to secrete testosterone. In particular, there are also a large number of enzymes and cytokines that regulate the testicular microenvironment in testicular acellular extracellular matrix hydrogel, which can effectively reduce testicular inflammation and play an important role in reversing testicular aging. In summary, acellular extracellular matrix has great application potential in the fields of treating testosterone deficiency and delaying testicular aging. Summary of the Invention

[0004] The object of the present invention is to overcome the disadvantages and deficiencies of the prior art, and to provide a thermosensitive hydrogel of porcine testicular acellular extracellular matrix and a preparation method thereof. The hydrogel prepared by this method can present a solution form in vitro and transform into a gel state after being injected into the testis for a period of time. At the same time, it can spontaneously form an appropriate shape according to the structure of the testicular interstitium without damaging the implanted testis. The prepared hydrogel reduces the content of cell components such as DNA to the lowest level, while retaining components such as collagen, laminin, and glycosaminoglycans to the greatest extent. These components play an important role in regulating cell proliferation and stem cell differentiation.

[0005] To achieve the above object, the technical solution provided by the present invention is: a preparation method of a thermosensitive hydrogel of porcine testicular acellular extracellular matrix, comprising the following steps:

[0006] 1) After subjecting the obtained porcine testicular tissue to multiple freeze-thaw cycles, removing the tunica albuginea and rete testis tissues, then chopping it into small tissue pieces, and successively placing the small tissue pieces in washing solution A, washing solution B, disinfectant solution C, and deionized water for stirring, washing, and sterilization to obtain a white tissue. Freeze-drying, grinding, and sterilizing the obtained white tissue to obtain acellular extracellular matrix powder; wherein, the washing solution A is composed of a mixed solution of 0.05%-0.5% Triton X-100 and 0.1%-1% penicillin-streptomycin, the washing solution B is composed of 1x PBS solution and 0.1%-1% penicillin-streptomycin mixed solution, and the disinfectant solution C is composed of a mixed solution of 4% ethanol and 0.1% compound peracetic acid disinfectant solution;

[0007] 2) Placing the obtained acellular extracellular matrix powder in digestive solution D for stirring and digestion. After digestion is completed, adjusting the pH and ion concentration to obtain a pre-gel solution of porcine testicular acellular extracellular matrix. Placing the pre-gel solution of porcine testicular acellular extracellular matrix in a warm bath environment for a period of time to obtain a thermosensitive hydrogel of porcine testicular acellular extracellular matrix; wherein, the digestive solution D is prepared by dissolving pepsin with 0.01M-0.1M hydrochloric acid.

[0008] Preferably, in step 1), the washing solution A and the washing solution B are replaced every 2-4 hours during stirring, and the disinfectant solution C is stirred and disinfected for 1 hour.

[0009] Preferably, in step 1), the volume of the small tissue pieces is 1 cm 3 -4 cm 3 , and during the stirring process, every 100 g of tissue is added to 1 L of the stirring solution. The particle size of the ground powder is 10-100 μm.

[0010] Preferably, in step 1), the freeze-drying is carried out at -80°C for 2-5 days, and the sterilization is carried out by 5-10 kGy gamma-ray irradiation.

[0011] Preferably, in step 2), the digestion process is carried out in a sterile room temperature environment with stirring for 2 - 5 days at a stirring rate of 600 - 800 rpm to digest the decellularized extracellular matrix powder.

[0012] Preferably, in step 2), the concentration of pepsin in the digestion solution D is 1 mg / ml - 4 mg / ml, and the concentration of the decellularized extracellular matrix solution obtained after adding the decellularized extracellular matrix powder is close to 1%.

[0013] Preferably, in step 2), the pH is adjusted by titrating the decellularized extracellular matrix solution with 1M - 5M sodium hydroxide solution to pH = 6.8 - 7.5.

[0014] Preferably, in step 2), the ion concentration is adjusted by diluting with 10xPBS solution to 1xPBS solution.

[0015] Preferably, in step 2), the porcine testicular decellularized extracellular matrix pre - gel solution is diluted to 0.5% - 1% and incubated at 34 - 38 °C for 20 - 50 min.

[0016] The present invention also provides a thermosensitive hydrogel of porcine testicular decellularized extracellular matrix prepared by the above method. This hydrogel is derived from natural tissue, has low immunogenicity and high biocompatibility, and forms a thermosensitive shape through collagen self - assembly. It has injectability and plasticity. Its main components, collagen and laminin, can effectively regulate the adhesion, proliferation and differentiation of stem cells. Its characteristic of being derived from testicular tissue has strong tissue specificity and has a good effect on the regulation of the local testicular microenvironment and the secretion of testosterone hormone.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] 1. The present invention constructs a biomedical material with high biosafety and low immunogenicity, which can form a stable hydrogel through incubation at a certain temperature.

[0019] 2. The decellularized extracellular matrix hydrogel has good plasticity and sensitive thermosensitivity, and can form a stable gel in the testicular interstitium after implantation.

[0020] 3. In order to retain the integrity of the biological components of the extracellular matrix to the greatest extent, a mild non - ionic detergent is used in the decellularization process, realizing a high retention of collagen, laminin and glycosaminoglycan.

[0021] 4. The retained biological components contain a large number of biological signal cues, which can recruit cells in the testis, so as to act on the differentiation of testicular interstitial stem cells into testicular interstitial cells and secrete testosterone. The hydrogel can provide more specific receptors for luteinizing hormone, thereby improving the efficiency of testosterone synthesis and further enhancing the reproductive ability of male individuals.

[0022] 5. A large number of cytokines and enzymes in the prepared acellular extracellular matrix hydrogel have a certain inhibitory effect on the inflammation of the testicular microenvironment.

[0023] 6. The acellular extracellular matrix material is inexpensive to prepare, easy to store in powder form, and has good degradability. It can be used as an excellent and multifunctional biomedical material with good clinical application prospects. Description of the Drawings

[0024] Figure 1 It is a roadmap for the hydrogel preparation experiment.

[0025] Figure 2 It is a physical diagram and SEM diagram of the hydrogel plasticity.

[0026] Figure 3 It is a rheological test curve graph of the hydrogel.

[0027] Figure 4 It is a quantitative statistical chart of the DNA content and collagen content of the hydrogel.

[0028] Figure 5 It is a result graph of the hydrogel proteomics analysis. Detailed Embodiments

[0029] The present invention will be further described in detail below with reference to the drawings and embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0030] Example 1

[0031] 1) After subjecting fresh porcine testicular tissue to freeze-thaw cycles, the tunica albuginea and rete testis on the surface of the testis are removed, and then the testis is cut into pieces with a volume of 1 cm using a knife and scissors. 3Small pieces are then weighed, and washing solution A is added at a density of 100 g / 1 L and stirred for washing for 24 h. Washing solution A is prepared by adding 10 ml of TritonX-100 solution and 10 ml of penicillin-streptomycin to 1 L of deionized water to form a 0.1% TritonX-100 and 0.1% penicillin-streptomycin solution, and stirred thoroughly. During the stirring process, washing solution A is changed every 2 h. Subsequently, the white tissue pieces are transferred to washing solution B and stirred for washing for 24 h. Washing solution B is prepared by adding 50 ml of 10xPBS solution and 10 ml of penicillin-streptomycin solution to 940 ml of deionized water to form 1xPBS and 0.1% penicillin-streptomycin solution. During the stirring process, washing solution B is changed every 2 h. Then the tissue is transferred to disinfectant solution C and stirred for 1 h. Disinfectant solution C is prepared by adding 1 ml of compound peracetic acid and 40 ml of absolute ethanol to 1 L of deionized water to form a solution with a concentration of 4% ethanol and 0.1% compound peracetic acid. Finally, the tissue is transferred to deionized water for cleaning for 24 h to thoroughly clean the disinfectant. Freeze-dry at -80 °C for 3 days, grind into particles with a size of 100 μm using a grinder, and then irradiate with gamma rays at a dose of 5 kGy for sterilization. Sterile acellular extracellular matrix powder is obtained.

[0032] 2) To the obtained acellular extracellular matrix powder, 20 ml of digestive solution D is added to every 200 mg of powder and stirred for digestion for 3 days at a stirring rate of 600 rpm. The whole process is carried out in a sterile environment. Digestive solution D is prepared by adding 100 mg of pepsin to 50 ml of 0.1 M hydrochloric acid to form a 2 mg / ml pepsin solution, and filtered and sterilized to obtain a sterile digestive solution. After digestion is completed, the solution is uniform and transparent. In a sterile environment, sterile 1 M sodium hydroxide is added drop by drop, and the pH of the solution is tested with pH test paper. When the pH reaches 7, the pH adjustment is completed. Subsequently, 2 ml of 10xPBS is added to adjust the ion concentration to obtain a porcine testicular acellular extracellular matrix pre-gel solution with a concentration of approximately 1%. Then, 1 ml of the pre-gel solution is aspirated and added to a 5 ml centrifuge tube, placed in a 38 °C water bath for 20 minutes, and then taken out and inverted. It can be seen that the gel is stably formed, and a porcine testicular acellular extracellular matrix thermosensitive hydrogel can be obtained.

[0033] The preparation process of this example is as Figure 1 shown. Porcine testes are chopped, acellularly washed, freeze-dried and ground, digested and volume-expanded, and the pH, ion concentration and temperature are adjusted, and finally an acellular extracellular matrix hydrogel is obtained. The SEM image of the acellular extracellular matrix hydrogel in this example is shown in Figure 2 shown. It can be seen from the electron microscope image that the hydrogel presents a collagen fiber structure, and the fiber filaments are thin and dense, indicating that the obtained hydrogel highly retains the microscopic structure of collagen. The physical image of the formed acellular extracellular matrix hydrogel in this example is shown in Figure 2As shown, the hydrogel can form triangles, squares, and circles according to the mold, and has good plasticity. The quantitative detection of the DNA content and collagen content of the decellularized extracellular matrix in this example is shown in Figure 3 As shown, the results show that after the decellularization treatment, the DNA content is effectively removed, while the collagen in the extracellular matrix is highly retained, and the collagen content is significantly increased compared with natural tissues. The rheological properties of the decellularized extracellular matrix hydrogel in this example are shown in Figure 4 As shown, the results show that the hydrogel has stable formability, thermosensitivity, and self-healing properties. The proteomics analysis of the decellularized extracellular matrix hydrogel in this example is shown in Figure 5 As shown, it can be seen that the main components of the hydrogel are composed of collagen, laminin, glycosaminoglycan, cytokines, and enzymes, and the hydrogel contains biological cues such as sex hormone secretion and sperm production.

[0034] Example 2

[0035] Different from Example 1, in step 1), the concentration of TritonX-100 in washing solution A is 0.5%, the washing solution is changed every 4 h, and freeze-dried at -80 °C for 2 days.

[0036] Example 3

[0037] Different from Example 1, in step 1), the concentration of TritonX-100 in washing solution A is 0.05%, the concentration of penicillin-streptomycin is 1%, and freeze-dried at -80 °C for 5 days.

[0038] Example 4

[0039] Different from Example 1, in step 2), the pepsin concentration is 1 mg / ml and the digestion time is 2 days.

[0040] Example 5

[0041] Different from Example 1, in step 2), the pepsin concentration is 4 mg / ml, the hydrochloric acid solution is 0.01 M, and the digestion time is 5 days.

[0042] Example 6

[0043] Different from Example 1, in step 2), the stirring rate is 800 rpm, the pepsin concentration is 3.3 mg / ml, the solvent used to adjust the PH is 5 M sodium hydroxide, and the solution PH is adjusted to 7.5.

[0044] Example 7

[0045] Different from Example 1, in step 1), the gamma-ray irradiation intensity is 10 kGy, the volume of the chopped testicular tissue block is 4 cm 3 , and the particle size of the ground powder is 10 μm.

[0046] Example 8

[0047] Differing from Example 1, in step 2), the pH of the acellular extracellular matrix pre-solution is adjusted to 6.8, diluted to 0.5%, the temperature of the warm bath is 34 °C, and the warm bath time is 50 min.

[0048] The examples of the present invention are merely examples given to clearly illustrate the present invention, rather than limitations on the implementation modes of the present invention. For professionals in the relevant field, based on the above examples, other different forms of changes or modifications can be made. It is not necessary and impossible to list all implementation modes here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A preparation method of a porcine testicular acellular extracellular matrix thermosensitive hydrogel, characterized in that, It includes the following steps: 1) After subjecting the obtained porcine testicular tissue to multiple freeze-thaw cycles, remove the tunica albuginea and rete testis tissue, then chop it into small tissue pieces, and successively place the small tissue pieces in washing solution A, washing solution B, disinfectant solution C, and deionized water for stirring, washing, and sterilization to obtain a white tissue. Freeze-dry, grind, and sterilize the obtained white tissue to obtain acellular extracellular matrix powder; wherein, the washing solution A is composed of a mixed solution of 0.05%-0.5% TritonX-100 and 0.1%-1% penicillin-streptomycin, the washing solution B is composed of 1x PBS solution and 0.1%-1% penicillin-streptomycin mixed solution, and the disinfectant solution C is composed of a mixed solution of 4% ethanol and 0.1% compound peracetic acid disinfectant; 2) Place the obtained acellular extracellular matrix powder in digestive solution D for stirring and digestion. After digestion is completed, adjust the pH and ionic concentration to obtain a porcine testicular acellular extracellular matrix pre-gel solution. Place the porcine testicular acellular extracellular matrix pre-gel solution in a warm bath environment for a period of time to obtain a porcine testicular acellular extracellular matrix thermosensitive hydrogel; wherein, the digestive solution D uses 0.01M-0.1M hydrochloric acid to dissolve pepsin.

2. The preparation method according to claim 1, characterized in that: In step 1), the washing solution A and the washing solution B are replaced every 2-4 h during stirring, and the disinfectant solution C is stirred and disinfected for 1 h.

3. The preparation method according to claim 1, characterized in that: In step 1), the volume of the small tissue block is 1 cm 3 - 4 cm 3 , during the stirring process, every 100 g of the tissue is added to 1 L of the stirring solution, and the particle size of the ground powder is 10 - 100 μm.

4. The preparation method according to claim 1, wherein: In step 1), the freeze-drying is carried out at -80°C for 2-5 days, and the sterilization is carried out by 5-10 kGy gamma ray irradiation.

5. The preparation method according to claim 1, characterized in that: In step 2), the digestion process is carried out in a sterile room temperature environment, stirred for 2-5 days, and the acellular extracellular matrix powder is digested at a stirring rate of 600-800 rpm.

6. The preparation method according to claim 1, wherein: In step 2), the concentration of pepsin in the digestive solution D is 1 mg / ml-4 mg / ml, and the concentration of the acellular extracellular matrix solution obtained after adding the acellular extracellular matrix powder is close to 1%.

7. The preparation method according to claim 1, characterized in that: In step 2), the pH is adjusted by titrating the acellular extracellular matrix solution with 1M-5M sodium hydroxide solution to pH = 6.8-7.

5.

8. The preparation method according to claim 1, wherein: In step 2), the ionic concentration is adjusted by diluting to 1x PBS solution with 10x PBS solution.

9. The preparation method according to claim 1, characterized in that: In step 2), the porcine testicular acellular extracellular matrix pre-gel solution is diluted to 0.5%-1% and warm-bathed at 34-38°C for 20-50 min.

10. The porcine testicular acellular extracellular matrix thermosensitive hydrogel prepared by the method according to any one of claims 1-9. This hydrogel is from natural tissue, has low immunogenicity and high biocompatibility, and uses collagen self-assembly thermosensitive molding, has injectability and plasticity. Its main components, collagen and laminin, can effectively regulate the adhesion, proliferation, and differentiation of stem cells. Its characteristics from testicular tissue have strong tissue specificity and have good effects on the regulation of the local testicular microenvironment and the secretion of testosterone hormone.

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