CN108465125A - The tendon composite muscle in natural tissues source takes off the preparation method of cell material - Google Patents

Abstract

The invention discloses the preparation methods that a kind of tendon composite muscle in natural tissues source takes off cell material, by the tendon composite muscle tissue of mammal arbitrary size through the PBS containing protease inhibitors, the PBS buffer solution containing KCl, the PBS buffer solution containing KI, the PBS buffer solution of the X containing Triton, the PBS buffer solution containing SDS, the PBS buffer solution processing containing DNA enzymatic, the tendon composite muscle for obtaining natural tissues source takes off cell material.The present invention can carry out structural cell complete decellularization processing, and mild condition, without ECM damages, fast and stable.The material of gained, the layered structure of natural tendon composite muscle is not only simulated to greatest extent, preferably remains extracellular matrix components, also there is extremely low immunogenicity, the advantages that stronger mechanical property, can be used for repairing clinically muscle damage, regression relevant disease caused by all kinds of causes of disease.

Description

Preparation method of tendon composite muscle decellularized material derived from natural tissue

technical field

The present invention mainly relates to the medical field for repairing and regenerating skeletal muscle tissue of the whole body, in particular to the preparation of a tendon composite muscle decellularized material derived from natural tissue. It retains the layered structure of the natural tendon compound muscle, extracellular matrix components, has strong mechanical properties, and the material can promote the repair and regeneration of muscle defects.

Background technique

Muscle injuries caused by trauma, unreasonable exercise, degenerative diseases, etc. are very common in clinical practice and seriously affect the quality of life of patients. The widespread application of tissue engineering has made some progress in overcoming this disease. Current research is mainly focused on preparing a simple muscle scaffold to induce muscle fiber regeneration, but the effect is not ideal. First: Under physiological conditions, muscle fibers are connected to tendons or myofascia, and it is through the connection of muscles and tendons that a network structure is formed, and a single muscle fiber forms a sum of contractions to produce efficient movement, while a simple muscle scaffold lacks this structure. Second: The mechanical properties of simple muscle scaffolds are poor (maximum stress is 0.1-5Mpa), and they are easy to rupture under high tension of large abdominal defects or limb muscle defects, while the maximum stress level of tendon scaffolds is 30-1000Mpa, which can Provide stable mechanical support. Therefore, the tendon composite muscle scaffold with a complete mechanical conduction system is more suitable for muscle tissue engineering.

Due to the complexity of the natural tendon-composite muscle tissue, especially the junction between the two, few studies have been able to create a scaffold that completely simulates the natural tendon-composite muscle structure, which cannot achieve good repair and regeneration of muscle defects. Decellularization technology can provide a potential solution. While removing immunogenic cellular components, decellularized scaffolds can better simulate the natural tissue structure and retain extracellular matrix components. Currently, decellularized scaffolds for tendon and muscle have not yet See report. Therefore, the present invention prepares a natural tendon composite muscle decellularized scaffold through innovative decellularization technology, which simulates the natural tendon composite muscle structure to the greatest extent while removing cells, and proves that the scaffold has extremely low immunogenicity , has strong mechanical properties, better retains the extracellular matrix components of the natural tendon compound muscle tissue, and proves that the scaffold can well repair muscle defects in vivo, and has a great role in muscle regeneration tissue engineering meaning.

Invention content:

The purpose of the present invention is to fill in the prior art, there is no report on the preparation of tendon composite muscle scaffolds, to provide one with extremely low immunogenicity, good mechanical properties, better retain extracellular matrix components, and can promote Preparation method of decellularized tendon composite muscle scaffold for muscle defect regeneration.

A method for preparing a tendon compound muscle decellularized material derived from natural tissue, the method specifically includes the following steps:

(1) Take the tendon compound muscle tissue of any mammal within 12 hours of slaughter, remove the fat on the muscle and tendon with scissors, rinse with PBS buffer containing protease inhibitors for 3 times, each time for 30 minutes, and remove blood and attached fat debris and other impurities;

(2) In PBS buffer containing protease inhibitors, shake at 100 rpm on a shaker at 4°C for 24-48 hours; then freeze and thaw with liquid nitrogen for 3 cycles;

(3) In the PBS buffer solution of KCL, add the mixed antibacterial solution, shake with a shaker at 100 rpm for 2-12 hours at a temperature of 4°C;

(4) Add the mixed antibacterial solution to the PBS buffer solution of KI, and shake it with a shaker at 100 rpm for 2-12 hours at a temperature of 4°C;

(5) In the PBS buffer solution containing Triton X, add the mixed antibacterial solution, and shake with a shaker at 100 rpm for 24-48 hours at a temperature of 4°C;

(6) In the PBS buffer solution containing SDS, add the mixed antibacterial solution, and shake with a shaker at 100 rpm for 24-48 hours at a temperature of 4°C;

(7) In the PBS buffer solution containing DNase, add the mixed antibacterial solution, and shake with a shaker at 120 rpm for 12-24 hours at a temperature of 37 ° C;

In steps (2)-(7), rinse with physiological saline for 8 hours after each step is completed.

As a preference, the protease inhibitor concentration in the PBS buffer containing the protease inhibitor is 10-50 KIU/ml.

Preferably, the PBS buffer containing KCL is a PBS buffer containing KCL at a concentration of 0.1M-1M.

Preferably, the KI-containing PBS buffer is a KI-containing PBS buffer with a concentration of 1M-1.5M.

Preferably, the PBS buffer solution containing Triton X is a PBS buffer solution with a concentration of 0.05%-2% Triton X-100 or Triton X-200.

Preferably, the PBS buffer containing SDS is a PBS buffer containing SDS at a concentration of 0.02%-0.25%.

Preferably, the PBS buffer solution containing DNase is a PBS buffer solution with a concentration of 1-2 mg/mL DNase.

As preferably, the concentrations of penicillin and streptomycin in the mixed antibacterial solution are 20K IU/ml and 20g/ml respectively; the ratio of penicillin and streptomycin is 1:1, and the volume ratio of the added mixed antibacterial solution to the original solution is 1:1.

Preferably, the temperature in the three cycles of freezing and thawing of liquid nitrogen is from -80°C to 37°C.

Aiming at the deficiencies and deficiencies in the materials and preparation methods currently used for muscle repair and regeneration, the inventors have established a method for the preparation of tendon composite muscle decellularized materials derived from natural tissues. This method composites tendons of any size in mammals Muscle tissue was treated with PBS buffer containing protease inhibitors, PBS buffer containing KCl, PBS buffer containing KI, PBS buffer containing Triton X, PBS buffer containing SDS, and PBS buffer containing DNase to obtain natural Tissue-derived tendon-composite muscle decellularized material. The invention can completely decellularize the cells on the tissue with mild conditions, no ECM damage, and rapid stability. The obtained material not only simulates the layered structure of the natural tendon compound muscle to the greatest extent, but also retains the extracellular matrix components better, and also has the advantages of extremely low immunogenicity and strong mechanical properties, which can be used for repairing Clinically, muscle damage and degeneration related diseases caused by various etiologies.

Compared with prior art, remarkable progress of the present invention is:

(1) The decellularized tendon composite muscle material prepared by the decellularization technology in the present invention can better retain the integrity of the original ECM while removing heterogeneous/alternative cells, and can simulate the layered structure of normal tendon composite muscle to the greatest extent. Extracellular matrix composition, microstructure.

(2) The material of the present invention does not contain antigenic substances such as cells, nuclei, and DNA, has extremely low immune rejection, and has good biocompatibility.

(3) The material of the present invention has strong biomechanical properties due to the composite tendon tissue.

(4) The material of the present invention can promote muscle regeneration in the model of muscle defect, and has great significance in muscle regeneration tissue engineering.

Description of drawings

Fig. 1 is the general appearance figure of material of the present invention;

Fig. 2 is a cell-free and cell-free nuclear component residue diagram of the material of the present invention stained with HE;

Fig. 3 is the material DNA quantitative detection of the present invention and almost does not contain the DNA component diagram;

Fig. 4 is a qualitative (Masson's trichrome staining) and quantitative detection of the collagen components of the material of the present invention to retain a large amount of collagen components;

Fig. 5 is a scanning electron microscope proof of the material of the present invention showing fiber arrangement and spatial three-dimensional structure retention;

Fig. 6 is the extremely strong biomechanical diagram proved by the detection of the biomechanical properties of the material of the present invention;

Fig. 7 is the excellent biocompatibility diagram proved by HE staining test of the material of the present invention in vitro;

Fig. 8 is the quantitative detection of myofiber production gene expression diagram by multiple cell PCR in vitro of the material of the present invention;

Figure 9 is a picture of subcutaneous embedding of this material in rats, 7 days and 30 days HE staining and CD86, CD163 immunohistochemical staining proves less inflammatory cell infiltration and extremely low immunogenicity;

Fig. 10 is a picture of HE and Masson staining of muscle fiber regeneration confirmed by the material of the present invention transplanted into rabbits with large area of muscle defect for 7 days and 30 days, which proves that it can promote muscle repair and regeneration in vivo.

specific implementation plan

The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but the implementation of the present invention is not limited thereto.

Example 1: Preparation method and regenerative repair research of tendon composite muscle decellularized material derived from natural tissue

1. Preparation of Tendon Composite Muscle Decellularized Material

(1) Materials: Take the tendon compound muscle material from the Achilles tendon of pigs, cut the tendon into length, width and thickness of 2*1.0*0.2cm according to the fiber direction, and the length, width and height of the muscle are 0.3cm. IU/ml protease inhibitor normal saline rinsed 3 times, 30min each time, fully rinsed with sterile PBS to remove blood and other impurities.

(2) The decellularization steps are as follows:

①. In the PBS buffer solution containing 10K IU/ml protease inhibitor, shake at 100 rpm on a low temperature (4°C) shaker for 48 hours, and then freeze and thaw with liquid nitrogen for 3 cycles (-80°C/37°C);

②. In the PBS buffer containing 0.1M KCL, add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1, and shake at 100rpm on a low-temperature (4°C) shaker for 6 hours;

③. In PBS buffer containing 1M KI, add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1, and shake at 100rpm on a low temperature (4°C) shaker for 6 hours;

④. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution in 0.05% Triton X-100 PBS buffer solution at a ratio of 1:1, shake at 100rpm on a low-temperature (4°C) shaker for 24 Hour;

⑤. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution in 0.02% SDS-containing PBS buffer solution at a ratio of 1:1, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;

⑥. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution in 1mg/ml DNase-containing PBS buffer solution at a ratio of 1:1, shake at 100rpm on a shaker at 37°C for 12 hours, that is Tendon composite muscle decellularized material is available.

Note: Rinse with saline for 8 hours after each step.

2. Evaluation of Cell Removal from Natural Tissue-Derived Tendon Composite Muscle Decellularized Materials

Fig. 1 is a general appearance diagram of the material of the present invention (* represents the tendon part; △ represents the muscle part); Fig. 2 is a cell-free and non-nuclear component residual image of the material of the present invention stained with HE; Fig. 3 is the DNA quantitative detection of the material of the present invention Almost no DNA composition map.

3. Evaluation of Extracellular Matrix Retention of Tendon-Compound Muscle Decellularized Materials

Fig. 4 is a graph of qualitative and quantitative detection of collagen components of the material of the present invention to retain a large amount of collagen components; Fig. 5 is a diagram of fiber arrangement and spatial three-dimensional structure retention of the material of the present invention as evidenced by scanning electron microscopy (arrows indicate the direction of fiber arrangement).

4. Evaluation of Biomechanical Properties of Tendon Composite Muscle Decellularized Materials

Fig. 6 is a very strong biomechanical diagram proved by the detection of the biomechanical properties of the material of the present invention (ultimate tensile stress, elastic coefficient and tension-pressure curve).

5. Determination of good biocompatibility and extremely low immunogenicity of tendon composite muscle acellular material

Fig. 7 is the excellent biocompatibility figure proved by the HE staining detection of the material of the present invention in vitro; Fig. 8 is the gene expression diagram of the quantitative detection of muscle fibers produced by PCR of the material of the present invention in vitro; Fig. 9 is the subcutaneous bag of the material in rats Buried, 7 days and 30 days HE staining and CD86, CD163 immunohistochemical staining proved less inflammatory cell infiltration, very low immunogenicity figure.

6. Tendon composite muscle decellularized material promotes the repair and regeneration of muscle defects

Fig. 10 is a picture of HE and Masson staining of muscle fiber regeneration confirmed by the material of the present invention transplanted into rabbits with large area of muscle defect for 7 days and 30 days, which proves that it can promote muscle repair and regeneration in vivo.

Example 2: Preparation and regenerative repair of decellularized tendon and muscle materials

1. Preparation of Tendon Composite Muscle Decellularized Material

(1) Materials: Take the tendon compound muscle material from the Achilles tendon of pigs, cut the tendon into length, width and thickness of 4.0*2.0*0.4cm according to the fiber direction, and the length, width and height of the muscle are 0.6cm. IU/ml protease inhibitor normal saline rinsed 3 times, 30min each time, fully rinsed with sterile PBS to remove blood and other impurities.

(2) The decellularization steps are as follows:

①. In the PBS buffer solution containing 30K IU/ml protease inhibitor, shake at 100 rpm for 48 hours on a low-temperature (4°C) shaker, and then freeze and thaw with liquid nitrogen for 3 cycles (-80°C/37°C);

②. In the PBS buffer containing 0.5M KCL, add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1, and shake at 100rpm on a low-temperature (4°C) shaker for 12 hours;

③. In the PBS buffer solution containing 1.3M KI, add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1, and shake at 100rpm on a low-temperature (4°C) shaker for 12 hours;

④. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution in 0.2% Triton X-100 PBS buffer solution at a ratio of 1:1, shake at 100rpm on a low temperature (4°C) shaker for 24 Hour;

⑤. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution in 0.05% PBS buffer solution containing SDS at a concentration of 0.05%, shake at 100rpm on a low temperature (4°C) shaker for 24 hours;

⑥. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1 to 1.5mg/ml DNase-containing PBS buffer, and shake at 100rpm for 12 hours on a shaker at 37°C. The tendon compound muscle decellularized material can be obtained. The rest were carried out with reference to the method of Example 1 to obtain the tendon composite muscle decellularized material derived from natural tissue.

Example 3: Preparation method and regenerative repair research of tendon composite muscle decellularized material derived from natural tissue

1. Preparation of Tendon Composite Muscle Decellularized Material

(1) Materials: The tendon compound muscle material was taken from the Achilles tendon of sheep, and the tendon was cut into length, width and thickness according to the fiber direction of 4.0*2.0*0.6cm, and the length, width and height of the muscle were 0.6cm. IU/ml protease inhibitor normal saline rinsed 3 times, 30min each time, fully rinsed with sterile PBS to remove blood and other impurities.

(2) The decellularization steps are as follows:

①. In the PBS buffer solution containing 30K IU/ml protease inhibitor, shake at 100 rpm for 36 hours on a low-temperature (4°C) shaker, and then freeze and thaw with liquid nitrogen for 3 cycles (-80°C/37°C);

②. In the PBS buffer containing 0.5M KCL, add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1, and shake at 100rpm on a low-temperature (4°C) shaker for 12 hours;

③. In the PBS buffer containing 1.3M KI, add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1, and shake at 100rpm on a low-temperature (4°C) shaker for 2 hours;

④. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution in 0.5% Triton X-100 PBS buffer solution at a ratio of 1:1. Hour;

⑤. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution in 0.1% PBS buffer containing SDS at a concentration of 1:1, shake at 100rpm on a low temperature (4°C) shaker for 30 hours;

⑥. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1 to 1.5mg/ml DNase-containing PBS buffer, and shake at 100rpm for 12 hours on a shaker at 37°C. The tendon compound muscle decellularized material can be obtained. The rest were carried out with reference to the method of Example 1 to obtain the tendon composite muscle decellularized material derived from natural tissue.

Example 4: Preparation method and regenerative repair research of tendon composite muscle decellularized material derived from natural tissue

1. Preparation of Tendon Composite Muscle Decellularized Material

(1) Materials: The tendon compound muscle material was taken from the Achilles tendon of pigs, and the tendon was cut into length, width and thickness according to the fiber direction to be 4*2.0*0.8cm in size, and the length, width, and height of the muscle were both 0.6cm. IU/ml protease inhibitor normal saline rinsed 3 times, 30min each time, fully rinsed with sterile PBS to remove blood and other impurities.

(2) The decellularization steps are as follows:

①. In the PBS buffer solution containing 50K IU/ml protease inhibitor, shake at 100rpm on a low temperature (4°C) shaker for 48 hours, and then freeze and thaw with liquid nitrogen for 3 cycles (-80°C/37°C);

②. In PBS buffer containing 1M KCL, add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1, and shake at 100rpm on a low temperature (4°C) shaker for 2 hours;

③. In the PBS buffer containing 1.5M KI, add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1, and shake at 100rpm on a low-temperature (4°C) shaker for 12 hours;

④. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1 to 2% Triton X-100 in PBS buffer, and shake at 100rpm on a low temperature (4°C) shaker for 36 Hour;

⑤. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution in 0.25% PBS buffer containing SDS at a concentration of 1:1, shake at 100rpm on a low temperature (4°C) shaker for 48 hours;

⑥. Add penicillin and streptomycin (20KIU/ml, 20g/ml) mixed antibacterial solution 1:1 to the PBS buffer solution containing DNase at a concentration of 2mg/ml, shake at 100rpm on a shaker at 37°C for 24 hours, that is Tendon composite muscle decellularized material is available. The rest were carried out with reference to the method of Example 1 to obtain the tendon composite muscle decellularized material derived from natural tissue.

Example 5: Preparation and regenerative repair of decellularized tendon and muscle materials

Take bovine tendon joint muscle tissue, step ④ add penicillin and streptomycin (20K IU/ml, 20g/ml) mixed antibacterial solution in 1:1 in the PBS buffer solution containing Triton X-200 with a concentration of 2%. (4° C.) Shake at 100 rpm for 8 hours. The rest were carried out with reference to the method of Example 1, and the decellularized material of tendon joint muscle was obtained.

The tendon composite muscle decellularized materials obtained in Examples 2-5 were subjected to histological evaluation, quantitative detection of antigenic components and mechanical detection, and the results were similar to those in Example 1, which indicated that a variety of tissue sources could be obtained through the above-mentioned various methods, and many Acellular tendon composite muscle material with different tissue sizes, and the histological evaluation and quantitative detection of antigenic components of the material showed that the material had completely removed cellular components and no obvious antigenic components remained. At the same time, the tendon composite muscle decellularized materials obtained by the above method all have extremely strong biomechanical properties and excellent biocompatibility. The tendon composite muscle decellularized material can be used as a safe, reliable, effective and rapid biomaterial for clinically promoting the repair and regeneration of muscle defects and lesions.

Claims (9)

1. the tendon composite muscle in natural tissues source takes off the preparation method of cell material, which is characterized in that this method is specifically wrapped Include following steps：

(1) the tendon composite muscle tissue for slaughtering arbitrary mammal within 12h is taken, is removed on muscle and tendon with scissors Fat, with containing protease inhibitors PBS buffer solution rinse 3 times, each 30min, remove blood, the fatty fragment of attaching and its His impurity；

(2) it in the PBS buffer solution containing protease inhibitors, is shaken 24-48 hours with shaking table 100rpm at a temperature of 4 DEG C；So 3 cycles of frozen-thawed are used afterwards；

(3) in the PBS buffer solution of KCL, mixing antimicrobial fluid is added, is shaken 2-12 hours with shaking table 100rpm at a temperature of 4 DEG C；

(4) in the PBS buffer solution of KI, mixing antimicrobial fluid is added, is shaken 2-12 hours with shaking table 100rpm at a temperature of 4 DEG C；

(5) in the PBS buffer solution of the X containing Triton, mixing antimicrobial fluid is added, with shaking table 100rpm concussions at a temperature of 4 DEG C 24-48 hours；

(6) in the PBS buffer solution containing SDS, mixing antimicrobial fluid is added, it is small with shaking table 100rpm concussions 24-48 at a temperature of 4 DEG C When；

(7) in the PBS buffer solution containing DNA enzymatic, mixing antimicrobial fluid is added, shakes 12- with shaking table 120rpm at a temperature of 37 DEG C 24 hours；

Normal saline flushing is used in step (2)-(7), after the completion of each step 8 hours.

2. the tendon composite muscle in natural tissues source according to claim 1 takes off the preparation method of cell material, special Sign is：A concentration of 10-50K IU/ml of protease inhibitors in the PBS buffer solution containing protease inhibitors.

3. the tendon composite muscle in natural tissues source according to claim 1 takes off the preparation method of cell material, special Sign is：The PBS buffer solution containing KCL is the PBS buffer solution of the KCL of concentration 0.1M-1M.

4. the tendon composite muscle in natural tissues source according to claim 1 takes off the preparation method of cell material, special Sign is：The PBS buffer solution containing KI is the PBS buffer solution of the KI of concentration 1M-1.5M.

5. the tendon composite muscle in natural tissues source according to claim 1 takes off the preparation method of cell material, special Sign is：The PBS buffer solution of the X containing Triton is Triton X-100 or the Triton X- of concentration 0.05%-2% 200 PBS buffer solution.

6. the tendon composite muscle in natural tissues source according to claim 1 takes off the preparation method of cell material, special Sign is：The PBS buffer solution containing SDS is the PBS buffer solution of the SDS of concentration 0.02%-0.25%.

7. the tendon composite muscle in natural tissues source according to claim 1 takes off the preparation method of cell material, special Sign is：The PBS buffer solution containing DNA enzymatic is a concentration of 1-2mg/mL DNA enzymatics PBS buffer solution.

8. the tendon composite muscle in natural tissues source according to claim 1 takes off the preparation method of cell material, special Sign is：The concentration of penicillin and streptomysin is respectively 20K IU/ml, 20g/ml in the mixing antimicrobial fluid；Penicillin and The volume ratio of streptomysin is 1：1, mixing antimicrobial fluid and the original solution volume ratio of addition are 1：1.

9. the tendon composite muscle in natural tissues source according to claim 1 takes off the preparation method of cell material, special Sign is：The temperature recycled in described 3 cycles of frozen-thawed is from -80 DEG C~37 DEG C.