CN114395525A - Preparation method of acellular and antigen-removed xenogenic tendon - Google Patents
Preparation method of acellular and antigen-removed xenogenic tendon Download PDFInfo
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- CN114395525A CN114395525A CN202111593093.XA CN202111593093A CN114395525A CN 114395525 A CN114395525 A CN 114395525A CN 202111593093 A CN202111593093 A CN 202111593093A CN 114395525 A CN114395525 A CN 114395525A
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- 210000002435 tendon Anatomy 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title abstract description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- 102000004190 Enzymes Human genes 0.000 claims abstract description 9
- 101000718529 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) Alpha-galactosidase Proteins 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 8
- 239000004094 surface-active agent Substances 0.000 claims abstract description 6
- 239000008213 purified water Substances 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 238000004140 cleaning Methods 0.000 claims description 26
- 229940088598 enzyme Drugs 0.000 claims description 8
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 6
- 238000004506 ultrasonic cleaning Methods 0.000 claims description 4
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 102000016911 Deoxyribonucleases Human genes 0.000 claims description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 claims description 2
- 239000000427 antigen Substances 0.000 abstract description 3
- 102000036639 antigens Human genes 0.000 abstract description 3
- 108091007433 antigens Proteins 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 8
- 230000000890 antigenic effect Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 206010061223 Ligament injury Diseases 0.000 description 2
- 208000021945 Tendon injury Diseases 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000024288 Rotator Cuff injury Diseases 0.000 description 1
- 206010039227 Rotator cuff syndrome Diseases 0.000 description 1
- 206010043248 Tendon rupture Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000001074 muscle attachment cell Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 238000009864 tensile test Methods 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/066—Tenocytes; Tendons, Ligaments
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a preparation method of a foreign tendon with cells removed and antigens removed. The method adopts the pig tendon raw material, and the pig tendon raw material is treated by a surfactant, NaOH solution and alpha-Gal antigen enzyme and DNA enzyme, so that the prepared pig tendon is complete in decellularization, good in cell compatibility, small in immune rejection, strong in mechanical property and wide in material source.
Description
Technical Field
The invention belongs to the technical field of tendon/ligament injury repair, and particularly relates to a preparation method of a acellular and antigen-removed heterogeneous tendon.
Background
Acute and chronic tendon injuries of different degrees caused by modern daily life include achilles tendon rupture, chronic rotator cuff injury, cruciate ligament injury and the like, and the traditional treatment mainly comprises suture, autograft, allograft, artificial synthetic materials and the like. The reconstruction of the autologous tendon has the advantages of early healing of the tendon, good tissue compatibility, rapid tissue shaping and the like, but the clinical application shows that the autologous tendon has the defects of uncontrollable length and diameter, complication at the tendon taking part, influence on the beauty and the like. Most of the artificially synthesized materials are macromolecular organic matters and are not easy to degrade, and meanwhile, degradation products can possibly cause the change of a local microenvironment, so that local inflammation is caused to be unfavorable for the healing of tissues. Allogeneic tendons present immune rejection and risk of infection, etc., and are also affected by the donor source.
Disclosure of Invention
The invention aims to provide a preparation method of a foreign tendon with cells removed and antigens removed.
A method for preparing a cell-free and antigen-removed xenogenic tendon comprises the following operation steps:
(1) taking a pig tendon raw material, adopting purified water for cleaning treatment, removing redundant tissues, then adopting a surfactant for treatment, and adopting purified water for ultrasonic cleaning after the treatment is finished;
(2) treating the NaOH solution for 0.5-2h by using a shaking table, and ultrasonically cleaning the NaOH solution by using purified water after the treatment is finished;
(3) carrying out shake bed treatment on the alpha-Gal antigen enzyme for 12-24h, and then ultrasonically cleaning by using purified water;
(4) treating for 12-24h by a DNA enzyme shaking table, and ultrasonically cleaning by purified water after the treatment is finished to obtain the acellular antigen-removed tendon.
The surfactant is SDS and/or triton, and the mass concentration is 1%.
And (2) after the treatment in the step (1) is finished, 0.5% of pancreatin is added for digestion for 1-3h, and after the treatment is finished, purified water is adopted for ultrasonic cleaning.
The concentration of the alpha-Gal antigen enzyme is 5 mg/mL.
The concentration of the DNase is 1 mg/mL.
The mass concentration of the NaOH solution is 1%.
The invention has the beneficial effects that: the invention relates to a preparation method of a foreign tendon with cells removed and antigen removed, which adopts a pig tendon raw material and is treated by a surfactant, NaOH solution and alpha-Gal antigen enzyme and DNA enzyme, and the prepared pig tendon has complete cell removal, good cell compatibility, small immune rejection, stronger mechanical property and wide material source.
Drawings
FIG. 1 shows the results of HE staining after decellularization;
in the figure, A is porcine tendon starting material, B is decellularized antigenic tendon of example 1, C is decellularized antigenic tendon of example 2, and D is decellularized antigenic tendon of example 3.
FIG. 2 shows the tenocyte cytotoxicity results after different processes.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1
A preparation method of a acellular and antigen-removed xenogenic tendon comprises the following operation steps:
(1) cleaning a pig tendon raw material by using purified water, removing redundant tissues, treating for 1h by using a 1% SDS shaking table, and ultrasonically cleaning by using the purified water after the treatment is finished; then digesting for 2 hours by adopting 0.5 percent pancreatin, and ultrasonically cleaning by adopting purified water after the treatment is finished;
(2) treating for 16h by adopting a 5mg/mL alpha-Gal antigen enzyme shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished;
(3) treating for 16h by adopting a 1mg/mL DNA enzyme shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished;
(4) and (3) treating for 1h by adopting a NaOH solution with the mass concentration of 1% in a shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished to obtain the acellular antigenic removed tendon.
Example 2
A preparation method of a acellular and antigen-removed xenogenic tendon comprises the following operation steps:
(1) cleaning a pig tendon raw material by using purified water, removing redundant tissues, treating for 72 hours by using a 1% triton shaking table, and ultrasonically cleaning by using the purified water after the treatment is finished; then treating for 24 hours by adopting a 1% triton shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished;
(2) treating for 16h by adopting a 5mg/mL alpha-Gal antigen enzyme shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished;
(3) treating for 16h by adopting a 1mg/mL DNA enzyme shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished;
(4) and (3) treating for 1h by adopting a NaOH solution with the mass concentration of 1% in a shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished to obtain the acellular antigenic removed tendon.
Example 3
A preparation method of a acellular and antigen-removed xenogenic tendon comprises the following operation steps:
(1) cleaning a pig tendon raw material by using purified water, removing redundant tissues, treating for 72 hours by using a 1% triton shaking table, and ultrasonically cleaning by using the purified water after the treatment is finished; then treating for 24 hours by adopting a 1% triton shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished;
(4) treating for 1h by adopting a NaOH solution with the mass concentration of 1% in a shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished;
(2) treating for 16h by adopting a 5mg/mL alpha-Gal antigen enzyme shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished;
(3) and (3) treating for 16h by adopting a 1mg/mL DNA enzyme shaking table, and ultrasonically cleaning by adopting purified water after the treatment is finished to obtain the acellular antigen-removed tendon.
Experimental example 1:
the porcine tendons prepared in examples 1-3 were fixed in 40g/L paraformaldehyde for 48h, dehydrated, embedded, sliced, deparaffinized, stained with hematoxylin-eosin, and observed under a microscope. As shown in fig. 1, the pig tendon prepared in example 1 and example 2 has a certain decellularization effect, the pig tendon prepared in example 3 has a thorough decellularization effect, and the effect is the best, while the procedure of example 2 is substantially the same as that of example 3, the example 3 is performed by shaking the NaOH solution before the enzyme solution treatment, and the example 2 is performed after the enzyme solution treatment, and the timing of shaking the NaOH solution is proved to be a key step.
Experimental example 2:
the cell compatibility of porcine tendon was evaluated using L929 cells. Samples of examples 1-3 (processes 1-3) and untreated porcine tendon (control) were added to the medium at a ratio of 0.2ml/1ml, and extracted at 37 ℃ for 24 hours to obtain an extract. Taking normally cultured L929 cells, adjusting cell density to 2 × 104Inoculating the culture medium to a 96-well culture plate, culturing for 24h, removing the original culture medium, and adding corresponding leaching liquor; after 72h, the original medium was discarded, 100ul of medium containing 10% CCK8 was added to each well, and absorbance was measured at a wavelength of 450nm after 30 min. The relative increment rates of the respective sample groups and the control group are shown in FIG. 2.
Experimental example 3:
the porcine tendons prepared in examples 1 to 3 and the untreated porcine tendon (control) were cut into test pieces using a tensile (compression) testing machine, and immediately after the cut pieces were left to stand at a relative humidity of 50% and a temperature of 22 ℃. + -. 2 ℃ for 2 hours, the test was conducted. And fixing the two ends of the sample on a chuck of a tensile testing machine, sequentially stretching outwards at the speed of 100mm/min until the sample is broken, and respectively testing the longitudinal sample and the transverse sample. The force at which the sample breaks is recorded in units of N and the results are shown in table 1:
TABLE 1
Note: represents P <0.05 compared to the example 1 group.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (6)
1. A method for preparing a acellular and antigen-removed xenogenic tendon is characterized by comprising the following operation steps:
(1) taking a pig tendon raw material, adopting purified water for cleaning treatment, removing redundant tissues, then adopting a surfactant for treatment, and adopting purified water for ultrasonic cleaning after the treatment is finished;
(2) treating the NaOH solution for 0.5-2h by using a shaking table, and ultrasonically cleaning the NaOH solution by using purified water after the treatment is finished;
(3) carrying out shake bed treatment on the alpha-Gal antigen enzyme for 12-24h, and then ultrasonically cleaning by using purified water;
(4) treating for 12-24h by a DNA enzyme shaking table, and ultrasonically cleaning by purified water after the treatment is finished to obtain the acellular antigen-removed tendon.
2. The method for preparing decellularized and deintigen xenogenic tendon according to claim 1, wherein the surfactant is SDS and/or triton and the concentration by mass is 1%.
3. The method for preparing acellular and antigen-removed xenogenic tendon according to claim 1, wherein the step (1) is performed by adding 0.5% pancreatin for 1-3h after the treatment, and the purified water is used for ultrasonic cleaning after the treatment.
4. The method for preparing a decellularized and antigenicized tendon according to claim 1 wherein the concentration of said α -Gal antigen enzyme is 5 mg/mL.
5. The method of claim 1, wherein the DNase is present at a concentration of 1 mg/mL.
6. The method of claim 1, wherein the NaOH solution is present at a concentration of 1% by mass.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115029298A (en) * | 2022-07-01 | 2022-09-09 | 北京德益达美医疗科技有限公司 | Antigen-removed tendon and preparation method thereof |
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