CN102166373B - Preparation method of human homologous tendon - Google Patents
Preparation method of human homologous tendon Download PDFInfo
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- CN102166373B CN102166373B CN 201010599384 CN201010599384A CN102166373B CN 102166373 B CN102166373 B CN 102166373B CN 201010599384 CN201010599384 CN 201010599384 CN 201010599384 A CN201010599384 A CN 201010599384A CN 102166373 B CN102166373 B CN 102166373B
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Abstract
The invention relates to a preparation method of human homologous tendon, which belongs to the technical field of biomedical materials. The invention mainly solves the technical problems of immunological rejection, bacterial infection, virus infection, complicated operation steps, and the like in the traditional method for transplanting homologous tendon. The preparation method of human homologous tendon comprises the following steps of: (1) cleaning the human homologous tendon; (2) freezing the cleaned human homologous tendon for 30-90 days; (3) trimming loose connective tissues surrounding the tendon; (4) immersing the trimmed tendon material in a mixed solution of peroxyacetic acid, ethanol and water, standing still for inactivation of viruses; (5) cleaning the inactivated tendon with water until the content of the peroxyacetic acid in the cleaning solution reaches a certain value for the last time; (6) trimming the tendon again, dripping the water on the surface of the tendon, vacuumizing the trimmed tendon under an aseptic condition, and packing twice by common packaging; and (7) radiating the packed tendon by using Cobalt-60 for disinfection again. The invention has the advantages of no immunological rejection, no bacterial or virus infection, easiness for operation, no drug residue, and the like.
Description
Technical field
The present invention relates to a kind of human homologous tendon preparation method, be specifically related to a kind of preparation method that can be used for making the allogeneic tendon, belong to the biomaterial for medical purpose technical field.
Background technology
Tendon is a kind of dense connective tissue of queueing discipline, is comprised of collagen, tendon cell and tendon substrate.Collagen is the key component of tendon, accounts for 70～90% of tendon dry weight, brings into play mastery reaction keeping on tendon intensity.Tendon cell is the special fibroblast of a kind of form, negligible amounts, the main carriers of antigen when being tendon transplantation of the same race.Tendon substrate provides structure carrier for collagen tissue, and its elasticity also helps to reduce frictional force and the shearing force that acts on tendon.Tendon connects skeletal muscle and muscular tissue, makes muscular tissue be attached to bone or fascia, and strength transmission to skeletal system, is produced joint motions.
Tendon/ligament is damaged is the common damage of motor system, because ligament is similar to tendon on forming, traditional method is multiplex repairs impaired tendon/ligament from the body tendon transplantation, but owing to having, limited amount not enough for the tendon volume from the body tendon transplantation, and to causing the new problems such as damage for the district, in recent years, the tendon transplantation of the same race of the more use of Chinese scholars replaces being used for the damaged treatment of tendon/ligament from the body tendon.For example in the anterior cruciate ligament revision procedure, than from the body tendon transplantation, scholars are more prone to tendon of the same race as first-selection, and in the anterior cruciate ligament revision procedure, tendon utilization rate of the same race reaches 90% at present; Hand Tendon Defection for another example, limited from body tendon source especially during many Tendon Defections, tendon of the same race is at present comparatively desirable from body tendon substitute.If but there is active tendon cell in tendon of the same race, can cause comparatively significantly immunological rejection after transplanting, and have potential antibacterial, viral infection.
Publication number CN101496912A discloses a kind of bio-derivative tendon repair material and preparation method thereof, that detecting through serology of legal donor source is qualified tendon, or confirm that after quarantine healthy animal tendon carries out Physical Processing and chemical treatment, remove soft tissue, sterilization, by removing fat, taking off cell and process, after rapid (2) pre-freeze of superledge and lyophilization, tissue is gone fine film sub-wire, again braiding is processed, and finally obtains keeping a novel Tendon Defection repair materials of class of tendon basic structure.Except xenogenic tendon, although the method has adopted tendon of the same race to do raw material, operating procedure is numerous and diverse, adopt many chemical reagent to tendon carry out disinfection, defat, deproteinization, be difficult to avoid fully drug residue; Through lyophilization, with processes such as tendon depolymerization, sub-wire restructuring, can cause the change of tendon original structure of the same race; Adopt 25kGy larger dose irradiation, easily cause the tendon Strength Changes, affect the tendon performance.
Summary of the invention
The objective of the invention is to have immunological rejection and antibacterial, viral infection for present tendon transplantation of the same race, and operating procedure is numerous and diverse, drug residue is many, tendon original structure and intensity change large technical problem, provide a kind of without immunological rejection and antibacterial, viral infection, and simple to operate, drug residue free, tendon original structure and the unaltered human homologous tendon preparation method of intensity.
The present invention for the technical scheme that realizes above-mentioned technical problem and take is:
A kind of human homologous tendon preparation method comprises the following steps:
1) extract mankind's donor tendon material and cleaning up with the bloodstain of normal saline with the tendon surface;
2) the tendon material that cleans up is placed in-60～-80 ℃ of freezing preservations 30～90 days;
3) get the tendon material of step (2) after freezing, under room temperature after rewarming, cut off the loose connective tissue around tendon;
4) pruning good tendon material, to immerse weight ratio be 0.05～2: 10～30: in the mixed solution of 70～90 peracetic acid, second alcohol and water, carried out inactivation of virus in standing 20～50 minutes;
5) to clean deactivation tendon peracetic acid content to the last cleanout fluid be 0.1～5ppm to water;
6) tendon that cleans up of shearing procedure (5) again, and the water on tendon surface is drained, after tendon was pruned in vacuum packaging under aseptic condition, ordinary packing was twice again;
7) make after the packaged tendon of employing cobalt-60 irradiation is sterilized again.
The dosage that described cobalt-60 irradiation adopts is 10～20kGy.
it is 15～50 years old that the present invention's tendon of the same race is taken from donor age, noninductive catch an illness history or health check-up are without infecting symptom, without the malignant tumor medical history, virus-free hepatitis, the prunus mume (sieb.) sieb.et zucc. poison, acquired immune deficiency syndrome (AIDS), tuberculosis or leprosy medical history, without autoimmune disease, without severe malnutrition, accept steroid therapy and be no more than 3 months, the heavy dose of application was no more than for 1 week, without medicine or other toxic poisoning history, and carry out the detection aspect following before obtaining the donor tendon: serologic test HIV I/II antibody, HIV antigen, the HIV polymerase chain reaction, HBsAg, the HBV surface antibody, the HBV core antibody, HCV antibody, human T lymphotropic virus's antibody (HTLV I/II antibody), the rapid plasma reagin test of syphilis, when above-mentioned detection is all negative at 10,000 grades of clean rooms, strict sterile working, obtain donor.
The present invention detects the pathogenic bacteria inactivation technology in the present invention, and testing result is as follows:
1) check result of PRV sees Table 1 and table 2:
The result of table 1 tendon repair material peracetic acid of the same race-ethanol deactivation PRV
Annotate: "-" representative fails to detect virus titer.
The pathological changes result of table 2 inactivation of virus sample in the passage process
Annotate: "-" representative has no specific cell pathological changes (CPE).
As can be seen from Table 1 and Table 2, tendon repair material of the same race is through after peracetic acid-ethanol deactivation, all without the virus that can detect, can make PRV titre decline 4 each more than Log.For the sample of failing to detect virus titer, get its minimum extension rate hole supernatant, the inoculation sensitive cells, in 3 generations of blind passage, result is not all observed the specific cell pathological changes.
2) check result of BVDV sees Table 3 and table 4:
The result of table 3 tendon repair material peracetic acid of the same race-ethanol deactivation BVDV
Annotate: "-" representative fails to detect virus titer.
The pathological changes result of table 4 inactivation of virus sample in the passage process
Annotate: "-" representative has no specific cell pathological changes (CPE).
Can find out from table 3 and table 4, tendon repair material of the same race is after peracetic acid-ethanol deactivation, and is all large to virus without detecting, and can make the titre of BVDV descend 4 more than Log.For the sample of failing to detect virus titer, get its minimum extension rate hole supernatant, inoculation is responsive, and in 3 generations of blind passage, result is not all observed the specific cell pathological changes.
3) check result of PPV sees Table 5 and table 6:
The result of table 5 tendon repair material peracetic acid of the same race-ethanol deactivation BVDV
Annotate: "-" representative fails to detect virus titer.
The pathological changes result of table 6 inactivation of virus sample in the passage process
Annotate: "-" representative has no specific cell pathological changes (CPE).
Can find out from table 5 and table 6, tendon repair material of the same race is after peracetic acid-ethanol deactivation, and is all large to virus without detecting, and can make the titre of PPV descend 4 more than Log.For the sample of failing to detect virus titer, get its minimum extension rate hole supernatant, inoculation is responsive, and in 3 generations of blind passage, result is not all observed the specific cell pathological changes.
4) conclusion
Tendon repair material of the same race is after peracetic acid-ethanol deactivation, and all without the PRV that can detect, BVDV and PPV, confirmation can make respectively the titre of PRV, BVDV and PPV all descend 4 more than Log.For the sample of failing to detect virus titer, in 3 generations of blind passage on sensitive cells, do not observe the specific cell pathological changes.Above result shows that tendon repair material of the same race is effective through peracetic acid-ethanol inactivation of viruses.
The present invention also detects the effect of peracetic acid in production technology-ethanol-water solution infusion method deactivation HIV, and testing result is as follows:
The result of table 7 three batch sample titration of virus
Table 8 blind passage three generations's result (CPE)
Annotate: the blind passage three generations is acellular pathological changes (CPE) all ,-represent acellular pathological changes.
Conclusion: can find out from table 7 and table 8, processed 30 minutes through peracetic acid and-alcohol-water mixed solution, cytopathy does not all appear in the sample titration, compare with virus control, virus titer 4.17 log that on average descend at least in 3 generations of sample blind passage, cytopathy all do not occur.
The present invention compared with prior art has following beneficial effect:
1, to adopt the mixed solution of peracetic acid-alcohol-water to kill the mechanism of action of microorganism in tendon be that Oxidation and acid effect by peracetic acid and ethanol causes microbial death in the present invention, particularly, the enzyme system of Oxidation destroy microorganisms, and peracetic acid all has efficient killing action to bacterial propagule, fungus, spore and virus, but acid effect coup injury microorganism, especially the present invention effectively controls the concentration range of peracetic acid and ethanol, both reach the effect of kill bacteria, virus, can also keep the mechanical property of tendon simultaneously;
2, adopt cobalt-60 irradiation sterilization, especially the irradiation of high dose (though＞30kGy) effective kill bacteria, kill virus, but easily cause the tendon Strength Changes, affect the tendon performance, the present invention is controlled at irradiation dose in 10～15kGy scope, can reach the function of sterilization, the mixed solution with peracetic acid-alcohol-water is used in conjunction with simultaneously, also can reach the effect to inactivation of virus, the more important thing is to have kept the original intensity of tendon, to the Effect on Mechanical Properties of tendon seldom;
Use chemicals when 3, the present invention processes tendon seldom, prevented that effectively number of chemical reagent from the drug residue that tendon is carried out disinfection, produce in defat, deproteinization process, having kept the initial quality of tendon;
4, the present invention has kept the stability of tendon structure of the same race in a word, reduced its transplantation immunity originality, solved the potential antibacterial that tendon transplantation of the same race may bring, the viral infection problem, the present invention adopts tendon of the same race as raw material, keeping adopting cryotherapy to make the tendon cell inactivation on constitutionally stable basis, peracetic acid-ethanol associating irradiation carries out inactivation of virus and sterilization, both guaranteed the safety of tendon graft of the same race, the organizational structure and the mechanical property that keep again tendon of the same race, for clinical tendon/ligament damaged provides a kind of repair materials of transplanting safely and effectively.
Specific implementation method
Embodiment 1
A kind of human homologous tendon preparation method in the present embodiment comprises the following steps:
1) extract mankind's donor tendon material and cleaning up with the bloodstain of normal saline with the tendon surface;
2) the tendon material that cleans up is placed in-60 ℃ of freezing preservations 30 days;
3) get the tendon material of step (2) after freezing, rewarming after 30 minutes under room temperature cuts off the loose connective tissue around tendon;
4) pruning good tendon material, to immerse weight ratio be in the mixed solution of peracetic acid, second alcohol and water of 0.05: 10: 70, carried out inactivation of virus in standing 20 minutes;
5) to clean deactivation tendon peracetic acid content to the last cleanout fluid be 0.1ppm to water;
6) tendon that cleans up of shearing procedure (5) again, and the water on tendon surface is drained, after tendon was pruned in vacuum packaging under aseptic condition, ordinary packing was twice again;
7) adopt cobalt-60 to make after under 10kGy dosage, the packaged tendon of irradiation is sterilized again.
Embodiment 2
A kind of human homologous tendon preparation method in the present embodiment comprises the following steps:
1) extract mankind's donor tendon material and cleaning up with the bloodstain of normal saline with the tendon surface;
2) the tendon material that cleans up is placed in-70 ℃ of freezing preservations 60 days;
3) get the tendon material of step (2) after freezing, rewarming after 45 minutes under room temperature cuts off the loose connective tissue around tendon;
4) pruning good tendon material, to immerse weight ratio be in the mixed solution of peracetic acid, second alcohol and water of 1: 15: 80, carried out inactivation of virus in standing 35 minutes;
5) to clean deactivation tendon peracetic acid content to the last cleanout fluid be 3ppm to water;
6) tendon that cleans up of shearing procedure (5) again, and the water on tendon surface is drained, after tendon was pruned in vacuum packaging under aseptic condition, ordinary packing was twice again;
7) adopt cobalt-60 to make after under 15kGy dosage, the packaged tendon of irradiation is sterilized again.
Embodiment 3
A kind of human homologous tendon preparation method in the present embodiment comprises the following steps:
1) extract mankind's donor tendon material and cleaning up with the bloodstain of normal saline with the tendon surface;
2) the tendon material that cleans up is placed in-80 ℃ of freezing preservations 90 days;
3) get the tendon material of step (2) after freezing, rewarming after 60 minutes under room temperature cuts off the loose connective tissue around tendon;
4) pruning good tendon material, to immerse weight ratio be in the mixed solution of peracetic acid, second alcohol and water of 2: 30: 90, carried out inactivation of virus in standing 50 minutes;
5) to clean deactivation tendon peracetic acid content to the last cleanout fluid be 5ppm to water;
6) tendon that cleans up of shearing procedure (5) again, and the water on tendon surface is drained, after tendon was pruned in vacuum packaging under aseptic condition, ordinary packing was twice again;
7) adopt cobalt-60 to make after under 20kGy dosage, the packaged tendon of irradiation is sterilized again.
Claims (2)
1. human homologous tendon preparation method is characterized in that comprising the following steps:
1) extract mankind's donor tendon material and cleaning up with the bloodstain of normal saline with the tendon surface;
2) the tendon material that cleans up is placed in-60～-80 ℃ of freezing preservations 30～90 days;
3) get the tendon material of step (2) after freezing, under room temperature after rewarming, cut off the loose connective tissue around tendon;
4) pruning good tendon material, to immerse weight ratio be 0.05～2: 10～30: in the mixed solution of 70～90 peracetic acid, second alcohol and water, carried out inactivation of virus in standing 20～50 minutes;
5) to clean deactivation tendon peracetic acid content to the last cleanout fluid be 0.1～5ppm to water;
6) tendon that cleans up of shearing procedure (5) again, and the water on tendon surface is drained, after tendon was pruned in vacuum packaging under aseptic condition, ordinary packing was twice again;
7) make after the packaged tendon of employing cobalt-60 irradiation is sterilized again.
2. human homologous tendon preparation method according to claim 1, is characterized in that the dosage that described cobalt-60 irradiation adopts is 10～20kGy.
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| CN 201010599384 CN102166373B (en) | 2010-12-13 | 2010-12-13 | Preparation method of human homologous tendon |
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| CN 201010599384 CN102166373B (en) | 2010-12-13 | 2010-12-13 | Preparation method of human homologous tendon |
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| CN102166373B true CN102166373B (en) | 2013-05-22 |
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| CN104888272B (en) * | 2015-04-27 | 2017-07-21 | 南昌大学第二附属医院 | One kind removes cell aorta petal support and its production and use |
| CN114522258A (en) * | 2022-03-04 | 2022-05-24 | 杭州倍荣生物科技有限公司 | Inactivation method of biological soft tissue virus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970095A (en) * | 2005-11-23 | 2007-05-30 | 曲彦隆 | Derivative tendon stent material and its preparing process |
| CN101496912A (en) * | 2008-01-30 | 2009-08-05 | 北京大清生物技术有限公司 | Bio-derivative tendon repair material and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2001137329A (en) * | 1999-11-15 | 2001-05-22 | Natl Inst For Res In Inorg Mater | Bio-tissue for tendon or ligament and method of manufacturing it |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970095A (en) * | 2005-11-23 | 2007-05-30 | 曲彦隆 | Derivative tendon stent material and its preparing process |
| CN101496912A (en) * | 2008-01-30 | 2009-08-05 | 北京大清生物技术有限公司 | Bio-derivative tendon repair material and preparation method thereof |
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| JP特开2001-137329A 2001.05.22 |
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