CN103301507A - Artificial biological tendon and preparation method thereof - Google Patents
Artificial biological tendon and preparation method thereof Download PDFInfo
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- CN103301507A CN103301507A CN201310203598XA CN201310203598A CN103301507A CN 103301507 A CN103301507 A CN 103301507A CN 201310203598X A CN201310203598X A CN 201310203598XA CN 201310203598 A CN201310203598 A CN 201310203598A CN 103301507 A CN103301507 A CN 103301507A
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Abstract
The invention provides an artificial biological tendon and a preparation method of the artificial biological tendon. Small intestinal sub mucosa tissues of inbred strain animals are adopted as raw materials of the artificial biological tendon and are removed with cell components and deoxyribonucleic acid (DNA) components, extracellular matrix components and structures are completely reserved, and the artificial biological tendon has a cellular structure. The preparation method of the artificial biological tendon comprises the following operation steps of: determining animal sources, preprocessing small intestine tissues, rough cleaning, viral inactivating, removing cells, clearing DNA, forming, packaging and sterilizing. The inbred strain animals are served as an animal source of the artificial biological tendon prepared by the method and have pure, stable and unite genetic characteristics, the stability and uniformity of different batches of products are basically ensured, the DNA residue of the animal source is small, the three-dimensional structure of the natural extracellular matrix component (ECM) is completely reserved, and the artificial biological tendon has low immunogenicity and strong anti-infection capability.
Description
Technical field
The present invention relates to animal derived Implantable Medical Device technical field, be specifically related to a kind of artificial bio-membrane's tendon and preparation method thereof.
Background technology
Tendon is a kind of viscoelasticity tissue, can the strength transmission of muscle generation to skeleton, cause the motion of limbs.The biology performance of tendon affects contractility and the sports achievement of muscle to a certain extent.Motion or disease cause tendon injury, can cause limbs disturbance if often in time repair.For the tendon that damaged damage is arranged, the method that adopts clinically comprises: (1) repairs Tendon Defection from the body tendon transplantation; (2) Tendon allograft; (3) xenogenic tendon is transplanted; 4) artificial tendon substitutes.Because there are the problems such as donor tendon shortage and immunological rejection in former three, along with the development of medical embedded material, artificial tendon has been widely used in clinical.According to chemical composition and the biological characteristics of materialogy, the artificial tendon material mainly contains not several classes of absorbing material, absorbable material and biomaterial at present.The developing direction of modern medical embedded material is degradable, have the initiatively biomaterial of induced tissue regeneration.
The extracellular matrix take animal tissue as raw material (extracellular matrix, ECM) material based on the tissue engineering principle is the main development direction.ECM is by compositions such as multiple macromolecular substances such as collagen, non-collagen sugar albumen, aminoglycan, Dan Baiduotang proteoglycan PG, elastin laminins, the organic three-dimension integrally of the complexity of building up with structure by a certain percentage, for the existence of various cells and movablely provide suitable place and microenvironment, can regulate growth, shape, metabolism, migration, propagation and the differentiation of various cells, and then organization of regulation control and organ dysfunction.A forfeiture that serious consequence is " soil "-ECM of tissue defect, this also is the reason that body self can't be realized tissue repair and regeneration.Natural ECM can be used as tissue regeneration " soil ", is desirable tissue renovation material.Remove the cell component of animal tissue and can remove most immunogenicity, and keep the ECM composition, can develop desirable artificial bio-membrane's tendon material.Be used at present clinical bioactive materials and comprised the ECM materials such as allogeneic dermis, trees-Osima jacoti, Osima excavata, pig dermis, embryo cattle corium.Wherein taking off cell submucous layer of small intestine (small intestinal submucosa, SIS) host material is the optimal tissue renovation material that academic circles at present is generally acknowledged.The artificial bio-membrane's tendon take trees-Osima jacoti, Osima excavata ECM as raw material of U.S. Cook Biotech Incorporated (trade name: Biodesign Surgisis) entered American-European countries market, and obtained preferably clinical effectiveness.
Biodesign Surgisis product has natural distinctive ECM structure and composition, initiatively induced tissue regeneration, and immunogenicity is low, and histocompatibility is good, the advantages such as degradable.But along with the increase of clinical practice, the problem of Biodesign Surgisis product also displays.Clinical studies show: the complication such as Biodesign Surgisis product has occurred in clinical practice that serosity in various degree is swollen, infection, chronic inflammatory reaction, organization healing are bad, wherein the swollen incidence rate of serosity is the highest.Complication may cause the recurrence of disease, even needs second operation to remove.In addition, the stability of the product of the anti-infection ability of product and different batches and uniformity are relatively poor.
On the one hand, the animal source DNA is residual is the major defect of Biodesign Surgisis product.The production technology of Biodesign Surgisis is not considered the residual link of removing DNA, and its product standard is not stipulated the content (reference standard: YZB/USA 0944-2010) of the residual control of DNA yet.Study verifiedly, serosity is swollen to be caused by the reaction of Th2 inflammatory cytokine, and this reaction is caused by the chronic immunoreation of animal source DNA exactly.On the other hand, Biodesign Surgisis product adopt common hybridization be pig as animal sources, hybridization is unstability and the heterogeneity that the individual variation of animal genetic background has caused the different batches product.Again on the one hand, the vacuum pressing lyophilizing moulding process that Biodesign Surgisis product adopts makes the space structure compression of SIS material, has destroyed natural ECM three dimensional structure, affects the anti-infection ability of product and promotes the tissue regeneration ability.
Summary of the invention
Technical problem to be solved by this invention provides a kind of artificial bio-membrane's tendon, this artificial bio-membrane's tendon with the inbred line animal as animal origin, inherited characteristic is pure, stable, unified, in the stability and the uniformity that have fundamentally guaranteed the different batches product, the residual few and complete three dimensional structure that keeps natural ECM of animal source DNA, immunogenicity is low, and anti-infection ability is strong.
A kind of artificial bio-membrane's tendon, it adopts the submucous layer of small intestine of inbred line animal to be organized as raw material, removes cell component and DNA composition, complete reservation extracellular matrix components and structure, and have microcellular structure.
Described inbred line animal is preferably inbred pig, more preferably Chinese wuzhishan miniature pig inbred-line.
The DNA residual quantity is 105 ~ 130pg/g behind described removal cell component and the DNA composition.
The aperture of described microcellular structure is 150 ~ 250um, and porosity is 85 ~ 90%.
Described artificial bio-membrane's tendon is rolled into cylindricality or is pressed into bar shaped by 8 ~ 12 layers of monolayer material by 2 ~ 4 layers of monolayer material axle, and has the penetrability aperture, and the aperture of affiliated penetrability aperture is 0.5 ~ 2mm, and span is 0.5 ~ 1cm.
Described penetrability aperture is for forming by the punching of laser micropore technology.
Another technical problem to be solved by this invention provides the preparation method of above-mentioned artificial bio-membrane's tendon, this preparation method is improved and has been optimized preparation technology, increase DNA and removed link, with mechanical oscillation and the effective combination of supersonic oscillations, with Freeze Drying Technique and the effective combination of laser micropore technology, thereby effectively removed the animal source DNA, complete structure and the composition that has kept natural ECM, the artificial biological tendon of the present invention is compared with existing similar products, the DNA residual quantity is low, and immunogenicity is low, anti-infection ability is high.
The preparation method of above-mentioned artificial bio-membrane's tendon comprises following operating procedure:
(1) zoogenously determines and pre-process and the previous cleaning of small intestine
Select the inbred line animal as animal origin, the method for definite employing patent ZL200510008994.2 defined of animal varieties is got small intestine's cleaning of fresh slaughtered animals, isolates submucous layer of small intestine, uses water for injection flushing 3 times.
(2) inactivation of virus
Adopt peracetic acid-alcoholic solution method inactivation of viruses, this step is carried out in the constant-temperature ultrasonic washer that rinse bath can vibrate, wherein the volumn concentration of peracetic acid is that 0.05 ~ 0.2%(is preferably 0.1%), inactivation time is that 1 ~ 2h(is preferably 1h), temperature range is 4 ~ 40 ℃, then in phosphate buffer, clean 2 ~ 5 times, each 15min, detect the pH value of the phosphate buffer after cleaning, after pH reaches 6.5-7.5, with the water for injection cleaning material that flows, detect electrical conductivity and reach 1.5um/s termination when following.
(3) take off cell
This step is carried out in the constant-temperature ultrasonic washer that rinse bath can vibrate, at first material is inserted in the rinse bath, then hydrogen injecting sodium hydroxide solution in rinse bath, open washer, scavenging period is that 5 ~ 30min(is preferably 20min), concentration of sodium hydroxide solution is that 5 ~ 100mmol/L(is preferably 5 ~ 20mmol/L, 10mmol/L more preferably), then close washer, sodium hydroxide solution is inclined to, inject phosphate buffer and clean, open washer, scavenging period is that 5 ~ 20min(is preferably 15min), phosphate buffer repeated washing 2 ~ 5 times, the pH value of the phosphate buffer after detection is cleaned is after pH reaches 6.5-7.5, with the water for injection cleaning material that flows, detect electrical conductivity and reach 1.5um/s termination when following.
(4) DNA removes and processes
This step is carried out in the constant-temperature ultrasonic washer that rinse bath can vibrate, sodium chloride solution is injected rinse bath, open washer, scavenging period is that 5 ~ 30min(is preferably 20min), concentration of sodium chloride solution is that 0.015mol/l or 2mol/L(are preferably 0.015mol/L), pH value is no more than 7.8, then the water for injection cleaning material to flow detects electrical conductivity and reaches 1.5um/s termination when following.
(5) molding
This step comprises that fixture fixes, lyophilization and 3 steps of laser micropore punching, 2 ~ 4 layers of axle of material are rolled into cylindricality (as shown in Figure 1) or 8 ~ 12 laminations are made bar shaped (as shown in Figure 2), be fixed on the fixture (being preferably the rustless steel fixture), then use the water for injection cleaning, the material that is fixed in fixture is put in the freezer dryer, lyophilizing flow process according in advance design is carried out lyophilization: pre-freeze to 25 ~ 50 ℃ (being preferably 25 ℃), be incubated 0.5 ~ 4 hour (being preferably 2h), be warming up to 15 ℃, be incubated 4 ~ 12 hours (being preferably 8h), heat up 15 ℃, be incubated 0.5 ~ 4 hour (being preferably 2h), be warming up to 25 ℃, be incubated 4 hours, after lyophilizing is finished, use laser micropore puncher punching (being the penetrability aperture), pore diameter range is 0.5 ~ 2mm, and span is 0.5 ~ 1cm.Described laser micropore punching refers to utilize laser technology that material is got micron-sized aperture, and using the punching of laser micropore puncher is in order to make material surface form hole, to be beneficial to tissue repair.
(6) packaging sterilizing
Pack under aseptic state, one deck adopts special strong defensive QI paper, another layer employing vinyon, and employing oxirane was sterilized after packing was finished.
The inbred line animal is preferably inbred pig in the described step (1), more preferably Chinese wuzhishan miniature pig inbred-line.Described Chinese wuzhishan miniature pig inbred-line refers to the animal varieties that ZL02149030.9 provides, and utilizes one male one brood female Wuzhi Mountain pig for being the ancestral, adopts " son joins mother ", " full ", the inbred pig population of cultivating.
As preferably, rinse bath frequency of oscillation is 100 ~ 300rpm, more preferably 200rpm in described step (2), (3), (4).
As preferably, ultrasonic frequency is 20 ~ 80KHZ, more preferably 45KHZ in described step (2), (3), (4).
The compound method of phosphate buffer described in the present invention is for claiming 7.9g NaCl, 0.2g KCl, 0.24g KH
2PO
4, 1.8g K
2HPO
4, be dissolved in the 800 ml distilled water, with the pH value to 7.4 of HCl regulator solution, then adding distil water is settled to 1 L.
The Application standard of water for injection is according to stipulating in the state-promulgated pharmacopoeia among the present invention.
The ultrasonic washing unit that rinse bath described in the present invention can vibrate is that the rinse bath with the conventional ultrasonic wave cleaning machine combines with mechanical oscillator, make rinse bath that machinery concussion can occur in ultrasonic waves for cleaning, realized that mechanical oscillation and ultrasonic waves for cleaning are united simultaneously to play a role.
Compared with prior art, the present invention has following remarkable advantage and beneficial effect:
The present invention selects the inbred line animal as animal origin, after further being preferably Chinese wuzhishan miniature pig inbred-line, make the coefficient of inbreeding of this kind up to 0.991, the gene high homogenous, has pure, stable, the unified advantage of inherited characteristic, in the stability and the uniformity that have fundamentally guaranteed the different batches product, make the genetic background difference between the individuality very little, thereby determined that immunogenic gene and the mankind have very high homology.It is that pig is the product of raw material that uniformity, stability and the low immunogenicity of the submucous layer of small intestine ECM material product of developing take this kind as animal origin are higher than to hybridize.
Preparation method of the present invention is improved and has been optimized preparation technology, has increased DNA and has removed link, has determined the standard that product dna is residual.Inactivation of virus of the present invention adopts low concentration peracetic acid-alcoholic solution in conjunction with mechanical oscillation and ultrasonic waves for cleaning, taking off cell adopts low-concentration sodium hydroxide solution in conjunction with mechanical oscillation and ultrasonic waves for cleaning, DNA removes and adopts sodium chloride solution in conjunction with mechanical oscillation and ultrasonic waves for cleaning, all adopts phosphate buffer and water for injection to clean after every step is finished.Inactivation of virus in the preparation method, take off the step such as cell and DNA removing all simultaneously in conjunction with mechanical oscillation and ultrasonic waves for cleaning, preparation method is with mechanical oscillation and the effective combination of supersonic oscillations, improved inactivation of virus, take off the efficient of cell and DNA removing technique, the DNA residual quantity significantly reduces, greatly reduce technique consuming time, simplified technological process, whole preparation process is only used peracetic acid-ethanol, three kinds of solution of sodium hydroxide and sodium chloride, and concentration is all well below existing technology of preparing, make the material of preparation remove immunogenicity fully, the effective ingredient such as the complete structure that has kept natural ECM and somatomedin, residual without poisonous and harmful substance; And, in moulding process, innovatively with Freeze Drying Technique and the effective combination of laser micropore technology, keeping natural ECM three dimensional structure, hole does not compress, and the anti-infection ability of product is high.
Description of drawings
Shown in Figure 1 is the structural representation of cylindricality artificial bio-membrane's tendon of the present invention;
Shown in Figure 2 is the structural representation of bar shaped artificial bio-membrane tendon of the present invention;
The sample that shown in Figure 3 is in the embodiment of the invention 2 is cut out schematic diagram;
The optical microscope figure that shown in Figure 4 is in the embodiment of the invention 2;
The Electronic Speculum ultrastructure figure that shown in Figure 5 is in the embodiment of the invention 2.
The specific embodiment
Below in conjunction with embodiment the present invention is further described in detail, but is not limited to this.
The preparation of embodiment 1:8 layer artificial bio-membrane tendon
Preparation method is as follows:
(1) zoogenously determines and pre-process and the previous cleaning of small intestine
Select Chinese wuzhishan miniature pig inbred-line as animal origin, the method of definite employing patent ZL200510008994.2 defined of animal varieties, get small intestine's cleaning of fresh Chinese wuzhishan miniature pig inbred-line of butchering, isolate submucous layer of small intestine, use water for injection flushing 3 times.
(2) inactivation of virus
Adopt peracetic acid-alcoholic solution method inactivation of viruses, this step is carried out in the constant-temperature ultrasonic washer that rinse bath can vibrate, wherein the volumn concentration of peracetic acid is that 0.05 ~ 0.2%(is preferably 0.1%), inactivation time is that 1 ~ 2h(is preferably 1h), temperature range is 4 ~ 40 ℃, then in phosphate buffer, clean 2 ~ 5 times, each 15min, detect the pH value of the phosphate buffer after cleaning, after pH reaches 6.5-7.5, with the water for injection cleaning material that flows, detect electrical conductivity and reach 1.5um/s termination when following.The rinse bath frequency of oscillation is that 100 ~ 300rpm(is preferably 200rpm), ultrasonic frequency is that 20 ~ 80KHZ(is preferably 45KHZ).
(3) take off cell
This step is carried out in the constant-temperature ultrasonic washer that rinse bath can vibrate, at first material is inserted in the rinse bath, then hydrogen injecting sodium hydroxide solution in rinse bath, open washer, scavenging period is that 5 ~ 30min(is preferably 20min), concentration of sodium hydroxide solution is that 5 ~ 100mmol/L(is preferably 5 ~ 20mmol/L, 10mmol/L more preferably), then close washer, sodium hydroxide solution is inclined to, inject phosphate buffer and clean, open washer, scavenging period is that 5 ~ 20min(is preferably 15min), phosphate buffer repeated washing 2 ~ 5 times, the pH value of the phosphate buffer after detection is cleaned is after pH reaches 6.5-7.5, with the water for injection cleaning material that flows, detect electrical conductivity and reach 1.5um/s termination when following.The rinse bath frequency of oscillation is that 100 ~ 300rpm(is preferably 200rpm), ultrasonic frequency is that 20 ~ 80KHZ(is preferably 45KHZ).
(4) DNA removes and processes
This step is carried out in the constant-temperature ultrasonic washer that rinse bath can vibrate, sodium chloride solution is injected rinse bath, open washer, scavenging period is that 5 ~ 30min(is preferably 20min), concentration of sodium chloride solution is that 0.015mol/l or 2mol/L(are preferably 0.015mol/L), pH value is no more than 7.8, then the water for injection cleaning material to flow detects electrical conductivity and reaches 1.5um/s termination when following.The rinse bath frequency of oscillation is that 100 ~ 300rpm(is preferably 200rpm), ultrasonic frequency is that 20 ~ 80KHZ(is preferably 45KHZ).
(5) molding
This step comprises that fixture fixes, lyophilization and 3 steps of laser micropore punching, material 8 laminations are made bar shaped, be fixed on the rustless steel fixture, then use the water for injection cleaning, the material that is fixed in fixture is put in the freezer dryer, and carry out lyophilization according to the lyophilizing flow process of in advance design: pre-freeze to 25 ~ 50 ℃ (being preferably 25 ℃) is incubated 0.5 ~ 4 hour (being preferably 2h), be warming up to 15 ℃, be incubated 4 ~ 12 hours (being preferably 8h), heat up 15 ℃, be incubated 0.5 ~ 4 hour (being preferably 2h), be warming up to 25 ℃, be incubated 4 hours, after lyophilizing is finished, use the punching of laser micropore puncher, pore diameter range is 0.5 ~ 2mm, and span is 0.5 ~ 1cm.Described laser micropore punching refers to utilize laser technology that material is got micron-sized aperture, and using the punching of laser micropore puncher is in order to make material surface form hole, to be beneficial to tissue repair.
(6) packaging sterilizing
Pack under aseptic state, one deck adopts special strong defensive QI paper, another layer employing vinyon, and employing oxirane was sterilized after packing was finished.
Embodiment 2: the physicochemical property of the material that embodiment 1 is prepared, histology, somatomedin and biology performance detect
1. 8 layer materials of preparation carried out the physical property detection, test item comprises porosity measurement, sews up retentivity, tensile strength, burst strength.
1) porosity measurement: adopt mercury injection method to measure the porosity of material, and with and compare with Biodesign Surgisis product.The porosity of the sample that result: embodiment 1 provides is that 87.8 ± 2.51, Biodesign Surgisis product porosity is 78.3 ± 6.38.
2) sewing up retentivity detects: method: be sewn to 2 millimeters places in artificial bio-membrane's tendon one end margin with the surgical sutures of 2-0 or the stainless steel silk of same diameter, the other end of suture or stainless steel silk and artificial bio-membrane's tendon is fixed on the tensiometer, speed with 20mm/min stretches, until stitch points is torn, the pulling force when recording stitch points and being torn.As stated above 3 batch samples are detected.Result: sew up tensile strength more than or equal to 10 ± 0.5N.
3) tensile strength detection method: method: (compression) testing machine that use to stretch, according to shown in Figure 3, artificial bio-membrane's tendon is cut into sample, be 40%-60% at relative humidity after the cutting, temperature is to test immediately behind the placement 2h under 22 ℃ ± 2 ℃ the condition.The sample two ends are fixed on the chuck of cupping machine, are pulled outwardly successively with the speed of 100mm/min and stretch sample fracture, longitudinal test piece and laterally sample test respectively.Power under take N as unit record during sample fracture.As stated above 3 batch samples are detected.Result: vertically: 300N, laterally: 150N.
4) burst strength detects: method: use (compression) testing machine that stretches, the square sample that material is cut into 23 * 23mm is for subsequent use, is 40%-60% at relative humidity, and temperature is to test immediately behind the placement 2h under 24 ℃ ± 2 ℃ the condition.With annular holder sample is fixed on the workbench of tensilometer, makes spheric probe pass sample with the speed of 750mm/min, record the power that probe is worn out sample.As stated above 3 batch samples are detected.The result: burst strength is greater than 500N.
2. chemical property detects, and test item comprises virus, acid-base value, endotoxin and DNA residual quantity.
1) test liquid preparation: test liquid preparation: the even thickness part of sample thief, be cut into 1cm
2Fragment, dry after washing, then add in the glass container, press the inside and outside total surface area (cm of sample
2) with the ratio of water (mL) be that the ratio of 5:1 adds water, add a cover to be placed in the pressure steam sterilizer, at 121 ℃ ± 1 ℃ heating 30min, with sample and fluid separation applications, be chilled to room temperature as test liquid after heating finishes.Get consubstantiality hydrops and place glass container, with the standby blank liquid of legal system.
2) virus detects: method: the selection Pseudorabies virus is indicator virus, adopts the real-time quantitative PCR method to detect the viral DNA copy number, detects 3 batch samples.Result: viral DNA copy number 0.
3) acid-base value detects: press 5.4.1(among the GB/T14233.1 " medical infusion, blood transfusion, instrument used for injection method of inspection part 1: chemical analysis method ") the middle method test of stipulating, the result: the difference of the pH value of test liquid and blank liquid is no more than 1.5.
4) endotoxin: press 6cm
2Sample adds the ratio of 1ml lixiviate medium, and 37 ± 1 ℃, 72 ± 2hr prepares experimental liquid, lixiviate medium: normal saline.By GB/T 14233.2-2005(" medical infusion, blood transfusion, instrument used for injection method of inspection part 2: biological test method ") regulation method carry out, detect 3 batch samples.The result: endotoxin content is less than 5EU/g.
5) DNA residues detection: according to biological preparation residual DNA detection method (" Chinese pharmacopoeia 2010, appendix IX-B Residual exogenous DNA quantitative determination method), adopt fluorescent staining method to detect the DNA residual quantity of the sample that embodiment 1 provides, and compare with Biodesign Surgisis product.The DNA residual quantity of the sample that result: embodiment 1 provides is 120 ± 15pg/g, and the DNA residual quantity of Biodesign Surgisis product is 250 ± 45pg/g.
3. histology
1) observation by light microscope: method: the capable hematoxylin of the material behind the paraffin peplos-Yihong dyeing, inverted phase contrast microscope is observed.The result: acellular residual with cell debris, the micro-continuous non-cracking of collagen, as shown in Figure 4.
2) Ultrastructural observation.The result: material is loose structure, the fiber non-cracking, and the aperture is even, and mean pore size is 200um, and porosity is greater than 85%, as shown in Figure 5.
4. somatomedin detects:
Press 6cm
2Sample adds the ratio of 1ml lixiviate medium, and 37 ± 1 ℃, 72 ± 2hr prepares experimental liquid, lixiviate medium: normal saline.Adopt the ELLISA method to detect lixiviating solution neutral and alkali somatomedin (bFGF) and VEGF (VEGF) content.The result: bFGF content is 121.8 ± 2.683ng/L, and VEGF content is 93.8 ± 3.033ng/L.
5. biology performance detects, and test item comprises cytotoxicity, delayed hypersensitivity, intradermoreaction.
1) cytotoxicity: method: press 6cm
2Sample adds the ratio of 1ml lixiviate medium, and 37 ± 1 ℃, 24 ± 2hr prepares experimental liquid, the lixiviate medium: the MEM culture medium that contains serum.Get experimental liquid according to GB/T16886.5-2003(" BiologicalEvaluationofMedicalDevice the tenth part: stimulate and the delayed hypersensitivity test ") in the test method of regulation test.The result: cell-cytotoxic reaction is less than or equal to 1 grade.
2) delayed hypersensitivity: method: press 6cm
2Sample adds the ratio of 1ml lixiviate medium, and 37 ± 1 ℃, 72 ± 2hr prepares experimental liquid, and the lixiviate medium is normal saline and Oleum Gossypii semen.According to CB/T 16886.10-2005(" BiologicalEvaluationofMedicalDevice the 10th part: stimulate and the delayed hypersensitivity test ") the test method regulation tests.Result: without delayed hypersensitivity.
3) intradermoreaction: press 6cm
2Sample adds the ratio of 1ml lixiviate medium, and 37 ± 1 ℃, 72 ± 2hr prepares experimental liquid, and the lixiviate medium is normal saline and Oleum Gossypii semen.According to CB/T 16886.10-2005(" BiologicalEvaluationofMedicalDevice the 10th part: stimulate and the delayed hypersensitivity test ") the test method regulation tests.The result: the difference that test specimen and solvent control are on average scored is less than 1.0.
The above embodiment of the present invention is can not be used for restriction the present invention to explanation of the present invention, and the implication suitable with claims of the present invention and any change in the scope all should be thought to be included in the scope of claims.
Claims (24)
1. artificial bio-membrane's tendon is characterized in that: adopts the submucous layer of small intestine of inbred line animal to be organized as raw material, removes cell component and DNA composition, and complete reservation extracellular matrix components and structure, and have microcellular structure.
2. a kind of artificial bio-membrane's tendon according to claim 1, it is characterized in that: described inbred line animal is inbred pig.
3. a kind of artificial bio-membrane's tendon according to claim 2, it is characterized in that: described inbred pig is Chinese wuzhishan miniature pig inbred-line.
4. a kind of artificial bio-membrane's tendon according to claim 1, it is characterized in that: the DNA residual quantity is 105 ~ 130pg/g behind described removal cell component and the DNA composition.
5. a kind of artificial bio-membrane's tendon according to claim 1, it is characterized in that: the aperture of described microcellular structure is 150 ~ 250um, porosity is 85 ~ 90%.
6. described a kind of artificial bio-membrane's tendon according to claim 1-5, it is characterized in that: be rolled into cylindricality or be pressed into bar shaped by 8 ~ 12 layers of monolayer material by 2 ~ 4 layers of monolayer material axle, and have the penetrability aperture, and the aperture of described penetrability aperture is 0.5 ~ 2mm, span is 0.5 ~ 1cm.
7. a kind of artificial bio-membrane's tendon according to claim 6 is characterized in that: described penetrability aperture is for forming by the punching of laser micropore technology.
8. the preparation method of a kind of artificial bio-membrane's tendon claimed in claim 1 is characterized in that comprising following operating procedure:
(1) zoogenously determines and pre-process and the previous cleaning of small intestine
Select the inbred line animal as animal origin, the method for definite employing patent ZL200510008994.2 defined of animal varieties is got small intestine's cleaning of fresh slaughtered animals, isolates submucous layer of small intestine, uses water for injection flushing 3 times;
(2) inactivation of virus
Adopt peracetic acid-alcoholic solution method inactivation of viruses, this step is carried out in the constant-temperature ultrasonic washer that rinse bath can vibrate, wherein the volumn concentration of peracetic acid is 0.05 ~ 0.2%, and inactivation time is 1 ~ 2h, and temperature range is 4 ~ 40 ℃, then in phosphate buffer, clean 2 ~ 5 times, each 15min, the pH value of the phosphate buffer after detection is cleaned is after pH reaches 6.5-7.5, with the water for injection cleaning material that flows, detect electrical conductivity and reach 1.5um/s termination when following;
(3) take off cell
This step is carried out in the constant-temperature ultrasonic washer that rinse bath can vibrate, at first material is inserted in the rinse bath, then hydrogen injecting sodium hydroxide solution in rinse bath, open washer, scavenging period is 5 ~ 30min, concentration of sodium hydroxide solution is 5 ~ 100mmol/L, then closes washer, and sodium hydroxide solution is inclined to, injecting phosphate buffer cleans, open washer, scavenging period is 5 ~ 20min, phosphate buffer repeated washing 2 ~ 5 times, detect the pH value of the phosphate buffer after cleaning, after pH reaches 6.5-7.5, with the water for injection cleaning material that flows, detect electrical conductivity and reach 1.5um/s termination when following;
(4) DNA removes and processes
This step is carried out in the constant-temperature ultrasonic washer that rinse bath can vibrate, sodium chloride solution is injected rinse bath, open washer, scavenging period is 5 ~ 30min, concentration of sodium chloride solution is 0.015mol/l or 2mol/L, pH value is no more than 7.8, the water for injection cleaning material to flow then, detects electrical conductivity and reaches 1.5um/s termination when following;
(5) molding
This step comprises that fixture fixes, lyophilization and 3 steps of laser micropore punching, 2 ~ 4 layers of axle of material are rolled into cylindricality or 8 ~ 12 laminations are made bar shaped, be fixed on the fixture, then use the water for injection cleaning, the material that is fixed in fixture is put in the freezer dryer, carries out lyophilization according to the lyophilizing flow process of in advance design: pre-freeze to 25 ~ 50 ℃ are incubated 0.5 ~ 4 hour), be warming up to 15 ℃, be incubated 4 ~ 12 hours, heat up 15 ℃, be incubated 0.5 ~ 4 hour, be warming up to 25 ℃, be incubated 4 hours, after lyophilizing is finished, use the punching of laser micropore puncher, pore diameter range is 0.5 ~ 2mm, and span is 0.5 ~ 1cm;
(6) packaging sterilizing
Pack under aseptic state, one deck adopts special strong defensive QI paper, another layer employing vinyon, and employing oxirane was sterilized after packing was finished.
9. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: the inbred line animal is inbred pig in the described step (1).
10. the preparation method of a kind of artificial bio-membrane's tendon according to claim 9, it is characterized in that: described inbred pig is Chinese wuzhishan miniature pig inbred-line.
11. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: the volumn concentration of peracetic acid is 0.1% in the described step (2).
12. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: inactivation time is 1h in the described step (2).
13. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: the scavenging period that with sodium hydroxide solution material is cleaned in the described step (3) is 20min.
14. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: concentration of sodium hydroxide solution is 5 ~ 20mmol/L in the described step (3).
15. the preparation method of a kind of artificial bio-membrane's tendon according to claim 14 is characterized in that: concentration of sodium hydroxide solution is 10mmol/L in the described step (3).
16. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: the scavenging period that with the phosphate-buffered liquor material is cleaned in the described step (3) is 15min.
17. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: the sodium chloride solution scavenging period is 20min in the described step (4).
18. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: concentration of sodium chloride solution is 0.015mol/L in the described step (4).
19. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: the rinse bath frequency of oscillation is 100 ~ 300rpm in described step (2), (3), (4).
20. the preparation method of a kind of artificial bio-membrane's tendon according to claim 19 is characterized in that: the rinse bath frequency of oscillation is 200rpm in described step (2), (3), (4).
21. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: ultrasonic frequency is 20 ~ 80KHZ in described step (2), (3), (4).
22. the preparation method of a kind of artificial bio-membrane's tendon according to claim 21 is characterized in that: ultrasonic frequency is 45KHZ in described step (2), (3), (4).
23. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: the fixture in the described step (5) is the rustless steel fixture.
24. the preparation method of a kind of artificial bio-membrane's tendon according to claim 8 is characterized in that: in the described step (5) in advance the lyophilizing flow process of design be: pre-freeze to 25 ℃, insulation 2h, be warming up to 15 ℃, insulation 8h heats up 15 ℃, insulation 2h is warming up to 25 ℃, is incubated 4 hours.
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| CN114344567A (en) * | 2021-12-01 | 2022-04-15 | 宁波大学医学院附属医院 | Achilles tendon repairing patch and preparation method thereof |
| CN114395525A (en) * | 2021-12-23 | 2022-04-26 | 北京鑫康辰医学科技发展有限公司 | Preparation method of acellular and antigen-removed xenogenic tendon |
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