CN103272274B - Biological repair tablet for herniae and preparation method thereof - Google Patents
Biological repair tablet for herniae and preparation method thereof Download PDFInfo
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Abstract
The invention provides a biological repair tablet for herniae and a preparation method thereof. The repair tablet uses small intestine submucosa tissues of inbred line animals without cell and DNA (deoxyribonucleic acid) components as the raw material, completely reserves the extracellular matrix component and structure, and has a micropore structure. The preparation method of the repair tablet comprises the following operation steps: determination of animal source, pretreatment and rough cleaning of small intestine tissues, virus inactivation, cell removal, DNA removal treatment, formation, packaging and sterilization. The biological repair tablet for herniae prepared by the method uses an inbred line animal as an animal source, and thus, the hereditary features are pure, stable and uniform, thereby radically ensuring the stability and uniformity of different batches of products; and the biological repair tablet for herniae has fewer animal source DNA residues, completely reserves the three-dimensional structure of natural ECM, and has the advantages of low immune source property and high infection resistance.
Description
Technical field
The present invention relates to animal derived Implantable Medical Device technical field, be specifically related to a kind of hernia Biological Repair sheet and preparation method thereof.
Background technology
Intraabdominal organ or tissue, from stomach wall weak area or the bulging of defect place, is medically called abdominal hernia, is frequently-occurring disease and the commonly encountered diseases of surgery.Weakness and the defect of the abdominal wall tissue that reason will cause various congenital and day after tomorrow are the causes of disease causing hernia.Operation is current unique effective therapeutic modality.Tension-free hernioplasty is the chief surgical mode of current hernia.Along with the fast development of biomaterial for medical purpose, various hernia patching material be widely used in clinical in.
According to chemical composition and the biological characteristics of materialogy, hernia patching material mainly contains not absorbing material, absorbable material, several large class of compound material at present.The hernia patch used clinically is at present based on macromolecular material, and after implanting, nonabsorable and degraded, can forever retain in body.Macromolecule sticking patch is inserted after in abdomen and is only played Intercepting effects, can not organically combine, meanwhile, as foreign body, can cause local organization inflammation and infection after inserting with body tissue.The developing direction of modern hernia surgical patching material is anti, antimicrobial biological activity sticking patch, can abdominal wall tissue be initiatively induced to regenerate, Absorbable rod and degraded, material property and structure more meet psychological need, complication is less, may be used for intraperitoneal to implant, for polluting or have the operation of contaminated risk.
Based on tissue engineering principle take animal tissue as extracellular matrix (extracellular matrix, the ECM) material of raw material is main development direction.ECM is as compositions such as collagen, non-collagen sugar albumen, aminoglycan, Dan Baiduotang proteoglycan PG, elastin laminins by multiple macromolecular substances, the organic three-dimension integrally of the complexity built up with structure by a certain percentage, for the existence of various cell and activity provide suitable place and microenvironment, the growth of various cell, shape, metabolism, migration, propagation and differentiation can be regulated, and then organization of regulation control and organ dysfunction.A serious consequence of tissue defect is the forfeiture of " soil "-ECM, and this is also the reason that body self cannot realize tissue repair and regeneration.Natural ECM can, as " soil " of tissue regeneration, be desirable tissue renovation material.The cell component removing animal tissue can remove most immunogenicity, and retains ECM composition, can develop desirable hernia patching material.Comprise the ECM materials such as allogeneic dermis, trees-Osima jacoti, Osima excavata, pig dermis, embryo cattle corium for clinical bioactive materials at present.Wherein U.S. Cook Biotech Incorporated with trees-Osima jacoti, Osima excavata small intestinal submucosa, SIS) ECM is the hernia Biological Repair sheet (trade name: Biodesign Surgisis) of raw material is the biological activity patch be most widely used at present, occupy the hernia patch market of 10 ~ 30% in American-European countries, and enter China market with the end of the year 2010.
Biodesign Surgisis product has natural distinctive ECM structure and composition, can initiatively inducing 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 is in various degree swollen, infection, chronic inflammatory reaction, organization healing are bad, wherein the serosity incidence rate that swells is the highest.Complication may cause disease palindromia, even needs second operation to remove the SIS material implanted.The anti-infection ability of product is poor, and for the operation polluting or have contaminated risk, its relapse rate is even up to 40 ~ 60%.In addition, the stability of the product of different batches and uniformity poor.
On the one hand, animal source DNA remains is the major defect of Biodesign Surgisis product.The production technology of BiodesignSurgisis is not considered and is removed the residual link of DNA, and its product standard does not specify that DNA remains the content (reference standard: YZB/USA 0944-2010) of control yet.Study verified, serosity swell by Th2 inflammatory cytokine react cause, and this reaction be exactly by animal source DNA Chronic immune react caused.On the other hand, Biodesign Surgisis product adopts standard hybridization system pig as animal sources, and hybridization is unstability and the heterogeneity that the individual variation of animal genetic background result in different batches product.Again on the one hand, the vacuum pressing lyophilizing moulding process that Biodesign Surgisis product adopts makes the space structure of SIS material compress, and destroys natural ECM three dimensional structure, affects the anti-infection ability of product and promotes tissue regeneration ability.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of hernia Biological Repair sheet, this hernia Biological Repair sheet is using inbred line animal as animal origin, inherited characteristic is pure, stable, unified, fundamentally ensure that stability and the uniformity of different batches product, animal source DNA remains less and the complete three dimensional structure remaining natural ECM, immunogenicity is low, and anti-infection ability is strong.
A kind of hernia Biological Repair sheet, it adopts the intestinal submucosa tissue of inbred line animal to be raw material, removes cell component and DNA composition, complete reservation extracellular matrix components and structure, and has microcellular structure.
Described inbred line animal is preferably inbred pig, more preferably Chinese wuzhishan miniature pig inbred-line.
After described removal cell component and DNA composition, DNA residual quantity is 105 ~ 130pg/g.
The aperture of described microcellular structure is 150 ~ 250um, and porosity is 85 ~ 90%.
Described hernia Biological Repair sheet is made up of 8 ~ 12 layers of monolayer, and shape is rectangle or square, and length is 7 ~ 20cm, width is 6 ~ 20cm, described hernia Biological Repair sheet has penetrability aperture, and the aperture of described penetrability aperture is 0.5 ~ 2mm, and span is 0.5 ~ 1cm.
As preferably, described hernia Biological Repair sheet is made up of 10 layers of monolayer.
Described penetrability aperture is for forming by the punching of laser micropore technology.
Another technical problem to be solved by this invention is to provide the preparation method of above-mentioned hernia Biological Repair sheet, this preparation method is improved and is optimized preparation technology, add DNA and remove link, mechanical oscillation is effectively combined with supersonic oscillations, Freeze Drying Technique and laser micropore technology effective are combined, thus effectively remove animal source DNA, complete structure and the composition remaining natural ECM, make hernia Biological Repair sheet of the present invention compared with existing similar products, DNA residual quantity is low, and immunogenicity is low, anti-infection ability is high.
The preparation method of above-mentioned hernia Biological Repair sheet, comprises following operating procedure:
(1) zoogenously to determine and the pre-process of small intestine and previous cleaning
Select inbred line animal as animal origin, the determination of animal varieties adopts the method for patent ZL200510008994.2 defined, gets small intestine's cleaning of fresh slaughtered animals, isolates submucous layer of small intestine, uses water for injection to rinse 3 times.
The method of patent ZL200510008994.2 defined is as described in its claims, specific as follows:
With the genomic DNA of pig to be measured for template, the pair of primers utilizing the reverse primer shown in the forward primer shown in sequence 2 and sequence 3 to form carries out pcr amplification, detect the band whether having a 88bp in amplified production, the pig choosing in amplified production the band having this 88bp is animal origin.Primer sequence is as follows:
Forward primer: 5 ' TCTGGAGCTAGCATAAGTGCC 3 ' (sequence 2),
Reverse primer: 5 ' GTGCAAGTACACATGCAGGG 3 ' (sequence 3).
(2) inactivation of virus
Adopt peracetic acid-alcoholic solution method inactivation of viruses, carry out in the constant-temperature ultrasonic washer that this step can be vibrated at rinse bath, wherein the volumn concentration of peracetic acid is 0.05 ~ 0.2% (being preferably 0.1%), inactivation time is 1 ~ 2h (being preferably 1h), temperature range is 4 ~ 40 DEG C, then clean 2 ~ 5 times in phosphate buffer, 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 of flowing, detect when electrical conductivity reaches below 1.5um/s and stop.
(3) de-cell
Carry out in the constant-temperature ultrasonic washer that this step can be vibrated at rinse bath, first material is inserted in rinse bath, then hydrogen injecting sodium hydroxide solution in rinse bath, open washer, scavenging period is 5 ~ 30min (being preferably 20min), concentration of sodium hydroxide solution is that 5 ~ 100mmol/L (is preferably 5 ~ 20mmol/L, more preferably 10mmol/L), then washer is closed, sodium hydroxide solution is inclined to, inject phosphate buffer to clean, open washer, scavenging period is 5 ~ 20min (being preferably 15min), 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 of flowing, detect when electrical conductivity reaches below 1.5um/s and stop.
(4) DNA removes process
Carry out in the constant-temperature ultrasonic washer that this step can be vibrated at rinse bath, sodium chloride solution is injected rinse bath, open washer, scavenging period is 5 ~ 30min (being preferably 20min), concentration of sodium chloride solution is 0.015mol/l or 2mol/L (being preferably 0.015mol/L), pH value is no more than 7.8, then with the water for injection cleaning material of flowing, detect when electrical conductivity reaches below 1.5um/s and stop.
(5) molding
This step comprises fixture and fixes, lyophilization and laser micropore punch 3 steps, add stacked for material 8 ~ 12 (being preferably 10 layers), be fixed on fixture (being preferably rustless steel fixture), then water for injection cleaning is used, the material being fixed on fixture is put in freezer dryer, lyophilization is carried out: pre-freeze is to-25 ~-50 DEG C (being preferably-25 DEG C) according to the lyophilizing flow process designed in advance, be incubated 0.5 ~ 4 hour (being preferably 2h), heat up 15 DEG C, be incubated 4 ~ 12 hours (being preferably 8h), heat up 15 DEG C, be incubated 0.5 ~ 4 hour (being preferably 2h), be warming up to 25 DEG C, be incubated 4 hours, after lyophilizing completes, use laser micropore puncher punching (i.e. penetrability aperture), pore diameter range is 0.5 ~ 2mm, span is 0.5 ~ 1cm.Described laser micropore punching refers to and utilizes laser technology that material is got micron-sized aperture, using the punching of laser micropore puncher to be form to make the hole run through between monolayer, being beneficial to tissue repair, and increasing the adhesion between monolayer.
(6) packaging sterilizing
Pack under aseptic conditions, one deck adopts special strong defensive QI paper, another layer to adopt vinyon, has packed rear employing oxirane and has carried out sterilizing.
In described step (1), inbred line animal is preferably inbred pig, 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 brood male female Wuzhi Mountain pig for being ancestral, adopts " son joins mother ", " full ", the inbred pig population cultivated.
As preferably, in described step (2), (3), (4), rinse bath frequency of oscillation is 100 ~ 300rpm, more preferably 200rpm.
As preferably, in described step (2), (3), (4), ultrasonic frequency is 20 ~ 80KHZ, more preferably 45KHZ.
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 800ml distilled water, regulate the pH value to 7.4 of solution with HCl, then adding distil water is settled to 1L.
In the present invention, the use standard of water for injection specifies according in state-promulgated pharmacopoeia.
The ultrasonic washing unit that rinse bath described in the present invention can vibrate is combined with mechanical oscillator by the rinse bath of conventional ultrasonic wave cleaning machine, enable rinse bath that machinery concussion occur while ultrasonic waves for cleaning, achieve mechanical oscillation and combine with ultrasonic waves for cleaning simultaneously and play a role.
Compared with prior art, the present invention has following remarkable advantage and beneficial effect:
The present invention selects inbred line animal as animal origin, after being further preferably Chinese wuzhishan miniature pig inbred-line, make the coefficient of inbreeding of this kind up to 0.991, gene high homogenous, there is the advantage that inherited characteristic is pure, stable, unified, fundamentally ensure that stability and the uniformity of different batches product, making the genetic background difference between individuality very little, thus determine immunogenic gene and the mankind have very high homology.The uniformity of the submucous layer of small intestine ECM material product developed for animal origin with this kind, stability and low immunogenicity are higher than the product be pig with hybridization being raw material.
Preparation method of the present invention is improved and is optimized preparation technology, adds DNA and removes link, determine 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, de-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 cleaning after every step completes.Inactivation of virus in preparation method, the de-step such as cell and DNA removing is all simultaneously in conjunction with mechanical oscillation and ultrasonic waves for cleaning, mechanical oscillation is effectively combined with supersonic oscillations by preparation method, improve inactivation of virus, de-cell and DNA remove the efficiency of technique, DNA residual quantity significantly reduces, greatly reduce technique consuming time, simplify technological process, whole preparation process only uses peracetic acid-ethanol, sodium hydroxide and sodium chloride 3 kinds of solution, and concentration is all well below existing technology of preparing, the material of preparation is made to completely eliminated immunogenicity, the complete effective ingredient such as structure and somatomedin remaining natural ECM, remain without poisonous and harmful substance, further, Freeze Drying Technique and laser micropore technology effective are combined innovatively in moulding process, retain natural ECM three dimensional structure, hole does not compress, and the anti-infection ability of product is high.
Accompanying drawing explanation
Shown in Fig. 1 is that schematic diagram cut out by sample in the embodiment of the present invention 2;
Shown in Fig. 2 is optical microscope figure in the embodiment of the present invention 2;
Shown in Fig. 3 is Electronic Speculum ultrastructure figure in the embodiment of the present invention 2.
Detailed description of the invention
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 hernia Biological Repair sheet
Preparation method is as follows:
(1) zoogenously to determine and the pre-process of small intestine and previous cleaning
Select Chinese wuzhishan miniature pig inbred-line as animal origin, the determination of animal varieties adopts the method for patent ZL200510008994.2 defined, 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 to rinse 3 times.
(2) inactivation of virus
Adopt peracetic acid-alcoholic solution method inactivation of viruses, carry out in the constant-temperature ultrasonic washer that this step can be vibrated at rinse bath, wherein the volumn concentration of peracetic acid is 0.05 ~ 0.2% (being preferably 0.1%), inactivation time is 1 ~ 2h (being preferably 1h), temperature range is 4 ~ 40 DEG C, then clean 2 ~ 5 times in phosphate buffer, 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 of flowing, detect when electrical conductivity reaches below 1.5um/s and stop.Rinse bath frequency of oscillation is 100 ~ 300rpm (being preferably 200rpm), and ultrasonic frequency is 20 ~ 80KHZ (being preferably 45KHZ).
(3) de-cell
Carry out in the constant-temperature ultrasonic washer that this step can be vibrated at rinse bath, first material is inserted in rinse bath, then hydrogen injecting sodium hydroxide solution in rinse bath, open washer, scavenging period is 5 ~ 30min (being preferably 20min), concentration of sodium hydroxide solution is that 5 ~ 100mmol/L (is preferably 5 ~ 20mmol/L, more preferably 10mmol/L), then washer is closed, sodium hydroxide solution is inclined to, inject phosphate buffer to clean, open washer, scavenging period is 5 ~ 20min (being preferably 15min), 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 of flowing, detect when electrical conductivity reaches below 1.5um/s and stop.Rinse bath frequency of oscillation is 100 ~ 300rpm (being preferably 200rpm), and ultrasonic frequency is 20 ~ 80KHZ (being preferably 45KHZ).
(4) DNA removes process
Carry out in the constant-temperature ultrasonic washer that this step can be vibrated at rinse bath, sodium chloride solution is injected rinse bath, open washer, scavenging period is 5 ~ 30min (being preferably 20min), concentration of sodium chloride solution is 0.015mol/l or 2mol/L (being preferably 0.015mol/L), pH value is no more than 7.8, then with the water for injection cleaning material of flowing, detect when electrical conductivity reaches below 1.5um/s and stop.Rinse bath frequency of oscillation is 100 ~ 300rpm (being preferably 200rpm), and ultrasonic frequency is 20 ~ 80KHZ (being preferably 45KHZ).
(5) molding
This step comprises fixture and fixes, lyophilization and laser micropore punch 3 steps, add stacked for material 8, be fixed on rustless steel fixture, then water for injection cleaning is used, the material being fixed on fixture is put in freezer dryer, lyophilization is carried out: pre-freeze is to-25 ~-50 DEG C (being preferably-25 DEG C) according to the lyophilizing flow process designed in advance, be incubated 0.5 ~ 4 hour (being preferably 2h), heat up 15 DEG C, be incubated 4 ~ 12 hours (being preferably 8h), heat up 15 DEG C, be incubated 0.5 ~ 4 hour (being preferably 2h), be warming up to 25 DEG C, be incubated 4 hours, after lyophilizing completes, use the punching of laser micropore puncher, pore diameter range is 0.5 ~ 2mm, span is 0.5 ~ 1cm.Described laser micropore punching refers to and utilizes laser technology that material is got micron-sized aperture, uses the punching of laser micropore puncher to be to make material surface form hole, being beneficial to tissue repair.
(6) packaging sterilizing
Pack under aseptic conditions, one deck adopts special strong defensive QI paper, another layer to adopt vinyon, has packed rear employing oxirane and has carried out sterilizing.
Embodiment 2: the physicochemical property of the material prepared by embodiment 1, histology, somatomedin and biology performance detect
1. 8 layer materials of pair preparation carry out physical property detection, and 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 contrast with Biodesign Surgisis product.Result: the porosity of the sample that embodiment 1 provides is 87.8 ± 2.51, Biodesign Surgisis product porosity is 78.3 ± 6.38.
2) sew up retentivity to detect: method: be sewn to 2 millimeters of places in patch 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 patch is fixed on tensiometer, stretch with the speed of 20mm/min, until stitch points is torn, record pulling force when stitch points is torn.As stated above 3 batch samples are detected.Result: sew up tensile strength and be more than or equal to 5 ± 0.5N.
3) tensile strength detection method: method: use (compression) testing machine that stretches, shown in Fig. 1, patch is cut into sample, and be 40%-60% at relative humidity after cutting, temperature is test immediately after placing 2h under the condition of 22 DEG C ± 2 DEG C.Be fixed on the chuck of cupping machine by sample two ends, stretch out until sample fracture successively with the speed of 100mm/min, longitudinal test piece and horizontal sample are tested respectively.Power during sample fracture is recorded in units of N.As stated above 3 batch samples are detected.Result: longitudinally: 15N, laterally: 8N.
4) burst strength detects: method: use (compression) testing machine that stretches, square sample material being cut into 23 × 23mm is for subsequent use, be 40%-60% at relative humidity, temperature is test immediately after placing 2h under the condition of 24 DEG C ± 2 DEG C.Be fixed on the workbench of tensilometer with annular holder by sample, make spheric probe with the speed of 750mm/min through sample, recording pops one's head in wears out the power of sample.As stated above 3 batch samples are detected.Result: burst strength is greater than 120N.
2. chemical property detects, and test item comprises virus, acid-base value, endotoxin and DNA residual quantity.
1) test liquid preparation: prepared by test liquid: the thickness uniform parts of sample thief, is cut into 1cm
2fragment, dry after washing, then add in glass container, by total surface area (cm inside and outside sample
2) being 5:1 with the ratio of water (mL), ratio adds water, add a cover and be placed in pressure steam sterilizer, at 121 DEG C ± 1 DEG C heating 30min, by sample and fluid separation applications after heating terminates, be chilled to room temperature as test liquid.Get consubstantiality hydrops and be placed in glass container, with legal system for blank liquid.
2) Viral diagnosis: method: selection Pseudorabies virus is indicator virus, adopts real-time quantitative PCR method to detect viral DNA copies number, detects 3 batch samples.Result: viral DNA copies number 0.
3) acid-base value detects: by the method test of regulation in 5.4.1 in GB/T14233.1 (" medical infusion, blood transfusion, instrument used for injection method of inspection part 1: chemical analysis method "), result: the difference of the pH value of test liquid and blank liquid is no more than 1.5.
4) endotoxin: by 6cm
2sample adds the ratio of 1ml lixiviate medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, lixiviate medium: normal saline.The method specified by GB/T 14233.2-2005 (" medical infusion, blood transfusion, instrument used for injection method of inspection part 2: BiologicalAssays Procedures ") is carried out, and detects 3 batch samples.Result: endotoxin content is less than 5EU/g.
5) DNA residues detection: according to biological preparation residual DNA detection method (" Chinese Pharmacopoeia " 2010, annex 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 contrast with Biodesign Surgisis product.Result: the DNA residual quantity of the sample that embodiment 1 provides is the DNA residual quantity of 120 ± 15pg/g, Biodesign Surgisis product is 250 ± 45pg/g.
3. histology
1) observation by light microscope: method: the capable hematoxylin-eosin stains of the material after paraffin peplos, inverted phase contrast microscope is observed.Result: acellular and cell debris remains, collagen is micro-continuous in fracture, as shown in Figure 2.
2) Ultrastructural observation.Result: material porous structure, fiber is without fracture, and uniform pore diameter, mean pore size is 200um, and porosity is greater than 85%, as shown in Figure 3.
4. somatomedin detects:
By 6cm
2sample adds the ratio of 1ml lixiviate medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, lixiviate medium: normal saline.ELLISA method is adopted to detect lixiviating solution neutral and alkali somatomedin (bFGF) and VEGF (VEGF) content.Result: bFGF content is 121.8 ± 2.683ng/L, VEGF content is 93.8 ± 3.033ng/L.
5. biology performance detects, and test item comprises cytotoxicity, delayed hypersensitivity, intradermoreaction.
1) cytotoxicity: method: by 6cm
2sample adds the ratio of 1ml lixiviate medium, and 37 ± 1 DEG C, 24 ± 2hr prepares experimental liquid, lixiviate medium: containing the MEM culture medium of serum.Get experimental liquid to test according to the test method specified in GB/T16886.5-2003 (" BiologicalEvaluationofMedicalDevice Part X: stimulate and test with delayed hypersensitivity ").Result: cell-cytotoxic reaction is less than or equal to 1 grade.
2) delayed hypersensitivity: method: by 6cm
2sample adds the ratio of 1ml lixiviate medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, and lixiviate medium is normal saline and Oleum Gossypii semen.Specify to test according to CB/T 16886.10-2005 (" BiologicalEvaluationofMedicalDevice the 10th part: stimulate and test with delayed hypersensitivity ") test method.Result: without delayed hypersensitivity.
3) intradermoreaction: by 6cm
2sample adds the ratio of 1ml lixiviate medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, and lixiviate medium is normal saline and Oleum Gossypii semen.Specify to test according to CB/T 16886.10-2005 (" BiologicalEvaluationofMedicalDevice the 10th part: stimulate and test with delayed hypersensitivity ") test method.Result: the difference of test specimen and solvent control mean score is less than 1.0.
The above embodiment of the present invention can not be used for limiting the present invention to explanation of the present invention, and any change in the implication suitable with claims of the present invention and scope, all should think to be included in the scope of claims.
Sequence table:
Sequence 2:
5’TCTGGAGCTAGCATAAGTGCC 3’
Sequence 3:
5’GTGCAAGTACACATGCAGGG 3’
Claims (16)
1. a preparation method for hernia Biological Repair sheet, is characterized in that comprising following operating procedure:
(1) zoogenously to determine and the pre-process of small intestine and previous cleaning
Select inbred pig as animal origin, the determination of animal varieties adopts following methods: with the genomic DNA of pig to be measured for template, the pair of primers utilizing the reverse primer shown in the forward primer shown in sequence 2 and sequence 3 to form carries out pcr amplification, detect the band whether having a 88bp in amplified production, the pig choosing in amplified production the band having this 88bp is animal origin, get small intestine's cleaning of fresh slaughtered animals, isolate submucous layer of small intestine, use water for injection to rinse 3 times;
(2) inactivation of virus
Adopt peracetic acid-alcoholic solution method inactivation of viruses, carry out in the constant-temperature ultrasonic washer that this step can be vibrated at rinse bath, wherein the volumn concentration of peracetic acid is 0.05 ~ 0.2%, and inactivation time is 1 ~ 2h, and temperature range is 4 ~ 40 DEG C, then clean 2 ~ 5 times in phosphate buffer, each 15min, detects the pH value of the phosphate buffer after cleaning, after pH reaches 6.5-7.5, with the water for injection cleaning material of flowing, detect when electrical conductivity reaches below 1.5um/s and stop;
(3) de-cell
Carry out in the constant-temperature ultrasonic washer that this step can be vibrated at rinse bath, first material is inserted in 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 ~ 20mmol/L, then washer is closed, sodium hydroxide solution is inclined to, inject phosphate buffer to clean, 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 of flowing, detect when electrical conductivity reaches below 1.5um/s and stop,
(4) DNA removes process
Carry out in the constant-temperature ultrasonic washer that this step can be vibrated at rinse bath, 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, then with the water for injection cleaning material of flowing, detects when electrical conductivity reaches below 1.5um/s and stop;
(5) molding
This step comprises fixture and fixes, lyophilization and laser micropore punch 3 steps, add stacked for material 8 ~ 12, be fixed on fixture, then water for injection cleaning is used, the material being fixed on fixture is put in freezer dryer, lyophilization is carried out: pre-freeze is to-25 ~-50 DEG C according to the lyophilizing flow process designed in advance, be incubated 0.5 ~ 4 hour, heat up 15 DEG C, be incubated 4 ~ 12 hours, heat up 15 DEG C, be incubated 0.5 ~ 4 hour, be warming up to 25 DEG C, be incubated 4 hours, after lyophilizing completes, use the punching of laser micropore puncher, pore diameter range is 0.5 ~ 2mm, span is 0.5 ~ 1cm,
(6) packaging sterilizing
Pack under aseptic conditions, one deck adopts special strong defensive QI paper, another layer to adopt vinyon, has packed rear employing oxirane and has carried out sterilizing.
2. the preparation method of a kind of hernia Biological Repair sheet according to claim 1, is characterized in that: described inbred pig is Chinese wuzhishan miniature pig inbred-line.
3. the preparation method of a kind of hernia Biological Repair sheet according to claim 1, is characterized in that: in described step (2), the volumn concentration of peracetic acid is 0.1%.
4. the preparation method of a kind of hernia Biological Repair sheet according to claim 1, is characterized in that: in described step (2), inactivation time is 1h.
5. the preparation method of a kind of hernia Biological Repair sheet according to claim 1, is characterized in that: be 20min with sodium hydroxide solution to the scavenging period that material cleans in described step (3).
6. the preparation method of a kind of hernia Biological Repair sheet according to claim 1, is characterized in that: in described step (3), concentration of sodium hydroxide solution is 10mmol/L.
7. the preparation method of a kind of hernia Biological Repair sheet according to claim 1, is characterized in that: be 15min with phosphate buffer to the scavenging period that material cleans in described step (3).
8. the preparation method of a kind of hernia Biological Repair sheet according to claim 1, is characterized in that: in described step (4), sodium chloride solution scavenging period is 20min.
9. the preparation method of a kind of hernia Biological Repair sheet according to claim 1, is characterized in that: in described step (4), concentration of sodium chloride solution is 0.015mol/L.
10. the preparation method of a kind of hernia Biological Repair sheet according to claim 1, is characterized in that: in described step (2), (3), (4), rinse bath frequency of oscillation is 100 ~ 300rpm.
The preparation method of 11. a kind of hernia Biological Repair sheets according to claim 9, is characterized in that: in described step (2), (3), (4), rinse bath frequency of oscillation is 200rpm.
The preparation method of 12. a kind of hernia Biological Repair sheets according to claim 1, is characterized in that: in described step (2), (3), (4), ultrasonic frequency is 20 ~ 80KHZ.
The preparation method of 13. a kind of hernia Biological Repair sheets according to claim 12, is characterized in that: in described step (2), (3), (4), ultrasonic frequency is 45KHZ.
The preparation method of 14. a kind of hernia Biological Repair sheets according to claim 1, is characterized in that: the fixture in described step (5) is rustless steel fixture.
The preparation method of 15. a kind of hernia Biological Repair sheets according to claim 1, is characterized in that: add stacked for material 10 in described step (5), be fixed on fixture.
The preparation method of 16. a kind of hernia Biological Repair sheets according to claim 1, is characterized in that: the lyophilizing flow process designed in advance in described step (5) is: pre-freeze, to-25 DEG C, is incubated 2h, heat up 15 DEG C, insulation 8h, heats up 15 DEG C, insulation 2h, is warming up to 25 DEG C, is incubated 4 hours.
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| CN106267347B (en) * | 2016-08-16 | 2019-07-12 | 北京大清生物技术股份有限公司 | Basin bottom biological sticking patch and preparation method thereof |
| CN106880872B (en) * | 2016-12-23 | 2019-10-29 | 北京大清生物技术股份有限公司 | Natural extracellular matrix biomembrane and the preparation method and application thereof |
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| CN106983918B (en) * | 2017-03-03 | 2020-05-05 | 北京博辉瑞进生物科技有限公司 | Biological anti-adhesion material, preparation method and application thereof |
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