CN103272275A - Biological repair tablet for endocranium and preparation method thereof - Google Patents
Biological repair tablet for endocranium and preparation method thereof Download PDFInfo
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- CN103272275A CN103272275A CN2013102035941A CN201310203594A CN103272275A CN 103272275 A CN103272275 A CN 103272275A CN 2013102035941 A CN2013102035941 A CN 2013102035941A CN 201310203594 A CN201310203594 A CN 201310203594A CN 103272275 A CN103272275 A CN 103272275A
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Abstract
The invention provides a biological repair tablet for endocranium 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 endocranium 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 endocranium 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 implantation technical field of medical instruments, be specifically related to biological patch of a kind of cerebral dura mater and preparation method thereof.
Background technology
Cerebral dura mater is a thick and tough and tensile duplicature.Skin is the periosteum of skull inner face, only loosely invests braincap, particularly adheres to more loosely at occipitalia and temples, is called periosteal layer; Internal layer is outer bed thickness and tough and tensile, with spinal dura mater at the Foramen magnum place continuous company, be called the meninges layer.Dural main effect is the protection cerebral tissue.Wound, tumor and neurosurgery etc. all can cause dura defect, it is the basic operational steps of neurosurgery that cerebral dura mater is rebuild, whether complete the and patient's of its reconstruction post-operative recovery be in close relations, also is the important measures of prevention cerebrospinal leak and intracranial infection; Traditional cerebral dura mater reconstruction biomaterials is mainly taken from autologous tissue, and material source is limited, and increases the weight of patient's misery, and the artificial dura mater patching material is widely accepted in recent years.
According to chemical constituent and the biological characteristics of materialogy, dural repairment material mainly contains not absorbing material, absorbable material, several big classes of biomaterial at present.The dural repairment material that uses clinically can not absorb and degrade after the implantation based on macromolecular material at present, can forever retain in the body, as foreign body, can cause local organization inflammation and infection after inserting.The developing direction of modern medical embedded material is degradable, have the initiatively biomaterial of induced tissue regeneration.
Based on the tissue engineering principle is that (extracellular matrix, ECM) material is main developing direction for the extracellular matrix of raw material with the animal tissue.ECM is by compositions such as multiple macromolecular substances such as collagen, non-collagen sugar albumen, aminoglycan, Dan Baijutang, 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 regulation and control tissue 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 cerebral dura mater biological material for repairing.Be used for clinical bioactive materials at present and comprised ECM materials such as allogeneic dermis, pig intestinal mucosa lower floor, pig dermis, embryo cattle corium.(small intestinal submucosa, SIS) host material is the optimal tissue renovation material that present academia is generally acknowledged wherein to take off the cell submucous layer of small intestine.U.S. Cook Biotech Incorporated's is the biological patch of the cerebral dura mater (trade name: Biodesign Surgisis) occupied 10 ~ 30% biological cerebral dura mater sheet market in American-European countries, and entered China market the end of the year 2010 of raw material with pig intestinal mucosa lower floor.
Biodesign Surgisis product has natural distinctive ECM structure and composition, initiatively induced tissue regeneration, and immunogenicity is low, and histocompatibility is good, advantages such as degradable.But along with the increase of clinical practice, the problem of Biodesign Surgisis product also displays.Clinical studies show: 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 does not consider and removes the residual link of DNA that 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 host material, has destroyed natural ECM three dimensional structure, influences 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 the biological patch of a kind of cerebral dura mater, the biological patch of this cerebral dura mater 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.
The biological patch of a kind of cerebral dura mater, 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 inbred line WZSP.
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%.
The biological patch of described cerebral dura mater is made of 4 ~ 12 layers of monolayer, is shaped as rectangle or square, and length is 7 ~ 20cm, width is 6 ~ 20cm, have the penetrability aperture on the biological patch of described cerebral dura mater, the aperture of described 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 the biological patch of above-mentioned cerebral dura mater, 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 biological patch of cerebral dura mater 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 the biological patch of above-mentioned cerebral dura mater 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, cleaning is cleaned by the small intestine of getting 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 thermostatic ultrasonic ripple 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 ℃, in phosphate buffer, clean then 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 thermostatic ultrasonic ripple washer that rinse bath can vibrate, at first material is inserted in the rinse bath, in rinse bath, inject sodium hydroxide solution then, 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), close washer then, 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 handles
This step is carried out in the thermostatic ultrasonic ripple 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, 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, with 4 ~ 12 stacked adding of material, be fixed on the fixture (being preferably the rustless steel fixture), clean clean then with water for injection, the material that is fixed in fixture is put in the freezer dryer, lyophilizing flow process according to design is in advance 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.The punching of described laser micropore 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 the hole that formations runs through between monolayer, to be beneficial to tissue repair, and the adhesion between the increase monolayer.
(6) packaging sterilizing
Pack under aseptic state, one deck adopts special strong defensive QI paper, another layer employing vinyon, adopts oxirane to sterilize after packing is finished.
The inbred line animal is preferably inbred pig in the described step (1), more preferably Chinese inbred line WZSP.Described Chinese inbred line WZSP refers to the animal varieties that ZL02149030.9 provides, and utilizes brood public 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, adding distil water is settled to 1 L then.
The use 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 take place 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 for use the inbred line animal as animal origin, after further being preferably Chinese inbred line WZSP, 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 immunogenic gene and the mankind to have very high homology.Be that uniformity, stability and the low immunogenicity of the submucous layer of small intestine ECM material product of animal origin exploitation be higher than to hybridize with this kind be that pig is the product of raw material.
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 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 technology, the DNA residual quantity significantly reduces, it is consuming time to greatly reduce technology, simplified technological process, whole process of preparation is only used peracetic acid-ethanol, 3 kinds of solution of sodium hydroxide and sodium chloride, and concentration is all well below the existing preparation technology, make the material of preparation remove immunogenicity fully, effective ingredient such as the complete structure that has kept natural ECM and somatomedin, no poisonous and harmful substance is residual; 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, the anti-infection ability height of product.
Description of drawings
The sample that shown in Figure 1 is in the embodiment of the invention 2 is cut out sketch map;
The optical microscope figure that shown in Figure 2 is in the embodiment of the invention 2;
The Electronic Speculum ultrastructure figure that shown in Figure 3 is in the embodiment of the invention 2.
The specific embodiment
Be further described in detail below in conjunction with the present invention of embodiment, but be not limited to this.
The preparation of the biological patch of embodiment 1:8 layer cerebral dura mater
Preparation method is as follows:
(1) zoogenously determines and pre-process and the previous cleaning of small intestine
Select Chinese inbred line WZSP as animal origin, the method of definite employing patent ZL200510008994.2 defined of animal varieties, the small intestine of getting fresh Chinese inbred line WZSP of butchering cleans clean, 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 thermostatic ultrasonic ripple 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 ℃, in phosphate buffer, clean then 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 thermostatic ultrasonic ripple washer that rinse bath can vibrate, at first material is inserted in the rinse bath, in rinse bath, inject sodium hydroxide solution then, 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), close washer then, 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 handles
This step is carried out in the thermostatic ultrasonic ripple 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, water for injection cleaning material to flow then 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, with 8 stacked adding of material, be fixed on the rustless steel fixture, clean clean then with water for injection, the material that is fixed in fixture is put in the freezer dryer, and carry out lyophilization according to the lyophilizing flow process of design in advance: 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, adopts oxirane to sterilize after packing is finished.
Embodiment 2: the physicochemical property of the material that embodiment 1 is prepared, histology, somatomedin and biology performance detect
1. 8 layer materials to preparation carry out the 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 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 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 the tensiometer, speed with 20mm/min stretches, be torn the pulling force when noting stitch points and being torn up to stitch points.As stated above 3 batch samples are detected.Result: sew up tensile strength more than or equal to 5 ± 0.5N.
3) tensile strength detection method: method: (compression) testing machine that use to stretch, according to shown in Figure 1, patch 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.Be the power during sample fracture under the unit record with N.As stated above 3 batch samples are detected.Result: vertically: 15N, laterally: 8N.
4) burst strength detects: method: use (compression) testing machine that stretches, the square sample that material is cut into 23 * 23mm is standby, 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, note the power that probe is worn out sample.As stated above 3 batch samples are detected.The 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: test liquid preparation: the thickness uniform parts of sample thief, be cut into 1cm
2Fragment, dry after washing, add then 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, be equipped with blank liquid with 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) the DNA residual quantity detects: according to biological preparation residual DNA detection method (" Chinese pharmacopoeia 2010, the exogenous DNA determination of residual amount of appendix IX-B 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 2.
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 3.
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(" medical apparatus and instruments biological assessment 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(" medical apparatus and instruments biological assessment the 10th part: stimulate and the delayed hypersensitivity test ") the test method regulation tests.Result: no 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(" medical apparatus and instruments biological assessment 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 kept the score 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 (23)
1. the biological patch of a cerebral dura mater 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. the biological patch of a kind of cerebral dura mater according to claim 1, it is characterized in that: described inbred line animal is inbred pig.
3. the biological patch of a kind of cerebral dura mater according to claim 2, it is characterized in that: described inbred pig is inbred line Wuzhi Mountain pig.
4. the biological patch of a kind of cerebral dura mater 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. according to the biological patch of the described a kind of cerebral dura mater of claim 1-4, it is characterized in that: constituted by 4 ~ 12 layers of monolayer, be shaped as rectangle or square, length is 7 ~ 20cm, width is 6 ~ 20cm, and have the penetrability aperture, and the aperture of described penetrability aperture is 0.5 ~ 2mm, span is 0.5 ~ 1cm.
6. the biological patch of a kind of cerebral dura mater according to claim 5, it is characterized in that: described penetrability aperture is for forming by the punching of laser micropore technology.
7. the preparation method of the biological patch of the described a kind of cerebral dura mater of 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, cleaning is cleaned by the small intestine of getting 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 thermostatic ultrasonic ripple 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 ℃, in phosphate buffer, clean then 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 thermostatic ultrasonic ripple washer that rinse bath can vibrate, at first material is inserted in the rinse bath, in rinse bath, inject sodium hydroxide solution then, open washer, scavenging period is 5 ~ 30min, concentration of sodium hydroxide solution is 5 ~ 100mmol/L, closes washer then, 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 handles
This step is carried out in the thermostatic ultrasonic ripple 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 that 0.015mol/l or 2mol/L, pH value are no more than 7.8, 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 is fixed, lyophilization and 3 steps of laser micropore punching, with 4 ~ 12 stacked adding of material, be fixed on the fixture, clean clean then with water for injection, the material that is fixed in fixture is put in the freezer dryer, carries out lyophilization according to the lyophilizing flow process of design in advance: 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, adopts oxirane to sterilize after packing is finished.
8. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: the inbred line animal is inbred pig in the described step (1).
9. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 8, it is characterized in that: described inbred pig is inbred line Wuzhi Mountain pig.
10. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: the volumn concentration of peracetic acid is 0.1% in the described step (2).
11. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: inactivation time is 1h in the described step (2).
12. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: the scavenging period that with sodium hydroxide solution material is cleaned in the described step (3) is 20min.
13. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: concentration of sodium hydroxide solution is 5 ~ 20mmol/L in the described step (3).
14. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: concentration of sodium hydroxide solution is 10mmol/L in the described step (3).
15. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: the scavenging period that with phosphate buffer material is cleaned in the described step (3) is 15min.
16. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: the sodium chloride solution scavenging period is 20min in the described step (4).
17. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: concentration of sodium chloride solution is 0.015mol/L in the described step (4).
18. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: the rinse bath frequency of oscillation is 100 ~ 300rpm in described step (2), (3), (4).
19. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 18, it is characterized in that: the rinse bath frequency of oscillation is 200rpm in described step (2), (3), (4).
20. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: ultrasonic frequency is 20 ~ 80KHZ in described step (2), (3), (4).
21. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 20, it is characterized in that: ultrasonic frequency is 45KHZ in described step (2), (3), (4).
22. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7, it is characterized in that: the fixture in the described step (5) is the rustless steel fixture.
23. the preparation method of the biological patch of a kind of cerebral dura mater according to claim 7 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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