CN104130437B - A kind of hard brain/spinal meninges biology patching material and its preparation method and application - Google Patents
A kind of hard brain/spinal meninges biology patching material and its preparation method and application Download PDFInfo
- Publication number
- CN104130437B CN104130437B CN201410373258.6A CN201410373258A CN104130437B CN 104130437 B CN104130437 B CN 104130437B CN 201410373258 A CN201410373258 A CN 201410373258A CN 104130437 B CN104130437 B CN 104130437B
- Authority
- CN
- China
- Prior art keywords
- spinal meninges
- hard brain
- preparation
- brain
- patching material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a kind of hard brain/spinal meninges biology patching material and its preparation method and application.The biology patching material for being obtained is particularly well-suited to the reparation of hard brain/spinal meninges defect, after being implanted into human body or animal body, can keep stablizing non-degradable in the year in June 2 in vivo, can gradually be degraded after more than 2 years, ensure that also there is biocompatibility while there is certain mechanical property and liquidproof seepage to act on, produce postoperative complications and the risk of side effect to substantially reduce, meet for the particular/special requirement for repairing hard brain/spinal meninges defect material.
Description
Technical field
The invention belongs to organizational engineering medical biomaterial technical field, and in particular to a kind of hard brain/spinal meninges is biological to be mended
Sheet material and its preparation method and application.
Background technology
Endocranium and endorchis are respectively function membrane tissues important between brain and skull, backbone and spinal cord, with phase
With biological characteristics seek peace function.Endocranium attaches cranial cavity inner face, loose with braincap link, is tightly combined with basis cranii；Endocranium
Stretch out at cranial nerve cranium, divide a word with a hyphen at the end of a line and with nerve by film combination, foramen magnum periphery to it is downward be endorchis.Hard brain
Film is consistent with the function and structure of endorchis, and endocranium is used to protect brain tissue, and endorchis is for protecting myeloid tissue.
Wound, tumour, inflammation, neurosurgery or other brainpan spinal diseases etc. can cause hard brain/spinal meninges defect.
The defect of hard brain/spinal meninges can cause the illnesss such as epilepsy, spinal fluid leakage, intracranial infection, meninx bulging and brain disorder.Such as in cranium
In the surgical operation of bottom, basis cranii endocranium relative thin and it is tightly combined with basion, once defect occur easily causes cerebrospinal rhinorrhea
And otorrhea.The tumours such as meningioma, the chordoma of basis cranii usually corrode infiltration to the skull and endocranium of surrounding, in tumor resection hand
During art, the tumour of excision can generally also attach part meninx and skull, cause postoperative generation leakage of cerebrospinal and intracranial infection.
To treat hard brain/spinal meninges defect, carried out frequently with hard brain/spinal meninges alternative materials implantation defect part in the prior art
Repair hard brain/spinal meninges defect.With the high speed development of medical technology, it is convex that dural substitutes are increasingly widely used in brain
Endocranium open decompression and some geneogenous factors are made in face operation, wound coup injury, the infiltration of tumour, surgical procedure
Into all many-sides such as dura defect, such as there is 10%~15% hard brain/spinal meninges of needs to replace in cranial surgery operation of opening cranium patient
Dura defect is repaired for material, also it has been reported that the ratio has been up to 30%.Therefore, in order to meet clinical needs, carry out
The suitable hard brain/spinal meninges repair materials of research are most important.
But, often there is many deficiencies in existing hard brain/spinal meninges alternative materials, usually cause tight during use
The latter problem of weight.For example in the problem of hard brain/spinal meninges alternative materials degradation speed control aspect after implanting, if firmly
The excessive velocities of brain/spinal meninges alternative materials degraded, tear edge declines, then cause liquidproof seepage to act on and decline, and causes hard brain/ridge
The illness such as film and surrounding tissue adhesion, infection, bleeding, leakage of cerebrospinal and epilepsy；If but when brain/spinal meninges alternative materials are long firmly
Between it is non-degradable, then cause itself and regeneration to mismatch again, poor biocompatibility is easily caused inflammatory reaction, connective group what is more
Knit the problems such as hyperplasia parcel, infection and bleeding.Therefore, for hard brain/spinal meninges alternative materials in implantation human body or animal body
Control in terms of degradation speed is very important.
However, in the technique that hard brain/spinal meninges alternative materials are produced with natural membrane material in the past, when membrane material is by lyophilized
After irradiation, the collagen in natural membrane material can be destroyed significantly, decline membrane material tear edge, and in order to more preferable
Cell and fat are sloughed, the destruction of the reagent of the de- cell, antigen and the fat that use to film is relatively large, makes natural membrane material
Other mechanical properties are greatly reduced, and ultimately result in the quickening of its degradation speed, and the effect of liquidproof seepage declines.In order to reduce biological group
The degradation speed of repair materials is knitted, membrane material is generally processed using crosslinking agent in the prior art, improve its mechanical property, reduce it
Degradation speed, make its in human body or animal body can for a long time it is non-degradable.But for for repairing hard brain/spinal meninges defect
For alternative materials, the group for processing other positions to be used in the prior art because its special physiological location and function are determined
Knit the crosslinking agent in the alternative materials of repair materials or hard brain/spinal meninges defect and be not particularly suited for hard brain/spinal meninges patching material, mainly
The problem of presence includes：(1) crosslinking agent treatment after, repair materials in human body or animal body for a long time it is non-degradable, cause its
After in implantation human body or animal body, poor biocompatibility is also easy to produce unfavorable side effect；(2) stability of crosslinking agent is low, mostly
Number crosslinking agent all has the group for being easier reaction, and this group causes crosslinking agent unstable, and then causes repair materials to exist
Degraded in the short time in human body or animal body, cause repair materials liquidproof seepage to act on and decline, and then trigger a series of arts
Infectious-related complication, brings detrimental effect；(3) crosslinking agent toxicity is high, it is difficult to control the final residual quantity on product of crosslinking agent, shadow
The human or animal's that sound is implanted is healthy.Therefore, exploitation one kind is needed badly in the prior art more to be repaiied suitable for hard brain/spinal meninges
Mend the biology patching material of degradation property requirement.
The content of the invention
Therefore, the technical problems to be solved by the invention are that hard brain/spinal meninges alternative materials are implanted into human body in the prior art
Degradation speed is too fast afterwards or is unfavorable for repairing the problem of hard brain/spinal meninges defect slowly excessively, and then provides a kind of suitable for hard brain/ridge
Hard brain/spinal meninges biology patching material that the degraded of film defect repair is required, and open its preparation method and application.
In order to solve the above technical problems, the invention provides a kind of preparation method of hard brain/spinal meninges biology patching material, bag
Include and take animal membrane and be organized as raw material, and be sequentially placed in surfactant solution, alkali lye, peroxide solutions, mass concentration and be
The step of being soaked in the glycidyl ether type epoxy cross-linking agent solution and phosphate buffer of 1-5wt%, and will be through upper
State after the membrane tissue material freeze-drying after the immersion of each step through irradiation sterilization, obtain final product.
The preparation method of described hard brain/spinal meninges biology patching material, the glycidyl ether type epoxy cross-linking agent solution
In the step of immersion, the crosslinking agent is propanetriol-diglycidyl-ether and/or DGEEG.
The preparation method of described hard brain/spinal meninges biology patching material, the glycidyl ether type epoxy cross-linking agent solution
In the step of immersion, the soak time is 15h-10d.
Preferably, the membrane tissue material soaks 20h in concentration is for the crosslinking agent of 2-4wt%.
It is furthermore preferred that the membrane tissue material soaks in concentration is for the crosslinking agent of 2wt%.
The preparation method of described hard brain/spinal meninges biology patching material, it is described the step of soaked containing surfactant solution
In, the mass concentration of the surfactant is 0.1-5wt%, and controls the solution temperature to be soaked for 0-25 DEG C.
In the step of preparation method of described hard brain/spinal meninges biology patching material, dipping by lye, the alkali lye
Concentration is 0.1-5mol/L, and controls the solution temperature to be soaked for 5-15 DEG C.
Preferably, in the step of the dipping by lye, it is the NaOH of 1mol/L that membrane tissue material is added to concentration
2h is soaked in solution, and it is 8 DEG C to control solution temperature.
The preparation method of described hard brain/spinal meninges biology patching material, the step of the peroxide solutions soak in, institute
The mass concentration for stating peroxide solutions is 1-5wt%.
Preferably, in the step of peroxide solutions soak, the membrane tissue material after immersion adds to concentration and is
Immersion treatment 1-5h in 2wt% peroxide solutions.
Preferably, the peroxide solutions are hydrogenperoxide steam generator.
The step of preparation method of described hard brain/spinal meninges biology patching material, irradiation sterilization is electron beam irradiation
Sterilizing, irradiation metering is 10-30KGy.
Preferably, the irradiation metering of the electron beam is 25KGy.
Preferably, in the step of surfactant solution soaks, the surfactant be TritonX_100,
At least one in Tween80 or Tween40.
Preferably, the animal membrane tissue raw material are the membrane tissue material of the pig or ox being born less than 6 months.
In the step of phosphate buffer soaks, the pH value of the phosphate buffer is 5.0-8.0.
Preferably, in the step of phosphate buffer soaks, it is 6.8 that the membrane tissue after the immersion adds pH
At least 5h is soaked in phosphate buffer.
The invention provides a kind of hard brain/spinal meninges biology patching material prepared by above-mentioned method.
Present invention also offers a kind of described hard brain/spinal meninges biology patching material tissue adhesion is prevented or reduces in preparation
Hard brain/the spinal meninges of material, the material for repairing impaired hard brain/mater tissue, treatment or prevention caused by wound or tumour
Application in leakage of cerebrospinal, infected material that the material of damage or defect, treatment or prevention cause by hard brain/spinal meninges defect.
It is used to prepare hard brain/spinal meninges biology patching material present invention also offers a kind of glycidyl ether type epoxy crosslinking agent
Purposes.
Technical solutions according to the invention have following advantage compared to existing technology：
(1) hard brain of the present invention/spinal meninges biology patching material preparation method, using mass concentration for 1-5wt% contracts
Water glycerine ethers epoxy crosslinking agent immersion treatment membrane tissue material, the biology patching material of acquisition is particularly well-suited to hard brain/spinal meninges
The reparation of defect, after being implanted into human body or animal body, can keep stablizing non-degradable in the year in June -2 in vivo, when super
Degraded after spending 2 years, it is ensured that also there is biocompatibility while being acted on certain mechanical property and liquidproof seepage, produce
Raw postoperative complications and the risk of side effect are substantially reduced, and are met for the spy for repairing hard brain/spinal meninges defect material
It is different to require；
(2) hard brain of the present invention/spinal meninges biology patching material preparation method, by selecting propanetriol-diglycidyl-ether
And/or in DGEEG be crosslinking agent, the hard brain/spinal meninges biological sticking patch cytotoxicity in itself will not be increased, drop
Low its brings the risk of side effect, in the sticking patch using above-mentioned crosslinking agent, though cytotoxicity has slightly lifted, its lifting width
Degree is smaller, can be ignored, and its toxicity is all in the range of 0 grade, and the human body that be implanted or animal can't be produced not
The influence of profit；
(3) hard brain of the present invention/spinal meninges biology patching material preparation method, is matched somebody with somebody by alkali lye and surfactant
Close substantially is reduced using the biology patching material fat content for obtaining, and substantially increases the security and validity of biological sticking patch.
Brief description of the drawings
In order that present disclosure is more likely to be clearly understood, below according to specific embodiment of the invention and combine
Accompanying drawing, the present invention is further detailed explanation, wherein
Fig. 1 is the stereoscan photograph of uncrosslinked membrane material；
Fig. 2 is the stereoscan photograph of the membrane material of crosslinking；
Fig. 3 is the control group photo of the uncrosslinked biological sticking patch of implantation；
Fig. 4 is the photo of the test group for being implanted into hard brain/spinal meninges biological sticking patch prepared by embodiment 1；
The HE colored graphs of biological sticking patch prepared by Fig. 5 embodiment of the present invention 1；
Fig. 6 is that the control group that hard brain/spinal meninges is repaired is not used；
Fig. 7 is the test group that the hard brain/spinal meninges biological sticking patch prepared using embodiment 1 is repaired.
Specific embodiment
Embodiment 1
The preparation method of the described hard brain/spinal meninges biological sticking patch of this implementation comprises the following steps：
(1) membrane tissue for taking out raw less than 6 months ox is raw material, is cleaned, and removal is not suitable for the part of processing,
It is standby；
(2) take the membrane tissue after above-mentioned cleaning and add mass concentration to be soaked in the TritonX_100 solution of 0.1wt%,
Concussion overnight, and controls solution temperature for 0 DEG C；
(3) take the membrane tissue after above-mentioned steps immersion and add concentration to soak 2h in the sodium hydroxide solution of 5mol/L, and
It is 15 DEG C to control solution temperature；
(4) it is immersion treatment in 1wt% hydrogenperoxide steam generators to take the membrane tissue after above-mentioned steps immersion and add mass concentration
1h；
(5) take the membrane tissue after above-mentioned steps immersion and add concentration to soak 15h in 1wt% propanetriol-diglycidyl-ethers；
(6) then take during the membrane tissue after the immersion adds the phosphate buffer that pH is 5.0 and soak at least 5h；
(7) the membrane tissue freeze-drying after above-mentioned steps are soaked, described freeze-drying program is：Product pre-freeze is first dropped
Vacuumized after extremely less than -60 DEG C of temperature, heated up, into sublimation stage；One time sublimation temperature rises to -25 DEG C, time by less than -35 DEG C
No less than 6 hours；Warming temperature rises to -15 DEG C by -25 DEG C, and the time is 1-3 hours；Secondary temperature elevation temperature is risen by -15 DEG C
To 25 DEG C, the time is 1-3 hours.It is then the electron beam radiation disinfection of 15KGy to irradiate metering, obtains final product.
Embodiment 2
The preparation method of the described hard brain/spinal meninges biological sticking patch of this implementation comprises the following steps：
(1) membrane tissue for taking out raw less than 6 months ox is raw material, is cleaned, standby；
(2) taking the membrane tissue after above-mentioned cleaning adds mass concentration to be soaked in the Tween80 solution of 5wt%, shakes
Night, and it is 25 DEG C to control solution temperature；
(3) take the membrane tissue after above-mentioned steps immersion and add concentration to soak 4h in the potassium hydroxide solution of 0.1mol/L,
And it is 5 DEG C to control solution temperature；
(4) it is immersion treatment in 5wt% peroxide solutions to take the membrane tissue after above-mentioned steps immersion and add mass concentration
5h；
(5) it is immersion in 1wt% DGEEG to take the membrane tissue after above-mentioned steps immersion and add mass concentration
15h；
(6) then take during the membrane tissue after the immersion adds the phosphate buffer that pH is 8.0 and soak at least 5h；
(7) the membrane tissue freeze-drying after above-mentioned steps are soaked, described freeze-drying program is：Product pre-freeze is first dropped
Vacuumized after extremely less than -60 DEG C of temperature, heated up, into sublimation stage；One time sublimation temperature rises to -25 DEG C, time by less than -35 DEG C
No less than 6 hours；Warming temperature rises to -15 DEG C by -25 DEG C, and the time was at 1-3 hours；Secondary temperature elevation temperature is risen by -15 DEG C
To 25 DEG C, the time was at 1-3 hours.It is then the electron beam radiation disinfection of 25KGy to irradiate metering, obtains final product.
Embodiment 3
The preparation method of the described hard brain/spinal meninges biological sticking patch of this implementation comprises the following steps：
(1) membrane tissue for taking out raw less than 6 months ox is raw material, is cleaned, standby；
(2) taking the membrane tissue after above-mentioned cleaning adds mass concentration to contain TritonX_100 and Tween40 for 2wt%
Soaked in solution, concussion overnight, and controls solution temperature for 8 DEG C；
(3) take the membrane tissue after above-mentioned steps immersion and add concentration to soak 2h in the sodium hydroxide solution of 1mol/L, and
It is 8 DEG C to control solution temperature；
(4) it is immersion treatment in 2wt% peroxide solutions to take the membrane tissue after above-mentioned steps immersion and add mass concentration
3h；
(5) take above-mentioned steps immersion after membrane tissue add mass concentration for 2wt% contain propanetriol-diglycidyl-ether and
20h is soaked in the mixed cross-linker of DGEEG；
(6) then take during the membrane tissue after the immersion adds the phosphate buffer that pH is 6.8 and soak at least 5h；
(7) the membrane tissue freeze-drying after above-mentioned steps are soaked, described freeze-drying program is：Product pre-freeze is first dropped
Vacuumized after extremely less than -60 DEG C of temperature, heated up, into sublimation stage；One time sublimation temperature rises to -25 DEG C, time by less than -35 DEG C
No less than 6 hours；Warming temperature rises to -15 DEG C by -25 DEG C, and the time was at 1-3 hours；Secondary temperature elevation temperature is risen by -15 DEG C
To 25 DEG C, the time was at 1-3 hours.It is then the electron beam radiation disinfection of 30KGy to irradiate metering, obtains final product.
Embodiment 4
The preparation method of the described hard brain/spinal meninges biological sticking patch of this implementation comprises the following steps：
(1) membrane tissue for taking out raw less than 6 months ox is raw material, is cleaned, standby；
(2) taking the membrane tissue after above-mentioned cleaning adds mass concentration to contain TritonX_100 and Tween80 for 3wt%
Soaked in solution, concussion overnight, and controls solution temperature for 0 DEG C；
(3) take the membrane tissue after above-mentioned steps immersion and add concentration to soak 4h in the sodium hydroxide solution of 1mol/L, and
It is 8 DEG C to control solution temperature；
(4) it is immersion treatment in 4wt% peroxide solutions to take the membrane tissue after above-mentioned steps immersion and add mass concentration
4h；
(5) take above-mentioned steps immersion after membrane tissue add mass concentration for 5wt% contain propanetriol-diglycidyl-ether and
10d is soaked in the mixed cross-linker of DGEEG；
(6) then take during the membrane tissue after the immersion adds the phosphate buffer that pH is 8.0 and soak at least 5h；
(7) the membrane tissue freeze-drying after above-mentioned steps are soaked, described freeze-drying program is：Product pre-freeze is first dropped
Vacuumized after extremely less than -60 DEG C of temperature, heated up, into sublimation stage；One time sublimation temperature rises to -25 DEG C, time by less than -35 DEG C
No less than 6 hours；Warming temperature rises to -15 DEG C by -25 DEG C, and the time was at 1-3 hours；Secondary temperature elevation temperature is risen by -15 DEG C
To 25 DEG C, the time was at 1-3 hours.It is then the electron beam radiation disinfection of 10KGy to irradiate metering, obtains final product.
Reference examples 1
The present embodiment is identical with the preparation method of embodiment 1, and it differs only in the step of not processed by crosslinking agent.
Reference examples 2
The present embodiment is identical with the preparation method of embodiment 2, and it differs only in the step of not processed by crosslinking agent.
Reference examples 3
The present embodiment is identical with the preparation method of embodiment 1, and it differs only in and is not soaked by the surfactant
The step of bubble.
Reference examples 4
The present embodiment is identical with the preparation method of embodiment 1, and it differs only in crosslinking agent used for glutaraldehyde cross-linking.
Effect example
The biological benefit of hard brain/spinal meninges prepared by following related embodiments of the invention and reference examples is investigated below by way of experimental example
Sheet material properties, and then prove that hard brain/spinal meninges biological sticking patch prepared by the present invention has notable technique effect.
1st, tear edge test
Test method：Use stretching (compression) testing machine, the biology that will be prepared according to embodiment 1-2, reference examples 1-2 methods
Patching material, the strip that specification is 1cm × 3cm is cut into by sample, is being worn with 4-0 sutures away from short side edge 3-5mm
Sample is crossed, doubling suture knots suture at the about 5cm at perforation, prevents suture from coming off；The above method is obtained
The sample for arriving, with water hydratable is purified 3-5 minutes, one end of non-threading is fixed on the bottom of tensile testing machine, by threading one end
The top of tensile testing machine is fixed on by hook, starts detection, until sample is torn, read the maximum of tensile load power
Value, the as tear edge of this sample.Result see the table below 1 and Fig. 1-2.
The tear edge test result of table 1
| Test group | Tear edge (N) |
| Embodiment 1 | 13.25 |
| Embodiment 2 | 10.65 |
| Reference examples 1 | 8.56 |
| Reference examples 2 | 7.35 |
| Reference examples 4 | 10.01 |
Conclusion：Upper table is three average values of experiment totally 30 flake products tear edges, embodiment 1,2 no matter 15KGy or
Under the dosage of 25KGy, relative to the situation of the reference examples 1,2 of cross-linking agent-free treatment, tear edge is obviously improved, and lifts ratio
Respectively 54.79% and 44.90%, and relative to the reference examples 4 processed using other crosslinking agents, relative to cross-linking agent-free treatment
Reference examples 1,2 situation, same tear edge is obviously improved, and lifting ratio is respectively 16.94% and 36.19%.Due to
There are a large amount of irregular gaps between the fiber that the collagenous fibres of membrane tissue material randomly weave and formed, porosity mainly takes
The certainly structure of natural collagen fibre, crosslinking agent can form new key between collagen and tense collagenous fibres and cause the slight of membrane material
Shrink, knowable to being contrasted from Fig. 1-2, before and after crosslinking, the collagenous fibres of biological sticking patch have obvious contraction, it was demonstrated that use the present invention
The crosslinking effect of crosslinking agent, have certain tensioning function to the collagenous fibres of film.
2nd, external degradation aspect
Test method：The biological sticking patch sub-cut that will be prepared according to embodiment 1-2, reference examples 3-4 methods is into (1-3) * (4-6)
The strip of mm sizes, is respectively placed in test tube, adds a certain amount of physiological saline, in being placed on 37 DEG C after 1d, changes one
Secondary solution, then places 3-4d, takes solution and is detected according to the detection method in medical device industry standard 16886, experiment
Result see the table below 2.
The degrading quality percentage of table 2
| Test group | First sub-sampling | Second sub-sampling |
| Embodiment 1 | 3.5% | 1.4% |
| Embodiment 2 | 6.2% | 2.4% |
| Reference examples 1 | 15.1% | 7.3% |
| Reference examples 2 | 21.8% | 7.6% |
| Reference examples 4 | 4.1% | 1.8 |
Table above shows the degrading quality percentage of respectively 15KGy and 25KGy irradiation doses biological sticking patch, by than
Relatively it can be seen that in the case of being acted at crosslinking dose, the degradation speed of biological sticking patch is decreased obviously.
3rd, internal degraded aspect
Test method：The biological sticking patch that will be prepared according to embodiment 1, reference examples 1, the method for reference examples 4, by above-mentioned preparation
In the rat body of biological sticking patch implantation health in situ, the Implantation Test method is as follows：Take 20 male SD rats, 3-4 week old,
Body weight 180-220g, is subcutaneously implanted the biological sticking patch of embodiment 1 after anesthesia, the biology of reference examples 1 or reference examples 4 is implanted into offside
Sticking patch, every material 1cm2, each 5 rats are put to death after choosing 3 week of implantation, 12 week, 30 week, take the week of the implantation region
Enclose tissue paraformaldehyde and fix photograph.Result of the test is shown in Table 3 and Fig. 3-4, after Fig. 3 is for 12 week of implantation of reference examples 1
Mouse photo, Fig. 4 is the mouse photo after 12 week of implantation of embodiment 1, it can be seen that Fig. 3 is degradable, figure
4 also have undegradable biological sticking patch to exist, and film form is than more complete, and film and surrounding tissue do not stick together situation, during degraded
Between substantially than it is uncrosslinked or from other crosslinking agents prepare biological sticking patch extend.Result of the test see the table below 3.
Degrading experiment result in the body of table 3
| Test group | 3 months | 12 months | 30 months |
| Embodiment 1 | It is undegraded | It is not degradable | It is degradable |
| Reference examples 1 | It is not degradable | It is degradable | —— |
| Reference examples 4 | It is undegraded | It is not degradable | It is not degradable |
4th, cytotoxicity aspect
Test method：The biological sticking patch prepared according to the method for embodiment 1, reference examples 1, reference examples 4 is extracted, is made
Standby leaching liquor；Prepare corresponding cell suspension；Former culture medium is discarded, leaching liquor, the feminine gender of debita spissitudo are added in 96 orifice plates
Control, positive control and blank, are placed in and cultivate 24h, 48h in 37 DEG C of CO2gas incubators respectively.In 24h and 48h
Afterwards, observation of cell upgrowth situation, discards the leaching liquor in 96 well culture plates, and add the μ L of MTT solution 50 of 1mg/ml.Add
96 orifice plates of MTT solution are placed in 37 DEG C of CO2gas incubators 2 hours.After 2 hours, the MTT solution in 96 orifice plates is discarded, and add
Enter the μ L of isopropanol 100, shake 96 orifice plate 5-10min, determine its absorbance at 492nm.The detection method of cytotoxicity
It is the detection carried out with reference to the mtt assay in cytotoxicity detection in 16886 professional standards.Result see the table below 4,
The cell toxicity test result of table 4
| Test group | Observation period 24h | Observation period 48h |
| Embodiment 1 | 103.12 | 104.07 |
| Reference examples 1 | 100.31 | 101.25 |
| Reference examples 4 | 92.02 | 90.84 |
Upper table is three average values of test products cytotoxicity, can be clearly seen that embodiment 1 uses crosslinking agent system
Standby product, cytotoxicity decreases, and all in the range of 0 grade, compared under the cytotoxicity embodiment 1 of reference examples 1
Drop ratio is 2.80% and 2.79%, and cytotoxicity change is little, after illustrate addition this crosslinking agent, will not increase product in itself
Cytotoxicity, but have certain increase compared to the cytotoxicity of the cytotoxicity controls example 4 of reference examples 1, and increase ratio
It is 9.01% and 11.46%, crosslinking agent used has certain cytotoxicity in illustrating reference examples 4.
5th, fat content aspect
Test method：The biological sticking patch prepared according to embodiment 1, the method for reference examples 3 is tested, the survey of fat content
The method of determining is foundation《Pharmacopoeia of People's Republic of China》Two " detections of pancreatin fat " are formulated, and measurement result see the table below 5.
The fat content result of the test of table 5
| Reference examples 3 | Embodiment 1 | Lifting ratio | |
| Fat content | 0.27% | 0.06% | 77.77% |
Upper table is three average values of experiment 0.1g biological sticking patch fat contents, can be clearly seen that the embodiment of the present invention
1 uses cooperatively the biological sticking patch for obtaining by alkali lye NaOH and surface-active agent TrironX-100, and fat content substantially drops
It is low.
6th, security and validity
6.1 securities
Test method：Biological sticking patch prepared by the embodiment of the present invention 1 carries out HE dyeing treatment, the HE dyeing treatment step
It is rapid as follows：By material graded ethanol it is descending enter water, successively cleaned with running water and distilled water afterwards, processed with 0.5% haematoxylin
Cleaned with running water afterwards, then with being cleaned with running water after 1% hydrochloride alcohol color separation, then with 1/400 ammoniacal liquor promote basket after clean, use
With 80% alcohol color separation after 1% eosin stains, gradient alcohol dehydration obtains resin mounting after dimethylbenzene is transparent.The present invention is implemented
The HE colored graphs of biology patching material prepared by example 1 are shown in Fig. 5, it can be seen that the biological sticking patch does not have cell and nucleus residual
Stay, it was demonstrated that the method for the present invention can effectively slough the debris such as the cell in membrane material, safe.
6.2 validity
Test method：As test group in biological sticking patch implantation healthy rabbit prepared by the embodiment of the present invention 1, not make
With pachymeninx repair rabbit as a control group, the test method is as follows：After anesthesia rabbit, rabbit cranium is opened, broken
Bad endocranium, adds biological sticking patch to substitute endocranium, and control group is not implanted into biological sticking patch；Then by skull reduction, suture wound
Mouthful.Experimental result is periodically checked, result of the test is shown in Fig. 6-7, and Fig. 6 is that the control group that pachymeninx is repaired is not used, and Fig. 7 is to use
The test group photo of hard brain/spinal meninges biological sticking patch prepared by the embodiment of the present invention 1, the as can be seen from the figure wound of two pictures
All fully recover, but scar is larger in Fig. 6 photos, and there is portion of tissue necrosis phenomena occur, and wound recovers good in Fig. 7 photos
It is good, and scar is smaller, wound is smoothed, and does not have necrosis phenomenon, illustrates repair tissue function well and without immunological rejection.
Obviously, above-described embodiment is only intended to clearly illustrate example, and not to the restriction of implementation method.It is right
For those of ordinary skill in the art, can also make on the basis of the above description other multi-forms change or
Change.There is no need and unable to be exhaustive to all of implementation method.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (9)
1. a kind of preparation method of hard brain/spinal meninges biology patching material, it is characterised in that be organized as former material including taking animal membrane
Material, and sequentially it is placed in the glycidol ethers that surfactant solution, alkali lye, peroxide solutions, mass concentration are 1-5wt%
The step of being soaked in epoxy cross-linking agent solution and phosphate buffer, and the film after being soaked through above steps
Through irradiation sterilization after organization material freeze-drying, obtain final product.
2. the preparation method of hard brain/spinal meninges biology patching material according to claim 1, it is characterised in that the shrink
In the step of glycerine ethers epoxy crosslinking agent solution soaks, the crosslinking agent is that propanetriol-diglycidyl-ether and/or ethylene glycol contract
Water glycerin ether, the soak time is 15h-10d.
3. the preparation method of hard brain/spinal meninges biology patching material according to claim 1 and 2, it is characterised in that the table
In the step of face activator solution soaks, the mass concentration of the surfactant is 0.1-5wt%, and controls the solution temperature
Spend for 0-25 DEG C is soaked.
4. the preparation method of hard brain/spinal meninges biology patching material according to claim 1 and 2, it is characterised in that the alkali
In the step of immersion is steeped, the concentration of the alkali lye is 0.1-5mol/L, and controls the solution temperature to be soaked for 5-15 DEG C.
5. the preparation method of hard brain/spinal meninges biology patching material according to claim 1 and 2, it is characterised in that the mistake
In the step of oxide solution soaks, the mass concentration of the peroxide solutions is 1-5wt%.
6. the preparation method of hard brain/spinal meninges biology patching material according to claim 1 and 2, it is characterised in that the phosphorus
In the step of phthalate buffer soaks, the pH value of the phosphate buffer is 5.0-8.0.
7. the preparation method of hard brain/spinal meninges biology patching material according to claim 1 and 2, it is characterised in that the spoke
The step of according to sterilizing is electron beam irradiation sterilization, and irradiation metering is 10-30KGy.
8. the hard brain/spinal meninges biology patching material for being prepared according to any described methods of claim 1-7.
9. the hard brain/spinal meninges biology patching material described in claim 8 prevents or reduces for preparation tissue adhesion's material, is used for
Hard brain/the spinal meninges of the material, treatment or prevention of impaired hard brain/mater tissue caused by wound or tumour is repaired to damage or defect
Material, treatment or the prevention leakage of cerebrospinal, the infected material that cause by hard brain/spinal meninges defect in purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410373258.6A CN104130437B (en) | 2014-07-31 | 2014-07-31 | A kind of hard brain/spinal meninges biology patching material and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410373258.6A CN104130437B (en) | 2014-07-31 | 2014-07-31 | A kind of hard brain/spinal meninges biology patching material and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104130437A CN104130437A (en) | 2014-11-05 |
| CN104130437B true CN104130437B (en) | 2017-05-31 |
Family
ID=51803308
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410373258.6A Active CN104130437B (en) | 2014-07-31 | 2014-07-31 | A kind of hard brain/spinal meninges biology patching material and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104130437B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106139247B (en) * | 2015-04-17 | 2019-11-12 | 天津市赛宁生物工程技术有限公司 | A kind of preparation method of artificial hard brain/spinal meninges sticking patch |
| CN105597151A (en) * | 2016-03-09 | 2016-05-25 | 烟台正海生物科技股份有限公司 | Surgery patch and preparing method thereof |
| CN107469145A (en) * | 2017-09-22 | 2017-12-15 | 北京华信佳音医疗科技发展有限责任公司 | A kind of preparation and its application of dura mater patching material |
| CN116531567B (en) * | 2023-05-09 | 2024-02-13 | 成都睿科美医疗科技有限公司 | Endometrial repair material |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4626286A (en) * | 1983-10-31 | 1986-12-02 | Schmid Laboratories, Inc. | Collagen gel and the process of making said gel |
| CN1162252A (en) * | 1994-10-21 | 1997-10-15 | 密执安大学 | Calcification-resitant bioprosthetic tissue and methods of making same |
| CN103239760A (en) * | 2013-05-22 | 2013-08-14 | 烟台正海生物技术有限公司 | Anti-adhesion surgical patch and preparation method thereof |
| CN103272272A (en) * | 2013-05-22 | 2013-09-04 | 烟台正海生物技术有限公司 | Natural skin patch with holes and preparation method thereof |
-
2014
- 2014-07-31 CN CN201410373258.6A patent/CN104130437B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4626286A (en) * | 1983-10-31 | 1986-12-02 | Schmid Laboratories, Inc. | Collagen gel and the process of making said gel |
| CN1162252A (en) * | 1994-10-21 | 1997-10-15 | 密执安大学 | Calcification-resitant bioprosthetic tissue and methods of making same |
| CN103239760A (en) * | 2013-05-22 | 2013-08-14 | 烟台正海生物技术有限公司 | Anti-adhesion surgical patch and preparation method thereof |
| CN103272272A (en) * | 2013-05-22 | 2013-09-04 | 烟台正海生物技术有限公司 | Natural skin patch with holes and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104130437A (en) | 2014-11-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105935454A (en) | Decellularized matrix-source tissue engineering scaffold and preparation method and application thereof | |
| US10426868B2 (en) | Method for preparing an animal decellularized tissue matrix material and a decellularized tissue matrix material prepared thereby | |
| CN106581770B (en) | A kind of dural patch, preparation method and the application in endocranium injury repair | |
| WO2016180259A1 (en) | Dry animal-derived collagen fiber tissue material and preparation method and bioprosthesis thereof | |
| CN104130437B (en) | A kind of hard brain/spinal meninges biology patching material and its preparation method and application | |
| CN103874762A (en) | Methods of sterilizing fetal support tissues | |
| CN106880872B (en) | Natural extracellular matrix biomembrane and the preparation method and application thereof | |
| CN103520780A (en) | Biological amnion and preparation method thereof | |
| CN103480042B (en) | Artificial dura mater spinalis, and preparation method and use method thereof | |
| US11529437B2 (en) | Biological tissue matrix material, preparation method therefor and use thereof in otological repair material | |
| CN107551312B (en) | Flocculent collagen hemostatic fiber and preparation method thereof | |
| CN107320776B (en) | Gel capable of promoting dermis regeneration and preparation method thereof | |
| CN108261565B (en) | Hernia patch, preparation method and application thereof in hernia repair | |
| CN100368534C (en) | Bioderived amnion, composite bioderived amnion and its preparation method | |
| CN115845141B (en) | Preparation method and application of dry amniotic membrane | |
| Van den Heever et al. | The effect of different treatment modalities on the calcification potential and cross-linking stability of bovine pericardium | |
| CN105963787A (en) | Preparation method of new biological patches | |
| CN106474548B (en) | Biological induction type artificial dura mater and preparation method thereof | |
| CN110420352A (en) | A kind of bion ocular tissue repair materials and preparation method thereof | |
| JP5763790B2 (en) | Biological transplant material derived from mammalian cartilage tissue | |
| CN106256384A (en) | A kind of biological sticking patch and preparation method | |
| CN110433340B (en) | Acellular matrix urethral suspension repair material and preparation method and application thereof | |
| CN108939157A (en) | The preparation method of natural tubulose small intestine acellular matrix film | |
| CN110496249A (en) | Vascular protection band and its preparation method and application | |
| US10052400B2 (en) | Method for preparing neutralized matrix of non-antigenic collagenous material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201225 Address after: 264006 No. 10, Yantai economic and Technological Development Zone, Shandong, Hengshan Road Patentee after: YANTAI ZHENGHAI BIO-TECH Co.,Ltd. Address before: Room 205, B2 / F, bio nano Park, 218 Xinghu street, Suzhou Industrial Park, 215000, Jiangsu Province Patentee before: SUZHOU ZHENGHAI BIOTECHNOLOGY Co.,Ltd. |
|
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 264006 No.7 Nanjing Street, Yantai Economic and Technological Development Zone, Yantai City, Shandong Province Patentee after: YANTAI ZHENGHAI BIO-TECH Co.,Ltd. Address before: 264006 No. 10, Yantai economic and Technological Development Zone, Shandong, Hengshan Road Patentee before: YANTAI ZHENGHAI BIO-TECH Co.,Ltd. |