CN106256381A - Repair materials and its production and use in order - Google Patents
Repair materials and its production and use in order Download PDFInfo
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- CN106256381A CN106256381A CN201510342202.9A CN201510342202A CN106256381A CN 106256381 A CN106256381 A CN 106256381A CN 201510342202 A CN201510342202 A CN 201510342202A CN 106256381 A CN106256381 A CN 106256381A
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Abstract
The present invention provides a kind of method preparing orderly repair materials, including: (1) makes fascia process in sodium hydroxide solution；(2) gains are made to process in detergent solution；(3) gains are made to be dried.The orderly repair materials that the inventive method prepares is linear collagen fiber.This linear fibre remains with the structure of natural collagen fibre, has the rough surface of beneficially cell attachment, has good biocompatibility and biodegradability, and it is more thorough to kill aspect on antibacterial and virus, and rejection is less.Therefore, the linear collagen fiber of the present invention can serve as tissue renovation material, in particular as nerve injury and the repair materials of tendon injury.
Description
Technical field
The present invention relates to a kind of orderly bioprosthetic material and its production and use.
Background technology
Spinal cord injury (Spinal cord injury, SCI) is a kind of serious nervous system trauma,
There is the crippling of height.Nowadays, the CO2 laser weld after central nervous system injury is proved to be possibility
, but the operation means of routine can only make the thorough rehabilitation of only a few people in these the wounded.Many
Item research shows, the internal microenvironment of spinal cord is not suitable for causing spinal cord regeneration ability extremely limited
Main cause.After spinal cord injury in a short period of time, cavity and scar are defined at damaged part
Trace, under such a hostile environment, aixs cylinder i.e. allows to growth, it is also difficult to go beyond so
An obstacle.Currently for spinal cord injury, application biological support bridges damage both sides
Spinal cord, in conjunction with neurotrophic factor or medicine or stem cell, thus promotes that axon regeneration is one
Important therapeutic strategy.When preparation is for the biologic bracket material of neuranagenesis, taking of material
Tropism is extremely important, because the growth of spinal nerves fiber (aixs cylinder) is directive.At present,
Repairing of neural injury material is mainly made up of the material of chemosynthesis, they often cell compatibilities
Bad, easily cause immune rejection problems.
Additionally, tendon injury is modal athletic injury, damage probability is high, and disability rate is the highest.
After tendon injury, if repairing in time, often can cause limbs disturbance, severe one is even
Maimed.Surgical intervention is generally required for more serious tendon injury, defect tendon is damaged
Wound generally require implantation dummy, such as: autologous tendon, tendon allograft, xenogenic tendon or
Artificial tendon.Former three is due to many reasons such as transplant limited source or rejections, no
Can meet clinical treatment demand, therefore exploitation artificial tendon has important practical significance.Tendon is only
Special biomechanical property is mainly due to the height sense of organization of Tenocyte cell epimatrix (ECM).
Extracellular matrix is mainly made up of I-type collagen, and the ECM in tendon is grade pencil and divides not
It is arranged in parallel in stromatin with aspect, tendon fibroblasts (the also referred to as tendon of prismatic
Cell) in longitudinal arrangement and a large amount of sheath like cell extends to extracellular matrix.Type i collagen can lure
The formation leading tendon extracellular matrix increases by 410%.Therefore, based on collagen and have certain orientation
Material be for tendon injury repair good material.
There is one layer of protective tissue, commonly referred to fascia in the muscle surface of cattle, belongs to prolonging of tendon
Extending portion is divided, and its main component, as tendon, is made up of with type III collagen fiber I type.This material
Material has filamentary structure arranged in parallel, as it is shown in figure 1, the most refreshing possible as one
Through guide material and tendon repair material.
Summary of the invention
On the one hand, the present invention relates to a kind of method preparing orderly repair materials, comprising:
(1) fascia is made to process in sodium hydroxide solution；
(2) gains are made to process in detergent solution；
(3) gains are made to be dried.
In embodiments, fascia can take from mammal, and such as cattle, sheep, horse etc., excellent
Elect cattle as.Fascia is fresh fascia.
In embodiments, before step (1), remove the tissue adhered on fascia tissue,
Such as muscular tissue etc..In embodiments, fascia group is removed before being additionally included in step (1)
Fat in knitting.
In embodiments, sodium hydroxide solution can be the aqueous solution of sodium hydroxide.Hydroxide
The concentration of sodium solution can be about 2～5N, e.g., from about 2N, 3N, 4N or 5N.Step (1)
Can carry out about 4 DEG C～16 DEG C, e.g., from about 4 DEG C, 5 DEG C, 6 DEG C, 7 DEG C, 8 DEG C, 9 DEG C, 10 DEG C,
11 DEG C, 12 DEG C, 13 DEG C, 14 DEG C, 15 DEG C or 16 DEG C.Additionally, step (1) can be carried out about
30 minutes to 300 minutes, e.g., from about 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70
Minute, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 150 minutes,
300 minutes etc..In step (1), the sodium hydroxide solution that can more renew, such as change
2～4 new sodium hydroxide solutions.
In embodiments, the detergent in step (2) can be chemical decontaminants, such as, tell
Temperature-80, tween 20, TritonX-100 etc..The concentration of detergent solution can be about 1～5%,
E.g., from about 1%, 2%, 3%, 4%, 5% etc..The solution of detergent can be detergent
Aqueous solution, buffer soln etc., wherein buffer can be phosphate buffer, Tris buffer
Deng.
In embodiments, before step (3), gains are fully cleaned.Wherein,
Heat source water can be spent material is carried out；Can also with other water such as distilled water, go from
Sub-water is carried out.
In embodiments, in step (3), cold drying can be used to make gains be dried,
Such as lyophilisation etc..After gains are dried, sterilizing, such as irradiation can be carried out
Sterilizing etc..
On the other hand, the present invention provides the orderly repair materials prepared by said method, its
For linear collagen fiber.These linear collagen fiber comprise type i collagen fiber and type III collagen fiber,
It remains with the structure of natural collagen fibre, it is possible to be gathered into the bundle of different-diameter as required
Shape, it is also possible to be trimmed to arbitrary shape and length.Additionally, these linear fibres have the thinnest
The rough surface that born of the same parents attach, and there is good biocompatibility and biodegradability.And,
Kill aspect on antibacterial and virus more thorough, and rejection is less.
Another further aspect, the present invention provides the most orderly repair materials as the use of tissue renovation material
On the way.
In embodiments, the orderly repair materials of the present invention may be used for the reparation of tissue, especially
It is neural and the reparation of tendon.
The present invention, based on natural fascia, combines common detergent-treatment with NaOH process,
I.e. can obtain the linear collagen fiber of ordered arrangement at short notice with the simple technique that processes.
Antibacterial and virus can be effectively killed in NaOH process, and reduce the rejection of final products.
These linear collagen fiber maintain the structure of natural collagen fibre, can constitute as required and appoint
The meaning lamellar of length or arbitrary diameter and the pencil of length.Additionally, these linear collagen-based materials tool
There is the orderly side that neurocyte and fibroblast etc. can be guided to grow up in collagen fiber side
Tropism, be beneficial to cell adhesion rough wearing fiber surface, be applied to organic good biofacies
Capacitive and biodegradability.Therefore, the linear collagen fiber of the present invention can serve as tissue and repair
Multiple material, in particular as nerve repair material and tendon repair material.
Accompanying drawing explanation
Fig. 1 illustrates the filamentary structure arranged in parallel of natural tendon Bovis seu Bubali film.
Fig. 2 A illustrates according to outside the macroscopic view of the linear collagen fiber of exemplary embodiment of the invention
See figure, be wherein cm chi below figure, it is seen that fiber length is about 7cm, Fig. 2 B and illustrates according to this
The scanning electron microscope (SEM) photograph of the linear collagen fiber of invention illustrative embodiments.
Fig. 3 A illustrates Dorsal Root Ganglion Neurons axon growth in Nostoc commune Vanch plate
Direction, Fig. 3 B illustrates that Dorsal Root Ganglion Neurons is according to exemplary embodiment of the invention
Linear collagen fiber on axon growth direction, Fig. 3 C illustrates that rat cerebellar granule cell is at root
According to the growing state on the linear collagen fiber of exemplary embodiment of the invention, in the most each figure
Scale be 200 μm.
Fig. 4 A illustrates that Rat Mesenchymal Stem Cells is at the line according to exemplary embodiment of the invention
Growing state on property collagen fiber, Fig. 4 B illustrates that human fibroblasts is according to example of the present invention
Growing state on the linear collagen fiber of property embodiment, the scale in the most each figure is 200
μm。
Detailed description of the invention
The close structure of natural fascia, composition is complex, is not suitable for attaching and the growth of cell,
And have the risk causing immunity of organism to repel.
The present invention combination by sodium hydroxide ablation and detergent-treatment, remove in fascia is thin
Born of the same parents' composition also retains extracellular matrix, obtain having ordered structure, good biocompatibility and
The linear collagen fiber of good biodegradability.And, this linear collagen fiber are carefully
It is more thorough that bacterium and virus kill aspect, and rejection is less.
The inventors discovered that, sodium hydroxide and detergent all can play cell free effect, but
Intensity is different.The process of detergent such as tween 80, TritonX-100 is the softest, the most not
Destroy natural structure and the composition of extracellular matrix, but the time of process is longer, and one can be lost
Albumen in part extracellular matrix.And the effect of NaOH is the most violent, but owing to comparing
Readily washing off, do not have residue problem on the final product, tissue and cell to repairing do not have
Toxicity.The antigenicity of fascia can be preferably removed in two kinds of combinations processed, and retain its I type and
Type III collagen fiber, main reservation type i collagen fiber.Compared with the process of independent detergent, NaOH
Formerly process can effectively kill antibacterial and virus, and reduce the rejection of final products.
Owing to NaOH is with low cost, and there is the effect above so that the product of the present invention is better than tradition side
The product that method obtains.Specifically, the present invention provides a kind of method preparing orderly repair materials,
Comprising:
(1) fascia is made to process in sodium hydroxide solution；
(2) gains are made to process in detergent solution；
(3) gains are made to be dried.
Fascia can be taken from mammal, the such as fresh fascia of cattle, sheep, horse etc..Due to
Source is more convenient and inexpensive, and the material obtained after processing does not has immunogenicity, therefore this
Bright repair materials can be widely used in clinic.
The sodium hydroxide solution used is the aqueous solution of sodium hydroxide, and concentration can be about
2～5N, e.g., from about 2N, 3N, 4N or 5N.When the concentration of sodium hydroxide is less than 2N, de-
Cell effect is bad, and when concentration is more than 5N, has the physical property of collagen-based materials necessarily
Destruction.Step (1) can be carried out in a low temperature of about 4 DEG C～16 DEG C, preferably from about 10 DEG C.
Additionally, step (1) can be carried out 30～300 minutes, and the most more renew
Sodium hydroxide solution, such as change 2～4 new sodium hydroxide solutions.During the process extended
Between and the renewal of solution treatment effect can be made more preferable, the most preferably remove cell component.
In step (2), the selection of detergent to consider that its intensity and its residual are to gained material
The impact of material application.After tested, tween 80, tween 20, TritonX-100 can be used in this
In invention, and there is good effect.And, skilled person will appreciate that, these are several goes
Dirty agent can replace use.The concentration of detergent is in the range of about 1～5%, and the too low meeting of concentration makes
Treatment effect is deteriorated, and excessive concentration can affect the performance of material.Additionally, the process of detergent
Time and temperature suitably can be determined by those skilled in the art.
In step (3), can use cold drying that gains are dried.Cryogenic vacuum
Being dried and can remove the moisture in gains and solution residual, intact holding fascia remains after processing
The structure of part.Cold drying can be done in freezing by the method for such as lyophilisation
Dry instrument is carried out.
If the gains of the inventive method medical application to be used for, need gains are filled
Distinguish and wash, and carry out sterilizing.Wherein it is possible to spend heat source water, material is carried out；
Can also be carried out and radiation sterilizing with other water such as distilled water, deionized water.Wherein,
Removing heat source water is the water without heat source substance.The preparation method removing heat source water is people in the art
Member is known, obtains as processed water etc. by methods such as ultrafilter membrane, reverse osmosis, chromatographies.
The orderly repair materials using the inventive method to prepare is linear collagen fiber.This line
Property collagen fiber remain with the structure of natural collagen fibre, can the most arbitrarily be gathered into not
Pencil with diameter, it is also possible to be trimmed to random length, is conveniently used for arbitrary size and any portion
The tissue repair of position.
The linear collagen fiber of the present invention also have directivity.This directivity is for nerve fiber
Growth with tendon fibroblasts is even more important.Wherein, the direction of nerve fiber (aixs cylinder)
Property be the accurate and effective basis of transmitting Nerve impulse, and the one-tenth fiber of the known tendon of those skilled in the art
Cell is longitudinal arrangement.The linear collagen fiber of the present invention not only give nerve fiber and tendon fibroblast
The growth of dimension cell provides to be supported, it is also possible to is used in combination with medicine etc. and promotes two kinds of cells
Growth.
It addition, the orderly repair materials of the present invention has good biocompatibility.The present invention's
Repair materials is made up of I type and type III natural collagen protein in order, predominantly I-type collagen,
There is no immunogenicity, therefore may apply in tissue repair.In the spinal cord of damage, this
Bright collagen-based materials can overlap the both sides of Spinal Cord, and the growth for aixs cylinder provides support, and
And neuranagenesis can be promoted in conjunction with neurotrophic factor or medicine etc..And nerve fiber of the present invention
Rough surface, the beneficially attaching of cell.Additionally, it turned out, the unique biological of tendon
Mechanical property is mainly due to the height sense of organization of Tenocyte cell epimatrix, and the present invention's is orderly
The type i collagen comprised in repair materials can induce the formation of Tenocyte cell epimatrix to increase
410%.
It is currently used for the collagen biomaterial that tissue injury repairs, mainly prepares from the molten collagen of acid,
Complex process, step is more, and main based on unordered tubulose and collagen gel.This
Bright processing method, based on the natural fascia of body, utilizes unique process technique, exploitation
Going out can be used for spinal cord and the new material of tendon injury reparation, it has excellent compatibility to cell,
Immunogenicity is low.The more important thing is, this material can make neuron or tendon regeneration kind careful
Born of the same parents along the direction ordering growth of collagen fiber, this for tendon and spinal cord injury reparation very
Important.Therefore, the orderly repair materials of the present invention has good potential applicability in clinical practice.
The specific embodiment of the present invention presented below, the embodiment merely illustrative explanation present invention, and
It does not limit the scope of the invention.
Preparation example. the preparation of linear collagen fiber
1. take the muscular tissue of the fresh Adult Bovine with white fascia, with 10 DEG C of deionized water punchings
Wash 3 times；
2. isolate fascia with tweezers and scalpel, remove the muscular tissue that internal layer adheres to as far as possible；
3. after the muscle vascular tissue of the fat of outer layer, attachment being removed, water-soluble with sodium hydroxide
Liquid processes (molar concentration of sodium hydroxide, temperature and process time see below table)；
Sodium hydroxide solution (molar concentration, temperature and process time and the previous step the most more renewed
Keep consistent), this step number of repetition is shown in below table；
5. with detergent solution (below table is shown in by the kind of detergent, concentration and solvent) 16
Process 60 minutes at DEG C；
6. spend heat source water material is fully cleaned；
7. material is used freeze drier lyophilizing.
With reference to below table, carry out the preparation of linear collagen fiber according to above-mentioned steps.
Use the muscular tissue from adult sheep, preparation example 14～26 executed as described above in step 1；
Use the muscular tissue from mature horses, preparation example 27～36 executed as described above in step 1.
Prepare comparative example. only use detergent to prepare linear collagen fiber
1. take the muscular tissue of the fresh Adult Bovine with white fascia, with 10 DEG C of deionized water punchings
Wash 3 times；
2. isolate fascia with tweezers and scalpel, remove the muscular tissue that internal layer adheres to as far as possible；
3. after the muscle vascular tissue of the fat of outer layer, attachment being removed, with the tween 20 of 5%
Aqueous solution processes 60 minutes at 16 DEG C；
4. spend heat source water material is fully cleaned；
5. material is used freeze drier lyophilizing.
Obtain comparing sample 1.
The morphologic observation of the linear collagen fiber of embodiment 1.
To each preparation example with prepare the sample that comparative example obtains and carry out morphologic observation.It will be seen that
Each preparation example and prepare the linear collagen fiber obtained in comparative example and all can be gathered into pencil,
As shown in Figure 2 A, its length and pencil diameter can arbitrarily regulate, and present directivity.
Afterwards, observe preparation example by scanning electron microscope and prepare the collagen fiber of comparative example, Qi Zhongtu
2B illustrates the wherein structure of of preparation example 13 collagen fiber of the present invention, finds wire collagen
Fiber has ordered structure, and its coarse surface texture is conducive to the attaching of cell.
The endotoxin assay of the linear collagen fiber of embodiment 2.
Although preparation example and prepare the sample that comparative example obtains and morphologically do not have a difference, but system
It is more thorough that the product that standby example obtains kills aspect on antibacterial and virus, and rejection is less.?
In the lixiviating solution of each preparation example sample, and endotoxin content < 0.5EU/ml, and comparative example will prepared
Sample processes in the lixiviating solution obtained equally, endotoxin content > 2.5EU/ml.
Endotoxin detection method:
The preparation of lixiviating solution: use injection water to press 6cm2/ ml ratio extraction material 24 hours, surveys
The endotoxin content of examination lixiviating solution.
1, the tachypleus amebocyte lysate selecting required specification (0.1ml) and sensitivity (0.25EU/ml) is (profound
This biological company limited of Jiang'an degree), bacterial endotoxin standard substance (study by Chinese food drug assay
Institute, lot number 250601-201174) and baterial endotoxin test water (2ml/ props up, and endotoxin contains
Amount is less than 0.003EU/ml, Zhanjiang Andusi Biology Co., Ltd.).
2, take endotoxin standard (dried frozen aquatic products) 1, be diluted to desired concn with after water dissolution
Standby.
3, tachypleus amebocyte lysate 8 is taken, 2 flag test sample inspection pipes, 2 flag negative control pipes, 2
Flag positive control pipe, 2 flag test sample positive control pipes.
4, negative control pipe adds 0.2ml inspection water, and remaining each pipe adds 0.1ml and checks use
Water；Every test sample inspection pipe separately adds 0.1ml sample；It is dense that positive control pipe adds 0.1ml
Degree is the endotoxin standard solution of 2 λ；It is 2 λ that test sample positive control pipe adds 0.1ml concentration
Endotoxic sample.
5, close the mouth of pipe, shake up gently, vertically put in 37 ± 1 DEG C of thermostats and hatch 60 ± 2
Minute, then observed result.Test tube should avoid any vibrations during hatching.
6, test tube is taken out from thermostat gently, slowly during reversing 180 °, if being formed in pipe
Gel, and gel is indeformable, is not positive from tube wall slippage person, be recorded as (+)；Do not formed
The gel of gel or formation is the most solid, deformation be feminine gender from tube wall slippage person, be recorded as (-).
Insulation and test tube process of taking should be avoided vibrated causing false negative result.
7, negative control pipe must be negative, and positive control pipe, test sample positive control pipe are necessary
Being positive, otherwise experimental result is invalid.
The growth on linear collagen fiber of embodiment 3. neuron
Separate the dorsal root ganglion of SD rat, be seeded in common plate or preparation example of the present invention respectively
On the linear collagen fiber of 13.
Specifically, under anatomic microscope, expose canalis spinalis and the intervertebral foramina of E15 tire Mus, use tweezer
The neuroganglion of related for spinal cord both sides is taken out by son together, extracts neuroganglion one by one with microforceps.With
Trypsin Sigma, the SH3004201B of 0.25% (vol/vol), is dissolved in PBS)
37 DEG C of digestion 30min, terminate reaction with about 2ml serum (invitrogen, 10099141).
300 eye mesh screens filter, and 1000rpm is centrifuged 5min and collects cell.PBS (pH 7.4) cleans
After one time, centrifugal collect, with appropriate DMEM/F12 complete culture solution (invitrogen,
11330032) blow and beat into single cell suspension, be seeded in common plate or preparation example of the present invention respectively
On the linear collagen fiber of 13.Be placed on after inoculation CO2 gas incubator (brand and model:
HERAcell 150i) and middle cultivation (37 DEG C, 5%CO2)。
After inoculating 7 days, examine under a microscope the growing state of dorsal root ganglion ball.Such as Fig. 3 A
Shown in, in common plate, the aixs cylinder four of dorsal root ganglion ball dissipates distribution, and linear in the present invention
On collagen fiber, the aixs cylinder of dorsal root ganglion is along the direction ordered distribution of collagen fiber, such as figure
Shown in 3B.
Hereafter, prepared as described below go out rat cerebellar granule cell.The SD rat being born one week,
Prolong seam in skull and open skull, separate cerebellum and be placed in cold PBS (pH 7.4).Brain will be separated
Meninges and the blood vessel exfoliation of tissue are clean.Shred cerebral tissue to 1mm3Fritter.By shred
Cerebral tissue be transferred to 2mL 0.25% (vol/vol) pancreatin (Sigma, SH3004201B,
It is dissolved in PBS) in, 37 DEG C digest 15 minutes, add 100 microlitre pancreatin inhibitors
(invitrogen, 17075-029) terminates digestion.The DMEM/F12 of 4mL is added after 5 minutes
1:1 basal medium, dispels tissue with glass dropper, in transfer supernatant to new centrifuge tube, to
The fresh basal medium adding 4mL in remaining precipitate continues piping and druming, after transfer supernatant again
Piping and druming is once.The all cleer and peaceful precipitation collected is combined, with the filter membrane of 40 μm (BD,
352340) filter.Liquid after filter is centrifuged 5 minutes in 500g, removes supernatant, will precipitation
With neuronal cell cultures base (DMEM-F12 containing 2%B27) (B27, invitrogen,
17504044；DMEM-12, invitrogen, 11330032) resuspended, it is seeded to after counting add
Added with preparation example 13 of the present invention linear collagen fiber be coated with poly-D-lysine (sigma,
P0899) on culture dish.
Specifically, by the rat cerebellar granule cell of gained with 5 × 104/mm3Density inoculation
On the linear collagen fiber of the present invention.CO2 gas incubator (brand and type it is placed on after inoculation
Number: cultivate (37 DEG C, 5%CO2) in HERAcell 150i).
After 2 days, it can be seen that neuronal cell grows along the machine direction of linear collagen fiber,
Present good order, as shown in Figure 3 C.
Embodiment 4. Rat Mesenchymal Stem Cells and human fibroblasts life on linear collagen fiber
Long
Prepared as described below go out Rat Mesenchymal Stem Cells.Choose the SD of 4 weeks after birth big
Mus, dislocation put to death, 75% alcohol-pickled 5min, aseptically take out bilateral tibial,
Femur, then removes two ends epiphysis and soft tissue, after exposing medullary cavity, serum-free
L-DMEM culture fluid (HYCLONE, SH3002101B), as flushing liquor, uses aseptic note
Emitter rinses bone marrow repeatedly to transparent, then the flushing liquor of gained is loaded 15mL and is centrifuged
Pipe；1000rpm is centrifuged 5min, abandons supernatant.Cultivate with 10mL serum-free L-DMEM
Liquid re-suspended cell, 200 mesh filter screens are filtered into unicellular.Use blood counting chamber counts, cell
With 1 × 106/ mL is inoculated in the L-DMEM culture medium containing 10% serum.Cell is placed in 5%
CO2Concentration, cultivates in 37 DEG C of incubators.24h half amount changes liquid, and 48h full dose changes liquid,
The most every replacing carrying out culture fluid for 3 days.When cell proliferation and being gradually fused to 90%,
Pancreatin with 0.25% carries out had digestive transfer culture, then with 1 × 106The density of/mL, in culture dish
Pass on, amplification cultivation.
Prepare human fibroblasts as follows.People's prepuce tissues PBS solution is rinsed three times,
Wash away the hemocyte of tissue surface attachment；Prepuce tissues is placed in the culture dish of PBS, with eye
Section cuts off the subcutaneous tissue allowancing for bark the attachment of skin inner surface；Prepuce tissues is placed in new filling again
In the 3.5cm culture dish of PBS, with eye scissors, tissue is cut into 1mm3Piece of tissue；1000
Rpm is centrifuged 2min；With 1ml DMEM culture medium (GIBCO, 11330032) resuspended tissue
Block, is transferred in T25 culture bottle (Corning, 430639), with Pasteur pipe (Fisher,
13-678-20A) piece of tissue is made to be uniformly distributed, and the careful unnecessary culture medium of sucking-off；T25 is trained
Support bottle to be inverted, be positioned in 37 DEG C of incubators；After about 20～24h, T25 culture bottle is overturn,
Add 1～1.5ml culture medium；The next day change liquid, after about 3～7 days, visible cell is around piece of tissue
Climb out of；Can carry out after about 10～14 days passing on (0.25%TE digestion).
Take the good rat MSC cell (P3 generation) of growth conditions or human fibroblasts (P5
Generation), after 0.25% trypsinization, about 1ml DMEM culture medium will be resuspended in after cell centrifugation
In (GIBCO, 11330032), it is inoculated on the linear collagen fiber of preparation example 13.It is placed in 37
DEG C, 5%CO2Incubator is placed 4～5h, adds appropriate DMEM culture medium, to flood
Linear collagen fiber are advisable.Afterwards, continue to cultivate in incubator.
After 2 days, can examine under a microscope, Rat Mesenchymal Stem Cells (Fig. 4 A) and
Human fibroblasts (Fig. 4 B) is good along material fiber direction on the linear collagen fiber of the present invention
Good growth, as shown in Figure 4.
Claims (13)
1. the method preparing orderly repair materials, including:
(1) fascia is made to process in sodium hydroxide solution；
(2) gains are made to process in detergent solution；
(3) gains are made to be dried.
Method the most according to claim 1, wherein, described fascia is taken from selected from cattle, horse
Mammal with sheep.
Method the most according to claim 1, wherein, described sodium hydroxide solution is concentration
It it is the sodium hydrate aqueous solution of 2～5N.
Method the most according to claim 1, wherein, step (1) is entered at 4 DEG C～16 DEG C
OK.
Method the most according to claim 1, wherein, step (1) carries out 30～300 points
Clock.
Method the most according to claim 1, wherein, described detergent solution is for selected from telling
Temperature-80, tween 20, the aqueous solution of TritonX-100 or buffer soln.
Method the most according to claim 6, wherein, described buffer delays selected from phosphate
Rush liquid and Tris buffer.
Method the most according to claim 1, wherein, the concentration of described detergent solution is
1～5%.
9. according to the method according to any one of claim 1～8, wherein, step (3) it
Before, gains are fully cleaned.
10. according to the method according to any one of claim 1～8, wherein, in step (3)
Afterwards, gains are carried out sterilizing.
11. 1 kinds of orderly repair materials, by the method system according to any one of claim 1～10
For obtaining, it is linear collagen fiber.
12. orderly repair materials according to claim 11 are as the use of tissue renovation material
On the way.
13. purposes according to claim 12, wherein, described tissue renovation material includes
Nerve repair material and tendon repair material etc..
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109771699A (en) * | 2017-11-10 | 2019-05-21 | 中国科学院苏州纳米技术与纳米仿生研究所 | Functionalization nerve regneration collagen scaffold, its preparation method and application |
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| CN1751747A (en) * | 2005-09-07 | 2006-03-29 | 中国科学院遗传与发育生物学研究所 | A kind of bioprosthetic material and preparation method thereof and application |
| CN1775189A (en) * | 2005-11-30 | 2006-05-24 | 烟台正海生物技术有限公司 | Acellular dermal matrix and its preparing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109771699A (en) * | 2017-11-10 | 2019-05-21 | 中国科学院苏州纳米技术与纳米仿生研究所 | Functionalization nerve regneration collagen scaffold, its preparation method and application |
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