CN109621011A - A kind of tendon Biological Repair mesh sheet and application thereof and preparation method - Google Patents
A kind of tendon Biological Repair mesh sheet and application thereof and preparation method Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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- A61L2430/00—Materials or treatment for tissue regeneration
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Abstract
The present invention discloses a kind of tendon Biological Repair mesh sheet and preparation method thereof, and using animal tissue as raw material, the immunizing compositions such as removal cell, DNA are twisted line after being cut into thin strips shape, are woven into sticking patch the tendon biological sticking patch.Xenogenesis takes off cell material and obtains bigger intensity at line by twisting to less be easily broken off, and because there is intertexture between line and line after weaving, the external force for stretching tearing is not acting on a point but on multiple intertwined points, therefore there is good mechanical property, solve the disadvantage that xenogenesis takes off cell material tendon biological sticking patch mechanical strength difference and needs introducing crosslinked agent or synthetic material, and the structure of the mesh sheet is made by weaving, the size in mesh sheet aperture can be adjusted by braided parameter, meet the needs of practical application to reach.Meanwhile the tendon biological sticking patch remains the ingredients such as original three-dimensional structure, non-collagen and growth factor in extracellular matrix, the functional reconstruction for promoting tendon and the healing after tendon repair.
Description
Technical field
The present invention relates to technical field of biological material, and in particular to a kind of tendon Biological Repair mesh sheet and application thereof and preparation side
Method.
Background technique
Tendon is the strand or membranaceous dense connective tissue at belly of muscle both ends, is adhered to convenient for muscle and fixed.Tendon is a kind of
The strength that muscle generates can be passed to bone, cause the movement of limbs by viscoelasticity tissue.The biology performance of tendon is certain
The convergent force and sports achievement of muscle are affected in degree.Movement or disease lead to tendon injury, if not giving repairing in time normal
It will lead to limbs disturbance.For the tendon for having defect to damage, the method clinically used is broadly divided into synthetic material sticking patch
With biomaterial sticking patch two major classes.
1. synthetic material sticking patch
The made tendon sticking patch main component of synthetic material is heteropolymer, including two class of degradable and non-degradable.It is non-can
Degradation-type synthetic material sticking patch tensile strength is good, can provide stable mechanics guarantee for tendon-bone interface, but because cannot be by group
Degradation is knitted, postoperative rejection is easily caused, the structural intergrity after retaining in vivo for a long time is destroyed, and can migrate to other groups
It knits, causes chronic inflammation and foreign body reaction, need to carry out revision procedure.And degradable synthetic material sticking patch, it is usually used
The synthesis such as polylactic acid, although mechanical property is fine, causes acute inflammatory reaction, followed by chronic inflammation, most after implanting
End form is at granulation tissue, fiber package;And high concentration lactic acid, hydroxyl that the materials such as polylactic acid are partially formed in degradation process
Acetic acid easily causes cytotoxicity.
Synthetic material mechanical strength is preferable, but biological property is poor, and is unable to inducing tissue regeneration and healing.Therefore synthesis is mended
Piece developing direction is imitation biochemistry, i.e., using different weaving methods and addition natural biologic material such as collagen, fiber egg
It is white etc., the tendon-bone healing tissue characteristics that are connect with bone of simulation tendon, so as to enhance in tendon healing potential, and then raising flesh
The success rate of tendon injury prosthesis.
2. biomaterial sticking patch
It is de- can be divided into autologous tissue's material, allohisto compatibility's material and xenogenesis mostly from organization material for biomaterial sticking patch
Cell material etc..
Autologous tissue's material is mostly derived from the tissue such as self fascia late, tendon of long head of biceps brachii, and advantage is have
Good biological attribute, does not cause body inflammatory to react, can be to bringing additional wound, shadow self when maximum disadvantage is to draw materials
Ring stability of joint etc..
Allogenic material is mostly derived from the product of human skin dermal tissue, although they, which have, promotes tendon repair
The ability of tendon-bone interface healing afterwards, but there are sources to lack, infectivity disease (such as AIDS) risk, so using
It is subject to certain restrictions.
Xenogenesis takes off cell material and is mainly derived from the tissue such as corium, small intestine, pericardium of animal, is by cell, DNA
The processing of equal immunizing compositions, so that is obtained remains with the material of original three-dimensional structure and collagenous fibres ingredient in extracellular matrix
Material.The ingredients such as three-dimensional structure, collagen, non-collagen and growth factor in extracellular matrix be host cell stick,
Proliferation, differentiation provide the environment adapted to, facilitate the functional reconstruction of the tissues such as muscle and tendon, and then promote tendon repair
Tendon-bone interface healing afterwards.
Cell material sticking patch is taken off in practical applications because of its own mechanical strength deficiency, needs to introduce ring in its process
The chemical cross-linking agents such as oxide or glutaraldehyde, or shared with synthetic material, it has had the disadvantage in that potential cytotoxicity, has dropped
It is slower to solve rate, is mismatched with regeneration, can lead to the reaction such as fibrosis, chronic inflammation.Application No. is
The Chinese patent of 201710862130.X, right-angled intersection ligament reproducibility implantation material and the preparation method and application thereof, although reaching
The mechanical requirements of tendon repair operation, but there is guidance tissue again by degradable high polymer and fibrinogen etc.
After raw biomaterial is blended, it is prepared using electrospinning technology.Application No. is the China of 201310203598.X specially
Benefit, a kind of artificial biological tendon and preparation method thereof, only simply curling or superposition, the active force very little between each layer,
Probably because the case where being layered and the inhomogenous and each layer of stress layer caused to rupture respectively.
For this purpose, on the basis of not introducing synthetic material or crosslinking agent, in order to enhance the mechanics of tendon biology patching material
Intensity, the present invention come into being.
Summary of the invention
It is an object of the invention to be directed to shortcoming of the biological sticking patch in the prior art in structural strength, one is provided
Kind tendon Biological Repair mesh sheet.
For this purpose, tendon Biological Repair mesh sheet provided by the invention, wherein the mesh sheet is compiled by xenogenesis acellular matrix material
It knits.
Further, the xenogenesis acellular matrix material by xenogenesis acellular matrix by cleaning slitting, inactivation of virus,
Degreasing, removes DNA and removes α-Gal antigen, sizing, is lyophilized de- cell.
Further, the acellular matrix includes but is not limited to the submucous layer of small intestine of mammal, mucous membrane of urinary bladder
One or more combinations of lower layer, submucous lamina of stomach, dermal matrix, pericardium, meninx, amnion, internal organs film, peritonaeum.
Further, the acellular matrix is the submucous layer of small intestine of animal.
Invention further provides the repairing healing purposes that a kind of above-mentioned tendon Biological Repair mesh sheet is damaged in tendon tissue.
The present invention also provides a kind of preparation methods of tendon Biological Repair mesh sheet, comprising:
(1) it pre-processes,
The animal tissue's cleaning for taking fresh slaughtered animals is placed in acetum immersion, strikes off the mucous membrane for removing animal tissue
Layer, muscle layer, placenta percreta, lymph node isolate submucosa, longitudinal to be uniformly cut into thin strips shape, and are rinsed with purified water, obtain flesh
Tendon biological material for repairing;
(2) inactivation of virus,
The mixed aqueous solution that tendon biological material for repairing is used to Peracetic acid and ethyl alcohol impregnates under room temperature in ultrasound, into
Row inactivation of virus is simultaneously cleaned by ultrasonic with purified water;
(3) degreasing,
Using ethanol solution, impregnates under ultrasound, normal temperature condition, be cleaned by ultrasonic later using water for injection;
(4) cell is taken off, DNA is removed and removes α-Gal antigen,
Using containing trypsase and containing the mixed aqueous solution of EDTA, impregnates under ultrasound condition, be cleaned by ultrasonic later using PBS;
Using the aqueous solution of the enzyme containing DNA, impregnated under ultrasound condition;Later using PBS drift ultrasonic cleaning;
Using the aqueous solution containing alpha-galactosidase, impregnated under ultrasound condition;It is cleaned by ultrasonic later using PBS;
Using NaOH aqueous solution, under ultrasound condition, PBS ultrasonic cleaning is used after soak at room temperature until neutral;
(5) it is formed, is lyophilized, braiding, sterilizing
By treated, fine strip shape submucosa is twisted line, is fixed on mold, after freeze-drying, is woven into the tendon of mesh sheet shape
Biological Repair mesh sheet, PET packaging bag carry out irradiation sterilization after packing.
Further, in pre-treatment step, the concentration of acetic acid is 0.01%-0.5%, soaking time 10-120min, is moved
The ratio of object tissue and acetum is 1:2-1:10.
Further, in viral inaction steps, the concentration of Peracetic acid is 0.5-1.5%, and the concentration of ethyl alcohol is 15-
25%, the ratio of tendon biological material for repairing and mixed aqueous solution is 1:2-1:10, soaking time 30-120min.
Further, in defatting step, the concentration of ethyl alcohol is 90-100%, the ratio of tendon biological material for repairing and ethyl alcohol
Example is 1:2-1:10, and the soak at room temperature time is 0.5-12h.
Further, in de- cell, remove DNA and go in α-Gal antigen step:
The content of trypsase and EDTA are respectively 0.01-0.10% and 0.01- 0.05% in mixed aqueous solution, and tendon biology is repaired
Material and trypsase/EDTA solution ratio are mended as 1:2-1:10, under ultrasound condition, impregnates 15- under the conditions of 36 ± 2 DEG C
40min;
In aqueous solution containing DNA enzymatic the content of DNA enzymatic be 0.05-10U/ml, tendon biological material for repairing with containing the water-soluble of DNA enzymatic
The ratio of liquid is 1:2-1:10, and soaking temperature is 36 ± 2 DEG C, soaking time 15-40min;
The content of alpha-galactosidase is 0.05-10U/ml, tendon biological material for repairing in aqueous solution containing alpha-galactosidase
Ratio with alpha-galactoside enzyme solutions is 1:2-1:10, and soaking temperature is 20-37 DEG C, soaking time 15-40min;
The concentration of NaOH aqueous solution is 5-40mM, and the ratio of tendon biological material for repairing and NaOH solution is 1:5-1:50, room temperature
Soaking time is 20-60min.
Compared with prior art, the present invention have following remarkable advantage and the utility model has the advantages that
The technical problem to be solved by the present invention is to be directed to the shortcoming of tendon biological sticking patch, a kind of novel tendon biology is provided
Repair mesh.
1. tendon Biological Repair mesh sheet provided by the invention is organized with corium, small intestine, pericardium of animal etc. as raw material,
The immunizing compositions such as cell, DNA are removed, line is twisted after being cut into thin strips shape, is woven into mesh sheet.Xenogenesis take off cell material by twisting at
Line obtains bigger mechanical strength to less be easily broken off, and is woven into after the sticking patch of mesh sheet shape because between line and line
There is intertexture, the external force for stretching tearing is not acting on a point but therefore has good mechanical property on multiple intertwined points
Can, solve the disadvantage that xenogenesis takes off cell material tendon biological sticking patch mechanical strength difference, and the structure of the mesh sheet is by weaving
Technique is made, and the size in mesh sheet aperture can be easy to adjust by braided parameter, meet the needs of practical application to reach.
2. tendon Biological Repair mesh sheet provided by the invention remains original three-dimensional structure, collagen in extracellular matrix
The ingredients such as fiber, non-collagen and growth factor have promoting healing effect, accelerate functional reconstruction and the tendon repairing of tendon
Postoperative healing.
3. tendon Biological Repair mesh sheet provided by the invention, ECM three-dimensional structure tool inducing cell and blood vessel grow into function,
It itself can gradually degrade while new tissue is grown into, the polypeptide moiety of catabolite has anti-microbial property, after can reducing implantation
The incidence of inflammation and infection.
4. tendon Biological Repair mesh sheet provided by the invention, introducing crosslinked agent and synthetic material, will not have potential
Cytotoxicity less will lead to the reaction such as fibrosis, chronic inflammation.
5. promoting healing can be added according to clinical demand in tendon Biological Repair mesh sheet provided by the invention during the preparation process
Substance or antibiotic can also load promoting healing substance or antibiotic by immersion way before implanting, thus into one
Step promotes Wound healing and reduces infection rate.
Detailed description of the invention
Fig. 1 is the pictorial diagram of tendon Biological Repair mesh sheet.
Fig. 2 is the structural schematic diagram of tendon Biological Repair mesh sheet.
Fig. 3 is that tendon Biological Repair mesh sheet is sliced HE colored graph.
Fig. 4 is the microgram of tendon Biological Repair mesh sheet.
Fig. 5 is the preparation flow figure of tendon Biological Repair mesh sheet.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
Embodiment 1
The preparation of trees-Osima jacoti, Osima excavata tendon Biological Repair mesh sheet, please refers to Fig. 5:
(1) it pre-processes
It takes the chitterlings tissue wash of fresh slaughtered animals clean, is placed in 0.5% acetum and impregnates 30min, chitterlings and acetic acid
The ratio of solution is 1:5, scrapes mucous layer, muscle layer, placenta percreta, the lymph node that division removes chitterlings jejunum, separation using physics
Submucosa out, it is longitudinal to be uniformly cut into thin strips shape, it is rinsed 3 times using purified water, obtains tendon biological material for repairing, i.e. small intestine
Submucosa, following abbreviation SIS materials.
(2) inactivation of virus
Using the mixed aqueous solution containing 1.0% Peracetic acid and 15% ethyl alcohol, the ratio of SIS material and mixed aqueous solution is 1:
10, under ultrasound condition, soaking at room temperature 100min is virus inactivated.It is cleaned by ultrasonic 3 times using purified water later.
(3) degreasing
The ethyl alcohol for the use of concentration being 95%, the ratio of SIS material and ethyl alcohol are 1:10, under ultrasound condition, soak at room temperature 2h.Later
It is cleaned by ultrasonic 3 times using water for injection.
(4) cell is taken off, DNA is removed and removes α-Gal antigen
Using containing 0.02% trypsase and containing the mixed aqueous solution of 0.02%EDTA, SIS material and trypsase/EDTA solution
Ratio be 1:5, under ultrasound condition, impregnate 30min under the conditions of 37 DEG C.It is cleaned by ultrasonic 3 times using PBS later.
Using the aqueous solution of the enzyme of DNA containing 5U/ml, the ratio of SIS material and DNA enzymatic solution is 1:5, under ultrasound condition,
20min is impregnated under the conditions of 37 DEG C.Later using PBS drift ultrasonic cleaning 3 times.
Using the aqueous solution of the alpha-galactosidase containing 5U/ml, the ratio of SIS material and alpha-galactoside enzyme solutions is 1:
5, under ultrasound condition, 20min is impregnated under the conditions of 30 DEG C.It is cleaned by ultrasonic 3 times using PBS later.
The NaOH aqueous solution for the use of concentration being 25mM, the ratio of SIS material and NaOH solution are 1:20, under ultrasound condition,
Soak at room temperature 50min.Later using PBS ultrasonic cleaning until neutral.
(5) it is formed, is lyophilized, braiding, sterilizing
By treated, band-like fine strip shape submucosa is twisted line, is fixed on mold, after freeze-drying, is woven by braider
At the tendon Biological Repair mesh sheet of the mesh sheet shape in Fig. 1, irradiation sterilization, tendon Biological Repair net are carried out after PET packaging bag packaging
The schematic diagram of piece is referring to figure 2..
Embodiment 2
The preparation of trees-Osima jacoti, Osima excavata tendon Biological Repair mesh sheet
(1) it pre-processes
It takes the chitterlings tissue wash of fresh slaughtered animals clean, is placed in 0.01% acetum and impregnates 120min, chitterlings and vinegar
The ratio of acid solution is 1:10, scrapes mucous layer, muscle layer, placenta percreta, the lymph node that division removes chitterlings jejunum using physics, point
Submucosa is separated out, segment is cut into, is rinsed 3 times using purified water.
(2) inactivation of virus
Using the mixed aqueous solution containing 0.5% Peracetic acid and 25% ethyl alcohol, the ratio of SIS material and mixed aqueous solution is 1:
15, under ultrasound condition, soaking at room temperature 120min is virus inactivated.It is cleaned by ultrasonic 3 times using purified water later.
(3) degreasing
The ethyl alcohol for the use of concentration being 90%, the ratio of SIS material and ethyl alcohol are 1:15, under ultrasound condition, soak at room temperature 4h.Later
It is cleaned by ultrasonic 3 times using water for injection.
(4) cell is taken off, DNA is removed and removes α-Gal antigen
Using containing 0.05% trypsase and containing the mixed aqueous solution of 0.01%EDTA, SIS material and trypsase/EDTA solution
Ratio be 1:10, under ultrasound condition, impregnate 20min under the conditions of 37 DEG C.It is cleaned by ultrasonic 3 times using PBS later.
Using the aqueous solution of the enzyme of DNA containing 1U/ml, the ratio of SIS material and DNA enzymatic solution is 1:10, under ultrasound condition,
30min is impregnated under the conditions of 37 DEG C.Later using PBS drift ultrasonic cleaning 3 times.
Using the aqueous solution of the alpha-galactosidase containing 1U/ml, the ratio of SIS material and alpha-galactoside enzyme solutions is 1:
10, under ultrasound condition, 30min is impregnated under the conditions of 30 DEG C.It is cleaned by ultrasonic 3 times using PBS later.
The NaOH aqueous solution for the use of concentration being 40mM, the ratio of SIS material and NaOH solution are 1:10, under ultrasound condition,
Soak at room temperature 30min.Later using PBS ultrasonic cleaning until neutral.
(5) it is formed, is lyophilized, braiding, sterilizing
By treated, fine strip shape submucous layer of small intestine is twisted line, is fixed on mold, after freeze-drying, is woven by braider
The tendon Biological Repair mesh sheet of networking sheet, PET packaging bag carry out irradiation sterilization after packing.
Embodiment 3
For the safety of sample, immunogenic substance detection is carried out to the sample that embodiment 1-2 is prepared.
(1) cell residue quantity measuring method: being fixed, paraffin embedding with 10% neutral formalin, is cut into 0.4 micron thin
Piece, through dimethylbenzene dewaxing, serial dehydration of alcohol, hematoxylin-eosin stains, microscopically observation cell residue situation and matrix
Fibre structure.
(2) DNA content detection method: according to YY/T 0606.25-2014, " animal derived biomaterial DNA residual is measured
Determine method: fluorescence colour " it is detected.
(3) α-Gal antigenic content detection method: after sample is fixed with paraformaldehyde, routine paraffin wax embedded section, piece thickness
3 microns.Immunohistochemical reaction is carried out using the special affinity characteristic of biotin labeling BSI-B4 and α-Gal antigen.Coloration result
Determine: dark brown yellow particle is strong positive (+++), and brown yellow granule is positive (++), and yellow particle is weakly positive (+), not
See and is colored as negative (-).
(4) lipid content detection method: referring to soxhlet extraction methods in " fatty measurement in 5009.6 food of GB/T " into
Row measurement.
As a result it see the table below:
Embodiment 4
Biology performance, histology, bacterial endotoxin and anti-microbial property inspection are carried out to the sample that embodiment 1-2 is prepared
It surveys.
(1) biology performance detects
Method: it is tested referring to GB/T16886 series methods.
As a result: cell-cytotoxic reaction is 1 grade;Without delayed allergy;Intradermal reaction shows test specimen and solvent
The difference of mean score is compareed less than 1.0;Without pyrogenicity;Without hemolytic reaction;Genetic toxicity test is the results show that mouse typhus sramana
Salmonella back mutation (Ames) test is negative, mouse lymphoma assay is negative, dye-free body distortion property;Without urgency
Property systemic toxicity reaction;Without sub- chronic generalized toxicity;Muscular grafting 30 days, 60 days, 90 days tissues with negative controls
Reaction is without significant difference.
(2) histology
1) optical microphotograph sem observation
Method: being fixed, paraffin embedding with 10% neutral formalin, is cut into 0.4 micron of thin slice, through dimethylbenzene dewaxing, series wine
Essence dehydration, hematoxylin-eosin stains, microscopically observation cell residue situation and matrix fiber structure.
As a result: cell-free and cell fragment residual;Collagenous fibres are continuous, no fracture, as shown in Figure 3.
2) Ultrastructural observation
Method: it is scanned using electron scanning mirror.
As a result: material porous structure, collagenous fibres are without fracture, as shown in Figure 4.
(3) detection of bacterial endotoxin
Method: it is detected referring to correlation technique in GB/T 14233.
As a result: being respectively less than 2.15EU/ part.
(4) anti-microbial property detects
Sample obtained by Examples 1 and 2 is ground respectively in the hydrochloric acid of 0.01M with grinding rod, until it is visible by naked eyes particle,
And adjusting its concentration is 100mg/10mL.Pepsin digestion is added, pepsin: sample ratio is 1:10.Continue at 25 DEG C
Stir 48h, after be cooled to 4 DEG C, the 0.1 M sodium hydroxide that 1/10 volume is added adjusts PH to 7.2-7.4.
Hybrid NC machine tool base plate is prepared, picks them separately a little cultured staphylococcus aureus and large intestine with oese
Bacteria suspension is made in sterile saline 5ml in bacillus inclined-plane culture substratess.It takes bacteria suspension 1.0ml and above-mentioned is degraded
Sample 1ml is added in the culture dish of sterilizing-drying, and addition is cooled to 50 DEG C or so of ordinary nutritional broth agar culture medium,
It shakes up, spare after sufficiently condensing, 35~37 DEG C of inversions are cultivated 24 hours, observe bacterial growth situation;Increase simultaneously and does not have to
Anti-biotic material and 5 μ g/mL antibacterial peptides of addition compare, and as a result see the table below:
Embodiment 5
Mechanics properties testing is carried out to the embodiment 1-2 sample being prepared and without the sample 1-2 of cutting and braiding.
(1) suture strength
Method: with the non-absorbing suture of 3-0 in sample both sides center far from being sutured at edge 2mm, by the suture other end and sample
The other end be separately fixed on tensiometer both ends, stretched with the speed of 20mm/min, until stitch points are torn, record
Maximal force.
(2) tensile strength
Method: it is 10mm shape that sample is cut to width respectively in both directions;In relative humidity 40%-60%, temperature after cutting
It is tested after being placed 2 hours in 22 ± 2 DEG C of environment of degree.Fixture spacing is 25mm, and sample both ends are fixed on cupping machine
Collet on, the maximal force with the speed tensile of 100mm/min, when record fracture.
(3) bursting strength
Method: according to the measurement method of " YY 0500-2004 cardiovascular implant artificial blood vessel " 8.3.3.2 probe rupture strength
9.5mm diameter probe is chosen to be detected.
As a result it see the table below:
Those skilled in the art can make a variety of variations to the present invention according to the above description.Thus, do not violating this hair
Under the premise of bright claim objective, certain details in embodiment should not constitute limitation of the invention, and the present invention will be with
The range that the appended claims define is as protection scope.
Claims (10)
1. a kind of tendon Biological Repair mesh sheet and application thereof and preparation method, it is characterised in that: the mesh sheet takes off cell by xenogenesis
Host material weaves.
2. tendon Biological Repair mesh sheet according to claim 1, it is characterised in that: the structure of the mesh sheet is by weaving
It is made, the size in mesh sheet aperture can be adjusted by braided parameter.
3. tendon Biological Repair mesh sheet according to claim 2, it is characterised in that: the acellular matrix includes but unlimited
Be formed on the submucous layer of small intestine of mammal, submucous layer of bladder, submucous lamina of stomach, dermal matrix, pericardium, meninx, amnion,
One or more combinations of internal organs film, peritonaeum.
4. tendon Biological Repair mesh sheet according to claim 1, it is characterised in that: the acellular matrix material can soak
Stain has promoting healing substance or antibiotic.
5. the repairing healing purposes that a kind of tendon Biological Repair mesh sheet as described in claim 1 ~ 4 is damaged in tendon tissue.
6. a kind of preparation method of tendon Biological Repair mesh sheet, which comprises the following steps:
(1) it pre-processes,
The animal tissue's cleaning for taking fresh slaughtered animals is placed in acetum immersion, strikes off the mucous membrane for removing animal tissue
Layer, muscle layer, placenta percreta, lymph node isolate submucosa, longitudinal to be uniformly cut into thin strips shape, and are rinsed with purified water, obtain flesh
Tendon biological material for repairing;
(2) inactivation of virus,
Tendon biological material for repairing is used into the mixed aqueous solution containing Peracetic acid and ethyl alcohol, is soaked under room temperature in ultrasound
Bubble, is virus inactivated and is cleaned by ultrasonic with purified water;
(3) degreasing,
Using ethanol solution, impregnates under ultrasound, normal temperature condition, be cleaned by ultrasonic later using water for injection;
(4) cell is taken off, DNA is removed and removes α-Gal antigen,
Using containing trypsase and containing the mixed aqueous solution of EDTA, impregnates under ultrasound condition, be cleaned by ultrasonic later using PBS;
Using the aqueous solution of the enzyme containing DNA, impregnated under ultrasound condition;Later using PBS drift ultrasonic cleaning;
Using the aqueous solution containing alpha-galactosidase, impregnated under ultrasound condition;It is cleaned by ultrasonic later using PBS;
Using NaOH aqueous solution, under ultrasound condition, PBS ultrasonic cleaning is used after soak at room temperature until neutral;
(5) it is formed, is lyophilized, braiding, sterilizing,
By treated, fine strip shape submucosa is twisted line, is fixed on mold, after freeze-drying, is woven into the tendon of mesh sheet shape
Biological Repair mesh sheet, PET packaging bag carry out irradiation sterilization after packing.
7. preparation method according to claim 6, which is characterized in that in pre-treatment step, the concentration of acetic acid is
The ratio of 0.01%-0.5%, soaking time 10-120min, animal tissue and acetum is 1:2-1:10.
8. preparation method according to claim 6, which is characterized in that in viral inaction steps, the concentration of Peracetic acid
For 0.5-1.5%, the concentration of ethyl alcohol is 15-25%, and the ratio of tendon biological material for repairing and mixed aqueous solution is 1:2-1:10,
Soaking time is 30-120min.
9. preparation method according to claim 6, which is characterized in that in defatting step, the concentration of ethyl alcohol is 90-
100%, the ratio of tendon biological material for repairing and ethyl alcohol is 1:2-1:10, and the soak at room temperature time is 0.5-12h.
10. preparation method according to claim 6, which is characterized in that in de- cell, remove DNA and go α-Gal antigen step
In:
The concentration of trypsase and EDTA are respectively 0.01-0.10% and 0.01- 0.05% in mixed aqueous solution, and tendon biology is repaired
Material and trypsase/EDTA solution ratio are mended as 1:2-1:10, under ultrasound condition, impregnates 15- under the conditions of 36 ± 2 DEG C
40min;
In aqueous solution containing DNA enzymatic the content of DNA enzymatic be 0.05-10U/ml, tendon biological material for repairing with containing the water-soluble of DNA enzymatic
The ratio of liquid is 1:2-1:10, and soaking temperature is 36 ± 2 DEG C, soaking time 15-40min;
The content of alpha-galactosidase is 0.05-10U/ml, tendon biological material for repairing in aqueous solution containing alpha-galactosidase
Ratio with alpha-galactoside enzyme solutions is 1:2-1:10, and soaking temperature is 20-37 DEG C, soaking time 15-40min;
The concentration of NaOH aqueous solution is 5-40mM, and the ratio of tendon biological material for repairing and NaOH solution is 1:5-1:50, room temperature
Soaking time is 20-60min.
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CN111407926A (en) * | 2020-03-13 | 2020-07-14 | 深圳兰度生物材料有限公司 | Biological repairing mesh and preparation method and application thereof |
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CN109453428B (en) | 2023-05-05 |
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