CN110141685A - A kind of vagina host material and preparation method thereof - Google Patents
A kind of vagina host material and preparation method thereof 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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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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
- A61L27/3687—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 characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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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
- A61L27/3691—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 characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/3641—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 characterised by the site of application in the body
- A61L27/3679—Hollow organs, e.g. bladder, esophagus, urether, uterus, intestine
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Abstract
The invention discloses a kind of preparation methods of vagina host material, include the following steps: the collection and sterilizing of step S1 raw material, the first processing of step S2 raw material, and step S3 takes off cell processing, and step S4 is cleaned, and step S5 is antibacterial modified.De-cell liquid used in de- cell treatment process is by each ingredient of following mass percent by being mixed evenly to prepare: aminotrimethylenephosphonic acid ethyl glycol ether surfactant 0.3%-0.5%, water-soluble ultrabranching polyaminoacid 0.1%-0.3%, leupeptin 0.1%, surplus is distilled water.The invention also discloses the vagina host materials being prepared according to the preparation method of the vagina host material.Vagina host material disclosed by the invention possesses the biological integrity of natural vaginal periplast, to vagina tissue missing, try to get to the heart of a matter damage and degenerative it is significant in efficacy, antibacterial anti-infection excellent performance, use is safe.
Description
Technical field
The present invention relates to medical material tech fields more particularly to a kind of vagina host material and preparation method thereof.
Background technique
The geneogenous absence of vagina of deformity occurs for ankylocolpos can be generally found in gender development to be in deformity or MRKH comprehensive
Simulator sickness (absence of vagina syndrome), congenital absence of uterus etc., such as AIS (the insensitivity syndrome of androgen) or other internal organs
Development occur abnormal etc., will lead to vagina tissue missing or try to get to the heart of a matter damage and degenerative, these diseases will cause pelvic cavity
Organ prolapse is the common illness of gynemetrics.While these illness bring pain to patient's body, also caused to patient huge
Big spirit and psychological pressure seriously affects the Health and Living quality of middle aged and aged women.Using effective measures to both
Illness carries out targetedly treatment and has very important significance.
At this stage, more to vagina tissue missing, the mode that damage and degenerative implementation are treated of trying to get to the heart of a matter, most common one
Kind therapeutic modality is to be rebuild using organizational project medical patch to vagina.Acellular matrix material is common organizational project
Medical patch, it is to handle host material by the method for physics, chemistry and biology, carries out de- cell processing, except decorrelation
Antigen after the material that obtains.It eliminates the cell component in natural material, remains matrix components, effectively reduces natural
The immunogenicity of material, while the basic structure and performance that are able to maintain host material are (such as macroscopical braiding structure, microscopic void knot
Structure, physical mechanical strength etc..It is applied can overcome traditional artificial reconstruction of vagina damage human body itself group in elytroplasty (colpoplasty
The shortcomings that knitting is a kind of potential method for rebuilding vagina.
There is also some problems in use for acellular matrix material.It is found that either long-term or short-term
It is retained in the intracorporal biomaterial of people, can often cause various bacterium infections, these infection once occur, even if using normal dose
The drug of hundreds of times can not be treated effectively, and the immune system of human body itself can not remove these bacteriums.Secondly, this material
There is also the risks of degradation in use.
The concentration of extracellular matrix made of the method that existing patent CN1284265A is recorded, lye is high, immersion when
Between it is long, treatment temperature is high, causes Fiber Digestion, occurs collapsing and void layer, this will cause cell when products application that cannot grow into,
The use of finished product is impacted.Its concentration of lye is low, soaking time is short, and treatment temperature is low, causes cell de- unclean, draws
The immune response for playing matrix causes product to use failure.
Therefore, a kind of bioactivity, biomechanical property are developed more preferably, to vagina tissue missing, try to get to the heart of a matter damage and function
It degenerates significant in efficacy, the vagina host material of antibacterial anti-infection excellent performance has very important significance.
Summary of the invention
In order to overcome the defects of the prior art, the present invention provides a kind of vagina host material and preparation method thereof, the system
Preparation Method is simple and easy, and preparation cost is cheap；It is original that the vagina host material being prepared remains tissue extracelluar matrix
Basic supporting structure, major biochemical ingredient and biomechanical strength；Having effectively removed in animal tissue can cause human body to be exempted from
The antigen of epidemic disease rejection；Improve the flexibility, drapability and wound curved surface integration performance of periplast, the de- cell of preparation
Host material is close with human vagina, possesses the biological integrity of natural vaginal periplast, to vagina tissue missing, damage of trying to get to the heart of a matter
Wound and degenerative are significant in efficacy, antibacterial anti-infection excellent performance, and use is safe.
To achieve the above object of the invention, the technical solution adopted by the present invention is that, a kind of preparation method of vagina host material,
Include the following steps:
The collection and sterilizing of step S1 raw material: the vaginal wall organization material of clip in Reconstructive pelvic is taken, is shredded, clearly
It washes, is filtered dry, reach germ-free condition using gamma Rays disinfection；
The first processing of step S2 raw material: the organization material being prepared by step S1 is added to water or physiological saline
In, it is homogenized, centrifugation, abandons supernatant, it is rear to use biological buffer 3-5 times, it is filtered dry, obtains lower-hierarchy；
Step S3 takes off cell processing: the lower-hierarchy being prepared by step S2 being placed in de-cell liquid, in double frequency
The de- cell processing of ultrasound, Frequency range are 20-40KHz in ultrasound environments, and low frequency handles 5-10min；Higher frequency is 70-
90KHz, high-frequency therapeutic treatment 10-20min, after at 35-40 DEG C concussion processing 10-15 days, be filtered dry, obtain take off cell tissue；
Step S4 cleaning: by the de- cell tissue being prepared by step S3 using cleaning solution in supersonic cleaning machine 18-
Carried out at 22 DEG C cleaning 3-5 times, it is 10-15 minutes each, after 3-5 times wash with distilled water, then obtained material is placed in
It, finally will be after freeze-drying in -65~-85 DEG C of freeze-drying 30-40h, and in 0 DEG C of vacuum drying 20-30h in freeze drier
Acellular matrix is placed in be saved backup at room temperature；
Step S5 is antibacterial modified: the acellular matrix material that will be prepared by step S4, at 50~80 DEG C, is added
PBS buffer solution handles 30~50min；Then modifying agent is added, handles 4~8 hours；Waste reaction solution is discarded, cleans, is filtered dry, then
- 80 DEG C of freeze-drying 30-40h in freeze drier are placed in, vagina host material is obtained；The modifying agent is by H- histidyl--essence ammonia
Sour (CAS:77369-21-2) is made with epoxychloropropane.
Further, shredding described in step S1 to shred to size is (1-3) cm × (0.5-1) cm.
Further, biological buffer described in step S2 is DIPSO buffer and Tris buffer 1:(3- in mass ratio
5) it mixes.
Further, the de-cell liquid is by each ingredient of following mass percent by being mixed evenly to prepare: amino front three
Pitch phosphonic acid base glycol ether based surfactants 0.3%-0.5%, water-soluble ultrabranching polyaminoacid 0.1%-0.3%, bright peptide
Element 0.1%, surplus is distilled water.
Preferably, the preparation method of the aminotrimethylenephosphonic acid ethyl glycol ether surfactant, including walk as follows
It is rapid:
I aminotrimethylenephosphonic acid, 3- chlorine-1,2-propylene glycol oleate are added in organic solvent, stir at 30-40 DEG C
Reaction 4-6 hours, solvent is evaporated off in back spin, and is washed 3-5 times with ether, then rotates removing ether, obtains ethyleneamino three
Methylenephosphonic acid；
II three methylenephosphonic acid of ethyleneamino, the 3- propyl- 2- alkene acyloxy propane -1- sulphur that will be prepared by step I
Sour sodium, 2- ethyleneoxy ethyl alcohol, initiator are added in tetrahydrofuran, are flowed back at nitrogen or 65-75 DEG C of atmosphere of inert gases
It is stirred to react, tetrahydrofuran is evaporated off in back spin.
Preferably, aminotrimethylenephosphonic acid described in step I, the quality of 3- chlorine-1,2-propylene glycol oleate, organic solvent
Than for 1:1.25:(7-10).
Preferably, the organic solvent is selected from least one of ether, acetone, methylene chloride, ethyl acetate.
Preferably, the methylenephosphonic acid of ethyleneamino three described in step II, 3- propyl- 2- alkene acyloxy propane -1- sodium sulfonate,
2- ethyleneoxy ethyl alcohol, initiator, tetrahydrofuran mass ratio be 0.2:1:2:(0.02-0.04): (10-15).
Preferably, the initiator is at least one of azodiisobutyronitrile, azobisisoheptonitrile；The inert gas
Selected from one of helium, neon, argon gas.
Further, lower-hierarchy described in step S3, de-cell liquid mass ratio be 1:(20-40).
Preferably, the temperature range of the de- cell processing of the ultrasound is 23-33 DEG C；Ultrasonic power is 5000W.
Preferably, oscillation frequency described in step S3 is 100-150r/min.
Preferably, the cleaning solution is PBS solution.
Preferably, acellular matrix material described in step S5, PBS buffer solution, modifying agent mass ratio be 1:(20-
50):0.1。
Preferably, the preparation method of the modifying agent includes the following steps: H- histidyl--arginine, epoxy chloropropionate
Alkane, basic catalyst are added in n,N-Dimethylformamide, are stirred to react at 30-50 DEG C 6-8 hours, after be filtered to remove not
Molten object, then rotate and remove n,N-Dimethylformamide and unreacted reactant, obtain modifying agent.
Preferably, the H- histidyl--arginine, epoxychloropropane, basic catalyst, n,N-Dimethylformamide matter
Amount is than being 1:1.78:(0.3-0.5): (10-15).
Preferably, the basic catalyst is selected from least one of sodium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide.
Further, a kind of vagina host material is made according to a kind of above-mentioned preparation method of vagina host material.
The beneficial effects of adopting the technical scheme are that
(1) vagina host material provided by the invention, preparation process is simple, and raw material is easy to get, and preparation cost is cheap, to equipment
It is of less demanding with reaction condition, it is able to satisfy large-scale production demand, Clinical practicability is strong.
(2) vagina host material provided by the invention remains the original basic supporting structure of tissue extracelluar matrix, master
Want biochemical composition and biomechanical strength；The anti-of human immunity rejection can be caused by having effectively removed in animal tissue
It is former；Improve the flexibility, drapability and wound curved surface integration performance of periplast, the same human body of acellular matrix material of preparation
Vagina is close, possesses the biological integrity of natural vaginal periplast, to vagina tissue missing, try to get to the heart of a matter damage and degenerative treatment
Effect is significant, antibacterial anti-infection excellent performance, and use is safe.
(3) vagina host material provided by the invention can be with de- cell since modifier molecules contain multiple epoxy groups
Amino in host material etc. generates multiple spot and combines, and forms the crosslinking of higher degree, to reduce the anti-of acellular matrix material
Originality improves its physical mechanical property and degradation resistance；Aminotrimethylenephosphonic acid ethyl glycol ether is added in de-cell liquid
Surfactant, water-soluble ultrabranching polyaminoacid, leupeptin, synergistic effect can not only make cell removing abundant, moreover it is possible to inhibit
The activity of dispase in subtractive process, guarantees the integrality of host material.
Specific embodiment
In order to make those skilled in the art more fully understand technical solution of the present invention, and make features described above of the invention,
Purpose and advantage are more clear understandable, and the present invention will be further explained with reference to the examples below.Embodiment is only used for
It is bright the present invention rather than limit the scope of the invention.
Wherein, the raw material being related in embodiment is commercially available.
Embodiment 1
A kind of preparation method of vagina host material, includes the following steps:
The collection and sterilizing of step S1 raw material: the vaginal wall organization material of clip in Reconstructive pelvic is taken, is shredded, clearly
It washes, is filtered dry, reach germ-free condition using gamma Rays disinfection；Described shred to shred to size is 1cm × 0.5cm；
The first processing of step S2 raw material: the organization material being prepared by step S1 is added to water or physiological saline
In, it is homogenized, centrifugation, abandons supernatant, it is rear to use biological buffer 3 times, it is filtered dry, obtains lower-hierarchy；The biological buffer is DIPSO
Buffer is mixed with Tris buffer 1:3 in mass ratio
Step S3 takes off cell processing: the lower-hierarchy being prepared by step S2 being placed in de-cell liquid, in double frequency
The de- cell processing of ultrasound, Frequency range are 20KHz in ultrasound environments, and low frequency handles 5min；Higher frequency is 70KHz, high
Frequency processing 10min, after at 35 DEG C concussion processing 10 days, be filtered dry, obtain de- cell tissue；The lower-hierarchy, de-cell liquid
Mass ratio be 1:20；The temperature range of the de- cell processing of ultrasound is 23 DEG C；Ultrasonic power is 5000W；The concussion frequency
Rate is 100r/min；
Step S4 cleaning: by the de- cell tissue being prepared by step S3 using PBS solution in supersonic cleaning machine 18
Carried out at DEG C cleaning 3 times, 10 minutes every time, after 3 times wash with distilled water, obtained material is then placed in freeze drier
In, in -65 DEG C of freeze-drying 30h, and in 0 DEG C of vacuum drying 20h, finally the acellular matrix after freeze-drying is placed in and is protected at room temperature
It deposits spare；
Step S5 is antibacterial modified: the acellular matrix material that will be prepared by step S4, and at 50 DEG C, PBS is added
Buffer handles 30min；Then modifying agent is added, handles 4 hours；Waste reaction solution is discarded, cleans, is filtered dry, then is placed in freezing and does
- 80 DEG C of freeze-drying 30h, obtain vagina host material in dry machine；The modifying agent is by H- histidyl--arginine and epoxychloropropane
It is made；The acellular matrix material, PBS buffer solution, modifying agent mass ratio be 1:20:0.1；
The de-cell liquid is by each ingredient of following mass percent by being mixed evenly to prepare: aminotrimethylenephosphonic acid base second
Glycol ethers based surfactants 0.3%, water-soluble ultrabranching polyaminoacid 0.1%, leupeptin 0.1%, surplus are distilled water.
The preparation method of the aminotrimethylenephosphonic acid ethyl glycol ether surfactant, includes the following steps:
I aminotrimethylenephosphonic acid 1kg, 3- chlorine-1,2-propylene glycol oleate 1.25kg is added in ether 7kg, at 30 DEG C
Under be stirred to react 4 hours, ether is evaporated off in back spin, and is washed 3 times with ether, then rotate removing ether, obtains ethyleneamino
Three methylenephosphonic acids；
II three methylenephosphonic acid 0.2kg, 3- propyl- 2- alkene acyloxy propane of ethyleneamino-that will be prepared by step I
1- sodium sulfonate 1kg, 2- ethyleneoxy ethyl alcohol 2kg, azodiisobutyronitrile 0.02kg are added in tetrahydrofuran 10kg, in nitrogen atmosphere
It encloses reflux at 65 DEG C to be stirred to react, tetrahydrofuran is evaporated off in back spin.
The preparation method of the modifying agent includes the following steps: H- histidyl--arginine 1kg, epoxychloropropane
1.78kg, sodium carbonate 0.3kg are added in n,N-Dimethylformamide 10kg, are stirred to react at 30 DEG C 6 hours, rear mistake filters out
Insoluble matter is removed, then rotates and removes n,N-Dimethylformamide and unreacted reactant, obtains modifying agent.
A kind of vagina host material is made according to a kind of above-mentioned preparation method of vagina host material.
Embodiment 2
A kind of preparation method of vagina host material, includes the following steps:
The collection and sterilizing of step S1 raw material: the vaginal wall organization material of clip in Reconstructive pelvic is taken, is shredded, clearly
It washes, is filtered dry, reach germ-free condition using gamma Rays disinfection；It is described shred for shred to size be 1.5cm ×
0.6cm；
The first processing of step S2 raw material: the organization material being prepared by step S1 is added to water or physiological saline
In, it is homogenized, centrifugation, abandons supernatant, it is rear to use biological buffer 4 times, it is filtered dry, obtains lower-hierarchy；The biological buffer is DIPSO
Buffer is mixed with Tris buffer 1:3.5 in mass ratio；
Step S3 takes off cell processing: the lower-hierarchy being prepared by step S2 being placed in de-cell liquid, in double frequency
The de- cell processing of ultrasound, Frequency range are 25KHz in ultrasound environments, and low frequency handles 6min；Higher frequency is 75KHz, high
Frequency processing 12min, after at 36 DEG C concussion processing 11 days, be filtered dry, obtain de- cell tissue；The lower-hierarchy, de-cell liquid
Mass ratio be 1:25；The temperature range of the de- cell processing of ultrasound is 25 DEG C；Ultrasonic power is 5000W；The concussion frequency
Rate is 115r/min.
Step S4 cleaning: by the de- cell tissue being prepared by step S3 using PBS solution in supersonic cleaning machine 19
Carried out at DEG C cleaning 4 times, 11 minutes every time, after 4 times wash with distilled water, obtained material is then placed in freeze drier
In, in -70 DEG C of freeze-drying 32h, and in 0 DEG C of vacuum drying 22h, finally the acellular matrix after freeze-drying is placed in and is protected at room temperature
It deposits spare；
Step S5 is antibacterial modified: the acellular matrix material that will be prepared by step S4, and at 60 DEG C, PBS is added
Buffer handles 35min；Then modifying agent is added, handles 5 hours；Waste reaction solution is discarded, cleans, is filtered dry, then is placed in freezing and does
- 80 DEG C of freeze-drying 32h, obtain vagina host material in dry machine；The modifying agent is by H- histidyl--arginine and epoxychloropropane
It is made；The acellular matrix material, PBS buffer solution, modifying agent mass ratio be 1:35:0.1；
The de-cell liquid is by each ingredient of following mass percent by being mixed evenly to prepare: aminotrimethylenephosphonic acid base second
Glycol ethers based surfactants 0.35%, water-soluble ultrabranching polyaminoacid 0.15%, leupeptin 0.1%, surplus are distilled water.
The preparation method of the aminotrimethylenephosphonic acid ethyl glycol ether surfactant, includes the following steps:
I aminotrimethylenephosphonic acid 1kg, 3- chlorine-1,2-propylene glycol oleate 1.25kg is added in acetone 8kg, at 33 DEG C
Under be stirred to react 4.5 hours, acetone is evaporated off in back spin, and is washed 3 times with ether, then rotate removing ether, obtains vinyl ammonia
Three methylenephosphonic acid of base；
II three methylenephosphonic acid 0.2kg, 3- propyl- 2- alkene acyloxy propane of ethyleneamino-that will be prepared by step I
1- sodium sulfonate 1kg, 2- ethyleneoxy ethyl alcohol 2kg, azobisisoheptonitrile 0.025kg are added in tetrahydrofuran 12kg, in helium
It flows back and is stirred to react at 68 DEG C of atmosphere, tetrahydrofuran is evaporated off in back spin.
The preparation method of the modifying agent includes the following steps: H- histidyl--arginine 1kg, epoxychloropropane
1.78kg, sodium carbonate 0.35kg are added in n,N-Dimethylformamide 12kg, are stirred to react at 35 DEG C 6.5 hours, rear mistake
Insoluble matter is filtered out, then rotates and removes n,N-Dimethylformamide and unreacted reactant, obtains modifying agent.
A kind of vagina host material is made according to a kind of above-mentioned preparation method of vagina host material.
Embodiment 3
A kind of preparation method of vagina host material, includes the following steps:
The collection and sterilizing of step S1 raw material: the vaginal wall organization material of clip in Reconstructive pelvic is taken, is shredded, clearly
It washes, is filtered dry, reach germ-free condition using gamma Rays disinfection；Described shred to shred to size is 2cm × 0.8cm；
The first processing of step S2 raw material: the organization material being prepared by step S1 is added to water or physiological saline
In, it is homogenized, centrifugation, abandons supernatant, it is rear to use biological buffer 4 times, it is filtered dry, obtains lower-hierarchy；The biological buffer is DIPSO
Buffer is mixed with Tris buffer 1:4 in mass ratio；
Step S3 takes off cell processing: the lower-hierarchy being prepared by step S2 being placed in de-cell liquid, in double frequency
The de- cell processing of ultrasound, Frequency range are 30KHz in ultrasound environments, and low frequency handles 8min；Higher frequency is 80KHz, high
Frequency processing 15min, after at 38 DEG C concussion processing 13 days, be filtered dry, obtain de- cell tissue；The lower-hierarchy, de-cell liquid
Mass ratio be 1:30；The temperature range of the de- cell processing of ultrasound is 27 DEG C；Ultrasonic power is 5000W；The concussion frequency
Rate is 130r/min；
Step S4 cleaning: by the de- cell tissue being prepared by step S3 using PBS solution in supersonic cleaning machine 20
Carried out at DEG C cleaning 4 times, 13 minutes every time, after 4 times wash with distilled water, obtained material is then placed in freeze drier
In, in -75 DEG C of freeze-drying 35h, and in 0 DEG C of vacuum drying 25h, finally the acellular matrix after freeze-drying is placed in and is protected at room temperature
It deposits spare；
Step S5 is antibacterial modified: the acellular matrix material that will be prepared by step S4, and at 70 DEG C, PBS is added
Buffer handles 40min；Then modifying agent is added, handles 6 hours；Waste reaction solution is discarded, cleans, is filtered dry, then is placed in freezing and does
- 80 DEG C of freeze-drying 35h, obtain vagina host material in dry machine；The modifying agent is by H- histidyl--arginine and epoxychloropropane
It is made；The acellular matrix material, PBS buffer solution, modifying agent mass ratio be 1:35:0.1.
The de-cell liquid is by each ingredient of following mass percent by being mixed evenly to prepare: aminotrimethylenephosphonic acid base second
Glycol ethers based surfactants 0.4%, water-soluble ultrabranching polyaminoacid 0.2%, leupeptin 0.1%, surplus are distilled water.
The preparation method of the aminotrimethylenephosphonic acid ethyl glycol ether surfactant, includes the following steps:
I aminotrimethylenephosphonic acid 1kg, 3- chlorine-1,2-propylene glycol oleate 1.25kg is added in methylene chloride 8.5kg,
It is stirred to react at 35 DEG C 5 hours, solvent is evaporated off in back spin, and is washed 4 times with ether, then rotate removing ether, obtains ethylene
Base aminotrimethylenephosphonic acid；
II three methylenephosphonic acid 0.2kg, 3- propyl- 2- alkene acyloxy propane of ethyleneamino-that will be prepared by step I
1- sodium sulfonate 1kg, 2- ethyleneoxy ethyl alcohol 2kg, azobisisoheptonitrile 0.03kg are added in tetrahydrofuran 13kg, in neon atmosphere
It encloses reflux at 69 DEG C to be stirred to react, tetrahydrofuran is evaporated off in back spin.
The preparation method of the modifying agent includes the following steps: H- histidyl--arginine 1kg, epoxychloropropane
1.78kg, sodium hydroxide 0.4kg are added in n,N-Dimethylformamide 13kg, are stirred to react at 40 DEG C 7 hours, rear to filter
Insoluble matter is removed, then rotates and removes n,N-Dimethylformamide and unreacted reactant, obtains modifying agent.
A kind of vagina host material is made according to a kind of above-mentioned preparation method of vagina host material.
Embodiment 4
A kind of preparation method of vagina host material, includes the following steps:
The collection and sterilizing of step S1 raw material: the vaginal wall organization material of clip in Reconstructive pelvic is taken, is shredded, clearly
It washes, is filtered dry, reach germ-free condition using gamma Rays disinfection；It is described shred for shred to size be 2.5cm ×
0.8cm；
The first processing of step S2 raw material: the organization material being prepared by step S1 is added to water or physiological saline
In, it is homogenized, centrifugation, abandons supernatant, it is rear to use biological buffer 5 times, it is filtered dry, obtains lower-hierarchy；The biological buffer is DIPSO
Buffer is mixed with Tris buffer 1:4.5 in mass ratio；
Step S3 takes off cell processing: the lower-hierarchy being prepared by step S2 being placed in de-cell liquid, in double frequency
The de- cell processing of ultrasound, Frequency range are 35KHz in ultrasound environments, and low frequency handles 9min；Higher frequency is 85KHz, high
Frequency processing 18min, after at 39 DEG C concussion processing 14 days, be filtered dry, obtain de- cell tissue；The lower-hierarchy, de-cell liquid
Mass ratio be 1:38；The temperature range of the de- cell processing of ultrasound is 31 DEG C；Ultrasonic power is 5000W；The concussion frequency
Rate is 140r/min；
Step S4 cleaning: by the de- cell tissue being prepared by step S3 using PBS solution in supersonic cleaning machine 21
Carried out at DEG C cleaning 5 times, 14.5 minutes every time, after 5 times wash with distilled water, obtained material is then placed in freeze-drying
In machine, in -80 DEG C of freeze-drying 39h, and in 0 DEG C of vacuum drying 29h, finally the acellular matrix after freeze-drying is placed at room temperature
It saves backup；
Step S5 is antibacterial modified: the acellular matrix material that will be prepared by step S4, and at 75 DEG C, PBS is added
Buffer handles 45min；Then modifying agent is added, handles 7.5 hours；Waste reaction solution is discarded, cleans, is filtered dry, then be placed in freezing
- 80 DEG C of freeze-drying 39h, obtain vagina host material in drying machine；The modifying agent is by H- histidyl--arginine and epoxy chloropropionate
Alkane is made；The acellular matrix material, PBS buffer solution, modifying agent mass ratio be 1:48:0.1.
The de-cell liquid is by each ingredient of following mass percent by being mixed evenly to prepare: aminotrimethylenephosphonic acid base second
Glycol ethers based surfactants 0.45%, water-soluble ultrabranching polyaminoacid 0.25%, leupeptin 0.1%, surplus are distilled water.
The preparation method of the aminotrimethylenephosphonic acid ethyl glycol ether surfactant, includes the following steps:
I aminotrimethylenephosphonic acid 1kg, 3- chlorine-1,2-propylene glycol oleate 1.25kg is added in ethyl acetate 9.5kg,
It is stirred to react at 39 DEG C 5.5 hours, ethyl acetate is evaporated off in back spin, and is washed 5 times with ether, then rotate removing ether, obtains
To three methylenephosphonic acid of ethyleneamino；
II three methylenephosphonic acid 0.2kg, 3- propyl- 2- alkene acyloxy propane of ethyleneamino-that will be prepared by step I
1- sodium sulfonate 1kg, 2- ethyleneoxy ethyl alcohol 2kg, initiator 0.035kg are added in tetrahydrofuran 14.5kg, in argon atmosphere
Reflux is stirred to react at 74 DEG C, and tetrahydrofuran is evaporated off in back spin；The initiator is azodiisobutyronitrile, azo two is different
Heptonitrile 3:5 in mass ratio is mixed.
The preparation method of the modifying agent includes the following steps: H- histidyl--arginine 1kg, epoxychloropropane
1.78kg, basic catalyst 0.48kg are added in n,N-Dimethylformamide 14.5kg, and it is small to be stirred to react 7.8 at 48 DEG C
When, after be filtered to remove insoluble matter, then rotate and remove n,N-Dimethylformamide and unreacted reactant, obtain modifying agent；Institute
Basic catalyst is stated to mix for sodium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide 1:2:4:3 in mass ratio.
A kind of vagina host material is made according to a kind of above-mentioned preparation method of vagina host material.
Embodiment 5
A kind of preparation method of vagina host material, includes the following steps:
The collection and sterilizing of step S1 raw material: the vaginal wall organization material of clip in Reconstructive pelvic is taken, is shredded, clearly
It washes, is filtered dry, reach germ-free condition using gamma Rays disinfection；Described shred to shred to size is 3cm × 1cm；
The first processing of step S2 raw material: the organization material being prepared by step S1 is added to water or physiological saline
In, it is homogenized, centrifugation, abandons supernatant, it is rear to use biological buffer 5 times, it is filtered dry, obtains lower-hierarchy；The biological buffer is DIPSO
Buffer is mixed with Tris buffer 1:5 in mass ratio；
Step S3 takes off cell processing: the lower-hierarchy being prepared by step S2 being placed in de-cell liquid, in double frequency
The de- cell processing of ultrasound, Frequency range are 40KHz in ultrasound environments, and low frequency handles 10min；Higher frequency is 90KHz, high
Frequency processing 20min, after at 40 DEG C concussion processing 15 days, be filtered dry, obtain de- cell tissue；The lower-hierarchy, de-cell liquid
Mass ratio be 1:40；The temperature range of the de- cell processing of ultrasound is 33 DEG C；Ultrasonic power is 5000W；The concussion frequency
Rate is 150r/min；
Step S4 cleaning: by the de- cell tissue being prepared by step S3 using PBS solution in supersonic cleaning machine 22
Carried out at DEG C cleaning 5 times, 15 minutes every time, after 5 times wash with distilled water, obtained material is then placed in freeze drier
In, in -85 DEG C of freeze-drying 40h, and in 0 DEG C of vacuum drying 30h, finally the acellular matrix after freeze-drying is placed in and is protected at room temperature
It deposits spare；
Step S5 is antibacterial modified: the acellular matrix material that will be prepared by step S4, and at 80 DEG C, PBS is added
Buffer handles 50min；Then modifying agent is added, handles 8 hours；Waste reaction solution is discarded, cleans, is filtered dry, then is placed in freezing and does
- 80 DEG C of freeze-drying 40h, obtain vagina host material in dry machine；The modifying agent is by H- histidyl--arginine and epoxychloropropane
It is made；The acellular matrix material, PBS buffer solution, modifying agent mass ratio be 1:50:0.1.
The de-cell liquid is by each ingredient of following mass percent by being mixed evenly to prepare: aminotrimethylenephosphonic acid base second
Glycol ethers based surfactants 0.5%, water-soluble ultrabranching polyaminoacid 0.3%, leupeptin 0.1%, surplus are distilled water.
The preparation method of the aminotrimethylenephosphonic acid ethyl glycol ether surfactant, includes the following steps:
I aminotrimethylenephosphonic acid 1kg, 3- chlorine-1,2-propylene glycol oleate 1.25kg is added in ethyl acetate 10kg,
It is stirred to react at 40 DEG C 6 hours, solvent is evaporated off in back spin, and is washed 5 times with ether, then rotate removing ether, obtains vinyl
Aminotrimethylenephosphonic acid；
II three methylenephosphonic acid 0.2kg, 3- propyl- 2- alkene acyloxy propane of ethyleneamino-that will be prepared by step I
1- sodium sulfonate 1kg, 2- ethyleneoxy ethyl alcohol 2kg, azodiisobutyronitrile 0.04kg are added in tetrahydrofuran 15kg, in nitrogen atmosphere
It encloses reflux at 75 DEG C to be stirred to react, tetrahydrofuran is evaporated off in back spin.
The preparation method of the modifying agent includes the following steps: H- histidyl--arginine 1kg, epoxychloropropane
1.78kg, potassium hydroxide 0.5kg are added in n,N-Dimethylformamide 15kg, are stirred to react at 50 DEG C 8 hours, rear to filter
Insoluble matter is removed, then rotates and removes n,N-Dimethylformamide and unreacted reactant, obtains modifying agent.
A kind of vagina host material is made according to a kind of above-mentioned preparation method of vagina host material.
Comparative example 1
A kind of vagina host material, according to the preparation method system of Chinese invention patent CN201510584734.3 embodiment 1
It is standby to form.
Comparative example 2
A kind of vagina host material, preparation method and formula are substantially the same manner as Example 1, the difference is that the de-cell liquid
It is middle to substitute aminotrimethylenephosphonic acid ethyl glycol ether surfactant with Triton X-100.
Comparative example 3
A kind of vagina host material, preparation method and formula are substantially the same manner as Example 1, the difference is that the de-cell liquid
In without addition water-soluble ultrabranching polyaminoacid.
Comparative example 4
A kind of vagina host material, preparation method and formula are substantially the same manner as Example 1, the difference is that without carrying out antibacterial
It is modified.
1-5 of the embodiment of the present invention and comparative example 1-4 products obtained therefrom are tested for the property, test method is as follows:
(1) residual cell detects: by the routine hematoxylin-eosin stains (× 200) of the vagina host material in each example and
DAPI (× 200) dyeing, the results showed that, cell-free knot in vagina host material described in embodiment 1-5, comparative example 1,3,4
Structure exists；With the presence of a little eucaryotic cell structure in vagina host material described in comparative example 2, it is seen that aminotrimethylenephosphonic acid base second two
Alcohol ether based surfactants to de- cell effect more preferably.
(2) residual DNA detects: by clip in the vagina host material and untreated Reconstructive pelvic in each example
Vaginal wall organization material is through DNA detection reagent and detection, the results showed that, the present invention
The method of embodiment 1-5 can significantly reduce nucleic acid content, further prove that the method for 1-5 of the embodiment of the present invention can be complete
Full removal cell.
(3) tensile strength detects: using (compression) testing machine is stretched, by material cutting sample into strips, in phase after cutting
To humidity 40%-60%, temperature is tested immediately after placing 2 hours under conditions of being 22 DEG C ± 2 DEG C.Sample both ends are fixed
On the collet of cupping machine, is successively stretched out with the speed of 100mm/min until sample fracture, recorded as unit of N
Power when lower sample fracture, embodiment 1-5 and comparative example 1-4 test result be followed successively by 210N, 220N, 225N, 232N, 240N,
180N,200N,206N,190N.As a result as it can be seen that vagina host material mechanical property of the present invention more preferably, and antibacterial modified is conducive to
Improve material mechanical performance.
(4) biology performance detect: detection project include pyrogen, cytotoxicity, delayed allergy, intradermal reaction,
Acute systemic toxicity, Salmonella reversion test, mouse lymphoma cell mutant test, chromosome aberration, implantation, subchronic toxicity, as a result
Show biology performance of the embodiment of the present invention more preferably.
(5) anti-microbial property detects: the material in each example is subjected to antibiotic property detection, antibiotic rate is followed successively by 98.4%,
99.0%, 99.5%, 99.7%, 100%, 90.1%, 97.0%, 96.8%, 91.0%, it is seen then that by antibacterial modified, this hair
Material anti-microbial property in bright is more preferably.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (10)
1. a kind of preparation method of vagina host material, which comprises the steps of:
The collection and sterilizing of step S1 raw material: taking the vaginal wall organization material of clip in Reconstructive pelvic, shred, and cleans, filter
It is dry, germ-free condition is reached using gamma Rays disinfection；
The first processing of step S2 raw material: the organization material being prepared by step S1 is added in water or physiological saline, even
Supernatant is abandoned in slurry, centrifugation, rear to use biological buffer 3-5 times, is filtered dry, is obtained lower-hierarchy；
Step S3 takes off cell processing: the lower-hierarchy being prepared by step S2 being placed in de-cell liquid, in double-frequency ultrasound
The de- cell processing of ultrasound, Frequency range are 20-40KHz in environment, and low frequency handles 5-10min；Higher frequency is 70-
90KHz, high-frequency therapeutic treatment 10-20min, after at 35-40 DEG C concussion processing 10-15 days, be filtered dry, obtain take off cell tissue；
Step S4 cleaning: by the de- cell tissue being prepared by step S3 using cleaning solution at 18-22 DEG C of supersonic cleaning machine
Under carry out cleaning 3-5 times, it is 10-15 minutes each, after 3-5 times wash with distilled water, obtained material is then placed in freezing
It, finally will be de- thin after freeze-drying in -65~-85 DEG C of freeze-drying 30-40h, and in 0 DEG C of vacuum drying 20-30h in drying machine
Cytoplasmic matrix is placed in be saved backup at room temperature；
Step S5 is antibacterial modified: the acellular matrix material that will be prepared by step S4, and at 50~80 DEG C, PBS is added
Buffer handles 30~50min；Then modifying agent is added, handles 4~8 hours；Waste reaction solution is discarded, cleans, is filtered dry, then set
- 80 DEG C of freeze-drying 30-40h, obtain vagina host material in freeze drier；The modifying agent is by H- histidyl--arginine
(CAS:77369-21-2) it is made with epoxychloropropane.
2. a kind of preparation method of vagina host material according to claim 1, which is characterized in that cut described in step S1
It is broken for shred to size be (1-3) cm × (0.5-1) cm；Biological buffer described in step S2 is DIPSO buffer and Tris
Buffer 1:(3-5 in mass ratio) it mixes.
3. a kind of preparation method of vagina host material according to claim 1, which is characterized in that the de-cell liquid by
Each ingredient of following mass percent is by being mixed evenly to prepare: aminotrimethylenephosphonic acid ethyl glycol ether surfactant
0.3%-0.5%, water-soluble ultrabranching polyaminoacid 0.1%-0.3%, leupeptin 0.1%, surplus is distilled water.
4. a kind of preparation method of vagina host material according to claim 3, which is characterized in that three methene of amino
The preparation method of phosphonic acid base glycol ether based surfactants, includes the following steps:
I aminotrimethylenephosphonic acid, 3- chlorine-1,2-propylene glycol oleate are added in organic solvent, are stirred to react at 30-40 DEG C
4-6 hours, solvent was evaporated off in back spin, and is washed 3-5 times with ether, then rotates removing ether, obtained three methene of ethyleneamino
Phosphonic acids；
II by three methylenephosphonic acid of ethyleneamino being prepared by step I, 3- propyl- 2- alkene acyloxy propane -1- sodium sulfonate,
2- ethyleneoxy ethyl alcohol, initiator are added in tetrahydrofuran, and return stirring is anti-at nitrogen or 65-75 DEG C of atmosphere of inert gases
It answers, tetrahydrofuran is evaporated off in back spin.
5. a kind of preparation method of vagina host material according to claim 4, which is characterized in that ammonia described in step I
The chloro- 1,2- propylene glycol oleate of three methylenephosphonic acid of base, 3-, organic solvent mass ratio be 1:1.25:(7-10)；It is described organic molten
Agent is selected from least one of ether, acetone, methylene chloride, ethyl acetate.
6. a kind of preparation method of vagina host material according to claim 4, which is characterized in that second described in step II
Three methylenephosphonic acid of alkenyl amino, 3- propyl- 2- alkene acyloxy propane -1- sodium sulfonate, 2- ethyleneoxy ethyl alcohol, initiator, tetrahydro furan
The mass ratio muttered is 0.2:1:2:(0.02-0.04): (10-15)；The initiator is azodiisobutyronitrile, azobisisoheptonitrile
At least one of；The inert gas is selected from one of helium, neon, argon gas.
7. a kind of preparation method of vagina host material according to claim 1, which is characterized in that under described in step S3
Layer tissue, de-cell liquid mass ratio be 1:(20-40)；The temperature range of the de- cell processing of ultrasound is 23-33 DEG C；Ultrasound
Power is 5000W；The oscillation frequency is 100-150r/min.
8. a kind of preparation method of vagina host material according to claim 1, which is characterized in that taken off described in step S5
Cell matrix materials, PBS buffer solution, modifying agent mass ratio be 1:(20-50): 0.1.
9. a kind of preparation method of vagina host material according to claim 1, which is characterized in that the system of the modifying agent
Preparation Method includes the following steps: H- histidyl--arginine, epoxychloropropane, basic catalyst being added to N, N- dimethyl methyl
In amide, be stirred to react at 30-50 DEG C 6-8 hours, after be filtered to remove insoluble matter, then rotate removing n,N-Dimethylformamide
With unreacted reactant, modifying agent is obtained；H- histidyl--the arginine, epoxychloropropane, basic catalyst, N, N- bis-
The mass ratio of methylformamide is 1:1.78:(0.3-0.5): (10-15)；The basic catalyst be selected from sodium carbonate, sodium carbonate,
At least one of sodium hydroxide, potassium hydroxide.
10. vagina base made of a kind of a kind of -9 described in any item preparation methods of vagina host material according to claim 1
Material.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114848912A (en) * | 2022-04-26 | 2022-08-05 | 北京桀亚莱福生物技术有限责任公司 | Acellular dermis and preparation method thereof |
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2019
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Cited By (2)
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| CN114848912A (en) * | 2022-04-26 | 2022-08-05 | 北京桀亚莱福生物技术有限责任公司 | Acellular dermis and preparation method thereof |
| CN114848912B (en) * | 2022-04-26 | 2024-02-20 | 北京桀亚莱福生物技术有限责任公司 | Acellular dermis and preparation method thereof |
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