CN108144124A - A kind of decellularized vascular matrix matrix and the application in mouth disease - Google Patents
A kind of decellularized vascular matrix matrix and the application in mouth disease Download PDFInfo
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- CN108144124A CN108144124A CN201810128740.1A CN201810128740A CN108144124A CN 108144124 A CN108144124 A CN 108144124A CN 201810128740 A CN201810128740 A CN 201810128740A CN 108144124 A CN108144124 A CN 108144124A
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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
- A61L27/3604—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
- A61L27/3633—Extracellular matrix [ECM]
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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
- A61L27/3604—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
- A61L27/362—Skin, e.g. dermal papillae
-
- 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
- A61L27/3683—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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- 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
- A61L27/3683—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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- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Abstract
The present invention relates to a kind of decellularized vascular matrix matrix and the applications in mouth disease.The invention belongs to the tissue engineering technique field of bio-medical material, application of specifically a kind of decellularized vascular matrix matrix after harelip and/Bone Graft, cleft palate, defect of oral mucosa reparation, gum defect, gingival recession reparation, bronchia mucosal reparation, Dental implant surgery, Root-bifurcating diseases reparation, impacted tooth are pulled out in terms of the prevention of the complication such as alveolitis.The decellularized vascular matrix matrix of the present invention is the product by process modification, and the mechanical performance of product is suitable for operation, suture strength 16N 18N；With soft texture after no immunological rejection, rapid induction regeneration, implantation, without profile sense, internal compatibility is good, the stent stablized or masterplate effect.
Description
Technical field
The invention belongs to the tissue engineering technique field of bio-medical material, specifically a kind of decellularized vascular matrix matrix
In harelip and/Bone Graft, cleft palate, defect of oral mucosa reparation, gum defect, gingival recession reparation, bronchia mucosal reparation, tooth
Implant operation, Root-bifurcating diseases reparation, impacted tooth pull out the application in terms of the prevention of the complication such as rear alveolitis.
Background technology
Defect of oral mucosa, which is repaired, mainly uses the neighbouring transfer mucosal flap in part, buccal fat pad graft, free skin transplantation etc., etc.
Self-tissue repair, although being easy to survive, there is also some defects：To self for area and by the function in area and the shadow of shape
It rings, postoperative clear scar deformity can be caused after healing；Second repair, operation wound is big, need to cut transplanting in other positions in body
Tissue, and often cause for area different degrees of dysfunction and complication, cause the negative effects such as patient suffering and the course of disease.
Tooth extraction is a kind of common oral surgery, suitable for odontopathies such as accessory tooth, difficult wisdom teeth cuttings.Extraction
Local alveolus ostosis reconstruction and absorption, bone amount reduce, cause later phase clinical repairing effect bad, how to reduce and prevention is pulled out afterwards
The absorption of alveolar bone and postexodontic complication are always the hot spot of Oral and Maxillofacial Surgery doctor concern after tooth.
Decellularized vascular matrix matrix sticking patch is derived from the sheet of mankind's allosome or membranaceous tissue, be by take off cell technology,
To biological corium carry out biological and chemical processing, remove completely it is various it is being identified by host, immunological rejection can be caused
Cell component, if the skin of transplanting is containing cell, then the immune response of endothelial cell may cause after transplanting
Tissue, once being handled by de- cell, is become the base of no cell by vessel retraction, tissue ischemia and tissue degeneratiaon's necrosis
Matter and stent, this material will generate immunologic inertia, therefore immunological rejection will not occur, while completely remain thin
Extracellular matrix components and its three dimensions frame structure.
Chinese invention patent CN1562388A is by the membranaceous or lamellar structure of the allosome of people or mammality object through taking off at cell
The extracellular matrix with three-D space structure is managed into, specific method is：Membranaceous group needed for being taken from people or mammal body
It knits or lamellar structure；The tissue of acquisition is fixed with glutaraldehyde and/or formaldehyde；Tissue is carried out at de- cell with alkaline solution
Reason；Tissue is washed with buffer solution, obtains extracellular matrix；Extracellular matrix is incubated in amino acid solution.
However, being fixed with glutaraldehyde and/or formaldehyde, because glutaraldehyde or formaldehyde have cellular toxicity, result in crosslinked using their progress
Biological tissue affects the growth of receptor normal structure, and then influence the healing of wound to a certain extent after human body is implanted into
With the recovery of function；The alkaline solution of use carries out tissue de- cell processing, and especially sodium hydroxide or potassium hydroxide are strong
Alkali can cause the collagen in decellularized vascular matrix matrix to hydrolyze under alkaline condition.
Based on disadvantages described above, research one kind is safe and non-toxic, decellularized vascular matrix matrix is complete, can be used in harelip
With/Bone Graft, cleft palate, defect of oral mucosa reparation, gum defect, gingival recession reparation, bronchia mucosal reparation, Dental implantion hand
Art, Root-bifurcating diseases reparation, impacted tooth pull out decellularized vascular matrix matrix in terms of the prevention of the complication such as rear alveolitis or
Oral cavity sticking patch has practical significance.
Invention content
In order to adapt to harelip and/Bone Graft, cleft palate, defect of oral mucosa reparation, gum defect, gingival recession reparation,
Hand in terms of the preventions of the complication such as rear alveolitis is pulled out in bronchia mucosal reparation, Dental implant surgery, Root-bifurcating diseases reparation, impacted tooth
Art needs, and provides suitable oral cavity patching material, and the present invention is ultrasonically treated by enzymatic treatment, surfactant, DNA degradation processing
Multi-steps cooperating is waited, invention is prepared for a kind of new decellularized vascular matrix host material, and is made into oral cavity benefit
Piece, for the prevention or treatment of above-mentioned disease.
To achieve the above object, the present invention provides following technical solution：
A kind of preparation method of decellularized vascular matrix matrix, includes the following steps：
Step 1 puts into allograft skin raw material in enzyme solutions, impregnates oscillation treatment, obtains semi-finished product A；The enzyme is phosphatide
Enzyme or the enzyme are phosphatidase and protease；
Semi-finished product A is taken out, is impregnated with physiological saline by step 2, and oscillation treatment obtains semi-finished product B；
Step 3 puts into semi-finished product B in surfactant solution, and ultrasonic immersion treatment obtains semi-finished product C；
Step 4 impregnates semi-finished product C with physiological saline, and oscillation treatment obtains semi-finished product D；
Step 5 fills semi-finished product D inputs in the container that DNA hydrolyzes enzyme solutions, and oscillation treatment obtains semi-finished product E；
Step 6 impregnates semi-finished product E with physiological saline, and oscillation treatment obtains semi-finished product F；
Step 7 by semi-finished product F water for injection cleaning and dippings, obtains finished product G；As decellularized vascular matrix matrix；It can
With directly as the use of oral cavity sticking patch or as the material for preparing oral cavity sticking patch.
Usually, decellularized vascular matrix matrix made from the above method can be stored in physiological saline；To extend it
Storage life also may be sterilized processing (such as irradiation sterilization).
For ease of the storage and transport of above-mentioned decellularized vascular matrix matrix, extend its term of validity, further, above-mentioned side
Method is further comprising the steps of：
Step 8 takes out finished product G, packs, and sterilizing obtains finished product H；Or take out finished product G, it is placed in containing frozen-dried protective
It is freeze-dried in the solution of agent；Packaging, sterilizing, obtains finished product H.
Further, protease described in step 1 includes trypsase, bromelain, papain, dispase
It is one or more in enzyme (neutral proteinase, dispase) etc..
In some embodiments, the enzyme in abovementioned steps one is phosphatidase or phosphatidase and trypsase, bromelain
It is one or more in enzyme, papain, dispase enzymes.Further, the enzyme solutions pH value is 7.0-8.0.It is preferred that
Ground, phospholipase concentration 0.1g/L-0.4g/L and/or the protease (or wherein trypsase, bromelain, pawpaw egg
White enzyme, dispase enzymes are any) a concentration of 0.1g/L-0.3g/L.Further, the phosphatidase is phospholipase A1, phosphorus
Lipase A2, phospholipase C, phospholipase D one or more, the preferred matter of phospholipase A1, phospholipase A2, phospholipase C, phospholipase D
Amount is than being 1:1:1:1.
In some embodiments, one oscillation treatment condition of abovementioned steps is oscillation 0.5-4 hours, and oscillation rotating speed is 10-
200rpm, temperature are 10-40 DEG C.
In some embodiments, the SDS (ten containing 0.1-0.3g/L in surfactant solution described in abovementioned steps three
Sodium dialkyl sulfate) or the surfactant solution in Triton X-100 (the polyethylene glycol octyl groups containing 0.1-0.3g/L
Phenyl ether).Further, ultrasonic soaking conditions are：40-80KHz, 100-400W, temperature is 1-20 DEG C, 3-8 minutes ultrasonic, leaching
Bubble 2-4 hours repeats 2-4 times.
In some embodiments, DNA described in abovementioned steps five hydrolyzes a concentration of 4-8g/L, pH7.0- of enzyme solutions
8.0；Preferred concentration is 4-6g/L.
In some embodiments, oscillation treatment condition is oscillation 2-8 hours in abovementioned steps five, and oscillation rotating speed is 10-
200rpm, temperature are 10-40 DEG C.
In some embodiments, it is described Step 2: Step 4: step 6 oscillation treatment condition for oscillation 1-2 hours, shake
Rotating speed is swung for 80-150rpm, replaces physiological saline, continues oscillation treatment, repeats operation 4-6 times, and temperature is 1-5 DEG C.
In some embodiments, the step 7 water for injection cleaning and dipping condition is oscillation 2 hours, and oscillation rotating speed is
80-150rpm, temperature are 1-5 DEG C, replace water for injection, continue oscillation treatment, repeat operation 4-6 times.
In some embodiments, frozen-dried protective agent solution described in step 8 include phosphate buffer, hyaluronic acid and
A concentration of 0.4-0.8mg/100mL of sugar, wherein hyaluronic acid, sugared a concentration of 10-20mg/100mL, the sugar are seaweed
One or more of sugar, lactose, sucrose, glycerine, mannitol, sorbierite, mannose, glucose etc..The phosphate-buffered
Solution (PBS) can be prepared by this field conventional method, preferably 7.0 phosphate buffers of 10mmol/LpH.
It is described freeze-drying step be specially：By finished product G in 5 DEG C of inlets, kept for 1 hour, cool to -40 DEG C and holding 1
Hour, it is warming up to -18 DEG C and is kept for 1 hour, be cooled to -35 DEG C again and kept for 1 hour, start vacuum pump, by drying box vacuum
Degree is extracted into 5-10Pa；Partition board is warming up to -30 DEG C with 2 hours, drying box vacuum degree is extracted into 1-5Pa, maintains 15h；It will with 1 hour
Partition board is warming up to 0 DEG C, and after maintaining 10h, measures pressure liter, until pressure, which rises, is less than 1pa；Partition board is warming up to 10 with 1 hour
DEG C, and 10h is maintained, pressure liter is measured, until pressure, which rises, is less than 1pa；Partition board was warming up to 25 DEG C, and maintain 8h with 0.5 hour,
Pressure liter is measured, until pressure, which rises, is less than 1pa；Preceding case vacuum degree should not be greater than 30Pa in entire drying process.
In some embodiments, abovementioned steps eight are packaged as finished product G being fitted into packaging bag, and sealing packaging finishes；
The sterilizing is irradiated for cobalt -60, exposure dose 20-30kGy.
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined with each other each preferably to get the present invention
Example.
The invention also includes decellularized vascular matrix matrix (can also be referred to as oral cavity sticking patch) prepared by the above method.
The suture strength of decellularized vascular matrix matrix prepared by the present invention is 16N-18N.
The decellularized vascular matrix matrix (oral cavity sticking patch) needs first to proceed as follows before use：20 DEG C of -35 DEG C of lifes
(rehydration) is completely soaked in reason brine 15 minutes.
Can appropriate size be made in above-mentioned decellularized vascular matrix matrix according to actual needs.
The invention also includes above-mentioned decellularized vascular matrix matrix answering in the repair materials for preparing treatment injury of mouth
With.
The injury of mouth includes：Harelip, Bone Graft, cleft palate, defect of oral mucosa reparation, gum defect, nasal cavity stick
Film reparation, Dental implant surgery, gingival recession reparation, Root-bifurcating diseases reparation, impacted tooth pull out the complication such as rear alveolitis.
Allograft skin raw material sources of the present invention are in the skin of voluntary donor (such as non-living body).
The preservation condition of decellularized vascular matrix matrix of the present invention is at shady and cool drying, and relative humidity is less than or equal to 45%.
Another aspect of the present invention provides a kind of kit for oral cavity repairing, contains aforementioned decellularized vascular matrix base
Matter (oral cavity sticking patch).The kit can also further include scalpel, operating scissors, haemostatic clamp, tweezers, sewing needle, can inhale
Receive surgical thread, the ampoule bottle containing water for injection, syringe etc..
The present invention is repaiied according to harelip and/Bone Graft, cleft palate, defect of oral mucosa reparation, gum defect, gingival recession
Multiple, bronchia mucosal reparation, Dental implant surgery, Root-bifurcating diseases reparation, impacted tooth pull out prevention of the complication such as rear alveolitis etc.
The needs of operation improve and optimize the preparation process of decellularized vascular matrix matrix.The product is the skin that human body is contributed to body
Skin is handled by special biotechnology, all cell clearances that host immune rejection can be caused to react in tissue is fallen, simultaneously
Completely retain the extracellular matrix identical with original institutional framework；And as needed, by freeze-drying process, make it more just
In storage and transport.By constantly furtheing investigate discovery to the product, due to while removal immune rejection ingredient,
Former organized three-dimensional bracket structure is completely remained, as cytoskeleton, it has the function of induced tissue generation, is planting
Autologous tissue can be identified as by human tissue cell after entering human body, has new vessels and fibroblast to grow into quickly, guiding is thin
Born of the same parents achieve the purpose that supplement, particularly quickly repair along its collagen frame ordering growth, it have good histocompatbility and
Mechanical property, can long-term existence, become the part of tissue, so as to complete the repair and reconstruction to tissue defect.
When this field is performed the operation using decellularized vascular matrix matrix, such as need to carry out gum defect repair operation
When, it needs to carry out gingival flap operation under local anesthesia, surgical wound surface thoroughly stops blooding, and takes the de- cellular allograft slightly larger than defect face
Dermal matrix, physiological saline soaking flushing, center are cut 1-2 small notch and are drained with profit, and the surface of a wound, sticking patch side are attached at rough surface
Interrupted suture between edge and edge of wound stays long line；After suture, iodoform oil gauze anti-package is pricked using stayed long line and is pressurizeed, makes benefit
Piece is close to the surface of a wound.This needs to improve sewing density, to meet the needs of operation, it is therefore desirable to decellularized vascular matrix matrix mouth
Chamber sticking patch has higher suture strength；And in order to meet the postoperative comfort level of patient, while need decellularized vascular matrix matrix
Oral cavity sticking patch has certain pliability, so that the foreign sense after human body is filled into of decellularized vascular matrix matrix oral cavity sticking patch.
Decellularized vascular matrix matrix of the present invention or oral cavity sticking patch can harelip and/Bone Graft, cleft palate, mucous membrane of mouth lack
After damage reparation, bronchia mucosal reparation, Dental implant surgery, gum defect or reparation of shrinking back, Root-bifurcating diseases reparation, impacted tooth are pulled out
The operation demand such as prevention of the complication such as alveolitis.The present invention by enzymatic treatment, surfactant solution supersound process combine into
The de- cell of row, can reach and take off cell effect well, and is smaller to the damage of dermal matrix, be conducive to improve tissue products machine
Tool performance obtains preferable elasticity and toughness, improves tear, the suture strength of tissue, and DNA is contributed to hydrolyze enzymatic treatment step
Remaining DNA in middle removing dermal matrix, reduces cytotoxicity and rejection.
To sum up, decellularized vascular matrix matrix of the invention or oral cavity sticking patch have following excellent as dental prosthetic material
Point：(1) for properties of product, decellularized vascular matrix matrix of the invention or oral cavity sticking patch are the productions by process modification
Product, the mechanical performance of product are suitable for operation, suture strength 16N-18N.(2) for product effect, de- cell of the invention
Allodermis Matrix matrix or oral cavity sticking patch with soft texture after no immunological rejection, rapid induction regeneration, implantation, without wheel
Exterior feature sense, the stent that internal compatibility is good, stable or masterplate effect.
Description of the drawings
Fig. 1 is picture before 1 corrective surgery of case；
Fig. 2 is that decellularized vascular matrix matrix specification picture is chosen in 1 corrective surgery of case；
Fig. 3 is 3 months follow-up pictures after 1 corrective surgery of case；
Fig. 4 is 2 corrective surgery process picture of case.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..It is not specified in embodiment specific
Technology or condition person carry out according to the described technology of document in the art or condition or according to product description.It is used
Production firm person is not specified in reagent or instrument, is the conventional products that can be commercially available by regular distributor.
The preparation method of 1 decellularized vascular matrix matrix of embodiment
Allodermis Matrix raw material is put into a concentration of 0.3g/L phosphatidases and 0.1g/L trypsin solutions, enzyme solutions pH value
It is 7.0, temperature is 37 DEG C, and 100rpm vibrates 2 hours, replaces an enzyme solutions, this process of repetition is primary, obtains semi-finished product A；Phosphatide
Enzyme is phospholipase A1：Phospholipase A2：Phospholipase C：Phospholipase D is 1 according to mass ratio:1:1:1 mixture.
Semi-finished product A is taken out, input fills is impregnated in the container of normal saline solution with physiological saline, oscillation 3 times, every time
Oscillation 2 hours, oscillation rotating speed are 80rpm, and temperature is 2 DEG C, obtains semi-finished product B.
Semi-finished product B is put into containing in 0.2g/LTritonX-100 solution, 50KHz, 220W, ultrasound 5 minutes impregnate 3
Hour, it repeats this process 3 times, obtains semi-finished product C.
Semi-finished product C is taken out, input fills is impregnated in the container of normal saline solution with physiological saline, oscillation 3 times, every time
Oscillation 2 hours, temperature are 2 DEG C, and oscillation rotating speed is 80rpm, obtains semi-finished product D.
Semi-finished product D is put into and fills a concentration of 5g/L, the DNA that pH is 7.0 is hydrolyzed in enzyme solutions, and temperature is 37 DEG C, is shaken
Processing 4 hours is swung, oscillation rotating speed is 20rpm, obtains semi-finished product E.
Semi-finished product E inputs are filled in the container of normal saline solution and impregnated with physiological saline, vibrated 3 times, vibrate 2 every time
Hour, temperature is 2 DEG C, and oscillation rotating speed is 80rpm, obtains semi-finished product F.
Semi-finished product F inputs are filled in the container of water for injection and impregnated with physiological saline, vibrated 3 times, oscillation 2 is small every time
When, temperature is 2 DEG C, and oscillation rotating speed is 80rpm, obtains finished product G.
Finished product G is taken out, is fitted into packaging bag, is paved, is sealed, cobalt -60 is carried out and irradiates, exposure dose 20-30kGy,
It takes out, obtains finished product H eventually.Suture strength is 17.7N.
The preparation method of 2 decellularized vascular matrix matrix of embodiment
Allodermis Matrix raw material is put into a concentration of 0.2g/L phosphatidases and 0.2g/L trypsin solutions, enzyme solutions pH value
It is 7.0, temperature is 37 DEG C, and 100rpm vibrates 2 hours, replaces an enzyme solutions, this process of repetition is primary, obtains semi-finished product A；Phosphatide
Enzyme is phospholipase A1, phospholipase A2, phospholipase C and phospholipase D according to mass ratio 1:1:1:1 mixture.
Semi-finished product A is taken out, input fills is impregnated in the container of normal saline solution with physiological saline, oscillation 3 times, every time
Oscillation 2 hours, oscillation rotating speed are 80rpm, and temperature is 2 DEG C, obtains semi-finished product B.
Semi-finished product B is put into containing in 0.2g/LSDS solution, 10mMEDTA, 50KHz, 220W, ultrasound 7 minutes are impregnated
It 2 hours, repeats this process 3 times, obtains semi-finished product C.
Semi-finished product C is taken out, input fills is impregnated in the container of normal saline solution with physiological saline, oscillation 3 times, every time
Oscillation 1 hour, oscillation rotating speed are 100rpm, and temperature is 4 DEG C, obtains semi-finished product D.
Semi-finished product D is put into and fills a concentration of 7g/L, the DNA that pH is 7.0 is hydrolyzed in enzyme solutions, and temperature is 30 DEG C, is shaken
Processing 3 hours is swung, oscillation rotating speed is 30rpm, obtains semi-finished product E.
Semi-finished product E inputs are filled in the container of normal saline solution and impregnated with physiological saline, vibrated 3 times, vibrate 1 every time
Hour, oscillation rotating speed is 100rpm, and temperature is 4 DEG C, obtains semi-finished product F.
Semi-finished product F inputs are filled in the container of water for injection and impregnated with physiological saline, vibrated 3 times, oscillation 2 is small every time
When, temperature is 2 DEG C, and oscillation rotating speed is 80rpm, obtains finished product G.
Finished product G is taken out, is placed in frozen-dried protective agent solution；In 5 DEG C of inlets, kept for 1 hour, cool to -40 DEG C and protect
It holds 1 hour, be warming up to -18 DEG C and kept for 1 hour, be cooled to -35 DEG C again and kept for 1 hour, start vacuum pump, drying box is true
Reciprocal of duty cycle is extracted into 5-10Pa；Partition board is warming up to -30 DEG C with 2 hours, drying box vacuum degree is extracted into 1-5Pa, maintains 15h；With 1 hour
Partition board is warming up to 0 DEG C, and after maintaining 10h, measures pressure liter, until pressure, which rises, is less than 1pa；Partition board is warming up to 1 hour
10 DEG C, and 10h is maintained, pressure liter is measured, until pressure, which rises, is less than 1pa；Partition board was warming up to 25 DEG C, and maintain with 0.5 hour
8h measures pressure liter, until pressure, which rises, is less than 1pa；Preceding case vacuum degree should not be greater than 30Pa in entire drying process；Obtain semi-finished product
G1。
The frozen-dried protective agent solution is by the phosphate buffer of 10mmol/L pH 7.0, hyaluronic acid and trehalose group
Into the wherein a concentration of 0.6mg/100mL of hyaluronic acid, a concentration of 10mg/100mL of trehalose.
Semi-finished product G1 is taken out, is fitted into packaging bag, is paved, is sealed, cobalt -60 is carried out and irradiates, exposure dose 20-
30kGy takes out, and obtains finished product H eventually.Suture strength is 16.8N.
1 decellularized vascular matrix matrix of experimental example (oral cavity sticking patch) is performed the operation for gum defect
Case 1
Lee, man, 59 years old, upper left incisor and upper right incisor gum defect, defect area maximum 1.0cm × 0.8cm were minimum
For 0.2cm × 0.1cm, gingival flap operation is carried out under local anaesthesia, surgical wound surface thoroughly stops blooding, and takes the reality slightly larger than defect face
The decellularized vascular matrix matrix of the preparation of example 1 is applied, physiological saline soaking flushing, center is cut 2 small notch and drained with profit, with coarse
Face paste invests the surface of a wound, and interrupted suture between sticking patch edge and edge of wound stays long line.It is using stayed long line that iodoform is oily after suture
Gauze anti-package pricks pressurization, and sticking patch is made to be close to the surface of a wound.Open iodoform packet within postoperative 10 days, dermal sutures out is shown in that sticking patch basal surface is dotted
Ruddy area, uneven color, edge Mild edema, 15 days after operation edema extinction.3 months surface of a wound no inflammations of Follow-up After, color
Uniformly close with offside gum, sticking patch outer rotating edge is smooth, patient's foreign sense.Experimental result is shown in Fig. 1-3.
Case 2
Liu, female, 47 years old, left 3,4 canine tooth gum defects, defect area about 0.1cm × 0.1cm, under local anaesthesia into
Row gingival flap operation, surgical wound surface thoroughly stop blooding, and take the decellularized vascular matrix base of the preparation of embodiment 2 slightly larger than defect face
Matter, physiological saline soaking flushing, center are cut 1 small notch and are drained with profit, and the surface of a wound, sticking patch edge and edge of wound are attached at rough surface
Between interrupted suture, stay long line.After suture, iodoform oil gauze anti-package is pricked using stayed long line and is pressurizeed, makes sticking patch and the surface of a wound
It is close to.Iodoform packet is opened within postoperative 10 days, dermal sutures out is shown in the dotted ruddy area of sticking patch basal surface, uneven color, edge is slight water
It is swollen, 15 days after operation edema extinction.3 months surface of a wound no inflammations of Follow-up After, color is uniformly close with offside gum, sticking patch flanging
Edge is smooth, patient's foreign sense.Experimental result is shown in Fig. 4.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (13)
1. a kind of preparation method of decellularized vascular matrix matrix, which is characterized in that include the following steps：
Step 1 puts into allograft skin raw material in enzyme solutions, impregnates oscillation treatment, obtains semi-finished product A；The enzyme for phosphatidase or
Enzyme described in person is phosphatidase and protease；
Semi-finished product A is taken out, is impregnated with physiological saline by step 2, and oscillation treatment obtains semi-finished product B；
Step 3 puts into semi-finished product B in surfactant solution, and ultrasonic immersion treatment obtains semi-finished product C；
Step 4 impregnates semi-finished product C with physiological saline, and oscillation treatment obtains semi-finished product D；
Step 5 fills semi-finished product D inputs in the container that DNA hydrolyzes enzyme solutions, and oscillation treatment obtains semi-finished product E；
Step 6 impregnates semi-finished product E with physiological saline, and oscillation treatment obtains semi-finished product F；
Step 7 by semi-finished product F water for injection cleaning and dippings, obtains finished product G；Alternatively, further, it is further comprising the steps of：
Step 8 takes out finished product G, packs, and sterilizing obtains finished product H；Or take out finished product G, it is placed in containing freeze drying protectant
It is freeze-dried in solution；Packaging, sterilizing, obtains finished product H.
2. preparation method according to claim 1, which is characterized in that phosphatidase described in step 1 is phospholipase A1, phosphorus
Lipase A2, phospholipase C, phospholipase D it is one or more, it is highly preferred that the phosphatidase for phospholipase A1, phospholipase A2, phosphorus
Lipase C, phospholipase D are 1 according to mass ratio:1:1:1 mixture；And/or
The protease includes one or more in trypsase, bromelain, papain, dispase enzymes.
3. preparation method according to claim 1 or 2, which is characterized in that enzyme solutions pH value described in step 1 are 7.0-
8.0；And/or
Preferably, phospholipase concentration 0.1g/L-0.4g/L；And/or
Preferably, a concentration of 0.1g/L-0.3g/L of the protease.
4. according to claim 1-3 any one of them preparation methods, which is characterized in that step 1 oscillation treatment condition is oscillation
0.5-4 hours, oscillation rotating speed was 10-200rpm, and temperature is 10-40 DEG C.
5. according to claim 1-4 any one of them preparation methods, which is characterized in that surfactant solution described in step 3
In the Triton X-100 containing 0.1-0.3g/L in the SDS containing 0.1-0.3g/L or described surfactant solutions；
Preferably, ultrasonic soaking conditions are：40-80KHz, 100-400W, temperature is 1-20 DEG C, 3-8 minutes ultrasonic, impregnates 2-4
Hour, it repeats 2-4 times.
6. according to claim 1-5 any one of them preparation methods, which is characterized in that DNA hydrolases described in step 5 is molten
A concentration of 4-8g/L of liquid, pH 7.0-8.0；Preferred concentration is 4-6g/L；And/or
Oscillation treatment condition is oscillation 2-8 hours in the step 5, and oscillation rotating speed is 10-200rpm, and temperature is 10-40 DEG C.
7. according to claim 1-6 any one of them preparation methods, which is characterized in that described Step 2: Step 4: step
6th, oscillation treatment condition is oscillation 1-2 hours, and oscillation rotating speed is 80-150rpm, replaces physiological saline, continues oscillation treatment, weight
Operation 4-6 times again, temperature are 1-5 DEG C；And/or
The step 7 water for injection cleaning and dipping condition is oscillation 2 hours, and oscillation rotating speed is 80-150rpm, temperature 1-5
DEG C, water for injection is replaced, continues oscillation treatment, repeats operation 4-6 times.
8. according to claim 1-7 any one of them preparation methods, which is characterized in that frozen-dried protective agent solution described in step 8
It is made of phosphate buffer, hyaluronic acid and sugar, wherein a concentration of 0.4-0.8mg/100mL of hyaluronic acid, sugared concentration
For 10-20mg/100mL, the sugar is trehalose, in lactose, sucrose, glycerine, mannitol, sorbierite, mannose, glucose
It is one or more of；Preferably, the phosphate buffering liquid concentration is 10mmol/L, pH 7.0.
9. according to claim 1-8 any one of them preparation methods, which is characterized in that freeze-drying includes described in step 8：
It by finished product G in 5 DEG C of inlets, is kept for 1 hour, cool to -40 DEG C and is kept for 1 hour, be warming up to -18 DEG C and kept for 1 hour, again
It is cooled to -35 DEG C to be kept for 1 hour, starts vacuum pump, drying box vacuum degree is extracted into 5-10Pa；Be warming up to partition board with 2 hours-
30 DEG C, drying box vacuum degree is extracted into 1-5Pa, maintains 15h；Partition board is warming up to 0 DEG C with 1 hour, and after maintaining 10h, measures pressure
Power liter, until pressure, which rises, is less than 1pa；Partition board was warming up to 10 DEG C, and maintain 10h with 1 hour, measures pressure liter, until pressure
It rises and is less than 1pa；Partition board was warming up to 25 DEG C, and maintain 8h with 0.5 hour, measures pressure liter, until pressure, which rises, is less than 1pa；It is whole
Preceding case vacuum degree should not be greater than 30Pa in a drying process.
10. decellularized vascular matrix matrix prepared by any one of claim 1-9 the methods；Preferably, the de- cell is different
The suture strength of body dermal matrix is 16N-18N.
11. a kind of decellularized vascular matrix matrix, which is characterized in that be that host can be caused to exempt from through removal completely by allograft skin raw material
All cells of epidemic disease rejection completely retain the extracellular matrix identical with original institutional framework and are made；Preferably, institute
The suture strength for stating decellularized vascular matrix matrix is 16N-18N.
12. the decellularized vascular matrix matrix of claim 10 or 11 answering in the repair materials for preparing treatment injury of mouth
With；Preferably, the injury of mouth includes：Harelip, Bone Graft, cleft palate, defect of oral mucosa reparation, gum defect, nasal cavity
Mucous rehabilitation, Dental implant surgery, gingival recession reparation, Root-bifurcating diseases reparation, impacted tooth pull out rear alveolitis complication.
13. a kind of kit for oral cavity repairing, which is characterized in that true containing the de- cellular allograft of claim 10 or 11
Scytoblastema matter；Alternatively, the kit can also further include scalpel, operating scissors, haemostatic clamp, tweezers, sewing needle, can absorb
One or more of surgical thread, the ampoule bottle containing water for injection, syringe.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109078222A (en) * | 2018-08-01 | 2018-12-25 | 青岛海洋生物医药研究院 | A kind of novel fish-skin source oral restoration film and preparation method thereof |
| CN109646718A (en) * | 2019-01-29 | 2019-04-19 | 北京颢美细胞基因生物技术有限公司 | Regenerating tissues base composition, preparation and application for micro-shaping |
| CN109821071A (en) * | 2019-02-22 | 2019-05-31 | 上海仁康科技有限公司 | A kind of hydrogel and preparation method thereof based on acellular dermal matrix |
| CN115845036A (en) * | 2023-02-20 | 2023-03-28 | 滨州益洁口腔有限公司 | Protease for oral implant gingival tissues and preparation method thereof |
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| CN1562388A (en) * | 2004-04-15 | 2005-01-12 | 深圳市清华源兴生物医药科技有限公司 | Patch of oral cavity tissue and preparation method and application |
| CN104888274A (en) * | 2015-05-19 | 2015-09-09 | 暨南大学 | Acellular matrix with natural level of glycosaminoglycan and preparation and application thereof |
| CN106581770A (en) * | 2016-12-29 | 2017-04-26 | 北京桀亚莱福生物技术有限责任公司 | Dura graft, preparation method and applications in dural damage repair thereof |
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| CN1562388A (en) * | 2004-04-15 | 2005-01-12 | 深圳市清华源兴生物医药科技有限公司 | Patch of oral cavity tissue and preparation method and application |
| CN104888274A (en) * | 2015-05-19 | 2015-09-09 | 暨南大学 | Acellular matrix with natural level of glycosaminoglycan and preparation and application thereof |
| CN106581770A (en) * | 2016-12-29 | 2017-04-26 | 北京桀亚莱福生物技术有限责任公司 | Dura graft, preparation method and applications in dural damage repair thereof |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109078222A (en) * | 2018-08-01 | 2018-12-25 | 青岛海洋生物医药研究院 | A kind of novel fish-skin source oral restoration film and preparation method thereof |
| CN109646718A (en) * | 2019-01-29 | 2019-04-19 | 北京颢美细胞基因生物技术有限公司 | Regenerating tissues base composition, preparation and application for micro-shaping |
| CN109821071A (en) * | 2019-02-22 | 2019-05-31 | 上海仁康科技有限公司 | A kind of hydrogel and preparation method thereof based on acellular dermal matrix |
| CN109821071B (en) * | 2019-02-22 | 2021-08-17 | 上海仁康科技有限公司 | Hydrogel based on acellular dermal matrix and preparation method thereof |
| CN115845036A (en) * | 2023-02-20 | 2023-03-28 | 滨州益洁口腔有限公司 | Protease for oral implant gingival tissues and preparation method thereof |
| CN115845036B (en) * | 2023-02-20 | 2023-05-09 | 滨州益洁口腔有限公司 | Protease for gingival tissue of oral implant and preparation method thereof |
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| CN108144124B (en) | 2021-03-30 |
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