CN106983918A - A kind of biological adherence preventing material, preparation method and applications - Google Patents
A kind of biological adherence preventing material, preparation method and applications Download PDFInfo
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- CN106983918A CN106983918A CN201710125136.9A CN201710125136A CN106983918A CN 106983918 A CN106983918 A CN 106983918A CN 201710125136 A CN201710125136 A CN 201710125136A CN 106983918 A CN106983918 A CN 106983918A
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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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/005—Ingredients of undetermined constitution or reaction products thereof
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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
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Abstract
The present invention provides a kind of biological adherence preventing material, preparation method and applications, it is characterised in that：The biological adherence preventing material is using the intestinal submucosa tissue material of animal as raw material, then remove immunogenicity and retain complete extracellular matrix components and be made, the biological adherence preventing material includes first surface, second surface and positioned at middle degradable absorbing structure, the first surface is shiny surface, the second surface is mat surface, the degradable absorbing structure that centre connects for gradual change.The mat surface of adherence preventing material of the present invention is conducive to guiding the growth of wound tissue cell, so that wound repairing；And shiny surface is then conducive to preventing the other tissues of these cytoadherences, so as to effectively prevent adhesion.Anti-adhesive available for the surface of a wound in abdominal cavity, thoracic cavity or pelvic cavity.
Description
Technical field
The present invention relates to medicine equipment technical field of biological material, specially it is a kind of be used to preventing the surface of a wound from sticking together prevent adhesion
Material, preparation method and applications.
Background technology
Adhesion be connective fiber band with adjacent tissue or organ be combined together formed by anomaly sxtructure.It is postoperative
Adhesion incidence is up to more than 90%, and thoracic cavity, abdominal cavity and operation on pelvis can all cause different degrees of adhesion.Face caused by adhesion
Bed severe complication includes intestinal obstruction, sterility, chronic pelvic pain etc., increases the probability of second operation.Even if moreover, carrying out
Second operation, still suffers from same postoperative adhesion again and occurs the potentiality of complication again.At present, there are two kinds of ways both at home and abroad
Footpath prevents postoperative tissue adhesion, and a kind of is the treatment method according to physiology/pharmacological mechanism, mainly medicine mitigates inflammatory reaction
And solution fibrin, another is the physical barrier Antiadhesive film of medical mechanical domain.Antiadhesive film is in the surface of a wound and perienchyma
Between form interim physical isolation barrier, the effect of preventing adhesion is played in tissue healing process, and can in vivo voluntarily
Degraded is absorbed.Antiadhesive film is generally manufactured using by materials such as hyaluronic acid, cellulose derivative, chitosan, PLAs
Polymer material is manufactured.But part high polymer material has subproblem, the catabolite of such as PLA is acidity, office
Portion's accumulation easily produces slight aseptic inflammation；Chitosan gel rubber or solution easily flow, and are difficult be partially formed higher concentration, and
The mechanical strength and toughness of spongy or film-form chitosan are inadequate.
Studies have found that the small intestinal submucosa tissue (SIS) for removing processing through immunogene is that a kind of acellular, nothing is immune
Originality, energy are biodegradable and with the good material of fibrous elasticity.Patent of invention (CN103272278A) once disclosed it is a kind of with
The method that animal derived immunogene removes small intestinal submucosa tissue preparation implantable biomaterial for medical purpose, is described by animal
The preposition processing separation of organization material, previous cleaning, inactivation of virus, immunogene removal, sodium chloride processing, shaping, packaging sterilizing are obtained
Be of different sizes, the curable product of thickness and mechanical strength.Immunogene can induce patient certainly after removing in matrix implants
Body cell is grown into, and is provided template for cellular reconstitution damaged tissues while providing temporary physical isolation barrier, is repaired as vascularization, work(
The tissue or organ of energyization, and immunogene remove matrix can progressively degrade, with rebuild tissue regeneration processes basic synchronization, and
It will not be sticked together with other tissues.
The content of the invention
The present invention provides a kind of biologies that can be used for preventing sticking together with other tissues in tissue surface of a wound recovery process and prevented
Adhering material.The adherence preventing material has good biocompatibility, suitable tissue adherence and certain mechanical strength
And pliability.In addition, the adherence preventing material has suitable RT in vivo, it can effectively prevent Adhesion formation and not influence wound
The normal healing of mouth.
The technical solution adopted by the present invention is：A kind of biological adherence preventing material, it is characterised in that：The biological adherence preventing material
Using the submucous layer of small intestine of animal as raw material, then remove immunogenicity and retain complete extracellular matrix components and be made；Should
Biological adherence preventing material includes first surface, second surface and positioned at middle degradable absorbing structure, and the first surface is
Shiny surface, the second surface mat surface, the degradable absorbing structure that centre connects for gradual change.
The above-mentioned biological adherence preventing material of the present invention, including multilayer submucous layer of small intestine (SIS) organization material, wherein according to baking
The process steps such as dry and freeze-drying cause the exposed first surface of intestinal submucosa tissue material to be the table of relative smooth
Face, the exposed second surface of intestinal submucosa tissue material is the surface of relative coarseness；Specifically：The first surface is
The surface that vacuum dried organization material is formed；The surface that the second surface is formed by freeze-dried organization material.
Animal of the present invention is mammal, preferably pig or ox.
Removal immunogenicity of the present invention refers to that cell residue amount is less than 10ng/mg, half less than 10, DNA residual quantities
Lactoside enzyme (α-Gal) clearance rate is more than 99%.
The complete extracellular matrix components of reservation of the present invention, which refer to retain, includes collagenous fibres, polysaccharide material, activity
The factor and growth factor composition.
Collagenous fibres of the present invention include I-type collagen and type III, IV types and VI collagen types.
Institute's polysaccharide material of the present invention includes chondroitin sulfate and hyaluronic acid.
Active factors of the present invention includes fibronectin splicing variants, laminin, integrin and its part.
Growth factor of the present invention includes basic fibroblast growth factor (bFGF) and VEGF (VEGF).
Sticking patch of the present invention is laminated structure, including 2-8 layer immunogene remove host materials and constituted, the laminated structure
Long 1-10cm, wide 1-7cm, thickness 0.1-1mm.
Shiny surface of the present invention is fine and close less porous, with gloss, and described mat surface is loose porous, in matt
Shape.
The degradation time of biological adherence preventing material of the present invention in vivo 1-3 months.
The tensile break strength of biological adherence preventing material of the present invention is more than 40N/cm, and suture confining force is more than 8N.
The present invention also provides a kind of preparation method of biological adherence preventing material, it is characterised in that：First processing including raw material,
Inactivation of virus, immunogene are removed, are dried in vacuo and are freeze-dried.
Specifically, the preparation method step includes：
(1) the preposition processing of tissue：Intestinal submucosa tissue material (SIS) is taken, cleans and is filtered dry water；
(2) inactivation of virus：Virus is carried out using Peracetic acid-alcohol solution dipping intestinal submucosa tissue material to go out
It is living；
(3) clean：Under ultrasound environments, handled respectively with PBS solution and water is cleaned；
(4) immunogene is removed：The intestinal submucosa tissue material obtained after step (3) is cleaned carries out immunogene and gone
Remove, including the first step uses NaOH solution, hydrochloric acid clean and change liquid is used in sonic oscillation processing after processing；Second step, which is used, contains pancreas
The PBS solution of protease and EDTA, multiple frequency ultrasonic vibration；3rd step uses NaCl solution, sonic oscillation；
(5) clean：Under ultrasound environments, cleaned respectively with PBS solution and water；
(6) it is dried in vacuo：The immunogene cleaned by step (5) removal material is fixed on mould and together dried；
(7) it is freeze-dried：It will be covered in and be obtained through step (6) by the cleaned immunogene removal organization material of step (5)
Vacuum drying after organization material on, be together freeze-dried.
The concentration of volume percent of Peracetic acid is 0.1%- in Peracetic acid-ethanol solution that inventive step (2) is used
5%th, the concentration of volume percent of ethanol is 5%-40% (being configured to solution with water), and Peracetic acid-ethanol solution glues with small intestine
The volume ratio of film lower-hierarchy material is (3-20) ︰ 1, inactivation time 2-4 hours, temperature range is 10-40 DEG C.
Inventive step (3) is cleaned：Cleaned, then cleaned with water to inspection with PBS solution in supersonic wave cleaning machine
Survey electrical conductivity to terminate for below 10 μ S/cm, obtain submucous layer of small intestine host material.Wherein PBS solution pH value range is 6-8,
Used water is the purified water Jing Guo purification process.
The immunogene that step (4) of the present invention uses removes liquid
(20-40)：1.
Step (4) first step of the present invention uses concentration for 0.1-0.5mol/L NaOH solution, sonic oscillation processing 1-
The volume ratio of 30min, 20-25 DEG C of temperature, solution and intestinal submucosa tissue material is 20-40：1, ultrasonic power exists
More than 3000W；The hydrochloric acid solution clean and change liquid for being 0.1-0.5mol/L with concentration after processing, and pH is adjusted to neutrality；Second step
The pH value for using the EDTA for being 0.1-1mmol/L for 0.01-0.2% trypsase and concentration containing mass percent concentration is
7 PBS solution, multiple frequency ultrasonic vibration 10-80min, multiple frequency ultrasonic at least includes two supersonic frequencies, and Frequency scope is
20-40KHz, higher frequency is 60-90KHz, wherein low frequency processing 5-40min, high-frequency therapeutic treatment 5-40min, 30 DEG C of temperature, solution
Volume ratio with intestinal submucosa tissue material is (30-40)：1, ultrasonic power is in more than 5000W；3rd step is adopted
The NaCl solution for being 0.8-1.5mol/L with concentration, sonic oscillation processing 10min, 20 DEG C of temperature, solution and submucous layer of small intestine
The volume ratio of organization material is 20-40：1, ultrasonic power is in more than 3000W.
Cleaning process is in the step (5)：Cleaned in supersonic wave cleaning machine with PBS solution, then with the note of cooling
Penetrate to be cleaned with water to the water for injection electrical conductivity difference before and after detection cleaning and terminated for below 1 μ S/cm, obtain submucous layer of small intestine base
Material.Wherein PBS solution pH value range is 6-8.Preferred 10-40 DEG C of water for injection temperature.
Drying described in step (6) of the present invention is that immunogene removal organization material is placed in into vacuum environment, 25-40 DEG C of temperature
Afterwards, the time is 8-16h, prepares shiny surface.
Freeze-drying described in step (7) of the present invention is：Pre-freeze is incubated 1-2 hours to -45 DEG C, then adjust the temperature to -
15 DEG C, 5-7 hour are incubated, then adjusts the temperature to 0 DEG C, 2 hours are incubated, 25 DEG C are finally adjusted the temperature to, 4 hours are incubated, formation
The coarse centre of one-sided smooth one side is the adherence preventing material of the degradable absorption of gradual change attachment structure.The formation of gradual change attachment structure
It is due to that in freeze-drying process, the organization material that immunogene that step (5) is cleaned, containing moisture is removed is covered in through step
(6) during the dried organization material obtained, moisture has permeated to vacuum drying organization material, is formed from unseasoned tissue
Material to the water content in the direction of dry organization material change, it is freeze-dried after, formed gradual change attachment structure.
The mat surface of adherence preventing material of the present invention is conducive to guiding the growth of wound tissue cell, so that wound repairing；And
Shiny surface is then conducive to preventing the other tissues of these cytoadherences, so as to effectively prevent adhesion.
The preparation method of above-mentioned biological adherence preventing material, can also include the steps of：(8) sterilizing parsing：Using epoxy
Ethane is sterilized, and sterilising conditions are：40 DEG C of temperature, soaking time 4 hours, humidity 60%, ethylene oxide concentration is 600mg/
L, sterilization time 6 hours；Resolving is carried out in the Resolution Room of ventilation, and temperature control is between 20 DEG C, 14 days time.
Compared with prior art, the present invention has following remarkable advantage and beneficial effect：
(1) trypsase and EDTA are used, the connection between cell and extracellular matrix is destroyed；Using low frequency ultrasound
Cell is crushed, while acting on broken cell and extracellular matrix using high frequency ultrasound, further makes cell detachment
Extracellular matrix, reaches de- cell purpose.Using aforesaid way, each step during whole cell detachment matrix is carried out
Reinforcing, makes cell be completely disengaged from from matrix.Reach optimal immunogene removal effect；
(2) multi-way immunogene removes technique：One journey low concentration alkaline solution grease removal simultaneously destroys cell membrane, and two Cheng Caiyong contain
The sonicated further elution cell of PBS solution of EDTA and enzyme, three journey NaCl solutions further remove residual DNA, simultaneously
Control reagent concentration protection extra-cellular matrix structure；
(3) molding technology thereof：Realize that sticking patch one-sided smooth, one side are thick by vacuum drying, freeze-drying two-step process
Rough bilateral structure, shiny surface is conducive to guiding epithelial cell to climb shifting reparation, and mat surface is conducive to guiding the group of smooth muscle cell
Knit reparation；
(4) mechanics reinforcement process：Strengthen the strong bottom of mechanics of adherence preventing material, mechanics stabilization by being dried in vacuo forming technique
Property, so as to improve resistance to enzymolysis ability of the material in vivo in environment.
Brief description of the drawings
Shown in Fig. 1 is the structure chart of biological adherence preventing material of the invention；
Shown in Fig. 2 is the microstructure SEM photograph of biological adherence preventing material of the invention.
Shown in Fig. 3 is the HE colored graphs of biological adherence preventing material of the invention
Embodiment
The present invention is further described in detail with reference to embodiments, but is not limited to this.
Described trees-Osima jacoti, Osima excavata organization material (SIS) or calf intestinal submucosa tissue material are applied to this hair
Bright following embodiments.
Embodiment 1：
The preparation method of the present embodiment animal sources implantable biology adherence preventing material, including following operating procedure：
(1) the preposition processing of tissue：
Take intestinal submucosa tissue material to be divided into given size wide 10cm, long 15cm, lymphoid tissue is rejected, with originally
Water is rinsed 3 times, then is rinsed with purified water to surface without spot, and the intestinal submucosa tissue material after cleaning then is placed in into sieve
On the water treatment plants such as net, more than five minutes are stood, water is filtered dry.
(2) inactivation of virus：
Inactivation of virus is carried out using Peracetic acid-alcohol solution dipping intestinal submucosa tissue material, the process can be
Carried out in stainless steel cask.The concentration (percent by volume) of Peracetic acid is the 1%, concentration of ethanol in Peracetic acid-ethanol solution
(percent by volume) is 24%, and the ratio (volume ratio) of Peracetic acid-ethanol solution and intestinal submucosa tissue material is 8：
1, inactivation time 2h, temperature range are 20 DEG C.
(3) clean：
Intestinal submucosa tissue material is cleaned using cleaning fluid, cleaning fluid is the PBS solution that pH value is 7.0, PBS solution
Temperature is 20 DEG C, and the ratio (volume ratio) of PBS solution and intestinal submucosa tissue material is 30 ︰ 1, preferably cleaning 3 times, every time
20 minutes；Then cleaned using 20 DEG C of purified water, purified water is 30 ︰ 1 with intestinal submucosa tissue material proportion, is extremely detected
Electrical conductivity terminates for below 10 μ S/cm；Cleaning process is carried out in supersonic wave cleaning machine, and the preferred 40kHz of frequency, power is preferred
More than 3000W.
(4) immunogene is removed：
Immunogene removal step uses concentration for 0.3mol/L NaOH solution including the first step, sonic oscillation processing
10min, 20 DEG C of temperature, solution is 30 with intestinal submucosa tissue material proportion (volume ratio)：1；It is with concentration after processing
0.3mol/L hydrochloric acid solution clean and change liquid, and pH is adjusted to neutrality, ultrasonic power 3000W；Second step, which is used, contains quality hundred
Divide the PBS solution that the pH value of the trypsase that specific concentration is 0.02% and the EDTA that concentration is 0.5mmol/L is 7-8, multifrequency surpasses
Sound oscillation 10min, multiple frequency ultrasonic at least includes two supersonic frequencies, and Frequency scope is 30KHz, and higher frequency is 80KHz,
Wherein low frequency processing 5min, high-frequency therapeutic treatment 5min, 30 DEG C of temperature, solution and intestinal submucosa tissue material proportion (volume ratio)
For (30-40)：1, ultrasonic power 5000W；3rd step uses concentration for 1mol/L NaCl solution, sonic oscillation processing 10min,
20 DEG C of temperature, solution is 40 with intestinal submucosa tissue material proportion (volume ratio)：1, ultrasonic power is in 3000W.
(5) clean；
Cleaned using cleaning fluid, cleaning process is carried out in supersonic wave cleaning machine, cleaning fluid is pH7 PBS solution,
PBS solution temperature is 20 DEG C, and the ratio (volume ratio) of PBS solution and intestinal submucosa tissue material is 30 ︰ 1, preferably cleans 3
It is secondary, 20 minutes every time；Then cleaned using 20 DEG C of water for injection, water for injection and intestinal submucosa tissue material proportion
(volume ratio) is 30 ︰ 1, and detection cleans water for injection and do not clean the difference of water for injection electrical conductivity less than 1 μ S/cm terminations, frequency
It is preferred that 40kHz, preferred more than the 3000W of power.
(6) it is dried in vacuo：
Vacuum drying chamber is preheated to after 40 DEG C, one or more layers immunogene that will be cleaned by step (5) removes material and consolidated
Due on mould and being together put in vacuum drying chamber and being dried, the time is 16 hours.There is light through dry organization material
Sliding surface.The mould includes band pin bottom plate, cover plate and briquetting, and its structure may be referred to patent of invention ZL201310203588.6
And ZL201310203602.2.
(7) it is freeze-dried：
One or more layers immunogene cleaned by step (5) is removed into material-paving in after the drying obtained by step (6)
Organization material, be together positioned in vacuum freeze drier, close cryodesiccation chamber door, open circulating pump about 1min, open pressure
Contracting machine is to freeze drying box refrigeration, by product pre-freeze to -45 DEG C, is incubated 2 hours, opens vavuum pump, and about -15 DEG C of product temperature of regulation rises
China, after 6 hours, adjusts 0 DEG C of product temperature, is incubated 2 hours, adjust 25 DEG C of product temperature, be incubated 4 hours, vacuum freeze drying
Complete.The side that the material of formation is relative with smooth surface has rough surface.
The preparation method can also include the steps of：
(8) sterilizing parsing：
Sterilized using oxirane, sterilising conditions are：40 DEG C of temperature, soaking time 4 hours, humidity 60%, epoxy
Ethane concentration is 600mg/L, sterilization time 6 hours；Resolving is carried out in the Resolution Room of ventilation, temperature control 20 DEG C it
Between, 14 days time.
Although the vacuum drying surface of the biological adherence preventing material obtained by the present invention also experienced frozen dried, by
In vacuum drying surface almost without moisture, thus influence of the frozen dried to the pattern on the surface can ignore.Vacuum drying
Surface is smooth and glossiness, even if being again still smooth and glossiness by freezing.
Structure of title compound of the present invention is as Figure 1-3.
Embodiment 2：
Performance detection is carried out to sample in embodiment 1, detection project is as follows with result：
1) DNA is remained：According to biological agent residual DNA detection method《Chinese Pharmacopoeia》Version the 4th in 2015, using glimmering
The sample DNA residual quantity that light decoration method detection embodiment 1 is provided, as a result：The DNA residual quantities for the sample that embodiment 1 is provided
Less than 2.89 ± 0.36ng/mg.
2) galactosidase (α-Gal) clearance rate：Take animal derived biomaterial Gal positive reference product, Gal antigen negatives
Each 2mg of reference material, plus lysate 1ml, crack 30-90min, are configured to 20,10,5,2.5,1.25,0.625 μ g Gal standards
Curve sample, the test article before and after test immunogene is removed respectively takes 50mg, plus lysate 2ml, cracks 30-90min；Take lysate
With the supernatant after M86 antibody responses, 96 orifice plates, plus secondary antibody, plus developer are added, extinction is detected using ELISA method 450nm
Angle value, is calculated the Gal values of sample by standard curve, and the Gal values that immunogene removes before processing material are 20.89 ± 2.22 ×
1014The Gal values of sample are 0.12 ± 0.01 × 10 in/mg, embodiment 114/ mg, galactosidase (α-Gal) clearance rate exists
More than 99.43%.
3) Viral diagnosis：Selection Pseudorabies virus is indicator virus, and the DNA for detecting virus using real-time quantitative PCR method is copied
Shellfish number, detects 3 batches of samples.As a result：Viral DNA copies number is 0.
4) bacterium endogenous toxic material：According to GB/T 14233.2-2005《Medical infusion, blood transfusion, the instrument used for injection method of inspection the 2nd
Point：BiologicalAssays Procedures》Detected, totally 3 batches of samples, as a result：Bacterium endogenous toxic material is packed less than 20EU/.
5) collagen neuraminidase：I, III, IV type and VI collagen types, 3 μm are detected using Immunohistochemical Staining
Thick serial section, dimethylbenzene dewaxing, graded ethanol dehydration.Section is moved into electric cooker water-bath and (includes 0.01mol/L, pH6.0
Citric acid trisodium buffer solution), temperature is maintained at 95-100 DEG C, boils 20min, carries out antigen retrieval, after taking-up at room temperature from
So cooling.Phosphate buffer (PBS) is washed, 5min × 3 time.Two step method SABC：I, III, IV type and VI are added dropwise respectively
Collagen type monoclonal antibody primary antibody, concentration 1：100,4 DEG C of refrigerator overnights are incubated 60min at room temperature, and PBS is washed 3 times.It is added dropwise
Envision reaction solutions, are incubated 30min at room temperature.PBS is washed 3 times.The 3 of 0.05%, 3 one diaminobenzidines+0.03%
H2O2 colour developings 5-10min.Flowing water is washed, haematine lining dye.Incremental gradient ethanol dehydration, dimethylbenzene is transparent, usual resins sealing.Knot
Fruit shows that the micro- all visible pale brown dyeing of four kinds of stained preparations of Microscopic observation is positive, show detectable I in sample, III,
IV types and VI collagen types.
6) polysaccharide material content detection：10 samples are taken, are sampled, extraction, with Biocolor chondroitin sulfate detection reagents
It is 5236 ± 185 μ g/g that box, which tests content of chondroitin sulfate average value in content of chondroitin sulfate, sample,；Detected with hyaluronic acid
Kit tests hyaluronic acid (HA) content, as a result shows, hyaluronic acid (HA) the reserved average value of sample is 296 ± 23 μ
g/g。
7) active factors species differentiates：After sample immersion PBS 24h, 4% paraformaldehyde 5-10min is fixed on, is used
0.1mol/LPBS is washed 3 times, and then each 5min is gone on the slide for scribbling poly-D-lysine with glass tubule, carries out immune group
Weave chemistry is dyed.LN antibody, FN antibody and integrin potency are 1: 100,0.5% pancreatin digestion 3-5min exposure antigens,
0.1%Triton X100 effects 10min increases the penetrability of antibody.Wrapped in the aobvious positive of immunohistochemical staining, surface sample
Fibre-bearing Fibronectin, laminin, integrin and its part etc. material.
8) growth factor residual quantity：ELISA method detection sample neutral and alkali growth factor is used to sample in embodiment 1
(bFGF) animal tissue is used as control before removing and VEGF (VEGF) content, and to immunogene.As a result find
Content is respectively 2137 ± 187ng/L, 1055 ± 114ng/L before and after basic fibroblast growth factor (bFGF) immunogene is removed, and retains life
The long factor more than 45%；VEGF (VEGF) content immunogene remove before and after content be respectively 748 ± 61ng/L,
315 ± 21ng/L, retains growth factor 4 more than 0%.
9) tensile strength is sutured：Sample is prepared according to embodiment 1, with 3-0 non-absorbing suture in adherence preventing material one end
At edge 2mm, the other end of suture and adherence preventing material is fixed on tensiometer, drawn with 20mm/min speed
Stretch, until stitch points are torn, record maximal force, as a result show, maximum is up to 12N.
10) tensile strength：Sample is prepared according to embodiment 1, sample is cut into 2 × 5cm sizes, is in relative humidity
40-60%, temperature be 22 ± 2 DEG C under conditions of place 2h after tested immediately.Sample two ends are fixed on cupping machine
Chuck on, stretched out successively until sample fracture with 100mm/min speed, longitudinal test piece and horizontal sample are carried out respectively
Experiment.Last measurement result shows machine direction tensile strength up to 48N/cm.
11) residual ethylene oxide：By GB/T14233.1-2008《Medical infusion, blood transfusion, the instrument used for injection method of inspection
1 part：Chemical analysis》In 9 defined method test, as a result：Product residual ethylene oxide is packed no more than 10 μ g/.
12) heavy metal inspection：Lead, chromium press 5.9.1 in GB/T 14233.1-2008《Medical infusion, blood transfusion, instrument used for injection
Method of inspection part 1：Chemical analysis》Defined method test, mercury, arsenic press 5.9.3 in GB/T 14233.1-2008《Doctor
With transfusion, blood transfusion, instrument used for injection method of inspection part 1：Chemical analysis》Lead in defined method test, examination and test of products liquid,
Chromium, mercury, arsenic total heavy metal content are less than 1 μ g/g.
Embodiment 3：
Biocompatibility experiment is carried out to sample in embodiment 1, detection project includes：Pyrogen, cytotoxicity, delayed surpass
Quick reaction, intradermal reaction, Acute systemic toxicity, Salmonella reversion test, mouse lymphoma cell mutant test, chromosome aberration, implantation,
Subchronic toxicity.
1) pyrogen
In mass ratio 1：The ratio of 5 extraction media, 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiology salt
Water.Method is carried out as defined in GB/T 14233.2-2005, the reaction of product apyrogeneity.
2) cytotoxicity
In mass ratio 1：The ratio of 5 extraction media, 37 ± 1 DEG C, 24 ± 2hr prepares experimental liquid, extracts medium：Containing serum
MEM culture mediums.Experimental liquid is taken to be tested according to test method specified in GB/T16886.5-2003, as a result the cell of product
Toxic reaction is not more than 1 grade.
3) delayed allergy
In mass ratio 1：The ratio of 5 extraction media, 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiological saline
And cottonseed oil.According to the parts of GB/T 16886.10-2005 the 10th:Stimulate and provide to carry out with delayed allergy test method
Experiment, as a result product is without delayed allergy.
4) intradermal reaction
In mass ratio 1：The ratio of 5 extraction media, 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiological saline
And cottonseed oil.According to the parts of GB/T 16886.10-2005 the 10th:Stimulate and delayed allergy experiment test method regulation
Tested, as a result：The difference of test specimen and solvent control mean score is less than 1.0.
5) Acute systemic toxicity
In mass ratio 1：The ratio of 5 extraction media, 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiological saline
And cottonseed oil.Experimental liquid is taken to be tested according to test method as defined in GB/T16886.11-2011, as a result：Product is without acute
General toxic reaction.
6) Salmonella reversion test
In mass ratio 1：The ratio of 5 extraction media, 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiological saline
And DMSO.Method is carried out as defined in GB/T16886.3-2008, as a result：The Salmonella reversion test of product is feminine gender.
7) mouse lymphoma cell mutant test
In mass ratio 1：The ratio of 5 extraction media, 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiological saline
And DMSO.Method is carried out as defined in GB/T16886.3-2008, as a result：The mouse lymphoma cell mutant test of product is the moon
Property result.
8) chromosomal aberration test
In mass ratio 1：The ratio of 5 extraction media, 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiological saline
And DMSO, method is carried out as defined in GB/T16886.3-2008, as a result：The chromosomal aberration test of product is feminine gender.
9) subchronic toxicity
Method is carried out as defined in GB/T 16886.11, as a result：Without subchronic toxicity reaction.
To sum up, a kind of biological adherence preventing material of the present invention：
(1) it is combined using kinds of processes, diluted alkaline, enzyme, salting liquid substep are carried out, and efficient removal cell efficiently removes residual
DNA and immunogenicity, DNA residual can reach below 10ng/mg, galactosidase clearance is higher, can reach 99% with
On；
(2) destruction to matrix structure is reduced, retains the active growth factor in matrix；
(3) sticking patch formation one-sided smooth is realized by vacuum drying, freeze-drying two-step process, simultaneously coarse two-sided knot
Structure, the strong bottom of mechanics of Antiadhesive film, mechanical stability, so as to improve resistance to enzymolysis ability of the material in vivo in environment.
(4) sterilization process：Degradation process in product body is adjusted by sterilization process, immunogene is removed matrix sticking patch progressively
Degraded, with rebuilding tissue regeneration processes basic synchronization, final immunogene removes matrix sticking patch and replaced completely by host tissue；
(5) bilateral structure on the one hand be conducive to tissue cell growth, on the other hand prevent different tissues stick together there is provided
Effective preventing adhesiving effect.
The above embodiment of the present invention is the description of the invention and cannot be used for the limitation present invention, the right with the present invention
Any change in claim suitable implication and scope, is all considered as being included within the scope of the claims.
Claims (9)
1. a kind of biological adherence preventing material, it is characterised in that：The biological adherence preventing material is with the intestinal submucosa tissue of animal
Material is raw material, then removes immunogenicity and retains complete extracellular matrix components and be made, the biological adherence preventing material bag
Include first surface, second surface and positioned at middle degradable absorbing structure, the first surface is shiny surface, second table
Face mat surface, the degradable absorbing structure that centre connects for gradual change.
2. biological adherence preventing material according to claim 1, it is characterised in that：The animal is mammal, preferably pig
Or ox.
3. biological adherence preventing material according to claim 1, it is characterised in that：The removal immunogenicity refers to that cell is residual
It is more than 99% that allowance is less than 10ng/mg, galactosidase clearance rate less than 10, DNA residual quantities.
4. biological adherence preventing material according to claim 1, it is characterised in that：The extracellular matrix includes collagen egg
In vain, polysaccharide material, active factors and growth factor；The collagen is I-type collagen and type III, IV types and VI type glue
The composition of former albumen；The polysaccharide material is the composition comprising chondroitin sulfate and hyaluronic acid；The active factors bag
Fibre-bearing Fibronectin, laminin, the composition of integrin and its part；The growth factor composition be alkaline growth because
Son and VEGF.
5. biological adherence preventing material according to claim 1, it is characterised in that：The first surface is vacuum dried group
Knit the surface that material is formed；The surface that the second surface is formed by freeze-dried organization material；Described is degradable
For degradation in vivo time 1-3 month；Described biological adherence preventing material tensile break strength is more than 40N/cm, sutures confining force
More than 8N.
6. a kind of preparation method of biological adherence preventing material, it is characterised in that：Step includes：
(1) the preposition processing of tissue：Intestinal submucosa tissue material is taken, cleans and is filtered dry water；
(2) inactivation of virus：Inactivation of virus is carried out using Peracetic acid-alcohol solution dipping intestinal submucosa tissue material；
(3) clean：Under ultrasound environments, handled respectively with PBS solution and water is cleaned；
(4) immunogene is removed：The intestinal submucosa tissue material obtained after step (3) is cleaned carries out immunogene removal, bag
Include the first step and use NaOH solution, hydrochloric acid clean and change liquid is used in sonic oscillation processing after processing；Second step, which is used, contains trypsase
With EDTA PBS solution, multiple frequency ultrasonic vibration；3rd step uses NaCl solution, sonic oscillation；
(5) clean：Under ultrasound environments, cleaned respectively with PBS solution and water；
(6) it is dried in vacuo：The immunogene cleaned by step (5) removal material is fixed on mould and drying；
(7) it is freeze-dried：The organization material that the immunogene cleaned by step (5) is removed is covered in through doing that step (6) is obtained
On organization material after dry, together it is freeze-dried.
7. the preparation method of biological adherence preventing material according to claim 6, it is characterised in that：
The concentration of volume percent of Peracetic acid is 0.1%-5%, ethanol in Peracetic acid-ethanol solution that step (2) is used
Concentration of volume percent be 5%-40%, the volume ratio of Peracetic acid-ethanol solution and intestinal submucosa tissue material is
(3-20) ︰ 1, inactivation time 2-4 hours, temperature range is 10-40 DEG C；
Step (3) is cleaned：Cleaned, then cleaned with water to detection electrical conductivity with PBS solution in supersonic wave cleaning machine
Terminated for below 10 μ S/cm, obtain submucous layer of small intestine host material.Wherein PBS solution pH value range is 6-8, used
Water is the purified water Jing Guo purification process；
Step (4) first step uses concentration for 0.1-0.5mol/L NaOH solution, sonic oscillation processing 1-30min, temperature 20-
25 DEG C, the volume ratio of solution and intestinal submucosa tissue material is 20-40：1, ultrasonic power is in more than 3000W；After processing
The hydrochloric acid solution clean and change liquid for being 0.1-0.5mol/L with concentration, and pH is adjusted to neutrality；Second step, which is used, contains quality percentage
The PBS solution that the pH value for the EDTA that the trypsase and concentration that specific concentration is 0.01-0.2% are 0.1-1mmol/L is 7, multifrequency
Sonic oscillation 10-80min, multiple frequency ultrasonic at least includes two supersonic frequencies, and Frequency scope is 20-40KHz, higher frequency
For 60-90KHz, wherein low frequency handles 5-40min, high-frequency therapeutic treatment 5-40min, 30 DEG C of temperature, solution and submucous layer of small intestine group
The volume ratio for knitting material is (30-40)：1, ultrasonic power is in more than 5000W；3rd step uses concentration for 0.8-
1.5mol/L NaCl solution, sonic oscillation processing 10min, 20 DEG C of temperature, the body of solution and intestinal submucosa tissue material
Product is than being 20-40：1, ultrasonic power is in more than 3000W；
Cleaning process is in the step (5)：Cleaned in supersonic wave cleaning machine with PBS solution, then with the injection of cooling
Water is cleaned to the water for injection electrical conductivity difference before and after detection cleaning and terminated for below 1 μ S/cm, obtains submucous layer of small intestine matrix material
Material；Wherein PBS solution pH value range is 6-8；
Drying described in step (6) is that immunogene removal organization material is placed in into vacuum environment, and environment temperature is 25-40 DEG C, when
Between be 8-16h, prepare shiny surface；
Freeze-drying described in step (7) is：Pre-freeze is incubated 1-2 hours to -45 DEG C, then adjusts the temperature to -15 DEG C, insulation
5-7 hours, then adjust the temperature to 0 DEG C, 2 hours are incubated, 25 DEG C are finally adjusted the temperature to, 4 hours are incubated, one-sided smooth one is formed
The coarse centre in face is the degradable biological adherence preventing material of gradual change attachment structure.
8. the preparation method of biological adherence preventing material according to claim 6, it is characterised in that：Step is also gone out including (8)
Fungi degradation is analysed：Sterilized using oxirane, sterilising conditions are：40 DEG C of temperature, soaking time 4 hours, humidity 60%, epoxy second
Alkane concentration is 600mg/L, sterilization time 6 hours；Resolving is carried out in the Resolution Room of ventilation, temperature control 20 DEG C it
Between, 14 days time.
9. a kind of application of biological adherence preventing material in implant, it is characterised in that：Described implant is used for abdominal cavity, thoracic cavity
Or in pelvic cavity the surface of a wound Anti-adhesive.
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| CN116617471A (en) * | 2023-07-24 | 2023-08-22 | 成都贝施美医疗科技股份有限公司 | Oral collagen membrane with double-layer structure and preparation method thereof |
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