CN102266585A - Biological composite patch for female pelvic floor and manufacturing method thereof - Google Patents
Biological composite patch for female pelvic floor and manufacturing method thereof Download PDFInfo
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- CN102266585A CN102266585A CN2011102030987A CN201110203098A CN102266585A CN 102266585 A CN102266585 A CN 102266585A CN 2011102030987 A CN2011102030987 A CN 2011102030987A CN 201110203098 A CN201110203098 A CN 201110203098A CN 102266585 A CN102266585 A CN 102266585A
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Abstract
The invention relates to a biological composite patch for female pelvic floor and a manufacturing method thereof, and according to the patch, a surface of a polypropylene mesh is coated with a bladder acellular matrix of an animal donor. A bladder obtained from an animal donor needs to be treated by decellularization and freeze-drying; after disinfection, the treated bladder is preserved by plastic packaging at a low temperature; then the obtained bladder acellular matrix is coated onto a surface of a polypropylene mesh. The invention effectively combines biomaterials with inorganic materials, and with the integration of the advantages of both materials, obviously excellent using effect is obtained. Thereinto, the external bladder matrix has the effect of insulating the polypropylene mesh from host tissue, and can significantly improve the biocompatibility. After the patch is implanted, fibrous tissue of a patient is induced to grow into the patch, and finally the restoration of the fibrous tissue of the patient itself is realized.
Description
Technical field
The present invention relates to biomedical materials field, particularly relate to a kind of sticking patch that is used to repair the female pelvic cavity tissue defects.
Background technology
The female pelvic functional disorder disease is commonly encountered diseases, the frequently-occurring disease of middle aged and aged women more than 50 years old, causes for the life of middle aged and aged women and health and seriously influences.Present Therapeutic Method is guipure or the sheet with some polypropylene for medical article class synthetic materials, carries out pelvic floor function by surgical operation and rebuilds.Relative biomaterial, inorganic material easily causes serious inflammatory reaction.Clinical effectiveness shows, polypropylene mesh has organizes rodent shortcoming, its biocompatibility is poor, easily produce not accommodating of foreign body stimulation and organize aggressivity, sexual anhedonia etc., and, simple polypropylene mesh is difficult and implant local the fusion, adds in vivo effects such as ageing hardening becomes fragile, friction, displacement, eating thrown, the normal bad complication such as bladder, vagina that pierce through.Once there was the scholar on probation to take off the cell cadaveric fascia for overcoming these drawbacks, carries out pelvic floor function from body fascia, TPR raw material and rebuild.But these materials all have peculiar separately defective to be difficult to overcome, and are all under test.And in the substitution material, xenogenesis acellular matrix material has ideal characterisitics most, its wide material sources, intensity is good, and contains bioactie agent, and histocompatibility is outstanding, it is ideal repair materials at the bottom of the following basin, unique defective is that natural degradation causes its late result instability, under the present stage technical conditions, still can't replace the status of polypropylene material.But can be used as polyacrylic auxiliary material, purpose is to improve biocompatibility, thereby reduces the polypropylene complication.
Summary of the invention
In order to overcome above-mentioned defective in the prior art, the invention provides a kind of sticking patch that is used to repair the female pelvic cavity tissue defects, with the auxiliary material of xenogenesis acellular matrix material as polypropylene mesh, with raising biocompatibility, reduction polypropylene complication, thus the tangible problem of inflammatory reaction of solution implant site.
Simultaneously, the present invention also provides the preparation method of above-mentioned sticking patch.
Technical scheme of the present invention is:
A kind of biological composite patch that is used for female pelvic is at the bladder acellular matrix that is coated with animal donor of polypropylene mesh.Wherein, described bladder acellular matrix has preferably been inoculated BMSCs.
Selectively, described polypropylene mesh is rectangle, square, circle or obform body.
Further, the machine direction of the orientation of described polypropylene fibre and bladder acellular matrix is the 25-75 degree, preferred 40-50 degree.
Further, described polypropylene mesh is built between two described bladder acellular matrixes, and absorbable suture connects two described bladder acellular matrixes, in polypropylene mesh is wrapped in.
The present invention also provides a kind of preparation method that is used for the biological composite patch of female pelvic, comprises the steps:
(1) preparation bladder acellular matrix
Get the bladder of animal donor, remove mucous layer and placenta percreta, obtain bladder substrate;
Bladder substrate is taken off cell processing and lyophilizing processing; After the sterilization, the low temperature plastic packaging is preserved;
(2) get polypropylene mesh, the bladder acellular matrix of above-mentioned acquisition is wrapped in the surface of described polypropylene mesh.
Further, described step (2) is specially: polypropylene mesh is built between two described bladder acellular matrixes, two described bladder acellular matrixes is sewed up with absorbable suture, make that polypropylene mesh is wrapped in.
Further, the cell processes that takes off of described bladder substrate is: adopt and to take off Cell sap and buffer and bladder substrate is soaked repeatedly and wash.
Preferably, describedly take off the mixed liquor that Cell sap is Tris and Phenylmethanesulfonyl fluoride.
The construction features of multifunctional bio sticking patch of the present invention adopts animal to take off cell bladder material (urinarybladder matrix, be called for short UBM), through certain working procedure, remove the cell component of animal's bladder, keep the acellular matrix of animal's bladder, and contain the various biotic components of cell growth, as angiogenesis factor, TGF-β, VFGF etc., with the polypropylene mesh parcel, constitute " sandwich " structure then.The present invention carries out effective combination with biomaterial and inorganic material, and comprehensively the advantage separately of the two has obtained obvious good result of use.Wherein, outer field bladder substrate can play the effect of isolating polypropylene mesh and host tissue, can significantly improve biocompatibility.Simultaneously, can also regulate immunoreation to the immunological adaptation sex reversal, effectively reduce its immune immunogenicity, organizing of flexibility and people is close, drawbacks such as no foreign body stimulation, eating thrown can induce patient's fibrous tissue to grow into after the implantation, form the reparation of patient self fibrous tissue at last, can be used for the bulging of vagina front and rear wall, uterine prolapse, the handicapped operative treatment of female pelvic such as stress incontinence.Simultaneously, biological composite patch of the present invention can improve the stability of sticking patch greatly through above-mentioned PROCESS FOR TREATMENT.
Description of drawings
Fig. 1 is the three-dimensional exploded view of the embodiment of the invention;
Fig. 2 is the structural representation of the embodiment of the invention;
Fig. 3 implants for different experiments group of the present invention and implants cd4 cell SABC testing result in the local organization under the vaginal mucosa;
Fig. 4 implants for different experiments group of the present invention and implants cd8 cell SABC testing result in the local organization under the vaginal mucosa;
Fig. 5 implants for different experiments group of the present invention and implants CCR4 cellular immunization group testing result in the local organization under the vaginal mucosa;
Fig. 6 implants for different experiments group of the present invention and implants CXCR3 cellular immunization group testing result in the local organization under the vaginal mucosa.
The specific embodiment
Below in conjunction with accompanying drawing technical scheme of the present invention is elaborated.
For the person of ordinary skill in the field, from detailed description of the invention, above-mentioned purpose of the present invention, feature and advantage will be apparent.
The invention provides a kind of biological composite patch that is used for female pelvic, sticking patch comprises that polypropylene mesh 1 and bladder take off acellular matrix 2.Wherein, polypropylene mesh is the commercially available prod, and the present invention does not limit the structure and the shape of polypropylene mesh 1, that is to say, polypropylene mesh 1 can adopt rectangle, square, circle or other obform body.When choosing the polypropylene mesh of clinical practice, can increase the porosity of this net sheet, be about to conventional 2mm and be increased to 4-5mm, to reduce the density of polypropylene material, this way can further reduce immunoreation intensity.The bladder that the present invention uses takes off acellular matrix 2 and comes from animal donor, and through taking off cell, sterilization processing, this animal donor includes but not limited to pig, cattle, sheep, preferred pig source property.SIS, AP, UBM, ADM, CEM are five kinds of the most frequently used pig acellular matrix materials, all can be used as selection of the present invention, wherein preferred UBM, detect from correlation properties, comprise that water absorption, degradation cycle, antibiotic property, cell compatibility, 5 aspects of biomechanical property see, found that UBM has the longest degradation cycle, the strongest antibiotic property, the highest water absorption, best biomechanical property and outstanding histocompatibility are very suitable for the special environment at the bottom of the basin.
As shown in Figure 1, 2, in the present embodiment, it is (clear for expressing that bladder takes off the UBM that acellular matrix 2 is selected from pig, below the UBM 2 of Chu Xianing all is bladder acellular matrixes 2), UBM 2 isolates composition as the biology of composite patch, UBM 2 and 1 two kinds of materials of polypropylene mesh is compound: that above-mentioned polypropylene mesh 1 is built between two described UBM 2, adopt suture way to connect two described UBM 2 with the absorbable suture (not shown), polypropylene mesh 1 is wrapped in the inside the most at last, as shown in Figure 2.Stitching thread can be any medical grade absorbable suture used in the prior art, the stitching thread of making such as the polymeric polyglycolide-polylactide material.As shown in Figure 1, described polypropylene mesh 1 adopts rectangle, and cardinal principle fiber 21 directions (being represented by dotted lines machine direction among the figure) of the orientation of the fiber 11 of polypropylene mesh 1 wherein and UBM 2 are the 40-50 degree, preferably 45 degree.Adopt the sticking patch of this structure, polypropylene fibre 11 plays the support effect of " reinforcing bar ", and UBM 2 occupy the outside, not only strengthened the intensity of UBM 2, and do not influence the histocompatibility of UBM 2, and the fiber orientation of polypropylene mesh 1 can not influence the compliance of sticking patch substantially, can reach the compliance level of simple UBM.
Above-mentioned UBM 2 is taken from the bladder of animal donor, obtains bladder substrate behind removal mucous layer and the placenta percreta; Then bladder substrate is taken off cell processing and lyophilizing processing; After the sterilization, adopt the low temperature plastic packaging to preserve.Wherein, when taking off cell and handling, need soak repeatedly bladder substrate and wash with taking off Cell sap and buffer.It will be appreciated by persons skilled in the art that this takes off Cell sap and buffer can be any suitable product, preferably take off the mixed liquor that Cell sap adopts Tris and Phenylmethanesulfonyl fluoride, the mixed weight ratio is controlled at 80-120: 1.
As preferred embodiment, the present invention can also be inoculated into stem cell and make up tissue engineering material on the biomaterial, be that above-mentioned UBM can also inoculate BMSCs (bone marrowmesenchymal stem cells mesenchymal stem cells MSCs), one week of In vitro culture by prior art.The biological assessment that will describe in detail also can be classified it as a contrast object below.
Provide the concrete manufacture method of UBM below, but,, also can adopt other feasible in prior art method as those skilled in the art not as restriction of the present invention.
1, the preparation of Vesica sus domestica acellular matrix
Get the fresh pig bladder specimen of enclosed grazing pig (the about 200kg of body weight) in half an hour after death, under super-clean bench, remove mucous layer and placenta percreta with mechanical means.
2, taking off cell handles
Handle for the first time: get the beaker of a clean 1000ml, take by weighing 12.11g Tris and Phenylmethanesulfonyl fluoride 0.0122g, add about 800ml deionized water, fully stir evenly dissolving, transfer pH value to 8.0.Solution to going in the clean 5000ml beaker, and is put into beaker with bladder substrate and soaked.Beaker placed on the magnetic stirring apparatus stirred 12 hours, spend the night.
Handle for the second time: get the beaker of a clean 1000ml, take by weighing 12.11g Tris and Phenylmethanesulfonyl fluoride 0.0122g, add about 800ml deionized water, fully stir evenly dissolving, transfer pH value to 8.0,, and bladder substrate is put into the beaker immersion then to going in the clean 5000ml beaker.Beaker placed on the magnetic stirring apparatus stirred 12 hours.
Handle for the third time: get the beaker of a clean 1000ml, take by weighing 12.11g Tris and Phenylmethanesulfonyl fluoride 0.0122g, add about 800ml deionized water, fully stir evenly dissolving, transfer pH value to 8.0,, and bladder substrate is put into the beaker immersion then to going in the clean 5000ml beaker.Beaker placed on the magnetic stirring apparatus stirred 12 hours, spend the night.
Handle for the 4th time: get the beaker of a clean 1000ml, take by weighing 12.11g Tris and Phenylmethanesulfonyl fluoride 0.0122g, add about 800ml deionized water, fully stir evenly dissolving, transfer pH value to 8.0,, and bladder substrate is put into the beaker immersion then to going in the clean 5000ml beaker.Beaker placed on the magnetic stirring apparatus stirred 12 hours.
Handle for the 5th time: get the beaker of a clean 500ml, take by weighing 12.11g Tris and Phenylmethanesulfonyl fluoride 0.0122g, draw 20mlTriton X-100, put into beaker, add suitable quantity of water, fully stirring and dissolving, be settled to 2000ml, and change solution over to the 5000ml beaker.With putting into the solution of configuration before after an amount of phosphate buffer washing bladder substrate, beaker is placed on the magnetic stirring apparatus stirred 12 hours, spend the night.
Handle for the 6th time: the beaker of getting a clean 500ml, take by weighing 12.11g Tris and Phenylmethanesulfonyl fluoride 0.0122g, draw 20mlTriton X-100, put into beaker, add suitable quantity of water, fully stirring and dissolving, be settled to 2000ml, and change solution over to the 5000ml beaker, and bladder substrate is put into beaker soak, beaker is placed on the magnetic stirring apparatus stirred 12 hours.
Handle for the 7th time: the beaker of getting a clean 500ml, take by weighing 12.11g Tris and Phenylmethanesulfonyl fluoride 0.0122g, draw 20mlTriton X-100, put into beaker, add suitable quantity of water, abundant stirring and dissolving, be settled to 2000ml, and change solution over to the 5000ml beaker, bladder substrate is put into beaker soak, beaker placed on the magnetic stirring apparatus stir, spend the night.
Handle for the 8th time: the beaker of getting a clean 500ml, take by weighing 12.11g Tris and Phenylmethanesulfonyl fluoride 0.0122g, draw 20mlTriton X-100, put into beaker, add suitable quantity of water, fully stirring and dissolving, be settled to 2000ml, and change solution over to the 5000ml beaker, and bladder substrate is put into beaker soak, beaker is placed on the magnetic stirring apparatus stirred 12 hours.
Handle for the 9th time: DNA enzyme and two enzymic digestion solution of RNA enzyme: the NaCl of 1mol/L (containing DNase and each 40U/ml of RNA enzyme) of configuration 2000ml, after an amount of phosphate buffer rinsing bladder substrate, bladder substrate is put into two enzymic digestion liquid soaked 12 hours, spend the night.
Handle for the tenth time: the DNA enzyme of configuration 2000ml and two enzymic digestion solution of RNA enzyme, and bladder substrate is put into two enzymic digestion liquid soaked 12 hours.
The tenth single treatment: get the beaker of a clean 500ml, take by weighing 12.11g Tris and 20g sodium lauryl sulphate, put into beaker, add suitable quantity of water, fully stirring and dissolving is settled to 2000ml.With putting into the solution of configuration before after an amount of phosphate buffer rinsing bladder substrate, beaker is placed on the magnetic stirring apparatus stirred 12 hours, spend the night.
The tenth after-treatment: the beaker of getting a clean 500ml, take by weighing 12.11g Tris and 20g sodium lauryl sulphate, put into beaker, add suitable quantity of water, abundant stirring and dissolving, be settled to behind the 2000ml in the beaker of going into 5000ml, bladder substrate put into beaker soak, beaker is placed on the magnetic stirring apparatus stirred 12 hours.
Handle for the 13 time: the beaker of getting a clean 500ml, take by weighing 12.11g Tris and 20g sodium lauryl sulphate, put into beaker, add suitable quantity of water, abundant stirring and dissolving is settled to behind the 2000ml in the beaker of going into 5000ml, bladder substrate is put into beaker soak, beaker placed on the magnetic stirring apparatus stirred 12 hours, spend the night.
Handle for the 14 time: the beaker of getting a clean 500ml, take by weighing 12.11g Tris and 20g sodium lauryl sulphate, put into beaker, add suitable quantity of water, abundant stirring and dissolving, be settled to behind the 2000ml in the beaker of going into 5000ml, bladder substrate put into beaker soak, beaker is placed on the magnetic stirring apparatus stirred 12 hours.
Handle for the 15 time: dispose the phosphate buffer of 2000ml, and bladder substrate was put into rinsing 12 hours, spend the night.
Handle for the 16 time: dispose the phosphate buffer of 2000ml, and bladder substrate was put into rinsing 12 hours.
Handle for the 17 time: dispose the phosphate buffer of 2000ml, and bladder substrate was put into rinsing 12 hours, spend the night.
Handle for the 18 time: dispose the phosphate buffer of 2000ml, and bladder substrate was put into rinsing 12 hours.
3, lyophilizing is handled:
Bladder after the above-mentioned processing has become and has taken off cell UBM, takes out back procedural cooling to-80 degrees centigrade 16 hours.
4, disinfect:
The degerming of CO60 radiation gamma, 4 ℃ of preservations are standby.
Be that experimental subject enters Implantation Test below with the animal, the composite patch material that the present invention obtains is done biologically evaluation in the body.Mainly comprise at the bottom of four kinds of different materials such as the UBM of UBM, composite polypropylene of simple UBM, inoculation BMSCs and simple polypropylene mesh are as basin and repair the biological reactivity that biological sticking patch implants.
One, laboratory animal
The SD rat: the SPF level, 200 ± 10g, Third Military Medical University's Experimental Animal Center provides.
Two, main agents and material
Three, main agents preparation
1.0.25% trypsin solution
Trypsin 0.25g through 0.22 μ m filtering with microporous membrane degerming, is distributed into the 4ml/ bottle with D-Hank`s liquid 100ml dissolving mixing, and-20 ℃ of preservations are standby.
2.PBS solution
Finished product PBS powder adds ddH2O 100ml, stirs and to make it to dissolve fully, changes in the 2000ml volumetric flask, adds ddH2O to graduation mark, mixing, and measuring pH value is 7.5.Divide to be filled in 500ml, the 100ml vial, after the disinfection with high pressure steam sterilization, 4 ℃ of refrigerators are preserved standby.
3. two anti-culture medium of penicillin, streptomycin
The preparation of penicillin solution: penicillin 800,000 units add normal saline to 80ml, and calculating its concentration according to formula is 10000U/ml, and the 0.22um micropore filter filters the back packing, and-20 ℃ store for future use.Ratio in 1% during use adds in the culture medium.The preparation of streptomycin solution: get streptomycin 1.0g, add physiological saline solution to 100ml, then concentration is: 10000 μ g/ml, and 0.22 μ m micropore filter filters the back packing, and-20 ℃ store for future use.Ratio in 1% during use adds in the culture medium.
4.DMEM-F12 full culture medium
DMEM-F12+10% hyclone+1% green grass or young crops/streptomycin
Four, test method
(1) implants sticking patch
1. experiment is divided into 5 groups, and every group of 12 rats are implanted UBM respectively, the UBM of inoculation BMSCs, the UBM of composite polypropylene net sheet (the foregoing description acquisition), simple polypropylene mesh, sham operated rats.
2. cut off vaginal mucosa with operating scissors, with UBM, the UBM of inoculation BMSCs, the UBM of composite polypropylene net sheet, simple polypropylene mesh is implanted the vaginal mucosa below, and sham operated rats is not implanted any material, and all the other operation sequences are identical;
3. the stitching wound is smeared chlortetracycline on wound, protects from infection.
4. 1,2,3,4 weeks were put to death rat after operation, and each 3, observe and implant the variation substantially that possesses, get implanting tissue and surrounding tissue, 10% neutral formalin is fixed, and carries out histology and SABC and detects.
(2) histology of implanting tissue and SABC detect
1. fixed tissue carries out paraffin section and HE dyeing, prepares white sheet simultaneously and carries out the SABC detection;
2. SABC detects
1) getting white toasted 20 minutes in 60 ℃ of calorstats;
2) tissue slice is placed dimethylbenzene soaked 10 minutes, soaking 10 minutes behind the replacing dimethylbenzene;
3) soaked five minutes in the dehydrated alcohol;
4) soaked five minutes in 95% ethanol;
5) soaked five minutes in 75% ethanol;
6) distilled water flushing is put PBS and was soaked 5 minutes;
7) with water-bath 0.01M sodium citrate buffer (pH6.0) is heated to about 95 ℃, puts into tissue slice heating 10-15 minute, carry out antigen retrieval;
8) drip 3%H2O2, incubated at room 15min is with the blocking-up endogenous peroxydase;
9) PBS flushing, each 2 minutes, totally 3 times;
10) add the sealing of 10% lowlenthal serum, incubated at room 10 minutes;
11) serum deprivation that inclines, drip one anti-, 37 ℃ hatch 2h after, 4 ℃ of wet boxes spend the night.
12) get the section of overnight incubation, PBS flushing, each 2 minutes, totally 3 times;
13) drip biotin labeling goat anti-rabbit igg (1%BSA-PBS dilution), hatched 20 minutes for 37 ℃;
14) PBS flushing, each 2 minutes, totally 3 times;
15) drip Radix Cochleariae officinalis enzyme labelling strepto-avidin (PBS dilution), hatched 20 minutes for 37 ℃;
16) PBS flushing, each 2 minutes, totally 3 times;
17) with diaminobenzidine (DAB) colour developing, haematoxylin redyeing, mirror are observed the colour developing result down;
18) tap water fully washes, gradient alcohol dehydration, and dimethylbenzene is transparent, the resinene mounting.
Five, result of the test
(1) gross examination of skeletal muscle
Implantation Test is after 1 week, as seen the simple UBM color of implanting is slightly yellowish, the UBM color that the UBM color of inoculation BMSCs is more simple is shallow slightly, and simple polypropylene mesh group has slight infection phenomenon, the UBM color of composite polypropylene net sheet is darker, and the perusal effect is poor than the UBM of UBM and inoculation BMSCs, but obviously is better than simple polypropylene mesh, sham operated rats is obviously not unusual, does not see infection.Implant the 2nd week of back, the UBM that implants and 1 all preceding no significant changes, there are fusion phenomenon in UBM and the rat autologous tissue of inoculation BMSCs, implanting tissue lightens little yellowish, simple polypropylene mesh group does not merge with rat tissue, boundary clear, the UBM of composite polypropylene net sheet becomes slightly yellowish, and polypropylene mesh and tissue are inlayed and are grown together.Implant the 3rd week of back, the simple UBM that implants can't obviously offer an explanation in vivo, implant part has slight yellowish variation, degraded may take place in the UBM major part of implanting, the UBM of inoculation BMSCs also can't distinguish, the implant part of simple polypropylene mesh has incrustation to occur, and the UBM sticking patch of composite polypropylene does not find the UBM of implantation, and it is local that visible polypropylene mesh is blended in implantation.The observed result in the 4th week and the 3rd all no significant differences.
(2) histology of implant site changes
Simple UBM implantation group is implanted a visible significantly inflammatory reaction in week back, prolongation in time after this, and inflammatory reaction alleviates gradually, but around the time, appointing has slight inflammatory reaction.Tangible inflammatory reaction also took place after the UBM of inoculation BMSCs implanted a week, but its degree is starkly lower than simple UBM implantation group, after this prolong in time, inflammatory reaction reduces gradually, in the time of around the, implant the inflammatory cell that minute quantity is only arranged in the partial tissue, in its each stage after implantation, inflammatory reaction all is starkly lower than simple UBM implantation group.Simple polypropylene mesh implantation group has stronger inflammatory reaction, and apparently higher than other each groups, and this intensive inflammatory reaction was followed in the whole test observation stage.The inflammatory reaction of the UBM implantation group of composite polypropylene net sheet is implanted category seemingly with simple UBM, and the initial stage is comparatively strong, after this reduces gradually.Sham operated rats is not seen tangible inflammatory reaction.
(3) SABC testing result
(1) CD4 SABC testing result
Simple UBM implantation group implanted for the 1st week and the 2nd all cd4 cell ratios are respectively 37.1 ± 9.4% and 27.9 ± 8.1%, and after this its ratio descends rapidly, and the 3rd week and the 4th all ratios are respectively 10.6 ± 5.1% and 8.3 ± 2.9%.The UBM implantation afterreaction of inoculation BMSCs and simple UBM category are seemingly, but cd4 cell each stage after implantation all is lower than the UBM group, the cd4 cell ratio that its 1st thoughtful the 4th week is implanted local organization is respectively: 28.5 ± 4.7%, 18.2 ± 3.5%, 8.3 ± 3.2% and 5.6 ± 2.4%.And simple polypropylene mesh group cd4 cell ratio in each stage after implantation all maintains a higher level, the cd4 cell ratio that its 1st thoughtful the 4th week is implanted local organization is respectively: 62.9 ± 11.7%, 43.5 ± 8.7%, 39.2 ± 7.4% and 38.7 ± 6.9%.The UBM of reaction tendency after the UBM group of composite polypropylene is implanted and UBM combination inoculation BMSCs is similar, but implant each stage cd4 cell ratio of back and all be higher than above two groups, and significantly be lower than simple polypropylene mesh group, the cd4 cell ratio that its 1st thoughtful the 4th week is implanted local organization is respectively: 46.3 ± 10.5%, 32.6 ± 5.4%, 28.9 ± 4.3% and 16.4 ± 3.5%.Matched group all significantly is lower than other each groups in the cd4 cell ratio in each stage, and the cd4 cell ratio that its 1st thoughtful the 4th week is implanted local organization is respectively: 5.6 ± 2.3%, 4.8 ± 2.7%, 6.4 ± 2.9% and 5.3 ± 1.7%.Referring to Fig. 3.
(2) CD8 SABC testing result
Simple UBM implantation group implanted for the 1st week and the 2nd all cd8 cell ratios are respectively 21.6 ± 3.3% and 21.5 ± 2.7%, and after this its ratio descends rapidly, and the 3rd week and the 4th all ratios are respectively 6.2 ± 2.4% and 5.5 ± 2.8%.The UBM of inoculation BMSCs implants afterreaction and simple UBM category seemingly, and cd8 cell ratio that its 1st thoughtful the 4th week is implanted local organization is respectively: 23.4 ± 3.9%, 21.3 ± 3.5%, 8.1 ± 2.7% and 7.5 ± 2.7%.And simple polypropylene mesh group cd8 cell ratio in each stage after implantation all maintains a higher level, the cd8 cell ratio that its 1st thoughtful the 4th week is implanted local organization is respectively: 37.3 ± 4.6%, 34.4 ± 3.5%, 32.6 ± 2.8% and 30.7 ± 3.6%.The UBM of reaction tendency after the UBM group of composite polypropylene is implanted and UBM combination inoculation BMSCs is similar, but implant each stage cd8 cell ratio of back and all be higher than above two groups, and significantly be lower than simple polypropylene mesh group, the cd8 cell ratio that its 1st thoughtful the 4th week is implanted local organization is respectively: 25.7 ± 2.9%, 23.4 ± 3.2%, 12.6 ± 2.7% and 10.4 ± 3.6%.Matched group all significantly is lower than other each groups in the cd8 cell ratio in each stage, and the cd8 cell ratio that its 1st thoughtful the 4th week is implanted local organization is respectively: 6.1 ± 2.3%, 5.4 ± 2.8%, 6.8 ± 1.9% and 6.3 ± 1.2%.Referring to Fig. 4.
(3) CCR4 SABC testing result
It is the 12.3 ± 2.1%, the 2nd week to be rapidly increased to 55.5 ± 5.6% that simple UBM implantation group is implanted the 1st all CCR4 cell proportions, and the 3rd week and the 4th week fall after rise, are respectively 13.2 ± 2.2% and 12.3 ± 3.7%.The UBM implantation afterreaction trend of inoculation BMSCs and simple UBM category are seemingly, the CCR4 cell proportion that its 1st thoughtful the 4th week is implanted local organization is respectively: 13.4 ± 2.3%, 62.3 ± 5.3%, 17.9 ± 2.5% and 15.7 ± 2.3%, except that first week, itself and each all ratio all are significantly higher than the UBM group.Simple polypropylene mesh group CCR4 cell proportion in each stage after implantation all maintains a lower level, the CCR4 cell proportion that its 1st thoughtful the 4th week is implanted local organization is respectively: 6.3 ± 1.4%, 8.4 ± 2.3%, 6.2 ± 2.7% and 7.3 ± 1.8%.The UBM of reaction tendency after the UBM group of composite polypropylene is implanted and UBM combination inoculation BMSCs is similar, but implant each stage CCR4 cell proportion of back and all be lower than above two groups, and be significantly higher than simple polypropylene mesh group, the CCR4 cell proportion that its 1st thoughtful the 4th week is implanted local organization is respectively: 9.3 ± 2.7%, 24.6 ± 3.5%, 9.6 ± 2.3% and 8.4 ± 3.1%.Matched group all significantly is lower than other each groups at the CCR4 in each stage cell proportion, and the CCR4 cell proportion that its 1st thoughtful the 4th week is implanted local organization is respectively: 2.3 ± 1.6%, 3.8 ± 2.4%, 1.9 ± 1.5% and 3.2 ± 1.2%.See Fig. 5.
(4) CXCR3 SABC testing result
It is that the 23.8 ± 4.6%, the 2nd circumference dropped down onto for the 16.6 ± 3.2%, the 3rd week and the 4th week was respectively 9.6 ± 2.4% and 5.3 ± 2.6% that simple UBM implantation group is implanted the 1st all CXCR3 cell proportions.The UBM implantation afterreaction trend of inoculation BMSCs and simple UBM category are seemingly, the CXCR3 cell proportion that its 1st thoughtful the 4th week is implanted local organization is respectively: 8.9 ± 2.3%, 7.8 ± 2.1%, 6.3 ± 2.1% and 3.2 ± 1.3%, each all ratio all significantly is lower than the UBM group.Simple polypropylene mesh group CXCR3 cell proportion in each stage after implantation all maintains a higher level, the CXCR3 cell proportion that its 1st thoughtful the 4th week is implanted local organization is respectively: 43.7 ± 6.4%, 38.9 ± 7.3%, 26.4 ± 5.4% and 17.6 ± 6.3%.Reaction tendency and UBM after the UBM group of composite polypropylene is implanted are similar, but implant each stage CXCR3 cell proportion of back and all be higher than the UBM group, and significantly be lower than simple polypropylene mesh group, the CXCR3 cell proportion that its 1st thoughtful the 4th week is implanted local organization is respectively: 32.6 ± 7.3%, 29.3 ± 5.7%, 18.6 ± 4.3% and 12.1 ± 2.5%.Matched group all significantly is lower than other each groups at the CXCR3 in each stage cell proportion, and the CXCR3 cell proportion that its 1st thoughtful the 4th week is implanted local organization is respectively: 4.6 ± 1.3%, 5.3 ± 1.8%, 4.3 ± 2.1% and 4.6 ± 1.8%.See Fig. 6.
Above-mentioned testing result shows: different materials are as pelvic floor repairing patch, and the fusion degree of itself and implanting tissue has nothing in common with each other.UBM to the three Zhou Shiyi of UBM and inoculation BMSCs can not distinguish from implant site, only can observe little xanthochromiaization at implant site, may be due to undegradable tissue and in-vivo tissue merge.The simple relatively UBM material of UBM of inoculation BMSCs, its easier and tissue implant site merge, may to be inoculation material that cell arranged implanting preliminary phase to the material of inoculating cell not to its reason, material internal has more cell, can express stromatins such as more fibronectin, therefore be easier to and implant local organization and merge.When finishing to the experimental observation phase, simple polypropylene material still can't merge with implant site, help the tissue of polypropylene material and implant site to merge between the UBM material and polypropylene material is clipped in by sandwich method, in second when week after implantation, just the tissue with implant site merges.Effective fusion of embedded material and implant site will help promoting the performance of embedded material repair, so the UBM of composite polypropylene net sheet, and simple relatively UBM or polypropylene material have tangible use advantage.
All there is certain inflammatory reaction at the material initial stage of implanting, but the inflammatory reaction of the UBM of UBM and inoculation BMSCs reduction gradually with the prolongation of the time of implantation, but simple polypropylene material all shows intensive inflammatory reaction in the whole implantation cycle.The inflammatory reaction of the UBM of composite polypropylene and UBM reaction tendency are transplanted, and its inflammatory reaction degree will significantly be lower than simple polypropylene material group, show that UBM has certain inflammation buffer action in the UBM material of composite polypropylene.Simple relatively UBM, the inflammatory reaction degree that inoculation has the UBM of BMSCs to cause is low, and reason may be the stem cell and different immunocyte interaction generation immunomodulating of implanting, and has suppressed the generation of immunologic rejection on the part degree.
Aspect the immunologic rejection that implants at material, the UBM of inoculation BMSCs has low immunoreactivity material in four kinds of different materials of this test evaluation, is simple UBM secondly, and the poorest is simple polypropylene mesh.By the cellular immunization group coloration result of implanting in the local organization is shown, the UBM group is higher at implantation initial stage cd4 cell ratio, reduce gradually to the 3rd Zhou Shicai, although the UBM of inoculation BMSCs raises to some extent in implantation initial stage cd4 cell ratio, but significantly be lower than the UBM group, simple polypropylene mesh group is all kept a higher CD4 ratio in the whole implantation phase, and the UBM of composite polypropylene net sheet implants back cd4 cell ratio trend and UBM is similar, but each stage all is significantly higher than UBM group after implantation, and significantly is lower than and occupies propylene sticking patch group merely.The trend and the CD4 of cd8 cell were similar after each group was implanted, but ratio significantly is lower than cd4 cell ratio.The result shows that UBM and the simple UBM group CCR4 cell ratio when second week of inoculating BMSCs are higher, and polypropylene mesh CCR4 cell ratio in each stage after implantation is all lower, the UBM of composite polypropylene net sheet is between between the two, CXCR3 cell ratio is just in time opposite with CCR4 in each group, show that UBM has the effect that adapts to of transplanting as pelvic floor repairing patch, and can utilize the immunological rejection of this improved properties polypropylene mesh of UBM, utilize the stem cell inoculation technique also can further induce embedded material to break up simultaneously to immunological adaptation.
In a word, UBM is less as inflammation and immunoreation that pelvic floor repairing patch has initiation, and the induction of immunity reaction changes to immunological adaptation, and UBM also can modify polypropylene mesh, play that inflammation is isolated and immunoregulatory effect, the UBM of the composite polypropylene net sheet among the present invention is a kind of outstanding selection of repair materials at the bottom of the basin.
It should be noted that; the above only is preferred embodiment of the present invention; be not so limit scope of patent protection of the present invention, the present invention can also carry out the improvement of material and structure to the structure of above-mentioned various parts, or adopts the technical equivalents thing to replace.So the equivalent structure that all utilizations description of the present invention and diagramatic content are done changes, or directly or indirectly apply to other correlative technology fields and all in like manner all be contained in the scope that the present invention contains.
Claims (10)
1. a biological composite patch that is used for female pelvic is characterized in that, at the bladder acellular matrix that is coated with animal donor of polypropylene mesh.
2. sticking patch according to claim 1 is characterized in that, described bladder acellular matrix has also been inoculated BMSCs.
3. sticking patch according to claim 1 is characterized in that, described polypropylene mesh is rectangle, square, circle or obform body.
4. according to arbitrary described sticking patch among the claim 1-3, it is characterized in that the machine direction of the orientation of described polypropylene fibre and bladder acellular matrix is the 25-75 degree.
5. sticking patch according to claim 4 is characterized in that, the machine direction of the orientation of the fiber of described polypropylene mesh and bladder acellular matrix is the 40-50 degree.
6. sticking patch according to claim 1 is characterized in that, described polypropylene mesh is built between two described bladder acellular matrixes, and absorbable suture connects two described bladder acellular matrixes, in polypropylene mesh is wrapped in.
7. a preparation method that is used for the biological composite patch of female pelvic is characterized in that: comprise the steps:
(1) preparation bladder acellular matrix
Get the bladder of animal donor, remove mucous layer and placenta percreta, obtain bladder substrate;
Bladder substrate is taken off cell processing and lyophilizing processing; After the sterilization, the low temperature plastic packaging is preserved;
(2) get polypropylene mesh, the bladder acellular matrix of above-mentioned acquisition is wrapped in the surface of described polypropylene mesh.
8. preparation method according to claim 7, it is characterized in that: described step (2) is specially: polypropylene mesh is built between two described bladder acellular matrixes, with absorbable suture two described bladder acellular matrixes are sewed up, make that polypropylene mesh is wrapped in.
9. preparation method according to claim 7 is characterized in that, the cell processes that takes off of described bladder substrate is: adopt and to take off Cell sap and buffer and bladder substrate is soaked repeatedly and wash.
10. preparation method according to claim 9 is characterized in that, describedly takes off the mixed liquor that Cell sap is Tris and Phenylmethanesulfonyl fluoride.
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| CN103316379B (en) * | 2013-06-18 | 2015-06-03 | 北京大学人民医院 | Patch for treating female pelvic floor dysfunctional disease and preparation method thereof |
| CN104323871A (en) * | 2014-10-22 | 2015-02-04 | 首都医科大学附属北京妇产医院 | Grid-shaped bracket composite membrane of pelvic floor repairing belt and use method thereof |
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| CN104721889A (en) * | 2015-02-10 | 2015-06-24 | 苏州大学 | Composite polypropylene mesh and preparation method thereof |
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| CN105311678A (en) * | 2015-11-24 | 2016-02-10 | 无锡中科光远生物材料有限公司 | Complex hernia patch and preparing method and application thereof |
| CN105311679A (en) * | 2015-11-24 | 2016-02-10 | 无锡中科光远生物材料有限公司 | Complex hernia patch and preparing method and application thereof |
| CN108096169A (en) * | 2017-12-27 | 2018-06-01 | 广州昕生医学材料有限公司 | A kind of secret repairs gel |
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| CN108939159A (en) * | 2018-07-27 | 2018-12-07 | 吉林大学 | Small intestine acellular matrix film combination metal mesh wall of the chest sticking patch preparation method |
| CN113621247A (en) * | 2021-08-16 | 2021-11-09 | 江苏优创生物医学科技有限公司 | Polymerization mesh-shaped composite patch |
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