CN104695205A - Preparation method of collagen fiber membrane and application thereof - Google Patents
Preparation method of collagen fiber membrane and application thereof Download PDFInfo
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- CN104695205A CN104695205A CN201510114478.1A CN201510114478A CN104695205A CN 104695205 A CN104695205 A CN 104695205A CN 201510114478 A CN201510114478 A CN 201510114478A CN 104695205 A CN104695205 A CN 104695205A
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Abstract
The invention provides a preparation method of a collagen fiber membrane and application thereof. The collagen fiber membrane prepared by the method is outstanding in biocompatibility, relatively high in mechanical strength, applicable to human soft tissue injury repair, and meets the suturing requirement in actual use.
Description
Technical field
The invention belongs to biomedicine field, specifically, the present invention relates to a kind of collagenous fibres membrane preparation method and application thereof.
Background technology
Collagenous fibres have excellent biological safety, and comparatively strong with cellular affinity, also having certain cell inducibility simultaneously, Cell Differentiation can be guided to rise in value, is a kind of prospect tissue engineering bracket material very.Be at present that the film product of raw material mainly contains acellular matrix, collagen freeze-drying sponge, collagen electrospinning film etc. with collagen, but they have self inevitable defect.
Acellular matrix: mechanical strength is good, and there is very reasonably gap structure, excellent environment can be provided for the growth of cell.But acellular matrix is for raw material is obtained by physicochemical method removal cell component by animal skin, pericardium, peritonaeum, little goldbeater's skin etc., this process, not by above-mentioned tissue dispersion, is difficult to likely to cause host that immunoreactive material occurs and removes clean.
Collagen freeze-drying sponge: obtained by the collagen solution freeze-drying of extracting from animal tissue.It has excellent biocompatibility, be conducive to the gap structure that cell grows into, but its intensity is lower, limit he as tooth film, hernia paster, the use of meninx etc. can be sewed up.
Collagen electrospinning film: obtained by electrostatic spinning by collagen solution.It has good biocompatibility, preferably mechanical property and certain gap structure, but collagen electrospinning can use the organic solvents such as hexafluoroisopropanol, its toxicity more greatly, not easily Ex-all.There is bibliographical information electrospinning can destroy the triple helix structure of collagen in addition.
Therefore, it is that product strength prepared by raw material is low with collagen that those skilled in the art are devoted to overcome existing, can not sew up, can not the defect of load.
Summary of the invention
The object of the present invention is to provide a kind of collagenous fibres membrane preparation method and application thereof.
A first aspect of the present invention, provide a kind of method preparing collagen fiber membrane, described method comprises step:
(1) collagenous fibres are dissolved in acid medium, obtain collagenous fibril solution;
(2) described collagenous fibril solution is poured in container, stifling under being placed on volatility alkali environment, obtained described collagen fiber membrane;
Wherein, the bottom of described container is filter structure; Described filter screen is 50-500 object filter screen, and described filter screen allows the water in described sample solution to flow out described container, and the collagen fiber matrix that described collagenous fibril is formed is retained on described filter screen.
In another preference, in described step (1), in described collagenous fibril solution, the concentration of collagenous fibril is 1g/L-200g/L, is preferably 10g/L-100g/L, is more preferably 15g/L-50g/L.
In another preference, the pH of described acid medium is 0-6; Preferably pH is 0.5-5.5; More preferably pH is 1.0-5.0; Optimally pH is 2.0-4.0.
In another preference, described acid medium is aqueous acid.
In another preference, described acid medium is selected from: aqueous hydrochloric acid solution, aqueous sulfuric acid, aqueous solution of nitric acid, aqueous acetic acid or its combination.
In another preference, described volatility alkali is selected from lower group: ammoniacal liquor.
In another preference, described volatility alkali is ammoniacal liquor; Preferably, described ammonia concn is 0.1% (w/w)-5% (w/w); More preferably, described ammonia concn is 0.5% (w/w)-2% (w/w).
In another preference, the fumigation time in described step (2) is 8h-48h; Preferably, described fumigation time is 12h-36h; More preferably, described fumigation time is 18h-30h.。
In another preference, in described step (1), also comprise dispersion steps, use refiner to carry out homogenate, collagenous fibres are fully dissolved in acid medium, form collagenous fibril solution; Preferably, rotating speed is 100r/min-5000r/min, is preferably 500r/min-3000r/min.
In another preference, described Homogenization time is 1min-30min, is preferably 5min-20min.
In another preference, in described step (1), also comprise centrifugal de-bubble step, use centrifuge to carry out centrifugal segregation bubble to described collagenous fibril solution; Preferably, centrifugal rotational speed is 1000r/min-10000r/min, is more preferably 2000r/min-8000r/min.
In another preference, described centrifugation time is 1min-30min, is more preferably 5min-20min.
In another preference, the bottom of described container is filter structure; Preferably, described filter screen is 50-500 object filter screen; More preferably described filter screen is 100-400 object filter screen; Most preferably, described filter screen is 150-250 object filter screen.
In another preference, in described step (2), described collagenous fibril solution is coated on described filter screen, stifling under being then placed on volatility alkali environment.
In another preference, in described step (2), the coating thickness of described collagenous fibril solution is 2-20mm; Preferably coating thickness is 4-15mm; More preferably coating thickness is 5-10mm.
In another preference, in described step (2), after having fumigated, described collagen fiber membrane is taken out dry.
In another preference, after described step (2), also comprise step:
(3) described collagen fiber membrane is placed in phosphate buffer swelling, pure water cleaning postlyophilization.
In another preference, described method also comprises step, and described collagen fiber membrane is carried out heat cross-linking.The method of described heat cross-linking is the conventional method in this area.
In another preference, described method also comprises step, by described collagen fiber membrane sterilization treatment.
In another preference, described collagenous fibres are obtain by the method that acidleach is carried.
A second aspect of the present invention, provides a kind of collagen fiber membrane, and described collagen fiber membrane is prepared by method according to claim 1 and obtains.
In another preference, described collagen fiber membrane TENSILE STRENGTH is between 5MPa-7MPa.
In another preference, the thickness of described collagen fiber membrane is 0.5mm-1.5mm.
A third aspect of the present invention, provide a kind of device preparing collagen fiber membrane, described device comprises: shuttle and volatility alkali container, and described shuttle is placed in the top of described volatility alkali container;
Wherein, described shuttle is set to for holding collagenous fibril sample solution, and the bottom of described shuttle is filter structure.
In another preference, described filter screen is selected from: nylon leaching net, stainless steel filtering net.
In another preference, described filter screen is 50-500 object filter screen; More preferably described filter screen is 100-400 object filter screen; Most preferably, described filter screen is 150-250 object filter screen.
In another preference, the sidewall of described shuttle is made up of stainless steel material.
In another preference, described volatility alkali container is arranged for and holds volatility aqueous slkali.
In another preference, described volatility alkali is ammoniacal liquor.
In another preference, described device also comprises housing, and described shuttle and described volatility alkali container are positioned at described enclosure interior.
In another preference, described housing comprises main body and lid, and when described cover cap closes on the body, described main body and described lid form an airtight space, and described shuttle and described volatility alkali container are positioned at described confined space.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows collagen fiber membrane biological safety (immunogenicity) testing result.
Fig. 2 is the device result schematic diagram preparing collagen fiber membrane of better embodiment of the present invention.
Fig. 3 A, Fig. 3 B respectively illustrate the collagen fiber membrane photo of preparation in embodiment 1.
Detailed description of the invention
The present inventor is by extensive and deep research, obtain a kind of preparation method of collagen fiber membrane, first collagenous fibres are dissolved in acidic aqueous solution, then volatility alkali is adopted to fumigate, induction collagenous fibril forms longer collagenous fibres, and distribute uniformly, air-dryly obtain hard collagem membrane, collagem membrane swelling rear freeze-drying in aqueous systems can obtain collagen fiber membrane finished product.Collagen gel after swelling first can be done crosslinking Treatment and then freeze-drying if needed.Adopt the activity that the collagem membrane prepared in this way saves collagen to greatest extent, have good mechanical property and pore structure simultaneously, and non-collagen tissue is easily removed totally, biological safety is excellent.On this basis, the present invention is completed.
Compared with traditional collagenous fibres membrane preparation method, method proposed by the invention can retain collagenous fibres length, most possibly the mechanical strength of reserved materials and biocompatibility.Tradition collagenous fibres film build method, will pulverize collagenous fiber bundle by homogenizer, obtain collagenous fibres turbid liquid, and then do post-processed.So longer collagenous fibres have been interrupted, destroy its shaping after mechanical strength, and strong mechanism has the risk of collagenous degeneration.
Method of the present invention adopts acid-treated mode to prepare collagen solution (collagenous fibril solution, sample solution), is then issued in the induction of other external conditions such as alkaline steam by collagenous fibril solution and is conigenous assembling, forms long fiber.Fiber solution is air-dry, then swelling, be optionally cross-linked, finally by sample freeze-drying packaging sterilizing or in being directly packaged in containing conserving liquid packaging material and sterilizing.
Can be very easy to according to the method prepare the collagen fiber membrane with better intensity, its thickness, porosity all can regulate and control according to demand.The product finally obtained can be used for the products such as endocranium sticking patch, tooth film, periosteum, nerve trachea, hernia paster.
The preparation method of collagen fiber membrane of the present invention, belongs to biomaterial for medical purpose field, can solve the problem that current collagenous fibres film-strength is low, expands the range of application of collagen fiber membrane.Adopt the collagen fiber membrane prepared by method in the present invention can be widely used in the repairing of various soft tissue in body, as endocranium sticking patch, hernia paster, tooth film, skin reparative membrane, neural sticking patch, tendon repairing etc.
In one preferably embodiment, method of operating of the present invention comprises step:
1. collagenous fibres are dissolved in acidity (pH0-4.8) aqueous solution, obtain homogeneous collagenous fibril solution example.
2. pour in stainless steel frame by sample, stainless steel frame bottom surface 200 order nylon leaching nets are closed.
3. sample is placed in closed container, bottom closed container, fills 1% ammoniacal liquor, until sample pH value to 6.0 ± 0.5 time take out (about placing 20h), air-dry, obtain collagen fiber membrane.
4. by swelling in the phosphate buffer of 0.01M, pH=7.2 for the collagen fiber membrane after air-dry, reach after about 1mm until thickness, take out, clean up with pure water, freeze-drying, can heat cross-linking be carried out if needed.
5. pack after sample cutting, sterilizing.
Present invention also offers a kind of device (as shown in Figure 2) preparing collagen fiber membrane, described device comprises: shuttle 1 and volatility alkali container 2, and described shuttle 1 is placed in the top of described volatility alkali container 2;
Wherein, described shuttle 1 is set to for holding collagenous fibril solution 11, and the bottom of described shuttle is filter structure; Described volatility alkali container 2 is arranged for and holds volatility aqueous slkali 21.
In present embodiment, it is inner that described shuttle 1 can hang on described volatility alkali container 2, and arranging a lid 3, when lid 3 covers on described volatility alkali container 2, forms an airtight space, avoid volatility alkali to leak.
In one preferably embodiment, described device also comprises housing, and described shuttle and described volatility alkali container are positioned at described enclosure interior; Preferably, described housing comprises main body and lid, and when described cover cap closes on the body, described main body and described lid form an airtight space, and described shuttle and described volatility alkali container are positioned at described confined space.
In other embodiments, a lid can be set above described shuttle, when above described cover cap is combined in described shuttle, forms an airtight space, avoid volatility alkali to leak.
Major advantage of the present invention is:
(1) method of the present invention can retain collagenous fibres length, most possibly the mechanical strength of reserved materials and biocompatibility;
(2) the collagem membrane excellent in mechanical performance that the collagen fiber membrane adopting method of the present invention to prepare is prepared compared with additive method, can be used for soft tissue repairing operation in body completely;
(3) the present invention is in collagenous fibril leaching process, the mode of homogenate is adopted to be dispersed in acid solution by collagenous fibril, after stifling can there is the thicker collagenous fiber bundle of self assembly formation in collagenous fibril, no longer homogenized is done to collagenous fiber bundle, and the processing process that traditional method is conveniently follow-up, homogenized to be carried out to collagenous fiber bundle before the forming.Method of the present invention adopt induction self assembly mode original position obtain collagenous fibres, the collagenous fibres obtained are evenly distributed, avoiding collagenous fibres homogenate in forming process causes longer collagenous fibres to be interrupted, remain its shaping after mechanical strength, and avoid strong mechanism and have the risk of collagenous degeneration.
Below in conjunction with specific embodiment, further detailed old the present invention.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.Unless otherwise indicated, otherwise percentage and number calculate by weight.Experiment material used in following examples and reagent all can obtain from commercially available channel if no special instructions.Experiment material used in the embodiment of the present invention all can obtain from commercially available channel if no special instructions.
The preparation of embodiment 1 collagen fiber membrane
Get 10g collagenous fibres sample (its preparation method can with reference to United States Patent (USP) 5997895), swelling at 500ml, in the acetum (pH2.5) of 0.1M, then with refiner, collagenous fibres are thoroughly disperseed, collagenous fibres are dissolved in acetum completely, (this process is collagenous fibril leaching process to obtain collagenous fibril solution, the mode of homogenate is adopted to be dispersed in acid solution by collagenous fibril, after stifling can there is the thicker collagenous fiber bundle of self assembly formation in collagenous fibril, no longer homogenized is done to collagenous fiber bundle, and traditional method will carry out homogenized to collagenous fiber bundle before the forming, so just be convenient to processing process below), collagenous fibril sample solution is obtained after centrifugal de-bubble.Be coated in by this sample solution on 200 object stainless steel filtering nets, THICKNESS CONTROL is at 5mm-7mm.
Filter screen is put in the airtight container of the ammoniacal liquor filling 1% and fumigates, after 24 hours, take out sample, and air-dry.After the PBS buffer solution of 0.01M is swelling, fully clean gained collagen fiber membrane with pure water, freeze drying, then cutting sample, irradiation after packaging.
The present embodiment, under the condition that ammoniacal liquor is fumigated, induce collagenous fibril to be assembled into thicker collagenous fibres, assemble the collagenous fibres obtained comparatively thick, electron microscopic observation has light and shade striped, there is good mechanical property, be comparatively close to the state of collagenous fibres normal presence in body.
The biological safety (cytotoxicity) of embodiment 2 collagen fiber membrane
1, leaching liquor makes
According to the regulation of GB/T 16886, the collagenous fibres membrane material of this test is because thickness is at below 5mm, and leaching liquor volume calculates according to material surface area, 6cm
2/ ml carries out lixiviate.Each class sample all needs the leaching liquor of 24h, 48h and 72h.Extraction temperature is 37 DEG C.The super-clean bench of operation all in cell room carries out.Leaching liquor is 1640 culture mediums containing 10%FBS being used to cultivate L929 cell.
2, cell chulture
Propose recovery the last week and cultivated L929 cell (purchased from Bai Li bio tech ltd, Shanghai), spread in 96 orifice plates after digestion, every hole 2 × 10
5individual cell.Culture medium in sucking-off next day orifice plate, adds leaching liquor, does 6 multiple holes.Control group, with the phenol-culture medium solution cultured cell containing 6.4%.
3, data monitoring
Adopt cck-8 method to detect and cultivate 48h detection, 72h detection and corresponding 2 control group plates.Detection mode is and measures 450nm absorbance on ELIASA.
4, data analysis
The computational methods of cell proliferation rate are (experimental group absorbance-blank group absorbance)/(ordinary culture medium cultivation group absorbance-blank group absorbance) * 100%, and the rate of increase is more than or equal to 80% explanation no cytotoxicity.
Final data is calculated as follows:
Result: use the positive controls cell growth rate of phenol to be respectively 4.6% and 4.3%.
The equal showed cell activity of the testing result obtained is greater than 80%, proves gained leaching liquor no cytotoxicity, thus illustrates that the collagen fiber membrane detected remains without soluble cell toxicant.And partial data is much larger than 100%, hint may have facilitation to cell metabolism.
Embodiment 3, collagen fiber membrane biological safety (immunogenicity)
1, plating cells: spread into RAW264.7 cell (grinding Science and Technology Ltd. purchased from upper sea valley) in 96 orifice plates in 6 × 4 square formations, every hole 100 μ l cell suspension, 2 × 10
4individual cell, is placed in overnight incubation in 37 DEG C of cell culture incubators.
2, immersion liquid makes: get collagenous fibres membrane sample to be measured and insert in centrifuge tube and make leaching liquor.The heavy 1.1g of sample, adds 11ml lixiviate stoste.The lixiviate stoste that this test uses is 1640 culture mediums.
3, application of sample stimulates: 4 arrange the leaching liquor or contrast liquid difference that add respectively: tunica fibrosa sample leaching liquor, blank (1640 culture medium), negative control (BSA, 40ug/ul) with positive control (ConA, 40ug/ul and 100ug/ml).Exhausted the same day cell culture fluid, after DPBS cleaning cell three times, adds respective sample, each sample 6 hole, every hole 100 μ l respectively in 8 row.Cultivate 4h again, then wash 3 times with DPBS, add the neutral red solution 100ul of 0.1%, continue to cultivate 30min.Neutral red solution is abandoned in suction, washs 3 times, adds lysate (glacial acetic acid and absolute ethyl alcohol=1:1) 200ul, hold over night.
4, absorbance detection: after 12 hours, cell cracking completely, ELIASA detects 540nm absorbance.
5, research process and result:
Testing result as shown in Figure 1, data display collagenous fibres membrane sample leaching liquor is all very low to the excitant of macrophage, close to 1640 culture mediums of blank, also lower than negative control BSA solution, four kinds of detected samples are described all without can the immunogenicity of stimulating expression of macrophage.
Embodiment 4 collagen fiber membrane intensity detection
With reference to the process of embodiment 1, the technical parameter designed according to table 1, prepares collagem membrane, and requires to measure its mechanical property (concrete assay method is as described in Example 5) according to national legislation, and its result is as shown in the table:
Table 1
Through repeatedly verifying, in other embodiments, the acid medium that pH is 2.0-4.0 is used to dissolve collagenous fibres, in collagenous fibril sample solution, the concentration of collagen is 10g/L-100g/L, use 100-400 object filter screen, be fumigate under the aqueous ammonia conditions of 0.5% (w/w)-2% (w/w) in concentration, fumigation time is 12h-36h, all can prepare performance preferably, meet the collagen fiber membrane of application claims.
Embodiment 5 collagenous fibres film-strength reappearance detects
Random selecting collagen fiber membrane (being prepared by the method for embodiment 1) and collagen sponge (adopt enzyme process to extract collagen from ox heel string, then collagen sponge is prepared in the mode of freeze-drying, preparation method's bibliography: the research of extracting collagen of bovine tendon with enzyme, Ye Yichun etc., " Chinese leather " the 34th volume the 7th phase) each 3, sample is cut into the strip that 10mm is wide and 80mm is long, is placed on temperature 23 DEG C, until sample is stablized under the environment of humidity 50%.
The nominal clamp distance of setting Tensile Tester is 50mm ± 1mm, in clamper center clamping style.Pretension is set as 2N.
Machine, with the constant elongation speed tensile sample of 10mm/min until fracture, record the brute force-extension curve of every block sample, calculate the ultimate strength (power that curve peak is corresponding) of sample.
TENSILE STRENGTH according to following formulae discovery product:
TENSILE STRENGTH=ultimate strength ÷ (batten wide × batten is thick)
Test result is as follows:
Above-mentioned testing result shows, its TENSILE STRENGTH of collagen fiber membrane prepared according to the methods of the invention is nearly 6 times of collagen sponge, illustrates that the collagen fiber membrane prepared by method of the present invention has high TENSILE STRENGTH.
Comparative example 1
Prepare collagen fiber membrane according to the method in embodiment 1, difference is, is coated in by sample solution after on 200 object stainless steel filtering nets, and use ammoniacal liquor to fumigate 30min, other condition is substantially identical, obtained 5 groups of collagem membranes.Detect gained collagen film strength according to method in embodiment 4, after testing, the mean intensity of gained collagem membrane is less than 2.5Mpa.
Comparative example 2
Prepare collagen fiber membrane according to the method in embodiment 1, difference is, be coated in by sample solution on 200 object stainless steel filtering nets, re-use ammoniacal liquor and fumigate 30min, then standing and drying 20h, other condition is substantially identical, obtained 5 groups of collagem membranes.Detect gained collagen film strength according to method in embodiment 5, after testing, the mean intensity of gained collagem membrane is less than 2Mpa.
Comparative example 3
Prepare collagen fiber membrane according to the method in embodiment 1, difference is, be coated in by sample solution on 200 object stainless steel filtering nets, standing and drying 20h, and then use ammoniacal liquor to fumigate 30min, other condition is substantially identical, obtained 5 groups of collagem membranes.Detect gained collagen film strength according to method in embodiment 5, after testing, the mean intensity of gained collagem membrane is less than 1.5Mpa.
Comparative example 4
Prepare collagen fiber membrane according to the method in embodiment 1, difference is, is coated in by sample solution and does not have on the flat board in hole, carries out the preparation of collagen fiber membrane, and other condition is substantially identical, obtained 5 groups of collagen fiber membranes.Detect gained collagenous fibres film strength according to method in embodiment 5, after testing, the mean intensity of gained collagen fiber membrane is less than 3Mpa; Repeating test finds to rupture in its multiple sample portion in the sample to which that fails, and shows its product uniformity poor, does not meet national legislation requirement, pass through perusal simultaneously, compared with method provided by the invention, its product thickness heterogeneity obtained, color evenness is also poor.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after having read above-mentioned instruction content of the present invention.
Claims (10)
1. prepare a method for collagen fiber membrane, it is characterized in that, described method comprises step:
(1) collagenous fibres are dissolved in acid medium, obtain collagenous fibril solution;
(2) described collagenous fibril solution is poured in container, stifling 8h-48h under being placed on volatility alkali environment, obtained described collagen fiber membrane;
Wherein, the bottom of described container is filter structure; Described filter screen is 50-500 object filter screen, and described filter screen allows the water in described sample solution to flow out described container, and the collagen fiber matrix that described collagenous fibril is formed is retained on described filter screen.
2. the method for claim 1, is characterized in that, in described step (1), in described collagenous fibril solution, the concentration of collagenous fibril is 10g/L-100g/L, is preferably 15g/L-50g/L.
3. the method for claim 1, is characterized in that, the pH of described acid medium is 0-6; Preferably pH is 0.5-5.5; More preferably pH is 1.0-5.0; Optimally pH is 2.0-4.0.
4. the method for claim 1, is characterized in that, described volatility alkali is ammoniacal liquor; Preferably, described ammonia concn is 0.1% (w/w)-5% (w/w); More preferably, described ammonia concn is 0.5% (w/w)-2% (w/w).
5. the method for claim 1, is characterized in that, the fumigation time in described step (2) is 12h-36h; Preferably, described fumigation time is 18h-30h.
6. the method for claim 1, is characterized in that, described filter screen is 100-400 object filter screen; Preferably, described filter screen is 150-250 object filter screen.
7. the method for claim 1, is characterized in that, in described step (2), is coated on described filter screen by described collagenous fibril solution, stifling under being then placed on volatility alkali environment.
8. a collagen fiber membrane, is characterized in that, described collagen fiber membrane is prepared by method according to claim 1 and obtains.
9. prepare a device for collagen fiber membrane, it is characterized in that, described device comprises: shuttle and volatility alkali container, and described shuttle is placed in the top of described volatility alkali container;
Wherein, described shuttle is set to for holding collagenous fibril sample solution, and the bottom of described shuttle is filter structure.
10. prepare the device of collagen fiber membrane as claimed in claim 9, it is characterized in that, described filter screen is 50-500 object filter screen; More preferably described filter screen is 100-400 object filter screen; Most preferably, described filter screen is 150-250 object filter screen.
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| CN106880872B (en) * | 2016-12-23 | 2019-10-29 | 北京大清生物技术股份有限公司 | Natural extracellular matrix biomembrane and the preparation method and application thereof |
| CN106880872A (en) * | 2016-12-23 | 2017-06-23 | 北京大清生物技术股份有限公司 | Natural extracellular matrix biomembrane and preparation method and application |
| US11214844B2 (en) | 2017-11-13 | 2022-01-04 | Modern Meadow, Inc. | Biofabricated leather articles having zonal properties |
| US11352497B2 (en) | 2019-01-17 | 2022-06-07 | Modern Meadow, Inc. | Layered collagen materials and methods of making the same |
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Address after: No. 4011, building 1, Haike Electronic Information Industrial Park, 319 Qingpi Avenue, cross strait science and Technology Industrial Development Park, Wenjiang District, Chengdu, Sichuan Province Patentee after: Chengdu Jingze biopharmaceutical Co.,Ltd. Address before: No. 4011, building 1, Haike Electronic Information Industrial Park, 319 Qingpi Avenue, cross strait science and Technology Industrial Development Park, Wenjiang District, Chengdu, Sichuan Province Patentee before: Chengdu Zeyan Biotechnology Co.,Ltd. |
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Denomination of invention: Preparation and application of collagen fiber membrane Effective date of registration: 20221116 Granted publication date: 20170222 Pledgee: Industrial Bank Limited by Share Ltd. Chengdu branch Pledgor: Chengdu Jingze biopharmaceutical Co.,Ltd. Registration number: Y2022510000307 |