CN102225219B - Bone tissue regeneration guiding membrane and preparation method thereof - Google Patents
Bone tissue regeneration guiding membrane and preparation method thereof Download PDFInfo
- Publication number
- CN102225219B CN102225219B CN201110145229.0A CN201110145229A CN102225219B CN 102225219 B CN102225219 B CN 102225219B CN 201110145229 A CN201110145229 A CN 201110145229A CN 102225219 B CN102225219 B CN 102225219B
- Authority
- CN
- China
- Prior art keywords
- tissue
- membrane tissue
- membrane
- concussion
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 35
- 239000012528 membrane Substances 0.000 title claims abstract description 22
- 230000017423 tissue regeneration Effects 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims description 29
- 102000008186 Collagen Human genes 0.000 claims abstract description 32
- 108010035532 Collagen Proteins 0.000 claims abstract description 32
- 229920001436 collagen Polymers 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 31
- 239000011248 coating agent Substances 0.000 claims abstract description 19
- 238000000576 coating method Methods 0.000 claims abstract description 19
- 239000000835 fiber Substances 0.000 claims abstract description 17
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 241000124008 Mammalia Species 0.000 claims abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 7
- 229920001184 polypeptide Polymers 0.000 claims abstract description 5
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 5
- 210000004379 membrane Anatomy 0.000 claims description 109
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 57
- 230000009514 concussion Effects 0.000 claims description 40
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 35
- 230000008569 process Effects 0.000 claims description 25
- 108010019160 Pancreatin Proteins 0.000 claims description 18
- 229940055695 pancreatin Drugs 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000011780 sodium chloride Substances 0.000 claims description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 14
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 claims description 14
- 239000000427 antigen Substances 0.000 claims description 13
- 102000036639 antigens Human genes 0.000 claims description 13
- 108091007433 antigens Proteins 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- 230000000890 antigenic effect Effects 0.000 claims description 12
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 claims description 12
- 230000002188 osteogenic effect Effects 0.000 claims description 11
- 229910000831 Steel Inorganic materials 0.000 claims description 7
- 239000008280 blood Substances 0.000 claims description 7
- 210000004369 blood Anatomy 0.000 claims description 7
- 239000010959 steel Substances 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 210000000577 adipose tissue Anatomy 0.000 claims description 5
- FFBHFFJDDLITSX-UHFFFAOYSA-N benzyl N-[2-hydroxy-4-(3-oxomorpholin-4-yl)phenyl]carbamate Chemical compound OC1=C(NC(=O)OCC2=CC=CC=C2)C=CC(=C1)N1CCOCC1=O FFBHFFJDDLITSX-UHFFFAOYSA-N 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 5
- 229910001220 stainless steel Inorganic materials 0.000 claims description 5
- 239000010935 stainless steel Substances 0.000 claims description 5
- 238000003491 array Methods 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 12
- 238000006731 degradation reaction Methods 0.000 abstract description 9
- 230000015556 catabolic process Effects 0.000 abstract description 8
- 230000008468 bone growth Effects 0.000 abstract description 7
- 230000011164 ossification Effects 0.000 abstract description 7
- 229920000642 polymer Polymers 0.000 abstract description 5
- 210000004872 soft tissue Anatomy 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000006698 induction Effects 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 230000000116 mitigating effect Effects 0.000 abstract 1
- 150000001413 amino acids Chemical group 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 238000001727 in vivo Methods 0.000 description 11
- 239000000463 material Substances 0.000 description 10
- 230000004888 barrier function Effects 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 7
- 230000007547 defect Effects 0.000 description 6
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 5
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 5
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 5
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 5
- 229940112869 bone morphogenetic protein Drugs 0.000 description 5
- 238000002955 isolation Methods 0.000 description 4
- 229920005615 natural polymer Polymers 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 102000009618 Transforming Growth Factors Human genes 0.000 description 3
- 108010009583 Transforming Growth Factors Proteins 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 229920000954 Polyglycolide Polymers 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 238000010382 chemical cross-linking Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000000635 electron micrograph Methods 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 210000003516 pericardium Anatomy 0.000 description 2
- 230000003239 periodontal effect Effects 0.000 description 2
- 210000004303 peritoneum Anatomy 0.000 description 2
- 229920000747 poly(lactic acid) Polymers 0.000 description 2
- 239000004633 polyglycolic acid Substances 0.000 description 2
- 239000004626 polylactic acid Substances 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 102100036597 Basement membrane-specific heparan sulfate proteoglycan core protein Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 241000282894 Sus scrofa domesticus Species 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 210000001909 alveolar process Anatomy 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000003037 histogenic effect Effects 0.000 description 1
- 102000058223 human VEGFA Human genes 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 108010049224 perlecan Proteins 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229950000845 politef Drugs 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000001846 repelling effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
Abstract
A bone tissue regeneration guiding membrane. The invention employs an antigen-extracted mammal tissue membrane, which has a double layer structure of a compact layer and a loosening layer. The compact layer is formed by tightly arranged collagen fibers, and an external side of the compact layer is composited with a collagen coating containing active polypeptides; the loosening layer is formed by interlaced collagen fibers and is composited with ossification active factors. The prepared guiding membrane can prevent soft tissue from evolving and promote new bone formation, has good biological compatibility, hydrophilism and bone induction capability, and can be completely degraded in body without generation of harmful substances. Besides, the guiding membrane has an effect of isolating cell evolving, a characteristic of slow degradation and a longer retaining time in body than a current guiding membrane prepared from natural high-molecular polymer, and can provide enough time and space for new bone growth. The method of the invention is simple, and raw materials have wide sources and low prices, so that the method is suitable for large-scale industrial production and beneficial to lowering medical costs and mitigating patient burdens.
Description
Technical field
The invention belongs to biomaterial for medical purpose technical field, be specifically related to a kind of biomembrane material with inducting osseous tissue regeneration effect and preparation method thereof.
Background technology
Film guide tissue regeneration (guided tissue regeneration, GTR) technology is widely used in bone tissue restoration field, its principle is the physical barriers effect by membrane material, stop soft tissue cells to enter osteogenesis district, for the new bone growth headspace of bone defect area, and guide skeletonization class cell to generate new bone in bone defect area.
The film of GTR technology initial stage use is nonabsorable in vivo, and as the GTR film of preparing with politef, essential second operation takes out in use, to patient, bring very big misery, and active because of its inanimate object, can not, for the tissue regeneration of larger damaged area, limit the scope in clinical use.Chinese scholars seeks to draw materials extensively always, preparation is simple, the effective membrane material of inducting osseous tissue regeneration.The degradable guiding film of exploitation can absorb in vivo in recent years, reduces the misery of patient's second operation and the risk of secondary infection.Therefore, the research and development of degradable guiding film cause extensive concern, and the GTR film that synthetic high molecular polymer (polylactic acid, polyglycolic acid etc.), natural polymers be main component of wherein take is more.
GRT film prepared by the synthetic high molecular polymer that polylactic acid, polyglycolic acid be representative of take, the advantage because degradable absorbs in vivo, mechanical strength is good, is employed.In Chinese patent application 201010105573.2,200910053789.6 and 03117481.7, all take synthetic high molecular polymer as raw material, preparation has the GTR film of certain space structure.But this type of GTR film also has deficiency, for example after its degradation in vivo, can produce acidic micro-environment, the reaction that causes inflammation, is unfavorable for organization healing.
The GTR film of being prepared by natural polymerses such as collagen protein, chitosans, can not produce any harmful substance after degradation in vivo, and biocompatibility is better than synthetic high molecular polymer, becomes gradually the primary raw material of preparation GTR film.But mechanical performance and the water repelling property of guiding film prepared by natural polymers be poor, it is too fast to degrade, and do not mate with bone growth speed.Chinese patent application 200410071932.1 discloses a kind of double-layer collagen base guide tissue regeneration material and preparation method thereof, for overcoming the collagen-based materials too fast defect of degrading, it improves the anti-degradation capability of material with chemical cross-linking agent, extend regeneration membrane degradation time in vivo; Because the residual meeting of the chemical cross-linking agents such as glutaraldehyde in product causes the repulsion of patient's body to product, and its preparation method is complicated, and appointed condition is had relatively high expectations, and makes cost up, and large-scale production difficulty is large.
Desirable bone tissue regeneration guiding membrane should have compacted zone and weaker zone double-decker, and compacted zone can prevent the soft tissue bone defect area of growing into, and is the growth headspace of new bone; Weaker zone can provide larger contact area, is conducive to the attaching growth of cytokine absorption and skeletonization class cell, accelerates new bone formation; Certainly, it also should have degradability, without second operation, takes out, and guarantees to maintain in vivo isolation barrier effect a period of time, for new bone growth provides grace time.
Summary of the invention
For the deficiencies in the prior art, the object of this invention is to provide a kind of bone tissue regeneration guiding membrane and preparation method thereof, prepared guiding film has compacted zone and weaker zone double-decker, and good biocompatibility, can extend degradation time in vivo, its catabolite is harmless.
Bone tissue regeneration guiding membrane proposed by the invention is characterised in that, described guiding film is the mammal membrane tissue that antigen is processed, and main component is collagen; Guiding film has compacted zone and weaker zone double-decker, wherein, compacted zone consists of collagenous fiber bundle close-packed arrays, in compacted zone outside, be compounded with collagenic coating, in collagenic coating, contain and can promote angiopoietic beading sample albumen the first Structure and function domain (PlnDL) aminoacid sequence or/and RGD peptide; Weaker zone forms by collagen fiber are interlaced, is compounded with the osteogenic activity factor in weaker zone.The described osteogenic activity factor is any or several combination of bone morphogenetic protein (BMP), platelet derived growth factor (PDGF) or transforming growth factor (TGF), specifically according to clinical practice, need to select.
The preparation method of bone tissue regeneration guiding membrane proposed by the invention, it is characterized in that, to go antigen to process to mammal membrane tissue, by dewatering and being dried the compactness extent that improves membrane tissue, in compacted zone surface recombination, contain PlnDL aminoacid sequence or/and the collagenic coating of RGD peptide again, and be combined into the bone active factor at weaker zone.The concrete steps of preparation method of the present invention comprise:
Antigenic membrane tissue is removed in step 1, preparation
1), pretreatment of raw material: get mammal membrane tissue (as pericardium, peritoneum, barrier film etc.), remove surperficial blood stains and fatty tissue, clean at least 3 times with normal saline concussion;
2), ungrease treatment: it is that in 0.1% NaCl solution, concussion was processed after half an hour that the membrane tissue after cleaning is inserted to mass volume ratio, then inserts in ether solvent supersound process 1 hour, the NaCl solution that is 2% with mass volume ratio flushing;
Because fat has very strong immunogenicity, should remove thoroughly; This link first adopts hypisotonic solution to make lax dispersion of collagen fiber in membrane tissue, then removes fat with ether solvent, finally adopts hyperosmotic solution that membrane tissue is shunk, and makes the three-D space structure of membrane tissue compacter.
3), compacted zone pancreatin processes: by the membrane tissue dense face after defat outwardly, be bundled in loose facing bag-shapedly, in bag, inject phosphate buffer (PBS); It is in 0.1%～0.4% pancreatin solution that bag-shaped membrane tissue is inserted to mass volume ratio, and half an hour is processed in concussion, then inserts the CaCl of 0.5M
2in solution, concussion was cleaned after 10 minutes, untie bag water and rinse, by membrane tissue from seal bag-shaped revert to membranaceous;
Because pancreatin can degradation of cell epimatrix, remove the antigenic component in membrane tissue; But pancreatin also can destroy the three dimensional structure of weaker zone.Because the architectural difference of compacted zone and weaker zone, is bundled into dense face sealing outwardly by membrane tissue bag-shaped, adopt pancreatin to process compacted zone, be both beneficial to the antigenic component of removing compacted zone, retained again the natural structure of membrane tissue.
4), NaOH processes: the membrane tissue after pancreatin is processed is inserted in the NaOH solution that concentration is 0.01～0.2M, and under 0～4 ℃ of condition, concussion is cleaned 1 hour, by pure water rinsing;
Because NaOH under room temperature can destroy the collagenous fiber bundle structure of natural membranes, shorten guiding film degradation time in vivo, therefore NaOH processes and must remain on 0～4 ℃ and complete.Use the NaOH solution of recommended density can remove in membrane tissue major part and there is remaining trace fat after immunogenic albumen and ungrease treatment, and can not destroy the double-decker of natural membranes tissue.
5), height oozes processing: it is that in 2%～5% NaCl solution, concussion is cleaned water after 2 hours and rinsed that the membrane tissue after NaOH is processed is inserted mass volume ratio; Obtain the natural membranes tissue of antigen;
After NaOH treatment step, adopt the high processing of oozing, can remove the solubility noncollagen protein between collagenous fiber bundle in membrane tissue, further reduce histogenic immunity originality.
Step 2, collagen fiber shrink process: the membrane tissue that removes antigen is inserted in acetone soln, and concussion was processed after 0.5～2 hour, with dehydrated alcohol concussion, cleaned 3 times;
Because the space between collagen fiber in membrane tissue is combined with a large amount of water, acetone can make tissue dewatering, causes that collagen fiber shrink, and improve the compactness extent of membrane tissue, the barrier action of strengthening membrane; Dehydrated alcohol can clean the acetone in membrane tissue, keeps the good biocompatibility of membrane tissue.
Step 3, dried: the membrane tissue after shrink process is laid in to medical stainless steel plate surface, makes dense face contact steel plate, keep 50 ℃ of freeze-day with constant temperature of steel plate after 2～10 minutes, film is proceeded to-80 ℃ of pre-freezes at least 3 hours; Through low-temperature freeze drying, obtain having the double-deck antigenic membrane tissue that goes again;
For the two-layer different structure of membrane tissue, for strengthening the compactness extent on compacted zone surface, keep the three-D space structure of weaker zone, adopt 50 ℃ of dry compacted zone surfaces that can not cause collagen fiber degeneration, then lyophilization membrane tissue; Make the compacted zone of prepared membrane tissue have isolation barrier effect, weaker zone three dimensional structure is conducive to adsorb blood and active factors promotes new bone formation.
Step 4, preparation are containing the collagenic coating of active polypeptide: the collagen solution of the pH7.2 that is 5～30mg/ml by concentration is evenly coated the compacted zone surface of membrane tissue after lyophilizing, dries; In this collagen solution, contain the PlnDL aminoacid sequence of 5～80 μ g/ml or/and the RGD peptide of 0.2～2pmol/ml;
Wherein, described even coating can adopt vacuum rotating technology for coating, by rotary speed and time, control the compactness extent of collagenic coating, rotary speed is 1000rmp～5000rpm, time is that (speed was higher in 0.5～2 minute, the compactness extent of film is higher, and the thickness of the longer film of time of filming is larger); The present invention, by the physical barriers effect of the fine and close collagenic coating in the preparation of compacted zone surface, can improve the ability that dense face stops that non-osteoblast is grown into, and guarantees the required space of bone defect area new bone growth.
Beading sample albumen the first structure function domain amino acid sequence (being called for short PlnDL aminoacid sequence) is a kind of aminoacid functional fragment, can under low concentration environment, promote vascularization, active high compared with VEGF; Acquisition methods can reference: Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells, BMC Biochemistry 2010,11:43.
RGD peptide is arginine-glycine-aspartic acid polypeptide, it is the binding site of ECM albumen and corresponding cell, it can be also combined with it with the cell membrane integrin subunit identification by adherent cell, promotes cellular metabolism and protein synthesis, strengthens cell attachment, propagation, differentiation; Be widely used in the surface treatment to biomaterial, acquisition methods can reference: bioactive short peptide RGD synthetic and in the research of pet sheet face grafting method, Sichuan University's Master's thesis, 2001.
Step 5, weaker zone are combined into the bone active factor: the membrane tissue weaker zone surface uniform after drying drips the PBS solution that contains 20～100mg/ml osteogenic activity factor, to saturation, after cryogenic vacuum lyophilizing, completes preparation.
Bone tissue regeneration guiding membrane of the present invention is natural membranes tissue, and main component is collagen, has good biocompatibility, hydrophilic and bone inducibility, in vivo can be degradable, can not produce harmful substance; Because raw material adopts the mammal membrane tissue with isolation barrier effect, through the densified to material, prepared guiding film has the feature of the isolated cell effect of growing into and slow degradation, the guiding film of preparing than existing natural polymers in vivo remaining time is long, can provide sufficient time and space for new bone growth.Zoopery proves, in subcutaneous rat, implant the collagen-based guiding film of guiding film of the present invention and prior art the same period, sampling is observed weekly, and in the time of 4 weeks, collagen-based guiding film starts degraded, to 6～7 weeks, degraded over half, guiding film no longer has isolation barrier effect; And guiding film of the present invention is since degraded in the 8th week, membrane tissue attenuation in the time of 16 weeks, but still can observe the existence of complete film structure, during to 22 weeks, membrane tissue is degradable, has no untoward reaction.
Guiding film of the present invention has the desired compacted zone of desirable bone tissue regeneration guiding membrane and weaker zone double-decker, can stop soft tissue to be grown into, and can promote new bone formation again; Weaker zone can adsorb the blood of surrounding tissue after implanting damaged site, forms blood clot, healing acceleration; In compacted zone surface recombination, have and promote angiopoietic PlnDL aminoacid sequence and promote the RGD peptide that cell attaches, be conducive to wound healing; PlnDL aminoacid sequence is more stable than the activity of VEGF, and can bring into play angiogenesispromoting effect at low concentration; RGD peptide can synthetic, non-immunogenicity, without taking viruliferous danger, cost is lower, at differing texture material surface, has higher stability, the impact that not changed by temperature and pH, easily storage transportation.Owing to having added the somatomedin that promotes new bone growth at weaker zone, there is bone-inducting active, can further promote osseous tissue to rebuild.Preparation method of the present invention is simple, raw material sources are extensive and cheap, is applicable to scale industry and produces, and is conducive to reduce medical treatment cost, alleviates patient's burden.
Accompanying drawing explanation
Accompanying drawing 1 is the cardinal principle photo of the prepared bone tissue regeneration guiding membrane of the inventive method; Accompanying drawing 2 is the compacted zone partial sweep electron micrograph of guiding film shown in Fig. 1, and visible compacted zone smooth surface is fine and close, can effectively stop soft tissue to be grown into; Accompanying drawing 3 is the weaker zone partial sweep electron micrograph of guiding film shown in Fig. 1, and visible weaker zone gap structure is obvious, can effectively promote new bone formation.
The specific embodiment
Below in conjunction with example, technical solution of the present invention is described in further detail.
Embodiment 1,
Antigenic membrane tissue is removed in step 1, preparation
1), pretreatment of raw material: get Cor Sus domestica peplos, remove surperficial blood stains and fatty tissue, clean 3 times each 15 minutes with normal saline concussion;
2), ungrease treatment: the membrane tissue after cleaning is inserted in the NaCl solution of 0.1% (M/V), and half an hour is processed in concussion, then inserts in ether (analytical pure) solvent supersound process 1 hour, with the NaCl solution flushing of 2% (M/V);
3), compacted zone pancreatin processes: by the membrane tissue dense face after defat outwardly, be bundled in loose facing bag-shapedly, in bag, inject PBS buffer; It is in 0.2% pancreatin solution that bag-shaped membrane tissue is inserted to mass volume ratio, and half an hour is processed in concussion, then inserts the CaCl of 0.5M
2in solution, concussion was cleaned after 10 minutes, untie bag water and rinse, by membrane tissue from seal bag-shaped revert to membranaceous;
4), NaOH processes: the membrane tissue after pancreatin is processed is inserted in the NaOH solution of 0.02M concentration, and under 0～4 ℃ of condition, concussion is cleaned 1 hour, uses pure water rinsing 1 hour;
5), height oozes processing: it is that in 2% NaCl solution, concussion is cleaned water after 2 hours and rinsed, and obtains the natural membranes tissue of antigen that the membrane tissue after NaOH is processed is inserted mass volume ratio;
Step 2, collagen fiber shrink process: the membrane tissue that removes antigen is inserted in acetone soln (analytical pure), and concussion was processed after half an hour, cleaned 3 times each 1 hour with dehydrated alcohol concussion;
Step 3, dried: the membrane tissue after shrink process is laid in to medical stainless steel plate surface, makes dense face contact steel plate, keep 50 ℃ of freeze-day with constant temperature after 2 minutes, rapidly membrane tissue is proceeded to-80 ℃ of low temperature pre-freezes 3 hours; Through low-temperature freeze drying, obtain having the double-deck antigenic membrane tissue that goes again;
Step 4, preparation contain the collagenic coating of PlnDL aminoacid sequence: the collagen solution of the pH7.2 that is 9mg/ml by concentration adopts vacuum rotating technology for coating (speed 2000rpm, 1 minute time), evenly coat the compacted zone surface of membrane tissue after lyophilizing, dry; The PlnDL aminoacid sequence that contains 20 μ g/ml in this collagen solution;
Step 5, weaker zone are combined into the bone active factor: the membrane tissue weaker zone surface uniform after drying drips the PBS solution that contains the 50mg/ml osteogenic activity factor, present saturation to weaker zone, the osteogenic activity factor adopts platelet derived growth factor (PDGF); After cryogenic vacuum lyophilizing, complete preparation.
Prepared bone tissue regeneration guiding membrane thickness approximately 400 μ m, one side contains short angiopoietic active amino acid sequence, one side is compounded with the active factors that promotes skeletonization, be applicable to periodontal tissue's guiding regeneration art, also can be used in conjunction with bone grafting material, the periodontal bone that treatment is caused by periodontitis is damaged.
Embodiment 2,
Antigenic membrane tissue is removed in step 1, preparation
1), pretreatment of raw material: get pig peritoneum, remove surperficial blood stains and fatty tissue, clean 3 times each 15 minutes with normal saline concussion;
2), ungrease treatment: the membrane tissue after cleaning is inserted in the NaCl solution of 0.1% (M/V), and concussion was processed after half an hour, then inserted in ether (analytical pure) solvent supersound process 1 hour, with the NaCl solution flushing of 2% (M/V);
3), compacted zone pancreatin processes: by the membrane tissue dense face after defat outwardly, be bundled in loose facing bag-shapedly, in bag, inject PBS solution; It is in 0.3% pancreatin solution that bag-shaped membrane tissue is inserted to mass volume ratio, and half an hour is processed in concussion, then inserts the CaCl of 0.5M
2in solution, concussion was cleaned after 10 minutes, untie bag water and rinse, by membrane tissue from seal bag-shaped revert to membranaceous;
4), NaOH processes: the membrane tissue after pancreatin is processed is inserted in the NaOH solution that concentration is 0.05M, and under 0～4 ℃ of condition, concussion is cleaned 1 hour, by pure water rinsing;
5), height oozes processing: it is that in 3% NaCl solution, concussion is cleaned water after 2 hours and rinsed that the membrane tissue after NaOH is processed is inserted mass volume ratio; Obtain the natural membranes tissue of antigen;
Step 2, collagen fiber shrink process: the membrane tissue that removes antigen is inserted to concussion in acetone soln (analytical pure) and process after 1 hour, with dehydrated alcohol (analytical pure) concussion, clean 3 times each 1 hour;
Step 3, dried: the membrane tissue after shrink process is laid in to medical stainless steel plate surface, makes dense face contact steel plate, keep 50 ℃ of freeze-day with constant temperature after 5 minutes, rapidly film is proceeded to-80 ℃ of low temperature pre-freezes 3.5 hours; Again through low-temperature freeze drying; Obtain having the double-deck antigenic membrane tissue that goes;
Step 4, preparation contain the collagenic coating of RGD peptide: the collagen solution of the pH7.2 that is 16mg/ml by concentration adopts vacuum rotating technology for coating (speed 2500rpm, time 80s), the dense face of evenly coating membrane tissue after lyophilizing, dries, in this collagen solution containing the RGD peptide of 1pmol/ml;
Step 5, weaker zone are combined into the bone active factor: the membrane tissue weaker zone surface uniform after drying drips the PBS solution that contains the 80mg/ml osteogenic activity factor, present saturation to weaker zone, the osteogenic activity factor adopts bone morphogenetic protein (BMP); After cryogenic vacuum lyophilizing, complete preparation.
Prepared bone tissue regeneration guiding membrane thickness approximately 1000 μ m, one side contains the RGD peptide that promotes surrounding tissue cell attaching growth, and one side is compounded with the active factors that promotes skeletonization, is applicable to alveolar ridge amplification in a big way and rebuilds.
Embodiment 3,
Antigenic membrane tissue is removed in step 1, preparation
1), pretreatment of raw material: get bovine pericardium, remove surperficial blood stains and fatty tissue, clean 3 times each 15 minutes with normal saline concussion;
2), ungrease treatment: the membrane tissue after cleaning is inserted in the NaCl solution of 0.1% (M/V), and concussion was processed after half an hour, then inserted in ether (analytical pure) solvent supersound process 1 hour, with the NaCl solution flushing of 2% (M/V);
3), compacted zone pancreatin processes: by the membrane tissue dense face after defat outwardly, be bundled in loose facing bag-shapedly, in bag, inject PBS solution; It is in 0.4% pancreatin solution that bag-shaped membrane tissue is inserted to mass volume ratio, and half an hour is processed in concussion, then inserts the CaCl of 0.5M
2in solution, concussion was cleaned after 10 minutes, untie bag water and rinse, by membrane tissue from seal bag-shaped revert to membranaceous;
4), NaOH processes: the membrane tissue after pancreatin is processed is inserted in the NaOH solution that concentration is 0.15M, and under 0～4 ℃ of condition, concussion is cleaned 1 hour, uses pure water rinsing 1 hour;
5), height oozes processing: it is that in 5% NaCl solution, concussion is cleaned water after 2 hours and rinsed that the membrane tissue after NaOH is processed is inserted mass volume ratio; Obtain the natural membranes tissue of antigen;
Step 2, collagen fiber shrink process: the membrane tissue that removes antigen is inserted to concussion in acetone soln (analytical pure) and process after 1.5 hours, with dehydrated alcohol (analytical pure) concussion, clean 3 times each 1 hour;
Step 3, dried: the membrane tissue after shrink process is laid in to medical stainless steel plate surface, makes dense face contact steel plate, keep 50 ℃ of freeze-day with constant temperature after 8 minutes, rapidly film is proceeded to-80 ℃ of low temperature pre-freezes 4 hours; Through low-temperature freeze drying, obtain having the double-deck antigenic membrane tissue that goes again;
Step 4, preparation are containing the collagenic coating of active polypeptide: the collagen solution of the pH7.2 that is 6mg/ml by concentration adopts vacuum rotating technology for coating (speed 1000rpm, 0.5 minute time), evenly coat the compacted zone surface of membrane tissue after lyophilizing, dry; In this collagen solution, contain the PlnDL aminoacid sequence of 8 μ g/ml and the RGD peptide of 0.5pmol/ml;
Step 5, weaker zone are combined into the bone active factor: the membrane tissue weaker zone surface uniform after drying drips the PBS solution that contains the 100mg/ml osteogenic activity factor, present saturation to weaker zone, the osteogenic activity factor adopts BMP, PDGF and TGF three's combination, and the ratio of three kinds of factors is 1: 1: 1; After cryogenic vacuum lyophilizing, complete preparation.
Prepared bone tissue regeneration guiding membrane thickness approximately 1500 μ m, two peptide species that the compound promotion vascularization of one side and cell attach, one side is combined into the bone active factor, is applicable to the wound surface outer protection after repair of cranial defects, can promote skull regeneration.
Claims (1)
1. a preparation method for bone tissue regeneration guiding membrane, is characterized in that, described guiding film is the mammal membrane tissue that antigen is processed, and main component is collagen; Guiding film has compacted zone and weaker zone double-decker, and wherein, compacted zone consists of collagenous fiber bundle close-packed arrays, in compacted zone outside, is compounded with collagenic coating, contains PlnDL aminoacid sequence or/and RGD peptide in collagenic coating; Weaker zone forms by collagen fiber are interlaced, is compounded with the osteogenic activity factor in weaker zone; Concrete steps comprise:
Antigenic membrane tissue is removed in step 1, preparation
1), pretreatment of raw material: get mammal membrane tissue, remove surperficial blood stains and fatty tissue, clean at least 3 times with normal saline concussion;
2), ungrease treatment: it is that in 0.1% NaCl solution, concussion was processed after half an hour that the membrane tissue after cleaning is inserted to mass volume ratio, then inserts in ether solvent supersound process 1 hour, the NaCl solution that is 2% with mass volume ratio flushing;
3), compacted zone pancreatin processes: by the membrane tissue dense face after defat outwardly, be bundled in loose facing bag-shapedly, in bag, inject PBS buffer; It is in 0.1%～0.4% pancreatin solution that bag-shaped membrane tissue is inserted to mass volume ratio, and half an hour is processed in concussion, then inserts the CaCl of 0.5M
2in solution, concussion was cleaned after 10 minutes, untie bag water and rinse, by membrane tissue from seal bag-shaped revert to membranaceous;
4), NaOH processes: the membrane tissue after pancreatin is processed is inserted in the NaOH solution that concentration is 0.01～0.2M, and under 0～4 ℃ of condition, concussion is cleaned 1 hour, by pure water rinsing;
5), height oozes processing: it is that in 2%～5% NaCl solution, concussion is cleaned water after 2 hours and rinsed that the membrane tissue after NaOH is processed is inserted mass volume ratio; Obtain the natural membranes tissue of antigen;
Step 2, collagen fiber shrink process: the membrane tissue that removes antigen is inserted in acetone soln, and concussion was processed after 0.5～2 hour, with dehydrated alcohol concussion, cleaned 3 times;
Step 3, dried: the membrane tissue after shrink process is laid in to medical stainless steel plate surface, makes dense face contact steel plate, keep 50 ℃ of freeze-day with constant temperature of steel plate after 2～10 minutes, film is proceeded to-80 ℃ of pre-freezes at least 3 hours; Through low-temperature freeze drying, obtain having the double-deck antigenic membrane tissue that goes again;
Step 4, preparation are containing the collagenic coating of active polypeptide: the collagen solution of the pH7.2 that is 5～30mg/mL by concentration is evenly coated the compacted zone surface of membrane tissue after lyophilizing, dries; In this collagen solution, contain the PlnDL aminoacid sequence of 5～80 μ g/mL or/and the RGD peptide of 0.2～2pmol/mL;
Step 5, weaker zone are combined into the bone active factor: the membrane tissue weaker zone surface uniform after drying drips the PBS solution that contains 20～100mg/mL osteogenic activity factor, to saturation, after cryogenic vacuum lyophilizing, completes preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110145229.0A CN102225219B (en) | 2011-06-01 | 2011-06-01 | Bone tissue regeneration guiding membrane and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110145229.0A CN102225219B (en) | 2011-06-01 | 2011-06-01 | Bone tissue regeneration guiding membrane and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102225219A CN102225219A (en) | 2011-10-26 |
| CN102225219B true CN102225219B (en) | 2014-01-29 |
Family
ID=44806245
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110145229.0A Active CN102225219B (en) | 2011-06-01 | 2011-06-01 | Bone tissue regeneration guiding membrane and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102225219B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103071189B (en) * | 2013-01-25 | 2014-09-03 | 广州华美康联生物科技有限公司 | Preparation method of collagen film for guided tissue regeneration |
| CN103961752B (en) * | 2013-02-02 | 2016-01-20 | 深圳兰度生物材料有限公司 | Tissue regeneration guiding film and preparation method thereof |
| CN104667349B (en) * | 2015-02-06 | 2017-01-25 | 福州大学 | Growth factor-loading silk fibroin/collagen bracket material and preparation method thereof |
| CN106693079B (en) * | 2015-11-17 | 2020-04-10 | 深圳兰度生物材料有限公司 | Guided tissue regeneration membrane and preparation method thereof |
| CN106693080B (en) * | 2015-11-17 | 2020-04-10 | 深圳兰度生物材料有限公司 | Guided tissue regeneration membrane and preparation method thereof |
| CN106693056B (en) * | 2015-11-17 | 2020-04-10 | 深圳兰度生物材料有限公司 | Cross-linking guided tissue regeneration membrane and preparation method thereof |
| CN107029296B (en) * | 2017-03-03 | 2020-09-29 | 北京博辉瑞进生物科技有限公司 | Periosteum repairing piece for guiding bone regeneration, preparation method and application |
| CN107029293B (en) * | 2017-03-03 | 2022-06-21 | 济南金泉生物科技有限公司 | Pericardium collagen membrane for guiding bone regeneration and preparation method and application thereof |
| CN107281552A (en) * | 2017-07-12 | 2017-10-24 | 上海白衣缘生物工程有限公司 | It is a kind of for composite membrane of Guided Bone Regeneration and preparation method thereof |
| CN109096520A (en) * | 2018-06-29 | 2018-12-28 | 四川大学 | A kind of modified collagen and preparation method thereof |
| CN113117150B (en) * | 2019-12-31 | 2022-07-19 | 广州迈普再生医学科技股份有限公司 | Guided tissue regeneration membrane and preparation method and application thereof |
| CN115006597B (en) * | 2022-06-02 | 2024-01-19 | 上海威高医疗技术发展有限公司 | Oral cavity repairing film and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5837278A (en) * | 1994-01-06 | 1998-11-17 | Ed Geistlich Sohne Ag Fur Chemische Industrie | Resorbable collagen membrane for use in guided tissue regeneration |
| CN1438041A (en) * | 2003-03-18 | 2003-08-27 | 四川大学 | Biological active bone tissue inducing regeneration film and preparation method |
-
2011
- 2011-06-01 CN CN201110145229.0A patent/CN102225219B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5837278A (en) * | 1994-01-06 | 1998-11-17 | Ed Geistlich Sohne Ag Fur Chemische Industrie | Resorbable collagen membrane for use in guided tissue regeneration |
| CN1438041A (en) * | 2003-03-18 | 2003-08-27 | 四川大学 | Biological active bone tissue inducing regeneration film and preparation method |
Non-Patent Citations (6)
| Title |
|---|
| )".《Biomaterials》.1997,第18卷第535-538页. * |
| AK. Schlegel et al.."Preclinical and clinical studies of a collagen membrane (Bio-Gide& * |
| AK. Schlegel et al.."Preclinical and clinical studies of a collagen membrane (Bio-Gide® |
| reg * |
| 任天斌等.可吸收引导组织再生膜.《化学进展》.2010,第22 卷(第1 期),第179-185页. |
| 可吸收引导组织再生膜;任天斌等;《化学进展》;20100131;第22 卷(第1 期);第179-185页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102225219A (en) | 2011-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102225219B (en) | Bone tissue regeneration guiding membrane and preparation method thereof | |
| US20230016722A1 (en) | Natural tissue scaffolds as tissue fillers | |
| CN103520780B (en) | Biological amnion and preparation method thereof | |
| CA2489156C (en) | Scaffold for regenerating hard/soft tissue interface | |
| CN101366975A (en) | Preparation method for cellfree intestinum tenue submucosa biological material | |
| van der Veen et al. | New dermal substitutes | |
| KR101377558B1 (en) | Tissue or Organ repair kit composed of growth factors, barriers and scaffolds | |
| CA3041719C (en) | Methods for isolating equine decellularized tissue | |
| CN106075584B (en) | Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof | |
| CA2625604C (en) | Biocompatible and biodegradable porous matrix in particular useful for tissue reconstruction | |
| AU2015334897B2 (en) | A biomaterial scaffold for regenerating the oral mucosa | |
| CN108837184A (en) | A kind of composite membrane and preparation method thereof for Guided Bone Regeneration | |
| CN202283366U (en) | Small intestinal submucosa (SIS) soft tissue recovery patch | |
| WO2006124946A2 (en) | Engineered extracellular matrices | |
| CN105524472A (en) | Keratin/silk fibroin blended membrane and preparation method thereof | |
| CN104857578B (en) | A kind of tissue regeneration membrane of high intensity and preparation method thereof | |
| CN104383601A (en) | Skeletal muscle acellular matrix biological patch and preparation method thereof | |
| CN1903382A (en) | Method for preparing heterogenic bone with cell-removing matrix | |
| CN1456363A (en) | Preparing method for heteroossein base materials | |
| CN103961752B (en) | Tissue regeneration guiding film and preparation method thereof | |
| EP2809340B1 (en) | Biomaterial for articular cartilage maintenance and treatment of arthritis | |
| KR20210005562A (en) | Decellularized bone biomaterial reinforced with a hydrogel containing decellularized extracellular bone matrix | |
| KR101403473B1 (en) | Dental membrane | |
| Zhang et al. | Self-assembling peptide and nHA/CTS composite scaffolds promote bone regeneration through increasing seed cell adhesion | |
| CN105311676A (en) | Hard tissue engineering scaffold material having bioactivity and preparation method of hard tissue engineering scaffold material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: SHAANXI BOAO REGENERATION MEDICAL CO., LTD. Free format text: FORMER OWNER: SHAANXI BOHONG BIOTECHNOLOGY CO., LTD. Effective date: 20141124 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhang Yongjie Inventor after: Yang Lu Inventor after: Huang Wentao Inventor after: Zhang Lijuan Inventor before: Yang Gaiye |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: YANG GAIYE TO: ZHANG YONGJIE YANG LU HUANG WENTAO ZHANG LIJUAN Free format text: CORRECT: ADDRESS; FROM: 710065 XI'AN, SHAANXI PROVINCE TO: 727100 TONGCHUAN, SHAANXI PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20141124 Address after: 727100 No. five, Datang eight, New District, Shaanxi, Tongchuan Patentee after: Shaanxi Boao Regeneration Medical Co., Ltd. Address before: 710065 Shaanxi city of Xi'an Province South Second Ring Road, Yanta District Sha Po Albert urban perspective 10C Patentee before: Shaanxi Bohong Biotechnology Co., Ltd. |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Bone tissue regeneration guiding membrane and preparation method thereof Effective date of registration: 20151221 Granted publication date: 20140129 Pledgee: Xianyang financing Company Limited by Guarantee Pledgor: Shaanxi Boao Regeneration Medical Co., Ltd. Registration number: 2015990001138 |
|
| PLDC | Enforcement, change and cancellation of contracts on pledge of patent right or utility model | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20170113 Granted publication date: 20140129 Pledgee: Xianyang financing guarantee Limited by Share Ltd Pledgor: Shaanxi Boao Regeneration Medical Co., Ltd. Registration number: 2015990001138 |
|
| PLDC | Enforcement, change and cancellation of contracts on pledge of patent right or utility model | ||
| PM01 | Change of the registration of the contract for pledge of patent right |
Change date: 20170113 Registration number: 2015990001138 Pledgee after: Xianyang financing guarantee Limited by Share Ltd Pledgee before: Xianyang financing Company Limited by Guarantee |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Bone tissue regeneration guiding membrane and preparation method thereof Effective date of registration: 20170420 Granted publication date: 20140129 Pledgee: Xianyang financing guarantee Limited by Share Ltd Pledgor: Shaanxi Boao Regeneration Medical Co., Ltd. Registration number: 2017990000327 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20180611 Granted publication date: 20140129 Pledgee: Xianyang financing guarantee Limited by Share Ltd Pledgor: Shaanxi Boao Regeneration Medical Co., Ltd. Registration number: 2017990000327 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Bone tissue regeneration guiding membrane and preparation method thereof Effective date of registration: 20180611 Granted publication date: 20140129 Pledgee: Xianyang financing guarantee Limited by Share Ltd Pledgor: Shaanxi Boao Regeneration Medical Co., Ltd. Registration number: 2018990000439 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20190701 Granted publication date: 20140129 Pledgee: Xianyang financing guarantee Limited by Share Ltd Pledgor: Shaanxi Boao Regeneration Medical Co., Ltd. Registration number: 2018990000439 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Bone tissue regeneration guiding membrane and preparation method thereof Effective date of registration: 20190701 Granted publication date: 20140129 Pledgee: Xianyang financing guarantee Limited by Share Ltd Pledgor: Shaanxi Boao Regeneration Medical Co., Ltd. Registration number: 2019990000643 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20200915 Granted publication date: 20140129 Pledgee: Xianyang financing guarantee Limited by Share Ltd. Pledgor: SHAANXI BIO REGENERATION MEDICINE Co.,Ltd. Registration number: 2019990000643 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A guiding membrane for bone tissue regeneration and its preparation method Effective date of registration: 20200915 Granted publication date: 20140129 Pledgee: Xianyang financing guarantee Limited by Share Ltd. Pledgor: SHAANXI BIO REGENERATION MEDICINE Co.,Ltd. Registration number: Y2020990001114 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20211119 Granted publication date: 20140129 Pledgee: Xianyang financing guarantee Limited by Share Ltd. Pledgor: SHAANXI BIO REGENERATIVE MEDICINE Co.,Ltd. Registration number: Y2020990001114 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |