CN105770991B - 生物源性静脉瓣膜的制备方法 - Google Patents
生物源性静脉瓣膜的制备方法 Download PDFInfo
- Publication number
- CN105770991B CN105770991B CN201610121967.4A CN201610121967A CN105770991B CN 105770991 B CN105770991 B CN 105770991B CN 201610121967 A CN201610121967 A CN 201610121967A CN 105770991 B CN105770991 B CN 105770991B
- Authority
- CN
- China
- Prior art keywords
- processing
- heparin
- biogenic
- preparation
- vein
- 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
- 210000003462 vein Anatomy 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 230000000035 biogenic effect Effects 0.000 title claims abstract description 18
- 229920000669 heparin Polymers 0.000 claims abstract description 50
- 238000000034 method Methods 0.000 claims abstract description 50
- 229960002897 heparin Drugs 0.000 claims abstract description 47
- 238000012545 processing Methods 0.000 claims abstract description 47
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 46
- 230000029087 digestion Effects 0.000 claims abstract description 13
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 11
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 11
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 11
- 238000005336 cracking Methods 0.000 claims abstract description 10
- 238000010494 dissociation reaction Methods 0.000 claims abstract description 9
- 230000005593 dissociations Effects 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 31
- 239000000463 material Substances 0.000 claims description 15
- 210000004731 jugular vein Anatomy 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000004132 cross linking Methods 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- ZNBNBTIDJSKEAM-UHFFFAOYSA-N 4-[7-hydroxy-2-[5-[5-[6-hydroxy-6-(hydroxymethyl)-3,5-dimethyloxan-2-yl]-3-methyloxolan-2-yl]-5-methyloxolan-2-yl]-2,8-dimethyl-1,10-dioxaspiro[4.5]decan-9-yl]-2-methyl-3-propanoyloxypentanoic acid Chemical compound C1C(O)C(C)C(C(C)C(OC(=O)CC)C(C)C(O)=O)OC11OC(C)(C2OC(C)(CC2)C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CC1 ZNBNBTIDJSKEAM-UHFFFAOYSA-N 0.000 claims description 3
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 3
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims description 3
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 claims description 3
- 102000005891 Pancreatic ribonuclease Human genes 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 claims description 3
- 229960001008 heparin sodium Drugs 0.000 claims description 3
- 229910000378 hydroxylammonium sulfate Inorganic materials 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000003599 detergent Substances 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 claims 1
- 239000006166 lysate Substances 0.000 claims 1
- 239000012620 biological material Substances 0.000 abstract description 13
- 230000002785 anti-thrombosis Effects 0.000 abstract description 3
- 239000003146 anticoagulant agent Substances 0.000 abstract description 2
- 210000003141 lower extremity Anatomy 0.000 description 8
- 206010046996 Varicose vein Diseases 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 7
- 208000027185 varicose disease Diseases 0.000 description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 238000002513 implantation Methods 0.000 description 4
- 210000001367 artery Anatomy 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 239000010836 blood and blood product Substances 0.000 description 2
- 229940125691 blood product Drugs 0.000 description 2
- 230000000747 cardiac effect Effects 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 238000003672 processing method Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- UWFRVQVNYNPBEF-UHFFFAOYSA-N 1-(2,4-dimethylphenyl)propan-1-one Chemical compound CCC(=O)C1=CC=C(C)C=C1C UWFRVQVNYNPBEF-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 241000248503 Euplotes woodruffi Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010039580 Scar Diseases 0.000 description 1
- 206010043595 Thrombophlebitis superficial Diseases 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000002308 calcification Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002118 epoxides Chemical class 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 230000001435 haemodynamic effect Effects 0.000 description 1
- VKYKSIONXSXAKP-UHFFFAOYSA-N hexamethylenetetramine Chemical compound C1N(C2)CN3CN1CN2C3 VKYKSIONXSXAKP-UHFFFAOYSA-N 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 238000007539 photo-oxidation reaction Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 210000002073 venous valve Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3625—Vascular tissue, e.g. heart valves
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/36—Materials or treatment for tissue regeneration for embolization or occlusion, e.g. vaso-occlusive compositions or devices
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Transplantation (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Vascular Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明属于生物医学工程学领域，具体指一种生物源性静脉瓣膜的制备方法，制备过程中是将生物材料清理消毒，经细胞裂解处理、细胞消化处理、核酸消化处理后结合肝素，结合过程采用了一套共价‑离子‑共价联合结合肝素的方法。采用本发明所述的方法制备的生物源性静脉瓣膜具有良好的生物物理性能，较强的抗血栓、抗钙化性，具有一定的临床应用推广前景。
Description
技术领域
本发明属于生物医学工程学领域，具体指一种生物源性静脉瓣膜的制备方法。
背景技术
下肢静脉曲张是临床上的常见病，易发于45岁以上的人群。患者早期很少出现明显的临床症状，但到后期则可能出现下肢浅静脉显露，伴酸、沉、胀、痛、易疲劳等症状，严重者可并发血栓性浅静脉炎、瘀积性皮炎、瘀积性溃疡、曲张静脉团出血。下肢静脉曲张的病因是静脉瓣功能不全，导致下肢静脉血液充盈，压力高，从而静脉曲张，静脉曲张导致局部血液循环障碍并影响肢体外观，目前治疗下肢静脉曲张最有效的方法是高位结扎和大隐静脉剥离，这类方法都仅仅是对症治疗，无法恢复静脉瓣的功能，没有消除静脉曲张的根本病因，并且对患者伤害大，术后患者恢复比较慢，复发率比较高，还会造成多个手术瘢痕的遗留。
生物材料在人体内表现出良好的生物相容性及血流动力学特征，目前尚未有用于治疗下肢静脉曲张的生物源性静脉瓣膜的相关报道。
发明内容
本发明旨在提供一种能够用于治疗下肢静脉曲张的生物源性静脉瓣膜的制备方法。
本发明所述生物源性静脉瓣膜的制备方法包括下述步骤：
a、剪取直径0.5～1.5cm，长3～5cm含有完整瓣膜的猪或牛的颈静脉管道，清理并消毒；
b、使用非离子型去污剂对步骤a中得到的材料进行细胞裂解处理，随后进行细胞消化及核酸消化处理；
c、将经过步骤b处理的材料浸入1mol/L羟胺硫酸盐溶液中，室温下处理12h后用蒸馏水漂洗，浸入heparin-EDC液中进行肝素交联处理，随后用PBS溶液冲洗；
d、将经过步骤c处理的材料浸入含1mg/mL PEI的0.15M NaCl水溶液中，室温下处理30min后用PBS溶液冲洗，浸入含0.5mg/mL肝素钠的PBS溶液中，室温下处理30min后用PBS溶液冲洗，再重复上述操作14遍；
e、对经过步骤d处理的材料重复进行1次步骤c所述处理过程，最后将成品置于70～80％的酒精中，室温下密封保存。
所述步骤b～e中的处理过程均在摇动转速为70～100rpm的摇床中进行。
所述步骤a中的消毒处理方式为在0.1％新洁尔灭溶液中浸泡30min。
所述步骤b中的细胞裂解处理使用含0.5％Triton X-100的PBS溶液处理24～48h。
所述步骤b中的细胞消化处理使用含0.025％胰蛋白酶及0.02％EDTA的PBS溶液37℃下处理30～120min。
所述步骤b中的核酸消化处理使用含30u/ml的DNase I及0.3mg/ml的RNase A的PBS溶液37℃下处理24～48h。
步骤b中将细胞裂解、细胞消化及核酸消化这三种处理方法联合使用，能够有效清除生物材料中的细胞组织，从而较好地实现消除钙化位点，降低免疫原性的目的。
所述步骤c中肝素交联的条件为37℃下摇动交联48～72h。
本发明所述制备方法中的肝素结合过程(对应于步骤c、d及步骤e中重复步骤c的部分)包括2次步骤c所述的共价键合肝素处理过程以及1次步骤d所述的离子结合肝素处理过程，构成一套共价-离子-共价联合结合肝素的方法，综合了两种肝素结合方式的优点，最大限度地提高了生物材料的肝素结合量，改善了生物材料的抗血栓性能。
本发明所述方法对生物材料损伤小，制备的产品中的瓣膜部分与人体静脉瓣膜质地相似，且组织相容性好，植入后不易发生急性免疫排斥反应或形成远期血栓，并能够持久保持通畅，有效防止静脉血管的近期或远期狭窄。将本发明制备的产品植入瓣膜功能不全的静脉中能够从根本上消除下肢静脉曲张的病因，由于瓣膜所附带的管道较短，临床应用中还可根据需要进行修剪，植入过程创伤较小，且操作较为简单、安全。
采用本发明提供的方法制备的生物源性静脉瓣膜具有良好的生物物理性能，较强的抗血栓、抗钙化性能，具有一定的临床应用推广前景。
附图说明
图1：本发明所制备的静脉瓣膜成品的外观图片；
图2：本发明所制备的静脉瓣膜成品的HE染色照片；
图3：与图2中静脉瓣膜成品相同来源且未作处理的生物材料的HE染色照片。
具体实施方式
下面结合实施例对本发明所述生物源性静脉瓣膜的制备方法作进一步说明。实施例一
a、取直径在0.5～1.5cm并带有瓣膜的屠宰场日常宰杀的新鲜牛颈静脉管道，翻面后找到瓣膜部分，剪取长3～5cm含有完整瓣膜的区段，翻回正面后修剪掉血管外周脂肪组织及多余的结缔组织，并用生理盐水顺行灌洗清除淤血块，再浸入0.1％新洁尔灭溶液中消毒30min后用PBS溶液冲洗干净备用。
b、使用含0.5％Triton X-100的PBS液处理步骤a中得到的材料48h，裂解细胞，并用PBS溶液冲洗。再浸入含0.025％胰蛋白酶及0.02％EDTA的PBS溶液(0.025g胰蛋白酶，0.02g EDTA溶于100mL PBS溶液中)中处理30min，消化细胞，并用PBS溶液冲洗。最后浸入含30u/ml DNase I及0.3mg/ml RNase A的PBS溶液中处理24h，消化核酸，并用PBS溶液冲洗。各处理过程均在37℃下，摇动转速70～100rpm的摇床中进行。
c、将经步骤b处理的材料浸入1mol/L羟胺硫酸盐溶液中，室温下以70～100rpm摇动转速处理12h后用蒸馏水漂洗3次，每次10min，浸入heparin-EDC液(1.67g EDC+0.835g肝素钠+0.05mol/L HCl 200ml，pH1.5)中在37℃，70～100rpm摇动转速下摇动交联48h后用PBS溶液冲洗3遍。
d、将经过步骤c处理的材料浸入含1mg/mL PEI的0.15M NaCl水溶液中，以70～100rpm摇动转速室温下处理30min后用PBS溶液冲洗3遍；再浸入含0.5mg/mL肝素的PBS溶液中，以70～100rpm摇动转速室温下处理30min后用PBS溶液冲洗3遍，再重复执行上述操作14遍。
e.对经过步骤d处理的材料重复进行1次步骤c所述处理过程，整个肝素结合过程可简记为c+d+c，最后得到的静脉瓣膜成品置于70～80％的酒精中，室温下密封保存。
实施例二
本实施例中静脉瓣膜的制备过程与实施例一基本相同，区别在于肝素结合过程仅包含1次步骤c所述的共价键合肝素处理过程，整个肝素结合过程可简记为c。得到的成品静脉瓣膜按照与实施例一相同的方法保存，即置于70～80％的酒精中，室温下密封保存。
实施例三
本实施例中静脉瓣膜的制备过程与实施例一基本相同，区别在于：1.步骤b中细胞裂解处理、细胞消化及核酸消化处理的时间分别为36h、60min和36h；2.肝素结合过程仅包含1次步骤d所述的离子结合肝素处理过程，整个肝素结合过程可简记为d。得到的成品静脉瓣膜置于70～80％的酒精中，室温下密封保存。
实施例四
本实施例中静脉瓣膜的制备过程与实施例一基本相同，区别在于：1.静脉瓣膜取材自新鲜猪颈静脉管道；2.步骤b中细胞裂解处理、细胞消化及核酸消化处理的时间分别为36h、90min和36h；3.肝素结合过程先进行1次步骤c所述的共价键合肝素处理过程，且肝素交联的条件为37℃下摇动交联60h，再进行1次步骤d所述的离子结合肝素处理过程，整个肝素结合过程可简记为c+d。得到的成品静脉瓣膜置于70～80％的酒精中，室温下密封保存。
实施例五
本实施例中静脉瓣膜的制备过程与实施例一基本相同，区别在于：1.静脉瓣膜取材自新鲜猪颈静脉管道；2.步骤b中细胞裂解处理、细胞消化及核酸消化处理的时间分别为24h、90min和48h；3.肝素结合过程在实施例一的基础上增加了1次步骤d所述的离子结合肝素处理过程，且步骤c中的肝素交联的条件为37℃下摇动交联60h，整个肝素结合过程可简记为c+d+c+d。得到的成品静脉瓣膜置于70～80％的酒精中，室温下密封保存。
实施例六
本实施例中静脉瓣膜的制备过程中与实施例一基本相同，区别在于：1.静脉瓣膜取材自新鲜猪颈静脉管道；2.步骤b中细胞裂解处理、细胞消化及核酸消化处理的时间分别为24h、120min和48h；3.肝素结合过程在实施例一的基础上先后增加了1次步骤d所述的离子结合肝素处理过程及1次步骤c所述的共价键合肝素处理过程，且步骤c中的肝素交联的条件为37℃下摇动交联72h，整个肝素结合过程可简记为c+d+c+d+c。得到的成品静脉瓣膜置于70～80％的酒精中，室温下密封保存。
实验部分
1.静脉瓣膜结合肝素方法的研究
通过电感耦合等离子体质谱仪(ICP-MS)测定实施例一至实施例六制备的成品静脉瓣膜的肝素浓度，比较其结合的肝素量。
六组实施例制备的静脉瓣膜测得肝素含量结果如表1所示，从表1可以看出，整合离子结合肝素及共价键合肝素这两种方法多次结合相较于单纯采用一种方法一次结合可以有效提高生物材料的肝素结合量。采用1次共价键合+1次离子结合+1次共价键合的实施例一制备的静脉瓣膜结合的肝素浓度达到最高值，在实施例一的基础上继续增加离子结合肝素及共价键合肝素的次数都无法再提高单位重量生物材料结合的肝素量。
表1：各组样品中肝素浓度的测定结果
| 组别 | 肝素含量(ug) | 血管重量(g) | 肝素浓度(ug/g) |
| 实施例二(c) | 29.51336 | 0.35 | 84.3239 |
| 实施例三(d) | 11.16994 | 0.16 | 69.81215 |
| 实施例四(c+d) | 35.87998 | 0.35 | 102.5142 |
| 实施例一(c+d+c) | 55.23197 | 0.17 | 324.894 |
| 实施例五(c+d+c+d) | 88.61461 | 0.42 | 210.9872 |
| 实施例六(c+d+c+d+c) | 77.53149 | 0.34 | 228.0338 |
2.静脉曲张模拟实验
将成年实验犬麻醉后在颈部做切口，显露颈静脉，使犬头低位，心脏静脉血液倒流入头部，模拟人体静脉曲张的原理，然后观察犬头低位颈静脉是否怒张，并监测压力，将实施例一制备的生物源性静脉瓣膜植入实验犬颈静脉，使实验犬处于同样头低位后，观察手术后的远端静脉是否仍然怒张，并测定其远端静脉张力。
植入实施例一制备的静脉瓣膜前，保持犬头低位，肉眼可见其颈静脉怒张，测量其静脉张力为15mmHg；植入后，保持犬头低位，远端颈静脉怒张程度明显减轻，测量其静脉张力为7mmHg。上述结果表明植入该静脉瓣膜后能明显减少犬头低位后心脏静脉血倒流入头部，即有效阻止静脉血倒流。
3.抗钙化性能研究
取长直径大致相同并带有完整瓣膜的猪颈静脉按照实施例一中步骤a和b所述方法进行处理后分别采用戊二醛交联法(A组)、光氧化交联法(B组)、环氧化合物交联法(C组)及实施例一所述的共价-离子-共价联合结合肝素法(D组)进行处理，每组制备10份样品。从处理好的每份样品中裁剪出1×1cm2带瓣叶的试片，植入SD大白鼠体内皮下包埋60天后取出，用原子吸收分光光度计测定其钙含量。各组样品的具体交联方法如下：
A组：采用0.625％戊二醛固定6小时，0.3％戊二醛保存；
B组：采用0.1％亚甲蓝作为介导剂，光照48小时，光照强度为4000流明小时，温度为10-15℃，消毒，75％酒精保存；
C组：采用4％乙二醇二缩水甘油醚溶液固定3天，漂洗后0.5％肝素钠溶液中浸泡1小时，75％酒精保存；
D组：与实施例一所述处理方法相同。
四组不同方法制备的样品试片皮下包埋后的钙含量检测结果如表2所示，从表2可以看出，实施例一所述方法制备的静脉瓣膜含钙量最低，抗钙化性能最佳。
表2：各组样品试片的皮下包埋实验钙含量测定结果
| 组别 | 钙含量测定(mg/g)(n＝10) |
| A | 187.2±30.5 |
| B | 96.3±21.1 |
| C | 128.5±29.1 |
| D | 23.6±5.1 |
4.HE染色检测脱细胞实验
将实施例一制备的静脉瓣膜成品和相同来源未作处理的生物材料进行HE染色后，在显微镜下观察。如图3所示，未作处理的生物材料中含有大量被染色的细胞核，即图3中的深色细斑。如图2所示，本发明将生物材料进行细胞裂解处理、细胞消化处理及核酸消化处理后，已见不到深色细斑，表明生物材料中的细胞组织已被完全脱去。
Claims (7)
1.一种生物源性静脉瓣膜的制备方法，其特征在于，包括下述步骤：
a、剪取直径0.5～1.5cm，长3～5cm含有完整瓣膜的猪或牛的颈静脉管道，清理并消毒；
b、使用非离子型去污剂对步骤a中得到的材料进行细胞裂解处理，随后进行细胞消化及核酸消化处理；
c、将经过步骤b处理的材料浸入1mol/L羟胺硫酸盐溶液中，室温下处理12h后用蒸馏水漂洗，浸入heparin-EDC液中进行肝素交联处理，随后用PBS溶液冲洗；
d、将经过步骤c处理的材料浸入含1mg/mL PEI的0.15M NaCl水溶液中，室温下处理30min后用PBS溶液冲洗，浸入含0.5mg/mL肝素钠的PBS溶液中室温下处理30min后用PBS溶液冲洗，再重复执行上述操作14遍；
e、对经过步骤d处理的材料重复进行1次步骤c所述处理过程，最后将成品置于70～80％的酒精中，室温下密封保存。
2.如权利要求1所述的生物源性静脉瓣膜的制备方法，其特征在于，所述步骤b～e中的处理过程均在摇动转速为70～100rpm的摇床中进行。
3.如权利要求2所述的生物源性静脉瓣膜的制备方法，其特征在于，所述步骤a中的消毒处理方式为在0.1％新洁尔灭溶液中浸泡30min。
4.如权利要求2所述的生物源性静脉瓣膜的制备方法，其特征在于，所述步骤b中的细胞裂解处理使用含0.5％Triton X-100的PBS溶液处理24～48h。
5.如权利要求2所述的生物源性静脉瓣膜的制备方法，其特征在于，所述步骤b中的细胞消化处理使用含0.025％胰蛋白酶及0.02％EDTA的PBS溶液37℃下处理30～120min。
6.如权利要求2所述的生物源性静脉瓣膜的制备方法，其特征在于，所述步骤b中的核酸消化处理使用含30u/ml的DNase I及0.3mg/ml的RNase A的PBS溶液37℃下处理24～48h。
7.如权利要求2所述的生物源性静脉瓣膜的制备方法，其特征在于，所述步骤c中肝素交联的条件为37℃下摇动交联48～72h。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610121967.4A CN105770991B (zh) | 2016-03-03 | 2016-03-03 | 生物源性静脉瓣膜的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610121967.4A CN105770991B (zh) | 2016-03-03 | 2016-03-03 | 生物源性静脉瓣膜的制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105770991A CN105770991A (zh) | 2016-07-20 |
| CN105770991B true CN105770991B (zh) | 2018-09-25 |
Family
ID=56387992
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610121967.4A Active CN105770991B (zh) | 2016-03-03 | 2016-03-03 | 生物源性静脉瓣膜的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105770991B (zh) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109316631B (zh) * | 2018-09-18 | 2021-04-20 | 曾祥军 | 含静脉瓣的生物源性静脉的制备方法 |
| CN109651627B (zh) * | 2018-12-18 | 2021-10-26 | 中国医学科学院生物医学工程研究所 | 天然聚合物交联剂及其在制备抗钙化生物瓣膜中的应用 |
| CN111445746A (zh) * | 2020-03-25 | 2020-07-24 | 陈显金 | 一种练习用的显微缝合材料的制作方法 |
| CN113499479B (zh) * | 2021-07-19 | 2023-03-17 | 科凯(南通)生命科学有限公司 | 改性生物材料的制备方法及得到的改性生物材料 |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1647777A (zh) * | 2003-11-20 | 2005-08-03 | 储晞 | 人工机械瓣膜的设计、材料及其制造方法 |
| CN2764324Y (zh) * | 2005-02-07 | 2006-03-15 | 赵珺 | 一种静脉瓣膜替代物 |
| CN1775304A (zh) * | 2005-11-30 | 2006-05-24 | 中国科学院上海硅酸盐研究所 | 具有生物活性的人工生物瓣瓣膜的制备方法 |
| EP1698356A1 (en) * | 2003-12-26 | 2006-09-06 | Cardio, Inc. | Decellularized tissue and method of preparing the same |
| CN101073678A (zh) * | 2007-06-26 | 2007-11-21 | 中国人民解放军第二军医大学 | 组织工程化静脉瓣及其制备方法 |
| CN101185770A (zh) * | 2007-12-27 | 2008-05-28 | 南京市儿童医院 | 猪带瓣血管脱细胞的支架的超高压制备方法 |
| CN201533878U (zh) * | 2008-09-27 | 2010-07-28 | 熊江 | 发夹型自膨式人工静脉瓣膜 |
| CN201790917U (zh) * | 2010-09-15 | 2011-04-13 | 上海索康医用材料有限公司 | 一种静脉瓣膜功能环 |
| CN102836464A (zh) * | 2011-06-20 | 2012-12-26 | 中国科学院上海硅酸盐研究所 | 一种生物源人工小口径血管及其制备方法 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10064948C1 (de) * | 2000-12-20 | 2002-07-11 | Auto Tissue Gmbh | Verfahren zur Dezellularisierung von Fremdmaterial zur Herstellung von Bioprothesen und Vorrichtung zur Durchführung des Verfahrens |
-
2016
- 2016-03-03 CN CN201610121967.4A patent/CN105770991B/zh active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1647777A (zh) * | 2003-11-20 | 2005-08-03 | 储晞 | 人工机械瓣膜的设计、材料及其制造方法 |
| EP1698356A1 (en) * | 2003-12-26 | 2006-09-06 | Cardio, Inc. | Decellularized tissue and method of preparing the same |
| CN2764324Y (zh) * | 2005-02-07 | 2006-03-15 | 赵珺 | 一种静脉瓣膜替代物 |
| CN1775304A (zh) * | 2005-11-30 | 2006-05-24 | 中国科学院上海硅酸盐研究所 | 具有生物活性的人工生物瓣瓣膜的制备方法 |
| CN101073678A (zh) * | 2007-06-26 | 2007-11-21 | 中国人民解放军第二军医大学 | 组织工程化静脉瓣及其制备方法 |
| CN101185770A (zh) * | 2007-12-27 | 2008-05-28 | 南京市儿童医院 | 猪带瓣血管脱细胞的支架的超高压制备方法 |
| CN201533878U (zh) * | 2008-09-27 | 2010-07-28 | 熊江 | 发夹型自膨式人工静脉瓣膜 |
| CN201790917U (zh) * | 2010-09-15 | 2011-04-13 | 上海索康医用材料有限公司 | 一种静脉瓣膜功能环 |
| CN102836464A (zh) * | 2011-06-20 | 2012-12-26 | 中国科学院上海硅酸盐研究所 | 一种生物源人工小口径血管及其制备方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105770991A (zh) | 2016-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105770991B (zh) | 生物源性静脉瓣膜的制备方法 | |
| EP3040088B1 (en) | Method for preparing an animal decellularized tissue matrix material and a decellularized tissue matrix material prepared thereby | |
| Hodde et al. | Effects of sterilization on an extracellular matrix scaffold: part I. Composition and matrix architecture | |
| CN101507837A (zh) | 生物植入材料及其制备方法 | |
| CN104055795B (zh) | 一种可注射植入剂及其制备方法 | |
| CN104023526B (zh) | 一种处理生物材料的方法及经该方法处理的生物材料 | |
| US20050008708A1 (en) | EB matrix production from fetal tissues and its use for tissue repair | |
| CN109651627A (zh) | 天然聚合物交联剂及其在制备抗钙化生物瓣膜中的应用 | |
| CN107233621A (zh) | 天然软组织去细胞基质的制备方法 | |
| CN101161293A (zh) | 异种生物组织材料的抗钙化改性方法 | |
| JP2022519407A (ja) | 外科的移植のための生体組織を調製する方法 | |
| CN101537207A (zh) | 一种组织工程化异种皮的制备方法 | |
| CN111084900A (zh) | 一种脱细胞鱼皮基质的制备方法及其应用 | |
| CN113476667A (zh) | 一种鱼皮脱细胞真皮基质支架及其制备方法和应用 | |
| JP2019162440A (ja) | 組織製品の酵素処理方法 | |
| Rao et al. | Sterilization of chitosan: implications | |
| CN109364298A (zh) | 一种脱细胞真皮基质材料的制备方法 | |
| CN112755247A (zh) | 一种脱细胞真皮基质及其制备方法 | |
| CN112618796B (zh) | 一种脱细胞真皮基质材料及其制备方法和应用 | |
| KR101269618B1 (ko) | 포유류의 연골조직에서 유래한 생체이식재 | |
| Kumar et al. | Effects of crosslinking treatments on the physical properties of acellular fish swim bladder | |
| WO2017067295A1 (zh) | 一种人工生物带瓣膜血管及其制备方法 | |
| Meuris et al. | Bioprosthetic tissue calcification: influence of blood contact and arterial pressure. an experimental study in rats and sheep | |
| WO2024001217A1 (zh) | 一种生物组织的处理方法及医用材料 | |
| RU2769248C1 (ru) | Способ получения ацеллюлярного дермального матрикса |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190430 Address after: 430075 No. 09 Huacheng Avenue, Donghu New Technology Development Zone, Wuhan City, Hubei Province Patentee after: Wuhan Weian Biotechnology Co.,Ltd. Address before: 430022 Asian Heart Disease Hospital 753 Jinghan Avenue, Wuhan City, Hubei Province Patentee before: Zeng Xiangjun |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230705 Address after: 234099 208, Building 5, Kangyuan Community, No. 530, Renmin South Road, Yongqiao District, Suzhou City, Anhui Province Patentee after: Zeng Xiangjun Address before: 430075 No. 09 Huacheng Avenue, Donghu New Technology Development Zone, Wuhan City, Hubei Province Patentee before: Wuhan Weian Biotechnology Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240317 Address after: 2nd Floor, R&D Center Building, No. 7266 Linquan Road, Haitang Street, Luyang District, Hefei City, Anhui Province, 230041 Patentee after: Anhui Maixin Medical Technology Co.,Ltd. Country or region after: China Address before: 234099 208, Building 5, Kangyuan Community, No. 530, Renmin South Road, Yongqiao District, Suzhou City, Anhui Province Patentee before: Zeng Xiangjun Country or region before: China |