CN112618797B - 一种交联抗生素的特定脱矿细胞外基质支架的制备方法 - Google Patents
一种交联抗生素的特定脱矿细胞外基质支架的制备方法 Download PDFInfo
- Publication number
- CN112618797B CN112618797B CN202011482219.1A CN202011482219A CN112618797B CN 112618797 B CN112618797 B CN 112618797B CN 202011482219 A CN202011482219 A CN 202011482219A CN 112618797 B CN112618797 B CN 112618797B
- Authority
- CN
- China
- Prior art keywords
- cancellous bone
- extracellular matrix
- solution
- bone
- sdecm
- 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
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 title claims abstract description 36
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 title claims abstract description 36
- 210000002744 extracellular matrix Anatomy 0.000 title claims abstract description 36
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 28
- 229940088710 antibiotic agent Drugs 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 238000004132 cross linking Methods 0.000 title claims abstract description 14
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 102
- 230000001954 sterilising effect Effects 0.000 claims abstract description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 10
- 239000000463 material Substances 0.000 claims description 77
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 claims description 66
- 108010059993 Vancomycin Proteins 0.000 claims description 55
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 claims description 55
- 229960003165 vancomycin Drugs 0.000 claims description 55
- 239000000243 solution Substances 0.000 claims description 45
- 238000005406 washing Methods 0.000 claims description 36
- 238000002791 soaking Methods 0.000 claims description 33
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 claims description 22
- 229920004890 Triton X-100 Polymers 0.000 claims description 22
- 239000008399 tap water Substances 0.000 claims description 22
- 235000020679 tap water Nutrition 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 13
- 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 11
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 229960002100 cefepime Drugs 0.000 claims description 11
- VAAUVRVFOQPIGI-SPQHTLEESA-N ceftriaxone Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1CSC1=NC(=O)C(=O)NN1C VAAUVRVFOQPIGI-SPQHTLEESA-N 0.000 claims description 11
- 229960004755 ceftriaxone Drugs 0.000 claims description 11
- 238000005520 cutting process Methods 0.000 claims description 11
- 238000004806 packaging method and process Methods 0.000 claims description 11
- 230000005855 radiation Effects 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 11
- 239000012498 ultrapure water Substances 0.000 claims description 11
- 230000003115 biocidal effect Effects 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 238000004108 freeze drying Methods 0.000 claims description 7
- 239000012984 antibiotic solution Substances 0.000 claims description 6
- 238000003860 storage Methods 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 2
- 241000124008 Mammalia Species 0.000 claims description 2
- 210000002449 bone cell Anatomy 0.000 claims description 2
- 238000011010 flushing procedure Methods 0.000 claims description 2
- 210000004197 pelvis Anatomy 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- HVFLCNVBZFFHBT-ZKDACBOMSA-N cefepime Chemical compound S([C@@H]1[C@@H](C(N1C=1C([O-])=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1C[N+]1(C)CCCC1 HVFLCNVBZFFHBT-ZKDACBOMSA-N 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 abstract description 36
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 24
- 239000003814 drug Substances 0.000 abstract description 14
- 238000001179 sorption measurement Methods 0.000 abstract description 13
- 239000002253 acid Substances 0.000 abstract description 12
- 229940079593 drug Drugs 0.000 abstract description 11
- 206010031252 Osteomyelitis Diseases 0.000 abstract description 7
- 230000008439 repair process Effects 0.000 abstract description 7
- 238000010382 chemical cross-linking Methods 0.000 abstract description 6
- 239000002131 composite material Substances 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 230000004044 response Effects 0.000 abstract description 3
- 238000005297 material degradation process Methods 0.000 abstract 1
- 230000002265 prevention Effects 0.000 abstract 1
- 239000000758 substrate Substances 0.000 abstract 1
- 230000001225 therapeutic effect Effects 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 35
- 230000007547 defect Effects 0.000 description 27
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 20
- 238000011156 evaluation Methods 0.000 description 19
- 108090000623 proteins and genes Proteins 0.000 description 16
- 230000000694 effects Effects 0.000 description 15
- 238000012360 testing method Methods 0.000 description 15
- 210000002997 osteoclast Anatomy 0.000 description 14
- 238000005115 demineralization Methods 0.000 description 13
- 230000002328 demineralizing effect Effects 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 238000010186 staining Methods 0.000 description 13
- 230000002924 anti-infective effect Effects 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 10
- HVFLCNVBZFFHBT-ZKDACBOMSA-O cefepime(1+) Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1C[N+]1(C)CCCC1 HVFLCNVBZFFHBT-ZKDACBOMSA-O 0.000 description 10
- 241000235395 Mucor Species 0.000 description 9
- 210000001991 scapula Anatomy 0.000 description 9
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 8
- 241000191967 Staphylococcus aureus Species 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- 230000002188 osteogenic effect Effects 0.000 description 7
- 230000010065 bacterial adhesion Effects 0.000 description 6
- 230000002458 infectious effect Effects 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 206010000269 abscess Diseases 0.000 description 5
- 125000003277 amino group Chemical group 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 230000011164 ossification Effects 0.000 description 5
- 210000000963 osteoblast Anatomy 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- 238000001356 surgical procedure Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 230000000181 anti-adherent effect Effects 0.000 description 4
- 239000001506 calcium phosphate Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 238000005538 encapsulation Methods 0.000 description 4
- 239000007943 implant Substances 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 238000002386 leaching Methods 0.000 description 4
- 210000002540 macrophage Anatomy 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000004445 quantitative analysis Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 4
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 3
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 3
- 241000194033 Enterococcus Species 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 239000003911 antiadherent Substances 0.000 description 3
- 239000003114 blood coagulation factor Substances 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 238000002329 infrared spectrum Methods 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000010287 polarization Effects 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 230000004853 protein function Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 229910000391 tricalcium phosphate Inorganic materials 0.000 description 3
- 235000019731 tricalcium phosphate Nutrition 0.000 description 3
- 229940078499 tricalcium phosphate Drugs 0.000 description 3
- 238000004506 ultrasonic cleaning Methods 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 102000004237 Decorin Human genes 0.000 description 2
- 108090000738 Decorin Proteins 0.000 description 2
- 208000005422 Foreign-Body reaction Diseases 0.000 description 2
- 238000002738 Giemsa staining Methods 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 102000014128 RANK Ligand Human genes 0.000 description 2
- 108010025832 RANK Ligand Proteins 0.000 description 2
- 208000035415 Reinfection Diseases 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 102000007000 Tenascin Human genes 0.000 description 2
- 108010008125 Tenascin Proteins 0.000 description 2
- 108010031318 Vitronectin Proteins 0.000 description 2
- 102100035140 Vitronectin Human genes 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000001733 anti-osteogenic effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000010478 bone regeneration Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000013329 compounding Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000009881 electrostatic interaction Effects 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229910010272 inorganic material Inorganic materials 0.000 description 2
- 239000011147 inorganic material Substances 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000010534 mechanism of action Effects 0.000 description 2
- 238000010603 microCT Methods 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 210000003625 skull Anatomy 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 229920002994 synthetic fiber Polymers 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 230000007730 Akt signaling Effects 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010051728 Bone erosion Diseases 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 208000006877 Insect Bites and Stings Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 101800004937 Protein C Proteins 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 102100036546 Salivary acidic proline-rich phosphoprotein 1/2 Human genes 0.000 description 1
- 101800001700 Saposin-D Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 102100029477 Vitamin K-dependent protein C Human genes 0.000 description 1
- 101710193900 Vitamin K-dependent protein C Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000010976 amide bond formation reaction Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000012237 artificial material Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000000560 biocompatible material Substances 0.000 description 1
- 230000032770 biofilm formation Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000002639 bone cement Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000010201 enrichment analysis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 210000001650 focal adhesion Anatomy 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000012482 interaction analysis Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012567 medical material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 238000007709 nanocrystallization Methods 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 210000000680 phagosome Anatomy 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000010118 platelet activation Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 230000005588 protonation Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Images
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
- A61L27/3633—Extracellular matrix [ECM]
-
- 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
-
- 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
- A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
-
- 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
- A61L27/3645—Connective tissue
- A61L27/365—Bones
-
- 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
- A61L27/36—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
- A61L27/3691—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 physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
-
- 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
- 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
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
- A61L2300/406—Antibiotics
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/41—Anti-inflammatory agents, e.g. NSAIDs
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
-
- 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
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
-
- 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)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Transplantation (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Vascular Medicine (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明公开了一种交联抗生素的特定脱矿细胞外基质支架的制备方法,属于骨感染治疗技术领域。本发明公开的一种交联抗生素的特定脱矿细胞外基质支架的制备方法,通过将特定脱矿脱细胞的松质骨细胞外基质支架(SDECM)作为底物,与抗生素以静电吸附和化学交联两种形式复合载药,通过在酸性环境中进行快速pH响应和伴随材料降解两种方式释放抗生素,通过抗生素抗感染并通过SDECM促修复,从而达到在感染早期酸性环境下pH敏感释放药物快速杀菌、中晚期生理环境中缓慢释放药物持续杀菌、无感染状态时仍具备永久杀菌能力防止感染复发的治疗效果,为骨感染的抗菌和修复治疗提供新思路和新工具。
Description
技术领域
本发明涉及骨感染治疗技术领域,更具体的说是涉及一种交联抗生素的特定脱矿细胞外基质支架的制备方法。
背景技术
尽管医疗水平不断提高,骨感染在临床工作中依然是一个巨大的难题,将近40%的患者面临着感染的复发与迁延不愈。在骨感染的病理过程中,细菌大量繁殖,在感染局部形成脓肿,躲避免疫系统追杀并保护其核心的活菌繁殖,脓肿里细菌分泌的因子以及酸性代谢产物,不仅促进破骨细胞的分化,降低成骨细胞的活性,同时有助于细菌粘附胶原或细胞膜上,促进生物膜的形成,导致骨感染迁延不愈。在脓肿、细菌粘附定植、生物膜形成、休眠细菌、及无抗菌活性的材料这5个导致感染难以治愈并由急性转为慢性的关键因素中,外科手段仅仅能解决脓肿及部分生物膜等因素,彻底的治愈仍需要依靠抗菌材料植入。
然而,植入的生物材料对于机体来说是一种“外来异物”,生物相容性差的材料在体内将由于异物反应(foreign body reaction,FBR)或细菌粘附导致植入失败,其在体内形成的包裹和纤维化,不仅阻碍植入物与宿主之间的整合,有利于细菌粘附,同时消耗大量的中性粒细胞,降低组织局部的抗感染能力,两者共同促进了感染的进展。因此,在具备抗感染能力的同时改进植入物的生物相容性,对于抗菌材料来说十分重要。如下设计的材料可能是解决的最佳方案:1)材料改性增加生物相容性的同时最大程度减少细菌的粘附能力;2)设计响应释放和按需释放的智能抗菌材料,在达到杀菌的同时最大程度减少组织及细胞的毒性,并且随着环境的改变再次释放抗菌剂以防止感染复发。
目前的可降解抗菌材料,主要是由无机材料如羟基磷灰石、磷酸三钙或者合成材料如聚乳酸-羟基乙酸共聚物(poly-lactic-co-glycolic acid,PLGA)等复合不同的抗生素、抗菌肽或金属离子组成。无机材料多在其表面进行有机物修饰以改进其生物相容性,而为避免交联对药物活性产生影响,抗生素、抗菌肽等生物制剂大多以混合的方式加入材料中以释放出来杀灭浮游细菌。这种混合方式有静电吸附载药、纳米化封装、凝胶化载药、脂质体封装、微球封装等等。金属离子既可加工成纳米颗粒吸附在材料中以杀灭浮游细菌,也可以交联在材料表面避免细菌粘附。申请号为201510882167.X的专利公开了一种抗感染磷酸钙复合骨水泥材料及其制备方法,得到了广谱抗菌效果;申请号为201210027387.0的专利公开了一种采用等离子技术制备抗感染医用材料的方法,对医用材料可进行广泛的抗菌涂层,尽管在近几十年中,这类材料的设计得以快速发展,部分达到了理想抗菌功能,仍有不少缺陷需要克服。首先,无机物与有机物两种介质之间由于表面自由能差异,这种修饰并不简单,不稳定的涂层随着时间推移和体内环境的变化,势必脱落导致材料的生物相容性下降;其次,随着抗生素或抗菌肽的完全释放,材料本身不再具备抗菌性能,成为细菌粘附的对象,且不能预防“潜伏”细菌的再次感染;最后,金属离子具有细胞毒性,不利于组织修复和成骨。
大量的研究表明,天然来源的细胞外基质支架(extracellular matrix,ECM)具有人工材料无法比拟的优势。首先,其完美的孔隙和良好的生物相容性,除了诱导间充质干细胞的生长分化以外,还可诱导趋化多种前体细胞和巨噬细胞,参与免疫调节;其次,细胞外基质支架含多种内源性蛋白及因子,可主动诱导巨噬细胞的极化,并且增强自身免疫系统的抗菌能力;第三、这些能力并不会如有机涂层改性的无机材料和合成材料一样,随着时间和环境的变化而减弱。
在智能抗菌研究领域,羧酸根的质子化被认为是酸敏感释放的一种有效策略,脱细胞化的细胞外基质含大量的活性羧基,不但可以静电作用吸附带正电荷的抗菌剂,而且可与其他分子的游离氨基以酰胺键形式交联。多种类的抗生素因含有氨基而带正电荷,同时可以与多种物质的羧基交联,不影响其抗菌性能。
因此,提供一种交联抗生素的特定脱矿细胞外基质支架的制备方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种交联抗生素的特定脱矿细胞外基质支架的制备方法,通过特定脱矿细胞外基质作为生物相容性支架,并以静电吸附和化学交联两种形式复合抗生素。
为了实现上述目的,本发明采用如下技术方案:
从动物富含松质骨部位获取松质骨颗粒,以优化的脱细胞方案进行脱细胞,制备特定脱矿的松质骨细胞外支架,并以静电吸附和化学交联两种形式复合抗生素,通过缓释及pH响应释放抗生素,达到长期的抗感染和促修复作用。
一种交联抗生素的特定脱矿细胞外基质支架的制备方法,具体步骤如下:
(1)从动物富含松质骨部位获取直径为4-8mm的松质骨颗粒;
(2)脱细胞:
①将步骤(1)获取的松质骨颗粒,切成2-4mm厚度的圆柱体,获得松质骨块;
②将步骤①获得的松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1h;
③将包埋盒转移至过滤除菌后的1%(v/v)Triton-X100溶液的烧瓶中,100rpm,4℃震动12-48h;
④用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;
⑤将松质骨块加入1%(w/v)十二烷基硫酸钠(SDS)水溶液中,100rpm,4℃震动12-48h,清洗包埋盒,获得松质骨ECM;
⑥将松质骨ECM浸泡于10%EDTA脱钙液中,放入VCare(上海颖穆)快速超声脱钙机中,在4℃下脱钙2-6h;
⑦将ECM骨块用自来水冲洗,辐射消毒,并冻干保存,获得松质骨细胞外支架;
(3)松质骨细胞外支架复合抗生素:
将30mg材料粉末加入到抗生素溶液中,浸泡1h,使两者充分混匀;加入等体积的EDC(1-ethyl-3-(3-dimethyl aminopropyl,1-乙基-3-(3-二甲基氨基丙基))-NHS(N-hydroxysuccinimide,N-羟基琥珀酰亚胺)混合溶液,所述EDC-NHS混合溶液中EDC浓度为16mM,NHS浓度为4mM;反应完成的材料用去离子水清洗三遍,9000rpm/min离心5min,弃去上清液,冻干保存,获得交联抗生素的特定脱矿细胞外基质支架;
所述抗生素溶液为2-20mg/ml的万古霉素溶液,或2-20mg/ml的头孢曲松溶液,或2-20mg/ml的头孢吡肟溶液。
进一步,步骤(1)所述动物为大型哺乳动物,包括牛、猪。
进一步,步骤(1)所述富含松质骨部位包括脊柱、肋骨、肩胛、骨盆。
进一步,步骤(2)③所述1%(v/v)Triton-X100用0.22μm滤头过滤除菌。
进一步,步骤(2)⑦所述辐射剂量为250kGy。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种交联抗生素的特定脱矿细胞外基质支架的制备方法,以静电吸附和化学交联两种形式复合抗生素,通过缓释及pH响应释放抗生素,为长期的骨植入物抗感染和促修复提供新思路和新方法。相比现有骨植入物抗感染和促修复的药物或材料,本发明的有益效果在于:
1)采用天然来源的细胞外基质材料,具有良好的生物相容性及生物活性;
2)对松质骨脱细胞材料进行特定脱矿,增强生物活性同时保留孔隙及机械强度;
3)分别采取静电吸附及共价交联的方式结合抗生素,以达到酸敏感释放和长期的降解引发释放效果;
4)体外实验验证其显著的游离杀菌和接触杀菌效果,对浮游细菌数量、细菌粘附、细菌总量指标均有明确的抑制作用;
5)在体外实验验证显著的抗感染和促成骨作用,对骨组织细菌引发的炎症及感染情况及破骨-成骨平衡介导的骨修复均有明确的改善作用;
6)作为一种基于细胞外基质的抗生素修饰材料,所修饰的细胞外基质基团及抗生素或其他生物活性分子均可替换,具有充足的可借鉴性。
本发明交联抗生素的特定脱矿细胞外基质支架可应用于防治骨感染,促进骨修复,具有明确的治疗效果。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明Van-SDECM支架合成与释放的原理示意图;
图2附图为本发明SDECM、Van、ECM和SDECM+Van(SDECM与Van吸附)的Zeta电位;
图3附图为本发明SDECM羧基与万古霉素氨基在不同pH条件下的静电相互作用示意图;
图4附图为本发明Van-SDECM合成时红外谱图(蓝色箭头表示NH伸缩带,红色箭头表示C=O伸缩带,黑色曲线指合成的Van-SDECM)和Van-SDECM降解6周后的红外谱图(Van-SDECM,红色曲线;原始材料,黑色曲线);
图5附图为本发明用不同的EDC和NHS浓度(SDECM粉末30mg,初始万古霉素浓度5mg/mL)制备的SDECM中万古霉素的量;
图6附图为本发明以不同EDC/NHS比例制备的SDECM中万古霉素的量;
图7附图为本发明在超声清洗不同的EDC/NHS浓度和比例制备的Van-SDECM后,材料中万古霉素的残留量;线c表示共价负载的万古霉素的大约量;线d表示SDECM EDC中万古霉素的总量=32mM;
图8附图为本发明单纯静电吸附制备的材料释放的万古霉素的累积量;虚线表示在不同材料中加载的万古霉素的初始量;
图9附图为本发明在静电吸附和化学交联条件下制备的材料释放的万古霉素的累积量;虚线表示在不同材料中加载的万古霉素的初始量;
图10附图为本发明当将支架与破骨细胞培养5天时,从Van-SDECM释放的万古霉素的量;
图11附图为本发明Van-SDECM(试验组)和SDECM(对照组)对金黄色葡萄球菌和肠球菌的抑菌环试验;
图12附图为本发明不同材料共培养24小时后,用平板法测定金黄色葡萄球菌悬液的细菌数;比例尺=10mm;
图13附图为本发明不同材料共培养24小时后,用平板法测定金黄色葡萄球菌悬液细菌数的定量分析;
图14附图为本发明超声清洗后的材料与金黄色葡萄球菌共培养24小时,扫描电镜观察各组样本表面的粘附细菌;细菌轮廓用photoshop作伪彩处理;可见经过交联的样本抗粘附细菌能力明显增加;比例尺=5μm;
图15附图为本发明超声清洗后的材料与金黄色葡萄球菌共培养24小时,各组样本表面的粘附细菌的定量分析;
图16附图为本发明粘附细菌经live/dead染色后的共聚焦显微镜图像;比例尺=1mm;
图17附图为本发明粘附细菌经live/dead染色后的定量分析;显著性差异为*(P<0.05),**(P<0.01);***(P<0.001);
图18附图为本发明CCK-8检测不同浓度万古霉素对MSCs的毒性;
图19附图为本发明CCK-8试验测定Van-SDECM浸出液对MSCs的毒性;
图20附图为本发明不同浓度万古霉素(1mg/mL、5mg/mL、10mg/mL)制备的Van-SDECM支架与MSCs共培养48h后,MSCs的live/dead染色结果;比例尺=100μm;
图21附图为本发明以初始万古霉素=5mg、EDC=NHS=16mM制备Van-SDECM支架,获取其浸提液与成骨细胞共培养4天,ALP染色结果;比例尺=100μm;
图22附图为本发明对感染性骨缺损的实验模型建立后1周、6周,分别获取各组别缺损区的软组织进行匀浆,涂布于平板上的细菌典型图片;Group I:单纯缺损组(Defect);Group II:缺损+感染组(Defect+infection);Group III:缺损+感染+SDECM组(Defect+infection+SDECM);Group IV:缺损+感染+万古霉素组(Defect+infection+Van);Group V:缺损+感染+Van-SDECM组(Defect+infection+Van-SDECM);比例尺=10mm;
图23附图为本发明感染性骨缺损的实验模型建立后1周、6周,涂布于平板上的细菌定量统计分析;
图24附图为本发明对感染性骨缺损的实验模型建立后1周、6周,分别行各组别Micro-CT检查的典型图片;比例尺=1mm;
图25附图为本发明对感染性骨缺损的实验模型建立后1周、6周,分别行各组别Micro-CT检查的半定量统计分析;
图26附图为本发明术后6周H&E观察材料的抗感染性能;比例尺=1mm(4X)、250μm(25X)、100μm(40X);
图27附图为本发明术后6周Masson染色观察材料的成骨性能;比例尺=1mm(4X)、250μm(25X)、100μm(40X);
图28附图为本发明流式细胞计数证实在pH 6.0下比在pH 7.4下凋亡的成骨细胞更多,水平轴为AV-FITC,垂直轴为PI;
图29附图为本发明TRAP染色表明破骨细胞在pH 6.0的RANKL+M-CSF培养基中比在pH 7.4分化和融合的更多;
图30附图为本发明在红色荧光中可以观察到破骨细胞和Van-SDECM之间的零星微环境,通过AIE pH探针可以看到破骨细胞的轮廓为蓝色;
图31附图为本发明术后1周和6周,Giemsa染色显示,与IV组和V组相比,II组和III组的细菌浸润(红点)显著增加(红色箭头);比例尺=10μm;
图32附图为本发明手术后一到六周,TRAP染色显示,II组和III组中活化的破骨细胞(红点)比IV组中多,其次是V组(绿色箭头);比例尺=50μm;
图33附图为本发明定量分析每个视野中的细菌计数;
图34附图为本发明定量分析每个视野中的破骨细胞计数;
图35附图为本发明蛋白质质谱;
图36附图为本发明利用KEGG数据库进行蛋白质功能富集分析;
图37附图为本发明富集蛋白的途径相互作用网络。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
交联万古霉素的特定脱矿细胞外基质支架(Van-SDECM)的制备方法,具体步骤如下:
(1)将新鲜的猪肩胛骨以直径6mm的空心骨钻获取松质骨,再切成3mm厚度的圆柱体;
(2)将松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1;1%(v/v)Triton-X100用0.22μm滤头过滤除菌后,再将包埋盒转移至Triton-X100溶液的烧瓶中,100rpm,4℃震动24h;用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;随后将松质骨块加入1%(w/v)十二烷基硫酸钠(SDS)水溶液中,100rpm,4℃震动36h,再次清洗包埋盒;
(3)将松质骨ECM浸泡于10%EDTA脱钙液中,放入在VCare(上海颖穆)快速超声脱钙机中,并在4℃下脱钙4h;此后,将ECM骨块用自来水冲洗,辐射消毒(剂量250kGy),并冻干;
(4)将30mg材料粉末加入到10mg/ml的万古霉素溶液中,浸泡1h,使两者充分混匀;加入同体积的EDC浓度16mM,NHS浓度4mM的EDC-NHS混合溶液,此时的万古霉素终浓度为5mg/ml,常温下反应12h;反应完成的材料以去离子水清洗三遍,9000rpm/min离心5min,弃去上清液,冻干保存。Van-SDECM支架合成与释放的原理示意图见图1。Van与SDECM之间以静电吸附和化学交联两种形式复合,在酸性环境中,吸附的万古霉素可以快速释放,同时材料在体内降解的时候仍可释放具有杀菌活性的万古霉素分子。由于其独特的作用机理,交联在支架上的万古霉素并不丧失其杀菌活性。
实施例2
交联万古霉素的特定脱矿细胞外基质支架(Van-SDECM)的制备方法,具体步骤如下:
(1)将新鲜的猪肩胛骨以直径4mm的空心骨钻获取松质骨,再切成2mm厚度的圆柱体;
(2)将松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1;1%(v/v)Triton-X100用0.22μm滤头过滤除菌后,再将包埋盒转移至Triton-X100溶液的烧瓶中,100rpm,4℃震动12h;用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;随后将松质骨块加入1%(w/v)十二烷基硫酸钠(SDS)水溶液中,100rpm,4℃震动12h,再次清洗包埋盒;
(3)将松质骨ECM浸泡于10%EDTA脱钙液中,放入在VCare(上海颖穆)快速超声脱钙机中,并在4℃下脱钙2h;此后,将ECM骨块用自来水冲洗,辐射消毒(剂量250kGy),并冻干;
(4)将30mg材料粉末加入到2mg/ml的万古霉素溶液中,浸泡1h,使两者充分混匀;加入同体积的EDC浓度16mM,NHS浓度4mM的EDC-NHS混合溶液,此时的万古霉素终浓度为1mg/ml,常温下反应18h;反应完成的材料以去离子水清洗三遍,9000rpm/min离心5min,弃去上清液,冻干保存。
实施例3
交联万古霉素的特定脱矿细胞外基质支架(Van-SDECM)的制备方法,具体步骤如下:
(1)将新鲜的猪肩胛骨以直径8mm的空心骨钻获取松质骨,再切成4mm厚度的圆柱体;
(2)将松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1;1%(v/v)Triton-X100用0.22μm滤头过滤除菌后,再将包埋盒转移至Triton-X100溶液的烧瓶中,100rpm,4℃震动48h;用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;随后将松质骨块加入1%(w/v)十二烷基硫酸钠(SDS)水溶液中,100rpm,4℃震动48h,再次清洗包埋盒;
(3)将松质骨ECM浸泡于10%EDTA脱钙液中,放入在VCare(上海颖穆)快速超声脱钙机中,并在4℃下脱钙6h;此后,将ECM骨块用自来水冲洗,辐射消毒(剂量250kGy),并冻干;
(4)将30mg材料粉末加入到20mg/ml的万古霉素溶液中,浸泡1h,使两者充分混匀;加入同体积的EDC浓度16mM,NHS浓度4mM的EDC-NHS混合溶液,此时的万古霉素终浓度为10mg/ml,常温下反应24h;反应完成的材料以去离子水清洗三遍,9000rpm/min离心5min,弃去上清液,冻干保存。
实施例4
交联头孢曲松的特定脱矿细胞外基质支架的制备方法,具体步骤如下:
(1)将新鲜的猪肩胛骨以直径6mm的空心骨钻获取松质骨,再切成3mm厚度的圆柱体;
(2)松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1h;1%(v/v)Triton-X100以0.22μm滤头过滤除菌后,再将包埋盒转移至Triton-X100溶液的烧瓶中,100rpm,4℃震动24h;用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;随后将松质骨块加入1%(w/v)十二烷基硫酸钠(SDS)水溶液中,100rpm,4℃震动36h,再次清洗包埋盒;
(3)将松质骨ECM浸泡于10%EDTA脱钙液中,放入在VCare(上海颖穆)快速超声脱钙机中,并在4℃下脱钙4h;此后,将ECM骨块用自来水冲洗,辐射消毒(剂量250kGy),并冻干;
(4)将30mg材料粉末加入到5mg/ml的头孢曲松中,浸泡1h,使两者充分混匀;加入同体积的EDC浓度16mM,NHS浓度4mM的EDC-NHS混合溶液,此时的头孢曲松终浓度为2.5mg/ml,常温下反应24h;反应完成的材料以去离子水清洗三遍,9000rpm/mi离心5min,弃去上清液,冻干保存。
实施例5
交联头孢曲松的特定脱矿细胞外基质支架的制备方法,具体步骤如下:
(1)将新鲜的猪肩胛骨以直径4mm的空心骨钻获取松质骨,再切成2mm厚度的圆柱体;
(2)将松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1;1%(v/v)Triton-X100用0.22μm滤头过滤除菌后,再将包埋盒转移至Triton-X100溶液的烧瓶中,100rpm,4℃震动12h;用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;随后将松质骨块加入1%(w/v)十二烷基硫酸钠(SDS)水溶液中,100rpm,4℃震动12h,再次清洗包埋盒;
(3)将松质骨ECM浸泡于10%EDTA脱钙液中,放入在VCare(上海颖穆)快速超声脱钙机中,并在4℃下脱钙2h;此后,将ECM骨块用自来水冲洗,辐射消毒(剂量250kGy),并冻干;
(4)将30mg材料粉末加入到2mg/ml的头孢曲松溶液中,浸泡1h,使两者充分混匀;加入同体积的EDC浓度16mM,NHS浓度4mM的EDC-NHS混合溶液,此时的头孢曲松终浓度为1mg/ml,常温下反应18h;反应完成的材料以去离子水清洗三遍,9000rpm/min离心5min,弃去上清液,冻干保存。
实施例6
交联头孢曲松的特定脱矿细胞外基质支架的制备方法,具体步骤如下:
(1)将新鲜的猪肩胛骨以直径8mm的空心骨钻获取松质骨,再切成4mm厚度的圆柱体;
(2)将松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1;1%(v/v)Triton-X100用0.22μm滤头过滤除菌后,再将包埋盒转移至Triton-X100溶液的烧瓶中,100rpm,4℃震动48h;用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;随后将松质骨块加入1%(w/v)十二烷基硫酸钠(SDS)水溶液中,100rpm,4℃震动48h,再次清洗包埋盒;
(3)将松质骨ECM浸泡于10%EDTA脱钙液中,放入在VCare(上海颖穆)快速超声脱钙机中,并在4℃下脱钙6h;此后,将ECM骨块用自来水冲洗,辐射消毒(剂量250kGy),并冻干;
(4)将30mg材料粉末加入到20mg/ml的头孢曲松溶液中,浸泡1h,使两者充分混匀;加入同体积的EDC浓度16mM,NHS浓度4mM的EDC-NHS混合溶液,此时的头孢曲松终浓度为10mg/ml,常温下反应24h;反应完成的材料以去离子水清洗三遍,9000rpm/min离心5min,弃去上清液,冻干保存。
实施例7
交联头孢吡肟的特定脱矿细胞外基质支架的制备方法,具体步骤如下:
(1)将新鲜的猪肩胛骨以直径6mm的空心骨钻获取松质骨,再切成3mm厚度的圆柱体;
(2)松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1h;1%(v/v)Triton-X100以0.22μm滤头过滤除菌后,再将包埋盒转移至Triton-X100溶液的烧瓶中,100rpm,4℃震动24h;用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;随后将松质骨块加入1%(w/v)十二烷基硫酸钠(SDS)水溶液中,100rpm,4℃震动36h,再次清洗包埋盒;
(3)将松质骨ECM浸泡于10%EDTA脱钙液中,放入在VCare(上海颖穆)快速超声脱钙机中,并在4℃下脱钙4h;此后,将ECM骨块用自来水冲洗,辐射消毒(剂量250kGy),并冻干。
(4)将30mg材料粉末加入到4mg/ml的头孢吡肟溶液中,浸泡1h,使两者充分混匀;加入同体积的EDC浓度16mM,NHS浓度4mM的EDC-NHS混合溶液,此时的头孢吡肟终浓度为2mg/ml,常温下反应12h;反应完成的材料以去离子水清洗三遍,9000rpm/min离心5min后弃去上清液,冻干保存。
实施例8
交联头孢吡肟的特定脱矿细胞外基质支架的制备方法,具体步骤如下:
(1)将新鲜的猪肩胛骨以直径4mm的空心骨钻获取松质骨,再切成2mm厚度的圆柱体;
(2)将松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1;1%(v/v)Triton-X100用0.22μm滤头过滤除菌后,再将包埋盒转移至Triton-X100溶液的烧瓶中,100rpm,4℃震动12h;用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;随后将松质骨块加入1%(w/v)十二烷基硫酸钠(SDS)水溶液中,100rpm,4℃震动12h,再次清洗包埋盒;
(3)将松质骨ECM浸泡于10%EDTA脱钙液中,放入在VCare(上海颖穆)快速超声脱钙机中,并在4℃下脱钙2h;此后,将ECM骨块用自来水冲洗,辐射消毒(剂量250kGy),并冻干;
(4)将30mg材料粉末加入到2mg/ml的头孢吡肟溶液中,浸泡1h,使两者充分混匀;加入同体积的EDC浓度16mM,NHS浓度4mM的EDC-NHS混合溶液,此时的头孢吡肟终浓度为1mg/ml,常温下反应18h;反应完成的材料以去离子水清洗三遍,9000rpm/min离心5min,弃去上清液,冻干保存。
实施例9
交联头孢吡肟的特定脱矿细胞外基质支架的制备方法,具体步骤如下:
(1)将新鲜的猪肩胛骨以直径8mm的空心骨钻获取松质骨,再切成4mm厚度的圆柱体;
(2)将松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1;1%(v/v)Triton-X100用0.22μm滤头过滤除菌后,再将包埋盒转移至Triton-X100溶液的烧瓶中,100rpm,4℃震动48h;用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;随后将松质骨块加入1%(w/v)十二烷基硫酸钠(SDS)水溶液中,100rpm,4℃震动48h,再次清洗包埋盒;
(3)将松质骨ECM浸泡于10%EDTA脱钙液中,放入在VCare(上海颖穆)快速超声脱钙机中,并在4℃下脱钙6h;此后,将ECM骨块用自来水冲洗,辐射消毒(剂量250kGy),并冻干;
(4)将30mg材料粉末加入到20mg/ml的头孢吡肟溶液中,浸泡1h,使两者充分混匀;加入同体积的EDC浓度16mM,NHS浓度4mM的EDC-NHS混合溶液,此时的头孢吡肟终浓度为10mg/ml,常温下反应24h;反应完成的材料以去离子水清洗三遍,9000rpm/min离心5min,弃去上清液,冻干保存。
对实施例1所得的Van-SDECM分别进行合成评估、载药量及释放效率评估、抗浮游和抗粘附细菌特性的评价、细胞毒性和成骨性能评价、抗感染和成骨性能评价、抑制破骨细胞性能评价、蛋白质功能分析
试验例1Van-SDECM的合成评估
(1)支架和万古霉素的电荷以Zeta电位法检测,结果见图2。
图2结果显示,支架(SDECM)呈现出明显的负电荷(-28.17±3.59mV),而万古霉素(Van)则表现为微弱的正电荷(0.54±0.08mV),意味着在万古霉素和支架之间存在强烈的静电作用。相比较于未脱矿的样本(ECM),经过特定脱矿的骨ECM支架(SDECM)负电荷值明显增加(-28.17±3.59mV VS.-14.53±1.94mV,P=0.0003),并且随着万古霉素的吸附(SDECM+Van)而减弱(-19.5±1.47mV,P=0.0078)。
(2)以图示的形式阐述了SDECM中羧基和万古霉素中氨基之间在不同pH环境中的作用机制,见图3。由于羧基和氨基的pKa之约为~5和~8,在生理环境中(pH7.4),大部分的羧基为去质子化的阴离子态(-COO-),带负电荷,而万古霉素为带正电荷的阳离子态,两者之间以氢键形式结合。这种静电吸引的方式,导致了药物在材料中的大量负载和缓慢释放。当材料存在于pH6.0左右的酸性环境中,SDECM材料表面质子化的羧酸(-COOH,带正电荷)增加,导致了药物的快速释放,这一现象也在后续实验中得到证实。
(3)Van-SDECM合成时红外光谱检测结果见图4,酰胺键的形成可以通过新的NH伸缩带(2910cm-1)的出现和C=O伸缩带(1640cm-1)的峰值相对下降来证实(蓝色箭头表示NH伸缩带,红色箭头表示C=O伸缩带,黑色曲线指合成的Van-SDECM)。通过红外光谱检测可以发现,酰胺键典型的C=O和NH伸缩带分别位于1640cm-1和2910cm-1处。新NH伸缩带的出现和C=O伸缩带的减少证实了酰胺键的形成。Van-SDECM降解6周后的红外谱图见图4,随着Van-SDECM的降解(红色曲线),相对于原始材料(黑色曲线),其NH伸缩带消失,C=O伸缩带相对峰高降得更低。
试验例2Van-SDECM的载药量及释放效率评估
(1)当SDECM粉末均为30mg,EDC/NHS=1:1,万古霉素为5mg时,EDC浓度在32mM可获得材料的最大载万古霉素含量(约85%),见图5;而且EDC/NHS之间的比率对于万古霉素载药量影响不是很明显,见图6。
(2)在不断的超声清洗后,记录不同清洗次数(横坐标)后材料上残留的万古霉素含量。单纯静电吸附的组别中,材料上残留万古霉素最少,而随着EDC浓度的增加,材料上残留的万古霉素逐渐增加,各组别均在3次清洗以后,万古霉素含量稳定于将近直线水平,结果见图7;以各组别与单纯吸附组的万古霉素含量差值表示交联量,并计算其交联率,交联速率计算为c除以d;可以得出在EDC=16mM和32mM时,两种交联率分别为37.5±5.6%和65.5±7.8%(P=0.0074)。
(3)单纯静电吸附制备的材料6周后,与生理状态(pH7.4)比较,酸性环境下(pH6.0)Van-SDECM中万古霉素的累积释放量大大增加,差异具有显著性(2285.0±105.6μgVS.686.3±43.6μg,P<0.0001)。而未脱矿的ECM酸敏感释放能力较差,磷酸三钙(TCP)则无酸敏感释放能力,见图8。
随着交联程度的增加,支架中这种酸敏感药物释放的能力逐渐减弱。未交联组表现出最明显的酸敏感性,而EDC=32mM组别则丧失了这一特性,EDC=16mM的组别则表现出中等的酸敏感释放能力。在初始万古霉素为5mg/mL、EDC为16mM的时候,Van-SDECM释放万古霉素的总时间超过了6周,而且最终释放浓度在酸碱环境中分别达到了101.1±13.8μg/mL和72.2±10.3μg/mL,均超过万古霉素的最低治疗剂量(20μg/mL),见图9。
(4)培养5天以后破骨细胞组(Van-SDECM+OC)中材料释放的万古霉素含量明显高于无细胞共培养的对照组(Van-SDECM)(4.0±0.5μg/mL VS.2.3±0.5μg/mL,P=0.0109),见图10,证实了其在感染模拟环境中的良好酸敏感释放能力。
试验例3Van-SDECM抗浮游和抗粘附细菌特性的评价
(1)抑菌环试验和材料/细菌共培养试验用来验证支架的杀游离细菌能力。将不同样本放置于生长金黄色葡萄球菌和肠球菌的MH平板上,48小时后可以观察到,Van-SDECM周围的抑菌圈分别为8mm(金黄色葡萄球菌)和2mm(肠球菌),明显大于对照组的2mm和0mm,见图11。
(2)细菌悬液与材料共培养后其上清液中残留细菌含量以涂布平板法(spread-plate method)定量测定,同时计算各不同样本对于浮游细菌的抗菌率。相比较于几乎无杀菌作用的的SDECM,Van-SDECM表现出优异的杀菌性能(98%以上),见图12和图13。EDC=0、16、32mM表示材料在初始万古霉素5mg/mL,EDC=NHS=0、16,32mM的条件下制备;选择不含万古霉素的SDECM作为对照组。
(3)扫描电镜显示,当制备材料的初始万古霉素浓度为5mg/mL,EDC为32mM时(Vanini=5mg/mL+EDC=32mM),样本表面的细菌量最少,与对照组相比,几乎达到98%的抗粘附细菌率,但与EDC为16mM时(Vanini=5mg/mL+EDC=16mM)组样本差异似乎无明显显著性,见图14和图15。
(4)激光共聚焦显示交联剂与材料抗粘附细菌能力的关系。EDC为32mM时制备的样本表面可见最高的红色荧光(死细菌)和最低的绿色荧光(活细菌),提示该组样本拥有最强的抗黏附细菌能力,EDC为16mM时制备的样本组次之,而未交联组样本无接触杀菌能力。各样本表面活/死细菌的比例显示,单纯材料组SDECM、药物直接混合组(EDC=0mM),中浓度EDC交联组(EDC=16mM)、高浓度EDC交联组(EDC=32mM)的活/死菌比例分别为7.8,6.0,1.8和0.3,表明随着交联剂浓度的增加,Van-SDECM支架的抗粘附细菌能力也随之增强,见图16和图17。用photoshop中的红绿像素值计算两者比例。
试验例4Van-SDECM的细胞毒性和成骨性能评价
(1)单纯万古霉素溶液的毒性检测表明高于3mg/ml可对骨髓间充质干细胞(MSCs)产生较为明显的毒性,见图18。
(2)以初始万古霉素浓度为1mg/ml、5mg/ml制备的Van-SDECM材料浸入培养基中,获取20%、50%、100%的浸提液,作为细胞毒性分析。CCK-8测试和live/dead染色均提示两种材料不同浓度的浸提液,对于MSCs增值无明显影响,见图19和图20。
(3)取Van=5mg/mL+EDC=16mM组,获取其浸提液测试成骨能力,可以发现,ALP染色提示其对于成骨细胞活性无明显不利影响,见图21。
试验例5Van-SDECM的体内抗菌与成骨性能评价
(1)颅骨感染感染模型第1周和第6周的涂布平板法显示,感染性骨缺损组(Defect+infection,II组)和感染+SDECM组(Defect+infection+SDECM,III组)的细菌数量远高于空白组(Defect,I组),而感染+Van-SDECM组(Defect+infection+Van-SDECM,V组)几乎在两个时间点都无明显细菌存在。相反,感染+Van组(Defect+infection+Van,IV组)中的残留菌落数量介于SDECM组和Van-SDECM组之间,见图22和图23。
(2)与术后1周的单纯缺损组相比,感染组、感染+SDECM组和感染+Van组的缺损周围均有大量虫咬样骨侵蚀区,而Van-SDECM组的侵蚀程度略低于其他各组。6周后,Van-SDECM组的新骨形成比对照组明显,骨再生率(BV/TV)达到90%以上,其次是万古霉素组(约60%)。感染+SDECM组的成骨能力与空白组(I组)相似(23.0±2.4%VS.20.5±3.1%,P=0.9055),两组均优于单纯感染组(II组)(10.9±1.4%),见图24和图25。
试验例6Van-SDECM的抗感染和成骨性能评价
(1)术后6周H&E观察材料的抗感染性能见图26,Ⅱ、Ⅲ、Ⅳ组内均可见大量中性粒细胞浸润,而Ⅴ组则明显减少,与Ⅰ组相似。
(2)Masson染色显示在Van-SDECM治疗的感染性颅骨缺损中新骨形成明显高于四个对照组。此外,经Van-SDECM处理后形成的新骨大部分为成熟的完全矿化松质骨(蓝色染色)。感染组和感染+SDECM组的缺损处未观察到成熟骨形成,而感染+van组仅观察到少量蓝色骨组织,见图27。
试验例7Van-SDECM的抑制破骨细胞性能评价
(1)TRAP染色可见pH6.0的RANKL+M-CSF培养基中破骨细胞的分化融合更加明显(73.7±5.0/well VS.45.3±5.0/well,P=0.0162),而流式细胞分析则证实在该环境下,成骨细胞群体的凋亡显著增加(89.9±0.6%VS.78.6±1.6%,P=0.0026),见图28和图29。
(2)术后1周,Giemsa染色显示感染组和感染+SDECM组骨缺损处的细菌浸润明显增加。同时,TRAP染色显示出类似的趋势。然而,Van-SDECM组在1周和6周后均未发现细菌和活化的破骨细胞,表明了细菌感染与破骨细胞活性之间的线性变化趋势,见图30-图34。
试验例8Van-SDECM的蛋白质功能分析
(1)蛋白质质谱显示在支架中含有大量有助于M2极化的因子,I型、II型、V型、XII型胶原(collagen I、II、V、XII)、凝血因子II、IX、X(coagulation factor II、IX、X)维生素K依赖性蛋白C(Vatimin K-dependent protein C)等,并且还含有协同抗菌的因子,如玻连蛋白(vitronectin)、纤维连接蛋白I(fibronectin I)、核心蛋白聚糖(decorin)和生腱蛋白(tenascin)等等,见图35。左边的红色条表示材料中具有辅助抗菌作用的蛋白质,右边的蓝色条表示促进再生能力的蛋白质;横坐标是各种蛋白质的百分比。
(2)利用KEGG数据库进行蛋白质功能富集分析。选择P值<0.05的功能分类进行下游分析。基因比值表示质谱检测到的蛋白质相对于某一功能类别的总蛋白项目的频率。在ECM蛋白质库中有几个炎症初期相关通路,如补体和凝血级联反应(校正P-value为9.27E-17;此后都用校正P-value)、血小板活化(P-value为1.05E-4),以及致病性感染反应(P-value为1.18E-4和5.13E-3),通过第一级反应促进单核细胞分化为抵御微生物的免疫细胞M1或免疫抑制型巨噬细胞(M2)。例如,我们发现在蛋白质组数据集中检测到许多凝血因子,包括F2、F7B、F9和F10,它们被证明是M2极化的刺激因素。这一过程促进了组织修复和骨再生的级联反应。涉及ECM与细胞膜相互作用的功能组别,包括PI3K-Akt信号途径(P-value4.74E-5)、ECM-受体相互作用(P-value 1.47E-10)、局灶性粘附(P-value 1.04E-6)和吞噬体(P-value 0.01),在ECM蛋白质组中显著富集,见图36。
(3)富集蛋白的途径相互作用网络,橙色圆圈表示功能通路,由相应的分类标题用粗体字标注;蓝色到红色圆圈表示通过对数转移质谱强度染色的蛋白质项目,并用其相应的基因符号标记。基因功能通路互作分析表明,功能簇彼此紧密聚集,包含许多强质谱信号的蛋白质,表明细胞基质因子可能触发巨噬细胞的细胞内重编程过程,见图37。
对实施例2-9所得的交联抗生素的特定脱矿细胞外基质支架分别进行合成评估、载药量及释放效率评估、抗浮游和抗粘附细菌特性的评价、细胞毒性和成骨性能评价、抗感染和成骨性能评价、抑制破骨细胞性能评价、蛋白质功能分析,结果与试验例1-8中Van-SDECM材料结果相似,这表明可通过上述优化后确定的支架类型和抗生素及制备条件的调整,实现效果类似的交联抗生素的特定脱矿细胞外基质支架的制备。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (5)
1.一种交联抗生素的特定脱矿细胞外基质支架的制备方法,其特征在于,具体步骤如下:
(1)从动物富含松质骨部位获取直径为4-8mm的松质骨颗粒;
(2)脱细胞:
①将步骤(1)获取的松质骨颗粒,切成2-4mm厚度的圆柱体,获得松质骨块;
②将步骤①获得的松质骨块用自来水冲洗1h,包装在包埋盒中,然后浸泡在0.6%(v/v)过氧乙酸超纯水中1h;
③将包埋盒转移至过滤除菌后的1%(v/v)Triton-X100溶液的烧瓶中,100rpm,4℃震动12-48h;
④用500ml灭菌水清洗包埋盒,并持续搅拌1h,重复两次;
⑤将松质骨块加入1%(w/v)SDS水溶液中,100rpm,4℃震动12-48h,清洗包埋盒,获得松质骨ECM;
⑥将松质骨ECM浸泡于10%EDTA脱钙液中,放入快速超声脱钙机中,在4℃下脱钙2-6h;
⑦将ECM骨块用自来水冲洗,辐射消毒,并冻干保存,获得松质骨细胞外支架;
(3)松质骨细胞外支架复合抗生素:
将30mg材料粉末加入到抗生素溶液中,浸泡1h,使两者充分混匀;加入等体积的EDC-NHS混合溶液,所述EDC-NHS混合溶液中EDC浓度为16mM,NHS浓度为4mM,常温下反应12-24h;反应完成的材料用去离子水清洗三遍,9000rpm/min离心5min,弃去上清液,冻干保存,获得交联抗生素的特定脱矿细胞外基质支架;
所述抗生素溶液为2-20mg/ml的万古霉素溶液,或2-20mg/ml的头孢曲松溶液,或2-20mg/ml的头孢吡肟溶液。
2.根据权利要求1所述的一种交联抗生素的特定脱矿细胞外基质支架的制备方法,其特征在于,步骤(1)所述动物为大型哺乳动物,包括牛、猪。
3.根据权利要求1所述的一种交联抗生素的特定脱矿细胞外基质支架的制备方法,其特征在于,步骤(1)所述富含松质骨部位包括脊柱、肋骨、肩胛、骨盆。
4.根据权利要求1所述的一种交联抗生素的特定脱矿细胞外基质支架的制备方法,其特征在于,步骤(2)③所述1%(v/v)Triton-X100用0.22μm滤头过滤除菌。
5.根据权利要求1所述的一种交联抗生素的特定脱矿细胞外基质支架的制备方法,其特征在于,步骤(2)⑦所述辐射剂量为250kGy。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011482219.1A CN112618797B (zh) | 2020-12-15 | 2020-12-15 | 一种交联抗生素的特定脱矿细胞外基质支架的制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011482219.1A CN112618797B (zh) | 2020-12-15 | 2020-12-15 | 一种交联抗生素的特定脱矿细胞外基质支架的制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112618797A CN112618797A (zh) | 2021-04-09 |
CN112618797B true CN112618797B (zh) | 2022-08-05 |
Family
ID=75313558
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011482219.1A Active CN112618797B (zh) | 2020-12-15 | 2020-12-15 | 一种交联抗生素的特定脱矿细胞外基质支架的制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112618797B (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116328039A (zh) * | 2023-02-24 | 2023-06-27 | 浙江狄赛生物科技有限公司 | 一种可调控炎症代谢的特定矿化度天然骨修复材料及其制备方法和应用 |
CN117138123B (zh) * | 2023-11-01 | 2024-02-23 | 北京大学口腔医学院 | 一种具有类骨结构的微米生物材料及其制备方法和应用 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017059418A1 (en) * | 2015-10-02 | 2017-04-06 | Thomas Jefferson University | Covalent modification of decellularized allogeneic grafts with active pharmaceuticals |
CN105435307A (zh) * | 2015-11-30 | 2016-03-30 | 广西医科大学 | 天然组织来源的脱细胞联合脱钙骨材料及其制备方法 |
CN110227182B (zh) * | 2019-01-17 | 2020-12-15 | 浙江大学医学院附属邵逸夫医院 | 一种梯度矿化骨细胞外基质材料的制备方法 |
-
2020
- 2020-12-15 CN CN202011482219.1A patent/CN112618797B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN112618797A (zh) | 2021-04-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Ghosh et al. | Arginine-presenting peptide hydrogels decorated with hydroxyapatite as biomimetic scaffolds for bone regeneration | |
Behzadi et al. | Nanomedicine for safe healing of bone trauma: opportunities and challenges | |
Huang et al. | Sustained zinc release in cooperation with CaP scaffold promoted bone regeneration via directing stem cell fate and triggering a pro-healing immune stimuli | |
Telgerd et al. | Enhanced osteogenic differentiation of mesenchymal stem cells on metal–organic framework based on copper, zinc, and imidazole coated poly‐l‐lactic acid nanofiber scaffolds | |
Zhou et al. | Improving osteogenesis of three-dimensional porous scaffold based on mineralized recombinant human-like collagen via mussel-inspired polydopamine and effective immobilization of BMP-2-derived peptide | |
Wenhao et al. | In vitro and in vivo evaluation of structurally-controlled silk fibroin coatings for orthopedic infection and in-situ osteogenesis | |
CN112618797B (zh) | 一种交联抗生素的特定脱矿细胞外基质支架的制备方法 | |
Jia et al. | Constructing multilayer silk protein/Nanosilver biofunctionalized hierarchically structured 3D printed Ti6Al4 V scaffold for repair of infective bone defects | |
WO2022012339A1 (zh) | 一种构建骨形态发生蛋白缓释系统的方法 | |
Cui et al. | Dual-functional composite scaffolds for inhibiting infection and promoting bone regeneration | |
RU2756164C2 (ru) | Нанокомпозитный слой на основе коллагеновых нановолокон и способ его изготовления | |
Wei et al. | Regenerating infected bone defects with osteocompatible microspheres possessing antibacterial activity | |
Jin et al. | Enhanced attachment, proliferation, and differentiation of human gingival fibroblasts on titanium surface modified with biomolecules | |
Chernozem et al. | Piezoelectric hybrid scaffolds mineralized with calcium carbonate for tissue engineering: Analysis of local enzyme and small-molecule drug delivery, cell response and antibacterial performance | |
Tian et al. | Chitosan-based biomaterial scaffolds for the repair of infected bone defects | |
Kim | Immunomodulation for maxillofacial reconstructive surgery | |
Deng et al. | Heterostructured metal–organic frameworks/polydopamine coating endows polyetheretherketone implants with multimodal osteogenicity and photoswitchable disinfection | |
Yuan et al. | The incorporation of strontium in a sodium alginate coating on titanium surfaces for improved biological properties | |
Ren et al. | Synergistic delivery of bFGF and BMP-2 from poly (l-lactic-co-glycolic acid)/graphene oxide/hydroxyapatite nanofibre scaffolds for bone tissue engineering applications | |
Fu et al. | Mussel-inspired gold nanoparticle and PLGA/L-lysine-g-graphene oxide composite scaffolds for bone defect repair | |
Xie | Bio‐inspired nanofunctionalisation of biomaterial surfaces: a review | |
Tang et al. | Dopamine/DOPAC-assisted immobilization of bone morphogenetic protein-2 loaded Heparin/PEI nanogels onto three-dimentional printed calcium phosphate ceramics for enhanced osteoinductivity and osteogenicity | |
Jayasuriya et al. | Rapid biomineralization of chitosan microparticles to apply in bone regeneration | |
Colorado et al. | Human recombinant cementum protein 1, dental pulp stem cells, and PLGA/hydroxyapatite scaffold as substitute biomaterial in critical size osseous defect repair in vivo | |
Nojehdehian et al. | Effect of poly-L-lysine coating on retinoic acid-loaded PLGA microspheres in the differentiation of carcinoma stem cells into neural cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
TR01 | Transfer of patent right |
Effective date of registration: 20231012 Address after: Room B2-303-17, No. 198 Qidi Road, Economic and Technological Development Zone, Xiaoshan District, Hangzhou City, Zhejiang Province, 311202 Patentee after: Hangzhou Yuanbao Biotechnology Co.,Ltd. Address before: 3 Qingchun East Road, Hangzhou, Zhejiang 310000 Patentee before: SIR RUN RUN SHAW HOSOITAL ZHEJIANG University SCHOOL OF MEDICINE |
|
TR01 | Transfer of patent right |