AU2020277195B1 - Protein-modified PLGA microsphere and tissue-engineered nerve constructed therewith - Google Patents
Protein-modified PLGA microsphere and tissue-engineered nerve constructed therewith Download PDFInfo
- Publication number
- AU2020277195B1 AU2020277195B1 AU2020277195A AU2020277195A AU2020277195B1 AU 2020277195 B1 AU2020277195 B1 AU 2020277195B1 AU 2020277195 A AU2020277195 A AU 2020277195A AU 2020277195 A AU2020277195 A AU 2020277195A AU 2020277195 B1 AU2020277195 B1 AU 2020277195B1
- Authority
- AU
- Australia
- Prior art keywords
- merr
- fat
- nerve
- tissue
- protein
- 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
- 239000004005 microsphere Substances 0.000 title claims abstract description 96
- 210000005036 nerve Anatomy 0.000 title claims abstract description 78
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 title claims abstract description 61
- 208000010886 Peripheral nerve injury Diseases 0.000 claims abstract description 13
- 239000013543 active substance Substances 0.000 claims abstract description 13
- 239000000284 extract Substances 0.000 claims description 109
- 210000004027 cell Anatomy 0.000 claims description 60
- 238000000034 method Methods 0.000 claims description 13
- 239000003431 cross linking reagent Substances 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 4
- 239000003102 growth factor Substances 0.000 claims description 4
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims description 3
- 238000010146 3D printing Methods 0.000 claims description 2
- 241000305492 Gastrodia Species 0.000 claims description 2
- 239000009429 Ginkgo biloba extract Substances 0.000 claims description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 claims description 2
- 102000007072 Nerve Growth Factors Human genes 0.000 claims description 2
- 230000010261 cell growth Effects 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 238000004132 cross linking Methods 0.000 claims description 2
- 238000010041 electrostatic spinning Methods 0.000 claims description 2
- 210000002744 extracellular matrix Anatomy 0.000 claims description 2
- 229940068052 ginkgo biloba extract Drugs 0.000 claims description 2
- 235000020686 ginkgo biloba extract Nutrition 0.000 claims description 2
- 150000004676 glycans Chemical class 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000003900 neurotrophic factor Substances 0.000 claims description 2
- 102000039446 nucleic acids Human genes 0.000 claims description 2
- 108020004707 nucleic acids Proteins 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 claims description 2
- 229920001184 polypeptide Polymers 0.000 claims description 2
- 229920001282 polysaccharide Polymers 0.000 claims description 2
- 239000005017 polysaccharide Substances 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 230000008929 regeneration Effects 0.000 abstract description 6
- 238000011069 regeneration method Methods 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 90
- 241001533159 Brucea javanica Species 0.000 description 59
- 235000010889 Rhus javanica Nutrition 0.000 description 59
- 238000013268 sustained release Methods 0.000 description 44
- 239000012730 sustained-release form Substances 0.000 description 44
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 40
- 108010085895 Laminin Proteins 0.000 description 31
- 239000002609 medium Substances 0.000 description 31
- 210000002569 neuron Anatomy 0.000 description 30
- 108010022355 Fibroins Proteins 0.000 description 28
- 230000000694 effects Effects 0.000 description 26
- 239000013642 negative control Substances 0.000 description 19
- 239000000243 solution Substances 0.000 description 17
- 241000700159 Rattus Species 0.000 description 15
- 210000004116 schwann cell Anatomy 0.000 description 15
- 210000003497 sciatic nerve Anatomy 0.000 description 15
- 210000003050 axon Anatomy 0.000 description 14
- 238000010186 staining Methods 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 13
- 239000000835 fiber Substances 0.000 description 13
- 230000028600 axonogenesis Effects 0.000 description 12
- 230000007547 defect Effects 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000011259 mixed solution Substances 0.000 description 9
- 210000000578 peripheral nerve Anatomy 0.000 description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 8
- 239000012091 fetal bovine serum Substances 0.000 description 8
- 230000012010 growth Effects 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 8
- 241000158621 Brucea Species 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 7
- 238000000605 extraction Methods 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 241000283707 Capra Species 0.000 description 6
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 6
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 6
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 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 6
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 5
- 108010035532 Collagen Proteins 0.000 description 5
- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 5
- 229920001436 collagen Polymers 0.000 description 5
- AZKVWQKMDGGDSV-UHFFFAOYSA-N genipin Natural products COC(=O)C1=COC(O)C2C(CO)=CCC12 AZKVWQKMDGGDSV-UHFFFAOYSA-N 0.000 description 5
- 230000023105 myelination Effects 0.000 description 5
- 210000002241 neurite Anatomy 0.000 description 5
- 230000008439 repair process Effects 0.000 description 5
- 102000016359 Fibronectins Human genes 0.000 description 4
- 108010067306 Fibronectins Proteins 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 239000004372 Polyvinyl alcohol Substances 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 229960000583 acetic acid Drugs 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 239000012362 glacial acetic acid Substances 0.000 description 4
- 210000003007 myelin sheath Anatomy 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 229920002451 polyvinyl alcohol Polymers 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 210000003594 spinal ganglia Anatomy 0.000 description 4
- 230000002459 sustained effect Effects 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 210000000689 upper leg Anatomy 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000263 cytotoxicity test Toxicity 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 210000003195 fascia Anatomy 0.000 description 3
- 239000000834 fixative Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000012760 immunocytochemical staining Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 230000002093 peripheral effect Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 241000283074 Equus asinus Species 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 208000028389 Nerve injury Diseases 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 206010033799 Paralysis Diseases 0.000 description 2
- -1 Poly(lactic acid) Polymers 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 229940124326 anaesthetic agent Drugs 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 2
- 229960002327 chloral hydrate Drugs 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000003193 general anesthetic agent Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 230000008764 nerve damage Effects 0.000 description 2
- 210000004498 neuroglial cell Anatomy 0.000 description 2
- 238000001543 one-way ANOVA Methods 0.000 description 2
- 229920001296 polysiloxane Polymers 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- IJDLCOIGFNUHAQ-UHFFFAOYSA-N 4,5-dihydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonic acid Chemical compound OC1=C2C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=C1N=NC1=CC=CC=C1 IJDLCOIGFNUHAQ-UHFFFAOYSA-N 0.000 description 1
- 241000394635 Acetomicrobium mobile Species 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101000979249 Homo sapiens Neuromodulin Proteins 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 102100023206 Neuromodulin Human genes 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 241001093962 Simaroubaceae Species 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 229940103272 aluminum potassium sulfate Drugs 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 238000005266 casting Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000002737 cell proliferation kit Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 235000019240 fast green FCF Nutrition 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 210000000609 ganglia Anatomy 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 229910000474 mercury oxide Inorganic materials 0.000 description 1
- UKWHYYKOEPRTIC-UHFFFAOYSA-N mercury(ii) oxide Chemical compound [Hg]=O UKWHYYKOEPRTIC-UHFFFAOYSA-N 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 238000007491 morphometric analysis Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000004126 nerve fiber Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 208000021090 palsy Diseases 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920001610 polycaprolactone Polymers 0.000 description 1
- 239000004632 polycaprolactone Substances 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 235000017550 sodium carbonate Nutrition 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0619—Neurons
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/16—Ginkgophyta, e.g. Ginkgoaceae (Ginkgo family)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/77—Sapindaceae (Soapberry family), e.g. lychee or soapberry
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/898—Orchidaceae (Orchid family)
- A61K36/8988—Gastrodia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
- A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1658—Proteins, e.g. albumin, gelatin
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/26—Mixtures of macromolecular compounds
-
- 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
- A61L27/28—Materials for coating prostheses
- A61L27/34—Macromolecular 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
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
-
- 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
- A61L2300/40—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
- A61L2300/414—Growth factors
-
- 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
- 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
- A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
- A61L2300/622—Microcapsules
-
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
-
- 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
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dermatology (AREA)
- Biomedical Technology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Neurology (AREA)
- Alternative & Traditional Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Nanotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Neurosurgery (AREA)
- Cell Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
Abstract
The present invention discloses a protein-modified PLGA microsphere and
tissue-engineered nerve constructed therewith. In the present invention, the microspheres
are loaded with active substances for treating peripheral nerve injury and are bound to
5 tissue-engineered nerves. Research shows that the prepared tissue-engineered nerve
effectively promotes nerve regeneration after peripheral nerve injury.
Description
Technical Field
The present invention relates to the technical field of biomedicine, and specifically to a protein-modified PLGA microsphere, and a tissue-engineered nerve constructed therewith that is useful in repairing peripheral nerve defects.
Related Art
Peripheral nerve injury is a common disease in clinic, which usually leads to paralysis, palsy and even loss of autonomous control of corresponding body parts, which seriously affects the quality of life of patients. At present, the surgical repair effect of simply suturing the severed nerve is not satisfactory, and autologous nerve transplantation, which is the gold standard for repairing peripheral nerve defects, is limited in clinical application due to the inadequate source of autologous nerve donors and secondary damage caused by the transplantation. Therefore, constructing suitable tissue-engineered nerves to replace autologous nerves to repair peripheral nerve defects has broad application prospects.
Tissue-engineered nerves are generally composed of nerve conduit scaffolds, combined with multiple growth factors or seed cells and other factors.
However, seed cells often suffer many problems such as inadequate source or immunogenicity of allogeneic cells, and thus have limitation in clinical application. Adding growth factors to a scaffold material requires consideration of maintaining stability and efficiency according to their physical and chemical properties.
Therefore, the development of new biologically active materials capable of repairing nerve damage and combining them with tissue-engineered nerves have a wider clinical application value.
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".
A preliminary study of the present invention (Chinese Patent Application No. .0 2020103158417) discloses a fat-soluble extract of BruceajavanicaL. Merr. It is found through research that the fat-soluble extract of Brucea javanica L. Merr promotes the growth of peripheral DRG neurons and neurites, and promotes the division and proliferation of the glial cells, that is, Schwann cells, of the peripheral nervous system, thus promoting peripheral nerve regeneration. In the present invention, a protein-modified PLGA microsphere is designed on .5 this basis, and the fat-soluble extract of BruceajavanicaL. Merr is used as an active substance to construct tissue-engineered nerves. In the present invention, the fat-soluble extract of Brucea javanica L. Merr is combined with laminin-modified PLGA microspheres to prepare laminin PLGA-fat-soluble extract of Brucea javanica L. Merr microspheres with sustained action of release, which can significantly promote the axon growth of DRG neurons in vitro. Further, the .0 sustained-release microspheres containing the fat-soluble extract of Brucea javanica L. Merr are bound to the silk fibroin fibers in the silkfibroin nerve conduit to construct a tissue engineered nerve containing laminin-PLGA-fat-soluble extract of Brucea javanica L. Merr microspheres. In-vivo experiments prove that the tissue-engineered nerve can effectively promote the myelination of the regenerated nerve after peripheral nerve injury, providing a new option for clinical treatment of peripheral nerve injury.
According to a first aspect, the present disclosure provides a protein-modified PLGA microsphere, wherein PLGA is cross-linked with laminin through a cross-linking agent, wherein the weight ratio of laminin to PLGA is 1:3.
According to a second aspect, the present disclosure provides the use of a protein modified PLGA microsphere according to the first aspect, when loaded with at least one active substance, in the preparation of a medicament for treating peripheral nerve injury or a tissue engineered nerve.
According to a third aspect, the present disclosure provides a method of treating peripheral nerve injury or a tissue-engineered nerve, the method comprising the step of administering to a subject, a protein-modified PLGA microsphere according to the first aspect, when loaded with at least one active substance.
According to a fourth aspect, the present disclosure provides a tissue-engineered nerve loaded with a protein-modified PLGA microsphere according to the first aspect, wherein the protein-modified PLGA microsphere is loaded on the inner/outer surface or inside of the tissue engineered nerve.
.0 A protein-modified PLGA microsphere is provided, where PLGA is cross-linked with one or more of collagen, fibronectin, silk fibroin, and laminin through a cross-linking agent. The crosslinking agent is a chemical crosslinking agent or a biological crosslinking agent, preferably a biological crosslinking agent, such as genipin, carbodiimide/N hydroxysuccinimide (EDC/NHS), and sodium alginate, preferably genipin.
.5 Preferably, the weight ratio of the protein to PLGA is 1:1-10, and more preferably, 1:3.
Preferably, the weight ratio of the fat-soluble extract of Brucea javanica L. Merr to PLGA is 1-10:1, and more preferably, 3-5:1.
A preferred embodiment of the present invention is laminin-modified PLGA sustained release microspheres.
zo The microspheres of the present invention can be loaded with active substances for treating peripheral nerve injury. The active substance is one or more selected from cells, polysaccharides, polypeptides, proteins, nucleic acids, compounds or natural extracts, for example, one or more of neurotrophic factors, cell growth factors, extracellular matrix, Ginkgo biloba extract, Gastrodia extract, and fat-soluble extract of BruceajavanicaL. Merr.
In a specific embodiment of the present invention, the fat-soluble extract of Brucea javanica L. Merr is used as the active substance. Preferably, the fat-soluble extract of Brucea javanica L. Merr is obtained by extraction with a mixed solution of absolute ethanol and ethyl acetate, where the percentage by volume of absolute ethanol is 45%-85%. More preferably, the percentage by volume of absolute ethanol in the mixed solution is 75%. Further, ultrasonic extraction can be used. Preferably, the ultrasonic frequency is 20kHz, the power is 750 W, the time is 60 min, and the material-to-liquid ratio is 1:5 g/mL.
The microspheres of the present invention have smooth surface and uniform particle size (bar=5 m); and the diameter of the microspheres is mostly concentrated at 2.5 [m.
The present invention also relates to use of the protein-modified PLGA microspheres in preparing medicines for treating peripheral nerve defects or tissue-engineered nerves. In the present invention, PLGA is modified with an active protein, which can not only improve the biocompatibility of PLGA sustained-release microspheres and the biological activity of the material and allow the prepared microspheres to have good sustained releasability, and also promote the adhesion and growth of nerve cells.
.0 The present invention investigates the effect of laminin modified PLGA-fat-soluble extract of Bruceajavanica L. Merr sustained-release microspheres prepared with laminin at different concentrations on the axon growth of DRG neurons cultured in vitro. The results show that the sustained-release microspheres prepared with 1-3 mg/ml laminin can promote the axon growth of DRG neurons, and the sustained-release microspheres prepared with 1 mg/ml laminin have .5 the most preferred effect in promoting the growth of DRG neurons, which is significantly better than the cases with a higher concentration of 9 mg/ml, and a lower concentration of 0.33 mg/ml and 0.11 mg/ml laminin.
The present invention further relates to a tissue-engineered nerve loaded with protein modified PLGA microspheres. Protein-modified PLGA microspheres loaded with active .0 substances for treating peripheral nerve injury are loaded on the inner/outer surface or inside of the tissue-engineered nerve by one or more of adsorption, coating, mixing, embedding, crosslinking with a crosslinking agent, three-dimensional printing and electrostatic spinning.
The tissue-engineered nerve can be prepared with materials commonly used in the prior art, such as silkfibroin, chitosan, polyglycolic acid, polycaprolactone, collagen, polylactic acid and gelatin, in shapes commonly used in the prior art, such as conduits, stents, surgical sutures, etc. Furthermore, the tissue-engineered nerve has pores or simulated nerve fibers inside.
Preferably, the tissue-engineered nerve of the present invention is a silk fibroin nerve conduit, and further, there are fibers in the conduit.
According to a preferred embodiment of the present invention, laminin modified PLGA fat-soluble extract of Brucea javanica L. Merr sustained-release microspheres are immobilized on silk fibroin fibers by a biological cross-linking agent genipin to obtain silk fibroin fibers with laminin modified PLGA-fat-soluble extract of Brucea javanicaL. Merr sustained-release microspheres. The silk fibroin fiber with laminin modified PLGA sustained-release microspheres was assembled into silk fibroin nerve conduit. The silk fibroin nerve conduit has a length of 10 mm, an outer diameter of about 2.2 mm and an inner diameter of about 1.5 mm. Generally, ten silk fibroin fibers containing the fat-soluble extract of BruceajavanicaL. Merr sustained-release microspheres were loaded into each silk fibroin nerve conduit in parallel, to .0 construct the tissue-engineered nerve containing the fat-soluble extract of Brucea javanica L. Merr sustained-release microspheres.
According to the present invention, the constructed tissue-engineered nerve containing
4a the fat-soluble extract of BruceajavanicaL. Merr sustained-release microspheres is used in repairing a rat sciatic nerve defect model, and myelination of regenerated nerve is observed 12 weeks after operation. The results show that the thickness of myelin sheath in the experimental group with treated microspheres prepared by loading 10 mL of 10 mg/mL fat-soluble extract of BruceajavanicaL. Merr on the microspheres is better than that in the negative control group. Compared with the negative control group treated with laminin modified PLGA sustained-release microsphere without the fat-soluble extract of Brucea javanica L. Merr, there are significant differences in axon diameter and myelin sheath thickness in the experimental group treated with microspheres prepared by loading 10 mL of 50 mg/mL fat-soluble extract of BruceajavanicaL. Merr on the microspheres.
The present invention has the following advantages.
1. In the present invention, PLGA with a larger molecular weight of 50-75 kDa is used to prepare microspheres. A larger molecular weight can effect a longer release period, which is suitable for the needs in the initial critical stage of peripheral nerve regeneration. By modifying PLGA with natural macromolecular proteins or extracellular matrix proteins, the repair of nerve injuries is promoted by taking advantage of the properties of these proteins. The prepared microspheres also have a sustained action of release, particularly laminin-modified PLGA microspheres have a long sustained-release period and the best sustained-release effect.
2. A preliminary study of the present invention finds that the fat-soluble extract of Brucea javanicaL. Merr prepared by extraction with a mixed solution of absolute ethanol and ethyl acetate at a specific ratio combined with ultrasound can significantly promote the axon growth of peripheral DRG neurons and promote the division and proliferation of peripheral glial cells, that is, Schwann cells, suggesting that the fat-soluble extract of Brucea javanica L. Merr can promote peripheral nerve regeneration. In the present invention, the fat-soluble extract of Brucea javanica L. Merr is combined with laminin-modified PLGA sustained-release microspheres to prepare laminin-PLGA-fat-soluble extract of Brucea javanica L. Merr sustained-release microspheres. The microspheres have the advantages of good biocompatibility, easy modification, uniform particle size, and regular morphology, and can achieve an effective sustained action of release with a long release period. Further, the laminin-PLGA-fat-soluble extract of Brucea javanica L. Merr sustained-release microspheres are bound to the silk fibroin fibers in the silk fibroin nerve conduit to successfully construct a tissue-engineered nerve containing the fat-soluble extract of Brucea javanicaL. Merr sustained-release microspheres. In-vivo experiments find that the tissue-engineered nerve can effectively promote the myelination of the regenerated nerve after peripheral nerve injury.
Fig. 1 shows the cytotoxicity of the fat-soluble extract of Brucea javanica L. Merr detected by CCK8.
Fig. 2 shows the effect of the fat-soluble extract of Brucea javanica L. Merr on the axon growth of DRG neurons detected by immunohistochemistry. (Fig. 2A shows the immunohistochemical staining of axon growth of DRG neurons promoted by various dosages of the fat-soluble extract of Brucea javanica L. Merr; and Fig. 2B is a histogram showing the statistical result of growing axon length of DRG neuron promoted by various dosages of the fat-soluble extract of BruceajavanicaL. Merr.
Fig. 3 shows EdU staining to detect the effect of the fat-soluble extract of Brucea javanica L. Merr on the proliferation of Schwann cells. (Fig. 3A shows the EdU staining of Schwann cell proliferation promoted by various dosages of the fat-soluble extract of Brucea javanica L. Merr; and Fig. 3B is a histogram showing the statistical result of Schwann cell proliferation promoted by various dosages of the fat-soluble extract of Brucea javanica L. Merr.
Fig. 4 shows the sciatic nerve function index in a rat sciatic nerve defect model after the fat-soluble extract of BruceajavanicaL. Merr is applied.
Fig. 5 shows release of the fat-soluble extract of BruceajavanicaL. Merr from various protein-modified PLGA sustained-release microspheres detected by HPLC.
Fig. 6 shows the effect of microspheres and different concentrations of laminin modified sustained-release microspheres on the axon growth of DRG neurons observed under an electron microscope (Fig. 6A is a representative transmission electron microscopy image of laminin-PLGA-fat-soluble extract of BruceajavanicaL. Merr sustained-release microspheres; Fig. 6B is a histogram of the diameter of the laminin-PLGA-fat-soluble extract of Brucea javanica L. Merr sustained-release microspheres; and Fig. 6C is a histogram of fluorescence immunocytochemical staining).
Fig. 7 shows trichrome staining of the nerve to observe the myelination of regenerated nerve after repair with a tissue-engineered nerve (Fig. 7A is a representative image of trichrome staining of the nerve; Fig. 7B shows the statistical result of trichrome staining of the nerve).
Specific steps of the present invention are described below based on embodiments, but the present invention is not limited to the embodiments.
The terms used in the present invention, unless otherwise stated, generally have the meaning commonly understood by a person of ordinary skill in the art.
The present invention is further described below in detail with reference to specific examples and data. It should be understood that the embodiments are only for describing the present invention by using examples, but do not limit the scope of the present invention in any manner.
In the following embodiments, various processes and methods that are not described in detail are common conventional methods in the art.
Example 1. Preparation of the fat-soluble extract of BruceajavanicaL. Merr
The fat-soluble extract of Brucea javanica L. Merr was prepared according to the method disclosed in CN2020103158417.
Brucea javanica L. Merr of family Simaroubaceae, purchased from the traditional Chinese medicine (TCM) pharmacy of Nantong Hospital of Traditional Chinese Medicine, was washed with deionized water, dried in an oven at 60-65°C for 2 days and then ground into powder (300 g). 50 g of dry BruceajavanicaL. Merr powder was accurately weighed, fed to an extraction vessel, and soaked in a mixed solution of absolute ethanol and ethyl acetate of various concentrations at room temperature (22°C) for 48 hrs. Groups: 95% absolute ethanol + 5% ethyl acetate; 85% absolute ethanol +15% ethyl acetate; 75% absolute ethanol + 25% ethyl acetate; 65% absolute ethanol + 35% ethyl acetate; 55% absolute ethanol + 4 5% ethyl acetate; 45% absolute ethanol + 55% ethyl acetate; 35% absolute ethanol + 6 5% ethyl acetate; 25% absolute ethanol + 75% ethyl acetate; 15% absolute ethanol + 85% ethyl acetate; 5% absolute ethanol + 95% ethyl acetate. The soak solutions of various concentrations were further ultrasonically extracted at an ultrasonic frequency of 20kHz, a power of 750 W, and a material-to-liquid ratio of 1:5 g/mL for a time of 60 min. The resulting solutions were filtered through Whatman No. 1 filter paper, and then evaporated in a rotary evaporator (Buchi rotavapor R-124) under reduced pressure and concentrated at 40°C. After most of the solvent was removed, the extract fractions A (about 18- 23 g) of Brucea javanica L. Merr seeds extracted with different extraction solutions were obtained, and stored in a refrigerator at -20°C for later use.
Example 2. Extraction effect of various concentrations of mixed solutions of absolute ethanol and ethyl acetate
In order to detect the effect of the fat-soluble extract of Brucea javanica L. Merr extracted with various concentrations of absolute ethanol and ethyl acetate on the growth of nerve cells, the nerve cell line PC12 cells were used as the observation model in the experiment.
PC12 cells were cultured in DMEM complete medium (containing 10% horse serum, 5% fetal bovine serum, 1OOU/ml penicillin, and 100 g/ml streptomycin) in a culture dish which was placed in an incubator at 5% CO2 and 37°C. The medium was refreshed every two days and the cells were subcultured when grown to 80% confluence. In the experiment, PC12 cells in logarithmic growth phase were inoculated at 5 x 10 4 /ml into a 24-well culture plate in 400 1 per well. After 24 hrs, the medium was replaced by a DMEM medium containing 1% horse serum and 1% fetal bovine serum. In the experiment, a negative control group with DMEM medium including 1% horse serum and 1% fetal bovine serum, and experimental groups treated with 100 ng/mL extract obtained by extraction with a mixed solution of different concentrations of absolute ethanol and ethyl acetate, were respectively set. After 3-d culture with the extract, the morphological changes of PCl2 cells were observed under an inverted microscope. 50 cells were randomly selected from each group, and the positive cell rate, axon length and number of axons were measured.
The results in Table 1 show that after the PC12 cells are treated 3 days with the fat-soluble extracts of Brucea javanica L. Merr prepared by using mixed solutions of absolute ethanol and ethyl acetate at different concentrations, the extract prepared by using a combination of 75% absolute ethanol + 25% ethyl acetate has the most significant effect of promoting the growth and differentiation of nerve cells (**p< 0.01*p<o.05) with respect to the positive cell rate, axon length and number of axons, compared with the negative control group with DMEM medium containing 1% horse serum and 1% fetal bovine serum. It is suggested that 75% absolute ethanol + 25% ethyl acetate is the most preferred concentration combination for extraction.
Table 1. Effects of extracts prepared by using mixed solutions of absolute ethanol and ethyl acetate at different concentrations on PC12 cells
Experiment group Positive cell Axon Number of rate/% length/ m axons Control 0.08+0.05 7.50+0.82 0.61+0.30 95% absolute ethanol +5% ethyl acetate 0.09+0.02 7.81±0.98 0.82+0.11 85% absolute ethanol +15% ethyl acetate 0.22+0.08 9.22+1.42 1.21+0.12* 75% absolute ethanol +25% ethyl acetate 0.85+0.09** 22.48+2.05** 2.82±0.31** 65% absolute ethanol +35% ethyl acetate 0.42+0.11* 12.34+1.64* 0.93+0.28 55% absolute ethanol +45% ethyl acetate 0.23+0.13 10.52+1.67 0.91+0.25 45% absolute ethanol +55% ethyl acetate 0.21+0.12 8.98+1.32 0.89+0.32 35% absolute ethanol +65% ethyl acetate 0.17+0.06 8.21+1.42 0.81±0.15 25% absolute ethanol +75% ethyl acetate 0.13+0.07 7.98±0.95 0.79+0.23 15% absolute ethanol +85% ethyl acetate 0.11+0.07 7.62+1.12 0.71+0.23 5% absolute ethanol +95% ethyl acetate 0.06±0.05 7.55±0.92 0.51+0.38
Example 3. Cytotoxicity test of the fat-soluble extract of BruceajavanicaL. Merr
The fat-soluble extract of Brucea javanica L. Merr prepared in Example 1 was lyophilized overnight (EYELA FDU-1200, Tokyo), and the prepared powder was fed to a 15 ml centrifuge tube and stored at 4°C. A 10 g/mL solution was formulated in sterile deionized water, and centrifuged at 8000 g for 10 min. Then the solution was diluted to each concentration required in the experiment, and then sterilized by filtering through a 0.2 mm nylon syringe filter (Millipore, USA) for cytotoxicity test in culturing PC12 cell line. The CCK8 kit was purchased from Dojindo Laboratories, Japan.
PC12 cells in the logarithmic growth phase were digested, counted and then re-suspended. The cells were adjusted to have a density of 5 x 10 5/mL, and inoculated in a 96-well plate in 100 L per well. After the cells were attached to the wall, the medium was discarded, and the cells were wash with 0.01 M PBS for 5 min (x2). The negative control group was a group treated with a DMEM medium containing 1% horse serum and 1% fetal bovine serum. The fat-soluble extract of Brucea javanica L. Merr in different concentrations of 20 ng/mL, 100 ng/mL and 500 ng/mL was added to each experimental group. and the cells were cultured for 24 hrs, 48 hrs and 72 hrs respectively. The medium was gently discarded, 10% CCK8 in medium was added in an amount of 100 LCCK8 per well (that is, 10 1 of CCK8 per 100 1 of medium was added), and the cells were continuously cultured for 2 hrs. The absorbency (OD value) of each group was measured on a microplate reader at 450 nm. The relative survival rate (%) was calculated according to the following formula: Relative survival rate (%) =[(OD of experimental group - OD of blank group)/(OD of control group - OD of blank group)] x 100%. Each group had 8 replicates.
Fig. 1 shows the cytotoxicity test result of the fat-soluble extract of BruceajavanicaL. Merr detected by CCK8. The test results by CCK8 show that the fat-soluble extract of Brucea javanicaL. Merr at a low concentration of 20 ng/mL and a medium concentration of 100 ng/mL has no obvious cytotoxicity within 72 hours of experimental observation, while the fat-soluble extract of Brucea javanica L. Merr at a high concentration of 500 ng/mL shows a significant cytotoxicity from the experimental observation timepoint of 24 hrs compared with the culture with the low concentration 20 ng/mL and the medium concentration 100 ng/mL, suggesting that high concentration of the fat-soluble extract of BruceajavanicaL. Merr is cytotoxic to nerve cells. The vertical coordinate shows the cell viability of PC12 cells cultured with each concentration of the fat-soluble extract of Brucea javanica L. Merr in the experimental group, which is expressed in viability percentage of PC12 cells cultured in the negative control group with DMEM medium containing 1% horse serum and 1% fetal bovine serum. ** p<0.01.
Example 4. Effect of the fat-soluble extract of BruceajavanicaL. Merr on the axon growth of DRG neurons
SD rats at 15 days of pregnancy were narcotized by intraperitoneally injecting with 10% chloral hydrate (0.2 mL/100 g), shaved, and disinfected by spraying 75% alcohol. The embryo was taken out of the womb and placed in a sterile dish filled with pre-cooled D-Hank's medium. Then the dish was placed on ice, the dorsal root ganglia was removed under a dissecting microscope, and the fascia on the surface of the ganglia was peeled off as much as possible. The dorsal root ganglia were cut to have a size of about 0.5 mm3 by ophthalmic scissors, digested with 0.25% trypsin at 37°C for 15 min, and then terminated with serum. After centrifugation, the single-cell suspension was inoculated at a cell density of 5 x 10 5/mL in a 24-well plate fitted with glass slides and pre-coated with PDL. The system was placed in an incubator at 37°C, and 5% C0 2 , and the cells were incubated with 10% FBS and 90% DMEM. After the cells were attached, the cells in the negative control group were cultured with 97% Neurobasal + 2% B27 + 1% GluMAX medium. The fat-soluble extract of BruceajavanicaL. Merr in different concentrations of 20 ng/mL, 100 ng/mL and 500 ng/mL was respectively added to each experimental group, and the DRG neurons were continuously cultured for 72 hrs.
After the DRG neurons were cultured for 72 hours according to the above method, the medium was aspirated, and the cells were washed once with 0.01 M PBS. 500 L of 4% paraformaldehyde was added, and the cells were fixed at room temperature for 30 min. The fixative was removed, and the cells were washed with 0.01 M PBS for 10 min (x3) at room temperature. The plate was blocked with 0.01 M PBS containing 10% goat serum and 0. 3 %
Triton X-100 at 37°C for 60 min, and then the blocking buffer was removed.
Fluorescence immunocytochemical analysis: The primary antibody (goat anti-GAP-43 polyclonal antibody, 1:200) was dripped, stood overnight at 4°C, and washed with 0.01 M PBS for 10min (x3). The secondary antibody (FITC donkey anti-goat IgG, 1:200) was dripped, and the cell nucleus was labeled with Hoechst33342 (5 [g/ml), stood at room temperature for 1 hr in the dark. The cells were then washed with 0.01 M PBS for 10 min (x3). A blank control group without primary antibody was set in the experiment. For the blank control group, the steps were the same as above except that in the step (3), the goat anti-GAP-43 polyclonal antibody was replaced by 0.01 M PBS. Under a laser confocal microscope (FITC excitation wavelength: 488 nm, observation wavelength: 500-535 nm; Hoechst33342 argon-ion Ar excitation wavelength: 353-364 nm, observation wavelength: 460-480 nm), the results of fluorescence immunocytochemical detection were observed.
Fig. 2 shows the effect of the fat-soluble extract of Brucea javanica L. Merr on the axon growth of DRG neurons detected by immunohistochemistry. (Fig. 2A shows the immunohistochemical staining of axon growth of DRG neurons promoted by various dosages of the fat-soluble extract of BruceajavanicaL. Merr; and Fig. 2B is a histogram showing the statistical result of growing axon length of DRG neuron promoted by various dosages of the fat-soluble extract of Brucea javanica L. Merr. The results in Fig. 2 show that compared with the negative control group treated with 97% Neurobasal + 2% B27 + 1% GluMAX medium and DRG neurons cultured with a low concentration of the extract, when the concentration of the fat-soluble extract of BruceajavanicaL. Merr. is OOng/ml, the number and length of axons marked with GAP-43 of DRG neurons increase significantly. The fat-soluble extract of BruceajavanicaL. Merr at a high concentration of 500ng/ml has no promotion on DRG neurons, and the growth promotion effect is even lower than the fat-soluble extract of Brucea javanica L. Merr. at a low concentration of 20ng/ml. It is suggested that the fat-soluble extract of BruceajavanicaL. Merr at an appropriate medium concentration of 1OOng/ml can significantly promote the axon growth of DRG neurons (**p<0.01 *p<0.05).
Example 5: Effect of the fat-soluble extract of Brucea javanica L. Merr on the proliferation of Schwann cells
1. Culture and purification of Schwann cells
1-2 day-old newborn SD rats were freeze narcotized and disinfect with 75% alcohol. The sciatic nerve was exposed and isolated through the posterolateral femoral muscle space, and soaked in pre-cooled HBSS solution. The epineurium was carefully removed under a dissecting microscope. The sciatic nerve was cut in 200 [ of1 mg/ml collagenase, digested for 30 min at 37°C. The collagenase was removed, and 0.125% trypsin was added for digestion at 37°C for 12 min. The digestion was terminated, the system was centrifuged and the supernatant was discarded. The cells were inoculated at a cell density of 1 x 106 to a plate pre-coated with PDL, and incubated in an incubator at 37°C and 5% CO2. The medium was replaced by a medium containing cytarabine (1:1000) within 24 hrs. The cells were purified after growth to 80% confluence. The cells were digested into a cell suspension with 0.125% trypsin, and then terminated with serum. Anti-thy1.1 (1:1000) was added and the cells were incubated on ice for 2 hrs. After centrifugation, the supernatant was discarded. A mixed solution containing 250 1 of rabbit complement and 750 1 of DMEM medium (1:3) was added and the cells were incubated at 37°C for 1 hr. The cells were re-suspended and then inoculated at a cell density of 3 x 105 to a plate pre-coated with PDL. Moreover, 2 M forsokolin and 10 ng/ml HRG were added to the dish, and the medium was refreshed every 3 days.
2. Detection of cell proliferation by EdU
Schwann cells in the 96-well plate were treated for 24 hrs with the fat-soluble extract of Bruceajavanica L. Merr at a different concentration of 20 ng/mL, 100 ng/mL, and 500 ng/mL. The cells were detected with the Cell-LightTM EdU DNA cell proliferation kit available from Guangzhou RiboBio Company following the experimental operation in the manual. The EdU solution (reagent A) was diluted with the cell culture medium at a ratio of 1:1000 to prepare an appropriate amount of 50 M EdU medium. 100 pL of 50 M EdU medium was added to each well and the cells were incubated at 37°C for 2 hrs. The medium was discarded, and the cells were washed with PBS for 5 min (x3). 50 L of 4% paraformaldehyde was added to each well to fix the cells for 15 min at room temperature, and then the fixative was discarded. 50 L of 2 mg/mL glycine was added to each well and the cells were incubated for 5 min. The glycine solution was discarded, and the cells were washed with PBS for 5 min (x3). 100 L of a penetrant (0.5% TritonX-100 in PBS) was added to each well and the cells were incubated for 10 min. The cells were washed with PBS for 5 min (x3). 100 L of 1x Apollo@ staining reaction solution was added to each well, and the cells were incubated for 30 min in the dark at room temperature. The staining reaction solution was discarded. 100 L of a penetrant (0.5% TritonX-100 in PBS) was added and the cells were washed on a decolorizing shaker for 10 min (x3). The penetrant was discarded. 100 L of methanol was added to each well and the cells were washed for 5 min (x3). 5 g/mL Hoechst 33342 reaction solution was added and the cells were incubated for 30 min. The staining reaction solution was discarded and the cells were washed with PBS for 10 min (x3). 5 low-magnification fields of each sample at 20x magnification were randomly selected, and the images were collected Leica DC 300 and analyzed by Leica QWin analysis software.
Fig. 3 shows EdU staining to detect the effect of the fat-soluble extract of Brucea javanica L. Merr on the proliferation of Schwann cells. (Fig. 3A shows the EdU staining of Schwann cell proliferation promoted by various dosages of the fat-soluble extract of Brucea javanica L. Merr; and Fig. 3B is a histogram showing the statistical result of Schwann cell proliferation promoted by various dosages of the fat-soluble extract of Brucea javanica L. Merr. The results show that for the cultured primary Schwann cells treated with different concentrations of the fat-soluble extract of Brucea javanicaL. Merr, the fat-soluble extract of Brucea javanica L. Merr at a concentration of OOng/ml can significantly promote the proliferation of Schwann cells compared with the negative control group and the group treated with a low concentration of 20ng/ml; however, when the concentration is 500ng/ml, it has a significant inhibitory effect on the proliferation of Schwann cells, suggesting that the fat-soluble extract of Brucea javanica L. Merr can significantly promote the proliferation of Schwann cells at an appropriate concentration of lOOng/ml (Scale bar 20 pm). (**p<0.0l*p<0.05).
Example 6. Promotion of the fat-soluble extract of Brucea javanica L. Merr on the recovery of nerve function in rat sciatic nerve defect model
Animal experiment and groups: SD rats were randomly divided into 4 groups (9 in each group), including a negative control group with saline, and experimental groups with different dosages of the fat-soluble extract of Brucea javanicaL. Merr, including 5 mg/kg, 15 mg/kg, 30 mg/kg and 45 mg/kg. Model preparation and drug treatment: A compound anaesthetic agent (0.2-0.3 ml/100 g) was intraperitoneally injected for anesthesia, and the surgical area of the left femur was routinely shaved, disinfected, and draped. A median incision was made at the back of the left thigh. The skin and fascia were cut in sequence, the sciatic nerve was fully exposed to form a 10 mm defect, and the sciatic nerve was bridged with a silicone tube. The control, namely, saline, was added to a silicone tube to serve as the negative control group, and 5 mg/kg, 15 mg/kg, 30 mg/kg and 45 mg/kg of the fat-soluble extract of BruceajavanicaL. Merr were respectively added to the experimental groups. The incision was closed by routine suture. The model preparation and subsequent breeding and observations were all carried out in an SPF-grade barrier system.
Sciatic nerve function index (SFI) is an intuitive and reliable indicator
used to evaluate the sciatic nerve regeneration and nerve function recovery. Footprint experiments were performed at 4 W, 8 W and 12 W after surgery. The rats were kept in a passage with a width of about 15 cm, a height of about 15 cm, and a length of about 80 cm. The white rice paper was folded to have the same length and width as the passage, and placed at the bottom of the wooden passage. The two hindfeet of rats were dipped in red ink, and then they were transferred to one end of the passage. The rats walked to the other end of the passage by themselves, leaving 4-5 footprints by each foot on the rice paper. The footprints at the clear normal side and the operation side were selected, and the toe spreads (including normal toe spread, NTS; and experimental toe spread, ETS), print length (including normal print length, NPL; and experimental print length, EPL), and intermediary toe spreads (including normal intermediary toe spread, NIT; and experimental intermediary toe spread, EIT) were measured. An SFI value of 0 indicates normal, and an SFI value of -100 indicates complete nerve disconnection. The SFI value is calculated according to a formula below:
SFI= -38.3 x [(EPL-NPL)/NPL]+ 109.5 x [(ETS-NTS)/NTS]+ 13.3 x [(EIT-NIT)/NIT]-8.8
Fig. 4 shows the sciatic nerve function index in a rat sciatic nerve defect model after different concentrations of the fat-soluble extract of Brucea javanica L. Merr are applied. The results show that a promotion on the recovery of nerve function is shown at a low dose of 5 mg/kg, the most significant effect of promoting the recovery of nerve function is achieved at a medium dose of 15 mg/kg, and a dose of 30 mg/kg still falls within the most preferred dose range, which all have significant effect than the negative control group with saline. However, there is no significant difference between the group treated with a high dosage of 45 mg/kg and the negative control group with saline. The most preferred dosage of the fat-soluble extract of Brucea javanica L. Merr in vivo to promote peripheral nerve regeneration is 15 mg/kg. (**p<0.0l*p<0.05).
Example 7. Preparation of various protein-modified PLGA-fat-soluble extract of BruceajavanicaL. Merr sustained-release microspheres
Poly(lactic acid)-glycolic acid copolymer (PLGA, molar ratio of lactic acid:ethanol 53:47, molecular weight [MW] 50-75 kDa, Sigma). 100 mg of PLGA was dissolved in 1 mL of dichloromethane to form an oil phase. The PLGA oil phase was emulsified to form 3 mL of 7% (w/v) polyvinyl alcohol (PVA) aqueous solution, which was ultrasonicated for 1 min to prepare an emulsion I. The emulsion I was further added to 50 mL of 1% (w/v) polyvinyl alcohol (PVA) aqueous solution (containing 2% isopropanol) by a two-step emulsification method, and ultrasonicated for 3 min to prepare an emulsion II. The emulsion II was slowly stirred at room temperature overnight, and centrifuged at 13,000 rpm for 5 min at 4°C to obtain PLGA microspheres.
A suitable amount of collagen, Fibronectin, silk fibroin or laminin was dissolved in sterile pure water to obtain an aqueous solution containing 1 mg/ml collagen, fibronectin, silk fibroin or laminin respectively. At room temperature, 0.4 mL of PLGA microsphere aqueous solution (10 mg/mL) was slowly added with stirring, 5 ml of genipin (0.8 mg/ml) was added as an auxiliary crosslinking agent, and the reaction was continued for 90 min. Then, 10 mL of 10 mg/ml fat-soluble extract of Brucea javanica L. Merr was added respectively to 20 mL of 1 mg/ml aqueous solution of the above four protein-modified
PLGA microspheres, mixed well and stirred overnight at 4°C, to form light-blue various protein-modified PLGA-fat-soluble extract of Brucea javanica L. Merr sustained-release microspheres separately.
The release rate of the fat-soluble extract of Brucea javanica L. Merr from various protein-modified PLGA-fat-soluble extract of Brucea javanica L. Merr was detected by HPLC. At room temperature, the leachate from each group of microspheres was collected on days 0, 1, 4, 7, 10 and 14, and stored at -20°C. After the samples at all the detection time points were collected, they were detected collectively. Chromatographic conditions: Shimadzu LC-6AD liquid chromatograph. The detection solution was added to 2 mL of deionized water: methanol (4:6, v/v). Chromatographic column X amide column with an inner wall diameter of 150 mm x 4.6 mm, particle diameter 5 m, and pore diameter 100 A. Mobile phase I: water, mobile phase II: acetonitrile, and mobile phase III: ammonium formate aqueous solution (100 mM, pH3.2). The detection parameters were set as follows: room temperature 22°C, methanol:water =6:4 (v/v) as mobile phase, gradient elution, detection wavelength 266 nm, volume of injection 20[L, and flow rate 1 mL/min. The result is as shown in Fig. 1.
The HPLC test results in Fig. 5 show that the release effect of collagen and fibronectin modified PLGA microspheres declines obviously from day 4, and silk fibroin modified PLGA microspheres show waterfall release in days 4-7. The sustained release effect of PLGA microspheres modified by the above three proteins is weak. Laminin-modified PLGA microspheres can release the fat-soluble extract of Brucea javanica L. Merr, at a release rate showing a slow and uniform gradient and reaching 79% at 7 days and still 43% at 14 days, with a long slow release period. The results show that the laminin-modified PLGA microspheres has the best effect in releasing the fat-soluble extract of Brucea javanicaL. Merr, and the sustained release effect is more desirable.
Example 8. Effect of different concentrations of laminin modified PLGA-fat-soluble extract of Brucea javanica L. Merr sustained-release microspheres on the axon growth of DRG neurons cultured in vitro SD rats at 15 days of pregnancy were conventionally narcotized by intraperitoneally injecting with 10% chloral hydrate (0.2 mL/100 g). Fetal rats were removed and placed in precooled D-Hank's solution. Dorsal root ganglions (DRGs) were removed under a dissecting microscope, digested with 0.25% trypsin at 37°C for 15min, and then terminated with a serum-containing medium. After centrifugation, the cells were adjusted to have a cell density of 5 x 10 5/mL, inoculated in a PDL-coated 24-well plate, and cultured with a DMEM complete medium containing 10% FBS in an incubator at 37°C and 5% C02. After the cells were attached, the medium was replaced by 97% Neurobasal + 2% B27 + 1% GluMAX neuron culture medium. The negative control group was a control group without sustained-release microspheres cultured with neuron culture medium. According to the method of Example 7 (prepared with 50 mg/ml fat-soluble extract of Brucea javanica L. Merr), DRG neurons in the experimental groups were respectively treated for another 72 hrs with laminin modified PLGA-fat-soluble extract of Brucea javanica L. Merr sustained-release microspheres prepared with 0.11 mg/ml, 0.33 mg/ml, 1mg/ml, 3 mg/ml and 9 mg/ml laminin.
After the DRG neurons were cultured for 72 hours according to the above method, the medium was aspirated, and the cells were washed once with 0.01 M PBS. 500 L of 4% paraformaldehyde was added, and the cells were fixed at room temperature for 30 min. The fixative was removed, and the cells were washed with 0.01 M PBS for 10 min (x3) at room temperature. The plate was blocked with 0.01 M PBS containing 10% goat serum and 0.3% Triton X-100 at 37°C for 60 min, and then the blocking buffer was removed. Fluorescence immunocytochemical analysis: The primary antibody (goat anti-NF-H polyclonal antibody, 1:250) was dripped, stood overnight at 4°C, and washed with 0.1 M PBS for 10min (x3). The secondary antibody (FITC donkey anti-goat IgG, 1:200) was dripped, and the cell nucleus was labeled with Hoechst33342 (5 [g/ml), stood at room temperature for 1 hr in the dark. The cells were then washed with 0.01 M PBS for 10 min (x3). A blank control group without primary antibody was set in the experiment. For the blank control group, the steps were the same as above except that in the step (3), the goat anti-NF-H polyclonal antibody was replaced by 0.01 M PBS. Under a laser confocal microscope (FITC excitation wavelength: 488 nm, observation wavelength: 500-535 nm; Hoechst33342 argon-ion Ar excitation wavelength: 353-364 nm, observation wavelength: 460-480 nm), the results of fluorescence immunocytochemical staining were observed. DRG neurites was measured by
ImageJ software, statistically calculated by One way ANOVA, and plotted.
Fig. 6 shows the morphology of microspheres observed under a transmission electron microscope, and the effects of different concentrations of laminin modified PLGA sustained-release microspheres on the growth of DRG neurites. Fig. 6A is a representative transmission electron microscope image of laminin-PLGA-the fat-soluble extract of Brucea javanica L. Merr sustained-release microspheres. The results show that the prepared sustained-release microspheres have smooth surface and uniform particle size (bar=5 m). Fig. 6B is a histogram of the diameter of the sustained-release microspheres, showing that the diameter of microspheres is mostly concentrated at 2.5 [m. Fig. 6C is a histogram of fluorescence immunocytochemical staining. The results show that the axon length of DRG neurites in the group with sustained-release microspheres prepared with 1 mg/ml laminin reaches 3.8 [m, which is significantly different from that in the control group without slow-release microspheres (**p<0.01). In the group with microspheres prepared with 3 mg/ml laminin, the axon length of DRG neurites is 2.2 [m, which is significantly different from that in the control group without slow-release microspheres (*p<0.05). They are obviously superior to the axon lengths in the groups with sustained-release microspheres prepared with a higher concentration of 9 mg/ml and lower concentrations of 0.11 mg/ml and 0.33 mg/ml laminin. This indicates that the PLGA sustained-release microspheres prepared with 1 mg/ml laminin have the most preferred effect in release of the fat-soluble extract of BruceajavanicaL. Merr.
Example 9. Construction of tissue-engineered nerve containing the fat-soluble extract of BruceajavanicaL. Merr
Bombyx mori silk was degummed fully by boiling in 0.05 M Na2CO3 aqueous solution for 30 min (x2), washed with deionized water, and air dried at room temperature to obtain silk fibroin fibers with a diameter of 10-30 m, which was sterilized at high temperature under high pressure for later use. Genipin having excellent biocompatibility was used as a crosslinking agent, and the bifunctional groups were respectively linked to the side-chain amino groups of silk fibroin and PLGA. Following the method of Example 7 (prepared with 1 mg/ml laminin), the laminin modified PLGA sustained-release microspheres without the fat-soluble extract of BruceajavanicaL. Merr for use in the negative control group, and the laminin modified PLGA sustained-release microspheres prepared with different concentrations of fat-soluble extract of Brucea javanica L. Merr, including 2 mg/mL, 10 mg/mL, 50 mg/mL and 250 mg/mL, were prepared respectively. 10 mL of sustained-release microspheres of each group were immobilized on silk fibroin fibers to obtain silk fibroin fibers loaded with laminin modified PLGA microspheres with different doses of the fat-soluble extract of BruceajavanicaL. Merr.
Also, the degummed silk fibroin fibers were dissolved in a ternary solvent system CaC 2/H 20/EtOH (molar ratio 1:8:2) at 80°C for 1 hr. The solution was placed in a cellulose tube (molecular weight cut-off: 12,000-14,000), dialyzed against distilled water, and allowed to stand at room temperature for 3 days. The dialyzed solution was concentrated by rotary evaporation at 40°C. The silk fibroin conduit was molded by a stainless steel casting mold. The mold was composed of a sleeve and a strut in the sleeve, and the distance between the sleeve and the inner strut determined the thickness of the conduit wall. The prepared silk fibroin nerve conduit is 10 mm in length, 2.2 mm in outer diameter and 1.5 mm in inner diameter. The silk fibroin fibers containing laminin modified PLGA-fat-soluble extract of Brucea javanica L. Merr sustained-release microspheres were fit into the silkfibroin nerve conduit. Generally, ten silk fibroin fibers containing the fat-soluble extract of BruceajavanicaL. Merr sustained-release microspheres were loaded into each silk fibroin nerve conduit in parallel, to construct the tissue-engineered nerve containing the fat-soluble extract of BruceajavanicaL. Merr.
Example 10. Use of tissue-engineered nerve containing the fat-soluble extract of BruceajavanicaL. Merr in repairing rat sciatic nerve defect model
Animal experiment and groups: SD rats were randomly divided into 5 groups (6 rats in each group). The negative control group was treated with tissue-engineered nerves constructed with laminin modified PLGA sustained-release microspheres without the fat-soluble extract of Brucea javanica L. Merr, and the experimental groups were respectively treated with tissue-engineered nerves constructed with laminin modified PLGA sustained-release microspheres loaded with different doses of fat-soluble extract of Brucea javanica L. Merr (including 2 mg/mL, 10 mg/mL, 50 mg/mL, and 250mg/mL fat-soluble extract of BruceajavanicaL. Merr) prepared in Example 3. Model preparation and drug treatment: A compound anaesthetic agent (0.3ml/100 g) was intraperitoneally injected for anesthesia, and the surgical area of the left femur was routinely shaved, disinfected, and draped. A median incision was made at the back of the left thigh. The skin and fascia were cut in sequence, the sciatic nerve was fully exposed to form a 10 mm defect. The tissue engineered nerves in the negative control group and the experimental groups were used to bridge the severed sciatic nerve, and the incision was closed by routine suture. The model preparation and subsequent breeding and observations were all carried out in an SPF-grade barrier system.
Twelve weeks after operation, trichrome staining of the nerve was performed. The following staining reagents were prepared. (1) Harris hematoxylin: 0.5 g of hematoxylin was added to and dissolved in 5 ml of absolute ethanol. 10 g of aluminum potassium sulfate to was added to and dissolved in 100 ml of double distilled water. The two solutions were mixed and heat to boiling. Then, 0.25 g of yellow mercury oxide was added, dissolved, and cooled in ice water. After filtering, 5ml of glacial acetic acid was added. (2) Trichrome staining solution: 0.3 g of solid green FCF, 0.6 g of Chromotrope 2R and 0.6 g of phosphotungstic acid were sequentially added to and dissolved in 100 ml of double distilled water. Then 2 ml of glacial acetic acid was added to adjust the pH to 3.4. (3) 0.3% glacial acetic acid solution: It was prepared immediately before use. Following appropriately modified experimental methods of Meyer et al., (1) the sections were conventionally deparaffinated into ddH 20; (2) the sections were stained in harris hematoxylin for 5 min; (3) then the sections were washed with double distilled water for 5 min, and the degree of bluing was controlled under a microscope; (4) the sections were stained with the trichrome staining solution for 30 min; (5) then the sections were washed with 0.3% glacial acetic acid twice for 20 sec each and then with running water for 5-10 min, until the sectioned tissue was not decolorized; and (7) the sections were dehydrated to become transparent and then encapsulated with a neutral gum. One-way ANOVA analysis was performed on the data for morphometric analysis of the trichrome staining of the nerve by STATA7 statistical analysis software. If the difference between groups is statistically significant (p<0.05), pairwise comparison was further performed by Turkey's test. The results are expressed as the mean standard deviation (X SD).
Fig. 7 shows trichrome staining of the nerve to observe the myelination of regenerated nerve after repair with a tissue-engineered nerve. Fig. 7A shows a representative graph of the trichrome staining results of each group of nerves, where a is the Control group, and the experimental groups are groups with tissue engineered nerves prepared with 2 mg/mL (b), 10 mg/mL (c), 50 mg/mL (d) and 250 mg/mL (e) fat-soluble extract of BruceajavanicaL. Merr (bar--5 m). The statistical results of the trichrome staining results of the nerve in Fig. 7B show that the myelin sheath thickness of regenerated nerve achieved by using the tissue-engineered nerve prepared with 10 mg/mL fat-soluble extract of BruceajavanicaL. Merr is better than that in the negative control group (**p< 0.01). The axon diameter and myelin sheath thickness of regenerated nerve achieved by using the tissue engineered nerve prepared with 50 mg/mL fat-soluble extract of Brucea javanica L. Merr are significantly different from those in the negative control group (**p<0.01, *p<0.05).
Claims (9)
1. A protein-modified PLGA microsphere, wherein PLGA is cross-linked with laminin through a cross-linking agent, wherein the weight ratio of laminin to PLGA is 1:3.
2. The protein-modified PLGA microsphere according to claim 1, wherein the microsphere is loaded with at least one active substance for treating peripheral nerve injury.
3. The protein-modified PLGA microsphere according to claim 2, wherein the at least one active substance is selected from the group consisting of cells, polysaccharides, polypeptides, proteins, nucleic acids, compounds or natural extracts.
4. The protein-modified PLGA microsphere according to claim 2 or 3, wherein the at least one active substance is selected from the group consisting of neurotrophic factors, cell growth factors, extracellular matrix, Ginkgo biloba extract, Gastrodia extract, and fat-soluble extract of BruceajavanicaL. Merr.
5. Use of a protein-modified PLGA microsphere according to any one of claims 1 to 4, when loaded with at least one active substance, in the preparation of a medicament for treating a peripheral nerve injury or a tissue-engineered nerve.
6. A method of treating a peripheral nerve injury or a tissue-engineered nerve, the method comprising the step of administering to a subject, a protein-modified PLGA microsphere according to any one of claims 1 to 4, when loaded with at least one active substance.
7. The use according to claim 5 or method according to claim 6, wherein the at least one active substance is a fat-soluble extract of BruceajavanicaL. Merr.
8. A tissue-engineered nerve loaded with a protein-modified PLGA microsphere according to any one of claims 1 to 4, wherein the protein-modified PLGA microsphere is loaded on the inner/outer surface or inside of the tissue-engineered nerve.
9. The tissue-engineered nerve according to claim 8, wherein the protein-modified PLGA microsphere is loaded on the inner/outer surface or inside of the tissue-engineered nerve by one or more of adsorption, coating, mixing, embedding, crosslinking with a crosslinking agent, three-dimensional printing and electrostatic spinning.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010407176.4 | 2020-05-14 | ||
| CN202010407176.4A CN111544648B (en) | 2020-05-14 | 2020-05-14 | Protein-modified PLGA microspheres and tissue-engineered nerves constructed by same |
| PCT/CN2020/097111 WO2021227199A1 (en) | 2020-05-14 | 2020-06-19 | Protein-modified plga microsphere and tissue-engineered nerve constructed thereby |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2020277195B1 true AU2020277195B1 (en) | 2021-04-08 |
Family
ID=75291601
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020277195A Active AU2020277195B1 (en) | 2020-05-14 | 2020-06-19 | Protein-modified PLGA microsphere and tissue-engineered nerve constructed therewith |
Country Status (3)
| Country | Link |
|---|---|
| US (1) | US11788058B2 (en) |
| EP (1) | EP4019058A4 (en) |
| AU (1) | AU2020277195B1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117771445A (en) * | 2023-05-16 | 2024-03-29 | 上海市第十人民医院 | Preparation method of chlorogenic acid-loaded GelMA microsphere composite nerve conduit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170292109A1 (en) * | 2016-04-06 | 2017-10-12 | Sutapa Barua | Method of forming microparticles for use in cell seeding |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1285725C (en) | 2004-05-18 | 2006-11-22 | 中山大学附属第一医院 | Tissue engineered peripheral nerve graft |
| US8263103B2 (en) | 2006-01-31 | 2012-09-11 | Boston Scientific Scimed, Inc. | Medical articles containing biodegradable polymers and acid-neutralizing cationic species |
| US8728817B2 (en) | 2007-05-04 | 2014-05-20 | University Of Virginia Patent Foundation | Compositions and methods for making and using laminin nanofibers |
| CN103364568B (en) | 2013-07-18 | 2015-05-27 | 博奥赛斯（天津）生物科技有限公司 | Laminin nano-magnetic particle chemiluminescent immunity quantitative detection kit and preparation method thereof |
| CN103894328B (en) | 2014-03-07 | 2016-01-06 | 西南交通大学 | The method of the nano particle of laminin and SDF-1 α is taken in the assembling of Ti material surface |
| CN104399131B (en) | 2014-10-21 | 2016-04-27 | 赵红斌 | There is the multi-channel nerve rehabilitating tube and mould of organizing inductivity function |
| WO2016077839A1 (en) | 2014-11-15 | 2016-05-19 | Tissuegen, Inc. | Device for induction of cellular activity and modification |
| CN107224571A (en) | 2016-03-23 | 2017-10-03 | 张巍 | A kind of PLGA complex microspheres with sciatic nerve defencive function |
| CN106215245A (en) | 2016-07-21 | 2016-12-14 | 青岛三帝生物科技有限公司 | Method and the artificial nerve catheter of artificial nerve catheter is prepared based on 3D printing |
| CN106619537A (en) | 2017-03-15 | 2017-05-10 | 苏州大学 | Ibuprofen-carried slow release nanoparticle, preparation method thereof and application |
| AU2018347514A1 (en) * | 2017-10-11 | 2020-05-07 | Illustris Pharmaceuticals, Inc. | Methods and compositions for topical delivery |
| CN108525017B (en) | 2018-03-21 | 2021-01-19 | 武汉理工大学 | Sustained-release hyaluronic acid-based injectable hydrogel and preparation method thereof |
| CN110227184B (en) * | 2019-07-16 | 2020-04-24 | 南通大学 | Differential tissue engineered nerves and applications |
-
2020
- 2020-06-19 US US17/596,647 patent/US11788058B2/en active Active
- 2020-06-19 EP EP20935070.1A patent/EP4019058A4/en active Pending
- 2020-06-19 AU AU2020277195A patent/AU2020277195B1/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170292109A1 (en) * | 2016-04-06 | 2017-10-12 | Sutapa Barua | Method of forming microparticles for use in cell seeding |
Non-Patent Citations (2)
| Title |
|---|
| Qiao, Feng, et al. (2017) "Silk fibroin-coated PLGA dimpled microspheres for retarded release of simvastatin." Colloids and Surfaces B: Biointerfaces, vol. 158, pages: 112-118. * |
| Zhang,T, et al.(2014) Colloids and Surfaces A: Physicochemical and Engineering Aspects vol. 452, p: 115-124 * |
Also Published As
| Publication number | Publication date |
|---|---|
| US20220213437A1 (en) | 2022-07-07 |
| US11788058B2 (en) | 2023-10-17 |
| EP4019058A4 (en) | 2022-12-14 |
| EP4019058A1 (en) | 2022-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10639399B2 (en) | Construction of MicroRNA gene-mediated novel tissue engineered nerve and applications thereof in repairing nerve defect | |
| WO2021227199A1 (en) | Protein-modified plga microsphere and tissue-engineered nerve constructed thereby | |
| Zhu et al. | Noncovalent bonding of RGD and YIGSR to an electrospun poly (ε‐caprolactone) conduit through peptide self‐assembly to synergistically promote sciatic nerve regeneration in rats | |
| CN105505854B (en) | Acquisition methods and application from the excretion body of human urine cell | |
| US9889233B2 (en) | Method of producing native components, such as growth factors or extracellular matrix proteins, through cell culturing of tissue samples for tissue repair | |
| JP2004523484A (en) | Electroprocessed collagen | |
| RU2483756C1 (en) | METHOD FOR PREPARING BIODEGRADED COMPOSITE MATRIX OF REGENERATED SILK FIBROIN Bombyx mori AND ITS USE | |
| AU2019317696A1 (en) | Method and composition for promoting cell growth and tissue repair | |
| CN114853852B (en) | Polypeptides and their use in promoting bone repair | |
| US11788058B2 (en) | Protein-modified PLGA microsphere and tissue-engineered nerve constructed therewith | |
| Gavrilova et al. | The effect of biodegradable silk fibroin-based scaffolds containing glial cell line-derived neurotrophic factor (GDNF) on the corneal regeneration process | |
| Yan et al. | Promising application of a novel biomaterial, light chain of silk fibroin combined with NT3, in repairment of rat sciatic nerve defect injury | |
| CN110464877A (en) | A kind of preparation method and its effect evaluation method of acellular nerve allografts | |
| CN111195370B (en) | High-magnesium microenvironment bone marrow stem cell microsphere carrier and preparation method and application thereof | |
| CN114832156B (en) | Novel medical and cosmetic shaping filler modified L-polylactic acid gel | |
| Vrehen et al. | Development of a fully synthetic corneal stromal construct via supramolecular hydrogel engineering | |
| US20220378844A1 (en) | Rotator cuff therapy using muscle fiber fragments | |
| Xuemei et al. | Biocompatibility studies of silk fibroin-based artificial nerve grafts in vitro and in vivo | |
| JP2010163435A (en) | Electroprocessed collagen | |
| CN106046340B (en) | A kind of polymer, the hydrogel containing the polymer and its application | |
| Larysz-Brysz et al. | Polymer hollow fiber-encapsulated peripheral nerve extracts change their activity towards injured hippocampal neurites in rats |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGA | Letters patent sealed or granted (standard patent) |