Udarbejdelse af termoresponsiv nanostrukturerede overflader for Tissue Engineering
Summary
Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.
Abstract
Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.
Introduction
Nanostrukturerede overflader har for nylig tiltrukket betydelig opmærksomhed på grund af deres forskellige anvendelsesmuligheder, herunder mønster, cellekultur, rengøring, og overflade skift. For eksempel superhydrophobic overflader inspireret af nanostruktur af lotus blade og andre responsive overflader er i stand til at reagere på ydre stimuli 1-4.
Langmuir film er en af de mest undersøgte polymerovertræk. En Langmuir film dannes ved at dryppe amfifile molekyler på en luft-vand grænsefladen 5-8. Filmen kan derefter overføres på en fast overflade ved fysisk eller kemisk adsorption, og den molekylære konformation på en fast overflade kan styres ved hjælp vertikale og horisontale overførselsmetoder 9-12. Densiteten af Langmuir film kan styres nøjagtigt ved at komprimere luft-vand-grænsefladen. For nylig er denne metode også vist sig effektiv til fremstilling nanoskalerede sea-island struktues ved at udnytte amfifile blokcopolymerer. Nanostrukturer antages at bestå af en kerne af hydrofobe segmenter og en skal af hydrofile segmenter 13-17. Desuden er antallet af nanostrukturer på en overflade reguleres ved at styre det areal pr molekyle (A m) af blokcopolymeren ved grænsefladen.
Vi har fokuseret på en original, unik stillads-fri vævsmanipulering tilgang, cellelaget teknik, ved anvendelse af en temperaturfølsom kultur overflade. Den udviklede teknologi er blevet anvendt på regenerative behandlinger for forskellige organer 18. En temperatur-responsive kultur overflade blev fremstillet ved podning af poly (N -isopropylacrylamide) (PIPAAm), en temperaturfølsom molekyle, på en overflade 19-27. PIPAAm og dets copolymerer udviser en lavere kritisk opløsningstemperatur (LCST) i vandige medier ved temperaturer nær 32 ° C. Kulturen overflade udviste også en temperaturfølsom alternati på mellem hydrofobicitet og hydrofilicitet. Ved 37 ° C, den PIPAAm-transplanterede overflade blev hydrofob, og cellerne let fastgøres og prolifererede på overfladen såvel som på konventionel vævskulturpolystyren. Når temperaturen blev sænket til 20 ° C, overfladen blev hydrofil, og cellerne spontant løsrevet fra overfladen. Derfor kunne dyrkede sammenflydende celler på overfladen høstes som et intakt ark ved at ændre temperaturen. Disse celleadhæsion og udstationeringsprocedurer egenskaber blev også vist ved en overflade fremstillet ved Langmuir filmcoating til laboratorie- demonstration 26, 27. En Langmuir film af blokcopolymerer bestående af polystyren (P (St)) og PIPAAm (St-IPAAm) blev fremstillet. Langmuir film med en specifik A m kunne horisontalt overført til en hydrofobisk modificeret glassubstrat. Desuden blev celleadhæsion på og adskillelse fra den forberedte overflade i afhængighed af temperatur evalueres.
_content "> Her beskriver vi protokoller til fremstilling af en nanostrukturerede Langmuir film bestående af termo-responsive amfifile blokcopolymerer på et glas substrat. Vores metode kan give en effektiv fabrikation teknik til økologiske nanofilms i forskellige områder af overfladen videnskab og kan lette mere effektiv kontrol af celleadhæsion på og spontan frigørelse fra en overflade.Protocol
Representative Results
Discussion
En temperatur-responsive overflade blev fremstillet ved Langmuir-Schaefer metoden, og overfladeegenskaberne for celleadhæsion / løsgørelse og cellelaget recovery blev optimeret. Når denne metode anvendes til fremstilling af overflader, adskillige trin er kritiske. Den molekylære sammensætning af St-IPAAm molekyler har en stor effekt på overfladen struktur og stabilitet af overfladen, og i forlængelse heraf, om celleadhæsion og løsrivelse. Navnlig bør de St-IPAAm molekyler har en snæver molekylvægtsfordeling…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This study was financially supported by the Creation of Innovation Centers for Advanced Interdisciplinary Research Program’s Project for Developing Innovation Systems “Cell Sheet Tissue Engineering Center (CSTEC)” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
Materials
| N-isopropylacrylamide | Kohjin | No catalog number | |
| Azobis(4-cyanovaleric acid) | Wako Pure Chemicals | 016-19332 | |
| Styrene | Sigma-Aldrich | S4972 | |
| 1,3,5-trioxane | Sigma-Aldrich | T81108 | |
| 1,4-Dioxane | Wako Pure Chemicals | 045-24491 | |
| DMEM | Sigma | D6429 | |
| PBS | Nakarai | 11482-15 | |
| Streptomycin | GIBCO BRL | 15140-163 | |
| Penicillin | GIBCO BRL | 15140-122 | |
| Trypsin-EDTA | Sigma | T4174 | |
| FBS | Japan Bioserum | JBS-11501 | |
| BAECs | Health Science Reserch Resources Bank | JCRB0099 | |
| Cover Glasses | Matsunami Glass Industry | C024501 | |
| AFM NanoScope V | Veeco | ||
| 1H NMR INOVA 400 | Varian, Palo Alto | ||
| ATR/FT-IR NICOLET 6700 | Thermo Scientific | ||
| GPC HLC-8320GPC | Tosoh | ||
| TSKgel Super AW2500, AW3000, AW4000 | Tosoh | ||
| Langmuir-Blodgett Deposition Troughs | KSV Instruments | KN 2002 | KSV NIWA Midium trough |
| Nikon ECLIPSE TE2000-U | Nikon |
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