Formation of Cell Sheets Based on Electrospun Fibrous PNIPA-AA/PCL Substrate
-
摘要: 将N-异丙基丙烯酰胺（NIPA）与丙烯酸（AA）通过自由基聚合得到AA接枝改性的聚N-异丙基丙烯酰胺（PNIPA-AA）。然后将PNIPA-AA与聚己内酯（PCL）混合电纺制备PNIPA-AA/PCL温敏纤维。采用核磁共振波谱与傅里叶红外光谱分析聚合物的化学结构，通过变温红外光谱与紫外-可见分光光谱检测聚合物的温敏性；通过扫描电镜与水接触角观察电纺纤维基底的形貌与温敏性；最后以小鼠成纤维细胞（C3H/10T1/2）为模型细胞，研究PNIPA-AA/PCL纤维基底对细胞膜片的形成与脱落的影响。结果表明，PNIPA-AA的低临界溶解温度（LCST）为33.6 °C，可电纺性较好；PNIPA-AA/PCL温敏纤维在高温（37 °C）下疏水程度更高，低温（20 °C）下水浸润速率更快；该温敏纤维基底可促进C3H/10T1/2细胞增殖和细胞外基质（ECM）分泌，仅需10 min降温即可使形成的细胞膜片完全分离，且脱落后膜片完整性与功能性保持完好。
-
关键词:
- 细胞膜片 /
- 电纺丝 /
- 温敏纤维 /
- 聚N-异丙基丙烯酰胺 /
- 组织工程
Abstract: Aimed to rapidly form high-quality cell sheets, poly(N-isopropylacrylamide-co-acrylic acid) (PNIPA-AA) was synthesized by free radical polymerization of N-isopropylacrylamide (NIPA) and acrylic acid (AA). Chemical structure of the synthesized PNIPA-AA was characterized by nuclear magnetic resonance spectroscopy (NMR) and Fourier infrared spectroscopy (FT-IR), and temperature sensitivity was detected by variable temperature infrared spectroscopy and ultraviolet-visible spectrophotometry. PNIPA-AA was subsequently blended with polycaprolactone (PCL) for electrospinning thermosensitive fibrous substrate of PNIPA-AA/PCL. Morphology of the electrospun fibrous PNIPA-AA/PCL substrate was observed by scanning electron microscopy (SEM) and its temperature sensitivity was confirmed through water contact angle measurement. Using mouse fibroblasts C3H/10T1/2 as model cells, effects of the fibrous PNIPA-AA/PCL substrate on the formation and detachment of cell sheets were examined. Results showed that the lower critical solution temperature (LCST) of PNIPA-AA was 33.6 °C. PNIPA-AA possessed better electrospinnability. Electrospun fibrous substrate of the PNIPA-AA/PCL showed higher hydrophobicity at high temperature (37 °C) and faster water infiltration rate at low temperature (20 °C). The fibrous PNIPA-AA/PCL substrate promoted cell proliferation and extracellular matrix (ECM) secretion in C3H/10T1/2. Moreover, it took merely 10 minutes for the formed cell sheets to be completely detached by cooling down the temperature to lower than LCST, e.g., 20 °C. And the integrity and function of the harvested cell sheets remained intact.-
Key words:
- cell sheet /
- electrospinning /
- thermosensitive fiber /
- poly(N-isopropylacrylamide) /
- tissue engineering
-
图 1 PNIPA-AA/PCL纤维基底的制备及细胞膜片获取示意图
Figure 1. Schematic of electrospun fibrous PNIPA-AA/PCL substrate formation and cell sheet harvest
图 2 （a）核磁共振氢谱图；（b）红外光谱图；（c）PNIPA-AA变温红外光谱图；（d）聚合物水溶液在不同温度下透光率曲线；（e）PNIPA和PNIPA-AA在不同温度下的溶液中析出/溶解宏观形貌变化
Figure 2. (a) 1H-NMR spectra; (b) FT-IR spectra; (c) Temperature-dependent FT-IR spectrum of PNIPA-AA; (d) Optical transmittance of aqueous polymer solutions at varied temperatures; (e) Macroscopic observation of morphological changes of precipitation/dissolution of PNIPA and PNIPA-AA
图 3 各组基底的（a）SEM形貌图；（b）水接触角；（c）20 ℃下的水浸润速率
Figure 3. (a) SEM images; (b) Water contact angles; (c) Water infiltration rate at 20 °C of different substrates
图 4 （a）第1 d、2 d、3 d细胞增殖情况；（b）第7 d时的 N-cad与Fn免疫荧光染色（细胞核蓝色和蛋白绿色）；（c）第7 d时的活细胞示踪剂染色；（d）细胞膜片的细胞脱落率统计；（e）第7 d时的细胞膜片脱落前后的细胞骨架形貌（细胞核蓝色和细胞骨架红色）；（f）细胞膜片实物图
Figure 4. (a) Cell proliferation at 1 d, 2 d, and 3 d; (b) Immunofluorescence staining of N-cad and Fn at 7 d (Nuclei blue and protein green); (c) Live cell tracking staining at 7 d; (d) Cell detachment rates; (e) Cytoskeletal morphology before and after detachment of cell sheets at 7 d (Nuclei blue and cytoskeleton red); (f) Photographs of detached cell sheets
图 5 （a）脱落后细胞膜片N-cad和Fn的免疫荧光染色（细胞核蓝色和蛋白绿色）；（b）分别对（a）中的荧光强度定量分析；（c）脱落后再黏附细胞活/死染色（活细胞绿色和死细胞红色）
Figure 5. (a) Immunofluorescence staining of N-cad and Fn after detachment of cell sheets (Nuclei blue and protein green); (b) Quantitative analysis of fluorescence intensity in (a); (c) Live/dead staining of adherent cells after being harvested (live cells green and dead cells red)
-
[1] YAMATO M, OKANO T. Cell sheet engineering [J]. Mater Today,2004,7(5):42-47. doi: 10.1016/S1369-7021(04)00234-2 [2] BAYOUSSEF Z, DIXON J E, STOLNIK S, SHAKESHEFF K M. Aggregation promotes cell viability, proliferation, and differentiation in an in vitro model of injection cell therapy [J]. J Tissue Eng Regen Med,2012,6(10):e61-e73. doi: 10.1002/term.482 [3] CERQUEIRA M T, PIRRACO R P, MARTINS A R, SANTOS T C, REIS R L, MARQUES A P. Cell sheet technology-driven re-epithelialization and neovascularization of skin wounds [J]. Acta Biomater,2014,10(7):3145-3155. doi: 10.1016/j.actbio.2014.03.006 [4] NISHIDA K, YAMATO M, HAYASHIDA Y, WATANABE K, MAEDA N, WATANABE H, YAMAMOTO K, NAGAI S, KIKUCHI A, TANO Y, OKANO T. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface [J]. Transplantation,2004,77(3):379-385. doi: 10.1097/01.TP.0000110320.45678.30 [5] KOMAE H, SEKINE H, DOBASHI I, MATSUURA K, ONO M, OKANO T, SHIMIZU T. Three-dimensional functional human myocardial tissues fabricated from induced pluripotent stem cells [J]. J Tissue Eng Regen Med,2017,11(3):926-935. doi: 10.1002/term.1995 [6] TSUMANUMA Y, IWATA T, WASHIO K, YOSHIDA T, YAMADA A, TAKAGI R, OHNO T, LIN K, YAMATO M, ISHIKAWA I, OKANO T, IZUMI Y. Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model [J]. Biomaterials,2011,32(25):5819-5825. doi: 10.1016/j.biomaterials.2011.04.071 [7] TANG Y, CHEN C, LIU F, XIE S, QU J, LI M, LI Z, LI X, SHI Q, LI S, LI X, HU J, LU H. Structure and ingredient-based biomimetic scaffolds combining with autologous bone marrow-derived mesenchymal stem cell sheets for bone-tendon healing [J]. Biomaterials,2020,241(C):119837. [8] TANG Z, OKANO T. Recent development of temperature-responsive surfaces and their application for cell sheet engineering [J]. Regen Biomater,2014,1(1):91-102. doi: 10.1093/rb/rbu011 [9] AKIYAMA Y, KIKUCHI A, YAMATO M, OKANO T. Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control [J]. Langmuir,2004,20(13):5506-5511. doi: 10.1021/la036139f [10] NASH M E, CARROLL W M, NIKOLOSKYA N, YANG R, O'CONNELL C, GORELOV A V, DOCKERY P, LIPTROT C, LYNG F M, GARCIA A, ROCHEV Y A. Straightforward, one-step fabrication of ultrathin thermoresponsive films from commercially available PNIPAm for cell culture and recovery [J]. ACS Appl Mater Inter,2011,3(6):1980-1990. doi: 10.1021/am200204j [11] TAKAHASHI H, MATSUZAKA N, NAKAYAMA M, KIKUCHI A, YAMATO M, OKANO T. Terminally functionalized thermoresponsive polymer brushes for simultaneously promoting cell adhesion and cell sheet harvest [J]. Biomacromolecules,2012,13(1):253-260. doi: 10.1021/bm201545u [12] COOPERSTEIN M A, NGUYEN P A, CANAVAN H E. Poly(N-isopropyl acrylamide)-coated surfaces: Investigation of the mechanism of cell detachment [J]. Biointerphases,2017,12(2):02C401. doi: 10.1116/1.4979920 [13] SAKULAUE P, SWE A Y Y, BENCHAPRATHANPHORN K, LERTVANITHPHOL T, VIRAVAIDYA-PASUWAT K, SIRIWATWECHAKUL W. Improving cell detachment from temperature-responsive poly(N-isopropylacrylamide-co-acrylamide)-grafted culture surfaces by spin coating [J]. ACS Omega,2018,3(12):18181-18188. doi: 10.1021/acsomega.8b02514 [14] YAMATO M, AKIYAMA Y, KOBAYASHI J, YANG J, KIKUCHI A, OKANO T. Temperature-responsive cell culture surfaces for regenerative medicine with cell sheet engineering [J]. Prog Polym Sci,2007,32(8-9):1123-1133. doi: 10.1016/j.progpolymsci.2007.06.002 [15] KWON O H, KIKUCHI A, YAMATO M, OKANO T. Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes [J]. Biomaterials,2003,24(7):1223-1232. doi: 10.1016/S0142-9612(02)00469-6 [16] YOUNG R E, GRAF J, MISEROCCHI I, VAN HORN R M, GORDON M B, ANDERSON C R, SEFCIK L S. Optimizing the alignment of thermoresponsive poly(N-isopropyl acrylamide) electrospun nanofibers for tissue engineering applications: A factorial design of experiments approach [J]. PLoS One,2019,14(7):e0219254. doi: 10.1371/journal.pone.0219254 [17] CICOTTE K N, REED J A, NGUYEN P A H, DE LORA J A, HEDBERG-DIRK E L, CANAVAN H E. Optimization of electrospun poly(N-isopropyl acrylamide) mats for the rapid reversible adhesion of mammalian cells [J]. Biointerphases,2017,12(2):02C417. doi: 10.1116/1.4984933 [18] WANG N, ZHAO Y, JIANG L. Low-cost, thermoresponsive wettability of surfaces: Poly(N-isopropylacrylamide)/polystyrene composite films prepared by electrospinning [J]. Macromol Rapid Comm,2008,29(6):485-489. doi: 10.1002/marc.200700785 [19] ALLEN A C B, BARONE E, CROSBY C O K, SUGGS L J, ZOLDAN J. Electrospun poly(N-isopropyl acrylamide)/poly(caprolactone) fibers for the generation of anisotropic cell sheets [J]. Biomater Sci,2017,5(8):1661-1669. doi: 10.1039/C7BM00324B [20] DAI Z, SHU Y, WAN C, WU C. Effects of culture substrate made of poly(N-isopropylacrylamide-co-acrylic acid) microgels on osteogenic differentiation of mesenchymal stem cells [J]. Molecules,2016,21(9):1192. doi: 10.3390/molecules21091192 [21] DZHOYASHVILI N A, THOMPSON K, GORELOV A V, ROCHEV Y A. Film thickness determines cell growth and cell sheet detachment from spin-coated poly(N-isopropylacrylamide) substrates [J]. ACS Appl Mater Inter,2016,8(41):27564-27572. doi: 10.1021/acsami.6b09711 [22] BAEK J, CHO Y, PARK H J, CHOI G, LEE J S, LEE M, YU S J, CHO S W, LEE E, IM S G. A surface-tailoring method for rapid non-thermosensitive cell-sheet engineering via functional polymer coatings [J]. Adv Mater,2020,32(16):1907225. doi: 10.1002/adma.201907225 [23] LIN X, TANG D, YU Z, FENG Q. Stimuli-responsive electrospun nanofibers from poly(N-isopropylacrylamide)-co-poly(acrylic acid) copolymer and polyurethane [J]. J Mater Chem B,2014,2(6):651-658. doi: 10.1039/C3TB21519A [24] PARK Y, KIM M, CHUNG H J, WOO A H, NODA I, JUNG Y M. The study of pH effects on phase transition of multi-stimuli responsive P(NiPAAm-co-AAc) hydrogel using 2D-COS [J]. Polymers,2021,13(9):1447. doi: 10.3390/polym13091447 [25] LI Y Y, ZHANG X Z, KIM G C, CHENG H, CHENG S X, ZHUO R X. Thermosensitive Y-shaped micelles of poly(oleic acid-Y-N-isopropylacrylamide) for drug delivery [J]. Small,2006,2(7):917-923. doi: 10.1002/smll.200600041 [26] CHEN M, DONG M, HAVELUND R, REGINA V R, MEYER R L, BESENBACHER F, KINGSHOTT P. Thermo-responsive core sheath electrospun nanofibers from poly (N-isopropylacrylamide)/polycaprolactone blends [J]. Chem Mater,2010,22(14):4214-4221. doi: 10.1021/cm100753r [27] GUO Z, LI S, WANG C, XU J, KIRK B, WU J, LIU Z, XUE W. Biocompatibility and cellular uptake mechanisms of poly(N-isopropylacrylamide) in different cells [J]. J Bioact Compat Pol,2016,32(1):17-31. [28] da SILVA R M, MANO J F, REIS R L. Smart thermoresponsive coatings and surfaces for tissue engineering: Switching cell-material boundaries [J]. Trends Biotechnol,2007,25(12):577-583. doi: 10.1016/j.tibtech.2007.08.014 -
下载:
点击查看大图
图(5)
计量
- 文章访问数: 26
- HTML全文浏览量: 8
- PDF下载量: 7
- 被引次数: 0
