JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Bioengenharia

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

Published: October 26, 2016
doi:
10.3791/53704
, , , , , , , ,
1Department of Chemical Engineering,Rowan University, 2Department of Biological Sciences,Rowan University, 3Department of Biomedical Engineering,Drexel University

Summary

聚（N-异丙基丙烯酰胺） – 接枝 – 硫酸软骨素组成的可注射的组织工程支架，制备（PNIPAAm的接枝CS）含藻酸盐微粒。粘合强度，溶胀性能和体外生物相容性在这项研究中进行分析。这里开发的表征技术可以适用于其他的热胶凝系统。

Abstract

可注射生物材料被定义为可被引入到体内的液体，并且在原位固化可植入材料。这种材料提供的植入微创侵入性，容易形成不规则形状的缺陷的空间填充物的临床优势。注射生物材料已被广泛研究作为组织工程支架。然而，对于某些承重区在体内的修复，如椎间盘，支架应该具有粘合性能。这将运动期间减少位错的风险，并确保与周围组织紧密接触，提供力的充分传输。在这里，我们描述了一个热敏感聚（N-异丙基）组成的支架的制备和表征接枝 – 硫酸软骨素（PNIPAAM-G-CS）和海藻酸钠微粒。所述的PNIPAAm-G-CS共聚物形成在RT在水中的粘性溶液，在其中alginatË颗粒悬浮以提高附着力。上面的低临界溶液温度（LCST），在30℃左右，该共聚物形成围绕微粒的固体凝胶。我们已经适应标准的生物材料的表征程序考虑到的PNIPAAm-G-CS的可逆相变。结果表明，50％或75毫克/毫升藻酸盐微粒中掺入5％（重量/体积）的PNIPAAm-G-CS溶液四倍的PNIPAAm-GCS的粘合抗拉强度单独（P <0.05）。藻酸盐微粒的掺入也显著增加的PNIPAAm-G-CS的溶胀容量（P <0.05），有助于保持组织缺损内的空间填充凝胶。最后， 体外毒理学测定试剂盒，2,3-双-的结果（2-甲氧基-4-硝基-5-磺苯基）-2H-四唑-5-羧酰（XTT）和活/死活力测定表明，该胶粘剂能够支持的生存和封装的人类胚胎肾细胞增殖（HEK）293ç厄尔超过5天。

Introduction

可注射生物材料是那些可方便地输送到身体中作为液体和原位固化。这样的材料已经在再生医学，在那里它们被用来封装细胞递送到受影响的部位1-4，并作为对电池单元5的三维临时的细胞外基质广泛应用。对于患者，因为用于植入的外科手术是微创和固相可以填充不规则形状的组织缺损，消除了对自定义尺寸的植入物的需要注射的生物材料是有利的。

注射能力可以通过各种机制来实现。外部因素，如pH值，已经研究作为用于封装细胞和生物活性分子6-8凝胶的形成的触发器。然而，pH值可能不是最方便的触发器中使用所有生理环境。另一个传统爱特纳略去对于实现可注射使用原位化学聚合或交联。 A组开发过硫酸铵和N，N，N'，N“四甲基乙二胺组成的水溶性氧化还原体系，并用它进行反应的聚乙二醇和聚（丙烯）二醇9,10-组成的大分子单体。咱等人 11发达注射壳聚糖聚乙烯醇网络交联用戊二醛。在这样的系统中，反应性组分的细胞毒性，必须考虑，尤其是对涉及细胞封装的应用程序。另外，放热聚合可以产生足够高的温度妥协周围组织，这已被报道用于聚合的骨水泥12,13。

仍然其它可注射的聚合物系统已经开发显示出从液体到固体的状态的变化与温度作为触发。被称为热胶系统，这些都是aqueo我们不要求化学刺激，单体或交联剂原位形成14来实现聚合物溶液。更确切地说，通常接近生理温度中发生的相变诱导物理交联的三维网络的形成。泊洛沙姆例如Pluronic F127是热胶化药物递送15-17和电池封装18,19研究最广泛的聚合物之一。然而，可以很好地接受，这些凝胶缺乏在生理条件下的稳定性。研究使用的扩链剂20或化学交联剂21,22展示更高的稳定性。然而，使用这些试剂的可能会限制电池封装的材料的潜力。

聚（N-异丙基丙烯酰胺）是已经接收显著关注组织工程和药物输送14的合成热胶凝聚合物。聚的水溶液（N- isoprop基丙烯酰胺）（PNIPAAm的）表现出较低的临界溶液温度（LCST），通常发生约32 – 34°C 23,24。下面的LCST，水保湿PNIPAAm的链。高于转变温度时，聚合物变得疏水，从而导致剧烈的相分离25-27和地层的固体凝胶，无需使用有毒的单体或交联剂。然而，PNIPAAm的均聚物表现出较差的弹性性能，并在生理温度下，由于疏水28容纳少量的水。在这项工作中，我们选择共价结合的硫酸软骨素入PNIPAAm的网络，该网络提供了酶促降解29，抗炎活性30,31，并增加水和营养的吸收32的潜力。通过在甲基丙烯酸酯官能化的CS的存在下，单体的NIPAAm聚合形成接枝共聚物（PNIPAAm的-G-CS）在我们实验室中制备的CS PNIPAAm的共聚物。 BEC该共聚物的低交联密度的澳洲英语，PNIPAAm的接枝CS形成在RT在水中的粘性溶液中，在生理温度下的弹性凝胶由于LCST 29。聚合物溶液由于过渡的可逆性成为再次冷却时低于LCST悬浮剂。

我们已经证明，PNIPAAm的接枝的CS具有以用作组织工程支架，由于机械特性可定制，降解，和人类胚胎肾（HEK）细胞相容293细胞29的潜力。然而，在某些承载区域，如椎间盘，组织工程支架应该具有形成与周围椎间盘组织大幅接口消除错位33的风险的能力。这个接口也是必需的力在整个植入物和组织33之间的界面处的适当的传输。在我们的工作中，我们已经暂停了一在的PNIPAAm-G-CS的水溶液lginate微粒，发现凝胶化本地化的微粒，其提供与周围组织34的粘合性。在本文中，我们列出的步骤准备的热胶，粘性聚合物。生物材料表征，细胞成像的生存力的标准技术，并测定法适于考虑到该聚合物的温度灵敏度和相转变的可逆性。在本文中所述的可注射的聚合物具有那些本文中所描述的以外的用于药物递送和组织工程应用宽的电位。此外，这里所描述的表征方法可以适用于其它的热胶凝系统。

Protocol

1.聚（N-异丙基丙烯酰胺）-g-硫酸软骨素合成之前的生物粘附凝胶的合成，纯化N-异丙基（的NIPAAm）单体和甲基丙烯酸酯硫酸软骨素（CS）。 称出至少10g的NIPAAm和在60℃下溶解在400ml正己烷的单体。定期搅动容器，直到完全溶解。再结晶在-20℃冷冻该溶液24小时。 从容器和真空过滤除去结晶单体使用布氏漏斗正己烷。置于在RT真空烘箱单体24小时以除去任何剩余的正己烷。储存在-…

Representative Results

一种热响应接枝共聚物已成功合成和表征其生物粘附强度，溶胀性能，并在体外细胞相容性。我们选择调查的褐藻胶因拥有完善粘膜粘附性。藻酸盐微粒，具有59.7±14.9微米的平均直径，用5％的共混（重量/体积）的PNIPAAm-G-CS在25，50，和75毫克/毫升的浓度。这些浓度是基于二分之一，等于，和在水溶液中的PNIPAAm-G-CS的等效干重的两倍。微粒浓度超过75毫克/毫升展出粘…

Discussion

有在合成水凝胶微粒复合材料和评价其粘合强度，溶胀能力和细胞生物相容性几个关键步骤。 PNIPAAm的接枝的CS的自由基聚合需要硫酸软骨素成功methacrylation，单体组分完全溶解，无氧的反应条件。被选择的NIPAAm单体甲基丙烯酸酯化的硫酸软骨素在反应混合物中的比率，因为它已被证明，在我们以前的工作，以产生具有机械性能类似于天然椎间盘组织29共聚物。在水凝胶增加硫酸软骨素的量…

Declarações

The authors have nothing to disclose.

Acknowledgements

笔者想衷心感谢詹妮弗Kadlowec博士的协助下在胶粘剂拉伸测试的协议的发展。

研究本出版物中报告是由关节肌肉骨骼及皮肤疾病研究所和生物医学成像和美国国立卫生研究院生物工程的下奖号1R15 AR 063920-01研究所的支持。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

Materials

N-isopropylacrylamide, 99%, pure, stabilized Acros Organics 2210-25-5 Refrigerate and remove stabilier with hexane
Chondroitin sulfate A sodium salt (from bovine trachea) Sigma-Aldrich 39455-18-0 Refrigerate
Hexanes Fisher Scientific H302-4 Store in a flammable cabinet
50% (w/w) sodium hydroxide Fisher Scientific SS254-1 Caustic in nature
Methacrylic anhydride Sigma-Aldrich 276685 Strong fumes; use in a fume hood
Acetone Fisher Scientific A18-4 Chill in a refrigerator prior to use
Nitrogen Gas Praxair 7727-37-9 Part Number: NI 4.8, cylinder style T, 99.998% pure nitrogen (Argon may be used as an alternative inert gas)
Tetramethylethylenediamine, 99% extra pure Acros Organics 110-18-9
Ammonium persulfate Sigma Aldrich A3678 Hygroscopic and degrades in the presence of water
Phosphate buffered saline tablets Fisher Scientific BP2944 Keep dry
Alginic acid, sodium salt Acros Organics 177775000 Use heat to aid in dissolving
Calcium chloride dihydrate Fisher Scientific C79
Canola oil Local store Obtain from a local store
Tween 20 Sigma-Aldrich 93773
70% (v/v) Isopropoanol Fisher Scientific A416-4
Porcine ears Haine's Pork Shop Obtain from a local butcher
Sodium Chloride Fisher Scientific S271-3
Human embryonic kidney 293 cells ATCC ATCC CRL-1573 Store in liquid nitrogen for long-term use
DMEM: 1X, high glucose, no pyruvate Life Technologies 11965126 Refrigerate
Fetal bovine serum Life Technologies 10082-147 Refrigerate
Penn Strep: 10,000 U/ml Life Technologies 15140-122 Refrigerate
Trypsin-EDTA: 0.5%, 10X Life Technologies 15400-054 Refrigerate
Methanol VWR AAA44571-K7
Live/Dead Cell viability kit Life Technologies L3224 Light sensitive, keep frozen
XTT cell viability kit Sigma Aldrich TOX2-1KT Light sensitive, keep frozen
Clear DMEM: 1X, high glucose, no phenol Life Technologies 21063-029 Refrigerate
Dulbecco's PBS: 1X Life Technologies 14190136 Refrigerate
Sodium citrate EMD SX0445-1
Positive displacement pipette BrandTech Scientific, INC 2702904 Dispenses 100 – 500 µL and comes with attachable tips
No 3. Stainless Steel scalpel handle Sigma Aldrich S2896
Miltex sterile surgical blades Fisher Scientific 12-460-440 Size 10
Power gem homogenizer Fisher Scientific 08-451-660 Model # 125
Porcelain mortar and pestle Sigma Aldrich Z247464 Holds 50 mL
FreeZone 1 L benchtop freeze dry system Labconco 7740020 Freeze samples prior to use
Oil sealed rotary vane pump Edwards A65301906 Model # RV5
Incubating orbital shaker VWR 12620-946 Model # 980153
Benchtop refrigerated centrifuge Forma Scientific, INC Model # 5682
Heated ovens VWR Model # 1235PC
2 N force gauge Shimpo FGV-0.5XY Model # FGV-0.5XY
E-force test stand Shimpo FGS-200PV Model # FGS-200PV
Tissue culture swinging bucket centrifuge Beckman Coulter 366830 Model #6S-6KR
Tissue culture microcentrifuge Eppendorf Model #5415C
Hemacytometer set Hausser Scientific 3720 Requires replacement cover glass slips
Slide warmer Lab Scientific XH-2022 Model # XH-2002
Portable heating lamp Underwriters Laboratories Helps to maintain polymer temperature at 37°C
Inverted fluorescent microscope Zeiss Model Axiovert 25 CFL
Heated water bath VWR Model # 1235PC
Rocking platform VWR Series 100
Multiskan FC microtiter plate reader Thermo Scientific Type 357
Cell culture incubator VWR Model # 2350T
Purifier class II biosafety cabinet Labconco Delta Series
DOWNLOAD MATERIALS LIST

Referências

  1. Bidarra, S.J., Barrias, C.C., & Granja, P.L. Injectable alginate hydrogels for cell delivery in tissue engineering. Acta Biomater. 10, 1646-1662 (2014).
  2. Choi, J. et al. Human extracellular matrix (ECM) powders for injectable cell delivery and adipose tissue engineering. J. Control. Release. 139, 2-7 (2009).
  3. Selvam, S., Pithapuram, M.V., Victor, S.P., & Muthu, J. Injectable in situ. forming xylitol-PEG-based hydrogels for cell encapsulation and delivery. Colloid Surface B. 126, 35-43 (2015).
  4. Park, K.M., Lee, S.Y., Joung, Y.K., Na, J.S., Lee, M.C., & Park, K.D. Thermosensitive chitosan-pluronic hydrogel as an injectable cell delivery carrier for cartilage regeneration. Acta Biomater. 5., 1956-1965 (2009).
  5. Ren, K., He, C., Xiao, C., Li, G., & Chen, X. Injectable glycopolypeptide hydrogels as biomimetic scaffolds for cartilage tissue engineering. Biomaterials. 51, 238-249 (2015).
  6. Chiu, Y.L., et al. pH-triggered injectable hydrogels prepared from aqueous N-palmitoyl chitosan: In vitro. characteristics and in vivo. biocompatibility. Biomaterials. 30, 4877-4888 (2009).
  7. Shim, W.S., et al. pH- and temperature-sensitive, injectable, biodegradable block copolymer hydrogels as carriers for paclitaxel. Int. J. Pharm. 331, 11-18 (2007).
  8. Singh, N.K., & Lee, D.S. In situ. gelling pH- and temperature-sensitive biodegradable block copolymer hydrogels for drug delivery. J. Control. Release. 193, 214-227 (2014).
  9. Wang, B., Zhu, W., Zhang, Y., Yang, Z., & Ding, J. Synthesis of a chemically-crosslinked thermo-sensitive hydrogel film and in situ encapsulation of model protein drugs. React. Funct. Polym. 66, 509-518 (2006).
  10. Zhu, W., & Ding, J. Synthesis and characterization of a redox-initiated, injectable, biodegradable hydrogel. J. Appl. Polym. Sci. 99, 2375-2383 (2006).
  11. Zan, J., Chen, H., Jiang, G., Lin, Y., & Ding, F. Preparation and properties of crosslinked chitosan thermosensitive hydrogel for injectable drug delivery systems. J. Appl. Polym. Sci. 101, 1892-1898 (2006).
  12. Togawa, D., Bauer, T.W., Lieberman, I.H., & Takikawa, S. Histologic evaluation of human vertebral bodies after vertebral augmentation with polymethyl methacrylate. Spine. 28, 1521-1527 (2003).
  13. Berman, A.T., Reid, J.S., Yanicko, D.R., Sih, G.C., & Zimmerman, M.R. Thermally induced bone necrosis in rabbits: relation to implant failure in humans. Clin. Orthop. Relat. R. 186, 284-292 (1984).
  14. Kretlow, J.D., Klouda, L., & Mikos, A.G. Injectable matrices and scaffolds for drug delivery in tissue engineering. Adv Drug. Deliver. Rev. 59, 263-273 (2007).
  15. Ye, F., Yaghmur, A., Jensen, H., Larsen, S.W., Larsen, C., & Ostergaard, J. Real-time UV imaging of drug diffusion and release from Pluronic F127 hydrogels. Eur. J. Pharm. Sci. 43, 236-243 (2011).
  16. Akash, M.S., & Rehman, K. Recent progress in biomedical applications of pluronic (PF127): Pharmaceutical perspectives. J. Control Release. 209, 120-138 (2015).
  17. Sellers, D.L., Kim, T.H., Mount, C.W., Pun, S.H., & Horner, P.J. Poly(lactic-co-glycolic) acid microspheres encapsulated in Pluronic F-127 prolong hirudin delivery and improve functional recovery from a demyelination lesion. Biomaterials. 35, 8895-8902 (2014).
  18. Jung, H., Park, K., & Han, D.K. Preparation of TGF-β1-conjugated biodegradable pluronic F127 hydrogel and its application with adipose-derived stem cells. J. Control. Release. 147, 84-91 (2010).
  19. Lee, S.Y., & Tae, G. Formulation and in vitro characterization of an in situ gelable, photo-polymerizable pluronic hydrogel suitable for injection. J. Control. Release. 119, 313-319 (2007).
  20. Chen, Y.Y., Wu, H.C., Sun, J.S., Dong, G.C., & Wang, T.W. Injectable and thermoresponsive self-assembled nanocomposite hydrogel for long-term anticancer drug delivery. Langmuir. 19, 3721-3729 (2013).
  21. Cellesi, F., Tirelli, N., & Hubbell, J.A. Materials for cell encapsulation via a new tandem approach combining reverse thermal gelation and covalent crosslinking. Macromol. Chem. Physic. 203, 1466-1472 (2002).
  22. Cellesi, F., Tirelli, N., & Hubbell, J.A. Towards a fully-synthetic substitute of alginate: development of a new process using thermal gelation and chemical cross-linking. Biomaterials. 25, 5115-5124 (2004).
  23. Hirokawa, Y., & Tanaka, T. Volume phase transition in a nonionic gel. J. Chem. Phys. 81, 6379-6380 (1984).
  24. Freitas, R., & Cussler, E.L. Temperature sensitive gels as extraction solvents. Chem. Eng. Sci. 42, 97-103 (1987).
  25. Schild, H., & Tirrel, D.A. Microcalorimetric detection of lower critical solution temperatures in aqueous polymer solutions. J. Chem. Phys. 94, 4352-4356 (1990).
  26. Yagi, Y., Inomata, H., & Saito, S. Solubility parameter of an N-isopropylacrylamide gel. Macromol. 25, 2997-2998 (1992).
  27. Illmain, F., Tanaka, T., & Kokufuta, E. Volume transition in a gel driven by hydrogen bonding. Nature. 349, 400-401 (1990).
  28. Vernengo, J., Fussell, G.W., Smith, N.G., & Lowman, A.M. Evaluation of novel injectable hydrogels for nucleus pulposus replacement. J. Biomed. Mater. Res. B. 84, 64-69 (2008).
  29. Wiltsey, C., et al. Characterization of injectable hydrogels based on poly(N-isopropylacrylamide)-g-chondroitin sulfate with adhesive properties for nucleus pulposus tissue engineering. J. Mater. Sci-Mater. M. 24, 837-847 (2013).
  30. Ronca, F., Palmieri, L., Panicucci, P., & Ronca G. Anti-inflammatory activity of chondroitin sulfate. Osteoarthr. Cart. 6, 14-21 (1998).
  31. Pipitone, V. Chondroprotection with chondroitin sulfate. Drug. Exp. Clin. Res. 17, 3-7 (1991).
  32. Moss, M., Kruger, G.O., & Reynolds, D.C. The effect of chondroitin sulfate on bone healing. Oral Surg. Oral Med. O. 20, 795-801 (1965).
  33. Nerurkar, N., Elliott, D.M., & Mauck, R.L. Mechanical design criteria fo intervertebral disc tissue engineering. J. Biomech. 43, 1017-1030 (2010).
  34. Wiltsey, C., et al. Thermogelling bioadhesive scaffolds for intervertebral disk tissue engineering: Preliminary in vitro comparison of aldehyde-based versus alginate microparticle-mediated adhesion. Acta Biomater. 16, 71-80 (2015).
  35. Xia, Y., Yin, X., Burke, N., & Stover, H. Thermal response of narrow-disperse poly(N-isopropylacrylamide) prepared by atom transfer radical polymerization. Macromol. 38, 5937-5943 (2005).
  36. Liu, Q., Zhang, P., Qing, A., Lan, Y., & Lu, M. Poly(N-isopropylacrylamide) hydrogels with improved shrinking kinetics by RAFT polymerization. Polymer. 47, 2330-2336 (2006).
  37. Lemoine, D., Wauters, F., Bouchend'homme, S., & Preat, V. Preparation and characterization of alginate microspheres containing a model antigen. Int. J. Pharm. 176, 9-19 (1998).
  38. Dang, T.D., & Joo, S.W. Preparation of tadpole-shaped calcium alginate microparticles with sphericity control. Colloid. Surface. B. 102, 766-771 (2013).
  39. Moebus, K., Siepmann, J., & Bodmeier, R. Novel preparation techniques for alginate-polaxamer microparticles controlling protein release on mucosal surfaces. Eur. J. Pharm. Sci. 45, 358-366 (2012).
  40. Lih, E., Lee, J.S., Park, K.M., Park, & K.D. Rapidly curable chitosan-PEG hydrogels as tissue adhesives for hemostasis and wound healing. Acta Biomater. 8, 3261-3269 (2012).
  41. Urban, J., & Maroudas, A. Swelling of the intervertebral disc in vitro. Connect. Tissue Res. 9, 1-10 (1981).
  42. Ma, H.L., Hung, S.C., Lin, S.Y., Chen, Y.L., & Lo, W.H., Chondrogenesis of human mesenchymal stem cells encapsulated in alginate beads. J. Biomed. Mater. Res. A. 64, 273-281 (2003).
  43. Leslie, S.K., et al. Controlled release of rat adipose-derived stem cells from alginate microbeads. Biomaterials. 34, 8172-8184 (2013).
  44. Peroglio, M., Eglin, D., Benneker, L.M., Alini, M., & Grad, S. Thermoreversible hyaluronan-based hydrogel supports in vitro. and ex vivo. disc-like differentiation of human mesenchymal stem cells. Spine J. 13, 1627-1639 (2013).
  45. Chen, J.P., & Cheng, T.H. Thermo-responsive chitosan-graft-poly(N-isopropylacrylamide) injectable hydrogel for cultivation of chondrocytes and meniscus cells. Macromol. Biosci. 6, 1026-1039 (2006).
  46. Schoichet, M.S., Li, R.H., White, M.L., & Winn, S.R. Stability of hydrogels used in cell encapsulation: an in vitro. comparison of alginate and agarose. Biotechnol. Bioeng. 50, 374-381 (1996).
  47. Dai, J., Wang, H., Liu, G., Xu, Z., Li, F., & Fang, H. Dynamic compression and co-culture with nucleus pulposus cells promotes proliferation and differentiation of adipose-derived mesenchymal stem cells. J. Biomech. 47, 966-972 (2014).
  48. Feng, G., et al. Effects of hypoxias and scaffold architecture on rabbit mesenchymal stem cell differentiation towards a nucleus pulposus-like phenotype. Biomaterials. 32, 8182-8189 (2011).
  49. Feng, G., et al. Hypoxia differentially regulates human nucleus pulposus and annulus fibrosus cell extracellular matrix production in 3D scaffolds. Osteoarth. Cartilage. 21, 582-588 (2013).

Tags

Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate CompositesAlginate MicroparticlesAdhesive PropertiesCellular BiocompatibilityHydrogel CompositeNucleus PulposusThermal Reversible HydrogelsCell EncapsulationInjectable Thermally-sensitive CompositeTensile Mechanical TestsCellular Viability AssaysBiocompatibility AssaysNIPAAm MonomerMCS PurificationDeionized Water Purification
check_url/pt/53704?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Christiani, T. R., Toomer, K., Sheehan, J., Nitzl, A., Branda, A., England, E., Graney, P., Iftode, C., Vernengo, A. J. Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering. J. Vis. Exp. (116), e53704, doi:10.3791/53704 (2016).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image