Tissue Regeneration: Spatial Control of Cell Gene Expression by siRNA Gradients in Biodegradable Hydrogels (Adv. Healthcare Mater. 5/2015)

To read the full-text of this research, you can request a copy directly from the authors.


By silencing specific gene expression, short interfering RNA (siRNA) is a potent biomolecule for regulating cell behavior in tissue engineering applications, and spatially patterning its presentation to cells may ultimately facilitate the engineering of complex tissues. The study by E. Alsberg and team on page 714 demonstrates a hydrogel system that presents a linear gradient of siRNA to encapsulated cells, inducing a spatial gradient of green fluorescent protein expression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

ResearchGate has not been able to resolve any citations for this publication.
We describe the development of a sensitive fluorescence-based solution assay for RNA using a new dye, RiboGreen RNA quantitation reagent. RiboGreen reagent exhibits >1000-fold fluorescence enhancement and high quantum yield (0.65) upon binding nucleic acids, with excitation and emission maxima near those of fluorescein. Unbound dye is essentially nonfluorescent and has a large extinction coefficient (67,000 cm-1 M-1). The RiboGreen assay allows detection of as little as 1.0 ng/ml RNA in a standard fluorometer, filter fluorometer, or fluorescence microplate reader-surpassing the sensitivity achieved with ethidium bromide by 200-fold. The linear quantitation range for RiboGreen reagent extends over three orders of magnitude in RNA concentration. Using 750 nM RiboGreen reagent, we quantitated 20 ng/ml to 1.0 microg/ml RNA. By diluting the reagent to 75 nM, we could quantitate 1.0 to 50 ng/ml RNA. Both assay ranges exhibited linear fluorescence increases versus RNA concentration (r2 = 0.999). Assay linearity was maintained in the presence of salts, protein, urea, ethanol, chloroform, agarose, and some detergents. Several different RNA types yielded similar signal intensities and detection sensitivities. The assay is easy to use, rapid, and readily adaptable for automation.
Hydrogels are cross-linked hydrophilic polymers that can imbibe water or biological fluids. Their biomedical and pharmaceutical applications include a very wide range of systems and processes that utilize several molecular design characteristics. This review discusses the molecular structure, dynamic behavior, and structural modifications of hydrogels as well as the various applications of these biohydrogels. Recent advances in the preparation of three-dimensional structures with exact chain conformations, as well as tethering of functional groups, allow for the preparation of promising new hydrogels. Meanwhile, intelligent biohydrogels with pH- or temperature-sensitivity continue to be important materials in medical applications.
New generations of synthetic biomaterials are being developed at a rapid pace for use as three-dimensional extracellular microenvironments to mimic the regulatory characteristics of natural extracellular matrices (ECMs) and ECM-bound growth factors, both for therapeutic applications and basic biological studies. Recent advances include nanofibrillar networks formed by self-assembly of small building blocks, artificial ECM networks from protein polymers or peptide-conjugated synthetic polymers that present bioactive ligands and respond to cell-secreted signals to enable proteolytic remodeling. These materials have already found application in differentiating stem cells into neurons, repairing bone and inducing angiogenesis. Although modern synthetic biomaterials represent oversimplified mimics of natural ECMs lacking the essential natural temporal and spatial complexity, a growing symbiosis of materials engineering and cell biology may ultimately result in synthetic materials that contain the necessary signals to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis.
  • J P Vacanti
  • R Langer
J. P. Vacanti, R. Langer, Lancet 1999, 354, S32.
  • F Berthiaume
  • T J Maguire
  • M L Yarmush
F. Berthiaume, T. J. Maguire, M. L. Yarmush, Annu. Rev. Chem. Biomol. Eng. 2011, 2, 403 ; b) K. Y. Lee, D. J. Mooney, Chem. Rev. 2001, 101, 1869.
  • F Ulloa
  • J Briscoe
F. Ulloa, J. Briscoe, Cell Cycle 2007, 6, 2640.
Cold Spring Harbor Perspec
  • F Wang
F. Wang, Cold Spring Harbor Perspec. Biol. 2009, 1, a002980.
  • K L Moffat
  • W.-H S Sun
  • P E Pena
  • N O Chahine
  • S B Doty
  • G A Ateshian
  • C T Hung
  • H H Lu
K. L. Moffat, W.-H. S. Sun, P. E. Pena, N. O. Chahine, S. B. Doty, G. A. Ateshian, C. T. Hung, H. H. Lu, Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 7947.
  • J Y Wong
  • A Velasco
  • P Rajagopalan
  • Q Pham
  • L R Carr
  • J E Krause
  • J.-R Ella-Menye
  • S Jiang
J. Y. Wong, A. Velasco, P. Rajagopalan, Q. Pham, Langmuir 2003, 19, 1908. [12] a) L. R. Carr, J. E. Krause, J.-R. Ella-Menye, S. Jiang, Biomaterials 2011, 32, 8456 ; b) X. Wang, E. Wenk, X. Zhang, L. Meinel, G. Vunjak-Novakovic, D. L. Kaplan, J. Controlled Release 2009, 134, 81.
  • O Jeon
  • D S Alt
  • S W Linderman
  • E Alsberg
O. Jeon, D. S. Alt, S. W. Linderman, E. Alsberg, Adv. Mater. 2013, 25, 6366.
  • A Fire
  • S Xu
  • M K Montgomery
  • S A Kostas
  • S E Driver
  • C C Mello
A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, C. C. Mello, Nature 1998, 391, 806.
  • M Ø Andersen
  • D Q S Le
  • M Chen
  • J V Nygaard
  • M Kassem
  • C Bünger
  • J Kjems
  • N E Van Dijk-Wolthuls
  • S K Y Tsang
  • J J Kettenes-Van Den Bosch
  • W E Hennink
  • K Raemdonck
  • T G Van Thienen
  • R E Vandenbroucke
  • N N Sanders
M. Ø. Andersen, D. Q. S. Le, M. Chen, J. V. Nygaard, M. Kassem, C. Bünger, J. Kjems, Adv. Funct. Mater. 2013, 23, 1. [24] a) W. N. E. van Dijk-Wolthuls, S. K. Y. Tsang, J. J. Kettenes-van den Bosch, W. E. Hennink, Polymer 1997, 38, 6235 ; b) K. Raemdonck, T. G. Van Thienen, R. E. Vandenbroucke, N. N. Sanders, J. Demeester, S. C. De Smedt, Adv. Funct. Mater. 2008, 18, 993 ;
  • L J Jones
  • S T Yue
  • C.-Y Cheung
  • V L Singer
L. J. Jones, S. T. Yue, C.-Y. Cheung, V. L. Singer, Anal. Biochem. 1998, 265, 368.
  • C D Hoemann
  • C.-H Lafantaisie-Favreau
  • V Lascau-Coman
  • G Chen
  • J Guzman-Morales
C. D. Hoemann, C.-H. Lafantaisie-Favreau, V. Lascau-Coman, G. Chen, J. Guzman-Morales, J. Knee Surg. 2012, 25, 85.
  • M R Doschak
  • R F Zernicke
M. R. Doschak, R. F. Zernicke, J. Musculoskelet. Neuronal Interact. 2005, 5, 35.
  • J Y Wong
  • A Velasco
  • P Rajagopalan
  • Q Pham
J. Y. Wong, A. Velasco, P. Rajagopalan, Q. Pham, Langmuir 2003, 19, 1908.
  • C T Lo
  • D J Throckmorton
  • A K Singh
  • A E Herr
C. T. Lo, D. J. Throckmorton, A. K. Singh, A. E. Herr, Lab Chip 2008, 8, 1273.
  • Z Gao
  • Z Wang
  • Y Shi
  • Z Lin
  • H Jiang
  • T Hou
  • Q Wang
  • X Yuan
  • Y Zhao
  • H Wu
  • Y Jin
Z. Gao, Z. Wang, Y. Shi, Z. Lin, H. Jiang, T. Hou, Q. Wang, X. Yuan, Y. Zhao, H. Wu, Y. Jin, Plast. Reconstr. Surg. 2006, 118, 1328.
  • H Motomura
  • H Niimi
  • K Sugimori
  • T Ohtsuka
  • T Kimura
  • I Kitajima
H. Motomura, H. Niimi, K. Sugimori, T. Ohtsuka, T. Kimura, I. Kitajima, Biochem. Biophys. Res. Commun. 2007, 357, 997 ;
  • M K Nguyen
  • O Jeon
  • M D Krebs
  • D Schapira
  • E Alsberg
M. K. Nguyen, O. Jeon, M. D. Krebs, D. Schapira, E. Alsberg, Biomaterials 2014, 35, 6278.
  • M D Krebs
  • O Jeon
  • E Alsberg
M. D. Krebs, O. Jeon, E. Alsberg, J. Am. Chem. Soc. 2009, 131, 9204 ; b) K. Nguyen, P. N. Dang, E. Alsberg, Acta Biomater. 2013, 9, 4487.
  • C E Nelson
  • A J Kim
  • E J Adolph
  • M K Gupta
  • F Yu
  • K M Hocking
  • J M Davidson
  • S A Guelcher
  • C L Duvall
C. E. Nelson, A. J. Kim, E. J. Adolph, M. K. Gupta, F. Yu, K. M. Hocking, J. M. Davidson, S. A. Guelcher, C. L. Duvall, Adv. Mater. 2014, 26, 607.
  • M Ø Andersen
  • D Q S Le
  • M Chen
  • J V Nygaard
  • M Kassem
  • C Bünger
  • J Kjems
M. Ø. Andersen, D. Q. S. Le, M. Chen, J. V. Nygaard, M. Kassem, C. Bünger, J. Kjems, Adv. Funct. Mater. 2013, 23, 1.
  • B Naeye
  • K Raemdonck
  • K Remaut
  • B Sproat
  • J Demeester
  • S C De
  • Smedt
B. Naeye, K. Raemdonck, K. Remaut, B. Sproat, J. Demeester, S. C. De Smedt, Eur. J. Pharm. Sci. 2010, 40, 342.
  • C.-C Lin
  • A T Metters
C.-C. Lin, A. T. Metters, Adv. Drug Delivery Rev. 2006, 58, 1379.
  • X Li
  • X Zhao
  • Y Fang
  • X Jiang
  • T Duong
  • C Fan
  • C.-C Huang
  • S R Kain
X. Li, X. Zhao, Y. Fang, X. Jiang, T. Duong, C. Fan, C.-C. Huang, S. R. Kain, J. Biol. Chem. 1998, 273, 34970.
  • M A Gosselin
  • W Guo
  • R J Lee
M. A. Gosselin, W. Guo, R. J. Lee, Bioconjugate Chem. 2001, 12, 989.