In Vitro Assessment of Fluorine Nanoemulsion-Labeled Hyaluronan-Based Hydrogels for Precise Intrathecal Transplantation of Glial-Restricted Precursors

  • Marcin Piejko
  • Piotr Walczak
  • Xiaowei Li
  • Jeff W. M. Bulte
  • Miroslaw JanowskiEmail author
Research Article



We studied the feasibility of labeling hydrogel scaffolds with a fluorine nanoemulsion for 19F- magnetic resonance imaging (MRI) to enable non-invasive visualization of their precise placement and potential degradation.


Hyaluronan-based hydrogels (activated hyaluronan, HA) with increasing concentrations of fluorine nanoemulsion (V-sense) were prepared to measure the gelation time and oscillatory stress at 1 h and 7 days after the beginning of gelation. All biomechanical measurements were conducted with an ARES 2 rheometer. Diffusion of fluorine from the hydrogel: Three hydrogels in various Vs to HA volumetric ratios (1:50, 1:10, and 1:5) were prepared in duplicate. Hydrogels were incubated at 37 °C. To induce diffusion, three hydrogels were agitated at 1000 rpm. 1H and 19F MRI scans were acquired at 1, 3, 7 days and 2 months after gel preparation on a Bruker Ascend 750 scanner. To quantify fluorine content, scans were analyzed using Voxel Tracker 2.0. Assessment of cell viability in vitro and in vivo: Luciferase-positive mouse glial-restricted progenitors (GRPs) were embedded in 0:1, 1:50, 1:10, and 1:5 Vs:HA mixtures (final cell concentration  =1 × 107/ml). For the in vitro assay, mixtures were placed in 96-wells plate in triplicate and bioluminescence was measured after 1, 3, 7, 14, 21, and 28 days. For in vivo experiments, Vs/HA mixtures containing GRPs were injected subcutaneously in SCID mice and BLI was acquired at 1, 3, 7, and 14 days post-injection.


Mixing of V-sense at increasing ratios of 1:50, 1:10, and 1:5 v/v of fluorine/activated hyaluronan (HA) hydrogel gradually elongated the gelation time from 194 s for non-fluorinated controls to 304 s for 1:5 V-sense:HA hydrogels, while their elastic properties slightly decreased. There was no release of V-sense from hydrogels maintained in stationary conditions over 2 months. The addition of V-sense positively affected in vitro survival of scaffolded GRPs in a dose-dependent manner.


These results show that hydrogel fluorination does not impair its beneficial properties for scaffolded cells, which may be used to visualize scaffolded GRP transplants with 19F MRI.

Key Words

Hydrogel Glial-restricted precursors Fluorine MRI Scaffold 


Funding Information

This work was supported by R01 EB023647, R56 NS098520, MSCRFD-3899, MSCRFII=2829, R01NS091110, R01NS091100, and R21NS106436. MP was supported by the KNOW program of Jagiellonian University, Cracow, Poland.

Compliance with Ethical Standards

The study was approved by our Institutional Animal Care and Use Committee at the Johns Hopkins University.

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Hess DC, Wechsler LR, Clark WM, Savitz SI, Ford GA, Chiu D, Yavagal DR, Uchino K, Liebeskind DS, Auchus AP, Sen S, Sila CA, Vest JD, Mays RW (2017) Safety and efficacy of multipotent adult progenitor cells in acute ischaemic stroke (masters): a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet Neurol 16:360–368CrossRefGoogle Scholar
  2. 2.
    Anderson KD, Guest JD, Dietrich WD, Bartlett Bunge M, Curiel R, Dididze M, Green BA, Khan A, Pearse DD, Saraf-Lavi E, Widerström-Noga E, Wood P, Levi AD (2017) Safety of autologous human Schwann cell transplantation in subacute thoracic spinal cord injury. J Nneurotrauma 34:2950–2963CrossRefGoogle Scholar
  3. 3.
    Gupta N, Henry RG, Strober J, Kang SM, Lim DA, Bucci M, Caverzasi E, Gaetano L, Mandelli ML, Ryan T, Perry R, Farrell J, Jeremy RJ, Ulman M, Huhn SL, Barkovich AJ, Rowitch DH (2012) Neural stem cell engraftment and myelination in the human brain. Sci Transl Med 4:155ra137CrossRefGoogle Scholar
  4. 4.
    Oliveira JM, Carvalho L, Silva-Correia J, Vieira S, Majchrzak M, Lukomska B, Stanaszek L, Strymecka P, Malysz-Cymborska I, Golubczyk D, Kalkowski L, Reis RL, Janowski M, Walczak P (2018) Hydrogel-based scaffolds to support intrathecal stem cell transplantation as a gateway to the spinal cord: clinical needs, biomaterials, and imaging technologies. NPJ Regen Med 3:8CrossRefGoogle Scholar
  5. 5.
    Windrem MS, Schanz SJ, Guo M, Tian GF, Washco V, Stanwood N, Rasband M, Roy NS, Nedergaard M, Havton LA, Wang S, Goldman SA (2008) Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell 2:553–565.CrossRefGoogle Scholar
  6. 6.
    Walczak P, All AH, Rumpal N, Gorelik M, Kim H, Maybhate A, Agrawal G, Campanelli JT, Gilad AA, Kerr DA, Bulte JWM (2011) Human glial-restricted progenitors survive, proliferate, and preserve electrophysiological function in rats with focal inflammatory spinal cord demyelination. Glia 59:499–510CrossRefGoogle Scholar
  7. 7.
    Lyczek A, Arnold A, Zhang J, Campanelli JT, Janowski M, Bulte JWM, Walczak P (2017) Transplanted human glial-restricted progenitors can rescue the survival of dysmyelinated mice independent of the production of mature, compact myelin. Exp Neurol 291:74–86CrossRefGoogle Scholar
  8. 8.
    Glass JD, Hertzberg VS, Boulis NM, Riley J, Federici T, Polak M, Bordeau J, Fournier C, Johe K, Hazel T, Cudkowicz M, Atassi N, Borges LF, Rutkove SB, Duell J, Patil PG, Goutman SA, Feldman EL (2016) Transplantation of spinal cord-derived neural stem cells for als: analysis of phase 1 and 2 trials. Neurology 87:392–400CrossRefGoogle Scholar
  9. 9.
    Jablonska A, Shea DJ, Cao S, Bulte JW, Janowski M, Konstantopoulos K, Walczak P (2017) Overexpression of vla-4 in glial-restricted precursors enhances their endothelial docking and induces diapedesis in a mouse stroke model. J Cereb Blood Flow Metab.
  10. 10.
    Guzman R, Janowski M, Walczak P (2018) Intra-arterial delivery of cell therapies for stroke. Stroke 49:1075–1082CrossRefGoogle Scholar
  11. 11.
    Slevin M, Kumar S, Gaffney J (2002) Angiogenic oligosaccharides of hyaluronan induce multiple signaling pathways affecting vascular endothelial cell mitogenic and wound healing responses. J Biol Chem 277:41046–41059CrossRefGoogle Scholar
  12. 12.
    Shu XZ, Liu Y, Palumbo F, Prestwich GD (2003) Disulfide-crosslinked hyaluronan-gelatin hydrogel films: a covalent mimic of the extracellular matrix for in vitro cell growth. Biomaterials 24:3825–3834CrossRefGoogle Scholar
  13. 13.
    Shu XZ, Liu Y, Luo Y, Roberts MC, Prestwich GD (2002) Disulfide cross-linked hyaluronan hydrogels. Biomacromolecules 3:1304–1311CrossRefGoogle Scholar
  14. 14.
    Ghosh K, Shu XZ, Mou R, Lombardi J, Prestwich GD, Rafailovich MH, Clark RAF (2005) Rheological characterization of in situ cross-linkable hyaluronan hydrogels. Biomacromolecules 6:2857–2865CrossRefGoogle Scholar
  15. 15.
    Prestwich GD (2011) Hyaluronic acid-based clinical biomaterials derived for cell and molecule delivery in regenerative medicine. J Control Release 155:193–199CrossRefGoogle Scholar
  16. 16.
    Liang Y, Walczak P, Bulte JW (2013) The survival of engrafted neural stem cells within hyaluronic acid hydrogels. Biomaterials 34:5521–5529CrossRefGoogle Scholar
  17. 17.
    Laurén P, Lou YR, Raki M, Urtti A, Bergström K, Yliperttula M (2014) Technetium-99m-labeled nanofibrillar cellulose hydrogel for in vivo drug release. Eur J Pharm Sci 65:79–88CrossRefGoogle Scholar
  18. 18.
    Kim H, Shin K, Park OK, Choi D, Kim HD, Baik S, Lee SH, Kwon SH, Yarema KJ, Hong J, Hyeon T, Hwang NS (2018) General and facile coating of single cells via mild reduction. J Am Chem Soc 140:1199–1202CrossRefGoogle Scholar
  19. 19.
    Hosoya H, Dobroff AS, Driessen WH, Cristini V, Brinker LM, Staquicini FI, Cardó-Vila M, D'Angelo S, Ferrara F, Proneth B, Lin YS, Dunphy DR, Dogra P, Melancon MP, Stafford RJ, Miyazono K, Gelovani JG, Kataoka K, Brinker CJ, Sidman RL, Arap W, Pasqualini R (2016) Integrated nanotechnology platform for tumor-targeted multimodal imaging and therapeutic cargo release. Proc Natl Acad Sci USA 113:1877–1882CrossRefGoogle Scholar
  20. 20.
    Liang Y, Bar-Shir A, Song X, Gilad AA, Walczak P, Bulte JWM (2015) Label-free imaging of gelatin-containing hydrogel scaffolds. Biomaterials 42:144–150CrossRefGoogle Scholar
  21. 21.
    Bulte JW (2005) Hot spot MRI emerges from the background. Nat Biotechnol 23:945–946CrossRefGoogle Scholar
  22. 22.
    Ahrens ET, Helfer BM, O'Hanlon CF, Schirda C (2014) Clinical cell therapy imaging using a perfluorocarbon tracer and fluorine-19 MRI. Magn Reson Med 72:1696–1701CrossRefGoogle Scholar
  23. 23.
    Muhammad G, Xu J, Bulte JWM, Jablonska A, Walczak P, Janowski M (2017) Transplanted adipose-derived stem cells can be short-lived yet accelerate healing of acid-burn skin wounds: a multimodal imaging study. Sci Report 7:4644.CrossRefGoogle Scholar
  24. 24.
    Rubinstein M, Colby R, Dobrynin AV, Joanny JF (1996) Elastic modulus and equilibrium swelling of polyelectrolyte gels. Macromolecules 29:398–406CrossRefGoogle Scholar
  25. 25.
    Bonnans C, Chou J, Werb Z (2014) Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 15:786–801CrossRefGoogle Scholar
  26. 26.
    Carey LE, Dearth CL, Johnson SA, Londono R, Medberry CJ, Daly KA, Badylak SF (2014) In vivo degradation of 14c-labeled porcine dermis biologic scaffold. Biomaterials 35:8297–8304CrossRefGoogle Scholar
  27. 27.
    Shu XZ, Liu Y, Palumbo FS (2004) In situ crosslinkable hyaluronan hydrogels for tissue engineering. Biomaterials 25:1339–1348CrossRefGoogle Scholar
  28. 28.
    Mohand-Kaci F, Assoul N, Martelly I, Allaire E, Zidi M (2013) Optimized hyaluronic acid-hydrogel design and culture conditions for preservation of mesenchymal stem cell properties. Tissue Eng Part C Methods 19:288–298CrossRefGoogle Scholar

Copyright information

© World Molecular Imaging Society 2019

Authors and Affiliations

  1. 1.Russell H. Morgan Department of Radiology and Radiological Science, Division of MR ResearchThe Johns Hopkins University School of MedicineBaltimoreUSA
  2. 2.Cellular Imaging Section and Vascular Biology Program, Institute for Cell EngineeringThe Johns Hopkins University School of MedicineBaltimoreUSA
  3. 3.3rd Department of General SurgeryJagiellonian University Medical CollegeKrakowPoland
  4. 4.Department of Neurology and NeurosurgeryUniversity of Warmia and MazuryOlsztynPoland
  5. 5.Translational Tissue Engineering CenterThe Johns Hopkins University School of MedicineBaltimoreUSA
  6. 6.Mary and Dick Holland Regenerative Medicine Program, Department of Neurological SciencesThe University of Nebraska Medical CenterOmahaUSA
  7. 7.Department of Biomedical EngineeringThe Johns Hopkins University School of MedicineBaltimoreUSA
  8. 8.Department of Chemical and Biomolecular Engineering, Whiting School of EngineeringThe Johns Hopkins UniversityBaltimoreUSA
  9. 9.Department of OncologyThe Johns Hopkins University School of MedicineBaltimoreUSA

Personalised recommendations