A methylcellulose and collagen based temperature responsive hydrogel promotes encapsulated stem cell viability and proliferation in vitro

Abstract

With the number of stem cell-based therapies emerging on the increase, the need for novel and efficient delivery technologies to enable therapies to remain in damaged tissue and exert their therapeutic benefit for extended periods, has become a key requirement for their translation. Hydrogels, and in particular, thermoresponsive hydrogels, have the potential to act as such delivery systems. Thermoresponsive hydrogels, which are polymer solutions that transform into a gel upon a temperature increase, have a number of applications in the biomedical field due to their tendency to maintain a liquid state at room temperature, thereby enabling minimally invasive administration and a subsequent ability to form a robust gel upon heating to physiological temperature. However, various hurdles must be overcome to increase the clinical translation of hydrogels as a stem cell delivery system, with barriers including their low tensile strength and their inadequate support of cell viability and attachment. In order to address these issues, a methylcellulose based hydrogel was formulated in combination with collagen and beta glycerophosphate, and key development issues such as injectability and sterilisation processes were examined. The polymer solution underwent thermogelation at ~36 °C as determined by rheological analysis, and when gelled, was sufficiently robust to resist significant disintegration in the presence of phosphate buffered saline (PBS) while concomitantly allowing for diffusion of methylene blue dye solution into the gel. We demonstrate that human mesenchymal stem cells (hMSCs) encapsulated within the gel remained viable and showed raised levels of dsDNA at increasing time points, an indication of cell proliferation. Mechanical testing showed the “injectability”, i.e. force required for delivery of the polymer solution through devices such as a syringe, needle or catheter. Sterilisation of the freeze-dried polymer wafer via gamma irradiation showed no adverse effects on the formed hydrogel characteristics. Taken together, these results indicate the potential of this gel as a clinically translatable delivery system for stem cells and therapeutic molecules in vivo.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Abbreviations

hMSCs:

human mesenchymal stem cells

MC:

methylcellulose

βGP:

beta-glycerophosphate

ID:

internal diameter

G’:

storage modulus

G”:

loss modulus

SEM:

standard error of mean

References

  1. 1.

    Orlando G, Wood KJ, Stratta RJ, Yoo JJ, Atala A, Soker S. Regenerative medicine and organ transplantation: past, present, and future. Transplantation. 2011;91(12):1310–7.

    Article  PubMed  Google Scholar 

  2. 2.

    Lau AN, Goodwin M, Kim CF, Weiss DJ. Stem cells and regenerative medicine in lung biology and diseases. Mol Ther. 2012;20(6):1116–30.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell. 2001;105(3):369–77.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    D. N. Kotton, B. Y. Ma, W. V Cardoso, E. A. Sanderson, R. S. Summer, M. C. Williams, and A. Fine, “Bone marrow-derived cells as progenitors of lung alveolar epithelium.,” Development, vol. 128, no. 24, pp. 5181–5188, 2001.

  5. 5.

    Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature. 2001;410(6829):701–5.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Antunes MA, Laffey JG, Pelosi P, Rocco PRM. Mesenchymal stem cell trials for pulmonary diseases. J Cell Biochem. 2014;115(6):1023–32.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    O’Neill HS, Gallagher LB, O’Sullivan J, Whyte W, Curley C, Dolan E, Hameed A, O’Dwyer J, Payne C, O’Reilly D, Ruiz-Hernandez E, Roche ET, O’Brien FJ, Cryan SA, Kelly H, Murphy B, Duffy GP. Biomaterial-enhanced cell and drug delivery: lessons learned in the cardiac field and future perspectives. Adv Mater. 2016:5648–61.

  8. 8.

    Ye Z, Zhou Y, Cai H, Tan W. Myocardial regeneration: roles of stem cells and hydrogels. Adv Drug Deliv Rev. 2011;63(8):688–97.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol. 2005;23(7):845–56.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol. 2001;33(5):907–21.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Malliaras K, Marbán E. Cardiac cell therapy: where we’ve been, where we are, and where we should be headed. Br Med Bull. 2011;98:161–85.

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Wang Q-D, Sjöquist P-O. Myocardial regeneration with stem cells: pharmacological possibilities for efficacy enhancement. Pharmacol Res. 2006;53(4):331–40.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Hastings CL, Roche ET, Ruiz-Hernandez E, Schenke-Layland K, Walsh CJ, Duffy GP. Drug and cell delivery for cardiac regeneration. Adv Drug Deliv Rev. 2014;84:85–106.

    Article  PubMed  Google Scholar 

  14. 14.

    Liu Z, Wang H, Wang Y, Lin Q, Yao A, Cao F, Li D, Zhou J, Duan C, Du Z, Wang Y, Wang C. The influence of chitosan hydrogel on stem cell engraftment, survival and homing in the ischemic myocardial microenvironment. Biomaterials. 2012;33(11):3093–106.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    A. C. Gaffey, M. H. Chen, C. M. Venkataraman, A. Trubelja, C. B. Rodell, P. V Dinh, G. Hung, J. W. MacArthur, R. V Soopan, J. A. Burdick, and P. Atluri, “Injectable shear-thinning hydrogels used to deliver endothelial progenitor cells, enhance cell engraftment, and improve ischemic myocardium.,” J Thorac Cardiovasc Surg, vol. 150, no. 5, pp. 1268–1277, 2015.

  16. 16.

    Hoare TR, Kohane DS. Hydrogels in drug delivery: Progress and challenges. Polymer (Guildf). 2008;49(8):1993–2007.

    CAS  Article  Google Scholar 

  17. 17.

    Klouda L, Mikos AG. Thermoresponsive hydrogels in biomedical applications. Eur J Pharm Biopharm. 2008;68(1):34–45.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Chenite A, Chaput C, Wang D, Combes C, Buschmann MD, Hoemann CD, Leroux JC, Atkinson BL, Binette F, Selmani A. Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials. 2000;21(21):2155–61.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Hastings CL, Kelly HM, Murphy MJ, Barry FP, O’Brien FJ, Duffy GP. Development of a thermoresponsive chitosan gel combined with human mesenchymal stem cells and desferrioxamine as a multimodal pro-angiogenic therapeutic for the treatment of critical limb ischaemia. J Control Release. 2012;161(1):73–80.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Zhang Z, Ni J, Chen L, Yu L, Xu J, Ding J. Biodegradable and thermoreversible PCLA-PEG-PCLA hydrogel as a barrier for prevention of post-operative adhesion. Biomaterials. 2011;32(21):4725–36.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Elstad NL, Fowers KD. OncoGel (ReGel/paclitaxel)--clinical applications for a novel paclitaxel delivery system. Adv Drug Deliv Rev. 2009;61(10):785–94.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Shive MS, Hoemann CD, Restrepo A, Hurtig MB, Duval N, Ranger P, Stanish W, Buschmann MD. BST-CarGel: in situ ChondroInduction for cartilage repair. Oper Tech Orthop. 2006;16(4):271–8.

    Article  Google Scholar 

  23. 23.

    Shalhoub J, Thapar A, Davies AH. The use of reverse thermosensitive polymer (LeGoo) for temporary vessel occlusion in clampless peripheral vascular surgery. Vasc Endovasc Surg. 2011;45(5):422–5.

    Article  Google Scholar 

  24. 24.

    Buwalda SJ, Boere KWM, Dijkstra PJ, Feijen J, Vermonden T, Hennink WE. Hydrogels in a historical perspective: from simple networks to smart materials. J Control Release. 2014;190:254–73.

  25. 25.

    Higuchi A, Sugiyama K, Yoon BO, Sakurai M, Hara M, Sumita M, Sugawara S, Shirai T. Serum protein adsorption and platelet adhesion on pluronic™-adsorbed polysulfone membranes. Biomaterials. 2003;24(19):3235–45.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Ruel-Gariépy E, Leroux J-C. In situ-forming hydrogels--review of temperature-sensitive systems. Eur J Pharm Biopharm. 2004;58(2):409–26.

    Article  PubMed  Google Scholar 

  27. 27.

    Yu L, Ding J. Injectable hydrogels as unique biomedical materials. Chem Soc Rev. 2008;37(8):1473–81.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Supper S, Anton N, Seidel N, Riemenschnitter M, Curdy C, Vandamme T. Thermosensitive chitosan/glycerophosphate-based hydrogel and its derivatives in pharmaceutical and biomedical applications. Expert Opin Drug Deliv. 2014;11(2):249–67.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Tang Y, Wang X, Li Y, Lei M, Du Y, Kennedy JF, Knill CJ. Production and characterisation of novel injectable chitosan/methylcellulose/salt blend hydrogels with potential application as tissue engineering scaffolds. Carbohydr Polym. 2010;82(3):833–41.

    CAS  Article  Google Scholar 

  30. 30.

    Chenite A, Buschmann M, Wang D, Chaput C, Kandani N. Rheological characterisation of thermogelling chitosan / glycerol-phosphate solutions. Carbohydr Polym. 2001;46:39–47.

    CAS  Article  Google Scholar 

  31. 31.

    Huang CC, Liao ZX, Chen DY, Hsiao CW, Chang Y, Sung HW. Injectable cell constructs fabricated via culture on a thermoresponsive methylcellulose hydrogel system for the treatment of ischemic diseases. Adv Healthc Mater. 2014;3(8):1133–48.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Thirumala S, Gimble J, Devireddy R. Methylcellulose based thermally reversible hydrogel system for tissue engineering applications. Cells. 2013;2:460–75.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Wallace D. Collagen gel systems for sustained delivery and tissue engineering. Adv Drug Deliv Rev. 2003;55(12):1631–49.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Wang L, Stegemann JP. Thermogelling chitosan and collagen composite hydrogels initiated with beta-glycerophosphate for bone tissue engineering. Biomaterials. 2010;31(14):3976–85.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Lee KY, Mooney DJ. Hydrogels for Tissue Engineering. Chem Rev. 2001;101(7):1869–80.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Camci-Unal G, Annabi N, Dokmeci MR, Liao R, Khademhosseini A. Hydrogels for cardiac tissue engineering. NPG Asia Mater. 2014;6(5):e99.

    CAS  Article  Google Scholar 

  37. 37.

    Liu X, Ma PX. Polymeric scaffolds for bone tissue engineering. Ann Biomed Eng. 2004;32(3):477–86.

    Article  PubMed  Google Scholar 

  38. 38.

    Burdick JA, Anseth KS. Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering. Biomaterials. 2002;23(22):4315–23.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Nichols JE, Kojima K, Bonassar LJ, Dargon P, Roy AK, Vacant MP, et al. Tissue-engineered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth. Tissue Eng. 2006;12(5):1213–25.

  40. 40.

    O’Brien F. Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds. Biomaterials. Mar. 2004;25(6):1077–86.

    Article  PubMed  Google Scholar 

  41. 41.

    Duffy GP, Ahsan T, O’Brien T, Barry F, Nerem RM. Bone marrow-derived mesenchymal stem cells promote angiogenic processes in a time- and dose-dependent manner in vitro. Tissue Eng. Part A. 2009;15(9):2459–70.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    C. of Europe, European Pharmacopoeia, 5.0., no. 1. Strasbourg, 2005.

  43. 43.

    Bramley K, Pisani MA, Murphy TE, Araujo KL, Homer RJ, Puchalski JT. Endobronchial ultrasound-guided cautery-assisted transbronchial forceps biopsies: safety and sensitivity relative to transbronchial needle aspiration. Ann Thorac Surg. 2016;101(5):1870–6.

    Article  PubMed  Google Scholar 

  44. 44.

    Nilsen T, Hermann M, Eriksen CS, Dagfinrud H, Mowinckel P, Kjeken I. Grip force and pinch grip in an adult population: reference values and factors associated with grip force. Scand J Occup Ther. 2012;19(3):288–96.

    Article  PubMed  Google Scholar 

  45. 45.

    Rungseevijitprapa W, Bodmeier R. Injectability of biodegradable in situ forming microparticle systems (ISM). Eur J Pharm Sci. 2009;36(4–5):524–31.

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Lutolf MP. Biomaterials: spotlight on hydrogels. Nat Mater. 2009;8(6):451–3.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    E. T. Roche, C. L. Hastings, S. A. Lewin, D. E. Shvartsman, Y. Brudno, N. V Vasilyev, F. J. O’Brien, C. J. Walsh, G. P. Duffy, and D. J. Mooney, “Comparison of biomaterial delivery vehicles for improving acute retention of stem cells in the infarcted heart.,” Biomaterials, vol. 35, no. 25, pp. 6850–6858, 2014.

  48. 48.

    Aguado BA, Mulyasasmita W, Su J, Lampe KJ, Heilshorn SC. Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers. Tissue Eng Part A. 2012;18(7–8):806–15.

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    O’Brien FJ. Biomaterials & scaffolds for tissue engineering. Mater Today. 2011;14(3):88–95.

    Article  Google Scholar 

  50. 50.

    Burdick JA, Mauck RL, Gerecht S. To serve and protect: hydrogels to improve stem cell-based therapies. Cell Stem Cell. 2016;18(1):13–5.

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Tate MC, Shear DA, Hoffman SW, Stein DG, LaPlaca MC. Biocompatibility of methylcellulose-based constructs designed for intracerebral gelation following experimental traumatic brain injury. Biomaterials. 2001;22(10):1113–23.

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Xu Y, Wang C, Tam KC, Li L. Salt-assisted and salt-suppressed sol-gel transitions of methylcellulose in water. Langmuir. 2004;20(3):646–52.

    Article  PubMed  Google Scholar 

  53. 53.

    Kobayashi K, Huang C, Lodge TP. Thermoreversible gelation of aqueous methylcellulose solutions. Macromolecules. 1999;32(21):7070–7.

    CAS  Article  Google Scholar 

  54. 54.

    Chen K, Baker AN, Vyazovkin S. Concentration effect on temperature dependence of gelation rate in aqueous solutions of methylcellulose. Macromol Chem Phys. 2009;210(3–4):211–6.

    CAS  Article  Google Scholar 

  55. 55.

    Yang Y-L, Leone LM, Kaufman LJ. Elastic moduli of collagen gels can be predicted from two-dimensional confocal microscopy. Biophys J. 2009;97(7):2051–60.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Helary C, Bataille I, Abed A, Illoul C, Anglo A, Louedec L, Letourneur D, Meddahi-Pellé A, Giraud-Guille MM. Concentrated collagen hydrogels as dermal substitutes. Biomaterials. 2010;31(3):481–90.

    CAS  Article  PubMed  Google Scholar 

  57. 57.

    Friess W. Collagen--biomaterial for drug delivery. Eur J Pharm Biopharm. 1998;45(2):113–36.

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Tang Y-F, Du Y-M, Hu X-W, Shi X-W, Kennedy JF. Rheological characterisation of a novel thermosensitive chitosan/poly(vinyl alcohol) blend hydrogel. Carbohydr Polym. 2007;67(4):491–9.

    CAS  Article  Google Scholar 

  59. 59.

    Edsman K, Carlfors J, Petersson R. Rheological evaluation of poloxamer as an in situ gel for ophthalmic use. Eur J Pharm Sci. 1998;6(2):105–12.

    CAS  Article  PubMed  Google Scholar 

  60. 60.

    Nowak AP, Breedveld V, Pakstis L, Ozbas B, Pine DJ, Pochan D, et al. Rapidly recovering hydrogel scaffolds from self-assembling diblock copolypeptide amphiphiles. Nature. 2002;417(6887):424–8.

  61. 61.

    Lin H-R, Sung K. Carbopol/pluronic phase change solutions for ophthalmic drug delivery. J Control Release. 2000;69(3):379–88.

    CAS  Article  PubMed  Google Scholar 

  62. 62.

    Jeong B, Kim SW, Bae YH. Thermosensitive sol–gel reversible hydrogels. Adv Drug Deliv Rev. 2002;54(1):37–51.

    CAS  Article  PubMed  Google Scholar 

  63. 63.

    Kleinman HK, Klebe RJ, Martin GR. Role of collagenous matrices in the adhesion and growth of cells. J Cell Biol. 1981;88(3):473–85.

    CAS  Article  PubMed  Google Scholar 

  64. 64.

    Gupta D, Tator CH, Shoichet MS. Fast-gelling injectable blend of hyaluronan and methylcellulose for intrathecal, localized delivery to the injured spinal cord. Biomaterials. 2006;27(11):2370–9.

    CAS  Article  PubMed  Google Scholar 

  65. 65.

    Lee DW, Choi WS, Byun MW, Park HJ, Yu Y-M, Lee CM. Effect of gamma-irradiation on degradation of alginate. J Agric Food Chem. 2003;51:4819–23.

    CAS  Article  PubMed  Google Scholar 

  66. 66.

    Wasikiewicz JM, Yoshii F, Nagasawa N, Wach RA, Mitomo H. Degradation of chitosan and sodium alginate by gamma radiation, sonochemical and ultraviolet methods. Radiat Phys Chem. 2005;73(5):287–95.

    CAS  Article  Google Scholar 

  67. 67.

    Sanina C, Hare JM. Mesenchymal stem cells as a biological drug for heart disease: where are We with cardiac cell-based therapy? Circ Res. 2015;117(3):229–33.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

A bursary to allow for this work to be carried out was provided by the School of Pharmacy, Royal College of Surgeons in Ireland.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Helena M. Kelly.

Ethics declarations

Conflict of interests

The authors declare that they have no conflict of interest.

Additional information

This research was funded by Science Foundation Ireland (SFI) Investigator Award 13/IA/840

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Payne, C., Dolan, E.B., O’Sullivan, J. et al. A methylcellulose and collagen based temperature responsive hydrogel promotes encapsulated stem cell viability and proliferation in vitro. Drug Deliv. and Transl. Res. 7, 132–146 (2017). https://doi.org/10.1007/s13346-016-0347-2

Download citation

Keywords

  • Mesenchymal stem cells
  • Thermoresponsive
  • Hydrogel
  • Methylcellulose
  • Collagen
  • Gamma irradiation
  • Injectability