13. Pressure needed to extrude cell-laden 3-3-3 paste may vary
according to the bioprinter used. A test with acellular printing
of 3-3-3 is recommended before using the cell-laden paste.
14. Cell viability, proliferation, and functionality studies typically
need a large amount of constructs. Stem cell-laden 3-3-3 can
be printed as a high-throughput process.
15. After crosslinking solution removal, a single wash with complete cell culture media can be performed to remove excess
CaCl 2 solution. Use enough cell culture media to completely
cover the entirely cell-laden scaffold.
Acknowledgments
The authors would like to thank Prof. Michael Gelinsky
(TU Dresden) for useful discussions and fruitful collaborations
over the last 3 years, Prof. Shoufeng Yang (KU Leuven) for discussions and access to the extrusion bioprinter, and Dr. Stuart Lanham
for useful discussions on methods. This work was supported by
grants from the Biotechnology and Biological Sciences Research
Council UK (BB/ L00609X and BB/LO21072/1) and University
of Southampton IfLS, FortisNet and Postgraduate awards
to ROCO.
References
1. Carretero MI, Pozo M (2009) Clay and
non-clay minerals in the pharmaceutical industry. Part I. Excipients and medical applications.
Appl Clay Sci 46:73–80
2. Carretero MI, Pozo M (2010) Clay and
non-clay minerals in the pharmaceutical and
cosmetic industries Part II. Active ingredients.
Appl Clay Sci 47:171–181
3. Dawson JI, Oreffo ROC (2013) Clay: New
opportunities for tissue regeneration and biomaterial design. Adv Mater 25:4069–4086
4. Ruzicka B, Zaccarelli E (2011) A fresh look at
the Laponite phase diagram. Soft Matter
7:1268–1286
5. Kroon M, Vos WL, Wegdam GH (1998) Structure and formation of a gel of colloidal disks.
Int J Thermophys 19:887–894
6. Abou B, Bonn D, Meunier J (2001) Aging
dynamics in a colloidal glass. Phys Rev E Stat
Phys Plasmas Fluids Relat Interdiscip Topics
64:6
7. Pignon F, Magnin A, Piau JM (1998) Thixotropic behavior of clay dispersions: combinations of scattering and rheometric techniques.
J Rheol 42:1349–1373
8. Dawson JI, Kanczler JM, Yang XB et al (2011)
Clay gels for the delivery of regenerative microenvironments. Adv Mater 23:3304–3308
9. Carrow JK, Cross LM, Reese RW et al (2018)
Widespread changes in transcriptome profile of
human mesenchymal stem cells induced by
two-dimensional nanosilicates. Proc Natl Acad
Sci U S A 115(17):E3905–E3913
10. Ho ¨lzl K, Lin S, Tytgat L et al (2016) Bioink
properties before, during and after 3D bioprinting. Biofabrication 8:032002
11. Gibbs DMR, Black CRM, Hulsart-Billstrom G
et al (2016) Bone induction at physiological
doses of BMP through localization by clay
nanoparticle gels. Biomaterials 99:16–23
12. Liu X, Bhatia SR (2015) Laponite® and
Laponite®-PEO hydrogels with enhanced elasticity in phosphate-buffered saline. Polym Adv
Technol 26:874–879
13. Viseras C, Aguzzi C, Cerezo P et al (2008)
Biopolymer–clay nanocomposites for controlled drug delivery. Mater Sci Technol
24:1020–1026
14. Gaharwar AK, Schexnailder PJ, Kline BP et al
(2011) Assessment of using Laponite crossClay Bioinks for Skeletal Regeneration
71
according to the bioprinter used. A test with acellular printing
of 3-3-3 is recommended before using the cell-laden paste.
14. Cell viability, proliferation, and functionality studies typically
need a large amount of constructs. Stem cell-laden 3-3-3 can
be printed as a high-throughput process.
15. After crosslinking solution removal, a single wash with complete cell culture media can be performed to remove excess
CaCl 2 solution. Use enough cell culture media to completely
cover the entirely cell-laden scaffold.
Acknowledgments
The authors would like to thank Prof. Michael Gelinsky
(TU Dresden) for useful discussions and fruitful collaborations
over the last 3 years, Prof. Shoufeng Yang (KU Leuven) for discussions and access to the extrusion bioprinter, and Dr. Stuart Lanham
for useful discussions on methods. This work was supported by
grants from the Biotechnology and Biological Sciences Research
Council UK (BB/ L00609X and BB/LO21072/1) and University
of Southampton IfLS, FortisNet and Postgraduate awards
to ROCO.
References
1. Carretero MI, Pozo M (2009) Clay and
non-clay minerals in the pharmaceutical industry. Part I. Excipients and medical applications.
Appl Clay Sci 46:73–80
2. Carretero MI, Pozo M (2010) Clay and
non-clay minerals in the pharmaceutical and
cosmetic industries Part II. Active ingredients.
Appl Clay Sci 47:171–181
3. Dawson JI, Oreffo ROC (2013) Clay: New
opportunities for tissue regeneration and biomaterial design. Adv Mater 25:4069–4086
4. Ruzicka B, Zaccarelli E (2011) A fresh look at
the Laponite phase diagram. Soft Matter
7:1268–1286
5. Kroon M, Vos WL, Wegdam GH (1998) Structure and formation of a gel of colloidal disks.
Int J Thermophys 19:887–894
6. Abou B, Bonn D, Meunier J (2001) Aging
dynamics in a colloidal glass. Phys Rev E Stat
Phys Plasmas Fluids Relat Interdiscip Topics
64:6
7. Pignon F, Magnin A, Piau JM (1998) Thixotropic behavior of clay dispersions: combinations of scattering and rheometric techniques.
J Rheol 42:1349–1373
8. Dawson JI, Kanczler JM, Yang XB et al (2011)
Clay gels for the delivery of regenerative microenvironments. Adv Mater 23:3304–3308
9. Carrow JK, Cross LM, Reese RW et al (2018)
Widespread changes in transcriptome profile of
human mesenchymal stem cells induced by
two-dimensional nanosilicates. Proc Natl Acad
Sci U S A 115(17):E3905–E3913
10. Ho ¨lzl K, Lin S, Tytgat L et al (2016) Bioink
properties before, during and after 3D bioprinting. Biofabrication 8:032002
11. Gibbs DMR, Black CRM, Hulsart-Billstrom G
et al (2016) Bone induction at physiological
doses of BMP through localization by clay
nanoparticle gels. Biomaterials 99:16–23
12. Liu X, Bhatia SR (2015) Laponite® and
Laponite®-PEO hydrogels with enhanced elasticity in phosphate-buffered saline. Polym Adv
Technol 26:874–879
13. Viseras C, Aguzzi C, Cerezo P et al (2008)
Biopolymer–clay nanocomposites for controlled drug delivery. Mater Sci Technol
24:1020–1026
14. Gaharwar AK, Schexnailder PJ, Kline BP et al
(2011) Assessment of using Laponite crossClay Bioinks for Skeletal Regeneration
71