Biofabrication and maturation of bone constructs is a long-term task that requires a high degree of specialization. This specialization falls onto the hierarchy complexity of the bone tissue that limits the transfer of this technology to the clinic. This work studied the effects of the short-term cryopreservation on biofabricated osteoblast-containing structures, with the final aim to make them steadily available in biobanks. The biological responses studied include the osteoblast post-thawing metabolic activity and the recovery of the osteoblastic function of 3D-bioprinted osteoblastic structures and beta tricalcium phosphate (β-TCP) scaffolds infiltrated with osteoblasts encapsulated in a hydrogel. The obtained structures were cryopreserved at -80 °C for 7 days using dimethyl sulfoxide (DMSO) as cryoprotectant additive. After thawing the structures were cultured up to 14 days. The results revealed fundamental biological aspects for the successful cryopreservation of osteoblast constructs. In summary, immature osteoblasts take longer to recover than mature osteoblasts. The pre-cryopreservation culture period had an important effect on the metabolic activity and function maintain, faster recovering normal values when cryopreserved after longer-term culture (7 days). The use of β-TCP scaffolds further improved the osteoblast survival after cryopreservation, resulting in similar levels of alkaline phosphatase activity in comparison with the non-preserved structures. These results contribute to the understanding of the biology of cryopreserved osteoblast constructs, approaching biofabrication to the clinical practice.

CEITEC Central European Institute of Technology Brno University of Technology Purkynova 123 612 00 Brno Czech Republic

Tissue Bioengineering Laboratory Faculty of Dentistry Universidad Nacional Autónoma de México Coyoacan Mexico City 04510 Mexico

Zobrazit více v PubMed

Groll J., Boland T., Blunk T., Burdick J.A., Cho D.W., Dalton P.D., Derby B., Forgacs G., Li Q., Mironov V.A., et al. Biofabrication: Reappraising the definition of an evolving field. Biofabrication. 2016;8:1–5. doi: 10.1088/1758-5090/8/1/013001. PubMed DOI

Langer R., Vacanti J.P. Tissue engineering. Science. 1993;260:920–926. doi: 10.1126/science.8493529. PubMed DOI

Marga F., Neagu A., Kosztin I., Forgacs G. Developmental biology and tissue engineering. Birth Defects Res. Part C Embryo Today Rev. 2007;81:320–328. doi: 10.1002/bdrc.20109. PubMed DOI

Gu Q., Tomaskovic-Crook E., Lozano R., Chen Y., Kapsa R.M., Zhou Q., Wallace G.G., Crook J.M. Functional 3D Neural mini-tissues from printed gel-based bioink and human neural stem cells. Adv. Healthc. Mater. 2016;5:1429–1438. doi: 10.1002/adhm.201600095. PubMed DOI

Murphy S.V., Atala A. 3D bioprinting of tissues and organs. Nat. Biotechnol. 2014;32:773–785. doi: 10.1038/nbt.2958. PubMed DOI

Tasoglu S., Demirci U. Bioprinting for stem cell research. Trends Biotechnol. 2013;31:10–19. doi: 10.1016/j.tibtech.2012.10.005. PubMed DOI PMC

Xu F., Wu J., Wang S., Durmus N.G., Gurkan U.A., Demirci U. Microengineering methods for cell-based microarrays and high-throughput drug-screening applications. Biofabrication. 2011;3:1–22. doi: 10.1088/1758-5082/3/3/034101. PubMed DOI PMC

Samavedi S., Joy N. 3D printing for the development of in vitro cancer models. Curr. Opin. Biomed. Eng. 2017;2:35–42. doi: 10.1016/j.cobme.2017.06.003. DOI

Taubenberger A.V. In vitro microenvironments to study breast cancer bone colonisation. Adv. Drug. Deliv. Rev. 2014;79:135–144. doi: 10.1016/j.addr.2014.10.014. PubMed DOI

Mir T.A., Nakamura M. Three-Dimensional Bioprinting: Toward the era of manufacturing human organs as spare parts for healthcare and medicine. Tissue Eng. Part B Rev. 2017;23:245–256. doi: 10.1089/ten.teb.2016.0398. PubMed DOI

Raman R., Bashir R. Biomimicry, biofabrication, and biohybrid systems: The emergence and evolution of biological desigin. Adv. Healthc. Mater. 2017;6:1–20. doi: 10.1002/adhm.201700496. PubMed DOI

Giannoudis P.V., Dinopoulos H., Tsiridis E. Bone substitutes: An update. Injury. 2005;36:S20–S27. doi: 10.1016/j.injury.2005.07.029. PubMed DOI

Shegarfi H., Reikeras O. Review Article: Bone transplantation and immune response. J. Orthop. Surg. 2016;17:206–211. doi: 10.1177/230949900901700218. PubMed DOI

Hunt C.J. Cryopreservation of human stem cells for clinical application: A review. Transfus. Med. Hemotherapy. 2011;38:107–123. doi: 10.1159/000326623. PubMed DOI PMC

Pogozhykh O., Prokopyuk V., Prokopyuk O., Kuleshova L., Goltsev A., Figueiredo C., Pogozhykh D. Towards biobanking technologies for natural and bioengineered multicellular placental constructs. Biomaterials. 2018;185:39–50. doi: 10.1016/j.biomaterials.2018.08.060. PubMed DOI

Kuleshova L.L., Gouk S.S., Hutmacher D.W. Vitrification as a prospect for cryopreservation of tissue-engineered constructs. Biomaterials. 2007;28:1585–1596. doi: 10.1016/j.biomaterials.2006.11.047. PubMed DOI

Dahl S.L., Chen Z., Solan A.K., Brockbank K.G., Niklason L.E., Song Y.C. Feasibility of vitrification as a storage method for tissue-engineered blood vessels. Tissue Eng. 2006;12:291–300. doi: 10.1089/ten.2006.12.291. PubMed DOI

Agudelo C.A., Iwata H. The development of alternative vitrification solutions for microencapsulated islets. Biomaterials. 2008;29:1167–1176. doi: 10.1016/j.biomaterials.2007.11.027. PubMed DOI

Malpique R., Osório L.M., Ferreira D.S., Ehrhart F., Brito C., Zimmermann H., Alves P.M. Alginate encapsulation as a novel strategy for the cryopreservation of neurospheres. Tissue Eng. Part C Methods. 2010;16:965–977. doi: 10.1089/ten.tec.2009.0660. PubMed DOI

Ahmad H.F., Sambanis A. Cryopreservation effects on recombinant myoblasts encapsulated in adhesive alginate hydrogels. Acta Biomater. 2013;9:6814–6822. doi: 10.1016/j.actbio.2013.03.002. PubMed DOI PMC

Zhao G., Liu X., Zhu K., He X. Hydrogel encapsulation facilitates rapid-cooling cryopreservation of stem cell-laden core-shell microcapsules as cell-biomaterial constructs. Adv. Healthc. Mater. 2017;6:1–26. doi: 10.1002/adhm.201700988. PubMed DOI PMC

Koebe H.G., Dunn J.C., Toner M., Sterling L.M., Hubel A., Cravalho E.G., Yarmush M.L., Tompkins R.G. A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration. Cryobiology. 1990;27:576–584. doi: 10.1016/0011-2240(90)90045-6. PubMed DOI

Umemura E., Yamada Y., Nakamura S., Ito K., Hara K., Ueda M. Viable cryopreserving tissue-engineered cell-biomaterial for cell banking therapy in an effective cryoprotectant. Tissue Eng. Part C Methods. 2011;17:799–807. doi: 10.1089/ten.tec.2011.0003. PubMed DOI

Teo K.Y., De Hoyos T.O., Dutton J.C., Grinnell F., Han B. Effects of freezing-induced cell-fluid-matrix interactions on the cells and extracellular matrix of engineered tissues. Biomaterials. 2011;32:5380–5390. doi: 10.1016/j.biomaterials.2011.04.008. PubMed DOI PMC

Chen F., Zhang W., Wu W., Jin Y., Cen L., Kretlow J.D., Gao W., Dai Z., Wang J., Zhou G., et al. Cryopreservation of tissue-engineered epithelial sheets in trehalose. Biomaterials. 2011;32:8426–8435. doi: 10.1016/j.biomaterials.2011.07.008. PubMed DOI

Kofron M.D., Opsitnick N.C., Attawia M.A., Laurencin C.T. Cryopreservation of tissue engineered constructs for bone. J. Orthop. Res. 2003;21:1005–1010. doi: 10.1016/S0736-0266(03)00103-7. PubMed DOI

Liu B.L., McGrath J. Vitrification solutions for the cryopreservation of tissue-engineered bone. Cell Preserv. Technol. 2004;2:133–143. doi: 10.1089/153834404774101972. DOI

Luo Y., Lode A., Akkineni A.R., Gelinsky M. Concentrated gelatin/alginate composites for fabrication of predesigned scaffolds with a favorable cell response by 3D plotting. RSC Adv. 2015;5:43480–43488. doi: 10.1039/C5RA04308E. DOI

Zehnder T., Sarker B., Boccaccini A.R., Detsch R. Evaluation of an alginate–gelatine crosslinked hydrogel for bioplotting. Biofabrication. 2015;7:025001. doi: 10.1088/1758-5090/7/2/025001. PubMed DOI

Duan B., Hockaday L.A., Kang K.H., Butcher J.T. 3D Bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels. J. Biomed. Mater. Res. Part A. 2013;101:1255–1264. doi: 10.1002/jbm.a.34420. PubMed DOI PMC

Xia Y., Mei F., Duan Y., Gao Y., Xiong Z., Zhang T. Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold. J. Biomed. Mater. Res. Part A. 2012;100:1044–1050. doi: 10.1002/jbm.a.33232. PubMed DOI

Stancu I.C., Dragusin D.M., Vasile E., Trusca R., Antoniac I., Vasilescu D.S. Porous calcium alginate-gelatin interpenetrated matrix and its biomineralization potential. J. Mater. Sci.: Mater. Med. 2011;22:451–460. doi: 10.1007/s10856-011-4233-7. PubMed DOI

Richards R., Czekanska E., Hayes J., Stoddart M. In search of an osteoblast cell model for in vitro research. Eur. Cells Mater. 2016;24:1–17. PubMed

Czekanska E.M., Stoddart M.J., Ralphs J.R., Richards R.G., Hayes J.S. A phenotypic comparison of osteoblast cell lines versus human primary osteoblasts for biomaterials testing. J. Biomed. Mater. Res. Part A. 2014;102:2636–2643. doi: 10.1002/jbm.a.34937. PubMed DOI

Billiet T., Gevaert E., De Schryver T., Cornelissen M., Dubruel P. 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. Biomaterials. 2014;35:49–62. doi: 10.1016/j.biomaterials.2013.09.078. PubMed DOI

Casas-Luna M., Tan H., Tkachenko S., Salamon D., Montufar E.B. Enhancement of mechanical properties of 3D-plotted tricalcium phosphate scaffolds by rapid sintering. J. Eur. Cer. Soc. 2019;39:4366–4374. doi: 10.1016/j.jeurceramsoc.2019.05.055. DOI

Freshney R.I. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. 6th ed. John Wiley & Sons, Inc.; New York, NY, USA: 2010. Cryopreservation; pp. 317–334.

Takeo T., Kondo T., Haruguchi Y., Fukumoto K., Nakagawa Y., Takeshita Y., Nakamuta Y., Tsuchiyama S., Shimizu N., Hasegawa T., et al. Short-term storage and transport at cold temperatures of 2-cell mouse embryos produced by cryopreserved sperm. J Am. Assoc. Lab. Anim. Sci. 2010;49:415–419. PubMed PMC

Mazur P., Leibo S.P., Chu E.H. A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. Exp. Cell. Res. 1972;71:345–355. doi: 10.1016/0014-4827(72)90303-5. PubMed DOI

Karlsson J.O., Toner M. Long-term storage of tissues by cryopreservation: Critical issues. Biomaterials. 1996;17:243–256. doi: 10.1016/0142-9612(96)85562-1. PubMed DOI

Whittingham D.G., Leibo S.P., Mazur P. Survival of Mouse Embryos Frozen to −196° and −269 °C. Science. 1972;178:411–414. doi: 10.1126/science.178.4059.411. PubMed DOI

Ji L., De Pablo L.L., Palecek S.P. Cryopreservation of adherent human embryonic stem cells. Biotechnol. Bioeng. 2004;88:299–312. doi: 10.1002/bit.20243. PubMed DOI

Goh B.C., Thirumala S., Kilroy G., Devireddy R.V., Gimble J.M. Cryopreservation characteristics of adipose-derived stem cells: Maintenance of differentiation potential and viability. J. Tissue Eng. Regen. Med. 2012;1:322–324. doi: 10.1002/term.35. PubMed DOI

Carroll J., Wood M.J., Whittingham D.G. Normal fertilization and development of frozen-thawed Mouse Oocytes: Protective action of certain macromolecules. Biol. Reprod. 1993;48:606–612. doi: 10.1095/biolreprod48.3.606. PubMed DOI

Sambu S., Xu X., Schiffter H.A., Cui Z.F., Ye H. RGDS-Fuctionalized alginates improve the survival rate of encapsulated embryonic stem cells during cryopreservation. Cryo-Letters. 2011;32:389–401. PubMed

Miranda P., Saiz E., Gryn K., Tomsia A.P. Sintering and robocasting of beta-tricalcium phosphate scaffolds for orthopaedic applications. Acta Biomater. 2006;2:457–466. doi: 10.1016/j.actbio.2006.02.004. PubMed DOI

Varghese S., Chien S., Gianneschi N.C., Kang H., Vecchio K.S., Caro E.J., Phadke A., Theodorakis E.A., Siu M., Hwang Y., et al. Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling. Proc. Natl. Acad. Sci. USA. 2014;111:990–995. PubMed PMC

Lee M.N., Hwang H.S., Oh S.O., Roshanzadeh A., Kim J.W., Song J.H., Kim E.S., Koh J.T. Elevated extracellular calcium ions promote proliferation and migration of mesenchymal stem cells via increasing osteopontin expression. Exp. Mol. Med. 2018;50:1–16. doi: 10.1038/s12276-018-0170-6. PubMed DOI PMC

Wang P., Zhao L., Liu J., Zhou X., Weir M.D. Bone tissue engineering via nanostructured calcium phosphate biomaterials and stem cells. Bone Res. 2014;2:1–13. doi: 10.1038/boneres.2014.17. PubMed DOI PMC

Pautke C., Schieker M., Tischer T., Kolk A., Neth P., Mutscher W., Milz S. Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2OS in comparison to human osteoblasts. Anticancer Res. 2004;24:3743–3748. PubMed

Park S., Nam J.S., Lee D.R., Kim H., Ahn C.W. Fetal bovine serum-free cryopreservation methods for clinical banking of human adipose-derived stem cells. Cryobiology. 2018;81:65–73. doi: 10.1016/j.cryobiol.2018.02.008. PubMed DOI

Liang X., Hu X., Hu Y., Zeng W., Zeng G., Ren Y., Liu Y., Chen K., Peng H., Ding H., et al. Recovery and functionality of cryopreserved peripheral blood mononuclear cells using five different xeno-free cryoprotective solutions. Cryobiology. 2019;86:25–32. doi: 10.1016/j.cryobiol.2019.01.004. PubMed DOI

Adaptability of Electrospun PVDF Nanofibers in Bone Tissue Engineering