Towards modular bone tissue engineering using Ti–Co-doped phosphate glass microspheres: cytocompatibility and dynamic culture studies

Abstract

The production of large quantities of functional vascularized bone tissue ex vivo still represent an unmet clinical challenge. Microcarriers offer a potential solution to scalable manufacture of bone tissue due to their high surface area-to-volume ratio and the capacity to be assembled using a modular approach. Microcarriers made of phosphate bioactive glass doped with titanium dioxide have been previously shown to enhance proliferation of osteoblast progenitors and maturation towards functional osteoblasts. Furthemore, doping with cobalt appears to mimic hypoxic conditions that have a key role in promoting angiogenesis. This characteristic could be exploited to meet the clinical requirement of producing vascularized units of bone tissue. In the current study, the human osteosarcoma cell line MG-63 was cultured on phosphate glass microspheres doped with 5% mol titanium dioxide and different concentrations of cobalt oxide (0%, 2% and 5% mol), under static and dynamic conditions (150 and 300 rpm on an orbital shaker). Cell proliferation and the formation of aggregates of cells and microspheres were observed over a period of two weeks in all glass compositions, thus confirming the biocompatibility of the substrate and the suitability of this system for the formation of compact micro-units of tissue. At the concentrations tested, cobalt was not found to be cytotoxic and did not alter cell metabolism. On the other hand, the dynamic environment played a key role, with moderate agitation having a positive effect on cell proliferation while higher agitation resulting in impaired cell growth. Finally, in static culture assays, the capacity of cobalt doping to induce vascular endothelial growth factor (VEGF) upregulation by osteoblastic cells was observed, but was not found to increase linearly with cobalt oxide content. In conclusion, Ti–Co phosphate glasses were found to support osteoblastic cell growth and aggregate formation that is a necessary precursor to tissue formation and the upregaulation of VEGF production can potentially support vascularization.

Keywords

Tissue engineering phosphate glass titanium cell culture bioprocessing biomaterials

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References

Mei

et al.

Modulating and modeling aggregation of cell-seeded microcarriers in stirred culture system for macrotissue engineering. J Biotechnol 2010; 150: 438–446.

Nichol

Khademhosseini

. Modular tissue engineering: engineering biological tissues from the bottom up. Soft Matter 2010; 5: 1312–1319.

Liu

Gartner

. Directing the assembly of spatially organized multicomponent tissues from the bottom up. Trends Cell Biol 2012; 22: 683–691.

Hong

Kim

. Preparation of porous bioactive ceramic microspheres and in vitro osteoblastic culturing for tissue engineering application. Acta Biomater 2009; 5: 1725–1731.

Chan

Hui

Wong

et al.

Mesenchymal stem cell-encapsulated collagen microspheres for bone tissue engineering. Tissue Eng Part C 2010; 16: 225–235.

Lakhkar

et al.

Titanium phosphate glass microspheres for bone tissue engineering. Acta Biomater 2012; 8: 4181–4190.

Perez

Riccardi

Altankov et al.

Dynamic cell culture on calcium phosphate microcarriers for bone tissue engineering applications. J Tissue Eng 2014; 5: 1–10.

Martin

Eldardiri

Lawrence-Watt

et al.

Microcarriers and their potential in tissue regeneration. Tissue Eng Part B 2011; 17: 71–80.

Park

J-H

et al.

Microcarriers designed for cell culture and tissue engineering of bone. Tissue Eng Part B 2013; 19: 172–190.

10.

Knowles

. Phosphate based glasses for biomedical applications. J Mater Chem 2003; 13: 2395–2395.

11.

Lakhkar

et al.

Bone formation controlled by biologically relevant inorganic ions: role and controlled delivery from phosphate-based glasses. Adv Drug Delivery Rev 2013; 65: 405–420.

12.

Lee

I-H

et al.

Development, characterisation and biocompatibility testing of a cobalt-containing titanium phosphate-based glass for engineering of vascularized hard tissues. Mater Sci Eng C 2013; 33: 2104–2112.

13.

et al.

Hypoxia-mimicking mesoporous bioactive glass scaffolds with controllable cobalt ion release for bone tissue engineering. Biomaterials 2012; 33: 2076–2085.

14.

Azevedo

et al.

Hypoxia inducible factor-stabilizing bioactive glasses for directing mesenchymal stem cell behavior. Tissue Eng Part A 2015; 21: 382–389.

15.

Guedes

et al.

TiO(2)-doped phosphate glass microcarriers: a stable bioactive substrate for expansion of adherent mammalian cells. J Biomater Appl 2013; 28: 3–11.

16.

Catelas

Petit

Zukor

et al.

Cytotoxic and apoptotic effects of cobalt and chromium ions on J774 macrophages – implication of caspase-3 in the apoptotic pathway. J Mater Sci Mater Med 2001; 12: 949–953.

17.

Fleury

et al.

Effect of cobalt and chromium ions on human MG-63 osteoblasts in vitro: morphology, cytotoxicity, and oxidative stress. Biomaterials 2006; 27: 3351–3360.

18.

Simonsen

Harbak

Bennekou

. Cobalt metabolism and toxicology – a brief update. Sci Total Environ 2012; 432: 210–215.

19.

Clover

Gowen

. Are MG-63 and HOS TE85 human osteosarcoma cell lines representative models of the osteoblastic phenotype? Bone 1994; 15: 585–591.

20.

Zhang

Williams-Dalson

Keshavarz-Moore

et al.

Computational-fluid-dynamics (CFD) analysis of mixing and gas-liquid mass transfer in shake flasks. Biotechnol Appl Biochem 2005; 41: 1–8.

21.

Zhang

Lamping

Pickering

SCR

et al.

Engineering characterisation of a single well from 24-well and 96-well microtitre plates. Biochem Eng J 2008; 40: 138–149.

22.

Zhang

et al.

Use of orbital shaken disposable bioreactors for mammalian cell cultures from the milliliter-scale to the 1,000-liter scale. Adv Biochem Eng Biotechnol 2010; 115: 33–53.

23.

Discacciati

et al.

Numerical simulation of orbitally shaken viscous fluids with free surface. Int J Numer Methods Fluids 2013; 71: 294–315.

24.

Klöckner

Büchs

. Advances in shaking technologies. Trends Biotechnol 2012; 30: 307–314.

25.

Weheliye

Yianneskis

Ducci

. On the fluid dynamics of shaken bioreactors-flow characterization and transition. AIChE J 2013; 59: 334–344.

26.

Pieralisi

Rodriguez

Micheletti

et al.

Microcarriers’ suspension and flow dynamics in orbitally shaken bioreactors. Chem Eng Res Des 2016; 108: 198–209.

27.

Abou Neel

et al.

Structure and properties of strontium-doped phosphate-based glasses. J R Soc Interface 2009; 6: 435–446.

28.

Lakhkar

. Phosphate glass microspheres as cell microcarrier substrates for bone tissue engineering applications, London: University College London, 2014.

29.

Brauer

Phosphate glasses. In: Jones

Clare

(eds). Bio-glasses: an introduction, John Wiley & Sons Ltd., 2012.

30.

Abou Neel

et al.

In vitro bioactivity and gene expression by cells cultured on titanium dioxide doped phosphate-based glasses. Biomaterials 2007; 28: 2967–2977.

31.

Abou Neel

Knowles

. Physical and biocompatibility studies of novel titanium dioxide doped phosphate-based glasses for bone tissue engineering applications. J Mater Sci Mater Med 2008; 19: 377–386.

32.

Wall

Donos

Carlqvist

et al.

Modified titanium surfaces promote accelerated osteogenic differentiation of mesenchymal stromal cells in vitro. Bone 2009; 45: 17–26.

33.

Kiani

et al.

Titanium-containing bioactive phosphate glasses. Philos Trans R Soc A Math Phys Eng Sci 2012, pp. 370: 1352–1375.

34.

Quinlan

et al.

Hypoxia-mimicking bioactive glass/collagen glycosaminoglycan composite scaffolds to enhance angiogenesis and bone repair. Biomaterials 2015; 52: 358–366.

35.

Navarro

et al.

Physicochemical degradation of titania-stabilized soluble phosphate glasses for medical applications. J Am Ceram Soc 2003; 86: 1345–1352.

36.

Bose

Fielding

Tarafder

et al.

Trace element doping in calcium phosphate ceramics to understand osteogenesis and angiogenesis. Trends Biotechnol 2013; 18: 1199–1216.

37.

Mitra

Tripathi

Sharma

et al.

Scaffolds for bone tissue engineering: role of surface patterning on osteoblast response. RSC Adv 2013; 3: 11073–11073.

38.

Brett

et al.

Roughness response genes in osteoblasts. Bone 2004; 35: 124–133.

39.

Lee

et al.

Tethering bi-functional protein onto mineralized polymer scaffolds to regulate mesenchymal stem cell behaviors for bone regeneration. J Mater Chem B 2013; 1: 2731–2731.

40.

Yuan

Hilliard

Ferguson

et al.

Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem 2003; 278: 15911–15916.

41.

Lum

et al.

The transcription factor HIF-1alpha plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis. Genes Dev 2007; 21: 1037–1049.

42.

Hsu

S-H

Chen

C-T

Wei

Y-H

. Inhibitory effects of hypoxia on metabolic switch and osteogenic differentiation of human mesenchymal stem cells. Stem Cells 2013; 31: 2779–2788.

43.

Wagner

Metabolic control of animal cell culture processes. In: Hauser

Wagner

(eds). Mammalian cell biotechnology in protein production, Germany: Walter de Gruyter, 1997.

44.

Higuera

et al.

Patterns of amino acid metabolism by proliferating human mesenchymal stem cells. Tissue Eng Part A 2012; 18: 654–664.

45.

Weheliye

Yianneskis

Ducci

. On the fluid dynamics of shaken bioreactors – flow characterization and transition. AIChE Journal 2012; 59: 1–11.