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.

Characterisation of osteogenic and vascular responses of hMSCs to Ti-Co doped phosphate glass microspheres using a microfluidic perfusion platform

“…In a previous study, we characterised the release of Co 2+ ions from CoO 0% and CoO 2% microspheres over a period of 21 days. 35 Although CoO concentrations used in this study were lower than those recognised to cause cytotoxic effects on human osteoblastic cells 15,42 and mesenchymal stem cells, 16 these results suggest that the soluble divalent Co 2+ ions may still interfere with Ca 2+ and Ti 4+ signalling that usually (as in the case of CoO 0% microspheres) leads to molecular events, such as increased expression of osteogenic transcription factors and subsequent osteogenic protein. 14,43,44 The observed lack of osteogenic induction on CoO 2% PG microspheres was expected to be somewhat compensated by clear improvements in vascular responses, an important consideration for creating 3D tissues that will require perfusion.…”

“…Material characterisation as well as degradation and ions release studies for the microspheres have been previously published by our group. 35…”

“…The molar concentration of titanium was chosen based on previous studies, showing cytocompatibility and osteogenic potential of 5% mol TiO 2 doped phosphate glass microspheres. 4 , 31 As cobalt-induced cytotoxicity has been reported 32 – 34 only one molar concentration previously tested in cobalt-releasing bioactive glass scaffolds was selected, that proved to be safe from a cytotoxic perspective 35 and to promote in vitro angiogenesis. 15 , 16 The effects of soluble ionic species on hMSC and endothelial cells responses were also assessed independently of the physical cell-material interaction.…”

A parameterised mathematical model to elucidate osteoblast cell growth in a phosphate-glass microcarrier culture

“…Experimental protocols are described in detail in Peticone et al 41 and De Silva Thompson et al, 42 and briefly overviewed here.…”

“…A previous study by Guedes et al 16 found that 5 and 7 mol% TiO 2 conferred a stable degradation rate of the phosphate carriers, which motivated the use of these particular concentrations. Two non-cytotoxic CoO concentrations were also examined: 2 and 5 mol%, 41 as CoO has been found to encourage in vitro angiogenesis. Spherical phosphate glass microcarriers were manufactured following the procedure set out in Guedes et al 16 and sieved down to spheres with diameters between 63 and 106 μm.…”