The inorganic phase of bone contains various bioinorganic compounds, apart from calcium, phosphate and carbonate, such as zinc, copper, magnesium, sodium, strontium and many others. These elements have been shown to affect bone mineral features, such as crystallinity, mechanical properties, and degradation. Among these compounds, zinc is an essential element of various enzymes and proteins, for instance, alkaline phosphatase (ALP), lactate dehydrogenase, carbonic anhydrase, and transcription factors. Osteoinductive biomaterials triggering osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells (hMSCs), achieved by the addition of growth factors or bioinorganics, are especially important in the treatment of critical-sized bone defects. In this study, we propose the use of a novel bioactive composite with high ceramic content composed of poly(ethyleneoxide terephthalate)/poly(butylene terephthalate) (1000PEOT70PBT30, PolyActive, PA) and 50% beta-tricalcium phosphate (β-TCP) with the addition of zinc in a form of a coating of the β-TCP particles. We hypothesise that the addition of zinc to the β-TCP will result in enhanced osteogenic properties.
To investigate the effect of zinc on osteogenic differentiation of hMSCs, β-TCP particles were coated by zinc by immersing them in a zinc ion solution with a concentration of 15 or 45mM before additive manufacture of porous 3D scaffolds composed of 1000PEOT70PBT30 and β-TCP in 1:1 ratio. Before in vitro testing, 1000PEOT70PBT30- β-TCP, 1000PEOT70PBT30- β-TCP +Zn15 and 1000PEOT70PBT30- β-TCP +Zn45 composites were sterilised and seeded with hMSCs. Osteogenic properties were evaluated based on the results of DNA content, ALP activity and osteogenic gene expression on days 3, 7, 14 and 28.
In vitro assessment of the osteogenic properties of the porous 3D composite scaffolds showed similar proliferation and ALP activity of hMSCS regardless of the type of scaffolds they were cultured on.
The addition of zinc to the composite resulted in increased expression of ALP, collagen I, osteocalcin and osteopontin at day 3 of culture. Furthermore, a comparison between the two concentrations of zinc showed higher expression of collagen I and osteocalcin on scaffolds coated by immersion in a 15mM zinc ion solution and higher ALP expression in the case of 45mM.
Regarding later time points of cell culture, the expression of collagen I was the highest on composites with 45mM of zinc on both days 14 and 28. Furthermore, the expression of osteocalcin and osteopontin were increased on day 14. On day 28 of culture, the presence of both concentrations of zinc resulted in increased collagen I expression in a concertation dependent manner and downregulation of osteocalcin and osteopontin expression.
The addition of zinc ions to 3D porous additive manufactured 1000PEOT70PBT30- β-TCP composites resulted in increased expression of several osteogenic markers. Interestingly, results showed that the expression of late osteogenic markers such as osteocalcin and osteopontin was upregulated at day 3 when cells were cultured on composite scaffolds containing zinc.