The aim of this project was to evaluate the effects of zinc in osteogenic media and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Zinc has a stimulatory effect on bone formation and mineralization in vivo and vitro (Seo et al. 2010 Nutr. Res. Pract. 4, 356–361). Our hypothesis was that the presence of zinc in the osteogenic media would positively influence both the speed of formation and the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided in 8 different treatments: 6 different concentrations of zinc in the osteogenic medium (8, 4, 0.8, 0.4, 0.08, and 0.04 mm) plus 2 control treatments (osteogenic medium without zinc and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 4 times. At the end of the culture period, cells were stained with Alizarin Red S. In each well, we counted the nodules and divided them in 2 categories: formed and forming nodules. The second evaluation that we did was to evaluate the diameter of the largest nodules (2/well) in each group. Data were analysed by ANOVA using the Generalized Linear Model procedure (SPSS, IBM Corp., Armonk, NY, USA). Bonferroni's post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results showed that the doses of zinc of both 4 and 8 mm were toxic to the whole cell populations in this treatment, which was indicated by cell death, whereas the concentrations of 0.8 and 0.4 mm were not cytotoxic but no nodules formed. Here we report the results that are greater than zero in Table 1. There is a positive effect on nodule formation when the zinc is added to the media. It is clear that the total number of nodules is different between the 0.08 mm zinc group and the control (P < 0.003). When we evaluated nodule diameter we found a direct correlation between the zinc concentration and the diameter of the nodules: 292.7 (±136.6) v. 366.8 (±218.7) v. 423.7 (±267.7) µm for the control, 0.04 mm zinc, and 0.08 mm zinc, respectively. The largest nodule was found in the 0.08 mm zinc treatment at 886.6 µm. These results confirmed the positive effect of this mineral on bone formation. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with zinc incorporated into their matrix. Table 1. The average number (SD in parentheses) of formed and forming osteoblast nodules compared between treatment groups

Group	Formed nodules	Forming nodules	Total nodules
0.08 mm zinc	10.3 (3.1)a	19.3 (4.2)A	29.7 (6.4)A,a
0.04 mm zinc	9.3 (2.9)b	7.3 (1.5)B	16.7 (3.5)b
Standard osteogenic medium	3 (0)c	5.3 (1.1)B	8.3 (1.1)B,b

^ABLeast square means within each column without common superscripts differ significantly (P < 0.01) ^abcLeast square means within each column without common superscripts differ significantly (P < 0.05)