Fig. 5. HMSCs spheroid fusion, proliferation and morphology after 48 hours. (A) The fusion of spheroids at 48 hours in BTA hydrogels. (B) Proliferating hMSCs cultured in BTA formulations stained for EdU. All formulations showed EdU positive cells in cell aggregates; however, the fewest EdU positive cells per spheroids were seen in the 0 : 100 formulation. Agarose control (allow free directed self-assembly of hMSCs) showed highest number of proliferating cells (EdU positive) compared to BTA formulations. (C) Quantitative analysis of EdU positive cells per aggregate in BTA based hydrogels versus agarose. EdU⁺ cells were counted in six aggregates. (D) Largest length of an aggregate in BTA based hydrogel versus agarose; aggregates mean length value was smaller in BTA based hydrogels compared to agarose after 48 hours. (E) Mean value of an aggregate area is smaller in BTA based hydrogels compared to agarose. (F) hMSCs aggregates are more circular in agarose compared to BTA based hydrogels. n = 3 biological replicates were used for D–F and between 20–40 aggregates were randomly selected and analysed. *p < 0.05, and **p < 0.005. Statistical significant test was performed using one-way ANOVA with Tukey's post-hoc test.