Data on in vitro and in vivo cell orientation on substrates with different topographies

This data article contains data related to the research article entitled “Substrate topography: A valuable in vitro tool, but a clinical red herring for in vivo tenogenesis” [1]. We report measurements on tenocyte viability, metabolic activity and proliferation on substrates with different topographies. We also report the effect of substrates with different topographies on host cells in a subcutaneous model.

This data article contains data related to the research article entitled "Substrate topography: A valuable in vitro tool, but a clinical red herring for in vivo tenogenesis" [1]. We report measurements on tenocyte viability, metabolic activity and proliferation on substrates with different topographies. We also report the effect of substrates with different topographies on host cells in a subcutaneous model. Two-dimensional substrates, with sub-micron to low micron features, may not be suitable for directional neotissue formation in vivo.
Three-dimensional constructs may be more effective tools for directional neotissue formation in vivo.

Data
Herein, we assessed tenocyte viability, metabolic activity and proliferation on substrates with different topographies. The substrates were poly(lactic-co-glycolic acid) (PLGA) based with constant groove and line width of 1911.42 737.50 nm and 2101.78 735.21 nm respectively and variable groove depth of 37.48 73.4 nm, 317.29 77.05 nm and 1988.27195.3 nm. Non-imprinted substrates were used as control. We also assessed these these substrates in a subcutaneous model.

Human tenocyte viability, metabolic activity and proliferation
Live/Dead s assay (BioSource International, Invitrogen, Ireland) was performed on days 1, 5 and 10 to assess cellular viability, as per manufacturer's protocol. Briefly, cells were washed 3 times with HBSS and exposed to the staining solution of calcein and ethidium homodimer. The cells were incubated at 37°C for 45 min. Following staining, the cells were viewed using the BX51 Olympus fluorescence microscope and analysed using ImageJ.
Cell metabolic activity was determined using alamarBlue s assay on days 1, 5, and 10, as per manufacturer's protocol. Briefly, alamarBlue s dye was diluted with HBSS to make a 10% (v/v) ala-marBlue s solution. Media was removed from each well and 0.5 ml alamarBlue s solution was added to each well. Cell were incubated for 3 h at 37°C; the absorbance of the alamarBlue s was measured at wavelengths of 550 nm and 595 nm using a microplate reader (Varioskan Flash, Thermo Scientific, UK). The level of metabolic activity was calculated using the simplified method of calculating % reduction, according to the supplier's protocol.
Cell proliferation was assessed on days 1, 5, and 10, by counting DAPI stained cell nuclei, using the BX51 Olympus fluorescence microscope.
All experiments (viability, metabolic activity and proliferation) were repeated in three independent experiments and each experiment was performed in triplicate.

in vivo study and analysis
The Animal Care Research Ethics Committee of NUI Galway approved all experimental protocols. For the subcutaneous study, female Lewis rats (200-250 g) were used, following a protocol described previously [2]. Briefly, surgery was performed on rats under general anaesthesia. Incisions were made at the back of each animal, allowing insertion of a 0.5 cm Â 0.5 cm structured substrate. The wound was then closed, using biodegradable sutures. Following euthanisation, the substrates were harvested at days 2 and 14 and were stained using DAPI and rhodamine conjugated phalloidin. Three animals were used per time point and at each animal all three structured substrates were implanted. Images were captured with an Olympus IX-81 inverted microscope (Olympus Corporation, Tokyo, Japan).