2.6.1. Alamarblue™ Assay

Duplicate samples were placed in 24 well plates (Nunc, Warrington, UK) and UV sterilised. Tissue Culture Plastic (TCP) was used as a control surface. Human Osteoblasts (HOBs) from bone chips of femoral heads of patients undergoing total hip arthroplasty were seeded into wells at a density of 40,000 cells cm<sup>−</sup><sup>2</sup> and incubated at 37 ◦C and 5% CO2. Media (Dulbecco/Vogt Modified Eagle's Minimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum (Gibco Life Technologies, Inchinnan, Scotland), 1% L-Glutamine, 2% HEPES Buffer, 1% non-essential amino acids, 2% penicillin and streptomycin (Invitrogen, Rugby, UK) and 75 mg ascorbic acid (Sigma, UK)) were replenished every two days. At each respective time point (1, 4, 7, 10 and 14 days) the media were removed and samples were washed three times in phosphate-buffered saline (PBS) solution. Then, a 1 mL dilution of AlamarBlue™ (Serotec, Kidlington, UK) and Hank's balanced salt solution (HBSS) (Gibco, Inchinnan, Scotland) in the ratio of 1:10 was added to each well, including unseeded TCP wells, and incubated for 80 min. The well plates were subsequently wrapped in foil and shaken at 300 rpm on a Heidolph Titramax 100 for 10 min in a dark environment. The AlamarBlue™ solution was then removed and 100 μm aliquots were transferred into a 96 well plate (Nunc, Warrington, UK). Fluorescence was measured using a Bio Tek Instruments FLx800 fluorescence plate reader (Swindon, UK) at 560 nm excitation and 590 nm emission filters. Unreduced AlamarBlue™ was subtracted from recorded values to remove background signal. The experiments were repeated twice.

### 2.6.2. Alkaline Phosphatase Assay

After each designated time point, media were removed from culture plates and washed three times in PBS solution. Aliquots of 1 mL of sterile distilled water were added to each well. A freeze/thaw method was employed to lyse the cells. The samples were frozen at −20 ◦C and then allowed to defrost at room temperature. This was repeated three times. 50 μL of lysate solution was added to a 96 well plate per sample which was mixed with 50 μL of <sup>4</sup>−nitrophenylphosphate (Sigma, UK) mixed with an appropriate quantity of diethanolamine buffer solution. Plates were shaken at 300 RPM for 1 min in a dark environment. The luminescence was then measured using a Bio Tek ELx800 luminescence plate reader with a primary wavelength of 405 nm and a reference wavelength of 630 nm.

### 2.6.3. DNA Hoechst Staining Assay

At each time point media were removed and HOBs were washed within PBS three times and submerged in 1 mL of sterile distilled water. A freeze/thaw cycle was carried out in triplicate to lyse HOB cell walls. Then, 100 μL of lysate was mixed with 100 μL of Hoechst 33,258 stain (Sigma, UK) and shaken at 300 RPM for 1 min in a dark environment. Fluorescence was then read on a Bio Tek Instruments FLx800 fluorescence plate reader with 360 nm excitation and 460 nm emission filters.

### 2.6.4. SEM Sample Preparation

At selected time points, media were removed and samples were washed with PBS thrice and replaced with 3% Glutaldehyde in 0.1 M sodium cacodylate buffer. This was replaced after 30 min with 7% sucrose solution in 0.1 M sodium cacodylate buffer. Specimens were then washed three times for 5 min periods with 0.1 M cacodylate buffer solution and then immersed in osmium tetraoxide for 1 h. Post fixing, cells were dehydrated using an ethanol/distilled water gradient (20% × 2 min, 40% × 5 min, 60% × 5 min, 80% × 5 min 90% × 5 min and 100% × 5 min × 2). Specimens were then submerged in hexamethyldisilazane (HMDS; Sigma, UK) for 5 min. This was then replaced with fresh HMDS and left to dry overnight. The samples were mounted on aluminium stubs with carbon adhesive tabs and gold/palladium coated (ca. 5 nm).
