Liquidación presupuestaria del ejercicio

3.4.1. Cell Culture and Maintenance

Human Osteoblast Cells (HOBs), from bone chips of the femoral heads of patients undergoing total hip arthoplasty were used in this study [238]. HOBs were cultured on tissue culture plastic (TCP) in 500 ml Dulbecco/Vogt Modified Eagle's Minimal Essential Medium (DMEM) supplemented with 10 % fetal bovine serum, 1 % L7Glutamine, 2% HEPES Buffer, 1 % non7essential amino acids, 2 % penicillin and streptomycin (Invitrogen, UK) and 75 mg ascorbic acid (Sigma, UK) and maintained at 37 °C and 5 % CO2. The medium was changed every two days.

Once confluent media was discarded, the cells were washed in phosphate buffer saline (PBS) and trypsanised for 4 mins at 37 °C with using 0.02 M bovine trypsin and scraped to remove from the flask base. An aliquot of 10 ml of fresh media was then added to the flask and the cells and media were transferred to a 20 ml universal and centrifuged in a Jouan CR422 centrifudge for 5 minutes at 1200 rpm creating approximately 2.4 g. The supernatant was then removed and the cell pellet was resuspended in 1 ml of fresh media. Cell number was assessed using a trypan blue exclusion stain which stains non7viable cells. An aliquot of 50 Ol of trypan blue was mixed with 50 Ol of cell suspension, applied to a haemocytometer and placed under a Nikon Eclipse TS100 microscope. A viable cell count was the calculated for the total number of cells. Appropriate adjustments were made to seed cells at the required cell density. Finally, 1 ml of media was used per well and plates were incubated at 37 °C for a given time period before an assay was applied.

3.4.2. Sample Sterilisation

Sterilisation was achieved by irradiating in uv light (λ = 320 7 280 nm) for 90 mins per sample side.

3.4.3. Elution Testing

Elution testing was performed to establish if selected samples were toxic or non7 toxic. Samples were immersed in media for 48 h. After 24 h HOBs were seeded on to Thermanox slides at a cell density of 40,000 cells cm72 _{in fresh media and}

left for one day. After a total of 48 h the media was changed for the media that had been on the samples. Metabolic activity of HOB cells was assessed by Alamarblue™ and DNA Hoechst staining 24 h after seeding. Cells were fixed and their morphology was viewed using SEM.

3.4.4. Initial Attachment

Samples were placed in 24 well plates (Nunc, UK) with one sample per well and 1 ml of media was added to each well. Tests were run in triplicate and repeated. Cells were seeded into wells at a cell density of 10,000 cells cm72_{. Plates were}

incubated for 90 minutes at 37 °C and 5 vol.% CO2. After this period media was

removed and the samples were washed in PBS three times and fixed in 4 wt.% paraformaldehyde for 10 min. Samples were washed again and then permealised for 5 min at 720 °C and washed. 0.1 wt.% propidium iodide was added for 172 s before washing and mounting samples on glass slides. A PBS solution containing 2 mg/ml of 1,47diazabicyclo[2.2.2]octane (DABCO) was prepared at pH 8.6 and combined with glycerol in a 9:1 ratio of glycerol:DABCO/PBS. This solution and a coverslip were then applied for imaging. A Lecia DMLB optical microscope with a mercury lamp used in fluorescence mode with a BG38 filter at x10 magnification with a Nikon DXM1200 digital camera was used to image 25 random visual fields per sample. Cell counting was performed manually.

3.4.5. Metabolic Activity Assay

An Alamarblue™ assay was undertaken to assess the metabolic activity of HOBs over a 14 day time period. When added to cell cultures, the oxidised form of the Alamarblue™ (tetrazolium dye) enters the cytoplasm and is converted to the reduced form proportionally to cell metabolism by mitochondrial enzyme activity by accepting electrons from NADPH, FADH, FMNH, NADH as well as from the cytochromes [239]. This redox reaction is accompanied by a shift in colour of the culture medium from indigo blue to fluorescent pink which can be read on a fluorescence plate reader.

Duplicate samples were placed in 24 well plates and sterilised. TCP was used as a control surface. HOBs were seeded into wells at a density of 40,000 cells cm72 _and

incubated at 37 °C and 5 vol.% CO2. Media were changed every two days. At

each respective time point (1, 4, 7, 10 and 14 days) the media was removed and samples were washed three times in PBS solution. 1 ml of a dilution of Alamarblue™ (Serotec, UK) and Hank’s balanced salt solution (HBSS) (Gibco, UK) in the ratio of 1:10 was added to each well, including unseeded TCP wells and incubated for 80 minutes. Well plates were subsequently wrapped in foil and shaken at 300 rpm on a Heidolph Titramax 100 for 10 minutes in a dark environment. The Alamarblue™ solution was then removed and 100 Om from each well was aliquoted into a 96 well plate. Fluorescence was read using a Bio Tek Instruments FLx800 fluorscence plate reader using 560 nm excitation and 590 nm emission filters. Unreduced Alamarblue™ was subtracted from recorded values to remove background signal. Experiments were repeated twice.

3.4.6. Alkaline Phosphatase Assay

they begin to enter the differentiation phase of the cell cycle. A substrate, 47 nitrophenlyphosphate mixed with diethanolamine buffer solution, was supplied with a Randox ALP alkaline phosphatase kit (Randox laboratories, UK) and mixed accordingly. This reacts with water in a dephosphorylation enzyme catalysed reaction:

Eqn 3.9. 47nitrophenlyphosphate + H20 phosphate + 47nitrophenolate

The production rate of 47nitrophenolate is then determined photometrically which is directly proportional to ALP activity of cells on the sample. Photometry is the measurement of light. The radiant power of a wavelength of light is measured giving a relative intensity.

After each designated time point media was 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 ensure lyses of cells. Samples were frozen at 720 °C and then allowed to defrost at room temperature. This was repeated three times. Aliquots of 50 Ol of solution were added to a 96 well plate per sample which was mixed with 50 Ol of 47nitrophenylphosphate mixed with an appropriate quantity of diethanolamine buffer solution. Plates were shaken at 300 rpm on a Heidolph Titramax 100 for 1 min in a dark environment. The eluminescence was measured using a Bio Tek ELx800 eluminescence plate reader with a primary wavelength of 405 nm and a reference wavelength of 630 nm.

3.4.7. DNA Hoechst Staining Assay

Cell proliferation can be determined by assaying the total DNA content at time points of 1, 4, 7, 10 and 14 days. DNA content is calculated by using standards based solutions of known DNA content.

At each time point media was removed and HOBs were washed in 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. 100 Ol of lysate was mixed with 100 Ol of Hoechst 33258 stain (Sigma, Dorset, UK) and shaken at 300 rpm on a Heidolph Titramax 100 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.

3.4.8. Scanning Electron Microscope Preparation

At selected time points media was removed and samples were washed three times in PBS and replaced with 3 wt.% glutaraldehyde in 0.1 M sodium cacodylate buffer. After 30 min this was replaced with 7 wt.% sucrose solution in 0.1 M sodium cacodylate buffer. Specimens were then washed three times for 5 min periods in 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 vol.% x 2 min, 40 vol.% x 5 min, 60 vol.% x 5 min, 80 vol.% x 5 min 90 vol.% x 5 min and 100 vol.% x 5 min x 2). Specimens were then submerged in hexamethyldisilazane (HMDS) for 5 mins. This was then replaced with fresh HMDS and left to dry overnight.

Samples were mounted on aluminum stubs with carbon adhesive tabs and gold/palladium coated for 2 min in argon using an Emscope SC500 sputtering

device, giving a gold thickness of approximately 5 nm. Samples were viewed on a Philips XL730 electron microscope as described in section 3.3.10.

3.4.9. Statistical Analysis

Statistical analysis was carried out using Prism® version 4.5.1 (Graphpad, California). Mean values, standard deviations (SD) and standard error of the mean (SE) were calculated using 3 replicates per time point. One way and two way analysis of the mean (ANOVA), with significance set at the 95 % confidence interval followed by a Tukey post test was used to measure significant differences between the control TCP, HA and SiHA surfaces.