Customised Fixation Plate Design

The merged model prepared in 3.2.1 was smoothed and exported as the non- uniform rational basis spline (NURBS) model, which is a mathematical model that describes curves and surfaces [177]. Modelling of a patient’s pelvis and fixation plates was performed in a CAD system. SolidWorks (Dassault Systemes S.A., French) was used to recognize the NURBS model and extract the surface to reconstruct the fixation plate mode. Curves extracted from the NURBS model were connected to construct the shape of a plate, and then the plate surface was thickened to form a 3D model. Screw holes were subsequently added at a position and angle that the screw can go through the cortical bone and away from the fracture line. The plate model was further exported as a .stl file for the 3D printing process.

3.2.4 3D Printing

Slicing

The .stl model prepared in 2.2.2 is not a recognizable file for a 3D printer. Before sending the data for 3D printing, the model needs to be sliced according to an algorithm to generate a file to guide the 3D printer to manufacture an item. The

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Figure 3-2 Symmetrical analysis, (a) segment of hemipelves of a non-fractured patient, (b) Mirror and superimpose hemipelves models, (c) DCM, and (d) filtered DCM that highlight areas that have deviation greater than 4 mm.

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a) Brim. The fringe of the model at each layer that the extruder scans along. The more brim loops are printed, the stronger the shell of a part is;

b) Infill. The inside area of the model, which is filled by a proposed algorithm by the selected pattern and density;

c) Support. The structure is not designed in the 3D model, but used to support the upper layer to maintain its spatial position while extruded as a melted material.

3D printing

An aluminium frame reinforced self-assembly deltabot 3D printer utilizing the FDM technique was selected to manufacture the polymer samples. A secure digital memory card is used to transfer the data to the 3D printer. A LED screen on the machine was used to navigate the location of the model to be printed. When a .gcode file was selected, the 3D printer worked automatically to fabricate the proposed physical model.

For metal 3D printing, this project selected the SLM technique to fabricate a medical implant product based on the previous study [128, 178]. The Magics software (Materialise, Belgium) was used to prepare the fixation plate model, which was sent to a SLM machine SLM solutions (SLM Solutions GmbH) to fabricate the metal fixation plate. The SLM system operates in argon protection with 100 W laser power, 375 mm/s laser speed, 0 mm focal offset and 0.13 hatch distance. Hand milling was performed as a post process with 700 grit sandpaper to remove the weakly adhered particles on the surface.

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Support removal

As per section 2.1, the support structure is necessary to maintain the spatial position of materials as it is solidified. After 3D printing, specimens were removed from substrate plate through mechanical tools by hand with particular care of the surface. The redundant structure was fragile and was manually removed by tearing away with pliers. Residue usually happens at the contact region between the support and the object, which gives rise to surface imperfections that require further surface finishing. This project used both laser surface modification and the classical grinding method for improving the surface quality of an object.

3.2.5 Laser Scanning

Theoretical laser settings

The primary subsequent surface treatment method used in this project is laser scanning. A 40-W CO2 industrial laser scanning machine (King Rabbit HX-40A, Shandong, China) is set up at a 38-mm focal length f of the lens with a spot size of 73 μm and the source laser spot size D of 2.5 mm. This project selected this machine because of its excellent energy absorption rate with polymers. The CO2 laser beam scans the layered surface produced using FDM 3D printing technique. The anisotropy polymer surface is melted and re-solidified to form a new surface profile. The melted areas are usually flattened by gravity and surface tension [179]. Theoretical energy deposition of a pulse laser is greatly influenced by the scan line gap, scan delay, and scan speed (the period the laser spot remains at an area).

In this study, the laser performance is subject to the working condition of a single machine and the great potential of improving the surface quality of FDM items might be demonstrated. Differences in laser scanning machines may require

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slightly different parameters to be set to achieve the same result.

To achieve shallow melt regime on the surface, the study used a set of the parameters of laser polishing treatment at 3W output power, 0.025 mm scan line gap, 30 ms delay between pulses, and 150 mm/s scan speed as previously described [180]. A square area that fully covered the sample is scanned by laser and each laser pulse scanned along the short side. Samples are post-processed at all sides of faces.

Machine setup

The cooling pump was turned on prior to running the laser scanning machine and the exhaust fan turned on afterwards. Figure 3-3 shows the control panel of the laser machine. A current indicator is used to adjust the scanning current that showed on the ampere meter. Pressing the laser test button to excite the laser radiation, and adjust the current regulator simultaneously to identify a proper emission current for the material to be polished.

3.2.6 Surface Grinding

The other post-processing method used in this project is surface grinding. A 700- grit sandpaper is applied with water as lubricant at a rotation speed of 150 r/min for mechanical surface grinding. Although the manually controlled grinding pressure could hardly be quantified to a standard procedure, this project optimised and set the grinding duration at 40-50 seconds on each corresponding side.

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3.2.7 Surface Validation

Optical profiler

The surface roughness of all samples before and after laser treatment was observed using an optical profiling system (Veeco Wyko NT1100, AZ, USA). Magnification at × 5 was set for the observation of overall surface (1.3 × 0.9 mm2) containing five protrusions. The magnification at ×50 was set for the local surface (126 × 94 μm2), which contains a single protrusion. Both conditions were without any stitching. The surface roughness (S_a) (µm) of the FDM-made PLA samples before and after post surface treatment [181], are shown below:

Sa[μm] =

1

A∬ |Z_A (x,y)|dxdy

The effectiveness of the topography optimization was evaluated by the roughness reduction of a surface (%), shown below:

S_areduction[%] =Sainitial_S− Sapolished

ainitial

× 100

Scan electron microscopy (SEM)

This project used a scanning electron microscopy (Zeiss UltraPlus analytical FESEM,) to provide a local description of 3D printing object surfaces. SEM image allowed a great visual comparison in term of quality, structure and melted matter.

3.2.8 Mechanical Test

An Instron mechanical tester (Instron®4505, MA, USA) was employed for investigating tensile, compression and flexure properties of the PLA parts at room temperature, 23 °C and 50% relative humidity according to the standards of ISO 527. For the tensile test, the loading speed was 5 mm/min and the specimens

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were loaded until they broke with a load cell of 5 kN. The same load cell was used for flexural modulus measurement at a speed of 2 mm/min. In a compression test, a 50 kN load cell was used and the 1.3 mm/min loading speed was selected. To quantify mass loss during the laser polishing treatment, the samples were weighed before and after the post-processing on an electronic balance with an accuracy of 0.0001g. For the compression test, cross-sectional dimensions after post processing were measured for the calculation of compression stress. The three-point bending method was used to calculate the flexural modulus.

3.2.9 Cell Culture

Sample preparation

Before a sample is used in a cell culture experiment, it needs to be thoroughly cleaned with a proper cleaning solution. In this project, ethanol was selected for the volume of the parts that needs to be cleaned. The item was placed into a beaker of an ultrasonic cleaner filled with the cleaning solution and cleaned for one minute at room temperature. The sample was rinsed with clean deionized water and dried afterwards.

OBs

To assess whether the different material discs influence on OB cell growth, the OB cells were seeded at a density of 5 × 103 cells per well in 1 mL of cell culture medium in 24-well plate containing testing samples. The cells were cultured at 37 °C, 5% CO2 for 3 days. The experiments were performed at triplicates in each

sample group. At the endpoint, the cells were washed, trypsinised, and resuspended in cold PBS. Three independent experiments were performed by two independent researchers.

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OCs

Approximately 2.5 × 105 _{OCs induced from “buffy coat” were placed in a well of}

12-well plate containing the testing sample in 1 mL of OC conditional culture medium. The medium was an α-MEM supplemented with 10% FCS and antibiotics, 10 × 10-9 _{M dexamethasone (Sigma-Aldrich, Australia), 25 ng mL}-1

human M-CSF (Merck Millipore, Australia), and 10 × 10-9 _{M 1,25(OH)2D3 (Sigma-}

Aldrich, Australia). The cell culture medium was refreshed every 2 days. The OC inductive medium was further supplemented with recombinant human RANKL (Merck Millipore) at 50 ng mL-1 _{and the induced OCs were measured at Day 8 of}

the culture.

Cell counting

The cells were counted using Vi-CELL™ Cell Viability Analyser (Beckman Coulter, IN, USA). Cell numbers represented the quantity of surviving cells on each treatment. The project sets a 70 µm filter for cells selected as induced matured/premature OCs.

3.2.10 Statistical Analysis

Experimental data was analysed using software Graphpad Prism7.0 (GraphPad Software, Inc. CA, USA). Student t-test was performed to analyse the differences between two means in the experiments. If the data exhibited a Gaussian distribution without equal standard deviations (SDs) for unpaired comparison, Welch’s correction was applied. The statistical significance was set at p ≤ 0.05. Three to six replicates of each experiment were performed.