Manufacturing of hOBMT

In vitro engineering of a bone metastases model allows for study of the effects of antiandrogen therapies in advanced prostate cancer

Authors

N. Bock,^1,2,3,4,5* T. Kryza,^1,2,3,† A. Shokoohmand,^1,2,3,4† J. Röhl,^1,2,3 A. Ravichandran,^2,3,4 M.-L. Wille,^2,5,6 C. C. Nelson,^1,2,3 D. W. Hutmacher,^1,3,4,5,6,7 J. A. Clements^1,2,3

Affiliations

¹School of Biomedical Sciences, Faculty of Health and Australian Prostate Cancer Research Centre (APCRC-Q) 

²Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), Brisbane, 4000, QLD, Australia

³Translational Research Institute (TRI), QUT, Woolloongabba, 4102, QLD, Australia.

⁴Centre in Regenerative Medicine, IHBI, QUT, Kelvin Grove, 4059, QLD, Australia.

⁵Australian Research Council (ARC) Training Centre for Multiscale 3D Imaging, Modelling and Manufacturing (M3D Innovation), QUT, Kelvin Grove, 4059, QLD, Australia

⁶Bone and Joint Disorders Program, School of Mechanical, Medical, and Process Engineering, Science and Engineering Faculty (SEF), QUT, Brisbane, 4000, QLD, Australia

⁷ARC Training Centre in Additive Biomanufacturing, QUT, Kelvin Grove, 4059, QLD, Australia.

Correspondence to: n.bock@qut.edu.au

Expanded Protocol for Manufacturing of hOBMT

3D scaffold manufacturing by melt electrowriting 

Microfiber scaffolds made of medical-grade polycaprolactone (mPCL, PURASORB PC12, 95-140 kDa, 2 mm pellets, Corbion Purac, The Netherlands) were manufactured by melt electrowriting (MEW) with an in-house-built apparatus (IHBI, QUT, Brisbane, Australia) and protocol described elsewhere.^1,2 Briefly, mPCL was loaded into a 3 CC clear Luer-Lock plastic syringe (Nordson EFD, Australia) and pre-heated to 60 ºC overnight in a bench oven (Labec, Australia). The syringe was fitted with a 23 G tapered needle (Nordson EFD) and set in the MEW block heaters, at 74 ºC and 85 ºC for the syringe and needle block heaters, respectively. After 2 hours, the extrusion pressure was regulated to 2.2 bar and the working distance between the needle tip and aluminum collector set to 9 mm. A G-code was loaded in the Mach 3 software (Artsoft, USA). The voltage was increased to 10.1 kV and the print was started after 15 min from voltage stabilization, lasting 2.5 days until completion. The resulting printed scaffold had dimensions favorable to cell growth and infiltration (600 µm thickness, 12 µm fiber diameter, 150 µm pore size) and was laser-cut (ILS12.75, Universal Laser Systems Inc., USA) in 10×10 mm small scaffolds prior to chemical treatment.

G-code

The following G-code (incremental type) was used to generate the layer-by-layer manufacturing of a microfiber polymeric scaffold with an overall size of 90 × 70 mm, spacing of 150 µm in between fibers, comprising 80 layers in the z direction in a ‘0 degree- 90 degree’ pattern, yielding approximately 600 µm in the z direction. In the following code, the sign ‘*’ indicates a new line in the code, i.e., all ‘*’ signs need to be replaced by pressing the ‘Enter’ key in the final code.

G-Code:

G17 G21 G40 G49 G54 G80 G94 F450 %G91 is relative coordinates! *F1000 *G91 *G1 x10 *M98 p1 l20 % trail print for jet stabilization *G1 y5 *M98 p1236 l40 % number of layers in overall scaffold construct--40X  and 40Y  *G1  x-10 **M30 **o1236 *M98 p1237 l300 % loop size/FD *G1 x90 *G1  y-90 *M98 p1238 l300 % loop size/FD *M99 **o1237 %x loops *G1 x90 *G1 y0.15 *G1 x-90 *G1 y0.15 *M99 **o1238 %y  loops  *G1 x-0.15 *G1 y90 *G1   x-0.15 *G1 y-90 *M99 **o1 % trail loop x100 *y0.2 *x-100 *y0.2 *M99 *M2.

3D scaffold surface modification

The laser-cut 10×10 mm mPCL scaffolds were treated with sodium hydroxide (NaOH) and calcium phosphate (CaP) for hydrophilicity and osteoinductive properties, as described elsewhere.³ All chemicals mentioned below were obtained from Sigma-Aldrich, Australia.

Surface activation

Scaffolds were immersed in 70% v/v ethanol under desiccator house-vacuum for 15 min. Scaffolds were rinsed twice with ddH₂O and transferred in a pre- warmed (37 ºC) 2 M NaOH solution in a 50 mL reaction tube. After 5 min under desiccator house- vacuum, the tube was placed in a water bath at 37ºC for 45 min. After NaOH solution removal, scaffolds were rinsed with ddH₂O for eight washes, and pH was controlled to be neutral (7).

Biomimetic mineralization

A 10× simulated body fluid solution (SBF 10×) was prepared by preparing Solution A, which involved dissolving the chemicals listed in Supplemental Table 1 in 300 mL ddH₂O in a 500 mL beaker, under magnetic stirring. In a second beaker, 710 mg of disodium hydrogen phosphate (Na₂HPO₄) was dissolved in 15 mL ddH₂O. Under magnetic stirring, the Na₂HPO₄ solution was added to Solution A dropwise, ensuring that pH remained at all times between 1.5 and 3.5 by HCl titration, until the final addition where pH reached 3.9. The final solution (SBF 10×) was topped-up to 500 mL with ddH₂O (final pH of 4). A 50 mL aliquot was placed in a 50 mL beaker and small amounts of sodium hydrogen carbonate (NaHCO₃) powder were gradually added until pH reached 6. The solution was 0.2 µm-filtered and placed in a 50 mL reaction tube with the mPCL scaffolds. After 5 min under desiccator house- vacuum, the tube was placed in a water bath at 37ºC for 30 min. A new 50 mL SBF aliquot was prepared, and the step was repeated once more. After rinsing once with ddH₂O, scaffolds were immersed in a pre-warmed (37 ºC) 0.5 M NaOH solution. After 5 min under desiccator house- vacuum, the tube was placed in a water bath at 37ºC for 30 min. Scaffolds were washed five times with ddH₂O until pH was neutral. Scaffolds were dried overnight in the desiccator and scanning electron microscopy was performed for quality control. The final treated scaffolds are referred to as ‘CaP-mPCL’ scaffolds.

Supplemental Table 1. Reagent amounts and order of use for simulated body fluid (SBF) 10×.

Sterilization

Prior to cell seeding, CaP-mPCL scaffolds were immersed in 70% v/v ethanol in Petri dishes in a biological safety cabinet class II (laminar flow), under aseptic conditions. After 20 min, ethanol was removed and the scaffolds were let to dry overnight inside the laminar flow cabinet. Scaffolds were exposed to the UV light from the cabinet for 20 min on each side and kept in the Petri dishes prior to cell seeding.

Primary cell isolation from bone tissue

Human primary osteoprogenitor cells were isolated from bone tissue obtained under informed consent from male donors undergoing hip and knee replacement surgery (QUT ethics approval number 1400001024), as described previously.4 Non- sclerotic, trabecular bone was collected either from the tibial plateau/femoral condyles from the knee or from the acetabular ground from the hip. Bone fragments (4-5 mg) were minced and washed in sterile phosphate buffer saline (PBS) without divalent ions (Gibco, Australia). Bone fragments were transferred to 175 cm² cell culture-treated flasks with 18 mL of growth media (GM), containing α-MEM with ribonucleosides, deoxyribonucleosides, phenol Red and L-glutamine (cat.12571), with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S, 10,000 U/mL stock solution), all from Gibco. Osteoprogenitor cell outgrowth occurred after 7-10 days. Cells were collected using 0.25% w/v  Trypsin – 1 mM EDTA (Gibco) at 80% confluence, and further expanded using GM. Cells from passages 3-4 were used for culture within the CaP-mPCL scaffolds and were tested free of mycoplasma.

Bioengineering of human osteoblast-derived microtissues

The sterilized CaP-mPCL scaffolds were placed in cell-culture treated 24-well plates. Primary osteoprogenitor cells were seeded at a density of 0.4×10⁶ cells/scaffold. Briefly, cells were first concentrated at 8×10⁶ cells/mL in GM. Five drops of 10 µL of cell suspension were seeded in each corner and in the center of each scaffold (total volume of 50µL/scaffold). Cell constructs were incubated for 1 hour in a humidified incubator (37 ºC, 95% air, 5% CO₂) before 50 µL of GM was added in the center of the scaffold. After 3 hours, 2 mL of GM was added to each well with media changes every 2-3 days. After 14 days, the cellular constructs were transferred to new 12-well plates each with 2 mL of osteogenic media (OM), containing GM + 10 mM β- glycerophosphate, 0.17 mM ascorbic acid, 100 nM dexamethasone (all from Sigma-Aldrich, Australia). Media was changed every 3-4 days and the resulting cellular microtissues were cultured for either 8 or 12 weeks in OM. The final bioengineered constructs are hereafter referred to as human osteoblast-derived microtissues (hOBMT).

References

1. Brown, T. D. et al. Design and fabrication of tubular scaffolds via direct writing in a melt electrospinning mode. Biointerphases 7, 13, doi:10.1007/s13758-011-0013-7 (2012).
2. Martine, L. C. et al. Engineering a humanized bone organ model in mice to study bone metastases. Nature Protocols 12, 639-663, doi:10.1038/nprot.2017.002 (2017).
3. Vaquette, C., Ivanovski, S., Hamlet, S. M. & Hutmacher, D. W. Effect of culture conditions and calcium phosphate coating on ectopic bone formation. Biomaterials 34, 5538-5551, doi:10.1016/j.biomaterials.2013.03.088 (2013).
4. Reichert, J. C. et al. Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment. Biomaterials 31, 7928-7936, doi:10.1016/j.biomaterials.2010.06.055 (2010).

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