(594b) 3D Polyurethane Scaffolds (3D-PURS) With Defined Architecture and Rigidity for Analysis of Tumor-Induced Bone Disease

Biomimetic 3D Polyurethane Scaffolds (3D-PURS) with Defined Architecture and Rigidity for Analysis of Tumor-Induced Bone Disease

Jonathan M. Page^a, Alyssa R. Merkel^b, Ushashi Dadwal^b, Scott Guelcher^a, Julie A. Sterling^b

^a Department of Chemical and Biomolecular Engineering, Vanderbilt University

^b Department of Cancer Biology, Vanderbilt University

Statement of Purpose: Breast cancer is known to have a predilection for metastasizing to bone, where it occurs in over 70% of patients with advanced disease [1].  Several factors have been described as important regulators of tumor-induced bone disease, however parathyroid hormone related protein (PTHrP) is the most well-known [2].  PTHrP has been shown to be overexpressed in bone metastases and is a key factor in the vicous cycle of bone disease in which the tumor cells stimulate bone destruction and release of growth factors that further stimulate tumor growth and the expression of proteins that stimulate bone destruction.  The expression of PTHrP is regulated by a complex interaction between tumor cells and the microenvironment via known mechanotransduction pathways.  Recently, we published that the rigidity of two dimensional (2D) substrates can influence tumor cell gene expression by generating polyurethane (PUR) films with modulus mimicking bone and soft tissues [3]. This work has been further developed by the generation of a model that uses a 3D polyurethane scaffold (3D-PURS) to better mimic the bone microenvironment.  3D-PURS are generated by reactive liquid molding of polyurethane precursors in a prefabricated polystyrene mold. Pore size is customizable and the molecular weight of the PUR precursors can generate a range of modulus spanning soft tissue to bone.  The goal of these studies is to analyze the effects of rigidity and pore size in three dimensions with MDA-MB-231 metastatic cancer cells that are known to invade bone.  3D-PURS were tested in vitro and in vivo to elucidate the driving mechanisms behind PTHrP expression of MDA-MB-231 cells.    

Methods:  Prefabricated 3D scaffolds were purchased from 3D Biotek, LLC.  The scaffolds are composed of transverse layers of polystyrene fibers with variable fiber diameter (FD) and spacing (SP), described in figure 1A-B.  The prefabricated scaffolds are placed in a teflon mold and a liquid reactive polyurethane is cast into the mold and allowed to cure around the prefabricated scaffolds.  The liquid polyurethane is a mixture of hexamethylene diisocyanate trimer (HDIt), a poly( e-caprolactone-co-glycolide-co-lactide) triol (polyol, Mn = 300 or 3000 Da), and iron(III) acetylacetonate catalyst.  After curing at 60^oC over 24 hours, the excess polyurethane is cut away from the prefabricated scaffolds and the polystyrene is leached out with dichloromethane.  The remaining 3D-PURS are the inverse of the prefabricated scaffold and is 100% interconnected, with a pore diameter equal to the FD, shown in figure 1C.  Elastic modulus data was obtained by nanoindentation (Agilent g200 nanoindentor). Cells can be easily seeded into 3D-PURS, shown in figure 1D-E.  In vitro experiments were applied to ascertain efficient seeding densities, cell growth, and PTHrP expression as a function of rigidity.   In vivo studies were conducted with MDA-MB-231 seeded scaffolds implanted into the mammary pads of nude mice.  Scaffolds were explanted after 21 days and analyzed for PTHrP expression.

Results: From nanoindentation data, the range of rigidity spanned by the 3D-PURS was 10 MPa for the 3000 Da polyol (soft) to 2600 MPa for the 300 Da polyol (hard).  This range accounts for the basement membrane of soft tissue and bone, respectively.  In vitro testing with 300 micron scaffolds seeded with MDA-MB-231 cells (1x10⁶ cells/scaffold) verified a significant increase in PTHrP expression with increasing rigidity after 24 hours in culture matching previous data shown in 2D films, figure 1G. RT-PCR data showed a larger difference in PTHrP expression between hard and soft scaffolds with 300 µm pores in vivo, figure 1H. Furthermore, both soft and hard scaffolds with 500 µm pores had significantly lower expression of PTHrP than the hard 300 µm scaffolds.

Conclusions: The changes in PTHrP expression with differences in rigidity and pore size point to an active response to environmental cues by metastatic cancer cells. This model is ideal for studies of the microenvironmental effects on tumor cell gene expression due to the customizable nature of both the scaffold architecture and mechanical properties.  Specifically, this model will allow for molecular signaling studies and testing of potential inhibitors of the mechanotransduction pathway.

References

1.Guise, T., et al., Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis. J Clin Invest, 1996. 98(7): p. 1544-1549.2.Powell, G.J., et al., Localization of parathyroid hormone-related protein in breast cancer metastases: increased incidence in bone compared with other sites. Cancer research, 1991. 51(11): p. 3059-3061.3.Ruppender NS, M.A., Martin TJ, Mundy GR, Sterling JA and Guelcher SA, Matrix Rigidity Induces Osteolytic Gene Expression of Metastatic Breast Cancer Cells. PLoS ne, 2010. 5(11): p. e15451.

Figure 1.  Analysis of metastatic cancer cells in a novel 3D polyurethane scaffold. (A&B) Diagram of prefabricated polystyrene scaffolds utilized as a mold for the reactive polyurethane; (C) SEM image of the 3D-polyurethane scaffold (3D-PURS) after the polystyrene mold is removed; (D) Fluorescent image of MDA-231 cells transfected with green fluorescent protein seeded into 3D-PURS; (E)  SEM image of MDA-231 cells adhered to channel walls of 3D-PURS; (F) Ex-vivo fluorescent images of MDA-231 cells in 3D-PURS after 21 days in vivo.  (G) In vitro PTHrP expression of MDA-231 cells culture on 3D-PURS (300 µm pores), the control is 2D tissue culture plastic; (H) In vivo PTHrP expression of MDA-231 cells culture on 3D-PURS after 21 days (5 = 500 micron pores, 3 = 300 micron pores). * p<0.05.

 
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