Jonathan M. Page

Biocompatibility and chemical reaction kinetics of injectable, settable polyurethane/allograft bone biocomposites

Injectable and settable bone grafts offer significant advantages over pre-formed implants due to their ability to be administered using minimally invasive techniques and to conform to the shape of the defect. However, injectable biomaterials present biocompatibility challenges due to the potential toxicity and ultimate fate of reactive components that are not incorporated in the final cured product. In this study the effects of stoichiometry and triethylenediamine (TEDA) catalyst concentration on the reactivity, injectability, and biocompatibility of two component lysine-derived polyurethane (PUR) biocomposites were investigated. Rate constants were measured for the reactions of water (a blowing agent resulting in the generation of pores), polyester triol, dipropylene glycol (DPG), and allograft bone particles with the isocyanate-terminated prepolymer using an in situ attenuated total reflection Fourier transform infrared spectroscopy technique. Based on the measured rate constants, a kinetic model predicting the conversion of each component with time was developed. Despite the fact that TEDA is a well-known urethane gelling catalyst, it was found to preferentially catalyze the blowing reaction with water relative to the gelling reactions by a ratio >17:1. Thus the kinetic model predicted that the prepolymer and water proceeded to full conversion, while the conversions of polyester triol and DPG were <70% after 24h, which was consistent with leaching experiments showing that only non-cytotoxic polyester triol and DPG were released from the reactive PUR at early time points. The PUR biocomposite supported cellular infiltration and remodeling in femoral condyle defects in rabbits at 8weeks, and there was no evidence of an adverse inflammatory response induced by unreacted components from the biocomposite or degradation products from the cured polymer. Taken together, these data underscore the utility of the kinetic model in predicting the biocompatibility of reactive biomaterials.

Wnt signaling induces gene expression of factors associated with bone destruction in lung and breast cancer.

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-β) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF-β in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by β-catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF-β and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched β-catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a β-catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF-β signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF-β signaling.

Fabrication of 3D Scaffolds with Precisely Controlled Substrate Modulus and Pore Size by Templated‐Fused Deposition Modeling to Direct Osteogenic Differentiation

Scaffolds with tunable mechanical and topological properties fabricated by templated-fused deposition modeling promote increased osteogenic differentiation of bone marrow stem cells with increasing substrate modulus and decreasing pore size. These findings guide the rational design of cell-responsive scaffolds that recapitulate the bone microenvironment for repair of bone damaged by trauma or disease.

Synthetic and tissue-derived models for studying rigidity effects on invadopodia activity.

Invasion by cancer cells through the extracellular matrix (ECM) of tissues is a critical step in cancer progression and metastasis. Actin-rich subcellular protrusions known as invadopodia are thought to facilitate this process by localizing proteinases which degrade the ECM and allow for cancer cell penetration. We have shown in vitro that invadopodia activity is regulated by the rigidity of the ECM, which suggests that matrix remodeling in vivo may also be regulated by the mechanical properties of tissues. In order to study rigidity effects on invadopodia activity in a controlled manner, we have developed assays in which we have conjugated degradable fluorescent matrix molecules to tunable synthetic substrates. In addition, we have also utilized ex vivo tissue-derived substrates to corroborate our findings. In this chapter, we present detailed protocols describing the synthesis and preparation of our synthetic substrates, polyacrylamide gels and polyurethane elastomers, for use in these matrix degradation assays as well as the steps required to utilize our tissue-derived substrates.

Design and development of reactive injectable and settable polymeric biomaterials

Injectable and settable biomaterials are a growing class of therapeutic technologies within the field of regenerative medicine. These materials offer advantages compared to prefabricated implants because of their ability to be utilized as part of noninvasive surgical procedures, fill complex defect shapes, cure in situ, and incorporate cells and other active biologics. However, there are significant technical barriers to clinical translation of injectable and settable biomaterials, such as achieving clinically relevant handling properties and benign reaction conditions. This review focuses on the engineering challenges associated with the design and development of injectable and chemically settable polymeric biomaterials. Additionally, specific examples of the diverse chemistries utilized to overcome these challenges are covered. The future translation of injectable and settable biomaterials is anticipated to improve patient outcomes for a number of clinical conditions.

Elucidation of adhesion-dependent spontaneous apoptosis in macrophages using phase separated PEG/polyurethane films

Circulating monocytes undergo spontaneous apoptosis when there is no activation stimulus, which is critical to population control for proper host response to implants. As activation and apoptosis of monocytes/macrophages are regulated by cell-cell and cell-matrix interactions, their regulatory mechanism was investigated in this study using polyethylene glycol (PEG)-containing polyurethane films in which PEG-rich and polyester-rich domains were phase separated. Human blood monocyte-derived macrophages (HBMs) preferentially adhered to PEG domains (cell-matrix interaction) due to the low molecular weight (600 g mol⁻¹), resulting in increased HBM density (cell-cell interaction). As both cell-cell and cell-matrix interactions were promoted, HBM apoptosis increased, while their activation as measured by phagocytosis, intracellular reactive oxygen species (ROS) level and matrix metalloproteinase-9 production decreased compared to PEG-free films. When cell seeding density and cell-adhesive gelatin coating on silicone films were controlled, a cooperative role of cell-matrix (adhesion) and cell-cell (density) interactions in inducing HBM apoptosis was observed. Expression of the macrophage adhesion molecule CD11b caused apoptosis in this context, which was mediated by tissue necrosis factor-α signaling but down-regulated by the ROS inhibitor diphenylene iodonium and the anti-inflammatory peptide Ac-SDKP, suggesting a new concept for the design of biomaterials that allows for cell adhesion without excessive inflammatory activation.

Injectable foams for regenerative medicine

The design of injectable biomaterials has attracted considerable attention in recent years. Many injectable biomaterials, such as hydrogels and calcium phosphate cements (CPCs), have nanoscale pores that limit the rate of cellular migration and proliferation. While introduction of macroporosity has been suggested to increase cellular infiltration and tissue healing, many conventional methods for generating macropores often require harsh processing conditions that preclude their use in injectable foams. In recent years, processes such as porogen leaching, gas foaming, and emulsion-templating have been adapted to generate macroporosity in injectable CPCs, hydrogels, and hydrophobic polymers. While some of the more mature injectable foam technologies have been evaluated in clinical trials, there are challenges remaining to be addressed, such as the biocompatibility and ultimate fate of the sacrificial phase used to generate pores within the foam after it sets in situ. Furthermore, while implantable scaffolds can be washed extensively to remove undesirable impurities, all of the components required to synthesize injectable foams must be injected into the defect. Thus, every compound in the foam must be biocompatible and noncytotoxic at the concentrations utilized. As future research addresses these critical challenges, injectable macroporous foams are anticipated to have an increasingly significant impact on improving patient outcomes for a number of clinical procedures.

Altering adsorbed proteins or cellular gene expression in bone-metastatic cancer cells affects PTHrP and Gli2 without altering cell growth.

The contents of this data in brief are related to the article titled “Matrix Rigidity Regulates the Transition of Tumor Cells to a Bone-Destructive Phenotype through Integrin β3 and TGF-β Receptor Type II”. In this DIB we will present our supplemental data investigating Integrin expression, attachment of cells to various adhesion molecules, and changes in gene expression in multiple cancer cell lines. Since the interactions of Integrins with adsorbed matrix proteins are thought to affect the ability of cancer cells to interact with their underlying substrates, we examined the expression of Integrin β1, β3, and β5 in response to matrix rigidity. We found that only Iβ3 increased with increasing substrate modulus. While it was shown that fibronectin greatly affects the expression of tumor-produced factors associated with bone destruction (parathyroid hormone-related protein, PTHrP, and Gli2), poly-l-lysine, vitronectin and type I collagen were also analyzed as potential matrix proteins. Each of the proteins was independently adsorbed on both rigid and compliant polyurethane films which were subsequently used to culture cancer cells. Poly-l-lysine, vitronectin and type I collagen all had negligible effects on PTHrP or Gli2 expression, but fibronectin was shown to have a dose dependent effect. Finally, altering the expression of Iβ3 demonstrated that it is required for tumor cells to respond to the rigidity of the matrix, but does not affect other cell growth or viability. Together these data support the data presented in our manuscript to show that the rigidity of bone drives Integrinβ3/TGF-β crosstalk, leading to increased expression of Gli2 and PTHrP.