Customizable biomaterials as tools for advanced anti-angiogenic drug discovery.

The inhibition of angiogenesis is a critical element of cancer therapy, as cancer vasculature contributes to tumor expansion. While numerous drugs have proven to be effective at disrupting cancer vasculature, patient survival has not significantly improved as a result of anti-angiogenic drug treatment. Emerging evidence suggests that this is due to a combination of unintended side effects resulting from the application of anti-angiogenic compounds, including angiogenic rebound after treatment and the activation of metastasis in the tumor. There is currently a need to better understand the far-reaching effects of anti-angiogenic drug treatments in the context of cancer. Numerous innovations and discoveries in biomaterials design and tissue engineering techniques are providing investigators with tools to develop physiologically relevant vascular models and gain insights into the holistic impact of drug treatments on tumors. This review examines recent advances in the design of pro-angiogenic biomaterials, specifically in controlling integrin-mediated cell adhesion, growth factor signaling, mechanical properties and oxygen tension, as well as the implementation of pro-angiogenic materials into sophisticated co-culture models of cancer vasculature.

Hydrogel Arrays and Choroidal Neovascularization Models for Evaluation of Angiogenic Activity of Vital Pulp Therapy Biomaterials.

INTRODUCTION: This study intended to evaluate the angiogenic properties of vital pulp therapy materials including white mineral trioxide aggregate (WMTA), calcium hydroxide (Ca[OH]2), Geristore (Den-Mat, Santa Maria, CA), and nano WMTA biomaterials. METHODS: WMTA, Ca(OH)2, Geristore, and nano WMTA disks were prepared, dispersed into 2 mL Milli-Q (Millipore, ThermoFisher, Hanover Park, IL) distilled water, and centrifuged to obtain 2 mL supernatant elution. Thirty-five wells of polyethylene glycol hydrogel arrays were prepared and divided into 5 groups of 7 (n = 7). Mice molar endothelial cells (ECs) were placed on hydrogel arrays. The elution prepared from each sample was diluted in growth medium (1:3) and added to the hydrogel arrays. The EC medium alone was used for the control. For the choroidal neovascularization (CNV) model, thirty-five 6-week-old female mice were lasered and divided into 5 groups, and elution from each sample (2 µL) or saline (control) was delivered by intravitreal injection on the day of the laser treatment and 1 week later. The mean number of nodes, the total length of the branches in the hydrogel arrays, and the mean area of CNV were calculated using ImageJ software (National Institutes of Health, Bethesda, MD) and analyzed by 1-way analysis of variance and post hoc Tukey honest significant difference tests. RESULTS: The comparison of results regarding the number of nodes showed the values of control > Geristore > nano WMTA > WMTA > Ca(OH)2. Regarding the total branch length and the CNV area, the comparison of results showed values of Geristore > control > nano WMTA > WMTA > Ca(OH)2. CONCLUSIONS: All tested materials showed minimal antiangiogenic activity, whereas Geristore and nano WMTA showed a higher proangiogenic activity than WMTA and Ca(OH)2.

Biomaterial arrays with defined adhesion ligand densities and matrix stiffness identify distinct phenotypes for tumorigenic and nontumorigenic human mesenchymal cell types.

Here, we aimed to investigate migration of a model tumor cell line (HT-1080 fibrosarcoma cells, HT-1080s) using synthetic biomaterials to systematically vary peptide ligand density and substrate stiffness. A range of substrate elastic moduli were investigated by using poly(ethylene glycol) (PEG) hydrogel arrays (0.34 - 17 kPa) and self-assembled monolayer (SAM) arrays (~0.1-1 GPa), while cell adhesion was tuned by varying the presentation of Arg-Gly-Asp (RGD)-containing peptides. HT-1080 motility was insensitive to cell adhesion ligand density on RGD-SAMs, as they migrated with similar speed and directionality for a wide range of RGD densities (0.2-5% mol fraction RGD). Similarly, HT-1080 migration speed was weakly dependent on adhesion on 0.34 kPa PEG surfaces. On 13 kPa surfaces, a sharp initial increase in cell speed was observed at low RGD concentration, with no further changes observed as RGD concentration was increased further. An increase in cell speed ~ two-fold for the 13 kPa relative to the 0.34 kPa PEG surface suggested an important role for substrate stiffness in mediating motility, which was confirmed for HT-1080s migrating on variable modulus PEG hydrogels with constant RGD concentration. Notably, despite ~ two-fold changes in cell speed over a wide range of moduli, HT-1080s adopted rounded morphologies on all surfaces investigated, which contrasted with well spread primary human mesenchymal stem cells (hMSCs). Taken together, our results demonstrate that HT-1080s are morphologically distinct from primary mesenchymal cells (hMSCs) and migrate with minimal dependence on cell adhesion for surfaces within a wide range of moduli, whereas motility is strongly influenced by matrix mechanical properties.

A quantitative comparison of human HT-1080 fibrosarcoma cells and primary human dermal fibroblasts identifies a 3D migration mechanism with properties unique to the transformed phenotype.

Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels ("synthetic extracellular matrix" or "synthetic ECM"). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced ß1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with ß1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.

Differential effects of a soluble or immobilized VEGFR-binding peptide.

Regulating endothelial cell behavior is a key step in understanding and controlling neovascularization for both pro-angiogenic and anti-angiogenic therapeutic strategies. Here, we characterized the effects of a covalently immobilized peptide mimic of vascular endothelial growth factor, herein referred to as VEGF receptor-binding peptide (VR-BP), on human umbilical vein endothelial cell (HUVEC) behavior. Self-assembled monolayer arrays presenting varied densities of covalently immobilized VR-BP and varied densities of the fibronectin-derived cell adhesion peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) were used to probe for changes in HUVEC attachment, proliferation and tubulogenesis. In a soluble form, VR-BP exhibited pro-angiogenic effects in agreement with previous studies, indicated by increases in HUVEC proliferation. However, when presented to cells in an insoluble context, covalently immobilized VR-BP inhibited several pro-angiogenic HUVEC behaviors, including attachment and proliferation, and also inhibited HUVEC response to soluble recombinant VEGF protein. Furthermore, substrates with covalently immobilized VR-BP also modulated HUVEC tubulogenesis when a matrigel overlay assay was used to provide cells with a pseudo-three dimensional environment. Taken together, these results demonstrate that the context in which ligands are presented to cell surface receptors strongly influences their effects, and that the same ligand can be an agonist or an antagonist depending on the manner of presentation to the cell.