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This study investigated the chondrogenic activity of encapsulated mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) and its impact on the mechanical properties of injectable poly(N-isopropylacrylamide)-based dual-network hydrogels loaded with poly(L-lysine) (PLL). To this effect, an ex vivo study model was employed to assess the behavior of the injected hydrogels - specifically, their surface stiffness and integration strength with the surrounding cartilage. The highest chondrogenic activity was observed from AC-encapsulated hydrogels, while the effect of PLL on MSC chondrogenesis was not apparent from biochemical analyses. Mechanical testing showed that there were no significant differences in either surface stiffness or integration strength among the different study groups. Altogether, the results suggest that the ex vivo model can allow further understanding of the relationship between biochemical changes within the hydrogel and their impact on the hydrogel's mechanical properties. This article is protected by copyright. All rights reserved.

Current in vitro methods for assessing cancer biology and therapeutic response rely heavily on monolayer cell culture on hard, plastic surfaces that do not recapitulate essential elements of the tumor microenvironment. While a host of tumor models exist, most are not engineered to control the physical properties of the microenvironment and thus may not reflect the effects of mechanotransduction on tumor biology. Utilizing coaxial electrospinning, we developed three-dimensional (3D) tumor models with tunable mechanical properties in order to elucidate the effects of substrate stiffness and tissue architecture in osteosarcoma. Mechanical properties of coaxial electrospun meshes were characterized with a series of macroscale testing with uniaxial tensile testing and microscale testing utilizing atomic force microscopy on single fibers. Calculated moduli in our models ranged over three orders of magnitude in both macroscale and microscale testing. Osteosarcoma cells responded to decreasing substrate stiffness in 3D environments by increasing nuclear localization of Hippo pathway effectors, YAP and TAZ, while downregulating total YAP. Additionally, a downregulation of the IGF-1R/mTOR axis, the target of recent clinical trials in sarcoma, was observed in 3D models and heralded increased resistance to combination chemotherapy and IGF-1R/mTOR targeted agents compared to monolayer controls. In this study, we highlight the necessity of incorporating mechanical cues in cancer biology investigation and the complexity in mechanotransduction as a confluence of stiffness and culture architecture. Our models provide a versatile, mechanically variable substrate on which to study the effects of physical cues on the pathogenesis of tumors. STATEMENT OF SIGNIFICANCE: The tumor microenvironment plays a critical role in cancer pathogenesis. In this work, we engineered 3D, mechanically tunable, coaxial electrospun environments to determine the roles of the mechanical environment on osteosarcoma cell phenotype, morphology, and therapeutic response. We characterize the effects of varying macroscale and microscale stiffnesses in 3D environments on the localization and expression of the mechanoresponsive proteins, YAP and TAZ, and evaluate IGF-1R/mTOR pathway activation, a target of recent clinical trials in sarcoma. Increased nuclear YAP/TAZ was observed as stiffness in 3D was decreased. Downregulation of the IGF-1R/mTOR cascade in all 3D environments was observed. Our study highlights the complexity of mechanotransduction in 3D culture and represents a step towards controlling microenvironmental elements in in vitro cancer investigations.

Biomaterials for tissue engineering that recapitulate the mechanical response and biological function of native tissue are highly sought after to lessen the burden of damaged or diseased tissue. Poly(ethylene glycol) diacrylate (PEGDA) hydrogels are a popular candidate because of their favorable bioactive properties. However, their mechanical behavior is very dissimilar to that of biological tissue, which behaves in a mechanically anisotropic, nonlinear, and viscoelastic fashion. It has been previously shown that PEGDA hydrogels can be patterned in alternating linear strips of different stiffnesses to generate anisotropic behavior, but these constructs still have a linear stress-strain response. In this study, we imparted nonlinear mechanical properties to PEGDA hydrogels by fabricating composite hydrogel constructs consisting of a stiff sinusoidal reinforcement embedded into a softer base matrix. This was achieved by polymerizing low molecular weight (MW) PEGDA hydrogel precursor into a stiff sinusoidal shape and then polymerizing this construct into a high MW precursor. Samples were generated with different relative stiffness between the two components and a range of sinusoid periodicities to assess the tunability of the resulting stress-strain curve. Tensile testing indicates that the sinusoidal patterning gives rise to nonlinear stress-strain behavior. Varying the relative stiffness was shown to tune the slope of the linear region of the stress-strain curve, and varying periodicity was shown to affect the length of the toe region of this curve. We conclude that composite hydrogels with stiff sinusoidally-patterned reinforcements display mechanical properties more similar to those of biological tissue than uniform or linearly-patterned hydrogels. Statement of Significance Hydrogel biomaterials are a popular candidate for engineering constructs that can mimic the properties of native tissue for disease modeling and tissue-engineering applications. Studies have shown that poly(ethylene) glycol diacrylate (PEGDA) hydrogels can be fabricated to display many biological aspects of native tissue. However, they are unable to recapitulate fundamental mechanical properties of such tissue, such as anisotropy and nonlinearity. Photolithographic techniques have been employed to generate anisotropic linear PEGDA hydrogels via patterned reinforcement. The present study indicates that such techniques can be modified to generate PEGDA constructs with a sinusoidal reinforcement that display a strongly nonlinear response to tensile loading. This work sets the stage for more intricate patterning for providing increased control over hydrogel mechanical response.

The past few decades have seen marked improvements in survival rates of osteosarcoma due to the advent of modern chemotherapy, radiotherapy, and surgical techniques. However, for patients presenting with metastatic disease, five-year survival rates have remained dismal, hovering around 20%. Repeated failures of clinical trials to confirm potential therapeutic options highlight the need for more accurate pre-clinical testing. Currently, the field of cancer research relies heavily on monolayer culture methods on hard plastic or glass in vitro models; there is a dearth of pre-clinical models that accurately recapitulate the tumor-microenvironment interactions. Matrix stiffness has been implicated in modulating intracellular signaling pathways that promote cancer cell survival, proliferation, and stem cell fate. We have developed a novel three-dimensional (3D) tumor model with variable mechanical properties in order to determine the effect of substrate stiffness and tissue architecture on osteosarcoma cell phenotype, plasticity, and response to therapy. We employed coaxial electrospinning techniques to fabricate highly porous fibrous mesh scaffolds that mimic the bone microenvironment. By controlling the ratio of poly(ϵ-caprolactone) (PCL) and gelatin (PCL:gelatin, core:shell, respectively) in constituent fibers, we were able to manipulate the range of tensile moduli of individual fibers over three orders of magnitude, from 68.91 ± 8.77 kPa to 66.05 ± 7.61 MPa. Osteosarcoma cells cultured in these variable mechanical environments responded by modulating the localization and expression of Hippo pathway regulators. YAP downregulation correlated with decreasing fiber stiffness while both YAP and TAZ had decreasing nuclear:cytoplasmic ratio in less stiff environments. Furthermore, the IGF-1/mTOR axis was downregulated in 3D conditions compared to monolayers and a strong upregulation of Sox2, a stem cell transcription factor, was observed in all 3D conditions. Correspondingly, in the presence of agents targeting the IGF-1/mTOR axis, dose response curves to doxorubicin indicated that IC50 values increase with decreasing substrate stiffness. These phenotypic changes indicate that osteosarcoma cells respond to both stiffness and architecture by modulating the Hippo and IGF-1R/mTOR pathways and increasing cancer stem cell qualities and chemoresistance. We sought to validate our model using osteosarcoma patient biopsies. Analysis of tumor samples from 36 osteosarcoma patients confirmed that YAP/TAZ localization and nuclear pIGF-1R/IGF-1R in our 3D models recapitulated phenotypes observed in patient samples. Our models highlight the need for incorporation of mechanical and architectural cues in the preclinical study of cancer biology as these signals have drastic impacts on osteosarcoma phenotypes and responses to therapy. Citation Format: Eric R. Molina, Letitia K. Chim, Maria C. Salazar, Shail M. Mehta, Brian A. Menegaz, Salah-Eddine Cherradi-Lamhamedi, Tejus Satish, David McCall, Sana Mohiuddin, Ana Maria Zaska, Katherine Jane Grande-Allen, Joseph A. Ludwig, Antonios G. Mikos. Mechanically tunable 3D microenvironments modulate tumor cell phenotype: Models of mechanotransduction and drug resistance in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-028.