Creating In Vitro Three-Dimensional Tumor Models: A Guide for the Biofabrication of a Primary Osteosarcoma Model.

Osteosarcoma (OS) is a highly aggressive primary bone tumor. The mainstay for its treatment is multiagent chemotherapy and surgical resection, with a 50-70% 5-year survival rate. Despite the huge effort made by clinicians and researchers in the past 30 years, limited progress has been made to improve patient outcomes. As novel therapeutic approaches for OS become available, such as monoclonal antibodies, small molecules, and immunotherapies, the need for OS preclinical model development becomes equally pressing. Three-dimensional (3D) OS models represent an alternative system to study this tumor: In contrast to two-dimensional monolayers, 3D matrices can recapitulate key elements of the tumor microenvironment (TME), such as the cellular interaction with the bone mineralized matrix. The advancement of tissue engineering and biofabrication techniques enables the incorporation of specific TME aspects into 3D models, to investigate the contribution of individual components to tumor progression and enhance understanding of basic OS biology. The use of biomaterials that mimic the extracellular matrix could also facilitate the testing of drugs targeting the TME itself, allowing a larger range of therapeutics to be tested, while averting the ethical implications and high cost associated with in vivo preclinical models. This review aims at serving as a practical guide by delineating the OS TME ("what it is like") and, in turn, propose various biofabrication strategies to create a 3D model ("how to recreate it"), to improve the in vitro representation of the OS tumor and ultimately generate more accurate drug response profiles.

Engineering microdent structures of bone implant surfaces to enhance osteogenic activity in MSCs.

Problems persist with the integration of hip and dental implants with host bone tissues, which may result in long-term implant failure. Previous studies have found that implants bearing irregular surfaces can facilitate osseointegration. An improvement to this approach would use implant surfaces harboring a well-defined surface microstructure to decrease variability in implant surfaces. In this study, we tested whether well-defined surfaces with arrays of microdents (each with depth approximately 3 µm) significantly affected the morphology, proliferation, and osteogenic activity of mesenchymal stem cells (MSCs). Arrays of microdents tested had diameters of 9 µm, 12 µm, and 18 µm, while spacing between arrays ranged from 8 µm to 34 µm. Effects on MSC morphology (cell spreading area) and proliferation were also quantified, with both significantly decreasing on micropatterned surfaces (p<0.05) on smaller and denser microdents. In contrast, MSCs were found to deposit more calcified matrix on smaller and denser arrays of microdents. MSCs on a pattern with arrays of microdents with a diameter of 9 µm and a spacing 8 µm deposited 3-4 times more calcified matrix than on a smooth surface (p<0.05). These findings show that well-defined surface microtopographies promote osteogenic activity, which can be used on implant surfaces to improve integration with the host bone tissue.

A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.

Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.