High-Throughput Screening Assay for the Identification of Compounds Enhancing Collagenous Extracellular Matrix Production by ATDC5 Cells

Introduction

Many different cell types secrete an extracellular matrix (ECM), which provides mechanical support and serves as a signaling hub conveying signals from the cells to the surrounding environment and vice versa. Some of these signals control important biological functions such as cell viability and cell fate.^1,2 In tissue engineering, a common procedure is to isolate cells from their native tissue while leaving behind the ECM. The cells are then expanded to increase numbers, and are reaggregated to mimic a three-dimensional (3D) environment, either by the use of a scaffold or simply by cell aggregation. Ideally, the cells in the tissue-engineered construct will adhere, proliferate, and secrete ECM. The nature of secreted ECM can be, to a certain extent, controlled by the signals provided by the scaffold (e.g., topographical cues) or by soluble signals to which the cells are exposed (e.g., growth factors or small molecules).^3–5

In bone tissue engineering through endochondral ossification, the amount of hypertrophic cartilage matrix in the tissue-engineered construct is crucial for successful bone formation. We reported previously a method to efficiently generate bone in vivo using mouse embryonic stem cells predifferentiated into hypertrophic chondrocytes in vitro.⁶ The amount of bone detected in the construct after implantation was highly correlated with the amount of hypertrophic cartilage observed in vitro. Interestingly, the capacity of hypertrophic cartilage matrix to induce bone formation in vivo is not only limited to living cartilage. In 1958, Bridge and Pritchard performed a series of experiments, in which devitalized tissues were placed under the kidney capsules or implanted subcutaneously in the ears of rabbits. Tissues containing hypertrophic cartilage, which were devitalized with alcohol, acetone, and HCl, or heated to 55°C consistently, formed bone.⁷ Later, Marshall Urist also observed new bone formation when a boiled fracture callus was transplanted into the anterior chamber of a rat's eye.^8–10 This interesting phenomenon can be exploited to produce off-the-shelf bone-inducing hypertrophic cartilage since devitalized allografts provoke a much milder immune response compared with viable tissue.^11,12 We attempt to repeat the devitalized cartilage experiment, but instead of using natural hypertrophic tissue, such as growth plate cartilage or fracture callus, we try to tissue engineer hypertrophic cartilage in vitro to better...