Abstract
Fibrous scaffolds are used for bone tissue engineering purposes with great success across a variety of polymers with different physical and chemical properties. It is now evident that the correct degree of curvature promotes increased cytoskeletal tension on osteoprogenitors leading to osteogenic differentiation. However, the mechanotransductive pathways involved in this phenomenon are not fully understood. To achieve a reproducible and specific cellular response, an increased mechanistic understanding of the molecular mechanisms driving the fibrous scaffold mediated bone regeneration must be understood. High throughput siRNA mediated screening technology has been utilized for dissecting molecular targets that are important in certain cellular phenotypes. In this study, we used siRNA mediated gene silencing to understand the osteogenic differentiation observed on fibrous scaffolds. A high-throughput siRNA screen was conducted using a library collection of 863 genes including important human kinase and phosphatase targets on pre-osteoblast SaOS-2 cells. The cells were grown on electrospun poly(methyl methacrylate) (PMMA) scaffolds with a diameter of 0.938 ± 0.304 µm and a flat surface control. The osteogenic transcription factor RUNX2 was quantified with an in-cell western (ICW) assay for the primary screen and significant targets were selected via two sample t-test. After selecting the significant targets, a secondary screen was performed to identify osteoinductive markers that also effect cell shape on fibrous topography. Finally, we report the most physiologically relevant molecular signaling mechanisms that are involved in growth factor free, fibrous topography mediated osteoinduction. We identified GTPases, membrane channel proteins, and microtubule associated targets that promote an osteoinductive cell shape on fibrous scaffolds.
Highlights
Surface topography can affect cellular functions by influencing the production of proteins that are secreted into the extracellular space to act as signals in the environment
Several studies reported to explain the global overview of important players in bone regeneration on biomaterial scaffolds, no groups have yet developed a global model of signaling molecules that drives osteogenicity on synthetic bone regenerative engineering scaffolds
High throughput screening (HTS) experiments have lead the new area of functional genomics and allowed researchers to study the extent of the genes that are only specific for a certain phenotype of interest
Summary
Surface topography can affect cellular functions by influencing the production of proteins that are secreted into the extracellular space to act as signals in the environment. Biomaterials play significant roles in regenerative therapies and various studies reported significantly increased bone regeneration with fibrous scaffolds[10,11]. Several studies reported to explain the global overview of important players in bone regeneration on biomaterial scaffolds, no groups have yet developed a global model of signaling molecules that drives osteogenicity on synthetic bone regenerative engineering scaffolds. High throughput screening (HTS) experiments have lead the new area of functional genomics and allowed researchers to study the extent of the genes that are only specific for a certain phenotype of interest. In an RNAi screening experiment a series of assay optimization steps are required to ensure the depletion of protein of interest and overall cell viability. Even though the experimental steps seem straightforward, the assay optimization is relatively cumbersome when compared to other high throughput experiments
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