Identification of Key Signaling Pathways Orchestrating Substrate Topography Directed Osteogenic Differentiation Through High-Throughput siRNA Screening

Tugba Ozdemir, Daniel T. Bowers, Xiang Zhan, Debashis Ghosh, Justin Lee Brown

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Article number1001
JournalScientific reports
Volume9
Issue number1
DOIs
StatePublished - Dec 1 2019

Fingerprint

Small Interfering RNA
Cell Shape
Bone Regeneration
GTP Phosphohydrolases
Gene Silencing
Polymethyl Methacrylate
Tissue Engineering
Osteoblasts
Ion Channels
Phosphoric Monoester Hydrolases
Microtubules
Libraries
Intercellular Signaling Peptides and Proteins
Polymers
Membrane Proteins
Transcription Factors
Phosphotransferases
Technology
Phenotype
Bone and Bones

All Science Journal Classification (ASJC) codes

  • General

Cite this

@article{0ba8110bfd2e4d74b48c14466c70afb4,
title = "Identification of Key Signaling Pathways Orchestrating Substrate Topography Directed Osteogenic Differentiation Through High-Throughput siRNA Screening",
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.",
author = "Tugba Ozdemir and Bowers, {Daniel T.} and Xiang Zhan and Debashis Ghosh and Brown, {Justin Lee}",
year = "2019",
month = "12",
day = "1",
doi = "10.1038/s41598-018-37554-y",
language = "English (US)",
volume = "9",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
number = "1",

}

Identification of Key Signaling Pathways Orchestrating Substrate Topography Directed Osteogenic Differentiation Through High-Throughput siRNA Screening. / Ozdemir, Tugba; Bowers, Daniel T.; Zhan, Xiang; Ghosh, Debashis; Brown, Justin Lee.

In: Scientific reports, Vol. 9, No. 1, 1001, 01.12.2019.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Identification of Key Signaling Pathways Orchestrating Substrate Topography Directed Osteogenic Differentiation Through High-Throughput siRNA Screening

AU - Ozdemir, Tugba

AU - Bowers, Daniel T.

AU - Zhan, Xiang

AU - Ghosh, Debashis

AU - Brown, Justin Lee

PY - 2019/12/1

Y1 - 2019/12/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85060852745&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85060852745&partnerID=8YFLogxK

U2 - 10.1038/s41598-018-37554-y

DO - 10.1038/s41598-018-37554-y

M3 - Article

C2 - 30700820

AN - SCOPUS:85060852745

VL - 9

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

M1 - 1001

ER -