Dataset Information

Post-transcriptional regulation in osteoblasts using localized delivery of miR-29a inhibitor from nanofibers to enhance extracellular matrix deposition.

ABSTRACT: MicroRNAs are important post-transcriptional regulators of skeletal biology, and miRNA-based therapeutics have the potential to aid bone repair. However, efficient tools for delivering miRNA mimics or inhibitors to specific target tissues are limited. Polymeric nanofibers closely mimic natural extracellular matrix (ECM) morphology, and are attractive candidates for supporting delivery of cells and bone-anabolic reagents. It is hypothesized that gelatin nanofibers could be used for the localized transient delivery of miRNA-based therapeutics, using miR-29a inhibitor as a prototype to increase ECM deposition. miR-29 family members are negative regulators of ECM synthesis, targeting the mRNAs of selected collagens and osteonectin/SPARC. Inhibiting miR-29 activity may therefore increase ECM production by cells. miR-29a inhibitor-loaded gelatin nanofibers, prepared by electrospinning, demonstrated continuous release of miRNA inhibitor over 72h. Pre-osteoblastic murine MC3T3-E1 cell line seeded on miR-29a inhibitor-loaded nanofibers synthesized more osteonectin, indicating efficient inhibitor delivery. These cells also displayed increased Igf1 and Tgfb1 mRNA. Moreover, primary bone marrow stromal cells from transgenic pOBCol3.6cyan reporter mice, grown on miR-29a inhibitor scaffolds, displayed increased col3.6 cyan expression as well as collagen production. This study demonstrates that ECM mimicking nanostructured scaffolds, in conjunction with bioactive miRNA-based therapeutics, may serve as a novel platform for developing biologically active localized cell delivery systems.

SUBMITTER: James EN

PROVIDER: S-EPMC4114157 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Post-transcriptional regulation in osteoblasts using localized delivery of miR-29a inhibitor from nanofibers to enhance extracellular matrix deposition.

James Eric N EN Delany Anne M AM Nair Lakshmi S LS

Acta biomaterialia 20140509 8

MicroRNAs are important post-transcriptional regulators of skeletal biology, and miRNA-based therapeutics have the potential to aid bone repair. However, efficient tools for delivering miRNA mimics or inhibitors to specific target tissues are limited. Polymeric nanofibers closely mimic natural extracellular matrix (ECM) morphology, and are attractive candidates for supporting delivery of cells and bone-anabolic reagents. It is hypothesized that gelatin nanofibers could be used for the localized ...[more]

PMID: 24816265

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Project description:Stem cell therapies have shown promise for regenerative treatment for musculoskeletal conditions, but their success is mixed. To enhance regenerative effects, growth factors are utilized to induce differentiation into native cell types, but uncontrollable in vivo conditions inhibit differentiation, and precise control of expressed matrix proteins is difficult to achieve. To address these issues, we investigated a novel method of enhancing regenerative phenotype through direct upregulation of major cartilaginous tissue proteins, aggrecan (ACAN), and collagen II (COL2A1) using dCas9-VPR CRISPR gene activation systems. We demonstrated increased expression and deposition of targeted proteins independent of exogenous growth factors in pellet culture. Singular upregulation of COL2A1/ACAN interestingly indicates that COL2A1 upregulation mediates the highest sulfated glycosaminoglycan (sGAG) deposition, in addition to collagen II deposition. Through RNA-seq analysis, this was shown to occur by COL2A1 upregulation mediating broader chondrogenic gene expression changes. Multiplex upregulation of COL2A1 and ACAN together resulted in the highest sGAG, and collagen II deposition, with levels comparable to those in chondrogenic growth factor-differentiated pellets. Overall, this work indicates dCas9-VPR systems can robustly upregulate COL2A1 and ACAN deposition without growth factors, to provide a novel, precise method of controlling stem cell phenotype for cartilage and intervertebral disc cell therapies and tissue engineering. Impact statement Stem cell therapies have come about as a potential regenerative treatment for musculoskeletal disease, but clinically, they have mixed results. To improve stem cell therapies, growth factors are used to aid a regenerative cell phenotype, but their effects are inhibited by in vivo musculoskeletal disease environments. This article describes CRISPR gene activation-based cell engineering methods that provide a growth factor-free method of inducing chondrogenic extracellular matrix deposition. This method is demonstrated to be as/more potent as growth factors in inducing a chondrogenic phenotype in pellet culture, indicating potential utility as a method of enhancing stem cell therapies for musculoskeletal disease.

Dataset Information

Post-transcriptional regulation in osteoblasts using localized delivery of miR-29a inhibitor from nanofibers to enhance extracellular matrix deposition.

Publications

Post-transcriptional regulation in osteoblasts using localized delivery of miR-29a inhibitor from nanofibers to enhance extracellular matrix deposition.

Similar Datasets

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