ABSTRACT
Silk-based bioresorbable medical devices, such as screws, plates, and rods, have been under investigation due to their promising properties for orthopedic repairs. Options to functionalize these new devices for enhanced control of bone regeneration would also exploit the compatible processing methods used to generate the devices. MicroRNAs are important regulators of bone maintenance and formation, and miRNA-based therapeutics have the potential to aid bone repair, utilizing a transient therapeutic approach with local bioactivity. We hypothesized that silk-based orthopedic devices could be used for the local delivery of miRNAs, using anti-sense miR-214 (AS-miR-214), to inhibit endogenous expression of osteoinductive antagonist and thereby supporting the upregulation of osteoinductive target molecules activating transcription factor 4 (ATF4) and Osterix (Osx). AS-miR-214 silk devices, prepared using surface coating, demonstrated continuous release of miRNA inhibitors up to 7 days in vitro. Additionally, human mesenchymal stem cells seeded on AS-miR-214 silk films expressed higher levels of osteogenic genes ATF4, Osx, Runx2, and Osteocalcin. Interestingly, these cells exhibited lower cell viability and DNA content over 21 days. Conversely, the cells demonstrated significantly higher levels of alkaline phosphatase expression and calcium deposition compared with cells seeded on silk films with nontargeting miRNA controls. The study demonstrated that the silk-based orthopedic devices, in conjunction with bioactive miRNA-based therapeutics, may serve as a novel system for localized bone tissue engineering, enhancing osteogenesis at the implant interface while avoiding detrimental systematic side effects.
Subject(s)
Biocompatible Materials , Bone Regeneration/drug effects , Mesenchymal Stem Cells/metabolism , Osteogenesis/drug effects , RNA, Antisense , Silk , Tissue Engineering/methods , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Cell Survival , Human Umbilical Vein Endothelial Cells , Humans , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , Orthopedics , RNA, Antisense/chemistry , RNA, Antisense/pharmacology , Silk/chemistry , Silk/pharmacologyABSTRACT
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.
Subject(s)
Extracellular Matrix/metabolism , MicroRNAs/antagonists & inhibitors , Nanocapsules/chemistry , Nanofibers/chemistry , Oligonucleotides/administration & dosage , Osteoblasts/physiology , Animals , BALB 3T3 Cells , Gene Expression Regulation/physiology , Mice , MicroRNAs/metabolism , Nanocapsules/administration & dosage , Nanocapsules/ultrastructure , Nanofibers/ultrastructure , Oligonucleotides/chemistry , Osteoblasts/cytology , Osteoblasts/drug effects , RNA Processing, Post-Transcriptional/drug effects , RNA Processing, Post-Transcriptional/physiologyABSTRACT
Lactoferrin loaded poly (epsilon-caprolactone) nanofibers were fabricated using the process of electrospinning and the osteocompatibility of the scaffolds were evaluated using MC3T3-E1 osteoblast-like cells. Morphology of rhLF/PCL scaffolds was determined by scanning electron microscopy. Fourier transform infrared spectroscopy and Energy-dispersive X-ray spectroscopy confirmed the incorporation of rhLF in the PCL nanofibers. The surface distribution of rhLF on the nanofibers was evaluated using fluorescently tagged (Far-red) rhLF. The presence of rhLF on rhLF/PCL nanofibers significantly increased the proliferation and viability of MC3T3-E1 cells compared to cells seeded on PCL nanofibers as evidenced from Ki67 immuno-staining and MTT assay. The study demonstrated the feasibility of incorporating different concentrations of rhLF in PCL nanofibers and the potential of the fiber matrix to support osteoblast cell functions.
