2016 Fiscal Year Research-status Report
Role of intra and extracellular miRNAs in stem cell-based bone regeneration
| Project/Area Number |
16K20492
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| Research Institution | Niigata University |
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Principal Investigator |
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| Project Period (FY) |
2016-04-01 – 2018-03-31
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| Keywords | miRNA / Mesenchymal Stem Cells / Bone |
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| Outline of Annual Research Achievements |
For implant placement, some patients require bone augmentation; recently a new method for tissue regeneration involves the use of microRNAs to regulate cell metabolism and differentiation. miRNAs play an important role regulating local cell to cell communication in situ, but also at distant sites moving throughout the blood stream. These research aims to elucidate the potential use of miRNA in bone regeneration through cell therapy as an alternative to the current more invasive methods.
BMCs derived cells were isolated from 4-week-old Wistar rat’s femur and tibia by flushing. Collected cells were expanded in a 100 mm plastic culture dish with alpha-MEM containing 10% Fetal bovine Serum, and 1% Penicillin/Streptomycin and maintained in 5% CO2 at 37 C until they reached 80% confluence.
After cell expansion, cells were seeded in 35 mm dish in concentration of 1 x 10＾5 ml. Two medium conditions were used. A normal condition (Control) and osteogenic induction condition (Induced). Control consisted in a medium containing alpha-MEM with 50 ug/ml of ascorbic acid (A.A); Osteogenic induction medium contained 2ml of alpha-MEM with 50 ug/ml of ascorbic acid (A.A), 2uM β-Glicerophosphate (β-G); and 10nM of Dexamethasone (Dex). Three time points (0 weeks, 1 week and 3 weeks) were selected to study the changes in cell characteristics.
Differentiation was analyzed by Alkaline phosphatase (ALP) Staining. Cells were fixed with 4% formaldehyde for 30 min., washed with DW and stained for 30 min with a solution of fast violet B and Naphthol AS-MX phosphate alkaline.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
To investigate the extent of specific miRNAs in bone regeneration a screening of osteogenic-related miRNAs in MSC is required. miRNA Microarray Screening results are insufficient to properly select relevant osteogenic related miRNA.
Osteogenic gene expression of selected miRNA are required to analyze, compared and determine differences in gene expression.
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| Strategy for Future Research Activity |
Preparation of transplants: To investigate the extent of specific miRNAs in bone regeneration; administration of the specific miRNAs in angiogenesis and endogenous stem cell homing will be analyzed by utilizing calvarial bone defect model. Either miRNA transfected or non-treated control cells will be prepared.
Defect Operation: Under deep anesthesia, 5 mm diameter circular defect will be created on calvaria of 8-week old male Wistar rats. Cell containing collagen gel will be transplanted to the defects and analyze after 1, 2, 4 and 8 weeks.
Micro CT analysis: After the experimental periods, analysis of the samples will be performed according to the guidelines using a ELE-Scan MicroCT machine to obtain images of the defect area. 3D reconstruction and analysis will be done using a specialized software (Tri-Bon software, Ratoc, Japan).
Histology and histomorphometry: Samples will be then cut into histological section using a microtome to a thickness of 5 um and placed onto glass slides.
By immunohistochemistry, Angiogenesis will be analyzed on histology preparations using anti-Endomucin and anti-CD31 antibody, and MSC recruitment using anti-CD29, anti-CD90 and anti-Stro-1 antibodies.
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| Causes of Carryover |
Microarray Screening results are insufficient to properly select relevant osteogenic related miRNA, thus subsequent steps and procedures where not performed due to this reason.
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| Expenditure Plan for Carryover Budget |
Screening of osteogenic-related miRNAs in MSC is required. miRNA Microarray Screening results must be obtained to properly select relevant osteogenic related miRNA.
Osteogenic gene expression of selected miRNA are required to analyze, compared and determine differences in gene expression in order to achieve the goals of the research.
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Research Products
(6 results)
