Abstract
Millions of patients each year suffer from challenging non-healing bone defects secondary to trauma or disease (e.g. cancer, osteoporosis or osteomyelitis). Tissue engineering approach to non-healing bone defects has been investigated over the past few decades in a search for a novel solution for critical size bone defects. The success of the tissue engineering approach relies on three main pillars, the right type of cells; and appropriate scaffold; and a biologically relevant biochemical/ biophysical stimuli. When it comes to cells the mesodermal origin of mesenchymal stem cells and its well demonstrated multipotentiality makes it an ideal option to be used in musculoskeletal regeneration.
For the presented set of experimental assays, fully characterised (passage 3 to 5)ovine adipose-derived mesenchymal stems cells (Ad-MSC) were cultured either in growth medium (GM) consisting of Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 10% (v/v) foetal bovine serum and 1% penicillin-streptomycin as a control or in osteogenic differentiation medium (DM), consisting of GM further supplemented with L- ascorbic acid (50 μg/ml), β-glycerophosphate (10 mM) and dexamethasone (100nM). Osteogenic differentiation was assessed biochemically by quantifying alkaline phosphatase (ALP) enzyme activity and alizarin red staining after 3, 7, 14 and 21 days in culture (where 1×105 cells/well were seeded in 24 well-plate, n=6/media type/ time point). Temporal patterns in osteogenic gene expression were quantified using real-time PCR for Runx-2, osteocalcin (OC), osteonectin (ON) and type 1 collagen (Col 1) at days 7, 15 and 21 (where 1×105 cells were seeded in T25 cell culture flasks for RNA extraction, n= 4 / gene/ media type/time point). The morphology of osteogenic cells was additionally evaluated by scanning electron microscopy (SEM) of cells seeded at low-density (1×102 cells) on glass coverslips for 2 weeks in GM or DM.
The level of ALP activity of cells grown in osteogenic DM was significantly higher than the control growing in the standard growth medium (p ≤ 0.05) at days 3, 7 and 14. At 21 days there was a sharp drop in ALP values in the differentiating cells. Mineralisation, as evidenced by alizarin red staining, increased significantly by day 14 and then peaked at day 21. Quantitative real-time PCR confirmed early increases in Runx-2, Col 1 and osteonectin, peaking in the second week of culture, while osteocalcin peaked at 21 days of culture. Taken as a whole, these data indicate that ovine-MSCs exhibit a tightly defined pathway of initial proliferation and matrix maturation (up to 14 days), followed by terminal differentiation and mineralisation (days 14 to 21). SEM analysis confirmed the flattened, roughened appearance of these cells and abandoned extracellular matrix which resembled mature osteoblasts.
Given the ready availability of adipose tissues, the use of Ad-MSCs as progenitors for bone tissue engineering applications is both feasible and reasonable. The data from this study indicate that Ad-MSCs follow a predictable pathway of differentiation that can be tracked using validated molecular and biochemical assays. Additional work is needed to confirm that these cells are osteogenic in vivo, and to identifying the best combination of scaffold materials and cell culture techniques (e.g. static versus dynamic) to accelerate or stimulate osteogenic differentiation for bone tissue engineering applications.
