Abstract
Introduction/Objectives
Clinical applications of Mesenchymal Stem Cells (MSCs) harvested from bone marrow (BM) are well established. Iliac crest (IC) is considered to be the gold standard. Vertebral body (VB) BM represents an alternative source, promoting bone fusion in spinal operations. Existing literature, comparing ICBM and VBBM MSCs, presents limitations. Density gradient centrifugation (lymphoprep, Lmp) for BM processing is used, despite the known effectiveness on ICBM, of the red cell lysis technique (ammonium chloride, AC). This study compared the yield and differentiation capacities of MSCs in VBBM and ICBM, and evaluated the effectiveness of two harvesting procedures on both BM sources.
Patient/Methods
Eighteen patients undergoing idiopathic scoliosis surgery were recruited (median age 15.4 years, range 13–17). BM was aspirated intraoperatively from IC (10ml) and 12th thoracic vertebra (10ml). Aspirates were processed using AC and Lmp. MSCs were enumerated using standard CFU-F assay. Colony areas, MSCs doubling rates and outcomes of the differentiation assays were statistically analyzed.
Results
AC yielded more leukocytes from ICBM (23x106cells/ml), compared with VBBM (14.4x106cells/ml, p = 0.013). Lmp yielded similar numbers of mononuclear cells from ICBM (7.5x106cells/ml) and VBBM (5x106cells/ml).
More MSCs were harvested from 1ml of BM aspirate using AC technique, from both anatomical sites (IC: Lmp 199MSCs/ml, AC 367MSCs/ml, p = 0.012; VB: Lmp 296MSCs/ml, AC 440MSCs/ml, p = 0.043). There were no significant differences in MSC numbers/ml and average colony areas between the sites (IC: AC 14mm2, Lmp 15mm2; VB: AC 11mm2, Lmp 13mm2) or MSCs doubling rates in days (IC: AC 2, Lmp 1.9, VB: AC 1.9, Lmp 1.8).
Calcium production, after 14 days of osteogenic induction, was higher in VBBM MSCs with both harvesting methods (AC: 15-fold, p = 0.003, Lmp: 6.6-fold). Adipogenic differentiation, measured by Nile-Red absorbance on day 21 post-induction, was not significantly different between two BM sources. Glycosaminoglycan (GAG) production, after 21 days of chondrogenic induction, was higher in VBBM MSCs (AC 2.1μg/ml, Lmp 3.7μg/ml) compared with ICBM (AC 1.2μg/ml, Lmp 2.9μg/ml), but not statistically significant.
Conclusion
AC results in more effective MSC isolation and does not affect MSCs’ proliferation and differentiation. Compared with ICBM, VBBM is richer in MSCs with osteo- and chondrogenic differentiation capacities and can therefore represent the first choice for MSC harvesting in spinal surgery.