Iliac crest bone marrow aspirate (ICBMA) is frequently cited as the ‘gold-standard’ source of MSCs. MSCs have been shown to reside within the intramedullary (IM) cavities of long-bones [Nelea, 2005] however a comparative assessment with ICBMA has not yet been performed and the phenotype of the latter compartment MSCs remains undefined in their native environment. Aspiration of the IM cavities of 6 patients' femurs with matched ICBMA was performed. The long-bone-fatty-bone-marrow (LBFBM) was filtered (70μm) to separate liquid and solid fractions and the solid fraction was briefly (60min, 37oC) digested with collagenase. MSC enumeration was performed using the colony-forming-unit-fibroblast (CFU-F) assay and quantification of cells with the CD45low CD271+ phenotype by flow-cytometry. [Jones 2002, Buhring 2007] MSCs were cultured and standard expansion media and passage 2 cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages.Introduction
Methods
Therapeutic exploitation of MSCs in orthopaedics has been tempered by their scarcity within ‘gold-standard’ iliac crest bone marrow aspirate (ICBMA) and the resulting need to expand cells in vitro. This is time-consuming, expensive and results in cells with a reduced differentiation capacity. [Banfi 2000] The RIA is a device that provides continuous irrigation and suction during reaming of long bones. Aspirated contents pass via a filter, trapping bony-fragments, before moving into a ‘waste’ bag, from which MSCs have been previously isolated. [Porter 2009] We hypothesised that ‘waste’ RIA bag contains more MSCs than a standard aspirated volume of ICBMA (30 ml). We further hypothesised than a fatty solid phase within this ‘waste bag’ contains many MSCs trapped within the adipocyte-rich stromal network and hence requiring an enzymatic digestion for their efficient release [Jones 2006]. The discarded filtrate ‘waste’ bag that contained saline from marrow cavity irrigation procedure from RIA reaming (7 patients) was filtered (70μm) and the solid fraction digested for 60min (37oC) with collagenase. MSC enumeration was performed using the colony-forming-unit-fibroblast (CFU-F). Following culture in standard expansion media, passage 2 cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages and their phenotype was assessed using flow cytometry. ICBMA from the same patients was used as controls.Introduction
Methods
