Abstract
Introduction
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].
Methods
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.
Results
The highest frequencies of MSCs/CFU-Fs (per 200μl of sample) were found within RIA-solid solid fraction (median 115, range 67-200) compared to ICBMA and RIA-liquid (8, 2-21 and 12, 4-41, respectively). Due to much larger volume of RIA-liquid, it contained the highest total yield of MSCs/CFU-Fs (114983, range 16500-477750), which was equivalent to ∼1137 ml of ICBMA. RIA-solid contained ∼10% of all MSCs within the “waste bag” (12785,7210-28475). MSCs isolated from the RIA phases were able to differentiate into osteogenic, chondrogenic and adipogenic lineages at least as well as matched ICBMA and had a phenotype consistent with MSCs (CD73+ CD90+ CD105+ CD33- CD34- CD45-).
Conclusions
The RIA filtrate bag contains massive numbers of MSCs that could potentially be therapeutically used without prior culture-expansion.
