Abstract
Introduction
MSCs have long promised benefits of synthesising bone/cartilage, treating non-unions and potentially accelerating fracture repair. This potential has been tempered by MSC scarcity in the ‘gold-standard’ iliac crest bone marrow aspirate (ICBMA) and the resulting need to expand numbers via cell-culture. Culture of MSCs is time-consuming, expensive and results in cells with a reduced differentiation capacity.
The reamer-irrigator-aspirator (RIA) is an innovation designed to reduce intra-medullary (IM) pressures during reaming of long-bones via continuous irrigation and suction. Aspirated contents are passed via a coarse filter, which traps bony-fragments before moving into a ‘waste’ bag - from which MSCs have been previously isolated. We examined liquid and solid phases found in this ‘waste’, performed a novel digestion of the solid phase and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA.
Methods
The filtrate ‘waste’ bag from RIA reaming (6 patients) was filtered (70μm) and the solid fraction digested for 60min (37°C) with collagenase. MSCs were isolated from liquid & solid fractions and from 10ml matched ICBMA. Enumeration of MSCs was achieved via colony-forming-unit-fibroblast (CFUF) assay and flow-cytometry on fresh sample using CD45low, CD271+. MSCs were cultured by virtue of their plastic adherence and passaged in standard, non-haematopoietic media. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages with their phenotype assessed with flow cytometry CD33 CD34 CD45 CD73 CD90 CD105.
Results
We found MSCs were in all fractions/patients. Using the CFU-F assay median number of colonies: ICBMA=8 (2–21), RIA-liquid=12 (4–41), RIA-solid=115 (67–200) per 200μl of sample. Total yield of cells was calculated from volume of sample: ICBMA=670 (228–4275), RIA-liquid=39000 (16500–83700), RIA-solid=9400 (7210–28475). MSC frequency as a percentage of total cells using flow-cytometry on fresh sample found similar frequencies. MSCs isolated from the RIA phases differentiated into osteogenic, chondrogenic and adipogenic lineages at least as well as ICBMA. Passaged (P2) cells, from all fractions/patients, had a phenotype consistent with other reported sources.
Discussion
The RIA filtrate bag is typically discarded at operation. These results show that this ‘waste’ represents a significant source of MSCs that could be isolated for autologous/allogenous use. Concentration of the liquid-phase/brief enzymatic digestion of the solid-phase offers the possibility of large numbers of MSCs being obtained without/with minimal culture expansion.
