MicroRNAs (miRNAs ) are small non-coding RNAs
that regulate gene expression. We hypothesised that the functions
of certain miRNAs and changes to their patterns of expression may
be crucial in the pathogenesis of nonunion. Healing fractures and
atrophic nonunions produced by periosteal cauterisation were created
in the femora of 94 rats, with 1:1 group allocation. At post-fracture
days three, seven, ten, 14, 21 and 28, miRNAs were extracted from
the newly generated tissue at the fracture site. Microarray and
real-time polymerase chain reaction (PCR) analyses of day 14 samples
revealed that five miRNAs, miR-31a-3p, miR-31a-5p, miR-146a-5p,
miR-146b-5p and miR-223-3p, were highly upregulated in nonunion.
Real-time PCR analysis further revealed that, in nonunion, the expression
levels of all five of these miRNAs peaked on day 14 and declined
thereafter. Our results suggest that miR-31a-3p, miR-31a-5p, miR-146a-5p,
miR-146b-5p and miR-223-3p may play an important role in the development
of nonunion. These findings add to the understanding of the molecular mechanism
for nonunion formation and may lead to the development of novel
therapeutic strategies for its treatment. Cite this article:
The scarcity of mesenchymal stem cells (MSCs) in iliac crest bone marrow aspirate (ICBMA), and the expense and time in culturing cells, has led to the search for alternative harvest sites. The reamer-irrigation-aspirator (RIA) provides continuous irrigation and suction during reaming of long bones. The aspirated contents pass via a filter, trapping bony fragments, before moving into a ‘waste’ bag from which MSCs have been previously isolated. We examined the liquid and solid phases, performed a novel digestion of the solid phase, and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA. The solid fraction from the filtrate was digested for 60 minutes at 37°C with collagenase. Enumeration was performed via the colony-forming unit fibroblast (CFU-F) assay. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages, and their phenotypes assessed using flow cytometry (CD33, CD34, CD45, CD73, CD90, and CD105). MSCs from the RIA phases were able to differentiate at least as well as those from ICBMA, and all fractions had phenotypes consistent with other established sources. The median number of colonies for the three groups was: ICBMA = 8.5 (2 to 86), RIA-liquid = 19.5 (4 to 90), RIA-solid = 109 (67 to 200) per 200 μl. The mean total yield of cells for the three groups was: ICBMA = 920 (0 to 4275), RIA-liquid = 114 983 (16 500 to 477 750), RIA-solid = 12 785 (7210 to 28 475). The RIA filtrate contains large numbers of MSCs that could potentially be extracted without enzymatic digestion and used for bone repair without prior cell expansion.
