Comparison of isolation techniques for bone marrow derived canine mesenchymal stem cells (MSCs) and the compatibility of MSCs loaded onto polycarpolactone hydroxyapatite

Abstract

Bone marrow was collected from 7 dogs and submitted to 3 different MSC isolation techniques (direct plating,
red blood cell lysis and gradient density). The number of cells, expression of MSC markers and in vitro the osteogenic
differentiation obtained from each technique were examined. In order to study in vivo the osteogenic capability of
derived MSCs, non-union ulna lesions (n=3) were firstly induced by transplantation of a composite of
polycarprolactone/ hydroxyapatite (PCL/HAp) scaffold experimentally on to a critical-size ulna bone defect. The
MSCs were injected into the lesions. The non-union sites were examined by radiography, angiography and histology
at 2, 4, 6, 8 and 12 weeks after MSC injection. Gradient density and RBC lysis techniques yielded higher numbers of
putative MSCs on day 7 of culture compared with the direct plating technique. A large proportion of isolated MSCs,
irrespective of the isolation techniques, expressed all MSC markers (CD 44 and CD 90). For all MSC transplanted dogs,
neither radiological changes at scaffold-ulna interface nor callus formation was observed, although all donors used
demonstrated in vitro osteogenesis. At 16 weeks after MSC injection, the angiogram indicated increased
neovascularization. This was confirmed by the histological finding that there was an improvement of vascularization
within the thick-fibrous tissue surrounding the scaffold. Gradient density and RBC lysis treatment are suitable MSC
isolation techniques, in terms of the numbers of cells obtained and also their MSC properties. However, potential use
of these MSCs following injection to a non-union bone site was compromised possibly because of a lack of osteogenic
stimulation.