Chondrogenic Priming at Reduced Cell Density Enhances Cartilage Adhesion of Equine Allogeneic MSCs - a Loading Sensitive Phenomenon in an Organ Culture Study with 180 Explants.
BACKGROUND
Clinical results of regenerative treatments for osteoarthritis are becoming increasingly significant. However, several questions remain UNANSWERED concerning mesenchymal stem cell (MSC) adhesion and incorporation into cartilage.


METHODS
To this end, peripheral blood (PB) MSCs were chondrogenically induced and/or stimulated with pulsed electromagnetic fields (PEMFs) for a brief period of time just sufficient to prime differentiation. In an organ culture study, PKH26 labelled MSCs were added at two different cell densities (0.5 x106 vs 1.0 x106). In total, 180 explants of six horses (30 per horse) were divided into five groups: no lesion (i), lesion alone (ii), lesion with naïve MSCs (iii), lesion with chondrogenically-induced MSCs (iv) and lesion with chondrogenically-induced and PEMF-stimulated MSCs (v). Half of the explants were mechanically loaded and compared with the unloaded equivalents. Within each circumstance, six explants were histologically evaluated at different time points (day 1, 5 and 14).


RESULTS
COMP expression was selectively increased by chondrogenic induction (p = 0.0488). PEMF stimulation (1mT for 10 minutes) further augmented COL II expression over induced values (p = 0.0405). On the other hand, MSC markers remained constant over time after induction, indicating a largely predifferentiated state. In the unloaded group, MSCs adhered to the surface in 92.6% of the explants and penetrated into 40.7% of the lesions. On the other hand, physiological loading significantly reduced surface adherence (1.9%) and lesion filling (3.7%) in all the different conditions (p < 0.0001). Remarkably, homogenous cell distribution was characteristic for chondrogenic induced MSCs (+/- PEMFs), whereas clump formation occurred in 39% of uninduced MSC treated cartilage explants. Finally, unloaded explants seeded with a moderately low density of MSCs exhibited greater lesion filling (p = 0.0022) and surface adherence (p = 0.0161) than explants seeded with higher densities of MSCs. In all cases, the overall amount of lesion filling decreased from day 5 to 14 (p = 0.0156).


CONCLUSIONS
The present study demonstrates that primed chondrogenic induction of MSCs at a lower cell density without loading results in significantly enhanced and homogenous MSC adhesion and incorporation into equine cartilage.
Jan H
JH
Spaas
Sarah Y
SY
Broeckx
Koen
K
Chiers
Stephen J
SJ
Ferguson
Marco
M
Casarosa
Nathalie
N
Van Bruaene
Ramses
R
Forsyth
Luc
L
Duchateau
Alfredo
A
Franco-Obregón
Karin
K
Wuertz-Kozak
D000818:Animals; D064236:Cartilage Oligomeric Matrix Protein / Q000378:metabolism; D002448:Cell Adhesion; D002452:Cell Count; D002454:Cell Differentiation; D002478:Cells, Cultured; D020219:Chondrogenesis; D024043:Collagen Type II / Q000378:metabolism; D004574:Electromagnetic Fields; D006736:Horses; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D009924:Organ Culture Techniques / Q000379:methods*
D064236:Cartilage Oligomeric Matrix Protein; D024043:Collagen Type II