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

Abstract

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). Conclusion: 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.

Tenogenically Induced Allogeneic Peripheral Blood Mesenchymal Stem Cells in Allogeneic Platelet-Rich Plasma: 2-Year Follow-up after Tendon or Ligament Treatment in Horses

“…Both tenogenically induced MSCs and PRP were prepared from peripheral blood (PB) from a 6-year-old German Warmblood horse according to good manufacturing practices and characterized as previously described ( 21 – 23 ).…”

“…All batches between P6 and P8 were seeded in T75 flasks and tenogenically induced by supplementing tendogenic growth factors to the expansion medium until 80% confluency was reached and tenogenic differentiation markers were upregulated ( 19 , 21 , 22 ). A higher passage was used to allow upscaling in future cell manufacturing processes, which is considered to be possible thanks to the phenotypic stability of PB-MSCs from P5 up to P10 ( 21 , 23 , 24 ). After trypsinization, all the cells were resuspended in 1 ml DMEM low glucose (Life Technologies) with 10% of dimethyl sulfoxide (Sigma).…”

Enhancement of mesenchymal stem cell chondrogenesis with short-term low intensity pulsed electromagnetic fields

“…The reported consequences of PEMF-stimulation over chondrogenesis, however, are largely inconsistent. Some studies report modest enhancements in the gene expression of Sox9, aggrecan, type II collagen (Col 2) as well as deposition of sulfated glycosaminoglycan (sGAG), typically on the order of 2-folds 35 , 36 , whereas other studies show little to no effect 38 – 41 . On the extreme end of the spectrum, Wang et al .…”

Tenogenically Induced Allogeneic Mesenchymal Stem Cells for the Treatment of Proximal Suspensory Ligament Desmitis in a Horse

“…Going to P7, 0.5 million MSCs were seeded in a T75 flask and tenogenically induced by supplementing tendogenic growth factors to the expansion medium until 80% confluency was reached, and tenogenic differentiation markers were upregulated (Gomiero et al, in review). A higher passage was used to allow upscaling in future cell manufacturing processes, which is considered to be possible thanks to the phenotypic stability of PB–MSCs from P5 up to P10 ( 23 , Gomiero et al, in review). After trypsinization, all the cells were resuspended in 1 ml of DMEM low glucose (Life Technologies) with 10% of dimethyl sulfoxide (DMSO, Sigma).…”

“…The samples were stored in −80°C until all quality controls were completed. The latter consisted of sterility testing and flow cytometry assessment for (stem) cell markers, as previously reported ( 22 , 23 ).…”