Spaas Jan H, Broeckx Sarah Y, Chiers Koen, Ferguson Stephen J, Casarosa Marco, Van Bruaene Nathalie, Forsyth Ramses, Duchateau Luc, Franco-Obregón Alfredo, Wuertz-Kozak Karin
Cell Physiol Biochem. 2015;37(2):651-65. doi: 10.1159/000430384.
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.
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).
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).
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.
骨关节炎再生治疗的临床效果日益显著。然而，关于间充质干细胞（MSC）黏附并整合到软骨中的几个问题仍未得到解答。
为此，对来自外周血（PB）的间充质干细胞进行软骨诱导和/或用脉冲电磁场（PEMF）刺激一小段时间，仅足以启动分化。在一项器官培养研究中，以两种不同的细胞密度（0.5×10⁶ 对 1.0×10⁶）添加 PKH26 标记的间充质干细胞。总共，将六匹马的 180 个外植体（每匹马 30 个）分为五组：无损伤组（i）、单纯损伤组（ii）、损伤加未处理的间充质干细胞组（iii）、损伤加软骨诱导的间充质干细胞组（iv）以及损伤加软骨诱导且经 PEMF 刺激的间充质干细胞组（v）。一半的外植体进行机械加载，并与未加载的对应外植体进行比较对照。在每种情况下，在不同时间点（第 1 天、第 5 天和第 14 天）对六个外植体进行组织学评估。
软骨诱导选择性地增加了 COMP 的表达（p = 0.0488）。PEMF 刺激（1mT，持续 10 分钟）使 COL II 的表达比诱导后的水平进一步增加（p = 0.0405）。另一方面，诱导后间充质干细胞标志物随时间保持恒定，表明其处于主要的预分化状态。在未加载组中，间充质干细胞在 92.6%的外植体中黏附于表面，并在 40.7%的损伤处渗透进去。另一方面，在所有不同条件下，生理加载显著降低了表面黏附（1.9%）和损伤填充（3.7%）（p < 0.0001）。值得注意的是，软骨诱导的间充质干细胞（无论是否经 PEMF 刺激）的特征是细胞分布均匀，而在 39%未诱导的间充质干细胞处理的软骨外植体中出现了细胞团块形成。最后，接种中等低密度间充质干细胞的未加载外植体比接种较高密度间充质干细胞的外植体表现出更大的损伤填充（p = 0.0022）和表面黏附（p = 0.0161）。在所有情况下，从第 5 天到第 14 天，损伤填充的总量均有所下降（p = 0.0156）。
本研究表明，在较低细胞密度下对间充质干细胞进行启动软骨诱导且不加载，会显著增强并使间充质干细胞均匀地黏附并整合到马软骨中。
