Intermittent hydrostatic pressure maintains and enhances the chondrogenic differentiation of cartilage progenitor cells cultivated in alginate beads

Abstract

The objective of this study was to explore the effects of intermittent hydrostatic pressure (IHP) on the chondrogenic differentiation of cartilage progenitor cells (CPCs) cultivated in alginate beads. CPCs were isolated from the knee joint cartilage of rabbits, and infrapatellar fat pad-derived stem cells (FPSCs) and chondrocytes (CCs) were included as the control cell types. Cells embedded in alginate beads were treated with IHP at 5 Mpa and 0.5 Hz for 4 h/day for 1, 2, or 4 weeks. The cells' migratory and proliferative capacities were evaluated using the scratch and Live/Dead assays, respectively. Hematoxylin and eosin staining, safranin O staining, and immunohistochemical staining were performed to determine the effects of IHP on the synthesis of extracellular matrix (ECM) proteins. Real-time polymerase chain reaction analysis was performed to measure the expression of genes related to chondrogenesis. The scratch and Live/Dead assays revealed that IHP significantly promoted the migration and proliferation of FPSCs and CPCs to different extents. The staining experiments showed greater production of cartilage ECM components (glycosaminoglycans and collagen II) by cells exposed to IHP, and the gene expression analysis demonstrated that IHP stimulated the expression of chondrocyte-related genes. Importantly, these effects of IHP were more prominent in CPCs than in FPSCs and CCs. Considering all of our experimental results combined, we conclude that CPCs demonstrated a stronger chondrogenic differentiation capacity than the FPSCs and CCs under stimulation with IHP. Thus, the use of CPCs, combined with mechanical stimulation, may represent a valuable strategy for cartilage tissue engineering.

Cells under pressure – the relationship between hydrostatic pressure and mesenchymal stem cell chondrogenesis

“…This indicates the importance of early matrix formation, especially pericellular matrix, to enable transmission of the compressive force (mechanotransduction), mimicking that produced in in vivo cartilage. Similar results have been found varying the point of hydrostatic loading on differentiating chondrogenic MSCs (Li et al, 2012;Li et al, 2016 In contrast to compressive and shear loading studies, application of hydrostatic pressure at the onset produces more matrix formation (GAG, collagens I, II) and increased mechanical properties (Young's and dynamic modulus) as compared to pre-differentiated MSCs. Despite no in-depth studies regarding the effect of different periods of pre-differentiation, it may be hypothesised that hydrostatic pressure has a larger effect on MSC chondrogenesis during its early stages as suggested in computational/theoretical models, whilst compressive loading exerts its influence on pre-differentiated MSCs, specifically at later stages of chondrogenesis.…”

“…In agreement with these results, Safeshaken et al 2012, using a pellet culture model, demonstrated no increase in chondroinduction without TGF-β addition using a loading regime (0-5 MPa at 0.5 Hz for 4 h/d) for 10 d only. Thus, whether hydrostatic pressure alone is sufficient to induce chondrogenesis to any significant degree remains unresolved (Carroll et al, 2014;Finger et al, 2007;Li et al, 2012;Li et al, 2009;Puetzer et al, 2013;Vinardell et al, 2012;Ye et al, 2014) (Table 2).…”

“…However, inhibition of TGF-β receptor I only partially inhibits the anabolic response caused by hydrostatic pressure, indicating that other pathways are involved (Li et al, 2012). Non-canonical SMAD pathways have also been investigated due to their association with TGF-β-induced chondrogenesis; specifically, mitogen-activated protein (MAP) kinases including extracellular-regulated protein kinase-1 (ERK1), p38 and c-Jun N-terminal kinases (JNK) (Li et al, 2009;Sakao et al, 2008;Zhao et al, 2016;Zhao et al, 2015). Sakao et al (2008) reported that application of hydrostatic pressure in the presence of ERK and p38 inhibitors does not inhibit expression of SOX9, COL2A1 and proteoglycan core protein but addition of a JNK inhibitor prevents upregulation of these genes.…”

Effects of hydrostatic pressure and deviatoric stress on human articular chondrocytes for designing neo‐cartilage construct

“…We demonstrated that the changes in HP alone, DS alone, and combined HP and DS followed by off‐loading altered the cell proliferation capacity and metabolism of ECM of hACs within an agarose hydrogel. In most studies conducted to clarify the effects of physical stresses on articular cartilage, a cartilage explant model has been used to apply compressive stresses ex vivo (Elder et al, ; Li et al, ; Sun et al, ). This explant model could reproduce compressive stresses within relatively short regimens; however, this model was often limited by its irregular sample shape due to deformation caused by overloading and/or frequent cycle of loading.…”

“…Thus, we sought to understand the roles of these stresses on metabolic turnover by chondrocytes. Recently, the effects of compressive stresses on metabolic functions in articular cartilage have been investigated extensively using alginate (Elder et al, ; Li, Zhou, Yang, Jiang, & Gui, ) or agarose (Chowdhury, Bader, Shelton, & Lee, ; Pingguan‐Murphy & Nawi, ) in vitro and ex vivo models (Kraft et al, ; Pascarelli, Collodel, Moretti, Cheleschi, & Fioravanti, ). However, these models could not adequately reproduce stress‐loaded cells and tissue to investigate the effects of pure HP, pure DS, and their combination on the metabolic profile of chondrocytes.…”