Redifferentiation of Dedifferentiated Bovine Articular Chondrocytes Enhanced by Cyclic Hydrostatic Pressure Under a Gas-Controlled System

Abstract

Hydrostatic pressure is one of the most frequently used mechanical stimuli in chondrocyte experiments. A variety of hydrostatic pressure loading devices have been used in cartilage cell experiments. However, no gas-controlled system with other than a low pressure load was used up to this time. Hence we used a polyolefin bag from which gas penetration was confirmed. Chondrocytes were extracted from bovine normal knee joint cartilage. After 3 passages, dedifferentiated chondrocytes were applied to form a pellet. These pellets were cultured in chemically defined serum-free medium with ITS+Premix for 3 days. Then 5 MPa of cyclic hydrostatic pressure was applied at 0.5 Hz for 4 h per day for 4 days. Semiquantitative reverse transcriptase-polymerase chain reaction showed a 5-fold increase in the levels of aggrecan mRNA due to cyclic hydrostatic pressure load (p<0.01). Type II collagen mRNA levels were also upregulated 4-fold by a cyclic hydrostatic pressure load (p<0.01). Type I collagen mRNA levels were similarly reduced in the cyclic hydrostatic pressure load group and in the control group. The partial oxygen pressure (PO2) and partial carbon dioxide pressure (PCO2) of the medium in the bag reached equilibrium in 24 h, and no significant change was observed for 3 days afterwards. PO2 and PCO2 were very well controlled. The loaded pellet showed better safranin O/fast green staining than did the control pellet. Metachromatic staining by Alcian blue staining was found to be stronger in the loaded than in the control pellets. The extracellular matrices excretion of loaded pellets was higher than that of control pellets. These results suggest that gas-controlled cyclic hydrostatic pressure enhanced the cartilaginous matrix formation of dedifferentiated cells differentiated in vitro.

Spontaneous Redifferentiation of Dedifferentiated Human Articular Chondrocytes on Hydrogel Surfaces

“…Such behavior has been observed when the cells were embedded in pellets, alginate beads, or polymer gels, even combined with pressure stimulation. [20][21][22][23][24][25] For tissue engineering applications, the redifferentiated cells have to be isolated and collected from the 3D scaffolds, which severely limits the choice of scaffolds. Biodegradable polymer materials are suitable 3D scaffolds.…”

Gene expression profiles of human chondrocytes during passaged monolayer cultivation

“…23 This stable expression of Sox9 in serially passaged chondrocytes suggests that other transcription factors may govern the dedifferentiation process, and that it may be reversible. [24][25][26][27] Chondrocytes maintain a homeostatic anabolic and catabolic balance of extracellular matrix (ECM) via regulation of secreted matrix proteinases and protease inhibitors to maintain the composition and structural integrity of ECM in articular cartilage. 28,29 In cultured chondrocytes, the expression levels of the matrix proteinases diminished as compared to that of intact cartilage, Figure 5.…”

A flow perfusion bioreactor with controlled mechanical stimulation: Application in cartilage tissue engineering and beyond

“…To investigate the effectiveness of the newly developed bioreactor in the context of scaffolds, we encapsulated primary bovine AChs in agarose hydrogels [109]. We chose bAChs as they have been widely used in ACTE [110,111], and represent an inexpensive, accessible cell source, which avoids challenges posed by human-derived cells which are limited in availability as they are harvested from patient joint replacements or cadavers. Moreover, agarose was used as the scaffold of choice because it supports growth and chondrogenic differentiation, and reverses dedifferentiation phenomena; that is, dedifferentiated chondrocytes (Chs) re-express the differentiated phenotype after encapsulation in agarose [112].…”