Sox9 expression of alginate‐encapsulated chondrocytes is stimulated by low cell density

Abstract

Recent research in tissue engineering for the treatment of cartilage defects have demonstrated that matrix-biomaterial, cell culture conditions, and cytokine-related factors influence the chondrogenic differentiation pattern, especially for the expression of matrix genes. However, little is known about the impact of cell seeding density in a three-dimensional environment on the key chondrogenic transcription factor Sox9. Here we investigated, whether the cell concentration of alginate encapsulated chondrocytes influences the Sox9 expression. Dedifferentiated passage-4 porcine chondrocytes were encapsulated in alginate beads at two different concentrations (4 x 10(6) versus 7 x 10(7) cells/mL) and cultivated for up to 4 weeks under TGF-ss stimulation. The expression of Sox9, Collagen I, II, and X was assessed via quantitative RT-PCR and compared to those observed in the initial monolayer culture. Cellular viability, cell morphology, and the sulphated glycosaminoglycan-production were monitored. Interestingly Sox9 expression was significantly upregulated in the low-cell-density group, whereas no difference between high-cell-density and monolayer culture group could be observed. Furthermore, the cellular survival and the sulphated glycosaminoglycan production were higher in the low-cell-density group. Collagen I expression was downregulated in the low-cell-density group whereas it was upregulated in the high-cell-density one. Surprisingly, only the high-cell-density group showed the expression of Collagen II, although it appeared not significant. Collagen X expression was upregulated in the low-cell-density group. Taken together our data indicate that a low concentration of cell seeding in a three-dimensional environment is beneficial for the overall chondrogenic development. However, this article reveals discrepancies between Sox9 and the chondrogenic pathway in redifferentiating chondrocytes that should be addressed in further work.

Osteoarthritis in the Knee Joints of Göttingen Minipigs after Resection of the Anterior Cruciate Ligament? Missing Correlation of MRI, Gene and Protein Expression with Histological Scoring

“…A variability of collagen II distribution in animals was also seen by Bernstein et al [23]. Another publication by Barley et al [133] reported an increased staining of collagen II and a decreased staining of collagen I in normal cartilage.…”

“…The pig and notably the Göttingen Minipig (GM) is often used in articular cartilage research [5,17,23,24] and is also a common model for the study of regeneration of focal cartilage defects [7,25,26] but the transection of the ACL has rarely been performed in this species up to now [6]. For the articular cartilage research skeletal mature individuals are required as older pigs may mirror the human condition better, as OA is mostly an affliction of older people whether or not there were previous injuries [27,28].…”

Safety of three different product doses in autologous chondrocyte implantation: results of a prospective, randomised, controlled trial

“…In a study conducted for approval of an autologous cell product by the European Medicines Agency (EMA), cell densities in this range were also used [7, 8]. However, there is still a debate as to whether an increased cell density can lead to better cartilage filling, better morphological structure biomechanical properties and/or a more favourable clinical outcome [9, 10]. Thus, there remains a crucial need for studies evaluating the efficacy and safety of different cell densities and product doses.…”

Enhancing Post-Expansion Chondrogenic Potential of Costochondral Cells in Self-Assembled Neocartilage

“…Using articular chondrocytes, high density 3D systems promote the formation of cartilaginous matrix and phenotypic stability [22]. In a variety of tissue engineering systems involving alginate, agarose, and fibrin scaffolds, it has been demonstrated that chondrocyte phenotype, matrix deposition, and mechanical properties depend on initial seeding density [23]–[27]. Significantly, in a study of cell concentration in alginate beads, SOX9 expression and sulfated glycosaminoglycan (GAG) production were upregulated in porcine ACs encapsulated at low density, compared with high density [23].…”