C3H10T1/2 Cells Utilize Wnt Signaling During Chondrocyte Differentiation in Scaffolds

Objective: Not only does proper TMJ function rely on smooth articulation provided for by cartilage found on articular surfaces, it is also important in the development of craniofacial and dental supportive structures. Understanding the molecular pathways involved during differentiation in both natural and manipulated systems is important for possible repair of damaged joints. As it is known that wnt signaling is important in cartilage development, we hypothesize wnt is important in chondrocyte differentiation of micromass cultures seeded in non-woven micro-fiber poly (caprolactone) electrospun scaffolds (MPS).

Methods: C3H10T1/2 cells, grown in micromass culture and within MPS, were cultured for 5 days. Cells grown in MPS were compressed 5, 10, 20 and 30% at 0.5 Hz for a duration of 4 hours. Each day and magnitude was assayed by PCR for mRNA expression of genes related to differentiation and wnt signaling.

Results: (i) Cells were characterized by the time needed to differentiate into chondrocytes. Markers chosen in defining differentiation included aggrecan, collagen type II and Sox-9. (ii) Under these conditions, cells seeded in micromass cultures on plates had an increase in these markers by the third day. However, cells seeded in scaffolds had a delayed differentiation time by two days. (iii) Wnts that are expressed in both naked and MPS systems include Wnt4, Wnt 5a, Wnt 7a and Wnt 11. (iv) iNOS was used to assess whether an inflammatory response was induced during compression. Magnitudes of 5 and 10 percent induced negligible iNOS expression over control. Magnitudes of 15, 20 and 30%, however, induced iNOS expression that suggested these magnitudes are pro inflammatory to cells in scaffolds.

Conclusion: Utilizing scaffolds allow the cells to differentiate and be compressed in a three dimensional environment. Scaffolds, more than naked micromass cultures, also have an increased potential for future clinical application. NIH DE017269, DE015399.