Figure 3.

Engineering hypertrophic cartilaginous tissue directly from human adipose tissue: (a) Human adipose tissue was harvested during liposuction surgery; the human adipose tissue was positive for COL IV but negative for fibronectin, COL II, and COL X. After 3 weeks of culture in proliferation medium, the adipose tissue was positive for COL IV and fibronectin but negative for COL II and COL X, indicating that proliferative culture results in more stromal cells in the adipose tissue. (b) Then, the cultured adipose tissue was subjected to endochondral priming. After 4 weeks of chondrogenic priming, the engineered constructs showed a cartilaginous phenotype, which was characterized by positive safranin O staining for GAG, weakly positive staining for COL I and COL X, and strongly positive staining for COL II. The chondrogenically primed constructs were cultured in HYM for 2 weeks, which resulted in strong positive staining for COL X, (c) The endochondrally primed constructs were subcutaneously implanted into nude mice for 12 weeks. MicroCT scanning of the retrieved constructs showed a bony shell around bone trabeculae inside. Bone tissue formation and morphological evidence of bone marrow in the retrieved constructs were identified histologically by hematoxylin and eosin (H&E) staining and osteocalcin (OCN) staining. Intensive bone resorption by osteoclasts, characterized as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, was observed in the inner margins of the bone marrow-like cavity surrounded by newly formed bone tissue.