SUMMARY The transcription factor Sox9 is necessary for early chondrogenesis, but its subsequent roles in the cartilage growth plate, a highly specialized structure that drives skeletal growth and endochondral ossification, remain unclear. Using a doxycycline-inducible Cre transgene and Sox9 conditional null alleles in the mouse, we show that Sox9 is required to maintain chondrocyte columnar proliferation and generate cell hypertrophy, two key features of functional growth plates. Sox9 keeps Runx2 expression and β-catenin signaling in check, and thereby inhibits not only progression from proliferation to prehypertrophy, but also subsequent acquisition of an osteoblastic phenotype. Sox9 protein outlives Sox9 RNA in upper hypertrophic chondrocytes, where it contributes with Mef2c to directly activate the major marker of these cells, Col10a1. These findings thus reveal that Sox9 remains a central determinant of the lineage fate and multi-step differentiation program of growth plate chondrocytes, and thereby illuminate our understanding of key molecular mechanisms underlying skeletogenesis.

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Abstract. Epiphyseal chondrocytes cultured in a medium containing 10% serum may be maintained as three dimensional aggregates and differentiate terminally into hypertrophic cells. There is an attendant expression of genes encoding type X collagen and high levels of alkaline phosphatase activity. Manipulation of the serum concentration to optimal levels of 0.1 or 0.01% in this chondrocyte pellet culture system results in formation of features of developing cartilage architecture which have been observed exclusively in growth cartilage in vivo. Cells are arranged in columns radiating out from the center of the tissue, and can be divided into distinct zones corresponding to the recognized stages of chondrocyte differentiation. Elimination of the optimal serum concentration in a chemically defined medium containing insulin eliminates the events of terminal differentiation of defreed cartilage architecture. Chondrocytes continue to enlarge into hypertrophic cells and synthesize type X collagen mRNA and protein, but in the absence of the optimal serum concentration, alkaline phosphatase activity does not increase and the cells retain a random orientation. Addition of thyroxine to the chemically defined medium containing insulin and growth hormone results in dose-dependent increases in both type X collagen synthesis and alkaline phosphatase activity, and reproduces the optimal serum-induced morphogenesis of chondrocytes into a columnar pattern. These experiments demonstrate the critical role of thyroxine in cartilage morphogenesis.T ERMINAL differentiation of chondrocytes into hypertrophic cells is an obligatory step in the endochondral ossification pathway that occurs during embryonic bone development, longitudinal bone growth, and fracture healing. This terminal differentiation process is marked by a several-fold increase in cell volume (11), synthesis of type X collagen, and high" levels of alkaline phosphatase activity. The net result of this developmental process is an increase in length of the growing bone and the mineralization of the surrounding cartilage matrix. This mineralized extracellular matrix of cartilage provides the scaffold for the deposition of new bone matrix by invading osteoblasts.The identification of factors regulating this critical developmental pathway of the skeleton has been hampered by lack of a suitable in vitro model. It is well-established that chondrocytes lose their polygonal morphology and dedifferentiate when placed in traditional monolayer cultures in vitro on tissue culture plastic, a problem which is exacerbated by low cell density or multiple passages (4)(5)(6)25 (3,6,25), the lack of a three-dimensional tissue architecture prevents morphogenesis of growing cartilage and its study as an organized tissue.We have resolved this dilemma by modification of a simple method of chondrocyte culture which maintains cells as an aggregated cell pellet, thereby more closely approximating the three-dimensional environment of developing cartilage in vivo under strict control of culture conditi...

Longitudinal growth of the skeleton is a result of endochondral ossification that occurs at the growth plate. Through a sequential process of cell proliferation, extracellular matrix synthesis, cellular hypertrophy, matrix mineralization, vascular invasion, and eventually apoptosis, the cartilage model is continually replaced by bone as length increases. The regulation of longitudinal growth at the growth plate occurs generally through the intimate interaction of circulating systemic hormones and locally produced peptide growth factors, the net result of which is to trigger changes in gene expression by growth plate chondrocytes. This review highlights recent advances in genetics and cell biology that are illuminating the important regulatory mechanisms governing the structure and biology of the growth plate, and provides selected examples of how studies of human mutations have yielded a wealth of new knowledge regarding the normal biology and pathophysiology of growth plate cartilage.

To assess the degree of success of anterior cruciate ligament (ACL) replacement using the patellar tendon (PT) autograft, 29 New Zealand white rabbits underwent ACL reconstruction using a medial one-third PT autograft. The femur-ligament-tibia complexes were evaluated at 0, 6, 30, and 52 weeks postoperatively for gross and histologic appearances and tensile load to failure properties. Grossly, the autografts did not resemble the control ACLs. Histologically, the autografts progressed from being hypercellular with a random collagen fiber bundle organization to having a near normal cellularity with a more parallel collagen fiber bundle pattern. Anteroposterior knee laxity was more than two times that of the control knees 52 weeks after reconstruction. Biomechanically, the PT autografts plateaued at 30 weeks postoperatively. The ultimate load and stiffness were 15 +/- 5% and 24 +/- 6% of the control ACLs, respectively. At 52 weeks, the appearance of the PT autograft had some general histologic similarities as compared with the native ACL. However, these similarities did not extend to the functional properties of the autograft.

Thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulation of the Wnt/β-catenin signaling pathway. IGF-1 has been described as a stabilizer of β-catenin, and thyroid hormone is a known stimulator of IGF-1 receptor expression. The purpose of this study was to test the hypothesis that IGF-1 signaling is involved in the interaction between the thyroid hormone and the Wnt/β-catenin signaling pathways in regulating growth plate chondrocyte proliferation and differentiation. The results show that IGF-1 and IGF1R stimulate Wnt-4 expression and β-catenin activation in growth plate chondrocytes. The positive effects of IGF-1/IGF1R on chondrocyte proliferation and terminal differentiation are partially inhibited by the Wnt antagonists sFRP3 and Dkk1. T3 activates IGF-1/IGF1R signaling and IGF-1-dependent PI3K/Akt/GSK3β signaling in growth plate chondrocytes undergoing proliferation and differentiation to prehypertrophy. T3-mediated Wnt-4 expression, β-catenin activation, cell proliferation and terminal differentiation of growth plate chondrocytes are partially prevented by the IGF1R inhibitor picropodophyllin as well as the PI3K/Akt signaling inhibitors LY294002 and Akti1/2. These data indicate that the interactions between thyroid hormone and β-catenin signaling in regulating growth plate chondrocyte proliferation and terminal differentiation are modulated by IGF-1/IGF1R signaling through both the Wnt and PI3K/Akt signaling pathways. While chondrocyte proliferation may be triggered by the IGF-1/IGF1R mediated PI3K/Akt/GSK3β pathway, cell hypertrophy is likely due to activation of Wnt/β-catenin signaling, which is at least in part initiated by IGF-1 signaling or the IGF-1-activated PI3K/Akt signaling pathway.

Thyroid hormone activates Wnt-4 expression and Wnt/␤-catenin signaling in rat growth plate chondrocytes. Wnt antagonists Frzb/sFRP3 and Dkk1 inhibit T3-induced Wnt/␤-catenin activation and inhibit the maturation-promoting effects of T3 in growth plate cells. This study indicates that thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulating Wnt/␤-catenin signaling.Introduction: Thyroid hormone is a potent regulator of skeletal maturation in the growth plate, yet the molecular mechanisms underlying this profound effect remain unknown. Wnt signaling has recently been recognized as an important signal transduction pathway in regulating chondrogenesis and terminal differentiation of growth plate chondrocytes. The objective of this study was to explore the interaction between the thyroid hormone and Wnt signaling pathways in the growth plate. Materials and Methods: Rat epiphyseal chondrocytes were maintained in 3D pellet culture and treated with triiodothyronine (T3). Activation of Wnt/␤-catenin signaling pathway in response to T3 was detected by measurement of the expression of Wnt-4 mRNA, the cellular accumulation of ␤-catenin, the transcriptional activity of TCF/LEF, and the expression of the Wnt/␤-catenin responsive gene Runx2/cbfa1. Terminal differentiation of the chondrocytes was assessed by measurement of alkaline phosphatase enzymatic activity and Col10a1 gene expression. Results: Thyroid hormone treatment of growth plate chondrocytes upregulated both Wnt-4 mRNA and protein expression, increased cellular accumulation of stabilized ␤-catenin, increased TCF/LEF transcriptional activity, and stimulated the expression of the Runx2/cbfa1 gene. Overexpression of either Wnt-4 or a stabilized form of ␤-catenin promoted growth plate chondrocyte terminal differentiation. Blocking Wnt ligand/receptor interactions with the secreted Wnt antagonists Frzb/sFRP3 or Dkk1 inhibited these T3-induced increases in ␤-catenin accumulation and Runx2 gene expression and inhibited the maturation-promoting effects of T3 in growth plate cells. Conclusions: These data suggest that thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulating canonical Wnt/␤-catenin signaling.