A stabilized tibia fracture model was used in young (8-week old) and aged (1-year old) mice to define the relative bone regenerative potential and the relative responsiveness of the periosteal progenitor population with aging and PTH 1-34 (PTH) systemic therapy. Bone regeneration was assessed through gene expressions, radiographic imaging, histology/histomorphometry, and biomechanical testing. Radiographs and microCT showed increased calcified callus tissue and enhanced bone healing in young compared to aged mice. A key mechanism involved reduced proliferation, expansion, and differentiation of periosteal progenitor cell populations in aged mice. The experiments showed that PTH increased calcified callus tissue and torsional strength with a greater response in young mice. Histology and quantitative histomorphometry confirmed that PTH increased callus tissue area due primarily to an increase in bone formation, since minimal changes in cartilage and mesenchyme tissue area occurred. Periosteum examined at 3, 5, and 7 days showed that PTH increased cyclin D1 expression, the total number of cells in the periosteum, and width of the periosteal regenerative tissue. Gene expression showed that aging delayed differentiation of both bone and cartilage tissues during fracture healing. PTH resulted in sustained Col10a1 expression consistent with delayed chondrocyte maturation, but otherwise minimally altered cartilage gene expression. In contrast, PTH 1-34 stimulated expression of Runx2 and Osterix, but resulted in reduced Osteocalcin. β-catenin staining was present in mesenchymal chondroprogenitors and chondrocytes in early fracture healing, but was most intense in osteoblastic cells at later times. PTH increased active β-catenin staining in the osteoblast populations of both young and aged mice, but had a lesser effect in cartilage. Altogether the findings show that reduced fracture healing in aging involves decreased proliferation and differentiation of stem cells lining the bone surface. While PTH 1-34 enhances the proliferation and expansion of the periosteal stem cell population and accelerates bone formation and fracture healing, the effects are proportionately reduced in aged mice compared to young mice. β-catenin is induced by PTH in early and late fracture healing and is a potential target of PTH 1-34 effects.

SummaryTAK1 is a MAP3K that mediates non-canonical TGF-b and BMP signaling. During the embryonic period, TAK1 is essential for cartilage and joint development as deletion of Tak1 in chondro-osteo progenitor cells leads to severe chondrodysplasia with defects in both chondrocyte proliferation and maturation. We have investigated the role of TAK1 in committed chondrocytes during early postnatal development. Using the Col2a1-CreER T2 ; Tak1 f/f mouse model, we induced deletion of Tak1 at postnatal day 7 and characterized the skeletal phenotypes of these mice at 1 and 3 months of age. Mice with chondrocyte-specific Tak1 deletion exhibited severe growth retardation and reduced proteoglycan and type II collagen content in the extracellular matrix of the articular cartilage. We found reduced Col2a1 and Acan expression, but increased Mmp13 and Adamts5 expression, in Tak1-deficient chondrocytes along with reduced expression of the SOX trio of transcription factors, SOX9, SOX5 and SOX6. In vitro, BMP2 stimulated Sox9 gene expression and Sox9 promoter activity. These effects were reduced; however, following Tak1 deletion or treatment with a TAK1 kinase inhibitor. TAK1 affects both canonical and non-canonical BMP signal transduction and we found that both of these pathways contribute to BMP2-mediated Sox9 promoter activation. Additionally, we found that ATF2 directly binds the Sox9 promoter in response to BMP signaling and that this effect is dependent upon TAK1 kinase activity. These novel findings establish that TAK1 contributes to BMP2-mediated Sox9 gene expression and is essential for the postnatal development of normal growth plate and articular cartilages.

RUNX2 is a transcription factor critical for chondrocyte maturation and normal endochondral bone formation. It promotes the expression of factors catabolic to the cartilage extracellular matrix and is upregulated in human osteoarthritic cartilage and in murine articular cartilage following joint injury. To date, in vivo studies of RUNX2 overexpression in cartilage have been limited to forced expression in osteochondroprogenitor cells preventing investigation into the effects of chondrocyte‐specific RUNX2 overexpression in postnatal articular cartilage. Here, we used the Rosa26Runx2 allele in combination with the inducible Col2a1CreERT2 transgene or the inducible AcanCreERT2 knock‐in allele to achieve chondrocyte‐specific RUNX2 overexpression (OE) during embryonic development or in the articular cartilage of adult mice, respectively. RUNX2 OE was induced at embryonic day 13.5 (E13.5) for all developmental studies. Histology and in situ hybridization analyses suggest an early onset of chondrocyte hypertrophy and accelerated terminal maturation in the limbs of the RUNX2 OE embryos compared to control embryos. For all postnatal studies, RUNX2 OE was induced at 2 months of age. Surprisingly, no histopathological signs of cartilage degeneration were observed even 6 months following induction of RUNX2 OE. Using the meniscal/ligamentous injury (MLI), a surgical model of knee joint destabilization and meniscal injury, however, we found that RUNX2 OE accelerates the progression of cartilage degeneration following joint trauma. One month following MLI, the numbers of MMP13‐positive and TUNEL‐positive chondrocytes were significantly greater in the articular cartilage of the RUNX2 OE joints compared to control joints and 2 months following MLI, histomorphometry and Osteoarthritis Research Society International (OARSI) scoring revealed decreased cartilage area in the RUNX2 OE joints. Collectively, these results suggest that although RUNX2 overexpression alone may not be sufficient to initiate the OA degenerative process, it may predetermine the rate of OA onset and/or progression following traumatic joint injury. © 2019 American Society for Bone and Mineral Research.

The ability to isolate primary chondrocytes from wild-type and genetically altered mice has provided tremendous advances in the understanding of signaling networks that regulate chondrocytes in health and disease. Isolation of chondrocytes from both growth plate tissues and articular cartilage has been challenging due to the cells being embedded within a highly organized tissue matrix. Here we describe highly reproducible methods for the isolation of pure populations of growth plate chondrocytes from the murine sternum and ribs and articular chondrocytes from the knee joint.

WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis.