Transforming growth factor β1 (TGFβ1) is a major mediator in the modulation of osteoblast differentiation. However, the underlying molecular mechanism is still not fully understood. Here, we show that TGFβ1 has a dual stage-dependent role in osteoblast differentiation; TGFβ1 induced matrix maturation but inhibited matrix mineralization. We discovered the underlying mechanism of the TGFβ1 inhibitory role in mineralization using human osteoprogenitors. In particular, the matrix mineralization-related genes of osteoblasts such as osteocalcin (OCN), Dickkopf 1 (DKK1), and CCAAT/enhancer-binding protein beta (C/EBPβ) were dramatically suppressed by TGFβ1 treatment. The suppressive effects of TGFβ1 were reversed with anti-TGFβ1 treatment. Mechanically, TGFβ1 decreased protein levels of C/EBPβ without changing mRNA levels and reduced both mRNA and protein levels of DKK1. The degradation of the C/EBPβ protein by TGFβ1 was dependent on the ubiquitin–proteasome pathway. TGFβ1 degraded the C/EBPβ protein by inducing the expression of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (SMURF1) at the transcript level, thereby reducing the C/EBPβ-DKK1 regulatory mechanism. Collectively, our findings suggest that TGFβ1 suppressed the matrix mineralization of osteoblast differentiation by regulating the SMURF1-C/EBPβ-DKK1 axis.

Histological and cytological observations of the human anterior cruciate ligament (ACL) had been described, but the differentiation potency based on their location is still unknown. To determine and compare proliferation and differentiation potential of cells derived from distal and middle thirds of the ACL remnant, ACL remnant was initially marked at the distal third (within 10 mm from the tibial insertion) and middle third (between 10-20 mm from the tibial insertion) and then dissected. Both the middle and distal third regions of ACL remnant were analyzed using CD34 + cell counting. Cell proliferation rate did not differ in both middle and distal third regions of ACL remnant, but they showed different characteristics in cell differentiation depending on their location. The distal third region of the ACL remnant had a tendency for chondrogenic differentiation with higher expression of CD34 + cells. On the other hand, the middle third region of ACL remnant had a strong tendency for osteogenic and ligamentous differentiation. Characteristics of the ACL remnant tissues should be considered when performing remnant-preserving or harvesting ACL remnants for tissue engineering. Anterior cruciate ligament reconstruction (ACLR) is one of the most common surgical procedures in the field of orthopaedic sports medicine, with more than 130,000 procedures performed annually in the United States alone 1,2. It has been well documented that a completely ruptured ACL does not spontaneously heal because of poor vascular supply and an unfavourable intra-articular environment 3. Given the importance of its biomechanical function, surgical treatment is generally accepted as the standard procedure for restoring knee stability. In most cases, non-augmented primary repair has been unsuccessful, and therefore ACL reconstruction is required 1,4,5. For surgical success, ACLR requires tendon graft healing in a surgically created bone tunnel and maturation (i.e., ligamentization) of the graft substance 4,6-9. Indeed, the lack of vascularity within the tendon graft induces degeneration or micro ruptures during the early postoperative period 10. To overcome these issues, tissue engineering using stem cells has been widely explored as a means to achieve early graft healing, tendon regeneration, and bone integration. Recently, reports have shown that ruptured human ACL tissues can possess numerous vascular-derived stem cells and that ACL-derived CD34 + cells can promote healing and have high expansion and multilineage differentiation potential 7,11-13. Mifune et al. 14 demonstrated that ACL-derived CD34 + cells contributed to tendon-bone healing after ACLR via angiogenesis and osteogenesis enhancements 15. Furthermore, Matsumoto et al. 16 found that incorporation of ruptured ACL tissues in autologous grafts reduced tunnel enlargement in ACLR 16,17. However, with regard to their clinical application, potential advantages of remnant-derived stem cells are still questionable. Histological observations of the uninjured human ACL have shown a differe...

Because damage to hyaline cartilage is irreversible, relieving progressive cartilage destruction is an important therapeutic approach for inflammatory arthritis. In the present study, human hyaline chondrocytes were isolated from total knee replacements of 15 patients with osteoarthritis (OA) and three with rheumatoid arthritis (RA). Synovial fluid of OA (n=25) and RA (n=34) were collected to measure tumor necrosis factor α (TNFα) using ELISA. Consistent with previous studies, the synovial fluid exhibited high TNFα levels and hyaline cartilage was severely destroyed in patients with RA. TNFα-treated chondrocytes were used as model for inflammatory arthritis. TNFα did not influence proliferation or extracellular matrix expression in chondrocytes, but induced matrix metalloproteinase (MMP)1, 3 and 13 expression levels in chondrocytes, which was accompanied by activation of nuclear factor-κB signaling. During chondrogenic differentiation, TNFα attenuated mRNA expression levels of anabolic factors (collagen type 2 and aggrecan) and enhanced mRNA expression of catabolic factors (MMP1, MMP3 and MMP13) in chondrocytes. Moreover, anti-TNFα agents (Golimumab) inhibited the TNFα-induced metabolic shift in chondrocytes and chondrogenic differentiation. The present study revealed a mechanism by which TNFα may induce metabolic shift in chondrocytes, leading to progressive chondrocyte destruction.

Dickkopf-1 (DKK1) is a secreted protein that acts as an antagonist of the canonical WNT/β-catenin pathway, which regulates osteoblast differentiation. However, the role of DKK1 on osteoblast differentiation has not yet been fully clarified. Here, we investigate the functional role of DKK1 on osteoblast differentiation. Primary osteoprogenitor cells were isolated from human spinal bone tissues. To examine the role of DKK1 in osteoblast differentiation, we manipulated the expression of DKK1, and the cells were differentiated into mature osteoblasts. DKK1 overexpression in osteoprogenitor cells promoted matrix mineralization of osteoblast differentiation but did not promote matrix maturation. DKK1 increased Ca+ influx and activation of the Ca+/calmodulin-dependent protein kinase II Alpha (CAMK2A)-cAMP response element-binding protein (CREB) and increased translocation of p-CREB into the nucleus. In contrast, stable DKK1 knockdown in SaOS2 cells exhibited reduced nuclear translocation of p-CREB and matrix mineralization. Overall, we suggest that manipulating DKK1 regulates the matrix mineralization of osteoblasts by Ca+-CAMK2A-CREB, and DKK1 is a crucial gene for bone mineralization of osteoblasts.