Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo

Nkx3.2 Promotes Primary Chondrogenic Differentiation by Upregulating Col2a1 Transcription

Background: The Nkx3.2 transcription factor promotes chondrogenesis by forming a positive regulatory loop with a crucial chondrogenic transcription factor, Sox9. Previous studies have indicated that factors other than Sox9 may promote chondrogenesis directly, but these factors have not been identified. Here, we test the hypothesis that Nkx3.2 promotes chondrogenesis directly by Sox9-independent mechanisms and indirectly by previously characterized Sox9-dependent mechanisms. Methodology/Principal Findings: C3H10T1/2 pluripotent mesenchymal cells were cultured with bone morphogenetic protein 2 (BMP2) to induce endochondral ossification. Overexpression of wild-type Nkx3.2 (WT-Nkx3.2) upregulated glycosaminoglycan (GAG) production and expression of type II collagen a1 (Col2a1) mRNA, and these effects were evident before WT-Nkx3.2-mediated upregulation of Sox9. RNAi-mediated inhibition of Nkx3.2 abolished GAG production and expression of Col2a1 mRNA. Dual luciferase reporter assays revealed that WT-Nkx3.2 upregulated Col2a1 enhancer activity in a dose-dependent manner in C3H10T1/2 cells and also in N1511 chondrocytes. In addition, WT-Nkx3.2 partially restored downregulation of GAG production, Col2 protein expression, and Col2a1 mRNA expression induced by Sox9 RNAi. ChIP assays revealed that Nkx3.2 bound to the Col2a1 enhancer element. Conclusions/Significance: Nkx3.2 promoted primary chondrogenesis by two mechanisms: Direct and Sox9-independen

P38 Mitogen-Activated Protein Kinase Inhibitor, FR167653, Inhibits Parathyroid Hormone Related Protein-Induced Osteoclastogenesis and Bone Resorption

p38 mitogen-activated protein kinase (MAPK) acts downstream in the signaling pathway that includes receptor activator of NF-κB (RANK), a powerful inducer of osteoclast formation and activation. We investigated the role of p38 MAPK in parathyroid hormone related protein (PTHrP)-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo. The ability of FR167653 to inhibit osteoclast formation was evaluated by counting the number of tartrate-resistant acid phosphatase positive multinucleated cells (TRAP-positive MNCs) in in vitro osteoclastgenesis assays. Its mechanisms were evaluated by detecting the expression level of c-Fos and nuclear factor of activated T cells c1 (NFATc1) in bone marrow macrophages(BMMs) stimulated with sRANKL and M-CSF, and by detecting the expression level of osteoprotegerin (OPG) and RANKL in bone marrow stromal cells stimulated with PTHrP in the presence of FR167653. The function of FR167653 on bone resorption was assessed by measuring the bone resorption area radiographically and by counting osteoclast number per unit bone tissue area in calvaria in a mouse model of bone resorption by injecting PTHrP subcutaneously onto calvaria. Whole blood ionized calcium levels were also recorded. FR167653 inhibited PTHrP-induced osteoclast formation and PTHrP-induced c-Fos and NFATc1 expression in bone marrow macrophages, but not the expression levels of RANKL and OPG in primary bone marrow stromal cells treated by PTHrP. Furthermore, bone resorption area and osteoclast number in vivo were significantly decreased by the treatment of FR167653. Systemic hypercalcemia was also partially inhibited. Inhibition of p38 MAPK by FR167653 blocks PTHrP-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo, suggesting that the p38 MAPK signaling pathway plays a fundamental role in PTHrP-induced osteoclastic bone resorption

Remote audit practice for inspection of structural and equipment standards for cell processing facilities under the act on the safety of regenerative medicine in Japan

Introduction: The Act on the Safety of Regenerative Medicine enforced in Japan in 2014, regulates the manufacture of cellular processed products. However, with regards to the manufacturing facilities at medical institutions, only the submission of necessary documents is required for a license, and the need for third-party inspection has been highlighted. Remote activities are becoming more prominent with the spread of the Severe Acute Respiratory Syndrome Coronavirus 2 infection; therefore, the current assessment of compliance with structural facility standards was conducted remotely. Methods: The entire process, including start-up meetings, preparation of the survey schedule, submission and review of preliminary materials, audits, and reporting of results, was conducted via e-mail and web conferencing systems. The survey was conducted remotely, to minimize the risk of contamination of the cell processing facility (CPF) and reduce the burden on surveyors, while contributing to the establishment of suitable structural facilities by identifying and highlighting the areas or items that were considered to be non-compliant with the regulations. The series of audits were completed in ten weeks, with a period of six weeks between the start-up meeting and the audit implementation. The audit was completed in approximately 3 h on the day of the inspection. Results: The audit results were delivered in the report, with four items requiring improvement and several other recommended items listed as non-conformities. Conclusions: We believe that this remote method allows the effective inspection of regenerative medicine manufacturing facilities and assessment of more cell culture processing facilities than the current in-person audit method, with limited human resources

The effect of FR167653 on PTHrP-induced bone resorption.

<p>The calvarial bone resorption was measured radiographically on the left side (area marked by yellow line) where treated by direct subcutaneous injection of PTHrP. A. Treatment by FR167653 reduced the bone resorption area (*: P<0.005). DW:distilled water. B. Representative X-rays of mouse calvaria with PTHrP-induced bone resorption. a: control group (treated with DW). b: FR167653 treatment group.</p

Interconnected porous hydroxyapatite ceramics for bone tissue engineering

Several porous calcium hydroxyapatite (HA) ceramics have been used clinically as bone substitutes, but most of them possessed few interpore connections, resulting in pathological fracture probably due to poor bone formation within the substitute. We recently developed a fully interconnected porous HA ceramic (IP-CHA) by adopting the ‘foam-gel’ technique. The IP-CHA had a three-dimensional structure with spherical pores of uniform size (average 150 μm, porosity 75%), which were interconnected by window-like holes (average diameter 40 μm), and also demonstrated adequate compression strength (10–12 MPa). In animal experiments, the IP-CHA showed superior osteoconduction, with the majority of pores filled with newly formed bone. The interconnected porous structure facilitates bone tissue engineering by allowing the introduction of mesenchymal cells, osteotropic agents such as bone morphogenetic protein or vasculature into the pores. Clinically, we have applied the IP-CHA to treat various bony defects in orthopaedic surgery, and radiographic examinations demonstrated that grafted IP-CHA gained radiopacity more quickly than the synthetic HA in clinical use previously. We review the accumulated data on bone tissue engineering using the novel scaffold and on clinical application in the orthopaedic field

Effects of FR167653 on osteoclast formation in M-CSF-dependent bone marrow macrophages treated with sRANKL.

<p>A. Osteoclast formation assay using M-CSF-dependent bone marrow macrophages (MDBMMs) prepared as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023199#s2" target="_blank">Materials and Methods</a> section. TRAP-positive MNCs with three or more nuclei were counted as osteoclasts. n = 6. *: p<0.001 versus the group treated only with sRANKL. B. TRAP staining for MDBMMs stimulated by sRANKL (100 ng/ml). a: No treatment. b: FR167653 (0.1 µM). c: FR167653 (1 µM). d: FR167653 (10 µM).</p

The effects of FR167653 on osteoclast number in calvaria from PTHrP-treated animals.

<p>A. Osteoclast number per unit bone area (OcN-BTA) was measured histologically as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023199#s2" target="_blank">Materials and Methods</a> section. Treatment of FR167653 reduces the number of osteoclasts (*: P<0.005). B. TRAP staining and counterstain with hematoxylin on calvaria sections. a: Histological section of animals treated with DW. b: Histological section of animals treated with FR167653. Osteoclasts are indicated by arrowheads.</p