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There is a worldwide epidemic of skeletal diseases causing not only a public health issue but also accounting for a sizable portion of healthcare expenditures. The vertebrate skeleton is known to be formed by mesenchymal cells condensing into tissue elements (patterning phase) followed by their differentiation into cartilage (chondrocytes) or bone (osteoblasts) cells within the condensations. During the growth and remodeling phase, bone is formed directly via intramembranous ossification or through a cartilage to bone conversion via endochondral ossification routes. The canonical pathway of the endochondral bone formation process involves apoptosis of hypertrophic chondrocytes followed by vascular invasion that brings in osteoclast precursors to remove cartilage and osteoblast precursors to form bone. However, there is now an emerging role for chondrocyte-to-osteoblast transdifferentiation in the endochondral ossification process. Although the concept of “transdifferentiation” per se is not recent, new data using a variety of techniques to follow the fate of chondrocytes in different bones during embryonic and post-natal growth as well as during fracture repair in adults have identified three different models for chondrocyte-to-osteoblast transdifferentiation (direct transdifferentiation, dedifferentiation to redifferentiation, and chondrocyte to osteogenic precursor). This review focuses on the emerging models of chondrocyte-to-osteoblast transdifferentiation and their implications for the treatment of skeletal diseases as well as the possible signaling pathways that contribute to chondrocyte-to-osteoblast transdifferentiation processes.

The expression of some chemokines and chemokine receptors is induced during the development of post-traumatic osteoarthritis (PTOA), but their involvement in the pathogenesis of the disease is unclear. The goal of this study was to test whether CCL21 and CXCL13 play a role in PTOA development. For this purpose, we evaluated the expression profiles of the chemokines Ccl21 and Cxcl13 , matrix metalloproteinase enzymes Mmp3 and Mmp13 , and inflammatory cell markers in response to partial medial meniscectomy and destabilization (MMD). We then assessed the effect of local administration of CCL21 neutralizing antibody on PTOA development and post-knee injury inflammation. The mRNA expression of both Ccl21 and Cxcl13 was induced early post-surgery, but only Ccl21 mRNA levels remained elevated 4 weeks post-surgery in rat MMD-operated knees compared to controls. This suggests that while both CXCL13 and CCL21 are involved in post-surgery inflammation, CCL21 is necessary for development of PTOA. A significant increase in the mRNA levels of Cd4 , Cd8 and Cd20 was observed during the first 3 days post-surgery. Significantly, treatment with CCL21 antibody reduced post-surgical inflammation that was accompanied by a reduction in the expression of Mmp3 and Mmp13 and post-MMD cartilage degradation. Our findings are consistent with a role for CCL21 in mediating changes in early inflammation and subsequent cartilage degeneration in response to knee injury. Our results suggest that targeting CCL21 signaling pathways may yield new therapeutic approaches effective in delaying or preventing PTOA development following injury.

In this study, the osteoinductive activity of a small-molecule NOTCH1 activator, Yhhu3792, and Poloxamer 407, an FDA-approved hydrogel, was evaluated independently regarding osteoblast functions in vitro using primary cultures of osteoblasts derived from C57BL/6J mice. We found that treatment with Yhhu3792 increased the number of NOTCH1-positive osteoblasts (36%) compared to the vehicle control (19%) after antibody staining, suggesting increased NOTCH1 signaling after Yhhu3792 treatment. Osteoblasts treated with varying doses (5, 10, and 20 μM) of Yhhu3792 and P407 (1–25%) stimulated both osteoblast proliferation and differentiation by 25–45% (p < 0.05) compared to the vehicle control. Accordingly, 10 µM Yhhu3792 treatment for 9 days increased the alizarin red-stained mineralized nodule area (8.69 ± 0.97 vs. 4.05 ± 1.51 arbitrary units; p < 0.05) compared to the vehicle treatment. Similarly, osteoblasts treated with 10% P407 also significantly increased mineralized nodule formation. The Cell Tox Green dye assay revealed that the dosage of Yhhu3792 used was not cytotoxic. Gene expression studies measured by real-time PCR revealed that a 24 h treatment with 10 µM Yhhu3792 significantly increased expression levels of bone formation markers (Vegf, Osteocalcin) and NOTCH1 targets (c-myc, Cox2, and Hes1) in osteoblasts. A low dose of P407 in combination with 10 µM Yhhu3792 stimulated a significant increase (>40%) in the proliferation of bone marrow stromal cells. In conclusion, our in vitro findings showing osteogenic effects of the small molecule Yhhu3792 and P407 hydrogel should be confirmed in vivo in animal fracture healing models.

[This corrects the article DOI: 10.1371/journal.pone.0247913.].[This corrects the article DOI: 10.1371/journal.pone.0247913.].

Retinol-binding protein 4 (RBP4), an adipokine secreted by adipose tissues, has been implicated in metabolic inflammation and insulin resistance. Type 2 diabetes (T2D) is a recognized risk factor for osteoarthritis, with both conditions characterized by chronic low-grade inflammation, suggesting potential links between metabolic disorder and joint degeneration. This study aimed to investigate whether inflammatory and metabolic stresses regulate RBP4 expression and function in joint-related cells. Murine immature chondrocyte cells (iMACs) and the mouse AT805 teratocarcinoma cell line, clone 5, that differentiates into chondrogenic cells (ATDC5), were used as in vitro models for chondrocyte cells. Rbp4 mRNA expression increased during differentiation of iMACs, with 3.6- and 2.2-fold elevations observed on days 7 and 14, respectively (p < 0.01 vs. undifferentiated controls). Inflammatory stimulation with interleukin-6 (IL-6) significantly increased Rbp4 mRNA expression in ATDC5 cells (p < 0.05 vs. vehicle), along with elevated expression of catabolic and inflammatory mediators, including monocyte chemoattractant protein-1 (Mcp1), cyclooxygenase-2 (Cox2), and matrix metalloproteinase-3 (Mmp3) (p < 0.05 vs. vehicle). Pharmacological inhibition of RBP4 using fenretinide (FEN) attenuated chondrogenic differentiation marker expression, reduced glycosaminoglycan synthesis during chondrogenic differentiation, and mitigated high-glucose-induced catabolic responses, as indicated by reduced Mcp2 (p = 0.04) and Mmp13 (p = 0.01) expression in ATDC5 cells treated with FEN compared with cells treated with the vehicle under high-glucose conditions. Furthermore, in RAW 264.7 cells, a murine macrophage cell line commonly used as an in vitro model for osteoclastogenesis, FEN significantly reduced the expression of osteoclast differentiation markers, dendritic cell-specific transmembrane protein (DC-Stamp), nuclear factor of activated T-cells, cytoplasmic 1 (Nf-atc1), cathepsin k (Cath.k), and tartrate-resistant acid phosphatase (Trap) under osteoclastogenic conditions (p < 0.01 vs. vehicle). Collectively, these findings suggest that RBP4 functions as a metabolic–inflammatory mediator influencing both cartilage and bone-remodeling processes. This study reveals a previously unrecognized role of RBP4 in regulating osteoclast-associated pathways. Targeting RBP4 may, therefore, represent a promising therapeutic strategy for delaying or preventing osteoarthritis progression, particularly in metabolically compromised conditions.

Global knockout (KO) of the Lrrk1 gene in mice causes severe osteopetrosis because of the failure of osteoclasts to resorb bone. The molecular mechanism of LRRK1 regulation of osteoclast function is not fully understood. Here, we performed a 2D DIGE phosphor-proteomics analysis to identify potential LRRK1 targets in osteoclasts. Splenocytes from Lrrk1 KO and wild-type (WT) mice were differentiated into osteoclasts for protein extraction. Lysates from Lrrk1 KO and WT cells were labeled with Cy3- and Cy5-dye, respectively. Labeled proteins were mixed and analyzed on the same 2D SDS PAGE for protein profiling. The same amounts of cellular protein were also labeled with Cy3-dye and ran on a 2D SDS PAGE. The gels were then stained using Pro-Q® Diamond Phosphoprotein Gel Stain for phosphoprotein profiling. Differentially phosphorylated protein spots between the two types of cells were collected, digested with trypsin, and identified by mass spectrometry. Seventeen phosphoproteins were identified, six of which are known to be involved in endosome/lysosome sorting, vacuolar protection, and trafficking. While five of these proteins (SNX2, VPS35, VTA1, CFL1, and CTSA) were significantly hypophosphorylated, SNX3 was hyperphosphorylated in LRRK1-deficient osteoclasts. The downregulation of VSP35 and CFL1 phosphorylation in LRRK1-deficient cells was validated by Phos-tag SDS PAGE analysis. Our results indicate that LRRK1 signaling regulates osteoclast function via modulating VPS35 and CFL1 phosphorylation critical for endosome/lysosome trafficking and dynamic cytoskeleton arrangement in osteoclasts.

The proximal and distal femur epiphyses of mice are both weight-bearing structures derived from chondrocytes but differ in development. Mineralization at the distal epiphysis occurs in an osteoblast-rich secondary ossification center (SOC), while the chondrocytes of the proximal femur head (FH), in particular, are directly mineralized. Thyroid hormone (TH) plays important roles in distal knee SOC formation, but whether TH also affects proximal FH development remains unexplored. Here, we found that TH controls chondrocyte maturation and mineralization at the FH in vivo through studies in thyroid stimulating hormone receptor ( Tshr -/- ) hypothyroid mice by X-ray, histology, transcriptional profiling, and immunofluorescence staining. Both in vivo and in vitro studies conducted in ATDC5 chondrocyte progenitors concur that TH regulates expression of genes that modulate mineralization ( Ibsp , Bglap2 , Dmp1 , Spp1 , and Alpl ). Our work also delineates differences in prominent transcription factor regulation of genes involved in the different mechanisms leading to proximal FH cartilage calcification and endochondral ossification at the distal femur. The information on the molecular pathways contributing to postnatal cartilage calcification can provide insights on therapeutic strategies to treat pathological calcification that occurs in soft tissues such as aorta, kidney, and articular cartilage.

Objectives The goal of this study was to evaluate the long-term impact of repeated (r) mild traumatic brain injury (mTBI) on the healing of fractures in a mouse model. Ten week-old male mice were subjected to r-mTBI once per day for 4 days followed by closed femoral fracture using a three-point bending technique, 1 week post impact and fracture healing phenotype evaluated at 20 weeks of age. Results Micro-CT analysis of the fracture callus region at nine weeks post fracture revealed reduced bone volume (30%, p  <  0.05 ) in the r-mTBI fracture group compared to the control-fracture group. The connectivity density of the fracture callus bone was reduced by 40% ( p  <  0.01) in the r-mTBI fracture group. Finite element analysis of the fracture callus region showed reduced failure load ( p  =  0.08 ) in the r-mTBI group compared to control group. There was no residual cartilage in the fracture callus region of either the r-mTBI or control fracture group. The reduced fracture callus bone volume and mechanical strength of fracture callus in r-mTBI mice 9 weeks post fracture are consistent with negative effects of r-mTBI on fracture healing over a long-term resulting in decreased mechanical strength of the fracture callus.

Vitamin C (ascorbic acid, AA) is a well-known regulator of bone and cartilage metabolism. However, the mechanisms of AA's action in these tissues are only partly understood. In this study, we confirmed that AA contributes to bone and cartilage metabolism by showing decreased articular cartilage and trabecular bone in AA-deficient spontaneous fracture (sfx) mutant mice. In vitro, we found that AA exerts differential effects on chondrocyte and osteoblast differentiation. Since AA is known to increase levels of 5-hydroxymethylcytosine (5-hmC) and induce DNA demethylation via the ten-eleven translocases (TETs), and since prolyl hydroxylase domain-containing protein 2 (PHD2), a known mediator of AA's effects in these tissues, is part of the same enzyme family as the TETs, we next investigated whether increases in 5-hmC might mediate some of these effects. All TETs and PHDs are expressed in chondrocytes and osteoblasts, and PHD2 is localized in both the cytoplasm and nucleus of the cell, lending plausibility to the hypothesis of altered 5-hmC content in these cells. We found that AA treatment increased levels of 5-hmC in both cell types globally, notably including promoter regions of osteoblast differentiation genes. Furthermore, inhibition of PHD2 decreased 5-hmC levels in chondrocyte differentiation gene promoters, and knockdown of Phd2 in chondrocytes reduced global 5-hmC levels, suggesting for the first time that PHD2 may itself directly mediate increases in 5-hmC in chondrocyte and osteoblast genes. Further investigation of this mechanism could lead to novel therapeutic approaches to treat debilitating diseases such as osteoarthritis and osteoporosis.

Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (Tfr1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin, and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of Tfr1 and Tfr1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of Tfr1 in osteoclast lineage cells in vivo and in vitro, we crossed Tfrc (encoding Tfr1)-floxed mice with Lyz2 (LysM)- Cre and Cathepsin K ( Ctsk )-Cre mice to generate Tfrc conditional knockout mice in myeloid osteoclast precursors (Tfr1 ΔLysM ) or differentiated osteoclasts (Tfr1 ΔCtsk ), respectively. Skeletal phenotyping by µCT and histology unveiled a significant increase in trabecular bone mass with normal osteoclast number in long bones of 10-week-old young and 6-month-old adult female but not male Tfr1 ΔLysM mice. Although high trabecular bone volume in long bones was observed in both male and female Tfr1 ΔCtsk mice, this phenotype was more pronounced in female knockout mice. Consistent with this gender-dependent phenomena, estrogen deficiency induced by ovariectomy decreased trabecular bone mass in Tfr1 ΔLysM mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts in vitro by attenuating mitochondrial respiration and activation of the Src-Rac1-WAVE regulatory complex axis, respectively, leading to decreased bone resorption with little impact on osteoclast differentiation. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling in a gender-dependent manner.