Abstract:Purpose
Endochondral ossification, which involves transdifferentiation of chondrocytes into osteoblasts, is an important process involved in the development and postnatal growth of most vertebrate bones as well as in bone fracture healing. To study the basic molecular mechanisms of this process, a robust and easy-to-use in vitro model is desirable. Therefore, we aimed to develop a standardized in vitro assay for the transdifferentiation of chondrogenic cells towards the osteogenic lineage.
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“…To the best of our knowledge, the here employed in vitro assay is the only currently available 2D culture model in which a chondrogenic cell line can be forced to transdifferentiate into osteoblasts. While using a monolayer cell line model for sure has several drawbacks, high reproducibility and standardization, as described earlier by our group 28 , represent strong and important advantages. Fig.…”
Section: Resultsmentioning
confidence: 94%
“…5a ) and conditioned medium (Fig. 5c ) from isolated CD11b + myeloid BM cells from SHC/CSC WT mice on chondrocyte-to-osteoblast transdifferentiation were assessed in a recently established in vitro transdifferentiation assay using the chondrogenic cell line ATDC5 28 . In agreement with the hypothesis that CSC indeed compromises chondrocyte-to-osteoblast differentiation via myeloid-derived CAs secreted locally in the BM and/or fracture hematoma, all tested synthetic CAs (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Next, we exposed C57BL/6N wild-type (WT) and TH flox/flox /CD11b-Cre + (referred to as TH flox /Cre + ) mice with a specific TH knockout (KO) in myeloid cells to either SHC/CSC conditions alone or to SHC/CSC conditions followed by standardized femur osteotomy, to reveal that CAs secreted locally by myeloid cells are indeed critical for the negative effects of mental trauma on bone metabolism and regeneration. To further show that locally secreted CAs directly act on chondrocytes and compromise their transdifferentiation towards osteoblasts, we employed synthetic CAs and conditioned media from myeloid cells of WT SHC/CSC mice, in the presence or absence of different CA receptor antagonists, in a novel in vitro assay, recently established by our group 28 . Finally, we exposed Adrb2 flox/flox /Col2a1-Cre + (referred to as Adrb2 flox /Cre + ) mice and their Cre - littermates (referred as Adrb2 flox /Cre - ) with a specific ß2-AR KO in chondrocytes to SHC/CSC conditions to confirm the critical role of ß2-AR signaling in chondrocytes shown in our in vitro assay.…”
Mental traumatization is associated with long-bone growth retardation, osteoporosis and increased fracture risk. We revealed earlier that mental trauma disturbs cartilage-to-bone transition during bone growth and repair in mice. Trauma increased tyrosine hydroxylase-expressing neutrophils in bone marrow and fracture callus. Here we show that tyrosine hydroxylase expression in the fracture hematoma of patients correlates positively with acknowledged stress, depression, and pain scores as well as individual ratings of healing-impairment and pain-perception post-fracture. Moreover, mice lacking tyrosine hydroxylase in myeloid cells are protected from chronic psychosocial stress-induced disturbance of bone growth and healing. Chondrocyte-specific β2-adrenoceptor-deficient mice are also protected from stress-induced bone growth retardation. In summary, our preclinical data identify locally secreted catecholamines in concert with β2-adrenoceptor signalling in chondrocytes as mediators of negative stress effects on bone growth and repair. Given our clinical data, these mechanistic insights seem to be of strong translational relevance.
“…To the best of our knowledge, the here employed in vitro assay is the only currently available 2D culture model in which a chondrogenic cell line can be forced to transdifferentiate into osteoblasts. While using a monolayer cell line model for sure has several drawbacks, high reproducibility and standardization, as described earlier by our group 28 , represent strong and important advantages. Fig.…”
Section: Resultsmentioning
confidence: 94%
“…5a ) and conditioned medium (Fig. 5c ) from isolated CD11b + myeloid BM cells from SHC/CSC WT mice on chondrocyte-to-osteoblast transdifferentiation were assessed in a recently established in vitro transdifferentiation assay using the chondrogenic cell line ATDC5 28 . In agreement with the hypothesis that CSC indeed compromises chondrocyte-to-osteoblast differentiation via myeloid-derived CAs secreted locally in the BM and/or fracture hematoma, all tested synthetic CAs (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Next, we exposed C57BL/6N wild-type (WT) and TH flox/flox /CD11b-Cre + (referred to as TH flox /Cre + ) mice with a specific TH knockout (KO) in myeloid cells to either SHC/CSC conditions alone or to SHC/CSC conditions followed by standardized femur osteotomy, to reveal that CAs secreted locally by myeloid cells are indeed critical for the negative effects of mental trauma on bone metabolism and regeneration. To further show that locally secreted CAs directly act on chondrocytes and compromise their transdifferentiation towards osteoblasts, we employed synthetic CAs and conditioned media from myeloid cells of WT SHC/CSC mice, in the presence or absence of different CA receptor antagonists, in a novel in vitro assay, recently established by our group 28 . Finally, we exposed Adrb2 flox/flox /Col2a1-Cre + (referred to as Adrb2 flox /Cre + ) mice and their Cre - littermates (referred as Adrb2 flox /Cre - ) with a specific ß2-AR KO in chondrocytes to SHC/CSC conditions to confirm the critical role of ß2-AR signaling in chondrocytes shown in our in vitro assay.…”
Mental traumatization is associated with long-bone growth retardation, osteoporosis and increased fracture risk. We revealed earlier that mental trauma disturbs cartilage-to-bone transition during bone growth and repair in mice. Trauma increased tyrosine hydroxylase-expressing neutrophils in bone marrow and fracture callus. Here we show that tyrosine hydroxylase expression in the fracture hematoma of patients correlates positively with acknowledged stress, depression, and pain scores as well as individual ratings of healing-impairment and pain-perception post-fracture. Moreover, mice lacking tyrosine hydroxylase in myeloid cells are protected from chronic psychosocial stress-induced disturbance of bone growth and healing. Chondrocyte-specific β2-adrenoceptor-deficient mice are also protected from stress-induced bone growth retardation. In summary, our preclinical data identify locally secreted catecholamines in concert with β2-adrenoceptor signalling in chondrocytes as mediators of negative stress effects on bone growth and repair. Given our clinical data, these mechanistic insights seem to be of strong translational relevance.
“…Alkaline phosphatase is an important marker of osteoblast differentiation and can be measured by alkaline phosphatase staining and quantitative real-time polymerase chain reaction (qRT-PCR) [ 29 ]. According to the results of different concentrations, the alkaline phosphatase staining in the Wnt3a group had the darkest positive staining at the concentration of 50 ng/mL ( Figure 1 A), and the ICG-001 group had the lightest staining at 20 μM ( Figure 1 B).…”
Growth factors were often used to improve the bioactivity of biomaterials in order to fabricate biofunctionalized bone grafts for bone defect repair. However, supraphysiological concentrations of growth factors for improving bioactivity could lead to serious side effects, such as ectopic bone formation, radiculitis, swelling of soft tissue in the neck, etc. Therefore, safely and effectively applying growth factors in bone repair biomaterials comes to be an urgent problem that needs to be addressed. In this study, an appropriate concentration (50 ng/mL) of Wnt3a was used to pretreat the 3D-bioprinting gelatin methacryloyl(GelMA)/polycaprolactone(PCL) scaffold loaded with bone marrow stromal cell line ST2 for 24 h. This pretreatment promoted the cell proliferation, osteogenic differentiation, and mineralization of ST2 in the scaffold in vitro, and enhanced angiogenesis and osteogenesis after being implanted in critical-sized mouse calvarial defects. On the contrary, the inhibition of Wnt/β-catenin signaling in ST2 cells reduced the bone repair effect of this scaffold. These results suggested that ST2/GelMA/PCL scaffolds pretreated with an appropriate concentration of Wnt3a in culture medium could effectively enhance the osteogenic and angiogenic activity of bone repair biomaterials both in vitro and in vivo. Moreover, it would avoid the side effects caused by the supraphysiological concentrations of growth factors. This functionalized scaffold with osteogenic and angiogenic activity might be used as an outstanding bone substitute for bone regeneration and repair.
“…ATDC5 cells were cultured in serum containing medium (S1a) [36]. During the experiments, cells were either cultured in chondrogenic differentiation medium (CDM) (S1a) for 7 d, or in serum-reduced medium with D (250 nM) + Q (5 µM) for 3 d. In a further experiment, the cells were cultured with D+Q stimulation and with or w/o ITS (3 d).…”
Cellular senescence is associated with various age-related disorders and is assumed to play a major role in the pathogenesis of osteoarthritis (OA). Based on this, we tested a senolytic combination therapy using Dasatinib (D) and Quercetin (Q) on aged isolated human articular chondrocytes (hACs), as well as in OA-affected cartilage tissue (OARSI grade 1-2). Stimulation with D+Q selectively eliminated senescent cells in both, cartilage explants and isolated hAC. Furthermore, the therapy significantly promoted chondroanabolism, as demonstrated by increased gene expression levels of COL2A1, ACAN, and SOX9, as well as elevated collagen type II and glycosaminoglycan biosynthesis. Additionally, D+Q treatment significantly reduced the release of SASP factors (IL6, CXCL1). RNA sequencing analysis revealed an upregulation of the anabolic factors, inter alia, FGF18, IGF1, and TGFB2, as well as inhibitory effects on cytokines and the YAP-1 signaling pathway, explaining the underlying mechanism of the chondroanabolic promotion upon senolytic treatment. Accordingly, stimulation of untreated hAC with conditioned medium of D+Q-treated cells similarly induced the expression of chondrogenic markers. Detailed analyses demonstrated that chondroanabolic effects could be mainly attributed to Dasatinib, while monotherapeutical application of Quercetin or Navitoclax did not promote the chondroanabolism. Overall, D+Q therapy restored the chondrogenic phenotype in OA hAC most likely by creating a pro-chondroanabolic environment through the reduction of SASP factors and upregulation of growth factors. This senolytic approach could therefore be a promising candidate for further testing as a disease-modifying osteoarthritis drug.
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