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Bone health-targeting drug development strategies still largely rely on inferior 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings. MC3T3-E1 pre-osteoblasts were cultured in α-MEM with 10% FCS, at 37 °C and 5% CO2 for up to 28 days, in non-adherent V-shaped plates to form uniformly sized 3D spheroids. Osteogenic differentiation was induced by 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid. Mineralization stages were assessed through studying expression of marker genes, alkaline phosphatase activity, and calcium deposition by histochemistry. Mineralization quality was evaluated by Fourier transformed infrared (FTIR) and scanning electron microscopic (SEM) analyses and quantified by micro-CT analyses. Expression profiles of selected early- and late-stage osteoblast differentiation markers indicated a well-developed 3D biomineralization process with strongly upregulated Col1a1, Bglap and Alpl mRNA levels and type I collagen- and osteocalcin-positive immunohistochemistry (IHC). A dynamic biomineralization process with increasing mineral densities was observed during the second half of the culture period. SEM–Energy-Dispersive X-ray analyses (EDX) and FTIR ultimately confirmed a native bone-like hydroxyapatite mineral deposition ex vivo. We thus established a robust and versatile biomimetic, and high-throughput compatible, cost-efficient spheroid culture model with a native bone-like mineralization for improved pharmacological ex vivo screenings. [ABSTRACT FROM AUTHOR]

Bone health-targeting drug development strategies still largely rely on inferior 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings. MC3T3-E1 pre-osteoblasts were cultured in α-MEM with 10% FCS, at 37 °C and 5% CO2 for up to 28 days, in non-adherent V-shaped plates to form uniformly sized 3D spheroids. Osteogenic differentiation was induced by 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid. Mineralization stages were assessed through studying expression of marker genes, alkaline phosphatase activity, and calcium deposition by histochemistry. Mineralization quality was evaluated by Fourier transformed infrared (FTIR) and scanning electron microscopic (SEM) analyses and quantified by micro-CT analyses. Expression profiles of selected early- and late-stage osteoblast differentiation markers indicated a well-developed 3D biomineralization process with strongly upregulated Col1a1, Bglap and Alpl mRNA levels and type I collagen- and osteocalcin-positive immunohistochemistry (IHC). A dynamic biomineralization process with increasing mineral densities was observed during the second half of the culture period. SEM–Energy-Dispersive X-ray analyses (EDX) and FTIR ultimately confirmed a native bone-like hydroxyapatite mineral deposition ex vivo. We thus established a robust and versatile biomimetic, and high-throughput compatible, cost-efficient spheroid culture model with a native bone-like mineralization for improved pharmacological ex vivo screenings.

Small teleost fishes such as zebrafish and medaka show remarkable regeneration capabilities upon tissue injury or amputation. To elucidate cellular mechanisms of teleost tissue repair and regeneration processes, the Cre/LoxP recombination system for cell lineage tracing is a widely used technique. In this chapter, we describe protocols used for inducible Cre/LoxP recombination-mediated lineage tracing of osteoblast progenitors during medaka fin regeneration as well as during the repair of osteoporosis-like bone lesions in the medaka vertebral column. Our approach can be adapted for lineage tracing of other cell populations in the regenerating teleost fin or in other tissues undergoing repair., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aim. Runx2 (AML3) transcription factor is the key regulator of osteoblastic lineage progression and is indispensable for the formation of mineral bones. Runx2 expression increases during differentiation of osteoblasts to induce osteoblast-specific genes necessary for the production and deposition of bone mineral matrix. However, Runx2 is also expressed at a lower level in early osteoprogenitors, where its function is less understood. Here we study how Runx2 determines the early stages of osteoblastic commitment using the model system of Runx2 re-introduction in mouse calvaria cells with Runx2 null background. Method. Affymetrix analysis, Western blot analysis and quantitative real-time reverse transcriptase PCR (qRT-PCR) analysis were employed. Results. Gene expression profiling by Affymetrix microarrays revealed that along with the induction of extracellular matrix and bone mineral deposition related phenotypic markers, Runx2 regulates several cell programs related to signaling and metabolism in the early osteoprogenitors. Particularly, Runx2 regulates transcription of genes involved in G-protein coupled signaling network, FGF and BMP/TGF beta signaling pathways and in biogenesis and metabolism pathways of steroid hormones. Conclusion. The data indicate that the lineage specific program, regulated by the master regulatory transcription factor, includes the regulation of cellular signaling and metabolism which may allow the committed cell to react and behave differently in the same microenvironment. [ABSTRACT FROM AUTHOR]

Abstract: The type I collagen promoter has been used to develop transgenic constructs that are able to mark different stages of osteoblastic differentiation. The pOBCol3.6 promoter is active in early mesenchymal progenitors, including preosteoblasts and osteoblasts, while the pOBCol2.3 promoter is more restricted, showing expression in mature osteoblasts and osteocytes. Transgenic mouse lines have been created that express various GFP reporters under the control of both promoters. These transgenic mice permit the tracking of osteoblastic lineage progression in vitro. They also represent a system to test lineage progression in vivo after the transplantation of progenitors. A parabiosis system was used in which pOBCol3.6GFP transgenic mice were surgically joined with mice bearing a Col2.3ΔTK transgene. The Col2.3ΔTK transgenic mouse bears a herpes thymidine kinase gene driven by the pOBCol2.3 promoter, and upon treatment with gancyclovir (GCV) displays extensive destruction of the bone lining cells. After a common circulation was established, parabiotic pairs were treated with GCV for 15 days. Histological analysis of their bones showed the clear presence of GFP positive cells in the Col2.3ΔTK parabionts, around trabecular bone and on the endosteal and periosteal surfaces. Stromal cell cultures from these Col2.3ΔTK parabionts did not display mineralized colonies coexpressing GFP. In contrast, scattered GFP positive clusters that contained large cells with morphology similar to osteoclast like cells (OCLs) were observed. These cells were also TRAP positive. They were readily detected in Col2.3ΔTK mice treated with GCV and transplanted with purified hematopoietic stem cells (HSCs) isolated from pOBCol3.6GFP mice. OCLs were also generated in vitro from osteoclast progenitor cells obtained from pOBCol3.6GFP mice that were defined by the B220− CD3− CD11b− c-fms+ phenotype. Molecular analysis showed that OCLs did not express type I collagen indicating that the Col3.6 promoter contains elements that are active during osteoclastogenesis and are not strictly related to collagen transcription. In summary, we demonstrate that pOBCol3.6 unexpectedly directs the expression of transgenes in the osteoclast lineage and this effect must be considered when utilizing this promoter to study of mesenchymal progenitor cells. [Copyright &y& Elsevier]

Abstract: Biglycan (bgn) is a small leucine-rich proteoglycan (SLPR) that is enriched in the extracellular matrix of skeletal tissues. Bgn-deficient mice develop age-related osteopenia with a phenotype that resembles osteoporosis. In order to identify sets of genes that play a key role in the skeletal abnormality, we determined the global gene expression patterns in bgn-deficient (bgn-KO) pre-osteoblasts using oligonucleotide microarray technology. Calvarial cells were harvested from newborn mice and cultured in the presence or absence of BMP-4 for 7 days. The total RNA was purified, labeled and hybridized to Affymetrix™ chips (U74A), and analyzed with a software program called GeneSpring™. Our data suggested that biglycan regulates the activity of osteoblastic progenitors through sets of genes associated with cell cycle, cell growth, and differentiation. The biological outcome from the altered expression of these genes could cause a defect in the quantity and quality of osteoblastic progenitors, which could contribute to the development of age-related osteopenia in bgn-KO mice. Moreover, the data from this approach also revealed that biglycan deficiency affected the genes that control inflammation, immune response, and growth of tumor cells. These are new and unexpected findings that lead to the formation of new paradigms for biglycan function. Based on these findings, we propose that the reduction of this small proteoglycan with aging may increase the risk of infection and autoimmune diseases, impair wound healing, and cause higher incidences of malignancy. This study provides a broad and deep foundation for understanding SLRP function at a more complex level. [Copyright &y& Elsevier]