Osteoblast Differentiation Assay Research Articles

Targeting EZH2 attenuates the ferroptosis-mediated osteoblast–osteoclast imbalance in rheumatoid arthritis

ObjectiveThe enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) can regulate osteogenesis and osteoclastogenesis. This study aimed to further explore the effects of EZH2 modification on ferroptosis and the osteoblast–osteoclast balance in rheumatoid arthritis (RA) in vitro and in vivo. MethodsBone marrow mesenchymal stromal cells were transfected with EZH2 overexpression (oeEZH2) and EZH2 shRNA (shEZH2) plasmids with or without ferrostatin-1 (Fer-1) treatment and subjected to an osteoblast differentiation assay. The cells were then cocultured with bone marrow-derived macrophages and subjected to an osteoclast differentiation assay. Collagen-induced arthritis (CIA) mice were generated and injected with shEZH2 adeno-associated virus (AAV). ResultsOeEZH2 repressed osteoblast differentiation, as reflected by decreased ALP and Alizarin Red S staining and increased OPN, RUNX2, OPG and RANKL; however, shEZH2 had the opposite effects. Besides, oeEZH2 promoted osteoblast ferroptosis, as suggested by increased MDA, Fe2+, ROS, and PTGS2 but decreased GPX4 and SLC7A11; these effects could be attenuated by Fer-1 treatment. In contrast, shEZH2 ameliorated osteoblast ferroptosis. OeEZH2 subsequently increased osteoclast differentiation, as indicated by increased TRAP+ multinucleated cells, NFATC1, CTSK, and c-FOS; however, shEZH2 had the opposite effect, except that it did not regulate CTSK. In CIA mice, shEZH2 AAV decreased the clinical symptom score, histological score of cartilage, and systemic inflammation (TNF-α and IL-6) and repressed bone ferroptosis and restored the osteoblast–osteoclast balance to some extent, as reflected by immunohistochemical staining of related markers. ConclusionTargeting EZH2 attenuates the ferroptosis-mediated osteoblast–osteoclast imbalance in RA, revealing its potential as a treatment target.

The Pseudo-Natural Product Tafbromin Selectively Targets the TAF1 Bromodomain 2.

Phenotypic assays detect small-molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule-target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4-g]quinoline (PQ) pseudo-natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA-box binding protein-associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains.

The Pseudo‐Natural Product Tafbromin Selectively Targets the TAF1 Bromodomain 2

AbstractPhenotypic assays detect small‐molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule‐target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4‐g]quinoline (PQ) pseudo‐natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA‐box binding protein‐associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains.

Abstract 2920: In vitro assays for investigating the effects of cancer therapeutics on bone

Abstract Bone microenvironment is an important factor in regulating bone metastasis. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Stimulatory factors released from the resorbed bone matrix in turn promote proliferation of cancer cells and this process, referred as the vicious cycle, often leads to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. The vicious cycle observed in osteolytic disease occurs also in osteoblastic metastases, and in addition cancer cells produce osteoblast-stimulating factors. Cancers with bone metastases often include both osteolytic and osteoblastic lesions. Our aim was to establish in vitro cell culture models to study the effects of cancer therapeutics on osteoblast and osteoclast differentiation and activity. In the osteoclast differentiation assay, human osteoclast precursor cells (Lonza) were cultured on bovine bone slices for 7 days. Denosumab was added in the cultures at day 0, and tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) activity was measured from the culture medium collected at day 7. Osteoclast activity was studied by allowing osteoclasts resorb bone after 7 days’ maturation period for additional 3 days in the presence of odanacatib. The amount of C-terminal cross-linked telopeptides of type I collagen (CTX-I) was measured in the culture medium collected at day 10 to quantitate osteoclast activity. Denosumab and odanacatib showed strong concentration dependent inhibition of osteoclast differentiation and activity, respectively. A mouse osteoblast progenitor cell line KS483 (Pharmatest Services) was used to study differentiation and activity of osteoblasts with test compounds bone morphogenetic protein 2 (BMP-2) and estradiol (E2). In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8. In the osteoblast activity assay, the cells were cultured for 13 days. N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to indicate the effects on organic bone formation and calcium deposition was determined at the end of the study as a marker of inorganic bone formation. BMP-2 and E2 stimulated osteoblast differentiation and activity indicated by the increase in ALP activity, PINP and calcium levels. We conclude that these culture systems can be used for studying the effects of cancer therapeutics and identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. Citation Format: Jenni H. Mäki-Jouppila, Mervi Ristola, Jukka Rissanen, Katja Fagerlund. In vitro assays for investigating the effects of cancer therapeutics on bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2920.

Vertical Vibration of Mouse Osteoblasts Promotes Cellular Differentiation and Cell Cycle Progression and Induces Aging In Vitro

This study aimed to investigate the effect of the vibration of osteoblasts on the cell cycle, cell differentiation, and aging. Primary maxilla osteoblasts harvested from eight-week-old mice were subjected to vibration at 3, 30, and 300 Hz once daily for 30 min; control group, 0 Hz. A cell proliferation assay and Cell-Clock Cell Cycle Assay were performed 24 h after vibration. Osteoblast differentiation assay, aging marker genes, SA-β-Gal activity, and telomere length (qPCR) were assayed two weeks post- vibration once every two days. Cell proliferation increased significantly at 30 and 300 Hz rather than 0 Hz. Several cells were in the late G2/M stage of the cell cycle at 30 Hz. The osteoblast differentiation assay was significantly higher at 30 Hz than at 0 Hz. Runx2 mRNA was downregulated at 30 Hz compared to that at 0 Hz, while osteopontin, osteocalcin, and sclerostin mRNA were upregulated. p53/p21, p16, and c-fos were activated at 30 Hz. SA-β-Gal activity increased significantly at 30 or 300 Hz. Telomere length was significantly lower at 30 or 300 Hz. The results suggest that providing optimal vibration to osteoblasts promotes cell cycle progression and differentiation and induces cell aging.

Synthesis and biological evaluation of substituted amide derivatives of C4-ageratochromene dimer analog

Substituted amide derivatives of C4-ageratochromene dimer analog (19) were synthesized through structural modification of precocene-I (4a), isolated from the essential oil of Ageratum conyzoides L. The target compounds (18–20, 23I-VI, 24I-VI, and 25I-VI) were evaluated for their bone-forming effect using osteoblast differentiation assay. Seven compounds (23I, 23II, 23IV, 23VI, 24III, 24VI, and 25VI) presented good activity within 1 pM–1 nM concentration. At 1 pM concentration, the most active compound i.e. 23II showed effective mineralization of osteoblast cells along with expression of osteogenic marker genes viz RUNX 2, BMP-2, and type 1 collagen (Type-1 col) without any toxicity towards osteoblast cells. Single crystal X-ray analysis of 18 and 20 revealed that the core nucleus of these molecules bear phenyl rings in a Trans-stilbenoid system and had a good structural correlation with 17β-estradiol (1) and diethylstilbestrol (DES, 3). In-silico study about 23II showed its structural complementarities with the LBD of estrogen receptor (ER) which indicated possible ER-mediated activity of compounds.

8. Modifiable polymer promotes a pro-osteogenic, M2-like macrophage phenotype and osteoblastic differentiation of progenitor cells

8. Modifiable polymer promotes a pro-osteogenic, M2-like macrophage phenotype and osteoblastic differentiation of progenitor cells

Abstract 2161: In vitro osteoblast assays for studying the effects of cancer therapeutics on bone biology

Abstract Bone cell activity is an important factor in regulating bone metastases from tumor cells in the skeletal environment. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Bone then releases stimulatory factors that in turn promote the growth and proliferation of cancer cells. This process is called the vicious cycle often leading to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. Osteolytic lesions are common in breast and lung cancer and multiple myeloma. There is a vicious cycle between cancer cells and bone cells also in the context of osteoblastic lesions. In osteoblastic metastases, the vicious cycle observed in osteolytic disease takes place but in addition to this cancer cells produce osteoblast-stimulating factors including bone morphogenetic protein (BMP), epidermal growth factor (EGF) and platelet derived growth factor (PDGF). Osteoblasts also influence osteoclasts by producing RANKL, which stimulates osteoclast differentiation. Many cancer patients with bone metastasis have both osteolytic and osteoblastic lesions. This is common for example in prostate cancer. Our aim was to establish an in vitro cell culture model to study the effects of compounds on osteoblast differentiation and activity. A mouse osteoblast progenitor cell line KS483 was used in the study. BMP-2 was used as a test compound and was added in the beginning of culture and simultaneously with culture medium change every 3-4 days. Ascorbic acid was added into the cell culture medium at day 4. In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8 from cell lysates. Total protein content was measured from the same samples. In the osteoblast activity assay, KS483 mouse osteoprogenitor cells were cultured for 13 days, during which N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to demonstrate effects on organic bone matrix formation. β-glycerophosphate was added to the culture at day 11. The cultures were stopped at day 13 by removing the culture media from the wells and adding hydrochloric acid. Calcium deposition, a marker of inorganic bone formation, was determined at the end of the study. BMP-2 stimulated osteoblast differentiation and activity shown by the increase in ALP, PINP and calcium levels. The results suggest that the KS483 cell line can be used for setting up reliable in vitro models of osteoblast differentiation and activity. These osteoblast assays can be used for studying the effects of compounds on bone formation and cancer related bone events. Citation Format: Jenni H. Mäki-Jouppila, Jussi M. Halleen, Katja Fagerlund. In vitro osteoblast assays for studying the effects of cancer therapeutics on bone biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2161.

SPARC inhibits breast cancer bone metastasis and may be a clinical therapeutic target.

Breast cancer is one of the most common types of cancer in females worldwide, and metastasis to bone is an important characteristic of malignancy. The present study aimed to investigate the molecular mechanism of breast cancer to bone metastasis of secreted protein acidic and rich in cysteine (SPARC). Immunohistochemistry was performed to examine the expression of SPARC in primary breast tumors and bone metastatic foci. Western blotting and reverse transcription-quantitative polymerase chain reaction were performed to detect the expression level of SPARC in several types of breast cancer cell. A Transwell filter assay was used to assess the effect of SPARC on breast cancer cell invasion ability, and an osteoblast differentiation assay was employed to analyze the effect of SPARC on the differentiation ability of mesenchymal stem cells. Clinical data revealed that decreased stromal SPARC expression is associated with breast cancer to bone metastasis. Gain- and loss-of-function studies reveal that SPARC inhibits the migration and invasion of breast cancer cells, and suppresses osteoclast activation in the breast cancer microenvironment. SPARC serves an important role in breast cancer bone metastasis and may be a promising therapeutic target for the treatment of breast cancer bone metastasis.

Fabrication of a Biodegradable Implant with Tunable Characteristics for Bone Implant Applications.

Biodegradable polymers are appealing material for the manufacturing of surgical implants as such implants break down in vivo, negating the need for a subsequent operation for removal. Many biocompatible polymers produce acidic breakdown products that can lead to localized inflammation and osteolysis. This study assesses the feasibility of fabricating implants out of poly(propylene carbonate) (PPC)-starch that degrades into CO2 and water. The basic compression modulus of PPC-starch (1:1 w/w) is 34 MPa; however, the addition of glycerol (1% w/w) and water as plasticizers doubles this value and enhances the surface wettability. The bioactivity and stiffness of PPC-starch blends is increased by the addition of bioglass microparticles (10% w/w) as shown by in vitro osteoblast differentiation assay and mechanical testing. MicroCT analysis confirms that the bioglass microparticles are evenly distributed throughout biomaterial. PPC-starch-bioglass was tested in vivo in two animal models. A murine subcutaneous pellet degradation assay demonstrates that the PPC-starch-bioglass blend's volume fraction loss is 46% after 6 months postsurgery, while it is 27% for poly(lactic acid). In a rat knee implantation model, PPC-starch-bioglass screws inserted into the distal femur show osseointegration with no localized adverse effects after 3 and 12 weeks. These data support the further development of PPC-starch-bioglass as a medical biomaterial.

Differentiation of skeletal osteogenic progenitor cells to osteoblasts with 3,4-diarylbenzopyran based amide derivatives: Novel osteogenic agents

A series of 3,4-diarylbenzopyran based amide derivatives was synthesized and evaluated for osteogenic activity in in vitro and in vivo models of osteoporosis. Compounds 17a, 21b–c and 22a–b showed significant osteogenic activity in osteoblast differentiation assay. Among the synthesized compounds, 22b was identified as lead molecule which showed significant osteogenic activity at 1 pM concentration in osteoblast differentiation assay and at 1 mg kg−1 body weight dose in estrogen deficient balb/c mice model. In vitro bone mineralization and expression of osteogenic marker genes viz BMP-2, RUNX-2, OCN, and collagen type 1 further confirmed the osteogenic potential of 22b. Gene expression study for estrogen receptor α and β (ER-α and ER-β) in mouse calvarial osteoblasts (MCOs) unveiled that possibly 22b exerted osteogenic efficacy via activation of Estrogen receptor-β preferentially. In vivo pharmacokinetic, estrogenicity and acute toxicity studies of 22b showed that it had good bioavailability and was devoid of uterine estrogenicity at 1 mg kg−1 and inherent toxicity up to 1000 mg kg−1 body weight dose respectively.

Comparison of osteoblast and cardiomyocyte differentiation in the embryonic stem cell test for predicting embryotoxicity in vivo

One of the most studied alternative embryotoxicity assays is the embryonic stem cell test, in which the effect of compounds on cardiomyocyte differentiation is evaluated (subsequently termed the ESTc). This single differentiation endpoint may limit the predictive value of the assay. We recently published a novel embryonic stem cell based osteoblast differentiation assay (subsequently termed the ESTo), in which we studied the effect of six embryotoxic compounds. Differentiation is monitored via the differential expression of three genes related to osteogenesis (Runx2, SPARC and collagen type I). In the current study, we evaluated the effect of 14 additional compounds in the ESTo, to assess its added value as compared to the ESTc. To this end, we compared the effects of the compounds in the ESTo to their effects in the ESTc and to their published in vivo developmental toxicity profiles. The results show that there is a high overall correlation between compound potencies as regards inhibition of osteoblast and cardiomyocyte differentiation. Moreover, the results in both the ESTo and ESTc showed a significant correlation to in vivo developmental toxicity potency ranking of compounds tested. Interestingly, the embryotoxic effect of TCDD could only be detected using the ESTo, which can be explained based on its mechanism of action and its known inhibitory effect on osteogenesis. The results of TCDD suggest that incorporating the ESTo into a testing battery together with the ESTc could improve the overall predictive value of the battery.

Mg ion implantation on SLA-treated titanium surface and its effects on the behavior of mesenchymal stem cell

Magnesium (Mg) is one of the most important ions associated with bone osseointegration. The aim of this study was to evaluate the cellular effects of Mg implantation in titanium (Ti) surfaces treated with sand blast using large grit and acid etching (SLA). Mg ions were implanted into the surface via vacuum arc source ion implantation. The surface morphology, chemical properties, and the amount of Mg ion release were evaluated by scanning electron microscopy (SEM), Auger electron spectroscopy (AES), Rutherford backscattering spectroscopy (RBS), and inductively coupled plasma-optical emission spectrometer (ICP-OES). Human mesenchymal stem cells (hMSCs) were used to evaluate cellular parameters such as proliferation, cytotoxicity, and adhesion morphology by MTS assay, live/dead assay, and SEM. Furthermore, osteoblast differentiation was determined on the basis of alkaline phosphatase (ALP) activity and the degree of calcium accumulation. In the Mg ion-implanted disk, 2.3×1016ions/cm2 was retained. However, after Mg ion implantation, the surface morphology did not change. Implanted Mg ions were rapidly released during the first 7days in vitro. The MTS assay, live/dead assay, and SEM demonstrated increased cell attachment and growth on the Mg ion-implanted surface. In particular, Mg ion implantation increased the initial cell adhesion, and in an osteoblast differentiation assay, ALP activity and calcium accumulation. These findings suggest that Mg ion implantation using the plasma source ion implantation (PSII) technique may be useful for SLA-treated Ti dental implants to improve their osseointegration capacity.

Novel oxysterols activate the Hedgehog pathway and induce osteogenesis

Localized induction of bone formation is essential during orthopedic procedures that involve skeletal repair, such as surgical treatment of non-union bone fractures and degenerative disk disease. Herein we disclose the synthesis and biological evaluation of novel oxysterol derivatives designed as anabolic bone growth agents. Structure–activity relationship studies of oxysterol 4 have identified analogues such as 18, 21 and 30. These new analogues are characterized by higher potency in an osteoblast differentiation assay and/or by increased metabolic stability in human liver microsomes. Oxysterols 4, 18 and 21 were evaluated in vivo in a rat spinal fusion model.

The effect of surface microgrooves and anodic oxidation on the surface characteristics of titanium and the osteogenic activity of human periodontal ligament cells

ObjectiveThe purpose of this study was to investigate the effect of titanium (Ti) surface microgrooves and anodic oxidation on the surface characteristics of titanium and the osteogenic activity of human periodontal ligament cells (PLCs) cultured on theses surfaces. DesignMechanically ground Ti was used as the control substratum (NE0). Truncated V-shaped microgrooves, 60μm-wide and 10μm-deep in cross-sections, were created on the Ti substrata by photolithography (NE60/10). Anodically oxidized Ti (NE0AO) and anodically oxidized microgrooved Ti (NE60/10AO) were also prepared. Scanning electron microscopy, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) were performed for surface characterization. Cell proliferation assay, osteoblast differentiation assay, and quantitative real-time PCR analysis were performed to compare the osteogenic activity of PLCs on NE0, NE60/10, NEAO, and NE60/10AO. ResultsA decrease in the microgroove-width of NE60/10AO compared to NE60/10 due to Ti oxide layer generation by anodic oxidation was detected with XRD and XPS. Cell proliferation, osteoblast differentiation, and osteo-related gene expression were enhanced on the NE60/10AO substrata compared with NE0, NE60/10, and NE0AO. ConclusionsThe combination of Ti surface microgrooves and subsequent anodic oxidation treatment synergistically upregulated osteo-related gene expression, despite showing limited ability to increase cell proliferation and osteoblast differentiation levels in PLCs.

Ent-Kaurane Diterpenoids from Croton tonkinensis Stimulate Osteoblast Differentiation

Four new ent-kaurane diterpenoids (1-4) were isolated from the leaves of Croton tonkinensis by bioactivity-guided fractionation using an in vitro osteoblast differentiation assay. Their structures were identified as ent-11β-acetoxykaur-16-en-18-ol (1), ent-11α-hydroxy-18-acetoxykaur-16-ene (2), ent-14β-hydroxy-18-acetoxykaur-16-ene (3), and ent-7α-hydroxy-18-acetoxykaur-16-ene (4). Compounds 1-4 significantly increased alkaline phosphatase activity and osteoblastic gene promoter activity. Compounds 1-3 also increased the levels of ALP and collagen type I alpha mRNA in C2C12 cells in a dose-dependent manner. These results suggest that ent-kaurane diterpenoids from C. tonkinensis have a direct stimulatory effect on osteoblast differentiation and may be potential therapeutic molecules against bone diseases such as osteoporosis.

Development of an osteoblast-based 3D continuous-perfusion microfluidic system for drug screening

In this work, we demonstrated that biological cells could be cultured in a continuous-perfusion glass microchip system for drug screening. We used mouse Col1a1GFP MC-3T3 E1 osteoblastic cells, which have a marker gene system expressing green fluorescent protein (GFP) under the control of osteoblast-specific promoters. With our microchip-based cell culture system, we realized automated long-term monitoring of cells and sampling of the culture supernatant system for osteoblast differentiation assay using a small number of cells. The system successfully monitored cells for 10 days. Under the 3D microchannel condition, shear stress (0.07 dyne/cm(2) at a flow rate of 0.2 microL/min) was applied to the cells and it enhanced the GFP expression and differentiation of the osteoblasts. Analysis of alkaline phosphatase (ALP), which is an enzyme marker of osteoblasts, supported the results of GFP expression. In the case of differentiation medium containing bone morphogenetic protein 2, we found that ALP activity in the culture supernatant was enhanced 10 times in the microchannel compared with the static condition in 48-well dishes. A combined system of a microchip and a cell-based sensor might allow us to monitor osteogenic differentiation easily, precisely, and noninvasively. Our system can be applied in high-throughput drug screening assay for discovering osteogenic compounds.

Cytotoxic cells against trophoblasts during human spontaneous abortions

One of the most studied alternative embryotoxicity assays is the embryonic stem cell test, in which the effect of compounds on cardiomyocyte differentiation is evaluated (subsequently termed the ESTc). This single differentiation endpoint may limit the predictive value of the assay. We recently published a novel embryonic stem cell based osteoblast differentiation assay (subsequently termed the ESTo), in which we studied the effect of six embryotoxic compounds. Differentiation is monitored via the differential expression of three genes related to osteogenesis (Runx2, SPARC and collagen type I). In the current study, we evaluated the effect of 14 additional compounds in the ESTo, to assess its added value as compared to the ESTc. To this end, we compared the effects of the compounds in the ESTo to their effects in the ESTc and to their published in vivo developmental toxicity profiles. The results show that there is a high overall correlation between compound potencies as regards inhibition of osteoblast and cardiomyocyte differentiation. Moreover, the results in both the ESTo and ESTc showed a significant correlation to in vivo developmental toxicity potency ranking of compounds tested. Interestingly, the embryotoxic effect of TCDD could only be detected using the ESTo, which can be explained based on its mechanism of action and its known inhibitory effect on osteogenesis. The results of TCDD suggest that incorporating the ESTo into a testing battery together with the ESTc could improve the overall predictive value of the battery.