Mineralization Of Osteoblastic Cells Research Articles

The screened compounds from Ligustri Lucidi Fructus using the immobilized calcium sensing receptor column exhibit osteogenic activity in vitro

Calcium sensing receptor (CaSR) has become the novel target of treating osteoporosis with herbal medicine Ligustri Lucidi Fructus (LLF), however, the bioactive compounds responsible for anti-osteoporosis are hard to clarify due to the complexity and diversity of chemical constituents in it. Herein, the immobilized CaSR column was packed with stationary phase materials, which were derived from integrating CLIP-tagged CaSR directly out of crude cell lysates onto the surface of silica gels (5.83 mg/g) in a site-specific covalent manner. The column had a great specificity of recognizing agonists and kept a good stability for at least 3 weeks. The two compounds from LLF extract were screened and identified as olenuezhenoside and ligustroflavone using the immobilized CaSR column in conjunction with mass spectrometry. Molecular docking predicted that both compounds were bound in venus flytrap (VFT) domain of CaSR by the formation of hydrogen bonds. Cellular results showed that both compounds exhibited the distinct osteogenic activity by enhancing the proliferation, differentiation and mineralization of osteoblastic cells. Our study demonstrated that, the immobilized protein column enables to screen the bioactive compounds rapidly from herbal extract, and the newly discovered natural product ligands towards CaSR, including olenuezhenoside and ligustroflavone, will be the candidates for the treatment of osteoporosis.

Modulation of calcium-influx by carboxymethyl tamarind‑gold nanoparticles promotes biomineralization for tissue regeneration

Gold nanoparticles (AuNPs) have been reported to modulate bone tissue regeneration and are being extensively utilized in biomedical implementations attributable to their low cytotoxicity, biocompatibility and simplicity of functionalization. Lately, biologically synthesized nanoparticles have acquired popularity because of their environmentally acceptable alternatives for diverse applications. Here we report the green synthesis of AuNPs by taking the biopolymer Carboxymethyl Tamarind (CMT) as a unique reducing as well as a stabilizing agent. The synthesized CMT-AuNPs were analyzed by UV–vis spectrophotometer, DLS, FTIR, XRD, TGA, SEM and TEM. These results suggest that CMT-AuNPs possess an average size of 19.93 ± 8.52 nm and have long-term stability. Further, these CMT-AuNPs promote the proliferation together with the differentiation and mineralization of osteoblast cells in a “dose-dependent” manner. Additionally, CMT-AuNPs are non-toxic to SD rats when applied externally. We suggest that the CMT-AuNPs have the potential to be a suitable and non-toxic agent for differentiation and mineralization of osteoblast cells in vitro and this can be tested in vivo as well.

Ultrasound stimulated piezoelectric barium titanate and boron nitride nanotubes in nonconductive poly-ε-caprolactone nanofibrous scaffold for bone tissue engineering

Nanomaterials can provide unique solutions for the problems experienced in tissue engineering by improving a scaffold’s physico-bio-chemical properties. With its piezoelectric property, bone is an active tissue with easy adaptation and remodeling through complicated mechanisms of electromechanical operations. Although poly(ε-caprolactone) (PCL) is an excellent polymer for bone tissue engineering, it is lack of conductivity. In this study, piezoelectric barium titanates (BaTiO3) and boron nitride nanotubes (BNNTs) are used as ultrasound (US) stimulated piezoelectric components in PCL to mimic piezoelectric nature of bone tissue. Electric-responsive Human Osteoblast cells on the scaffolds were stimulated by applying low-frequency US during cell growth. Biocompatibility, cell adhesion, alkaline phosphatase activities and mineralization of osteoblast cells on piezo-composite scaffolds were investigated. BaTiO3 or BNNTs as reinforcement agents improved physical and mechanical properties of PCL scaffolds. In vitro studies show that the use of BaTiO3 or BNNTs as additives in non-conductive scaffolds significantly induces and increases the osteogenic activities even without US stimulation. Although BaTiO3 is one of the best piezoelectric materials, the improvement is more dramatic in the case of BNNTs with the increased mineralization, and excellent chemical and mechanical properties.

Abstract 3828: Herbal extracts from lycii radicis corex and achyranthes japonica prevent multiple myeloma progression

Abstract Multiple Myeloma (MM) is a plasma cell malignancy that, despite advances in treatment, remains incurable. In over 90% of MM patients, aberrant bone remodeling occurs and results in osteolytic lesions. Korean traditional medicine has a long-standing interest in healthy foods to enhance the immune system, energy-boost, and Yin-and-Yang balance. Traditional medicine has used herbal extracts (HE) from natural plants with fewer side effects and long-term treatment tolerance. In earlier studies, lycii radices cortex (LRC) and achyranthes japonica (AJ) containing HE have demonstrated the ability to enhance cell growth and mineralization of osteoblast cells while also inhibiting osteoclast differentiation. The current study investigated the effects of LRC+AJ containing HE on murine 5TGM1 MM-bearing mice.This HE significantly increased the mouse survivals of both Treatment before Transplant (TbT) and Treatment after Transplant (TaT) groups with median survivals of undetermined and 52.5 days, respectively, while MM control had a median survival of 42 days. The Mantel-Cox test found that TbT and TaT mice were significantly different from the control group (P=0.0014 and 0.0182, respectively). In histomorphometric analysis, the spines were scanned by micro-CT and revealed that TbT and TaT groups had significantly increased bone volume over total volume (BV/TV) than control. To see if HE affects cell growth of myeloma cells and osteoblast cells, we further investigated the HE on preosteoblast cell line MC3T3-E1, mouse MM cell line 5TGM1-Luc, and human MM cell line U-266. Cells were treated with HE, and viability was assessed at 48 and 96 hours post-treatment. Remarkably, LRC+AJ containing HE increased MC3T3-E1 cell growth while it decreased 5TGM1 and U-266 cell viability. In addition, we found the DAP3G in HE used in this study.Our results demonstrated that LRC+AJ HE prevents MM and promotes bone formation in 5TGM1 engrafted NSG mice. We also found that LRC+AJ HE suppresses myeloma cell growth and enhances osteoblast cell survival. Although it is yet to be defined, a correlation of osteoblast activity and myeloma cell inhibition suggests a possible connection of these events to prevent MM progression. Citation Format: Syed Hassan Mehdi, Sun-OK Lee, Dam Huh, Donghoon Yoon. Herbal extracts from lycii radicis corex and achyranthes japonica prevent multiple myeloma progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3828.

The fabrication of multifunctional sodium alginate scaffold incorporating ibuprofen-loaded modified PLLA microspheres based on cryogenic 3D printing

A strategy to develop a multifunctional sodium alginate personalized scaffold with enhanced mechanical stability, osteogenesis activity and excellent anti-inflammatory activity by cryogenic 3 D printing combined with subsequent crosslinking with Sr2+ is proposed in this study. The ink for 3 D printing was prepared by dispersing modified PLLA droplets containing ibuprofen into sodium alginate aqueous solution using lecithin as stabilizer. The results showed that the drug-loaded microspheres formed from the low-temperature solidifying of the modified PLLA droplets were homogeneously dispersed in sodium alginate substrate, and the scaffold displayed a sustained drug release performance toward ibuprofen which endowed the scaffold with persistent anti-inflammatory effects. In vitro cell culture indicated that the lecithin not only acted as the stabilizer, but also stimulated the proliferation and mineralization of osteoblastic cells on the scaffold. Sr2+-crosslinking improved the mechanical properties and osteogenic activity of the scaffold.

Herbal Extracts from Lycii Radicis Corex and Achyranthes japonica Prevent Multiple Myeloma

IntroductionMultiple Myeloma (MM) is a plasma cell malignancy that, despite advances in treatment, remains incurable. In over 90% of MM patients, aberrant bone remodeling occurs and results in osteolytic lesions. It severely reduces the patient's quality of life and increases mortality. Korean traditional medicine has a long-standing interest in healthy foods to enhance the immune system, energy-boost, and Yin-and-Yang balance. A healthy diet in Tradition medicine is based on accumulated observations from countless cases over multi-centuries. Traditional medicine has used herbal extracts (HE) from natural plants with fewer side effects and long-term treatment tolerance. In earlier studies, lycii radices cortex (LRC) and achyranthes japonica (AJ) containing herbal extracts (HE) have demonstrated the ability to enhance cell growth and mineralization of osteoblast cells while also inhibiting osteoclast differentiation. Furthermore, a sesquiterpene glucoside, (1'R,3'S,5'R,8'S,2 Z,4 E)-dihydrophaseic acid 3'-O-β-d-glucopyranoside (DPA3G) were isolated from the LRC ethanol extract and shown to be a bioactive compound for enhancement of cell growth and bone anabolic activity (Park et al). The current study investigated the effects of LRC+AJ containing HE on murine 5TGM1 MM-bearing mice.MethodsWe transplanted 1x10 6 cells of the enforced luciferase-expressing 5TGM1 (5TGM1-Luc) into 8~12-week-old Nod-Scid-IL2Rg null (NSG) mice via tail vein (representing an equal number of each sex). For LRC+AJ containing HE treatment, mice were divided into three groups; i) PBS group; ii) treatment group at 300 mg/kg, gavage, TIW started two weeks before MM transplant (TbT); and iii) treatment group at 300 mg/kg, gavage, TIW two weeks after MM transplant (TaT). We assessed tumor burden by weekly bioluminescence imaging (IVIS 200 Imager, Perkin Elmer). The spine was extracted from carcass and scanned at postmortem by Dual Energy X-ray Absorptiometry using PIXImus Densitometer (G.E. Lunar, Madison, USA) and micro-computed tomography (microCT; Scanco Medical AG, Switzerland).HE effects in cell growth were tested in the myeloma cells (5TGM1 and U266) and preosteoblast cells (MC3T3-E1). The cells were grown in various concentrations (8, 80, 800 ug/ml) of HE up to 96 hours. In addition, Chromatogram and Mass Spectrometry were conducted to identify the DPA3G.ResultsAs shown in Figure 1A, this HE significantly increased the mouse survivals of both TbT and TaT groups with median survivals of undetermined and 52.5 days, respectively, while MM control had a median survival of 42 days. The Mantel-Cox test found that TbT and TaT mice were significantly different from the control group (P=0.0014 and 0.0182, respectively). Furthermore, DEXA scans at postmortem showed a significant increase in bone mineral density (BMD) and bone mineral content (BMC) in TbT and TaT groups than control MM mice. For a histomorphometry analysis, the spines were scanned by micro-CT and revealed that TbT and TaT groups had significantly increased bone volume over total volume (BV/TV) than control. To see if HE affects cell growth of myeloma cells and osteoblast cells, we further investigated the HE on preosteoblast cell line MC3T3-E1, mouse MM cell line 5TGM1-Luc, and human MM cell line U-266. Cells were treated with HE in various concentrations, and viability was assessed at 48 and 96 hours post-treatment. Remarkably, LRC+AJ containing HE increased MC3T3-E1 cell growth while it decreased 5TGM1 and U-266 cell viability. We identified the DAP3G and many other compounds in HE used in this study.ConclusionsOur results demonstrated that LRC+AJ HE prevents MM and promotes bone formation in 5TGM1 engrafted NSG mice. We also found that LRC+AJ HE suppresses myeloma cell growth and enhances osteoblast cell survival. Although it is yet to be defined, a correlation of osteoblast activity and myeloma cell inhibition suggests a potential mechanism of the HE action to prevent MM progression.1. Park E, Kim J, Yeo S, Lim E, Choi CW, Choi S, Li WY, Lee JW, Park JH, Huh D, Jeong SY. Anti-Osteoporotic Effects of Combined Extract of Lycii Radicis Cortexand Achyranthes japonica in Osteoblast and Osteoclast Cells and Ovariectomized Mice. Nutrients. 2019 Nov 9;11(11):2716. doi: 10.3390/nu11112716. PMID: 31717518; PMCID: PMC6893723.' [Display omitted] DisclosuresHuh: Dongwoodang Pharmacy Company: Current Employment. OffLabel Disclosure:Herbal Extracts from Lycii Radicis Corex and Achyranthes Japonica

Phenanthrenoid Coelogin Isolated from Coelogyne cristata Exerts Osteoprotective Effect Through MAPK-Mitogen-Activated Protein Kinase Signaling Pathway.

Osteoporosis is a major health problem in postmenopausal women globally. This study determined the mechanism through which coelogin stimulates osteoblastogenesis and its osteoprotective and bone regenerating potential. Coelogin effect on primary calvarial osteoblast cells was determined by measuring alkaline phosphatase activity, mineralization, osteoblast survival, and apoptosis and protein expression studies. The osteoprotective effect of coelogin was also evaluated on osteopenic adult female Swiss mice. At autopsy, bones were collected for dynamic and histomorphometry studies. Serum samples were also collected for assessment of serum parameters. Coelogin treatment led to increased osteoblast proliferation, survival, differentiation, and mineralization in osteoblast cells. Coelogin supplementation to Ovx mice promoted new bone formation, prevented Ovx-induced deterioration of bone microarchitecture, and enhanced bone regeneration. In addition, signaling studies revealed that coelogin treatment activates the ER-Erk and Akt-dependent signaling pathways which stimulate the osteoblastogenesis in osteoblast cells.

Prevalence and associated factors of radiographic vertebral fractures in men: Rural population cross-sectional observation study in Japan

BackgroundOsteoporotic fractures constitute a major health concern in both women and men, particularly with the aging population. Many potential risk factors of vertebral fractures (VFx) have been identified in women, but not in men. Several reports have focused on the quality of life (QOL) of clinically diagnosed VFx, but not much has been reported on quantified QOL scores of incidental radiographic VFx in men. This cross-sectional study aimed to investigate the associated factors and the influence of incident radiographic VFx on QOL of men. MethodsA total of 401 volunteer men aged 40 years or older (40–92 years) participated in this study. The mean age was 60.5 ± 11.8 years. Prevalent VFx were identified on the lateral lumbar spinal radiographs using the semiquantitative method. Bone mineral density, body composition, smoking, alcohol intake, past medical history, Mini-Mental State Examination, grip strength, gait speed, and biochemical markers were examined. QOL was evaluated using the 36-Item Short-Form Health Survey and the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire. ResultsOf the 401 subjects, 35 men (8.7%) had the prevalence of VFx. After adjustment for age, the presence of VFx was significantly associated with IGF-1 (odds ratio 0.985; p = 0.02). QOL scores were not significantly associated with VFx after adjustment for age. ConclusionsIGF-1 is the mediator of growth hormone, and IGF-1 stimulates the proliferation, differentiation, and mineralization of osteoblastic cells. The prevalence of VFx in men was 8.7%, and IGF-1 was significantly lower in the VFx group than in the non-VFx group.

Osteogenesis activity of fractions extracted from <i> Clinacanthus nutans</i> (Burm. F.) Lindau

Osteogenesis activity of fractions extracted from Clinacanthus nutans was evaluated on an in vitro model using osteoblast MC3T3-E1 cells. The results showed that all fractions, including ethanol (EtOH), n-hexane, ethyl acetate (EtOAc) and butanol (BuOH) were not significantly toxic to the osteoblast cells at the test concentrations of 5, 10, 25, and 50 µg/mL. The EtOH and EtOAc fractions exhibited the highest osteogenesis activity in terms of enhancement of alkaline phosphatase (ALP) and mineralization activity of MC3T3-E1 cells, especially the EtOAc fraction which increased ALP activity up to > 30% and mineralization activity up to > 100%. Thus, the EtOAc fraction shows osteogenesis activity through stimulating activites of the two markers for bone generation including ALP and mineralization in osteoblast cells. The fraction is now under extensive investigation to isolate and fully understand the modes of action of the active compounds.

Fabrication and Characterization of Chitosan-Polypyrrole/Strontium-Magnesium Substituted Hydroxyapatite Biocomposite with Potential Application in Tissue Engineering Scaffolds

Biocomposite scaffolds of strontium-magnesium substituted hydroxyapatite (SMHA) with a mixture of chitosan/polypyrrole (CS-PPY) have been prepared by solvent casting method. The synthesized SMHA nanoparticles and biocomposite scaffold were characterized by FTIR, XRD and SEM techniques. The SEM morphology revealed the porous structure of the scaffolds designed for the muticomponents. The biomineralization and cell viability of the biocomposite were assessed via alkaline phosphatase activity and MTT assay on the osteoblast cell line. The study demonstrated improved differentiation and mineralization of osteoblast cells in the designed biocomposite scaffolds. The dye stained fluorescent microscopic photographs apparent the good scattering and permeability of cells onto the scaffolds. However, biocomposite demonstrated good antibacterial activity and the excellent biocompatibility against osteoblast cells offers a possible route for the production of a biocomposite as a viable replacement for regenerative medicine scaffolding substance.

The role of orthosilicic acid-induced autophagy on promoting differentiation and mineralization of osteoblastic cells.

Silicon, an element, has been reported to possess various osteogenic biological functions. However, the precise cellular action mechanism of silicon in osteogenesis is elusive. Here, we examine whe...

α-melanocyte stimulating hormone (α-MSH) promotes osteoblast differentiation of MC3T3-E1 cells

α-melanocyte stimulating hormone (α-MSH) is a member of the melanocortin family, that has displayed important biological functions in diverse cells and tissues. The purpose of this study is to test the effect of α-MSH on the differentiation and mineralization of osteoblast cells. The expression of the α-MSH membrane receptor MC1R but not MC2R, MC3R, or MC4R increased distinctively during the osteogenic differentiation from 3, 7 to 14 d. Treatment with α-MSH promoted the differentiation and mineralization of MC3T3-E1 cells by increasing the activity of ALP, enhancing Alizarin Red S staining, and stimulating the expression of osteogenic genes, including ALP1, osteocalcin (Bglap2), and osterix (Sp7). Importantly, we found that α-MSH increased the expression of Runx-2, a master transcriptional factor of osteogenic differentiation. Mechanically, we found that the activation of ERK1/2 was involved in this process. Using the small interfering (si) RNA knockdown experiment, we proved that the effects of α-MSH on differentiation and mineralization of osteoblast cells are mediated by MC1R. The present study proposes α-MSH as a potential therapeutic agent to stimulate bone formation for osteoporosis and bone defect. Meanwhile, MC1R could also be a target candidate for the treatment of bone metabolism diseases.

Combined Treatment with Low-Level Laser and rhBMP-2 Promotes Differentiation and Mineralization of Osteoblastic Cells under Hypoxic Stress.

The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia. MC3T3-E1 cells were cultured under 1% oxygen tension for 72h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48h. Cells were treated with rhBMP-2 at 50ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved. The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha (HIF-1α), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation. Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxic-cultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

Development of nanocomposite scaffolds based on biomineralization of N,O-carboxymethyl chitosan/fucoidan conjugates for bone tissue engineering

Bone tissue engineering holds great promise and clinical efficacy for the regeneration of bone defects. In this study, an amphoteric N,O-carboxymethyl chitosan (NOCC) and fucoidan (FD) were covalently cross-linked via an amidation reaction to synthesize NOCC/FD composite hydrogels. The hydrogels were lyophilized and then three-dimensional scaffolds with interconnected macropores were obtained. To enhance the mechanical properties and osteogenic activity, the NOCC/FD scaffolds were biomineralized for the growth of hydroxyapatite crystals. A comparative assessment of the structures, morphologies, and physical properties of the original and mineralized scaffolds were performed by SEM, EDS, X-ray diffraction and FT-IR analysis. FD regulated the growth of hydroxyapatite nanocrystallites (n-HAp) and thus the NOCC/FD scaffolds showed better mineralization efficiency than NOCC scaffolds. The compressive strength of the scaffolds was greatly enhanced after mineralization with n-HAp. The n-HAp/NOCC/FD scaffolds enhanced the proliferation, ALP activity, and mineralization of osteoblast cells more strongly than the original and mineralized NOCC scaffolds. Hence, the n-HAp-mineralized NOCC/FD scaffolds may prove to be an excellent and versatile scaffold for bone tissue engineering.

Demethylbelamcandaquinone B (Dmcq B) Is the Active Compound of Marantodes pumilum var. alata (Blume) Kuntze with Osteoanabolic Activities.

Phytoestrogens have attracted considerable attention for their potential in the prevention of postmenopausal osteoporosis. Recently, a phytoestrogen-rich herbal plant, Marantodes pumilum var. alata (Blume) Kuntze was reported to protect against bone loss in ovariectomized rat. However, the bioactive compound responsible for these effects and the underlying mechanism were not known. Through bioassay-guided isolation, demethylbelamcandaquinone B (Dmcq B) was isolated and identified from Marantodes pumilum var. alata leaf extract. In terms of its bone anabolic effects, Dmcq B was at par with 17β-estradiol (E2), in promoting the proliferation, differentiation and mineralization of osteoblast cells. Dmcq-B increased early differentiation markers, collagen content and enzymatic ALP activity. It was demonstrated to regulate BMP2 signaling pathway which further activated the transcription factor, osterix. Subsequently, Dmcq B was able to increase the osteocalcin expression which promoted matrix mineralization as evidenced by the increase in calcium deposition. Dmcq B also reduced the protein level of receptor activator of NF-κβ ligand (RANKL) and promoted osteoprotegerin (OPG) protein expression by osteoblast cells, therefore hastening bone formation rate by decreasing RANKL/OPG ratio. Moreover, Dmcq B was able to increase ER expression, postulating its phytoestrogen property. As the conclusion, Dmcq B is the active compound isolated from Marantodes pumilum var. alata leaves, regulating osteoanabolic activities potentially through the BMP2 and ER signaling pathways.

Benzofuran-pyran hybrids: A new class of potential bone anabolic agents

Benzofuran moiety is an important pharmacophore showing positive effects on bone health. In the present study, sixteen benzofuran-pyran hybrids were synthesized and were evaluated for their osteogenic effects on primary osteoblast cells isolated from calvaria. Compounds 22 and 24 were found potent in stimulating osteoblast differentiation as assessed by the alkaline phosphatase activity. These compounds were also found to be nontoxic to osteoblast cells as compared to the control cells in MTT assay. Further, Alizarin Red-S staining for visualization of calcium nodules demonstrated compounds 22 and 34 as active in enhancing mineralization in osteoblast cells. Additionally, transcriptional analysis of these compounds on osteoblast cells revealed that compound 22 up-regulated the expression of osteogenic genes RUNX2, BMP-2, COL-1, thus substantiating that compound 22 having two geminal methyl groups in its R3 position is a potent osteogenic agent. Additionally, compound 22 enhanced the ability of bone marrow stromal cells to differentiate towards osteoblast lineage and therefore can be further studied in vivo in bone loss model.

Limonene attenuates methylglyoxal-induced dysfunction in MC3T3-E1 osteoblastic cells

ABSTRACTLimonene is a common natural terpene with powerful antioxidative properties. This study investigated the effects of limonene, a terpene found in citrus fruits, on the function of the murine pre-osteoblast cell line, MC3T3-E1 cells. The results showed that limonene treatment significantly elevated collagen synthesis, alkaline phosphatase activity, osteocalcin synthesis, and mineralization in osteoblastic cells. Methylglyoxal (MG), a highly reactive dicarbonyl metabolite, is a major precursor of advanced glycation end products, which are involved in the pathogenesis of diabetic osteopathy. We therefore investigated the effects of limonene on MG-induced cytotoxicity. Pre-treatment of MC3T3-E1 cells with limonene prevented MG-induced cell death and apoptosis. Limonene also reduced MG-triggered endoplasmic reticulum (ER) stress, as indicated by decreases in the levels of the ER-localized transmembrane signal transducers ATF-6 and IRE1. Furthermore, limonene treatment significantly reduced MG-induced autophagic activity and reactive oxygen species release. These results suggest that limonene may prevent the development of diabetic osteopathy.

How much medium do you use for cell culture? Medium volume influences mineralization and osteoclastogenesis invitro.

Bone is maintained by a balance between bone formation and resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. The present invitro study examined the impact of medium volume, using 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0ml/well in a 24‑well plate, on the differentiation of osteoblasts and osteoclasts. There were no differences in the alkaline phosphatase activity of osteoblasts amongst the groups; however, the area of mineral deposition was decreased in a media volume‑dependent manner. A co‑culture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrate‑resistant acid phosphatase (TRAP)‑positive multinuclear cells (≥2 nuclei), whereas the formation of large osteoclastic TRAP‑positive multinuclear cells (≥8 nuclei) was increased, in a media volume‑dependent manner. There were also no differences in receptor activator of nuclear factor‑κB ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volume‑dependent manner. In conclusion, the present study demonstrated that the suppression of mineralization in osteoblastic cells and the stimulation of osteoclast fusion are dependent on the medium volume, indicating that media volume is an important factor in invitro cell culture systems.

Azadirachta indica triterpenoids promote osteoblast differentiation and mineralization in vitro and in vivo

Terpenoids were isolated using chromatographic purification through solvent purification technique and identified as Azadirone (1), Epoxyazadiradione (2) Azadiradione (3) Gedunin (4) Nimbin (5) Salannin (6) Azadirachtin A (7) and Azadirachtin B (8) from Azadirachta indica. Out of eight compounds, only three compounds had osteogenic activity and enhanced osteoblast proliferation, differentiation and mineralization in osteoblast cells. Active compounds stimulated osteogenic genes ALP, RunX-2 and OCN expressions in vitro, but Azadirachtin A had a maximum ability to stimulate osteoblast differentiation and mineralization compared to other two active compounds. For in vivo study, Azadirachtin A injected subcutaneously in pups, which enhanced osteogenic gene expressions and promoted bone formation rate significantly. Here, we conclude that active compounds of Azadirachta indica have osteogenic activity and Azadirachtin A has a beneficial effects on bone.

Biocompatibility and mineralization of osteoblastic cells in contact with niobium

Biocompatibility and mineralization of osteoblastic cells in contact with niobium