Murine Pre-osteoblast Cell Line Research Articles

High-Throughput Pre-Osteoblastic Spheroids Elevate FGF23 via PTH Signaling Pathway.

Fibroblast Growth Factor 23 (FGF23) plays a crucial role in managing renal phosphate and the synthesis of 1,25-(OH)2-vitamin D3, which is essential for bone homeostasis. Developing robust in vitro systems to study FGF23 regulating mechanisms is crucial for advancing our knowledge and identifying potential therapeutic targets. Traditional in vitro 2D culture system results in relatively low expression of FGF23, complicating further exploration of its regulatory mechanisms and potential therapeutic targets. Herein, we reported a high-throughput approach to generate pre-osteoblastic cell spheroids with enhanced FGF23 production. For this purpose, murine pre-osteoblast cell line (MC3T3-E1) were cultured in our previously reported non-adherent micro-wells (200�m in-diameter, 148�m in-depth, and 100�m space in between), and self-assembled into spheroids with a diameter of 92.3�15.0 �m after 24hrs. Compared to monolayer culture, the MC3T3-E1 spheroids showed a significant upregulation of FGF23 in both gene and protein levels after 24h serum-free induction. RNA sequencing and western blotting analysis further suggested that the enhanced FGF23 production in MC3T3-E1 spheroids was attributed to the activation of the parathyroid hormone (PTH)/PTH1R signaling pathway. Impressively, inhibition of PTH signaling through small molecular inhibitors or short hairpin RNA targeting PTH1R effectively reduced FGF23 production. In summary, the current study revealed the efficacy of the high-throughput formation of pre-osteoblast cell spheroid in stimulating FGF23 expression for mechanistic studies. Importantly, our findings highlight the potential of the current 3D spheroid system in target identification and drug discovery.

Enhancement of Local Osseointegration and Implant Stability of Titanium Implant in Osteoporotic Rats by Biomimetic Multilayered Structures Containing Catalpol.

This study examined the surface modification of titanium (Ti) implants to enhance early-stage osseointegration, which reduced the failure rate of internal fixation in osteoporotic fractures that inherently decrease in bone mass and strength. We employed a layer-by-layer electroassembly technique to deposit catalpol-containing hyaluronic acid/chitosan multilayers onto the surface of Ti implants. To evaluate the in vitro osteoinductive effects of catalpol-coated Ti implants, the robust osteoblast differentiation capacity of the murine preosteoblast cell line, MC3T3-E1, was employed. Furthermore, the performance of these implants was evaluated in vivo through femoral intramedullary implantation in Sprague-Dawley rats. The engineered implant effectively regulated catalpol release, promoting increased bone formation during the initial stages of implantation. The in vitro findings demonstrated that catalpol-coated Ti surfaces boosted ALP activity, cell proliferation as measured by CCK-8, and osteogenic protein expression via WB analysis, surpassing the uncoated Ti group (P < 0.05). In vivo micro-computed tomography (CT) and histological analyses revealed that catalpol-coated Ti significantly facilitated the formation and remodeling of new bone in osteoporotic rats at 14 days post-implantation. This study outlines a comprehensive and straightforward methodology for the fabrication of biofunctional Ti implants to address osteoporosis.

3D-printed Sr2ZnSi2O7 scaffold facilitates vascularized bone regeneration through macrophage immunomodulation.

Biomaterial-based bone grafts are emerged as an effective strategy for the treatment of large bone defects, especially for the scaffolds with enhanced osteogenic and angiogenic bioactivities. However, most studies focused on the direct interactions between scaffolds and bone-related cells such as osteoblasts and endothelial cells, and ignored the effects of material-triggered immunomodulation and the subsequent immune-regulated bone regeneration process. In this study, we developed a silicate bioceramic (Sr2ZnSi2O7, SZS) scaffold with well-defined pore structures using a three-dimensional (3D) printing technique. The prepared scaffolds were biodegradable, and the released bioactive ions were beneficial for immunomodulation, which stimulated macrophages to release more pro-healing cytokines and less pro-inflammatory cytokines. The obtained scaffold/macrophage conditioned medium further promoted the proliferation and osteogenic differentiation of a murine preosteoblast cell line (MC3T3-E1), as well as the angiogenic activity of human umbilical vein endothelial cells (HUVECs). Moreover, the in vivo experiments of critical-sized calvarial defects in rats revealed that the 3D printed SZS scaffolds could facilitate more vascularized bone regeneration than the 3D printed β-tricalcium phosphate (β-TCP, a typical clinically used bioceramic) scaffolds, suggesting that the 3D-printed SZS scaffolds hold the potential as implantable biomaterials with favorable osteoimmunomodulation for bone repair.

A silane-based coupling strategy for enhancing the mechanical properties of proanthocyanidin nanocoatings on Ti dental implants

Typical strategies used to improve the osteo-inductive and antioxidative capacities of titanium and its alloys involve loading with functional molecules, such as proanthocyanidin (PAC), by physical adsorption, which limits the bonding strength and durability of the coating. Accordingly, in the present study, (3-aminopropyl) triethoxysilane (APTES) and glutaraldehyde (GA) were used to prepare covalently immobilized PAC and chitosan-encapsulated PAC (CS-PAC) coatings on Ti surface. Quantum chemistry and X-ray photoelectron spectroscopy analyses confirmed the chemical grafting of PAC and CS-PAC onto the Ti surfaces. Their superior wear and compression resistances were demonstrated by friction and nanoindentation testing. SEM observation of the surface revealed that the improvement of mechanical properties is not only related to the formation of chemical bonds, but also to the presence of nanoparticles that act as roller bearings. Contact angle measurement and TEM observation were also used for surface analysis. The murine pre-osteoblast cell line MC3T3-E1 was used to assess the osteo-inductive and antioxidative activities of the coatings under H2O2-induced oxidative stress in vitro. The cell-adhesion-, proliferation-, and differentiation-promoting properties as well as antioxidant properties of PAC are retained after the formation of chemical bonds to APTES. Thus, this silane coupling strategy can enhance the mechanical properties of PAC nanocoatings while preserving their biological activity.

Intercellular signaling between ameloblastoma and osteoblasts

Ameloblastoma is an odontogenic tumor located in the bone jaw with clinical characteristics of extensive bone resorption. It is a locally invasive tumor with a high recurrence rate despite adequate surgical removal. In bone disease, tumors and other cells including osteoblasts, osteoclasts, and osteocytes in the bone microenvironment contribute to the pathogenesis of tumor growth. However, the effect of osteoblasts on ameloblastoma cells is not well-understood, and there has been limited research on interactions between them.This study investigated interactions between ameloblastoma cells and osteoblasts using a human ameloblastoma cell line (AM-3 ameloblastoma cells) and a murine pre-osteoblast cell line (MC3T3-E1 cells). We treated each cell type with the conditioned medium by the other cell type. We analyzed the effect on cytokine production by MC3T3-E1 cells and the production of MMPs by AM-3 cells. Treatment with AM-3-conditioned medium induced inflammatory cytokine production of IL-6, MCP-1, and RANTES from MC3T3-E1 cells. The use of an IL-1 receptor antagonist suppressed the production of these inflammatory cytokines by MC3T3-E1 cells stimulated with AM-3-conditioned medium. The MC3T3-E1-conditioned medium triggered the expression of MMP-2 from AM-3 cells. Furthermore, we have shown that the proliferation and migration activity of AM-3 cells were accelerated by MC3T3-E1 conditioned media.In conclusion, these intercellular signalings between ameloblastoma cells and osteoblasts may play multiple roles in the pathogenesis of ameloblastoma.

Application of Temporary, Cell-Containing Alginate Microcarriers to Facilitate the Fabrication of Spatially Defined Cell Pockets in 3D Collagen Hydrogels.

Mimicking the complexity of natural tissue is a major challenge in the field of tissue engineering. Here, a facile 2-step fabrication method to prepare 3D constructs with distinct regions of high cell concentrations and without the need for elaborate equipment is proposed. The initial incorporation of cells in a sacrificial alginate matrix allows the addition of other, cell relevant biopolymers, such as, collagen to form a spatially confined, interpenetrating network at the microscale. A layered structure at the macroscale can be achieved by incorporating these cell-containing microspheres in thin collagen layers. Cells are locally released by de-gelling the alginate matrix and their attachment to the collagen hydrogel layers has been studied. The use of the murine pre-osteoblast cell line MC3T3-E1 as an example cell line shows that the cells behave differently in their cell migration pattern based on the initial composition of the alginate microspheres.

Spinning of Endless Bioactive Silicate Glass Fibres for Fibre Reinforcement Applications

Bioactive glasses have been used for many years in the human body as bone substitute. Since bioactive glasses are not readily available in the form of endless thin fibres with diameters below 20 µm, their use is limited to mainly non-load-bearing applications in the form of particles or granules. In this study, the spinnability of four bioactive silicate glasses was evaluated in terms of crystallisation behaviour, characteristic processing temperatures and viscosity determined by thermal analysis. The glass melts were drawn into fibres and their mechanical strength was measured by single fibre tensile tests before and after the surface treatment with different silanes. The degradation of the bioactive glasses was observed in simulated body fluid and pure water by recording the changes of the pH value and the ion concentration by inductively coupled plasma optical emission spectrometry; further, the glass degradation process was monitored by scanning electron microscopy. Additionally, first in vitro experiments using murine pre-osteoblast cell line MC3T3E1 were carried out in order to evaluate the interaction with the glass fibre surface. The results achieved in this work show up the potential of the manufacturing of endless bioactive glass fibres with appropriate mechanical strength to be applied as reinforcing fibres in new innovative medical implants.

Post-treatments of polydopamine coatings influence cellular response

Polydopamine (PDA) is the final oxidation product of dopamine or other catecholamines. Since the first reports of PDA coatings starting around 2007, these coatings have been widely studied as a versatile and inexpensive one-step coating option for biomaterial functionalization. The coating attach to a wide range of materials and can subsequently be modified with biomolecules or nanoparticles. However, as a strong candidate for biomaterial research and even clinical use, it is important to unravel the changes in physico-chemical properties and the cell-PDA interaction as a function of heat sterilization procedures and shelf storage periods. Four groups were examined in this study: titanium (Ti), PDA-coated Ti samples and PDA-coated Ti samples either stored for up to two weeks at room temperature or heated at 121 °C for 24 h, respectively. We used X-ray Photoelectron Spectroscopy (XPS), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Water contact angle (WCA) to characterize chemical composition and surface properties of the groups. Cell adhesion and proliferation was examined by three different cell types: human primary dermal fibroblasts (hDF), human epidermal keratinocytes (HaCaTs) and a murine preosteoblastic cell line (MC3T3-E1), respectively. Cells were cultured on PDA coated samples for 4 h, 3 days and 5 days. Both thermal treatment of PDA at 121℃ for 24 h and storage of the samples for 2 weeks increased the amount of quinone groups at the surface and decreased the amount of primary amine groups as detected by XPS and ToF-SIMS. Even though these surface reactions increased the WCA of the PDA coating, we found that the post-treatments increased cell proliferation for both hDFs, HaCaTs and MC3T3-E1 s as compared to pristine PDA. This emphasizes the importance of post-treatment and shelf-time for PDA coatings.

Biofabrication of gold nanoparticles with bone remodeling potential: an in vitro and in vivo assessment

Recent thrust in the biomaterials research has shifted towards developing bioactive and resorbable scaffolds with nanotechnological interventions. Next-generation bone scaffolds need to be multifunctional with desired osteo-integration ability, conformed resorption rate and in vivo imaging potential. In this study, we synthesized biogenic multifunctional gold nanoparticles (AuNPs) using immobilized crude enzyme (ImCE) of Bacillus licheniformis. The multifunctional characteristics of AuNPs and reusability of ImCE for AuNP synthesis can make the process and application cost-effective. ImCE catalyzed the bioreduction of gold ions to AuNPs of 18.4 ± 3.3 nm. AuNPs showed good hemocompatibility and antioxidant property without any toxic effects. Further, AuNPs demonstrated the increase in alkaline phosphatase activity and calcium deposition through MC3T3-E1 murine preosteoblast cell line and rat bone marrow stromal cells (BMSCs). The osteoinductive effect of AuNPs was also investigated in vivo wherein the composite cryogel scaffold functionalized with bone morphogenic protein-2 (rhBMP-2), zoledronic acid (ZA), and AuNPs was implanted in a muscle pouch of Wistar rats. AuNPs had synergistic effect on bone formation along with rhBMP-2 and ZA as seen from micro-computed tomography and histological analysis. The AuNPs were found to exhibit autofluorescence property and enhanced the degradation of scaffold at the implanted site in vivo. The developed process of using ImCE presents a novel approach for biogenic fabrication of AuNPs with the potential orthopedic application.

Enhancement of local bone formation on titanium implants in osteoporotic rats by biomimetic multilayered structures containing parathyroid hormone (PTH)-related protein

Osteoporosis is a severe health problem causing bone fragility and consequent fracture. Titanium (Ti) implants, used in patients with osteoporotic fractures, are prone to failure because of the decreased bone mass and strength. Therefore, it is of utmost importance to fabricate implants possessing osteogenic properties to improve implant osseointegration. To improve the long-term survival rate of Ti implants in osteoporotic patients, hyaluronic acid/ϵ-polylysine multilayers containing the parathyroid hormone (PTH)-related protein (PTHrP) were deposited on Ti implants by a layer-by-layer (LBL) electro assembly technique. The murine pre-osteoblast cell line MC3T3-E1, possessing a high potential of osteoblast differentiation, was used to evaluate the osteo-inductive effects of Ti-LBL-PTHrP in vitro. In addition, the performance of the Ti (Ti-LBL-PTHrP) implant was evaluated in vivo in a femoral intramedullary implantation in Sprague Dawley rats. The Ti-LBL-PTHrP implant regulated the release of the loaded PTHrP to increase bone formation in the early stage of implantation. The in vitro results revealed that cells on Ti-LBL-PTHrP did not show any evident proliferation, but a high level of alkaline phosphatase activity and osteoblast-related protein expression was found, compared to the uncoated Ti group (p < 0.05). In addition, in vivo micro-CT and histological analysis demonstrated that the Ti-LBL-PTHrP implants could significantly promote the formation and remodeling of new bone in osteoporotic rats at 14 d after implantation. Overall, this study established a profound and straightforward methodology for the manufacture of biofunctional Ti implants for the treatment of osteoporosis.

Integration of Human Umbilical Cord Mesenchymal Stem Cells-Derived Exosomes with Hydroxyapatite-Embedded Hyaluronic Acid-Alginate Hydrogel for Bone Regeneration.

The treatment of bone defects has plagued clinicians. Exosomes, the naturally secreted nanovesicles by cells, exhibit great potential in bone defect regeneration to realize cell-free therapy. In this work, we successfully revealed that human umbilical cord mesenchymal stem cells-derived exosomes could effectively promote the proliferation, migration, and osteogenic differentiation of a murine calvariae preosteoblast cell line in vitro. Considering the long period of bone regeneration, to effectively exert the reparative effect of exosomes, we synthesized an injectable hydroxyapatite (HAP)-embedded in situ cross-linked hyaluronic acid-alginate (HA-ALG) hydrogel system to durably retain exosomes at the defect sites. Then, we combined the exosomes with the HAP-embedded in situ cross-linked HA-ALG hydrogel system to repair bone defects in rats in vivo. The results showed that the combination of exosomes and composite hydrogel could significantly enhance bone regeneration. Our experiment provides a new strategy for exosome-based therapy, which shows great potential in future tissue and organ repair.

Preventive Effects of Chrysanthemum coronarium L. Extract on Bone Metabolism In Vitro and In Vivo.

Osteoporosis is characterized by decreased bone mass and bone microarchitectural failure, leading to an enhanced risk of bone fractures. Chrysanthemum coronarium L. (CC) is a natural plant with powerful antioxidant activity. This study investigated the antiosteoporotic effects of CC extracts in in vitro cell cultures and in vivo bone loss animal models. CC stimulated osteoblast differentiation and mineralized bone formation by osteoblasts by increasing the expression of bone formation markers (alkaline phosphatase, osteoprotegerin, and osteoprotegerin/receptor activator nuclear factor-κB ligand ratio) in the murine preosteoblastic cell line MC3T3-E1. Additionally, CC was found to inhibit osteoclast differentiation by downregulating bone resorption markers (tartrate-resistant acid phosphatase, cathepsin K, dendritic cell-specific transmembrane protein, and calcitonin receptor) in the murine macrophage-like cell line RAW264.7. CC prevented ovariectomy-induced bone loss, preserved trabecular microarchitecture, and improved serum bone turnover markers in an osteoporotic mouse model. These findings suggest that CC extract may be considered as a natural therapeutic or preventive agent for osteoporotic bone loss.

Phlorofucofuroeckol A from Edible Brown Alga Ecklonia Cava Enhances Osteoblastogenesis in Bone Marrow-Derived Human Mesenchymal Stem Cells.

The deterioration of bone formation is a leading cause of age-related bone disorders. Lack of bone formation is induced by decreased osteoblastogenesis. In this study, osteoblastogenesis promoting effects of algal phlorotannin, phlorofucofuroeckol A (PFF-A), were evaluated. PFF-A was isolated from brown alga Ecklonia cava. The ability of PFF-A to enhance osteoblast differentiation was observed in murine pre-osteoblast cell line MC3T3-E1 and human bone marrow-derived mesenchymal stem cells (huBM-MSCs). Proliferation and alkaline phosphatase (ALP) activity of osteoblasts during differentiation was assayed following PFF-A treatment along extracellular mineralization. In addition, effect of PFF-A on osteoblast maturation pathways such as Runx2 and Smads was analyzed. Treatment of PFF-A was able to enhance the proliferation of differentiating osteoblasts. Also, ALP activity was observed to be increased. Osteoblasts showed increased extracellular mineralization, observed by Alizarin Red staining, following PFF-A treatment. In addition, expression levels of critical proteins in osteoblastogenesis such as ALP, bone morphogenetic protein-2 (BMP-2), osteocalcin and β-catenin were stimulated after the introduction of PFF-A. In conclusion, PFF-A was suggested to be a potential natural product with osteoblastogenesis enhancing effects which can be utilized against bone-remodeling imbalances and osteoporosis-related complications.

3152 The role of myostatin in diabetic bone disease

OBJECTIVES/SPECIFIC AIMS: Our primary objective is to determine the mechanism of action of myostatin on osteoblasts by measuring markers of osteoblast differentiation. With these experiments we will evaluate the effects of myostatin on an osteoblastic cell line (MC3T3 cells) and primary murine osteoblasts during baseline and hyperglycemic conditions and assess whether these effects are altered in the presence of a hyperglycemic environment. METHODS/STUDY POPULATION: Primary osteoblasts from calvaria of WT mice will be isolated and cultured per previously published protocol. MC3T3 cells (murine pre-osteoblast cell line) and primary osteoblasts will be plated in 6-well plates until they reach confluency. They will subsequently be stimulated with or without myostatin at various concentrations under control and hyperglycemic conditions. Additional experiments will assess myostatin stimulation during cell differentiation/maturation in the presence of osteogenic induction medium. Subsequently, cells will be lysed and processed for gene analysis with qPCR. Genes of interest (e.g., myostatin, RUNX2, osteocalcin etc.) will be assessed. Additionally, cells will be collected and processed for protein quantification with western blot to assess myostatin-related pathways, such as Smad2/3 and MAPK signaling. RESULTS/ANTICIPATED RESULTS: We have demonstrated that the receptor for myostatin (Activin receptor 2b, AcvR2b) is present in MC3T3 cells and we have evidence of Smad2 phosphorylation in MC3T3 cells as a result of myostatin stimulation, confirming that myostatin can exert intracellular signaling events in bone cells (Fig 1). We anticipate to observe negative effects of myostatin on differentiation of primary osteoblasts and MC3T3 cells. Specifically, we anticipate suppression of Runt-related transcription factor 2 (RUNX-2), a transcription factor known as the “ master regulator” of osteogenic gene expression and programming, as a result of signaling downstream of Smad 2/3. Additionally we anticipate downregulation of osterix and osteocalcin, two essential genes for osteoblast differentiation and activity. We anticipate that hyperglycemia will potentiate the negative effects of myostatin on osteoblastogenesis. DISCUSSION/SIGNIFICANCE OF IMPACT: We have demonstrated that myostatin can directly act on osteoblastic cells. As myostatin is a negative regulator or bone mass, its direct effects on bone cells can be detrimental to the bone health of patients with elevated myostatin levels and/or activity. There is evidence suggesting that myostatin is elevated in Type 1 diabetes, and its effects might be potentiated in a hyperglycemic environments. Future experiments will be evaluating the role of myostatin on a diabetic animal model and in humans. Our experiments provide an additional mechanism by which muscle-bone interactions could be contributing to the development of diabetic bone disease.

Abstract A81: Heme oxygenase 1 alters the adhesive properties of prostate tumor cells growing in coculture with bone cells

Abstract Prostate cancer (PCa) is the second leading type of cancer in men. PCa cells display abnormalities in their adhesive properties which result in an augmented capacity to resist chemotherapy and colonize other organs such as bone. We have recently shown that heme-oxygenase 1 (HO¬-1) has a strong anti¬tumoral effect in PCa through its interaction with cytoskeletal proteins, which regulate filopodia formation towards a more adhesive phenotype. Given the osteomimetic properties of PCa cells, we used a coculture system where tumoral cells and bone cells share the medium but are not in direct physical contact. PC3 cells pretreated or not with hemin (HO-1 pharmacologic inducer, 50μΜ, 24h) were cocultured with the murine preosteoblastic (MC3T3) or preosteoclastic (RAW) cell line. The conditioned media (CM) from the cocultures were collected and added to new PC3 cultures in order to study changes in tumor cells' adhesiveness. After 24 h with the different CM, cells were fixed and stained with rhodamine-phalloidin to analyze contact among cells and filopodia in each cell individually. Results show that the CM from the coculture between PC3 cells and MC3T3 or RAW cells have a negative impact both in the number of filopodia per cell and contacts among them. However, when PC3 cells were pretreated with hemin prior to coculture, the number of filopodia and cell contacts are reestablished. Interestingly, PC3 cells cultured with CM from the coculture system displayed more covered area after 6 h of exposure in a wound healing assay, compared to CM from cocultures with PC3 cells pretreated with hemin. Finally, to identify the released factors in the coculture responsible for changing the adhesive properties of PC3 cells, we performed a mass spectrometry analysis followed by gene ontology analysis using DAVID software. Our analysis revealed more than 30 proteins released differentially in the coculture CM involved in the adhesiveness of the cell, such as cadherins (PCDHGA10, FAT4, CDH1, CDH10), collagen (COL2A1), and several proteins associated to cell detachment (GATAD2A, KANK2, BCAS3, CAMSAP1, EPPK1, FLNC, TMPPE). Altogether, our findings demonstrate that HO-1 modulation in PCa cells induces morphologic changes towards a less invasive phenotype through different proteins released as a consequence of the communication between PCa and bone cells. These data also show the relevance of the cytoskeleton regulating proteins as potential therapeutic targets against the aggressive and metastatic disease. Citation Format: Alejandra Veronica Paez, Nicolas Anselmino, Pia Valacco, Elba Susana Vazquez, Geraldine Gueron. Heme oxygenase 1 alters the adhesive properties of prostate tumor cells growing in coculture with bone cells [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A81.

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.

Intermittent compressive stress regulates Notch target gene expression via transforming growth factor-β signaling in murine pre-osteoblast cell line

ObjectiveDifferent mechanical stimuli regulate behaviors of various cell types, including osteoblasts, osteocytes, and periodontal ligament fibroblasts. Notch signaling participates in the mechanical stress-regulated cell responses. The present study investigated the regulation of Notch target gene and sclerostin (Sost) expression in murine pre-osteoblast cell line (MC3T3-E1) under intermittent compressive stress. MethodsMC3T3-E1 were subjected to the intermittent compressive force under the computerized controlled machine. In some experiments, cells were pretreated with chemical inhibitors for Notch and transforming growth factor (TGF)-β signaling prior to mechanical stimuli. To evaluate role of Notch signaling in MC3T3-E1 cells under unloaded condition, cells were seeded on indirect immobilized Notch ligand (Jagged1). Gene expression was determined using real-time quantitative polymerase chain reaction. ResultsThe intermittent compressive stress significantly upregulated Notch target gene expression (Hes Family BHLH transcription factor 1; Hes1 and Hairy/enhancer-of-split related with YRPW motif protein1; Hey1). The intermittent stress-induced Hes1 and Hey1 mRNA expression could be inhibited by a γ-secretase inhibitor (DAPT) or a TGF-β superfamily type I activing receptor-like kinase receptors inhibitor (SB431542). The results imply that intermittent compressive stress regulates Notch signaling via TGF-β pathway. Further, the intermittent compressive stress reduced Sost mRNA expression and this phenomenon could be rescued by a DAPT pretreatment, implying the involvement of Notch signaling. However, activation of Notch signaling under the unloaded condition resulted in the increase of Sost expression and the reduction of osteogenic marker genes. ConclusionsThese results imply the involvement of Notch signaling in the homeostasis maintaining of osteogenic cells under mechanical stress stimuli.

Tonsil-Derived Mesenchymal Stem Cells Promote Bone Mineralization and Reduce Marrow and Visceral Adiposity in a Mouse Model of Senile Osteoporosis.

Osteoporosis is a disease that affects 35% women and 20% men aged more than 65 years. Reduction in bone formation and increased bone resorption are known factors that drive osteoporosis, but recent studies suggest a positive correlation between bone marrow adipose tissue (MAT) and osteoporosis. Previously, we have observed that tonsil-derived mesenchymal stem cells (T-MSCs) reduce MAT in a mouse model of bone marrow depletion. That prompted us to investigate on the senile osteoporosis to characterize the bone-forming effect, as well as MAT-reducing effect of T-MSCs. In a mouse model of senescence-accelerated mouse prone 6 (SAMP6), we injected T-MSCs or T-MSC conditioned medium (CM) through tail vein and examined changes in bone microstructure using micro-CT scan and hematoxylin & eosin (H&E) staining. Biochemical markers of osteoporosis, deoxypyridinoline (DPD) and osteocalcin, were examined by ELISA. Results demonstrated attenuation in the progression of osteoporosis, in part, by sustaining osteocalcin production and by blocking MAT accumulation. Increase in matrix mineralization was determined using in vitro culture of murine preosteoblast cell line by treatment of T-MSC CM. Interestingly, T-MSC CM induced continuous weight loss and selectively reduced visceral adipose tissue mass. Finally, antiadipogenic effects of T-MSC CM were determined in vitro. In conclusion, regulation of bone together with MAT could be considered as a new therapeutic option for the treatment of senile osteoporosis and this report may provide a framework for future cell therapy using T-MSCs.

Ethanol Extract of Cissus quadrangularis Enhances Osteoblast Differentiation and Mineralization of Murine Pre-Osteoblastic MC3T3-E1 Cells.

Traditional medicinal literature and previous studies have reported the possible role of Cissus quadrangularis (CQ) as an anti-osteoporotic agent. This study examines the effectiveness of CQ in promoting osteoblast differentiation of the murine pre-osteoblast cell line, MC3T3-E1. Ethanolic extract of CQ (CQ-E) was found to affect growth kinetics of MC3T3-E1 cells in a dosage-dependent manner. High concentrations of CQ-E (more than 10 μg/ml) have particularly adverse effects, while lower concentrations of 0.1 and 1 µg/ml were non-toxic and did not affect cell viability. Notably, cell proliferation was significantly increased at the lower concentrations of CQ-E. CQ-E treatment also augmented osteoblast differentiation, as reflected by a substantial increase in expression of the early osteoblast marker ALP activity, and at later stage, by mineralization of extracellular matrix compared to the control group. These findings suggest dose-dependent effect of CQ-E with lower concentrations exhibiting anabolic and osteogenic properties. J. Cell. Physiol. 232: 540-547, 2017. © 2016 Wiley Periodicals, Inc.

Evaluation of osteogenic markers in the differentiation of murine preosteoblasts during the interaction with polyurethane/nanohydroxyapatite biomaterials

Event Abstract Back to Event Evaluation of osteogenic markers in the differentiation of murine preosteoblasts during the interaction with polyurethane/nanohydroxyapatite biomaterials Georgina Carbajal-De La Torre1, Ana Edith A. Higareda-Mendoza2, Ana Beatriz A. Martinez-Valencia1, Marco Aurelio Pardo Galvan3, Tania Saluen Gaytan Compean2, Lucia Marquez-Perez1 and Marco Antonio Espinosa-Medina1 1 Universidad Michoacana de San Nicolas de Hidalgo, Bioengineering, Mexico 2 Universidad Michoacana de San Nicolás de Hidalgo, Division of Graduate Studies, Faculty of Medical and Biological Sciences Dr. Ignacio Chavez, Mexico 3 Universidad Michoacana de San Nicolás de Hidalgo, Institute of Chemical Biology Research, Mexico In humans, the regenerative capacity of tissues is limited; consequently, there are bone structural damages for which it is appropriate to devise therapeutics that promote regeneration. The materials used currently as bone grafts have the main purpose of replacing the bone to fulfill a support function and not for tissue regeneration. The most common are organic, polymeric and metallic implants, each with advantages and disadvantages; among the latter, the main thing is that most are not biocompatible and resorbable, generating an immune rejection response in the body[1]. Tissue engineering has the challenge of developing bone substitutes that, besides serving as a support, promote bone regeneration; for this, the material must promote cell adhesion, proliferation and differentiation, have a temporary function, and gradually be resorbed and replaced by newly formed bone tissue[2]. For this purpose, the use of polyurethane/nano-hydroxyapatite hybrids (PU/nHAp) was proposed in this project[3]-[5]. Objective: To determine in vitro the optimal composition of the PU/nHAp hybrid that presents the best cell differentiation and bone mineralization stimulus. Methodology: PU/nHAp hybrid with compositions of 5, 10, and 20 weight% nHAp were evaluated. A murine preosteoblast cell line, MC3T3-E1 subclone 4, was used as the cellular model. To evaluate cell viability, trypan blue and alamar blue assays were used. Scanning Electron Microscopy (SEM) was used to analyze the surface of biomaterial samples. Expression of osteogenic markers was evaluated by RT-PCR and gel electrophoresis in 1% agarose. Results: None of the PU/nHAp biomaterials are cytotoxic, and neither have inhibitory effects on cell growth. Cell viability and the degree of mineralization are directly linked to the percentage of nHAp in the biomaterial composition. SEM micrographs at 0, 7, 10 and 14 days show cell adhesion and proliferation on the surface of biomaterial with PU-20%nHAp. The data suggests that the PU/20%nHAp composite might stimulate the osteogenic process in a greater degree, compared to the other composites tested. In control cultures, MC3T3-E1 in osteogenic medium and without the presence of biomaterials, the obtained osteocalcin, osterix and type I collagen gene expression profiles agree with previous reports using these genes as osteogenic markers; in cell cultures contacting biomaterials, the osteoinductive capability and bioactivity of PU/nHAp biomaterials was evident, without the need for an external osteogenic stimulus. Conclusions: The working hypothesis is confirmed: Biomaterials compounds of polyurethane and nano-hydroxyapatite, with compositions 20 weight % or lower of hydroxyapatite, stimulate cell differentiation and mineralization of newly formed bone tissue. This work was supported by CONACYT for the project financing Number: 243236 “, CB-2014-02, I0017 Fund.