p62/SQSTM1 indirectly mediates remote multipotent mesenchymal cells and rescues bone loss and bone marrow integrity in ovariectomized rats.

Intramuscular administration of p62/SQSTM1 (sequestosome1)-encoding plasmid demonstrated an anticancer effect in rodent models and dogs as well as a high safety profile and the first evidence of clinical benefits in humans. Also, an anti-inflammatory effect of the plasmid was reported in several rodent disease models. Yet, the mechanisms of action for the p62 plasmid remain unknown. Here, we tested a hypothesis that the p62-plasmid can act through the modulation of bone marrow multipotent mesenchymal cells (MSCs). We demonstrated that a p62 plasmid can affect MSCs indirectly by stimulating p62-transfected cells to secrete an active ingredient(s) sensed by untransfected MSCs. When we transfected MSCs with the p62-plasmid, collected their supernatant, and added it to an untransfected MSCs culture, it switched the differentiation state and prompt osteogenic responses of the untransfected MSCs. According to an accepted viewpoint, ovariectomy leads to bone pathology via dysregulation of MSCs, and restoring the MSC homeostasis would restore ovariectomy-induced bone damage. To validate our in vitro observations in a clinically relevant in vivo model, we administered the p62 plasmid to ovariectomized rats. It partially reversed bone loss and notably reduced adipogenesis with concurrent reestablishing of the MSC subpopulation pool within the bone marrow. Overall, our study suggests that remote modulation of progenitor MSCs via administering a p62-encoding plasmid may constitute a mechanism for its previously reported effects and presents a feasible disease-preventing and/or therapeutic strategy.

Autophagic Mediators in Bone Marrow Niche Homeostasis.

The bone marrow serves as a reservoir for a multifunctional assortment of stem, progenitor, and mature cells, located in functional anatomical micro-areas termed niches. Within the niche, hematopoietic and mesenchymal progenies establish a symbiotic relationship characterized by interdependency and interconnectedness. The fine-tuned physical and molecular interactions that occur in the niches guarantee physiological bone turnover, blood cell maturation and egression, and moderation of inflammatory and oxidative intramural stressful conditions. The disruption of bone marrow niche integrity causes severe local and systemic pathological settings, and thus bone marrow inhabitants have been the object of extensive study. In this context, research has revealed the importance of the autophagic apparatus for niche homeostatic maintenance. Archetypal autophagic players such as the p62 and the Atg family proteins have been found to exert a variety of actions, some autophagy-related and others not; they moderate the essential features of mesenchymal and hematopoietic stem cells and switch their operational schedules. This chapter focuses on our current understanding of bone marrow functionality and the role of the executive autophagic apparatus in the niche framework. Autophagic mediators such as p62 and Atg7 are currently considered the most important orchestrators of stem and mature cell dynamics in the bone marrow.

Archetypal autophagic players through new lenses for bone marrow stem/mature cells regulation.

The bone marrow landscape consists of specialized and stem/progenitor cells, which coordinate important tissue-related and systemic physiological features. Within the marrow cavity, stem/progenitor and differentiated hematopoietic and skeletal cells congregate into dynamic functional assemblies throughout specific anatomical regions, termed niches. There is a need for better understanding of the bone marrow microareas, through exploration of the intramural physical and molecular interactions of the distinctive cell populations. The elective liaisons established among the mesenchymal/stromal stem cell and hematopoietic stem cell lineage trees play a key role in orchestrating the stem/mature cell behavior and customized hierarchies within bone marrow cell populations. Recently, the autophagic apparatus has been discovered to be an important feature of bone marrow homeostasis. Autophagy-related factors involved in the labyrinthic and highly dynamic bone marrow workshop redesign the niche framework by coordinating the operational schedule of pluripotent stem and mature cells. The following report summarizes the most recent breakthroughs in our understanding of the intramural relationships between bone marrow cells and key autophagic mediators. Doubtless, the consideration of the autophagy-related and unrelated functions of main players, such as p62, Atg7, Atg5, and Beclin-1 remains a compelling task to thoroughly understand the complex relations between the heterogenic cell types that populate bone marrow.

The effects of 808-nm near-infrared laser light irradiation on actin cytoskeleton reorganization in bone marrow mesenchymal stem cells.

Tailoring the cell organelles and thus changing cell homeostatic behavior has permitted the discovery of fascinating metabolic features enabling enhanced viability, differentiation, or quenching inflammation. Recently, photobiomodulation (PBM) has been accredited as an effective cell manipulation technique with promising therapeutic potential. In this prospective, in vitro results revealed that 808-nm laser light emitted by a hand-piece with a flat-top profile at an irradiation set up of 60 J/cm2 (1 W, 1 W/cm2; 60 s, continuous wave) regulates bone marrow stromal cell (BMSC) differentiation toward osteogenesis. Considering the importance of actin cytoskeleton reorganization, which controls a range of cell metabolic activities, comprising shape change, proliferation and differentiation, the aim of the current work is to assess whether PBM therapy, using a flat-top hand-piece at higher-fluence irradiation on BMSCs, is able to switch photon signals into the stimulation of biochemical/differentiating pathways involving key activators that regulate de novo actin polymerization. Namely, for the first time, we unearthed the role of the flat-top hand-piece at higher-fluence irradiation on cytoskeletal characteristics of BMSCs. These novel findings meet the needs of novel therapeutically protocols provided by laser treatment and the manipulation of BMSCs as anti-inflammatory, osteo-inductive platforms.

Photobiomodulation by Near-Infrared 980-nm Wavelengths Regulates Pre-Osteoblast Proliferation and Viability through the PI3K/Akt/Bcl-2 Pathway.

BACKGROUND: bone tissue regeneration remains a current challenge. A growing body of evidence shows that mitochondrial dysfunction impairs osteogenesis and that this organelle may be the target for new therapeutic options. Current literature illustrates that red and near-infrared light can affect the key cellular pathways of all life forms through interactions with photoacceptors within the cells' mitochondria. The current study aims to provide an understanding of the mechanisms by which photobiomodulation (PBM) by 900-nm wavelengths can induce in vitro molecular changes in pre-osteoblasts. METHODS: The PubMed, Scopus, Cochrane, and Scholar databases were used. The manuscripts included in the narrative review were selected according to inclusion and exclusion criteria. The new experimental set-up was based on irradiation with a 980-nm laser and a hand-piece with a standard Gaussian and flat-top beam profile. MC3T3-E1 pre-osteoblasts were irradiated at 0.75, 0.45, and 0.20 W in continuous-wave emission mode for 60 s (spot-size 1 cm2) and allowed to generate a power density of 0.75, 0.45, and 0.20 W/cm2 and a fluence of 45, 27, and 12 J/cm2, respectively. The frequency of irradiation was once, three times (alternate days), or five times (every day) per week for two consecutive weeks. Differentiation, proliferation, and cell viability and their markers were investigated by immunoblotting, immunolabelling, fluorescein-FragELTM-DNA, Hoechst staining, and metabolic activity assays. RESULTS AND CONCLUSIONS: The 980-nm wavelength can photobiomodulate the pre-osteoblasts, regulating their metabolic schedule. The cellular signal activated by 45 J/cm2, 0.75 W and 0.75 W/cm2 consist of the PI3K/Akt/Bcl-2 pathway; differentiation markers were not affected, nor do other parameters seem to stimulate the cells. Our previous and present data consistently support the window effect of 980 nm, which has also been described in extracted mitochondria, through activation of signalling PI3K/Akt/Bcl-2 and cyclin family, while the Wnt and Smads 2/3-ß-catenin pathway was induced by 55 J/cm2, 0.9 W and 0.9 W/cm2.

Loss of p62 impairs bone turnover and inhibits PTH-induced osteogenesis.

The p62 (also named sequestosome1/SQSTM1) is multidomain and multifunctional protein associated with several physiological and pathological conditions. A number of studies evidenced an involvement of p62 on the disruptive bone scenarios due to its participation in the inflammatory/osteoclastogenic pathways. However, so far, information regarding the function of p62 in the fine-tuned processes underpinning the bone physiology are not well-defined and are sometime discordant. We, previously, demonstrated that the intramuscular administration of a plasmid coding for p62 was able to contrast bone loss in a mouse model of osteopenia. Here, in vitro findings showed that the p62 overexpression in murine osteoblasts precursors enhanced their maturation while the p62 depletion by a specific siRNA, decreased osteoblasts differentiation. Consistently, the activity of osteoblasts from p62-/- mice was reduced compared with wild-type. Also, morphometric analyses of bone from p62 knockout mice revealed a pathological phenotype characterized by a lower turnover that could be explained by the poor Runx2 protein synthesis in absence of p62. Furthermore, we demonstrated that the parathyroid hormone (PTH) regulates p62 expression and that the osteogenic effects of this hormone were totally abrogated in osteoblasts from p62-deficient mice. Therefore, these findings, for the first time, highlight the important role of p62 both for the basal and for PTH-stimulated bone remodeling.

Interpenetrating Hydrogel Networks Enhance Mechanical Stability, Rheological Properties, Release Behavior and Adhesiveness of Platelet-Rich Plasma.

Platelet-rich plasma (PRP) has attracted much attention for the treatment of articular cartilage defects or wounds due to its intrinsic content of growth factors relevant for tissue repair. However, the short residence time of PRP in vivo, due to the action of lytic enzymes, its weak mechanical properties and the consequent short-term release of bioactive factors has restricted its application and efficacy. The present work aimed at designing new formulation strategies for PRP, based on the use of platelet concentrate (PC)-loaded hydrogels or interpenetrating polymer networks, directed at improving mechanical stability and sustaining the release of bioactive growth factors over a prolonged time-span. The interpenetrating hydrogels comprised two polymer networks interlaced on a molecular scale: (a) a first covalent network of thermosensitive and biodegradable vinyl sulfone bearing p(hydroxypropyl methacrylamide-lacate)-polyethylene glycol triblock copolymers, tandem cross-linked by thermal gelation and Michael addition when combined with thiolated hyaluronic acid, and (b) a second network composed of cross-linked fibrin. The PC-loaded hydrogels, instead, was formed only by network (a). All the designed and successfully synthesized formulations greatly increased the stability of PRP in vitro, leading to significant increase in degradation time and storage modulus of PRP gel. The resulting viscoelastic networks showed the ability to controllably release platelet derived growth factor and transforming growth factr ß1, and to improve the tissue adhesiveness of PRP. The newly developed hydrogels show great potential for application in the field of wound healing, cartilage repair and beyond.

P62 deficiency shifts mesenchymal/stromal stem cell commitment toward adipogenesis and disrupts bone marrow homeostasis in aged mice.

With advancing age have been observed bone and bone marrow phenotypic alterations due to the impaired bone tissue homeostatic features, involving bone remodeling, and bone marrow niche ontogeny. The complex "inflamm-aging" pathological scenario that culminates with osteopenia and mesenchymal/stromal and hematopoietic stem cell commitment breakdown, is controlled by cellular and molecular intramural components comprising adapter proteins such as the sequestosome 1 (p62/SQSTM1). p62, a "multiway function" protein, has been reported as an effective anti-inflammatory, bone-building factor. In this view, we considered for the first time the involvement of p62 in aging bone and bone marrow of 1 year and 2 years p62-/- mice. Interestingly, p62 deficiency provoked accelerated osteopenia and impaired niche operational activities within the bone marrow. The above findings unearthed the importance of p62 in mesenchymal stem cell maintenance/differentiation schedule in old animals and provide, at least in part, a mechanistic scenario of p62 action.

Thermosensitive hybrid hyaluronan/p(HPMAm-lac)-PEG hydrogels enhance cartilage regeneration in a mouse model of osteoarthritis.

Osteoarthritis (OA), due to cartilage degeneration, is one of the leading causes of disability worldwide. Currently, there are not efficacious therapies to reverse cartilage degeneration. In this study we evaluated the potential of hybrid hydrogels, composed of a biodegradable and thermosensitive triblock copolymer cross-linked via Michael addition to thiolated hyaluronic acid, in contrasting inflammatory processes underlying OA. Hydrogels composed of different w/w % concentrations of hyaluronan were investigated for their degradation behavior and capacity to release the polysaccharide in a sustained fashion. It was found that hyaluronic acid was controllably released during network degradation with a zero-order release kinetics, and the release rate depended on cross-link density and degradation kinetics of the hydrogels. When locally administered in vivo in an OA mouse model, the hydrogels demonstrated the ability to restore, to some extent, bone remineralization, proteoglycan production, levels of Sox-9 and Runx-2. Furthermore, the downregulation of proinflammatory mediators, such as TNF-α, NFkB, and RANKL and proinflammatory cytokines was observed. In summary, the investigated hydrogel technology represents an ideal candidate for the potential encapsulation and release of drugs relevant in the field of OA. In this context, the hydrogel matrix could act in synergy with the drug, in reversing phenomena of inflammation, cartilage disruption, and bone demineralization associated with OA.

Bone and bone marrow disruption by endocrine-active substances.

Bone is a multifaceted dynamic tissue, involved in mobility, mineral metabolism, and mesenchymal or stromal and hematopoietic progenitor or stem cells breading. Recently, an endocrine role has been attributed to bone due to its ability to produce at least two hormones (osteocalcin and fibroblast growth factor 23) and to participate directly or indirectly in leptin, insulin, estrogens, and serotonin signaling; regulation; and action. Also, bearing in mind the enormous amounts of substances secreted by the different bone marrow cell types, it becomes understandable the contribution of bone tissue to systemic homeostasis. Besides, bone is a well-known estrogen-responsive tissue, reacting to environmental influences. Thus, it has been coined as a critical target of environmental xenoestrogens, known as endocrine-disrupting chemicals (EDCs). The exposure to EDCs results to disruption or imbalance of the systemic hormonal regulation of the skeleton including bone modeling and remodeling, local hormones, and cytokine or chemokine release. The present report highlights the harmful EDCs effects on bone tissue and provides up-to-date information of xenoestrogen action on proliferation, maturation, and homing of bone marrow inhabitants.

Prostaglandin F2α: a bone remodeling mediator.

Prostaglandin F2α (PGF2α) plays multiple roles on bone metabolism by regulating a wide range of signaling pathways. PGF2α, via activation of PKC, stimulates Na-dependent inorganic phosphate (Pi) transport system in osteoblasts; up-regulates interleukin (IL)-6 synthesis; increases vascular endothelial growth factor (VEGF). In addition, PGF2α acts as a strong mitogenic and survival agent on osteoblasts, and these effects are, at least in part, mediated by the binding of fibroblast growth factor-2 (FGF-2) to the specific receptor FGFR1. The understanding of PGF2α intracellular network, albeit complex to clarify, provides molecular bases useful to identify the players of osteoblast proliferation, apoptosis, and the associated angiogenic processes. Indeed, the molecular mechanism that underline PGF2α-regulated bone metabolism may be a promising platform for the development of novel targeted therapies in the treatment of bone disorders and disease.

FGF-2 enhances Runx-2/Smads nuclear localization in BMP-2 canonical signaling in osteoblasts.

Bone morphogenetic protein 2 (BMP-2) is one of the most potent regulators of osteoblast differentiation and bone formation. R-Smads (Smads 1/5/8) are the major transducers for BMPs receptors and, once activated, they are translocated in the nucleus regulating transcription target genes by interacting with various transcription factors. Runx-2 proteins have been shown to interact through their C-terminal segment with Smads and this interaction is required for in vivo osteogenesis. In particular, recruitment of Smads to intranuclear sites is Runx-2 dependent, and Runx-2 factor may accommodate the dynamic targeting of signal transducer to active transcription sites. Previously, we have shown, by in vitro and in vivo experiments, that BMP-2 up-regulated FGF-2 which is important for the maximal responses of BMP-2 in bone. In this study, we found that endogenous FGF2 is necessary for BMP-2 induced nuclear accumulation and co-localization of Runx-2 and phospho-Smads1/5/8, while Runx/Smads nuclear accumulation and co-localization was reduced in Fgf2-/- osteoblasts. Based on these novel data, we conclude that the impaired nuclear accumulation of Runx-2 in Fgf2-/- osteoblasts reduces R-Smads sub-nuclear targeting with a consequent decreased expression of differentiating markers and impaired bone formation in Fgf2 null mice.

The dual face of parathyroid hormone and prostaglandins in the osteoimmune system.

The microenvironment of bone marrow, an extraordinarily heterogeneous and dynamic system, is populated by bone and immune cells, and its functional dimension has been at the forefront of recent studies in the field of osteoimmunology. The interaction of both marrow niches supports self-renewal, differentiation, and homing of the hematopoietic stem cells and provides the essential regulatory molecules for osteoblast and osteoclast homeostasis. Impaired signaling within the niches results in a pathological tableau and enhances disease, including osteoporosis and arthritis, or the rejection of hematopoietic stem cell transplants. Discovering the anabolic players that control these mechanisms has become warranted. In this review, we focus on parathyroid hormone (PTH) and prostaglandins (PGs), potent molecular mediators, both of which carry out a multitude of functions, particularly in bone lining cells and T cells. These two regulators proved to be promising therapeutic agents when strictly clinical protocols on dose treatments were applied.

Endocrine disruptors and bone metabolism.

Bone microenvironment is a complex dynamic equilibrium between osteoclasts and osteoblasts and is modulated by a wide variety of hormones and osteocyte mediators secreted in response to physiological and pathological conditions. The rate of remodeling involves tight coupling and regulation of both cells population and is regulated by a wide variety of hormones and mediators such as parathyroid hormone, prostaglandins, thyroid hormone, sex steroids, etc. It is also well documented that bone formation is easily influenced by the exposure of osteoblasts and osteoclasts to chemical compounds. Currently, humans and wildlife animals are exposed to various environmental xenoestrogens typically at low doses. These compounds, known as endocrine disruptor chemicals (EDCs), can alter the systemic hormonal regulation of the bone remodeling process and the skeletal formation. This review highlights the effects of the EDCs on mammalian bone turnover and development providing a macro and molecular view of their action.

Tackling Inequalities in Oral Health: Bone Augmentation in Dental Surgery through the 3D Printing of Poly(ε-caprolactone) Combined with 20% Tricalcium Phosphate.

The concept of personalized medicine and overcoming healthcare inequalities have become extremely popular in recent decades. Polymers can support cost reductions, the simplicity of customized printing processes, and possible future wide-scale expansion. Polymers with ß-tricalcium phosphate (TCP) are well known for their synergy with oral tissues and their ability to induce osteoconductivity. However, poor information exists concerning their properties after the printing process and whether they can maintain an unaffected biological role. Poly(ε-caprolactone) (PCL) polymer and PCL compounded with TCP 20% composite were printed with a Prusa Mini-LCD-®3D printer. Samples were sterilised by immersion in a 2% peracetic acid solution. Sample analyses were performed using infrared-spectroscopy and statical mechanical tests. Biocompatibility tests, such as cell adhesion on the substrate, evaluations of the metabolic activity of viable cells on substrates, and F-actin labelling, followed by FilaQuant-Software were performed using a MC3T3-E1 pre-osteoblasts line. PCL+ß-TCP-20% composite is satisfactory for commercial 3D printing and appears suitable to sustain an ISO14937:200937 sterilization procedure. In addition, the proper actin cytoskeleton rearrangement clearly shows their biocompatibility as well as their ability to favour osteoblast adhesion, which is a pivotal condition for cell proliferation and differentiation.

Multipotent Mesenchymal Cells Homing and Differentiation on Poly(ε-caprolactone) Blended with 20% Tricalcium Phosphate and Polylactic Acid Incorporating 10% Hydroxyapatite 3D-Printed Scaffolds via a Commercial Fused Deposition Modeling 3D Device.

As highlighted by the 'Global Burden of Disease Study 2019' conducted by the World Health Organization, ensuring fair access to medical care through affordable and targeted treatments remains crucial for an ethical global healthcare system. Given the escalating demand for advanced and urgently needed solutions in regenerative bone procedures, the critical role of biopolymers emerges as a paramount necessity, offering a groundbreaking avenue to address pressing medical needs and revolutionize the landscape of bone regeneration therapies. Polymers emerge as excellent solutions due to their versatility, making them reliable materials for 3D printing. The development and widespread adoption of this technology would impact production costs and enhance access to related healthcare services. For instance, in dentistry, the use of commercial polymers blended with ß-tricalcium phosphate (TCP) is driven by the need to print a standardized product with osteoconductive features. However, modernization is required to bridge the gap between biomaterial innovation and the ability to print them through commercial printing devices. Here we showed, for the first time, the metabolic behavior and the lineage commitment of bone marrow-derived multipotent mesenchymal cells (MSCs) on the 3D-printed substrates poly(e-caprolactone) combined with 20% tricalcium phosphate (PCL + 20% ß-TCP) and L-polylactic acid (PLLA) combined with 10% hydroxyapatite (PLLA + 10% HA). Although there are limitations in printing additive-enriched polymers with a predictable and short half-life, the tested 3D-printed biomaterials were highly efficient in supporting osteoinductivity. Indeed, considering different temporal sequences, both 3D-printed biomaterials resulted as optimal scaffolds for MSCs' commitment toward mature bone cells. Of interest, PLLA + 10% HA substrates hold the confirmation as the finest material for osteoinduction of MSCs.

P62/SQSTM1 beyond Autophagy: Physiological Role and Therapeutic Applications in Laboratory and Domestic Animals.

Inflammation is the preceding condition for the development of mild and severe pathological conditions, including various forms of osteopenia, cancer, metabolic syndromes, neurological disorders, atherosclerosis, cardiovascular, lung diseases, etc., in human and animals. The inflammatory status is induced by multifarious intracellular signaling cascades, where cytokines, chemokines, arachidonic acid metabolites, adhesion molecules, immune cells and other components foster a "slow burn" at a local or systemic level. Assuming that countering inflammation limits the development of inflammation-based diseases, a series of new side-effects-free therapies was assessed in experimental and domestic animals. Within the targets of the drug candidates for quenching inflammation, an archetypal autophagic gear, the p62/sqstm1 protein, has currently earned attention from researchers. Intracellular p62 has been recently coined as a multi-task tool associated with autophagy, bone remodeling, bone marrow integrity, cancer progression, and the maintenance of systemic homeostasis. Accordingly, p62 can act as an effective suppressor of inflamm-aging, reducing oxidative stress and proinflammatory signals. Such an operational schedule renders this protein an effective watchdog for degenerative diseases and cancer development in laboratory and pet animals. This review summarizes the current findings concerning p62 activities as a molecular hub for cell and tissues metabolism and in a variety of inflammatory diseases and other pathological conditions. It also specifically addresses the applications of exogenous p62 (DNA plasmid) as an anti-inflammatory and homeostatic regulator in the treatment of osteoporosis, metabolic syndrome, age-related macular degeneration and cancer in animals, and the possible application of p62 plasmid in other inflammation-associated diseases.

Steering the multipotent mesenchymal cells towards an anti-inflammatory and osteogenic bias via photobiomodulation therapy: How to kill two birds with one stone.

The bone marrow-derived multipotent mesenchymal cells (MSCs) have captured scientific interest due to their multi-purpose features and clinical applications. The operational dimension of MSCs is not limited to the bone marrow reservoir, which exerts bone-building and niche anabolic tasks; they also meet the needs of quenching inflammation and restoring inflamed tissues. Thus, the range of MSC activities extends to conditions such as neurodegenerative diseases, immune disorders and various forms of osteopenia. Steering these cells towards becoming an effective therapeutic tool has become mandatory. Many laboratories have employed distinct strategies to improve the plasticity and secretome of MSCs. We aimed to present how photobiomodulation therapy (PBM-t) can manipulate MSCs to render them an extraordinary anti-inflammatory and osteogenic instrument. Moreover, we discuss the outcomes of different PBM-t protocols on MSCs, concluding with some perplexities and complexities of PBM-t in vivo but encouraging and feasible in vitro solutions.

Highly Potent and Selective Dopamine D4 Receptor Antagonists Potentially Useful for the Treatment of Glioblastoma.

To better understand the role of dopamine D4 receptor (D4R) in glioblastoma (GBM), in the present paper, new ligands endowed with high affinity and selectivity for D4R were discovered starting from the brain penetrant and D4R selective lead compound 1-(3-(4-phenylpiperazin-1-yl)propyl)-3,4-dihydroquinolin-2(1H)-one (6). In particular, the D4R antagonist 24, showing the highest affinity and selectivity over D2R and D3R within the series (D2/D4 = 8318, D3/D4 = 3715), and the biased ligand 29, partially activating D4R Gi-/Go-protein and blocking ß-arrestin recruitment, emerged as the most interesting compounds. These compounds, evaluated for their GBM antitumor activity, induced a decreased viability of GBM cell lines and primary GBM stem cells (GSC#83), with the maximal efficacy being reached at a concentration of 10 µM. Interestingly, the treatment with both compounds 24 and 29 induced an increased effect in reducing the cell viability with respect to temozolomide, which is the first-choice chemotherapeutic drug in GBM.

Molecular mediators involved in Ferulago campestris essential oil effects on osteoblast metabolism.

This study was performed to investigate the effects of the essential oil obtained from fruits of Ferulago campestris (FC) on primary calvarial mouse osteoblasts (COBs). The composition of the oil was dominated by monoterpene hydrocarbons (78.8-80.3%), with myrcene (33.4-39.7%), α-pinene (22.7-23.0%), and γ-terpinene (8.1-10.9%) as the major components. Owing to their lipophilic properties, these compounds easily cross cell membranes and affect bone cell function by stimulating or inhibiting specific molecular pathways. We demonstrated, for the first time, that FC oil increased osteoblast proliferation by MAP kinase activation; in addition, oils enhanced the protein kinase AKT, which is known to be critical for control of cell survival, also in presence of the MEK-1 inhibitor PD98059, and this effect was accompanied with a down-regulation of pro-apototic molecules such as Bax and caspases. Interestingly, FC oil significantly increased Runx2 (Runx2/Pebp2αA/AML3) and phospho-Smad1/5/8 protein level, the master regulators of osteoblast differentiation, and their nuclear localization. PD98059 pre-treatment further improved Runx2/phospho-Smads up-regulation. Thus, FC oils influence osteoblast metabolism probably using alternative signaling pathways depending also on the maturation stage of the cells. Taken together our data delineate a positive function of FC oil on osteoblast metabolism, suggesting its possible use as a dietetic integrator in the prevention or in the therapy of pathologies due to impaired bone remodeling.