Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca²⁺ and P_i, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.     

Matrix Vesicle-Mediated Mineralization and Osteocytic Regulation of Bone Mineralization

Bone mineralization entails two mineralization phases: primary and secondary mineralization. Primary mineralization is achieved when matrix vesicles are secreted by osteoblasts, and thereafter, bone mineral density gradually increases during secondary mineralization. Nearby extracellular phosphate ions (PO₄³⁻) flow into the vesicles via membrane transporters and enzymes located on the vesicles’ membranes, while calcium ions (Ca²⁺), abundant in the tissue fluid, are also transported into the vesicles. The accumulation of Ca²⁺ and PO₄³⁻ in the matrix vesicles induces crystal nucleation and growth. The calcium phosphate crystals grow radially within the vesicle, penetrate the vesicle’s membrane, and continue to grow outside the vesicle, ultimately forming mineralized nodules. The mineralized nodules then attach to collagen fibrils, mineralizing them from the contact sites (i.e., collagen mineralization). Afterward, the bone mineral density gradually increases during the secondary mineralization process. The mechanisms of this phenomenon remain unclear, but osteocytes may play a key role; it is assumed that osteocytes enable the transport of Ca²⁺ and PO₄³⁻ through the canaliculi of the osteocyte network, as well as regulate the mineralization of the surrounding bone matrix via the Phex/SIBLINGs axis. Thus, bone mineralization is biologically regulated by osteoblasts and osteocytes.     

Effects of Microvesicles Derived from NK Cells Stimulated with IL-1β on the Phenotype and Functional Activity of Endothelial Cells

Microvesicles (MVs) are plasma extracellular vesicles ranging from 100 (150) to 1000 nm in diameter. These are generally produced by different cells through their vital activity and are a source of various protein and non-protein molecules. It is assumed that MVs can mediate intercellular communication and modulate cell functions. The interaction between natural killer cells (NK cells) and endothelial cells underlies multiple pathological conditions. The ability of MVs derived from NK cells to influence the functional state of endothelial cells in inflammatory conditions has yet to be studied well. In this regard, we aimed to study the effects of MVs derived from NK cells of the NK-92 cell line stimulated with IL-1β on the phenotype, caspase activity, proliferation and migration of endothelial cells of the EA.hy926 cell line. Endothelial cells were cultured with MVs derived from cells of the NK-92 cell line after their stimulation with IL-1β. Using flow cytometry, we evaluated changes in the expression of endothelial cell surface molecules and endothelial cell death. We evaluated the effect of MVs derived from stimulated NK cells on the proliferative and migratory activity of endothelial cells, as well as the activation of caspase-3 and caspase-9 therein. It was established that the incubation of endothelial cells with MVs derived from cells of the NK-92 cell line stimulated with IL-1β and with MVs derived from unstimulated NK cells, leads to the decrease in the proliferative activity of endothelial cells, appearance of the pan leukocyte marker CD45 on them, caspase-3 activation and partial endothelial cell death, and reduced CD105 expression. However, compared with MVs derived from unstimulated NK cells, a more pronounced effect of MVs derived from cells of the NK-92 cell line stimulated with IL-1β was found in relation to the decrease in the endothelial cell migratory activity and the intensity of the CD54 molecule expression on them. The functional activity of MVs is therefore mediated by the conditions they are produced under, as well as their internal contents.     

Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated into Chondrocytes Generates Pi but Not PPi

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP_i). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PP_i or the mineralization stimulator inorganic phosphate (P_i), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and β-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using ³¹P-NMR and IR spectroscopy. (3) Results: ATP and PP_i hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PP_i. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate P_i and not PP_i, which may exacerbate plaque calcification.     

PGS/HAp Microporous Composite Scaffold Obtained in the TIPS-TCL-SL Method: An Innovation for Bone Tissue Engineering

In this research, we synthesize and characterize poly(glycerol sebacate) pre-polymer (pPGS) (¹H NMR, FTiR, GPC, and TGA). Nano-hydroxyapatite (HAp) is synthesized using the wet precipitation method. Next, the materials are used to prepare a PGS-based composite with a 25 wt.% addition of HAp. Microporous composites are formed by means of thermally induced phase separation (TIPS) followed by thermal cross-linking (TCL) and salt leaching (SL). The manufactured microporous materials (PGS and PGS/HAp) are then subjected to imaging by means of SEM and µCT for the porous structure characterization. DSC, TGA, and water contact angle measurements are used for further evaluation of the materials. To assess the cytocompatibility and biological potential of PGS-based composites, preosteoblasts and differentiated hFOB 1.19 osteoblasts are employed as in vitro models. Apart from the cytocompatibility, the scaffolds supported cell adhesion and were readily populated by the hFOB1.19 preosteoblasts. HAp-facilitated scaffolds displayed osteoconductive properties, supporting the terminal differentiation of osteoblasts as indicated by the production of alkaline phosphatase, osteocalcin and osteopontin. Notably, the PGS/HAp scaffolds induced the production of significant amounts of osteoclastogenic cytokines: IL-1β, IL-6 and TNF-α, which induced scaffold remodeling and promoted the reconstruction of bone tissue. Initial biocompatibility tests showed no signs of adverse effects of PGS-based scaffolds toward adult BALB/c mice.     

Effect of Melatonin on the Extracellular-Regulated Kinase Signal Pathway Activation and Human Osteoblastic Cell Line hFOB 1.19 Proliferation

It has been shown that melatonin may affect bone metabolism. However, it is controversial whether melatonin could promote osteoblast proliferation, and the precise molecular mechanism of melatonin on osteoblast proliferation is still obscure. In this study, the results of the CCK-8 assay showed that melatonin significantly promoted human osteoblastic cell line hFOB 1.19 cell proliferation at 1, 2.5, 5, 10, 25, 50 and 100 µM concentrations for 24 h, but there were no significant differences among the groups. Western blot demonstrated that 10 µM melatonin significantly promoted ERK1/2 phosphorylation. Furthermore, we also detected the phosphorylation of c-Raf, MEK1/2, p90RSK and MSK1, and all of them increased with 10 µM melatonin. U0126 (a selective inhibitor of MEK that disrupts downstream activation of ERK1/2) downregulated the phosphorylation of ERK1/2, p90RSK and MSK1. U0126 also attenuated the proliferation of osteoblasts stimulated by melatonin. In conclusion, this study for the first time indicates that melatonin (10 nM–100 µM) promotes the proliferation of a human osteoblastic cell line hFOB 1.19 through activation of c-Raf, MEK1/2, ERK1/2, p90RSK and MSK1.     

In Vitro Proliferation and Anti-Apoptosis of the Papain-Generated Casein and Soy Protein Hydrolysates towards Osteoblastic Cells (hFOB1.19)

Casein and soy protein were digested by papain to three degrees of hydrolysis (DH) 7.3%–13.3%, to obtain respective six casein and soy protein hydrolysates, aiming to clarify their in vitro proliferation and anti-apoptosis towards a human osteoblastic cell line (hFOB1.19 cells). Six casein and soy protein hydrolysates at five levels (0.01–0.2 mg/mL) mostly showed proliferation as positive 17β-estradiol did, because they conferred the osteoblasts with cell viability of 100%–114% and 104%–123%, respectively. The hydrolysates of higher DH values had stronger proliferation. Casein and soy protein hydrolysates of the highest DH values altered cell cycle progression, and enhanced cell proportion of S-phase from 50.5% to 56.5% and 60.5%. The two also antagonized etoposide- and NaF-induced osteoblast apoptosis. In apoptotic prevention, apoptotic cells were decreased from 31.6% to 22.6% and 15.6% (etoposide treatment), or from 19.5% to 17.7% and 12.4% (NaF treatment), respectively. In apoptotic reversal, soy protein hydrolysate decreased apoptotic cells from 13.3% to 11.7% (etoposide treatment), or from 14.5% to 11.0% (NaF treatment), but casein hydrolysate showed no reversal effect. It is concluded that the hydrolysates of two kinds had estradiol-like action on the osteoblasts, and soy protein hydrolysates had stronger proliferation and anti-apoptosis on the osteoblasts than casein hydrolysates.     

The hEag1 K+ Channel Inhibitor Astemizole Stimulates Ca2+ Deposition in SaOS-2 and MG-63 Osteosarcoma Cultures

The hEag1 (Kv10.1) K⁺ channel is normally found in the brain, but it is ectopically expressed in tumor cells, including osteosarcoma. Based on the pivotal role of ion channels in osteogenesis, we tested whether pharmacological modulation of hEag1 may affect osteogenic differentiation of osteosarcoma cell lines. Using molecular biology (RT-PCR), electrophysiology (patch-clamp) and pharmacology (astemizole sensitivity, IC₅₀ = 0.135 μM) we demonstrated that SaOS-2 osteosarcoma cells also express hEag1 channels. SaOS-2 cells also express to KCa1.1 K⁺ channels as shown by mRNA expression and paxilline sensitivity of the current. The inhibition of hEag1 (2 μM astemizole) or KCa1.1 (1 mM TEA) alone did not induce Ca²⁺ deposition in SaOS-2 cultures, however, these inhibitors, at identical concentrations, increased Ca²⁺ deposition evoked by the classical or pathological (inorganic phosphate, Pi) induction pathway without causing cytotoxicity, as reported by three completer assays (LDH release, MTT assay and SRB protein assay). We observed a similar effect of astemizole on Ca²⁺ deposition in MG-63 osteosarcoma cultures as well. We propose that the increase in the osteogenic stimuli-induced mineral matrix formation of osteosarcoma cell lines by inhibiting hEag1 may be a useful tool to drive terminal differentiation of osteosarcoma.     

The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization

KCNQ (K_V7) channels are voltage-gated potassium (K_V) channels, and the function of K_V7 channels in muscles, neurons, and sensory cells is well established. We confirmed that overall blockade of K_V channels with tetraethylammonium augmented the mineralization of bone-marrow-derived human mesenchymal stem cells during osteogenic differentiation, and we determined that K_V7.3 was expressed in MG-63 and Saos-2 cells at the mRNA and protein levels. In addition, functional K_V7 currents were detected in MG-63 cells. Inhibition of K_V7.3 by linopirdine or XE991 increased the matrix mineralization during osteoblast differentiation. This was confirmed by alkaline phosphatase, osteocalcin, and osterix in MG-63 cells, whereas the expression of Runx2 showed no significant change. The extracellular glutamate secreted by osteoblasts was also measured to investigate its effect on MG-63 osteoblast differentiation. Blockade of K_V7.3 promoted the release of glutamate via the phosphorylation of extracellular signal-regulated kinase 1/2-mediated upregulation of synapsin, and induced the deposition of type 1 collagen. However, activation of K_V7.3 by flupirtine did not produce notable changes in matrix mineralization during osteoblast differentiation. These results suggest that K_V7.3 could be a novel regulator in osteoblast differentiation.     

Detection of Bacterial Membrane Vesicles by NOD-Like Receptors

Bacterial membrane vesicles (BMVs) are nanoparticles produced by both Gram-negative and Gram-positive bacteria that can function to modulate immunity in the host. Both outer membrane vesicles (OMVs) and membrane vesicles (MVs), which are released by Gram-negative and Gram-positive bacteria, respectively, contain cargo derived from their parent bacterium, including immune stimulating molecules such as proteins, lipids and nucleic acids. Of these, peptidoglycan (PG) and lipopolysaccharide (LPS) are able to activate host innate immune pattern recognition receptors (PRRs), known as NOD-like receptors (NLRs), such as nucleotide-binding oligomerisation domain-containing protein (NOD) 1, NOD2 and NLRP3. NLR activation is a key driver of inflammation in the host, and BMVs derived from both pathogenic and commensal bacteria have been shown to package PG and LPS in order to modulate the host immune response using NLR-dependent mechanisms. Here, we discuss the packaging of immunostimulatory cargo within OMVs and MVs, their detection by NLRs and the cytokines produced by host cells in response to their detection. Additionally, commensal derived BMVs are thought to shape immunity and contribute to homeostasis in the gut, therefore we also highlight the interactions of commensal derived BMVs with NLRs and their roles in limiting inflammatory diseases.     

Bacterial Membrane Vesicles in Pneumonia: From Mediators of Virulence to Innovative Vaccine Candidates

Pneumonia due to respiratory infection with most prominently bacteria, but also viruses, fungi, or parasites is the leading cause of death worldwide among all infectious disease in both adults and infants. The introduction of modern antibiotic treatment regimens and vaccine strategies has helped to lower the burden of bacterial pneumonia, yet due to the unavailability or refusal of vaccines and antimicrobials in parts of the global population, the rise of multidrug resistant pathogens, and high fatality rates even in patients treated with appropriate antibiotics pneumonia remains a global threat. As such, a better understanding of pathogen virulence on the one, and the development of innovative vaccine strategies on the other hand are once again in dire need in the perennial fight of men against microbes. Recent data show that the secretome of bacteria consists not only of soluble mediators of virulence but also to a significant proportion of extracellular vesicles—lipid bilayer-delimited particles that form integral mediators of intercellular communication. Extracellular vesicles are released from cells of all kinds of organisms, including both Gram-negative and Gram-positive bacteria in which case they are commonly termed outer membrane vesicles (OMVs) and membrane vesicles (MVs), respectively. (O)MVs can trigger inflammatory responses to specific pathogens including S. pneumonia, P. aeruginosa, and L. pneumophila and as such, mediate bacterial virulence in pneumonia by challenging the host respiratory epithelium and cellular and humoral immunity. In parallel, however, (O)MVs have recently emerged as auspicious vaccine candidates due to their natural antigenicity and favorable biochemical properties. First studies highlight the efficacy of such vaccines in animal models exposed to (O)MVs from B. pertussis, S. pneumoniae, A. baumannii, and K. pneumoniae. An advanced and balanced recognition of both the detrimental effects of (O)MVs and their immunogenic potential could pave the way to novel treatment strategies in pneumonia and effective preventive approaches.     

The Effect of Pore Size Distribution and l-Lysine Modified Apatite Whiskers (HAP) on Osteoblasts Response in PLLA/HAP Foam Scaffolds Obtained in the Thermally Induced Phase Separation Process

In this research, we prepared foam scaffolds based on poly(l-lactide) (PLLA) and apatite whiskers (HAP) using thermally induced phase separation technique supported by the salt leaching process (TIPS-SL). Using sodium chloride having a size of (a) 150–315 μm, (b) 315–400 μm, and (c) 500–600 μm, three types of foams with different pore sizes have been obtained. Internal structure of the obtained materials has been investigated using SEM as well as μCT. The materials have been studied by means of porosity, density, and compression tests. As the most promising, the composite prepared with salt size of 500–600 μm was prepared also with the l-lysine modified apatite. The osteoblast hFOB 1.19 cell response for the scaffolds was also investigated by means of cell viability, proliferation, adhesion/penetration, and biomineralization. Direct contact cytotoxicity assay showed the cytocompatibility of the scaffolds. All types of foam scaffolds containing HAP whiskers, regardless the pore size or l-lysine modification induced significant stimulatory effect on the cal-cium deposits formation in osteoblasts. The PLLA/HAP scaffolds modified with l-lysine stimulated hFOB 1.19 osteoblasts proliferation. Compared to the scaffolds with smaller pores (150–315 µm and 315–400 µm), the PLLA/HAP foams with large pores (500–600 µm) promoted more effective ad-hesion of osteoblasts to the surface of the biomaterial.     

From Stem Cells to Bone-Forming Cells

Bone formation starts near the end of the embryonic stage of development and continues throughout life during bone modeling and growth, remodeling, and when needed, regeneration. Bone-forming cells, traditionally termed osteoblasts, produce, assemble, and control the mineralization of the type I collagen-enriched bone matrix while participating in the regulation of other cell processes, such as osteoclastogenesis, and metabolic activities, such as phosphate homeostasis. Osteoblasts are generated by different cohorts of skeletal stem cells that arise from different embryonic specifications, which operate in the pre-natal and/or adult skeleton under the control of multiple regulators. In this review, we briefly define the cellular identity and function of osteoblasts and discuss the main populations of osteoprogenitor cells identified to date. We also provide examples of long-known and recently recognized regulatory pathways and mechanisms involved in the specification of the osteogenic lineage, as assessed by studies on mice models and human genetic skeletal diseases.     

Biohybrid Bovine Bone Matrix for Controlled Release of Mesenchymal Stem/Stromal Cell Lyosecretome: A Device for Bone Regeneration

SmartBone^® (SB) is a biohybrid bone substitute advantageously proposed as a class III medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic, and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed. SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell (MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs (38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other hand, the process was still present, but tissue formation was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless, SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β.     

The Evaluation of the Possibilities of Using PLGA Co-Polymer and Its Composites with Carbon Fibers or Hydroxyapatite in the Bone Tissue Regeneration Process �C in Vitro and in Vivo Examinations

Synthetic polymers belonging to the aliphatic polyester group have become highly promising biomaterials for reconstructive medicine. The purpose of the present work is a biological evaluation of lactide-glycolide co-polymer (PLGA) and its composites with carbon fibers (PLGA+CF) or hydroxyapatite (PLGA+HA). The cytotoxicity of the evaluated materials towards hFOB 1.19 human osteoblast-like cells was assessed. Moreover, during the one-year contact of the assessed materials with living osseous tissue, the progress of bone formation was analyzed and the accompanying process of the materials’ degradation was evaluated. The materials under evaluation proved to be biocompatible.     

Different Contribution of Monocyte- and Platelet-Derived Microvesicles to Endothelial Behavior

Several contributions of circulating microvesicles (MVs) to the endothelial dysfunction have been reported in the past; a head-to-head comparison of platelet- and monocyte–derived MVs has however never been performed. To this aim, we assessed the involvement of these MVs in vessel damage related processes, i.e., oxidative stress, inflammation, and leukocyte-endothelial adhesion. Platelets and monocytes isolated from healthy subjects (HS, n = 15) were stimulated with TRAP-6 and LPS to release MVs that were added to human vascular endothelial cell (hECV) culture to evaluate superoxide anion production, inflammatory markers (IL-6, TNFα, NF-κB mRNA expression), and hECV adhesiveness. The effects of the MVs-induced from HS were compared to those induced by MVs spontaneously released from cells of patients with ST-segment elevation myocardial infarction (STEMI, n = 7). MVs released by HS-activated cells triggered a threefold increase in oxidative burst in a concentration-dependent manner. Only MVs released from monocytes doubled IL-6, TNFα, and NF-κB mRNA expression and monocyte-endothelial adhesion. Interestingly, the effects of the MVs isolated from STEMI-monocytes were not superimposable to previous ones except for adhesion to hECV. Conversely, MVs released from STEMI-platelets sustained both redox state and inflammatory phenotype. These data provide evidence that MVs released from activated and/or pathologic platelets and monocytes differently affect endothelial behavior, highlighting platelet-MVs as causative factors of impaired endothelial function in the acute phase of STEMI.     

Response of osteoblast-like MG63 cells to TiO2 layer prepared by micro-arc oxidation and electric polarization

Micro-arc oxidation (MAO) is one of the useful surface modifications of titanium implants to improve bioactivity. Also, electric polarization treatment enhances bioactivity of calcium phosphate. The aim of this study was to evaluate the effect of the combination of two surface modifications, micro-arc oxidation (MAO) with electric polarization, on the behavior of osteoblast-like osteosarcoma MG63 cells. MAO-treated materials had a surface geometry that was favored by MG63 cells as determined using scanning electron microscopy and X-ray diffraction; additionally, electric polarization induced surface electric fields, which were measured using thermally stimulated depolarization currents. The results of assays to study cell–material interactions suggest that these two approaches could regulate cell attachment, spreading, proliferation, and differentiation without the addition of other reagents. This new surface modification processes produce materials with a good surface geometry, generate surface electric fields and enhance the osteopromotive ability of osteoblasts.