Crafting Stable Antibiotic Nanoparticles via Complex Coacervation of Colistin with Block Copolymers.

To combat the ever-growing increase of multidrug-resistant (MDR) bacteria, action must be taken in the development of antibiotic formulations. Colistin, an effective antibiotic, was found to be nephrotoxic and neurotoxic, consequently leading to a ban on its use in the 1980s. A decade later, colistin use was revived and nowadays used as a last-resort treatment against Gram-negative bacterial infections, although highly regulated. If cytotoxicity issues can be resolved, colistin could be an effective option to combat MDR bacteria. Herein, we investigate the complexation of colistin with poly(ethylene oxide)-b-poly(methacrylic acid) (PEO-b-PMAA) block copolymers to form complex coacervate core micelles (C3Ms) to ultimately improve colistin use in therapeutics while maintaining its effectiveness. We show that well-defined and stable micelles can be formed in which the cationic colistin and anionic PMAA form the core while PEO forms a protecting shell. The resulting C3Ms are in a kinetically arrested and stable state, yet they can be made reproducibly using an appropriate experimental protocol. By characterization through dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), we found that the best C3M formulation, based on long-term stability and complexation efficiency, is at charge-matching conditions. This nanoparticle formulation was compared to noncomplexed colistin on its antimicrobial properties, enzymatic degradation, serum protein binding, and cytotoxicity. The studies indicate that the antimicrobial properties and cytotoxicity of the colistin-C3Ms were maintained while protein binding was limited, and enzymatic degradation decreased after complexation. Since colistin-C3Ms were found to have an equal effectivity but with increased cargo protection, such nanoparticles are promising components for the antibiotic formulation toolbox.

Targeting a therapeutically relevant concentration of alendronate for in vitro studies on osteoblasts.

OBJECTIVE: Bisphosphonates like alendronate mainly exert their effects on osteoclasts. However, osteoblasts are also affected, but exposed to a much lower concentration in vivo than the osteoclasts. Given that the effects are dose-dependent, the intention of the study was to identify a therapeutically relevant concentration of alendronate for in vitro studies on osteoblasts. MATERIALS AND METHODS: Primary human osteoblasts were incubated with alendronate (5, 20 and 100 µM) for 1, 3, 7 and 14 days. Proliferation and viability were assessed, and the effects on cellular growth and function were evaluated by multianalyte profiling of selected proteins in cell culture media using the Luminex 200TM. RESULTS: The viability was not affected by any of the dosages. Exposure to 5 µM alendronate had a neutral effect on osteoblast proliferation, and on secretion of osteogenic and inflammatory markers, while enhancing synthesis of a marker of angiogenesis. 20 µM alendronate induced a decline in proliferation and affected angiogenic and osteogenic biomarkers adversely. 100 µM alendronate reduced proliferation dramatically, and this dosage was excluded from further experiments. CONCLUSION: A concentration of 5 µM alendronate exerted effects on human osteoblasts that may translate to those observed in vivo and could therefore be relevant for in vitro studies.

LIGHT/TNFSF14 regulates estrogen deficiency-induced bone loss.

Bone loss induced by ovariectomy is due to the direct activity on bone cells and mesenchymal cells and to the dysregulated activity of bone marrow cells, including immune cells and stromal cells, but the underlying mechanisms are not completely known. Here, we demonstrate that ovariectomy induces the T-cell co-stimulatory cytokine LIGHT, which stimulates both osteoblastogenesis and osteoclastogenesis by modulating osteoclastogenic cytokine expression, including TNF, osteoprotegerin, and the receptor activator of nuclear factor-κB ligand (RANKL). Predictably, LIGHT-deficient (Tnfsf14-/- ) mice are protected from ovariectomy-dependent bone loss, whereas trabecular bone mass increases in mice deficient in both LIGHT and T and B lymphocytes (Rag -/- Tnfsf14 -/- ) and is associated with an inversion of the TNF and RANKL/OPG ratio. Furthermore, women with postmenopausal osteoporosis display high levels of LIGHT in circulating T cells and monocytes. Taken together, these results indicate that LIGHT mediates bone loss induced by ovariectomy, suggesting that patients with postmenopausal osteoporosis may benefit from LIGHT antagonism. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Biological responses to physicochemical properties of biomaterial surface.

Biomedical scientists use chemistry-driven processes found in nature as an inspiration to design biomaterials as promising diagnostic tools, therapeutic solutions, or tissue substitutes. While substantial consideration is devoted to the design and validation of biomaterials, the nature of their interactions with the surrounding biological microenvironment is commonly neglected. This gap of knowledge could be owing to our poor understanding of biochemical signaling pathways, lack of reliable techniques for designing biomaterials with optimal physicochemical properties, and/or poor stability of biomaterial properties after implantation. The success of host responses to biomaterials, known as biocompatibility, depends on chemical principles as the root of both cell signaling pathways in the body and how the biomaterial surface is designed. Most of the current review papers have discussed chemical engineering and biological principles of designing biomaterials as separate topics, which has resulted in neglecting the main role of chemistry in this field. In this review, we discuss biocompatibility in the context of chemistry, what it is and how to assess it, while describing contributions from both biochemical cues and biomaterials as well as the means of harmonizing them. We address both biochemical signal-transduction pathways and engineering principles of designing a biomaterial with an emphasis on its surface physicochemistry. As we aim to show the role of chemistry in the crosstalk between the surface physicochemical properties and body responses, we concisely highlight the main biochemical signal-transduction pathways involved in the biocompatibility complex. Finally, we discuss the progress and challenges associated with the current strategies used for improving the chemical and physical interactions between cells and biomaterial surface.

Bulk Fill Composites Have Similar Performance to Conventional Dental Composites.

The aim of the study was to perform comprehensive characterization of two commonly used bulk fill composite materials (SDR Flow (SDR) and Filtek™ Bulk Fill Flowable Restorative (FBF) and one conventional composite material (Tetric EvoCeram; TEC). Eleven parameters were examined: flexural strength (FS), flexural modulus (FM), degree of conversion, depth of cure, polymerisation shrinkage (PS), filler particle morphology, filler mass fraction, Vickers hardness, surface roughness following simulated toothbrush abrasion, monomer elution, and cytotoxic reaction of human gingival fibroblasts, osteoblasts, and cancer cells. The degree of conversion and depth of cure were the highest for SDR, followed by FBF and TEC, but there was no difference in PS between them. FS was higher for bulk fill materials, while their FM and hardness were lower than those of TEC. Surface roughness decreased in the order TEC→SDR→FBF. Bisphenol A-glycidyl methacrylate (BisGMA) and urethane dimethacrylate were found in TEC and FBF eluates, while SDR released BisGMA and triethylene glycol dimethacrylate. Conditioned media accumulated for 24h from FBF and TEC were cytotoxic to primary human osteoblasts. Compared to the conventional composite, the tested bulk fill materials performed equally or better in most of the tests, except for their hardness, elastic modulus, and biocompatibility with osteoblasts.

Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties.

Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel-the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN-its oligomerization ability-is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca2+)-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca2+ may explain the role of AMBN in biomineralization and more generally in Ca2+ homeostasis processes.

Effects of ionizing irradiation and interface backscatter on human mesenchymal stem cells cultured on titanium surfaces.

Radiotherapy to the head and neck region negatively influences the osseointegration and survival of dental implants. The effects of cobalt 60 (60 Co) ionizing radiation and the impact of backscatter rays were investigated on human mesenchymal stem cells cultured on titanium surfaces. Bone marrow-derived human mesenchymal stem cells were seeded on titanium (Ti), fluoride-modified titanium (TiF), and tissue culture plastic. Cells were exposed to ionizing γ-radiation in single doses of 2, 6, or 10 Gy using a 60 Co source. Density and distribution of cells were evaluated using confocal laser-scanning microscopy, 21 d post-irradiation. Lactate dehydrogenase concentration and the levels of total protein and cytokines/chemokines were measured in the cell-culture medium on days 1, 3, 7, 14, and 21 post-irradiation. Unirradiated cells were used as the control. Irradiation had no effect on cell viability, collagen and actin expression, or cell distribution, but induced an initial increase in the secretion of interleukin (IL)-6, IL-8, monocyte chemotactic protein 1 (MCP-1), and vascular endothelial growth factor (VEGF), followed by a decrease in secretion after 3 or 7 d. Irradiation resulted in secretion of a lower amount of all analytes examined compared with controls on day 21, irrespective of radiation dose and growth surface. Backscattering from titanium did not influence the cell response significantly, suggesting a clinical potential for achieving successful osseointegration of dental implants placed before radiotherapy.

Changes in Human Foetal Osteoblasts Exposed to the Random Positioning Machine and Bone Construct Tissue Engineering.

Human cells, when exposed to both real and simulated microgravity (s-µg), form 3D tissue constructs mirroring in vivo architectures (e.g., cartilage, intima constructs, cancer spheroids and others). In this study, we exposed human foetal osteoblast (hFOB 1.19) cells to a Random Positioning Machine (RPM) for 7 days and 14 days, with the purpose of investigating the effects of s-µg on biological processes and to engineer 3D bone constructs. RPM exposure of the hFOB 1.19 cells induces alterations in the cytoskeleton, cell adhesion, extra cellular matrix (ECM) and the 3D multicellular spheroid (MCS) formation. In addition, after 7 days, it influences the morphological appearance of these cells, as it forces adherent cells to detach from the surface and assemble into 3D structures. The RPM-exposed hFOB 1.19 cells exhibited a differential gene expression of the following genes: transforming growth factor beta 1 (TGFB1, bone morphogenic protein 2 (BMP2), SRY-Box 9 (SOX9), actin beta (ACTB), beta tubulin (TUBB), vimentin (VIM), laminin subunit alpha 1 (LAMA1), collagen type 1 alpha 1 (COL1A1), phosphoprotein 1 (SPP1) and fibronectin 1 (FN1). RPM exposure also induced a significantly altered release of the cytokines and bone biomarkers sclerostin (SOST), osteocalcin (OC), osteoprotegerin (OPG), osteopontin (OPN), interleukin 1 beta (IL-1ß) and tumour necrosis factor 1 alpha (TNF-1α). After the two-week RPM exposure, the spheroids presented a bone-specific morphology. In conclusion, culturing cells in s-µg under gravitational unloading represents a novel technology for tissue-engineering of bone constructs and it can be used for investigating the mechanisms behind spaceflight-related bone loss as well as bone diseases such as osteonecrosis or bone injuries.

The myokine irisin increases cortical bone mass.

It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 µg kg(-1). We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg kg(-1) per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes, Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulated Atf4, Runx2, Osx, Lrp5, ß-catenin, Alp, and Col1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursor Fndc5 was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressed Fndc5, albeit at low levels. Furthermore, muscle fibers from r-irisin-injected mice displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle-bone connectivity.

Effects of the Histamine 1 Receptor Antagonist Cetirizine on the Osteoporotic Phenotype in H(+) /K(+) ATPase Beta Subunit KO Mice.

Epidemiological studies suggest increased fracture risk in patients using proton pump inhibitors (PPIs). We have previously shown that the H(+) /K(+) ATPase beta subunit knockout (KO) mouse, which is a model of PPI-use, have lower bone mineral density (BMD) and impaired bone quality compared to wild type (WT) mice. Like PPI users, these KO mice display elevated gastric pH and hypergastrinemia, which in turn stimulates gastric histamine release. Previous studies have suggested a negative effect of histamine on bone, thus, we wanted to study whether a histamine 1 receptor (H1R) antagonist could improve bone quality in KO mice. Female KO and WT mice aged 8 weeks received either an H1R antagonist (cetirizine) or polyethylene glycol (PEG) for 6 months. At the end of the study, KO mice displayed elevated plasma histamine levels compared to WT. As demonstrated previously, the KO mice also exhibited lower whole body BMD, reduced mechanical bone strength, and impaired bone quality assessed by µCT. No significant differences, however, were found between the KO groups receiving cetirizine or PEG for any of the measured bone parameters. In vitro gene expression analyses of histamine receptors revealed the presence of H1R and H2R both in osteoblasts and osteoclasts, and H3R in late stage osteoblasts. In conclusion, administration of the H1R antagonist cetirizine in a concentration of 3 mg/kg did not rescue the osteoporotic phenotype in H(+) /K(+) ATPase beta subunit KO mice. It can, however, not be ruled out that histamine may influence bone via other receptors. J. Cell. Biochem. 117: 2089-2096, 2016. © 2016 Wiley Periodicals, Inc.

Alendronate alters osteoblast activities.

OBJECTIVE: Due to accumulation in the bone matrix and a half-life of at least 10 years, it is important to understand the cellular impact of bisphosphonates (BPs). This study assessed the effects of alendronate (ALN) on human primary osteoblasts. MATERIAL AND METHODS: Osteoblasts were incubated with ALN (5, 20 and 100 µM), and both cells and cell culture media were harvested after d 1, 3, 7 or 14. Proliferation was evaluated by 3H-thymidine incorporation and tetrazolium dye (MTT) colorimetric assay, and viability by the lactate dehydrogenase (LDH) activity in the medium. Differentiation was evaluated using protein Luminex multiplex assays and RT-PCR. RESULTS: ALN had no significant effects on cell viability. The lower concentrations enhanced the proliferation, whereas 100 µM diminished the proliferation. mRNA expression of osteocalcin (OC), alkaline phosphatase (ALP) and α-1 type 1 collagen were reduced, whereas ALN enhanced the expression of leptin mRNA and the secretion of interleukin-8 (IL-8) and regulated on activation normal T cell expressed and secreted (RANTES). CONCLUSIONS: ALN enhanced the secretion of immune factors from human osteoblasts. Combined with a lower rate of proliferation and a decline in differentiation, this indicates that higher dosages or accumulation may cause undesirable local changes in bone.

Hypoxia increases the expression of enamel genes and cytokines in an ameloblast-derived cell line.

The aim of this study was to investigate the effect of hypoxic conditions on the expression of enamel genes and on the secretion of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), cytokines, and interleukins by an ameloblast-derived cell line. Murine ameloblast-derived cells (LS-8 cells) were exposed to 1% oxygen for 24 and 48 h and harvested after 1, 2, 3, and 7 d. The effect of culture in hypoxic conditions on the expression of structural enamel matrix genes and on the secretion of cytokines and interleukins, as well as ALP and LDH, into the cell-culture medium was calculated relative to the expression and secretion of these factors by untreated cells (controls) at each time point. Hypoxia increased expression of the structural enamel matrix genes amelogenin (Amelx), ameloblastin (Ambn), and enamelin (Enam), and the enamel protease matrix metalloproteinase-20 (Mmp20). Expression of hypoxia-inducible factor 1-alpha (Hif1α), and secretion of several vascularization factors and pro-inflammatory factors, were increased after 24 and 48 h of hypoxia. The ALP activity was reduced after 24 and 48 h of hypoxia, whereas the LDH level in the cell-culture medium was higher after 24 h of hypoxic conditions compared with 48 h. In conclusion, hypoxic exposure may disrupt the controlled fine-tuned expression and processing of enamel genes, and promote the secretion of pro-inflammatory factors.

Simvastatin coating of TiO2 scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

Bone tissue engineering requires an osteoconductive scaffold, multipotent cells with regenerative capacity and bioactive molecules. In this study we investigated the osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) on titanium dioxide (TiO2) scaffold coated with alginate hydrogel containing various concentrations of simvastatin (SIM). The mRNA expression of osteoblast-related genes such as collagen type I alpha 1 (COL1A1), alkaline phosphatase (ALPL), osteopontin (SPP1), osteocalcin (BGLAP) and vascular endothelial growth factor A (VEGFA) was enhanced in hAD-MSCs cultured on scaffolds with SIM in comparison to scaffolds without SIM. Furthermore, the secretion of osteoprotegerin (OPG), vascular endothelial growth factor A (VEGFA), osteopontin (OPN) and osteocalcin (OC) to the cell culture medium was higher from hAD-MSCs cultured on scaffolds with SIM compared to scaffolds without SIM. The TiO2 scaffold coated with alginate hydrogel containing SIM promote osteogenic differentiation of hAD-MSCs in vitro, and demonstrate feasibility as scaffold for hAD-MSC based bone tissue engineering.

Sulcus fluid bone marker levels and the outcome of surgical treatment of peri-implantitis.

AIM: To analyse change in selected bone markers in peri-implant sulcus fluid (PISF) sampled before treatment and after 12 months and test correlation with change in disease progression. MATERIALS AND METHODS: Peri-implant sulcus fluid was sampled from 32 patients in a randomized, clinical study comparing peri-implant defect re-construction with or without porous titanium granules. Matrix metalloproteinase 8 levels were measured using the Quantikine Human Total MMP-8 (DMP800) ELISA. Multianalyte profiling of the level of bone markers [interleukin-6, osteprotegerin (OPG), osteocalcin, leptin, osteopontin, parathyroid hormone, tumour necrosis factor-α, adiponectin and insulin] was performed by Luminex using Human Bone Panel IB. Changes in bone marker levels were compared and correlation with clinical findings was tested. RESULTS: No differences in clinical parameter or bone marker levels between test and control group were found. When comparing bone marker levels irrespective of treatment allocation between baseline and 12 months, a significant reduction in total protein, matrix metalloproteinase -8, interleukin-6, OPG, leptin and adiponectin were demonstrated. Positive correlations were found between the reduction in interleukin-6 (r = 0.43), insulin (r = 0.38) and matrix metalloproteinase-8 (r = 0.47) concentration, and probing pocket depth reduction. CONCLUSION: Peri-implantitis surgical treatment induced some reduction of the studied bone markers. Conclusive evidence for correlation between change in bone marker concentrations with disease resolution was not found.

The effect of hydrofluoric acid treatment of titanium and titanium dioxide surface on primary human osteoblasts.

OBJECTIVE: The aim of the study was to investigate solely the effect of fluoride on the surface chemistry of polycrystalline ceramic titanium dioxide (TiO2 ) and metallic titanium (Ti) and its effect on proliferation and differentiation of primary human osteoblasts (NHO). MATERIALS AND METHODS: The NHO cells were exposed to fluoride-modified and unmodified samples for 1, 3, 7, 14 and 21 days. The fluoride effect on the mRNA expression was quantified and measured. The secretion of cytokines and interleukins in the cell culture medium was measured by Luminex, gene expression by RT-PCR, and compared with untreated controls. The effect on cell growth after 1 and 3 days in culture was measured using [(3) H]-thymidine incorporation. Fluoride release was measured using an ion-selective electrode. The surfaces were examined by X-ray photoelectron spectroscopy and profilometry. RESULTS: The fluoride release study detected that fluoride content easily washed off in TiO2 coins when compared with Ti coins. No increase in cell proliferation was found among fluoride-modified TiO2 surfaces compared with controls, except for washed Ti coins with fluoride modification. The cell differentiation with regard to gene expression showed no significant differences in both fluoride-modified and unmodified samples and less effect on protein release for all groups. CONCLUSIONS: The fluoride from hydrofluoric acid treatment on Ti and TiO2 surfaces gave no specific effect on primary human osteoblast cells. The study indicates that the released fluoride is not the unique factor for the bioactivity of Ti and TiO2 surfaces.

Enamel matrix derivative promote primary human pulp cell differentiation and mineralization.

Enamel matrix derivative (EMD) has been found to induce reactive dentin formation; however the molecular mechanisms involved are unclear. The effect of EMD (5-50 µg/mL) on primary human pulp cells were compared to untreated cells and cells incubated with 10⁻8 M dexamethasone (DEX) for 1, 2, 3, 7, and 14 days in culture. Expression analysis using Affymetrix microchips demonstrated that 10 µg/mL EMD regulated several hundred genes and stimulated the gene expression of proteins involved in mesenchymal proliferation and differentiation. Both EMD and DEX enhanced the expression of amelogenin (amel), and the dentinogenic markers dentin sialophosphoprotein (DSSP) and dentin matrix acidic phosphoprotein 1 (DMP1), as well as the osteogenic markers osteocalcin (OC, BGLAP) and collagen type 1 (COL1A1). Whereas, only EMD had effect on alkaline phosphatase (ALP) mRNA expression, the stimulatory effect were verified by enhanced secretion of OC and COL1A from EMD treated cells, and increased ALP activity in cell culture medium after EMD treatment. Increased levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant proteins (MCP-1) in the cell culture medium were also found. Consequently, the suggested effect of EMD is to promote differentiation of pulp cells and increases the potential for pulpal mineralization to favor reactive dentine formation.

Revisiting ameloblastin; addressing the EMT-ECM axis above and beyond oral biology.

Ameloblastin (AMBN) is best characterized for its role in dental enamel formation, regulating cell differentiation and mineralization, and cell matrix adhesion. However, AMBN has also been detected in mesenchymal stem cells in addition to bone, blood, and adipose tissue. Using immunofluorescence in a pilot scheme, we identified that AMBN is expressed in different parts of the gastrointestinal (GI) tract. AMBN mRNA and protein detection in several tissues along the length of the GI tract suggests a role for AMBN in the structure and tissue integrity of the extracellular matrix (ECM). Intracellular AMBN expression in subsets of cells indicates a potential alternative role in signaling processes. Of note, our previous functional AMBN promoter analyses had shown that it contains epithelial-mesenchymal transition (EMT) regulatory elements. ΑΜΒΝ is herein presented as a paradigm shift of the possible associations and the spatiotemporal regulation of the ECM regulating the EMT and vice versa, using the example of AMBN expression beyond oral biology.

Early evolution of enamel matrix proteins is reflected by pleiotropy of physiological functions.

Highly specialized enamel matrix proteins (EMPs) are predominantly expressed in odontogenic tissues and diverged from common ancestral gene. They are crucial for the maturation of enamel and its extreme complexity in multiple independent lineages. However, divergence of EMPs occured already before the true enamel evolved and their conservancy in toothless species suggests that non-canonical functions are still under natural selection. To elucidate this hypothesis, we carried out an unbiased, comprehensive phenotyping and employed data from the International Mouse Phenotyping Consortium to show functional pleiotropy of amelogenin, ameloblastin, amelotin, and enamelin, genes, i.e. in sensory function, skeletal morphology, cardiovascular function, metabolism, immune system screen, behavior, reproduction, and respiratory function. Mice in all KO mutant lines, i.e. amelogenin KO, ameloblastin KO, amelotin KO, and enamelin KO, as well as mice from the lineage with monomeric form of ameloblastin were affected in multiple physiological systems. Evolutionary conserved motifs and functional pleiotropy support the hypothesis of role of EMPs as general physiological regulators. These findings illustrate how their non-canonical function can still effect the fitness of modern species by an example of influence of amelogenin and ameloblastin on the bone physiology.

Decreased bone mineral density and reduced bone quality in H(+) /K(+) ATPase beta-subunit deficient mice.

Proton pump inhibitors (PPIs) are widely used against gastroesophageal reflux disease. Recent epidemiological studies suggest that PPI users have an increased risk of fractures, but a causal relationship has been questioned. We have therefore investigated the skeletal phenotype in H(+) /K(+) ATPase beta-subunit knockout (KO) female mice. Skeletal parameters were determined in 6- and 20-month-old KO mice and in wild-type controls (WT). Whole body bone mineral density (BMD) and bone mineral content (BMC) were measured by dual energy X-ray absorptiometry (DXA). Femurs were examined with µCT analyses and break force were examined by a three-point bending test. Plasma levels of gastrin, RANKL, OPG, osteocalcin, leptin, and PTH were analyzed. KO mice had lower whole body BMC at 6 months (0.53 vs. 0.59 g, P = 0.035) and at 20 months (0.49 vs. 0.74 g, P < 0.01) compared to WT as well as lower BMD at 6 months (0.068 vs. 0.072 g/cm(2) , P = 0.026) and 20 months (0.067 vs. 0.077 g/cm(2) , P < 0.01). Mechanical strength was lower in KO mice at the age of 20 months (6.7 vs. 17.9 N, P < 0.01). Cortical thickness at 20 months and trabecular bone volume% at 6 months were significantly reduced in KO mice. Plasma OPG/RANKL ratio and PTH was increased in KO mice compared to controls. H(+) /K(+) ATPase beta subunit KO mice had decreased BMC and BMD, reduced cortical thickness and inferior mechanical bone strength. Whereas the mechanism is uncertain, these findings suggest a causal relationship between long-term PPI use and an increased risk of fractures.

Human monocyte responses to lipopolysaccharide and 9-cis retinoic acid after laparoscopic surgery for colon cancer.

Surgery, even modern minimal invasive laparoscopic surgery, induces an initial inflammatory and acute phase response which is followed by a period of immunosuppression rendering surgical patients more susceptible to infection. Here, we aimed to study changes in monocyte inflammatory responses and inflammatory modulation mechanisms following laparoscopic colorectal surgery for colon cancer. Blood samples were collected from 19 colon cancer patients before, directly after and daily for 3 days following surgery. Blood cells were exposed ex vivo to bacterial lipopolysaccharide (LPS) or the inflammatory modulator 9-cis retinoic acid (9cisRA). In blood samples taken prior to surgery, we found significant pro-inflammatory responses to LPS, indicating classical monocyte activation. Directly after surgery, LPS induced significantly less early pro-inflammatory cytokines and monocyte/granulocyte-attracting chemokines. The LPS-mediated release of interleukin (IL)-1ß was still significantly attenuated 3 days after surgery. In patient monocytes collected after surgery, we found increased levels of suppressors of cytokine signaling (SOCS)1 and SOCS3 mRNA, reported to be associated with polarization towards resolving macrophages. The retinoic acid isomer 9cisRA, reported to attenuate LPS-mediated inflammatory responses and alter chemokine responses in cultured monocytes, had a similar effect in patient blood. Three days after surgery, 9cisRA still attenuated pro-inflammatory responses, but the induction of monocyte chemoattractive protein (MCP)-1/CCL2 mRNA in monocytes was reduced. This study indicates changes in monocyte responses that last for at least 3 days after laparoscopic surgery.