Apigenin and Rutaecarpine reduce the burden of cellular senescence in bone marrow stromal stem cells.

Introduction: Osteoporosis is a systemic age-related disease characterized by reduced bone mass and microstructure deterioration, leading to increased risk of bone fragility fractures. Osteoporosis is a worldwide major health care problem and there is a need for preventive approaches. Methods and results: Apigenin and Rutaecarpine are plant-derived antioxidants identified through functional screen of a natural product library (143 compounds) as enhancers of osteoblastic differentiation of human bone marrow stromal stem cells (hBMSCs). Global gene expression profiling and Western blot analysis revealed activation of several intra-cellular signaling pathways including focal adhesion kinase (FAK) and TGFß. Pharmacological inhibition of FAK using PF-573228 (5 µM) and TGFß using SB505124 (1µM), diminished Apigenin- and Rutaecarpine-induced osteoblast differentiation. In vitro treatment with Apigenin and Rutaecarpine, of primary hBMSCs obtained from elderly female patients enhanced osteoblast differentiation compared with primary hBMSCs obtained from young female donors. Ex-vivo treatment with Apigenin and Rutaecarpine of organotypic embryonic chick-femur culture significantly increased bone volume and cortical thickness compared to control as estimated by µCT-scanning. Discussion: Our data revealed that Apigenin and Rutaecarpine enhance osteoblastic differentiation, bone formation, and reduce the age-related effects of hBMSCs. Therefore, Apigenin and Rutaecarpine cellular treatment represent a potential strategy for maintaining hBMSCs health during aging and osteoporosis.

KIAA1199 deficiency enhances skeletal stem cell differentiation to osteoblasts and promotes bone regeneration.

Upon transplantation, skeletal stem cells (also known as bone marrow stromal or mesenchymal stem cells) can regulate bone regeneration by producing secreted factors. Here, we identify KIAA1199 as a bone marrow stromal cell-secreted factor in vitro and in vivo. KIAA1199 plasma levels of patients positively correlate with osteoporotic fracture risk and expression levels of KIAA1199 in patient bone marrow stromal cells negatively correlates with their osteogenic differentiation potential. KIAA1199-deficient bone marrow stromal cells exhibit enhanced osteoblast differentiation in vitro and ectopic bone formation in vivo. Consistently, KIAA1199 knockout mice display increased bone mass and biomechanical strength, as well as an increased bone formation rate. They also exhibit accelerated healing of surgically generated bone defects and are protected from ovariectomy-induced bone loss. Mechanistically, KIAA1199 regulates osteogenesis by inhibiting the production of osteopontin by osteoblasts, via integrin-mediated AKT and ERK-MAPK intracellular signaling. Thus, KIAA1199 is a regulator of osteoblast differentiation and bone regeneration and could be targeted for the treatment or management of low bone mass conditions.

Impaired Bone Fracture Healing in Type 2 Diabetes Is Caused by Defective Functions of Skeletal Progenitor Cells.

The mechanisms of obesity and type 2 diabetes (T2D)-associated impaired fracture healing are poorly studied. In a murine model of T2D reflecting both hyperinsulinemia induced by high-fat diet and insulinopenia induced by treatment with streptozotocin, we examined bone healing in a tibia cortical bone defect. A delayed bone healing was observed during hyperinsulinemia as newly formed bone was reduced by -28.4 ± 7.7% and was associated with accumulation of marrow adipocytes at the defect site +124.06 ± 38.71%, and increased density of SCA1+ (+74.99 ± 29.19%) but not Runx2+ osteoprogenitor cells. We also observed increased in reactive oxygen species production (+101.82 ± 33.05%), senescence gene signature (≈106.66 ± 34.03%), and LAMIN B1- senescent cell density (+225.18 ± 43.15%), suggesting accelerated senescence phenotype. During insulinopenia, a more pronounced delayed bone healing was observed with decreased newly formed bone to -34.9 ± 6.2% which was inversely correlated with glucose levels (R2 = 0.48, P < .004) and callus adipose tissue area (R2 = .3711, P < .01). Finally, to investigate the relevance to human physiology, we observed that sera from obese and T2D subjects had disease state-specific inhibitory effects on osteoblast-related gene signatures in human bone marrow stromal cells which resulted in inhibition of osteoblast and enhanced adipocyte differentiation. Our data demonstrate that T2D exerts negative effects on bone healing through inhibition of osteoblast differentiation of skeletal stem cells and induction of accelerated bone senescence and that the hyperglycemia per se and not just insulin levels is detrimental for bone healing.

Protein Expression of AEBP1, MCM4, and FABP4 Differentiate Osteogenic, Adipogenic, and Mesenchymal Stromal Stem Cells.

Mesenchymal stem cells (MSCs) gain an increasing focus in the field of regenerative medicine due to their differentiation abilities into chondrocytes, adipocytes, and osteoblastic cells. However, it is apparent that the transformation processes are extremely complex and cause cellular heterogeneity. The study aimed to characterize differences between MSCs and cells after adipogenic (AD) or osteoblastic (OB) differentiation at the proteome level. Comparative proteomic profiling was performed using tandem mass spectrometry in data-independent acquisition mode. Proteins were quantified by deep neural networks in library-free mode and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. We analyzed 4108 proteins across all samples, which revealed a distinct clustering between MSCs and cell differentiation states. Protein expression profiling identified activation of the Peroxisome proliferator-activated receptors (PPARs) signaling pathway after AD. In addition, two distinct protein marker panels could be defined for osteoblastic and adipocytic cell lineages. Hereby, overexpression of AEBP1 and MCM4 for OB as well as of FABP4 for AD was detected as the most promising molecular markers. Combination of deep neural network and machine-learning algorithms with data-independent mass spectrometry distinguish MSCs and cell lineages after adipogenic or osteoblastic differentiation. We identified specific proteins as the molecular basis for bone formation, which could be used for regenerative medicine in the future.

Identification of a clinical signature predictive of differentiation fate of human bone marrow stromal cells.

BACKGROUND: Transplantation of human bone marrow stromal cells (hBMSCs) is a promising therapy for bone regeneration due to their ability to differentiate into bone forming osteoblastic cells. However, transplanted hBMSCs exhibit variable capacity for bone formation resulting in inconsistent clinical outcome. The aim of the study was to identify a set of donor- and cell-related characteristics that detect hBMSCs with optimal osteoblastic differentiation capacity. METHODS: We collected hBMSCs from 58 patients undergoing surgery for bone fracture. Clinical profile of the donors and in vitro characteristics of cultured hBMSCs were included in uni- and multivariable analysis to determine their predictive value for osteoblastic versus adipocytic differentiation capacity assessed by quantification of mineralized matrix and mature adipocyte formation, respectively. RESULTS: We identified a signature that explained > 50% of variation in osteoblastic differentiation outcome which included the following positive predictors: donor sex (male), absence of osteoporosis diagnosis, intake of vitamin D supplements, higher fraction of CD146+, and alkaline phosphate (ALP+) cells. With the exception of vitamin D and ALP+ cells, these variables were also negative predictors of adipocytic differentiation. CONCLUSIONS: Using a combination of clinical and cellular criteria, it is possible to predict differentiation outcome of hBMSCs. This signature may be helpful in selecting donor cells in clinical trials of bone regeneration.

Elevation of Pro-Inflammatory Cytokine Levels Following Intra-Articular Fractures-A Systematic Review.

Introduction: Intra-articular fractures are a major cause of post-traumatic osteoarthritis (PTOA). Despite adequate surgical treatment, the long-term risk for PTOA is high. Previous studies reported that joint injuries initiate an inflammatory cascade characterized by an elevation of synovial pro-inflammatory cytokines, which can lead to cartilage degradation and PTOA development. This review summarizes the literature on the post-injury regulation of pro-inflammatory cytokines and the markers of cartilage destruction in patients suffering from intra-articular fractures. Methods: We searched Medline, Embase, and Cochrane databases (1960-February 2020) and included studies that were performed on human participants, and we included control groups. Two investigators assessed the quality of the included studies using Covidence and the Newcastle-Ottawa Scale. Results: Based on the surveyed literature, several synovial pro-inflammatory cytokines, including interleukins (IL)-1ß, IL-2, IL-6, IL-8, IL-12p70, interferon-y, and tumor necrosis factor-α, were significantly elevated in patients suffering from intra-articular fractures compared to the control groups. A simultaneous elevation of anti-inflammatory cytokines such as IL-10 and IL-1RA was also observed. In contrast, IL-13, CTX-II, and aggrecan concentrations did not differ significantly between the compared cohorts. Conclusions: Overall, intra-articular fractures are associated with an increase in inflammation-related synovial cytokines. However, more standardized studies which focus on the ratio of pro- and anti-inflammatory cytokines at different time points are needed.

In Situ Voltammetric Sensor of Potentially Bioavailable Inorganic Mercury in Marine Aquatic Systems Based on Gel-Integrated Nanostructured Gold-Based Microelectrode Arrays.

The development and field validation of newly designed nanostructured gold-plated gel-integrated microelectrode (Au-GIME) arrays applied to the direct in situ square wave anodic stripping voltammetry (SWASV) quantification of the potentially bioavailable inorganic mercury (Hg(II)) species in the coastal area are presented. The Au-GIME consists of arrays of 100-500 interconnected iridium (Ir)-based microdisks that are electroplated with renewable Au nanoparticles (AuNPs) or Au nanofilaments (AuNFs) and covered with an agarose gel. The gel protects the sensor surface from fouling and ensures that mass transport of analytes toward the sensor surface is by pure diffusion only and therefore independent of the ill-controlled convective conditions of the media. The responses of these sensors to direct SWASV measurements of inorganic Hg(II) at near-neutral pH were investigated first in synthetic media and then in UV-irradiated marine samples. The analytical responses were found to be correlated to the number of interconnected microelectrodes and the morphology of the nanostructured Au deposits and independent of the media composition for chloride concentration ≥0.2 M (salinity S ≥ 13) and pH ranging from 7 to 8.5. The AuNF-GIMEs have detection and quantification limits at a low pM level, fulfilling the requirement of sentinel tools for real-time monitoring of the dynamic fraction of Hg(II) in coastal area. The AuNF-GIMEs were incorporated in an in-house advanced multichannel sensing probe for remote in situ high-resolution trace metal monitoring. Field evaluation and validation were successfully performed as a part of a field study in Arcachon Bay (France), from which environmental data are presented. This work marks the first time that an autonomous electrochemical sensing probe successfully measures Hg(II) and its hourly temporal variation in situ without chemical modification of the sample.

Resveratrol inhibits adipocyte differentiation and cellular senescence of human bone marrow stromal stem cells.

Bone marrow adipose tissue (BMAT) is a unique adipose depot originating from bone marrow stromal stem cells (BMSCs) and regulates bone homeostasis and energy metabolism. An increased BMAT volume is observed in several conditions e.g. obesity, type 2 diabetes, osteoporosis and is known to be associated with bone fragility and increased risk for fracture. Therapeutic approaches to decrease the accumulation of BMAT are clinically relevant. In a screening experiment of natural compounds, we identified Resveratrol (RSV), a plant-derived antioxidant mediating biological effects via sirtuin- related mechanisms, to exert significant effects of BMAT formation. Thus, we examined in details the effects RSV on adipocytic and osteoblastic differentiation of tolermerized human BMSCs (hBMSC-TERT). RSV (1.0 µM) enhanced osteoblastic differentiation and inhibited adipocytic differentiation of hBMSC-TERT when compared with control and Sirtinol (Sirtuin inhibitor). Global gene expression profiling and western blot analysis revealed activation of a number of signaling pathways including focal adhesion kinase (FAK). Pharmacological inhibition of FAK using (PF-573228) and AKT inhibitor (LY-294002) (5µM), diminished RSV-induced osteoblast differentiation. In addition, RSV reduced the levels of senescence-associated secretory phenotype (SASP), gene markers associated with senescence (P53, P16, and P21), intracellular ROS levels and increased gene expression of enzymes protecting cells from oxidative damage (HMOX1 and SOD3). In vitro treatment of primary hBMSCs from aged patients characterized with high adipocytic and low osteoblastic differentiation ability with RSV, significantly enhanced osteoblast and decreased adipocyte formation when compared to hBMSCs from young donors. RSV targets hBMSCs and inhibits adipogenic differentiation and senescence-associated phenotype and thus a potential agent for treating conditions of increased BMAT formation.

Single-cell high-content imaging parameters predict functional phenotype of cultured human bone marrow stromal stem cells.

Cultured human bone marrow stromal (mesenchymal) stem cells (hBM-MSCs) are heterogenous cell populations exhibiting variable biological properties. Quantitative high-content imaging technology allows identification of morphological markers at a single cell resolution that are determinant for cellular functions. We determined the morphological characteristics of cultured primary hBM-MSCs and examined their predictive value for hBM-MSC functionality. BM-MSCs were isolated from 56 donors and characterized for their proliferative and differentiation potential. We correlated these data with cellular and nuclear morphological features determined by Operetta; a high-content imaging system. Cell area, cell geometry, and nucleus geometry of cultured hBM-MSCs exhibited significant correlation with expression of hBM-MSC membrane markers: ALP, CD146, and CD271. Proliferation capacity correlated negatively with cell and nucleus area and positively with cytoskeleton texture features. In addition, in vitro differentiation to osteoblasts as well as in vivo heterotopic bone formation was associated with decreased ratio of nucleus width to length. Multivariable analysis applying a stability selection procedure identified nuclear geometry and texture as predictors for hBM-MSCs differentiation potential to osteoblasts or adipocytes. Our data demonstrate that by employing a limited number of cell morphological characteristics, it is possible to predict the functional phenotype of cultured hBM-MSCs and thus can be used as a screening test for "quality" of hBM-MSCs prior their use in clinical protocols.

Biomimetic Planar Polymer Membranes Decorated with Enzymes as Functional Surfaces.

Functional surfaces were generated by a combination of enzymes with polymer membranes composed of an amphiphilic, asymmetric block copolymer poly(ethyleneglycol)- block-poly(γ-methyl-ε-caprolactone)- block-poly[(2-dimethylamino)ethylmethacrylate]. First, polymer films formed at the air-water interface were transferred in different sequences onto silica solid support using the Langmuir-Blodgett technique, generating homogeneous monolayers and bilayers. A detailed characterization of these films provided insight into their properties (film thickness, wettability, topography, and roughness). On the basis of these findings, the most promising membranes were selected for enzyme attachment. Functional surfaces were then generated by the adsorption of two model enzymes that can convert phenol and its derivatives (laccase and tyrosinase), well known as high-risk pollutants of drinking and natural water. Both enzymes preserved their activity upon immobilization with respect to their substrates. Depending on the properties of the polymer films, different degrees of enzymatic activity were observed: bilayers provided the best conditions in terms of both overall stability and enzymatic activity. The interaction between amphiphilic triblock copolymer films and enzymes is exploited to engineer "active surfaces" with specific functionalities and high efficacy resulting from the intrinsic activity of the biomolecules that is preserved by an appropriate synthetic environment.

Comparison of Regenerative Tissue Quality following Matrix-Associated Cell Implantation Using Amplified Chondrocytes Compared to Synovium-Derived Stem Cells in a Rabbit Model for Cartilage Lesions.

Known problems of the autologous chondrocyte implantation motivate the search for cellular alternatives. The aim of the study was to test the potential of synovium-derived stem cells (SMSC) to regenerate cartilage using a matrix-associated implantation. In an osteochondral defect model of the medial femoral condyle in a rabbit, a collagen membrane was seeded with either culture-expanded allogenic chondrocytes or SMSC and then transplanted into the lesion. A tailored piece synovium served as a control. Rabbit SMSC formed typical cartilage in vitro. Macroscopic evaluation of defect healing and the thickness of the regenerated tissue did not reveal a significant difference between the intervention groups. However, instantaneous and shear modulus, reflecting the biomechanical strength of the repair tissue, was superior in the implantation group using allogenic chondrocytes (p < 0.05). This correlated with a more chondrogenic structure and higher proteoglycan expression, resulting in a lower OARSI score (p < 0.05). The repair tissue of all groups expressed comparable amounts of the collagen types I, II, and X. Cartilage regeneration following matrix-associated implantation using allogenic undifferentiated synovium-derived stem cells in a defect model in rabbits showed similar macroscopic results and collagen composition compared to amplified chondrocytes; however, biomechanical characteristics and histological scoring were inferior.

SCHeMA EU Project Summer School Report (Bilbao June 16 - 17, 2016).

This conference report describes the training activities that took place in the frame of the Integrated in Situ Chemical MApping probe (SCHeMA) summer school organized from the 14th to the 16th of June 2016 in Bilbao (Spain).

Bile acid effects are mediated by ATP release and purinergic signalling in exocrine pancreatic cells.

BACKGROUND: In many cells, bile acids (BAs) have a multitude of effects, some of which may be mediated by specific receptors such the TGR5 or FXR receptors. In pancreas systemic BAs, as well as intra-ductal BAs from bile reflux, can affect pancreatic secretion. Extracellular ATP and purinergic signalling are other important regulators of similar secretory mechanisms in pancreas. The aim of our study was to elucidate whether there is interplay between ATP and BA signalling. RESULTS: Here we show that CDCA (chenodeoxycholic acid) caused fast and concentration-dependent ATP release from acini (AR42J) and duct cells (Capan-1). Taurine and glycine conjugated forms of CDCA had smaller effects on ATP release in Capan-1 cells. In duct monolayers, CDCA stimulated ATP release mainly from the luminal membrane; the releasing mechanisms involved both vesicular and non-vesicular secretion pathways. Duct cells were not depleted of intracellular ATP with CDCA, but acinar cells lost some ATP, as detected by several methods including ATP sensor AT1.03(YEMK). In duct cells, CDCA caused reversible increase in the intracellular Ca(2+) concentration [Ca(2 +)]i, which could be significantly inhibited by antagonists of purinergic receptors. The TGR5 receptor, expressed on the luminal side of pancreatic ducts, was not involved in ATP release and Ca(2+) signals, but could stimulate Na(+)/Ca(2+) exchange in some conditions. CONCLUSIONS: CDCA evokes significant ATP release that can stimulate purinergic receptors, which in turn increase [Ca(2+)]i. The TGR5 receptor is not involved in these processes but can play a protective role at high intracellular Ca(2+) conditions. We propose that purinergic signalling could be taken into consideration in other cells/organs, and thereby potentially explain some of the multifaceted effects of BAs.

Hybrid polymer-lipid films as platforms for directed membrane protein insertion.

Hybrids composed of amphiphilic block copolymers and lipids constitute a new generation of biological membrane-inspired materials. Hybrid membranes resulting from self-assembly of lipids and polymers represent adjustable models for interactions between artificial and natural membranes, which are of key importance, e.g., when developing systems for drug delivery. By combining poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) amphiphilic copolymers (PDMS-b-PMOXA) with various phospholipids, we obtained hybrid films with modulated properties and topology, based on phase separation, and the formation of distinct domains. By understanding the factors driving the phase separation in these hybrid lipid-polymer films, we were able to use them as platforms for directed insertion of membrane proteins. Tuning the composition of the polymer-lipids mixtures favored successful insertion of membrane proteins with desired topological distributions (in polymer or/and lipid regions). Controlled insertion and location of membrane proteins in hybrid films make these hybrids ideal candidates for numerous applications where specific spatial functionality is required.

"Active surfaces" formed by immobilization of enzymes on solid-supported polymer membranes.

In various domains ranging from catalysis to medical and environmental sciences, there is currently much focus on the design of surfaces that present active compounds at the interface with their environments. Here, we describe the design of "active surfaces" based on solid-supported monolayers of asymmetric triblock copolymers, which serve as templates for the attachment of enzymes. A group of poly(ethylene glycol)-block-poly(γ-methyl-ε-caprolactone)-block-poly[(2-dimethylamino) ethyl methacrylate] amphiphilic copolymers, with different hydrophilic and hydrophobic domains (PEG45-b-PMCLx-b-PDMAEMAy) was selected to generate solid-supported polymer membranes. The behavior of the copolymers in terms of their molecular arrangements at the air-water interface was established by a combination of Langmuir isotherms and Brewster angle microscopy. Uniform thin layers of copolymers were obtained by transferring films onto silica solid supports at optimal surface pressure. These solid-supported polymer membranes were characterized by assessing various properties, such as monolayer thickness, hydrophilic/hydrophobic balance, topography, and roughness. Laccase, used as an enzyme model, was successfully attached to copolymer membranes by stable interactions as followed by quartz crystal microbalance with dissipation measurements, and its activity was preserved, as indicated by activity assays. The interaction between the amphiphilic triblock copolymer films and immobilized enzymes represents a straightforward approach to engineer "active surfaces", with biomolecules playing the active role by their intrinsic bioactivity.

Functional surface engineering by nucleotide-modulated potassium channel insertion into polymer membranes attached to solid supports.

Planar solid-supported membranes based on amphiphilic block copolymers represent promising systems for the artificial creation of structural surfaces. Here we introduce a method for engineering functional planar solid-supported membranes through insertion of active biomolecules. We show that membranes based on poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) amphiphilic diblock copolymers, which mimic natural membranes, are suitable for hosting biomolecules. Our strategy allows preparation of large-area, well-ordered polymer bilayers via Langmuir-Blodgett and Langmuir-Schaefer transfers, and insertion of biomolecules by using Bio-Beads. We demonstrate that a model membrane protein, the potassium channel from the bacterium Mesorhizobium loti, remains functional after insertion into the planar solid-supported polymer membrane. This approach can be easily extended to generate a platform of functional solid-supported membranes by insertion of different hydrophobic biomolecules, and employing different types of solid substrates for desired applications.

A general strategy for creating self-defending surfaces for controlled drug production for long periods of time.

Infections associated with bacterial adhesion and subsequent biofilm formation constitute a grave medical issue for which conventional antibiotic therapies remain ineffective. Here, we introduce a new strategy employing nanotechnology to create smart surfaces with self-defending properties that result in controlled drug production and controlled release for long periods of time. Self-defending surfaces on solid supports are prepared by immobilizing polymer nanoreactors containing an encapsulated biocatalyst that can convert non-antibiotic substrates to an abiotic drug. For medical applications and biosensing, the immobilization method must fulfill specific criteria, and these were achieved by an immobilization strategy based on Schiff base formation between aldehyde groups on the outer surface of nanoreactors and amino groups on the solid support surface, followed by reductive amination. The resulting self-defending surfaces allow control of drug production at a specific rate for a specific period of time by adding predetermined amounts of substrate to the outer medium, minimization of dosages and therefore systemic toxicity, and limitation of the immune response. Such self-defending surfaces producing drugs offer a versatile strategy for the development of smart surfaces with improved stability and efficacy (by changing the biocatalyst) to serve as biosensors, antifouling surfaces, or smart packages.

Maf1-mediated repression of RNA polymerase III transcription inhibits tRNA degradation via RTD pathway.

tRNA precursors, which are transcribed by RNA polymerase III, undergo end-maturation, splicing, and base modifications. Hypomodified tRNAs, such as tRNA(Val(AAC)), lacking 7-methylguanosine and 5-methylcytidine modifications, are subject to degradation by a rapid tRNA decay pathway. Here we searched for genes which, when overexpressed, restored stability of tRNA(Val(AAC)) molecules in a modification-deficient trm4Δtrm8Δ mutant. We identified TEF1 and VAS1, encoding elongation factor eEF1A and valyl-tRNA synthetase respectively, which likely protect hypomodified tRNA(Val(AAC)) by direct interactions. We also identified MAF1 whose product is a general negative regulator of RNA polymerase III. Expression of a Maf1-7A mutant that constitutively repressed RNA polymerase III transcription resulted in increased stability of hypomodified tRNA(Val(AAC)). Strikingly, inhibition of tRNA transcription in a Maf1-independent manner, either by point mutation in RNA polymerase III subunit Rpc128 or decreased expression of Rpc17 subunit, also suppressed the turnover of the hypomodified tRNA(Val(AAC)). These results support a model where inhibition of tRNA transcription leads to stabilization of hypomodified tRNA(Val(AAC)) due to more efficient protection by tRNA-interacting proteins.