Gouqi-derived nanovesicles (GqDNVs) inhibited dexamethasone-induced muscle atrophy associating with AMPK/SIRT1/PGC1α signaling pathway.

With the increasing trend of global aging, sarcopenia has become a significant public health issue. Goji berry, also known as "Gou qi zi" in China, is a traditional Chinese herb that can enhance the structure and function of muscles and bones. Otherwise, previous excellent publications illustrated that plant-derived exosome-like nanoparticles can exert good bioactive functions in different aging or disease models. Thus, we issued the hypothesis that Gouqi-derived nanovesicles (GqDNVs) may also have the ability to improve skeletal muscle health, though the effect and its mechanism need to be explored. Hence, we have extracted GqDNVs from fresh berries of Lycium barbarum L. (goji) and found that the contents of GqDNVs are rich in saccharides and lipids. Based on the pathway annotations and predictions in non-targeted metabolome analysis, GqDNVs are tightly associated with the pathways in metabolism. In muscle atrophy model mice, intramuscular injection of GqDNVs improves the cross-sectional area of the quadriceps muscle, grip strength and the AMPK/SIRT1/PGC1α pathway expression. After separately inhibiting AMPK or PGC1α in C2C12 cells with dexamethasone administration, we have found that the activated AMPK plays the chief role in improving cell proliferation induced by GqDNVs. Furthermore, the energy-targeted metabolome analysis in the quadriceps muscle demonstrates that the GqDNVs up-regulate the metabolism of amino sugar and nucleotide sugar, autophagy and oxidative phosphorylation process, which indicates the activation of muscle regeneration. Besides, the Spearman rank analysis shows close associations between the quality and function of skeletal muscle, metabolites and expression levels of AMPK and SIRT1. In this study, we provide a new founding that GqDNVs can improve the quality and function of skeletal muscle accompanying the activated AMPK/SIRT1/PGC1α signaling pathway. Therefore, GqDNVs have the effect of anti-aging skeletal muscle as a potential adjuvant or complementary method or idea in future therapy and research.

Atorvastatin exerts a preventive effect against steroid-induced necrosis of the femoral head by modulating Wnt5a release.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a prevalent form of osteonecrosis in young individuals. More efficacious clinical strategies must be used to prevent and treat this condition. One of the mechanisms through which SONFH operates is the disruption of normal differentiation in bone marrow adipocytes and osteoblasts due to prolonged and extensive use of glucocorticoids (GCs). In vitro, it was observed that atorvastatin (ATO) effectively suppressed the impact of dexamethasone (DEX) on bone marrow mesenchymal stem cells (BMSCs), specifically by augmenting their lipogenic differentiation while impeding their osteogenic differentiation. To investigate the underlying mechanisms further, we conducted transcriptome sequencing of BMSCs subjected to different treatments, leading to the identification of Wnt5a as a crucial gene regulated by ATO. The analyses showed that ATO exhibited the ability to enhance the expression of Wnt5a and modulate the MAPK pathway while regulating the Wnt canonical signaling pathway via the WNT5A/LRP5 pathway. Our experimental findings provide further evidence that the combined treatment of ATO and DEX effectively mitigates the effects of DEX, resulting in the upregulation of osteogenic genes (Runx2, Alpl, Tnfrsf11b, Ctnnb1, Col1a) and the downregulation of adipogenic genes (Pparg, Cebpb, Lpl), meanwhile leading to the upregulation of Wnt5a expression. So, this study offers valuable insights into the potential mechanism by which ATO can be utilized in the prevention of SONFH, thereby holding significant implications for the prevention and treatment of SONFH in clinical settings.

Cilostazol Stimulates Angiogenesis and Accelerates Fracture Healing in Aged Male and Female Mice by Increasing the Expression of PI3K and RUNX2.

Fracture healing in the aged is associated with a reduced healing capacity, which often results in delayed healing or non-union formation. Many factors may contribute to this deterioration of bone regeneration, including a reduced 'angiogenic trauma response'. The phosphodiesterase-3 (PDE-3) inhibitor cilostazol has been shown to exert pro-angiogenic and pro-osteogenic effects in preclinical studies. Therefore, we herein analyzed in a stable closed femoral fracture model whether this compound also promotes fracture healing in aged mice. Forty-two aged CD-1 mice (age: 16-18 months) were daily treated with 30 mg/kg body weight cilostazol (n = 21) or vehicle (control, n = 21) by oral gavage. At 2 and 5 weeks after fracture, the femora were analyzed by X-ray, biomechanics, micro-computed tomography (µCT), histology, immunohistochemistry, and Western blotting. These analyses revealed a significantly increased bending stiffness at 2 weeks (2.2 ± 0.4 vs. 4.3 ± 0.7 N/mm) and an enhanced bone formation at 5 weeks (4.4 ± 0.7 vs. 9.1 ± 0.7 mm3) in cilostazol-treated mice when compared to controls. This was associated with a higher number of newly formed CD31-positive microvessels (3.3 ± 0.9 vs. 5.5 ± 0.7 microvessels/HPF) as well as an elevated expression of phosphoinositide-3-kinase (PI3K) (3.6 ± 0.8 vs. 17.4 ± 5.5-pixel intensity × 104) and runt-related transcription factor (RUNX)2 (6.4 ± 1.2 vs. 18.2 ± 2.7-pixel intensity × 104) within the callus tissue. These findings indicate that cilostazol accelerates fracture healing in aged mice by stimulating angiogenesis and the expression of PI3K and RUNX2. Hence, cilostazol may represent a promising compound to promote bone regeneration in geriatric patients.

Human milk extracellular vesicles enhance muscle growth and physical performance of immature mice associating with Akt/mTOR/p70s6k signaling pathway.

Extracellular vesicles (EVs) play an important role in human and bovine milk composition. According to excellent published studies, it also exerts various functions in the gut, bone, or immune system. However, the effects of milk-derived EVs on skeletal muscle growth and performance have yet to be fully explored. Firstly, the current study examined the amino acids profile in human milk EVs (HME) and bovine milk EVs (BME) using targeted metabolomics. Secondly, HME and BME were injected in the quadriceps of mice for four weeks (1 time/3 days). Then, related muscle performance, muscle growth markers/pathways, and amino acids profile were detected or measured by grip strength analysis, rotarod performance testing, Jenner-Giemsa/H&E staining, Western blotting, and targeted metabolomics, respectively. Finally, HME and BME were co-cultured with C2C12 cells to detect the above-related indexes and further testify relative phenomena. Our findings mainly demonstrated that HME and BME significantly increase the diameter of C2C12 myotubes. HME treatment demonstrates higher exercise performance and muscle fiber densities than BME treatment. Besides, after KEGG and correlation analyses with biological function after HME and BME treatment, results showed L-Ornithine acts as a "notable marker" after HME treatment to affect mouse skeletal muscle growth or functions. Otherwise, L-Ornithine also significantly positively correlates with the activation of the AKT/mTOR pathway and myogenic regulatory factors (MRFs) and can also be observed in muscle and C2C12 cells after HME treatment. Overall, our study not only provides a novel result for the amino acid composition of HME and BME, but the current study also indicates the advantage of human milk on skeletal muscle growth and performance.

The circadian rhythm gene Bmal1 ameliorates acute deoxynivalenol-induced liver damage.

Deoxynivalenol (DON) is widely emerging in various grain crops, milk, and wine products, which can trigger different toxic effects on humans and animals by inhalation or ingestion. It also imposes a considerable financial loss on the agriculture and food industry each year. Previous studies have reported acute and chronic toxicity of DON in liver, and liver is not only the main detoxification organ for DON but also the circadian clock oscillator directly or indirectly regulates critical physiologically hepatic functions under different physiological and pathological conditions. However, researches on the association of circadian rhythm in DON-induced liver damage are limited. In the present study, mice were divided into four groups (CON, DON, Bmal1OE, and Bmal1OE + DON) and AAV8 was used to activate (Bmal1) expression in liver. Then mice were gavaged with 5 mg/kg bw/day DON or saline at different time points (ZT24 = 0, 4, 8, 12, 16, and 20 h) in 1 day and were sacrificed 30 min after oral gavage. The inflammatory cytokines, signal transducers, and activators of transcription Janus kinase/signal transducers and activator of transcription 3 (JAKs/STAT3) pathway and bile acids levels were detected by enzyme-linked immunosorbent assay (ELISA), western blotting, and target metabolomics, respectively. The DON group showed significantly elevated interleukin-1ß (IL-1ß), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) levels (P < 0.05 for both) and impaired liver function with rhythm disturbances compared to the CON and Bmal1OE groups. At the molecular level, expressions of some circadian clock proteins were significantly downregulated (P < 0.05 for both) and JAKs/STAT3 pathway was activated during DON exposure, accompanied by indicated circadian rhythm disturbance and inflammatory damage. Importantly, Bmal1 overexpression attenuated DON-induced liver damage, while related hepatic bile acids such as cholic acid (CA) showed a decreasing trend in the DON group compared with the CON group. Our study demonstrates a novel finding that Bmal1 plays a critical role in attenuating liver damage by inhibiting inflammatory levels and maintaining bile acids levels under the DON condition. Therefore, Bmal1 may also be a potential molecular target for reducing the hepatotoxic effects of DON in future studies.

NET Formation Was Reduced via Exposure to Extremely Low-Frequency Pulsed Electromagnetic Fields.

Fracture-healing is a highly complex and timely orchestrated process. Non-healing fractures are still a major clinical problem and treatment remains difficult. A 16 Hz extremely low-frequency pulsed electromagnetic field (ELF-PEMF) was identified as non-invasive adjunct therapy supporting bone-healing by inducing reactive oxygen species (ROS) and Ca2+-influx. However, ROS and Ca2+-influx may stimulate neutrophils, the first cells arriving at the wounded site, to excessively form neutrophil extracellular traps (NETs), which negatively affects the healing process. Thus, this study aimed to evaluate the effect of this 16 Hz ELF-PEMF on NET formation. Neutrophils were isolated from healthy volunteers and exposed to different NET-stimuli and the 16 Hz ELF-PEMF. NETs were quantified using Sytox Green Assay and immunofluorescence, Ca2+-influx and ROS with fluorescence probes. In contrast to mesenchymal cells, ELF-PEMF exposure did not induce ROS and Ca2+-influx in neutrophils. ELF-PEMF exposure did not result in basal or enhanced PMA-induced NET formation but did reduce the amount of DNA released. Similarly, NET formation induced by LPS and H2O2 was reduced through exposure to ELF-PEMF. As ELF-PEMF exposure did not induce NET release or negatively affect neutrophils, the ELF-PEMF exposure can be started immediately after fracture treatment.

The vascularization paradox of non-union formation.

Despite major research efforts to elucidate mechanisms of non-union formation, failed fracture healing remains a common complication in orthopedic surgery. Adequate vascularization has been recognized as a crucial factor for successful bone regeneration, as newly formed microvessels guarantee the supply of the callus tissue with vital oxygen, nutrients, and growth factors. Accordingly, a vast number of preclinical studies have focused on the development of vascularization strategies to stimulate fracture repair. However, recent evidence suggests that stimulation of blood vessel formation is an oversimplified approach to support bone regeneration. This review discusses the role of vascularization during bone regeneration and delineates a phenomenon, for which we coin the term "the vascularization paradox of non-union-formation". This view is based on the results of a variety of experimental studies that suggest that the callus tissue of non-unions is indeed densely vascularized and that pro-angiogenic mediators, such as vascular endothelial growth factor, are sufficiently expressed at the facture site. By gaining further insights into the molecular and cellular basis of non-union vascularization, it may be possible to develop more optimized treatment approaches or even prevent the non-union formation in the future.

In vitro modeling of liver fibrosis in 3D microtissues using scalable micropatterning system.

Liver fibrosis is the late consequence of chronic liver inflammation which could eventually lead to cirrhosis, and liver failure. Among various etiological factors, activated hepatic stellate cells (aHSCs) are the major players in liver fibrosis. To date, various in vitro liver fibrosis models have been introduced to address biological and medical questions. Availability of traditional in vitro models could not fully recapitulate complicated pathology of liver fibrosis. The purpose of this study was to develop a simple and robust model to investigate the role of aHSCs on the progression of epithelial to mesenchymal transition (EMT) in hepatocytes during liver fibrogenesis. Therefore, we applied a micropatterning approach to generate 3D co-culture microtissues consisted of HepaRG and human umbilical cord endothelial cells (HUVEC) which co-cultured with inactivated LX-2 cells or activated LX-2 cells, respectively, as normal or fibrotic liver models in vitro. The result indicated that the activated LX-2 cells could induce EMT in HepaRG cells through activation of TGF-ß/SMAD signaling pathway. Besides, in the fibrotic microtissue, physiologic function of HepaRG cells attenuated compared to the control group, e.g., metabolic activity and albumin secretion. Moreover, our results showed that after treatment with Galunisertib, the fibrogenic properties decreased, in the term of gene and protein expression. In conclusion, it is proposed that aHSCs could lead to EMT in hepatocytes during liver fibrogenesis. Furthermore, the scalable micropatterning approach could provide enough required liver microtissues to prosper our understanding of the mechanisms involved in the progression of liver fibrosis as well as high throughput (HT) drug screening.

Optimisation of the HepaRG cell line model for drug toxicity studies using two different cultivation conditions: advantages and limitations.

The HepaRG cell line represents a successful model for hepatotoxicity studies. These cells are of human origin and are differentiated in vitro into mature and functional hepatocyte-like cells. The objective of this research was to compare two different culture protocols, Sison-Young et al. 2017 (hereinafter referred as Sison) and Gripon et al. 2002 (hereinafter referred as Biopredic) for HepaRG cells in order to optimise this model for drug metabolism and toxicity testing studies. HepaRG cells obtained from the same batch were cultured according to the described protocols. Using both protocols, differentiated HepaRG cells retained their drug metabolic capacity (major phase I/II enzymes) and transporters, as well as their morphological characteristics. Morphologically, HepaRG cells cultured after the Biopredic protocol formed more apical membranes and small ductular-like structures, than those cultivated using the Sison protocol. Also, the efflux activity of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1) as well as the activity of uridine-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) were significantly reduced in HepaRG cultured using the Sison protocol. Applying well-established drug cocktails to measure cytochrome P450 (CYPs) activity, we found that production of the corresponding metabolites was hampered in Sison-cultured HepaRG cells, indicating that the activity of CYP1A2, CYP2C9, CYP3A4, CYP2B6 and CYP2C19 was significantly reduced. Moreover, HepaRG sensitivity to well-known drugs, namely diclofenac, amiodarone, imipramine and paracetamol, revealed some differences between the two culture protocols. Furthermore, the HepaRG cells can be maintained with higher viability and sufficient CYPs activity and expression (i.e. CYP3A4, CYP1A2 and CYP2B6) as well as liver-specific functions, using Biopredic compared with the Sison culture protocol. These maintained liver-specific functions might be dependent on the prolongation of the culture conditions in the case of the Biopredic protocol. In conclusion, based on the metabolic activity of HepaRG cells using the standard protocol from Biopredic, we believe that this protocol is optimal for investigating drug metabolism and pharmacokinetic screening studies.

Febrile-Range Hyperthermia Can Prevent Toxic Effects of Neutrophil Extracellular Traps on Mesenchymal Stem Cells.

Fracture healing is characterized by an inflammatory phase directly after fracture which has a strong impact on the healing outcome. Neutrophils are strong contributors here and can release neutrophil extracellular traps (NETs). NETs are found after trauma, originally thought to capture pathogens. However, they can lead to tissue damage and impede wound healing processes. Their role in fracture healing remains unclear. In this study, the effect of isolated NETs on the function of bone-forming mesenchymal stem cells (SCP-1 cells) was examined. NETs were isolated from stimulated healthy neutrophils and viability, migration, and differentiation of SCP-1 cells were analyzed after the addition of NETs. NETs severely impaired the viability of SCP-1 cells, induced necrosis and already nontoxic concentrations reduced migration significantly. Short-term incubation with NETs had a persistent negative effect on osteogenic differentiation, as measured by AP activity and matrix formation. The addition of DNase or protease inhibitors failed to reverse the negative effect of NETs, whereas a short febrile-range temperature treatment successfully reduced the toxicity and membrane destruction. Thus, the possible modification of the negative effects of NETs in fracture hematomas could be an interesting new target to improve bone healing, particularly in patients with chronic diseases such as diabetes.

Development of an ischemic fracture healing model in mice.

BACKGROUND AND PURPOSE: In fracture healing, ischemia caused by vascular injuries, chronic vascular diseases, and metabolic comorbidities is one of the major risk factors for delayed union and non-union formation. To gain novel insights into the molecular and cellular pathology of ischemic fracture healing, appropriate animal models are needed. Murine models are of particular interest, as they allow to study the molecular aspects of fracture healing due to the availability of both a large number of murine antibodies and gene-targeted animals. Thus, we present the development of an ischemic fracture healing model in mice. MATERIAL AND METHODS: After inducing a mild ischemia by double ligature of the deep femoral artery in CD-1 mice, the ipsilateral femur was fractured by a 3-point bending device and stabilized by screw osteosynthesis. In control animals, the femur was fractured and stabilized without the induction of ischemia. The femora were analyzed at 2 and 5 weeks after fracture healing by means of radiology, biomechanics, histology, and histomorphometry. RESULTS: The surgically induced ischemia delayed and impaired the process of fracture healing. This was indicated by a lower Goldberg score, decreased bending stiffness, and reduced bone callus formation in the ischemic animals when compared with the controls. INTERPRETATION: We introduce a novel ischemic femoral fracture healing model in mice, which is characterized by delayed bone healing. In future, the use of this model may allow both the elucidation of the molecular aspects of ischemic fracture healing and the study of novel treatment strategies.

A simple method for decellularizing a cell-derived matrix for bone cell cultivation and differentiation.

The extracellular matrix regulates cell survival, proliferation, and differentiation. In vitro two-dimensional cell experiments are typically performed on a plastic plate or a substrate of a single extracellular matrix constituent such as collagen or calcium phosphate. As these approaches do not include extracellular matrix proteins or growth factors, they fail to mimic a complex cell microenvironment. The cell-derived matrix is an alternative platform for better representing the in vivo microenvironment in vitro. Standard decellularization of a cell-derived matrix is achieved by combining chemical and physical methods. In this study, we compared the decellularization efficacy of several methods: ammonium hydroxide, sodium dodecyl sulfate (SDS), or Triton X-100 with cold or heat treatment on a matrix of Saos-2 cells. We found that the protocols containing SDS were cytotoxic during recellularization. Heat treatment at 47 °C was not cytotoxic, removed cellular constituents, inactivated alkaline phosphatase activity, and maintained the levels of calcium deposition. Subsequently, we investigated the differentiation efficiency of a direct bone coculture system in the established decellularized Saos-2 matrix, an inorganic matrix of calcium phosphate, and a plastic plate as a control. We found that the decellularized Saos-2 cell matrix obtained by heat treatment at 47 °C enhanced osteoclast differentiation and matrix mineralization better than the inorganic matrix and the control. This simple and low-cost method allows us to create a Saos-2 decellularized matrix that can be used as an in vivo-like support for the growth and differentiation of bone cells.

Altered Secretome of Diabetic Monocytes Could Negatively Influence Fracture Healing-An In Vitro Study.

Diabetes mellitus is a main risk factor for delayed fracture healing and fracture non-unions. Successful fracture healing requires stimuli from different immune cells, known to be affected in diabetics. Especially, application of mononuclear cells has been proposed to promote wound and fracture healing. Thus, aim was to investigate the effect of pre-/diabetic conditions on mononuclear cell functions essential to promote osteoprogenitor cell function. We here show that pre-/diabetic conditions suppress the expression of chemokines, e.g., CCL2 and CCL8 in osteoprogenitor cells. The associated MCP-1 and MCP-2 were significantly reduced in serum of diabetics. Both MCPs chemoattract mononuclear THP-1 cells. Migration of these cells is suppressed under hyperglycemic conditions, proposing that less mononuclear cells invade the site of fracture in diabetics. Further, we show that the composition of cytokines secreted by mononuclear cells strongly differ between diabetics and controls. Similar is seen in THP-1 cells cultured under hyperinsulinemia or hyperglycemia. The altered secretome reduces the positive effect of the THP-1 cell conditioned medium on migration of osteoprogenitor cells. In summary, our data support that factors secreted by mononuclear cells may support fracture healing by promoting migration of osteoprogenitor cells but suggest that this effect might be reduced in diabetics.

3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics.

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.

Exposure to 16 Hz Pulsed Electromagnetic Fields Protect the Structural Integrity of Primary Cilia and Associated TGF-ß Signaling in Osteoprogenitor Cells Harmed by Cigarette Smoke.

Cigarette smoking (CS) is one of the main factors related to avoidable diseases and death across the world. Cigarette smoke consists of numerous toxic compounds that contribute to the development of osteoporosis and fracture nonunion. Exposure to pulsed electromagnetic fields (PEMF) was proven to be a safe and effective therapy to support bone fracture healing. The aims of this study were to investigate if extremely low frequency (ELF-) PEMFs may be beneficial to treat CS-related bone disease, and which effect the duration of the exposure has. In this study, immortalized human mesenchymal stem cells (SCP-1 cells) impaired by 5% cigarette smoke extract (CSE) were exposed to ELF-PEMFs (16 Hz) with daily exposure ranging from 7 min to 90 min. Cell viability, adhesion, and spreading were evaluated by Sulforhodamine B, Calcein-AM staining, and Phalloidin-TRITC/Hoechst 33342 staining. A migration assay kit was used to determine cell migration. Changes in TGF-ß signaling were evaluated with an adenoviral Smad2/3 reporter assay, RT-PCR, and Western blot. The structure and distribution of primary cilia were analyzed with immunofluorescent staining. Our data indicate that 30 min daily exposure to a specific ELF-PEMF most effectively promoted cell viability, enhanced cell adhesion and spreading, accelerated migration, and protected TGF-ß signaling from CSE-induced harm. In summary, the current results provide evidence that ELF-PEMF can be used to support early bone healing in patients who smoke.

Synthesis and Characterization of a Novel Biocompatible Alloy, Ti-Nb-Zr-Ta-Sn.

Many current-generation biomedical implants are fabricated from the Ti-6Al-4V alloy because it has many attractive properties, such as low density and biocompatibility. However, the elastic modulus of this alloy is much larger than that of the surrounding bone, leading to bone resorption and, eventually, implant failure. In the present study, we synthesized and performed a detailed analysis of a novel low elastic modulus Ti-based alloy (Ti-28Nb-5Zr-2Ta-2Sn (TNZTS alloy)) using a variety of methods, including scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and tensile test. Additionally, the in vitro biocompatibility of the TNZTS alloy was evaluated using SCP-1, SaOs-2, and THP-1 cell lines and primary human osteoblasts. Compared to Ti-6Al-4V, the elastic modulus of TNZTS alloy was significantly lower, while measures of its in vitro biocompatibility are comparable. O2 plasma treatment of the surface of the alloy significantly increased its hydrophilicity and, hence, its in vitro biocompatibility. TNZTS alloy specimens did not induce the release of cytokines by macrophages, indicating that such scaffolds would not trigger inflammatory responses. The present results suggest that the TNZTS alloy may have potential as an alternative to Ti-6Al-4V.

Use of in vitro bone models to screen for altered bone metabolism, osteopathies, and fracture healing: challenges of complex models.

Approx. every third hospitalized patient in Europe suffers from musculoskeletal injuries or diseases. Up to 20% of these patients need costly surgical revisions after delayed or impaired fracture healing. Reasons for this are the severity of the trauma, individual factors, e.g, the patients' age, individual lifestyle, chronic diseases, medication, and, over 70 diseases that negatively affect the bone quality. To investigate the various disease constellations and/or develop new treatment strategies, many in vivo, ex vivo, and in vitro models can be applied. Analyzing these various models more closely, it is obvious that many of them have limits and/or restrictions. Undoubtedly, in vivo models most completely represent the biological situation. Besides possible species-specific differences, ethical concerns may question the use of in vivo models especially for large screening approaches. Challenging whether ex vivo or in vitro bone models can be used as an adequate replacement for such screenings, we here summarize the advantages and challenges of frequently used ex vivo and in vitro bone models to study disturbed bone metabolism and fracture healing. Using own examples, we discuss the common challenge of cell-specific normalization of data obtained from more complex in vitro models as one example of the analytical limits which lower the full potential of these complex model systems.

Heme oxygenase-1 regulates autophagy through carbon-oxygen to alleviate deoxynivalenol-induced hepatic damage.

Deoxynivalenol (DON) cannot be totally removed due to its stable chemical characteristics and chronic exposure to low doses of DON causes significant toxic effects in humans and animals. However, the potential hazard of such low-dose exposure in target organs still remains not completely understood, especially in liver, which is mainly responsible for detoxification of DON. In the present study, we demonstrated for the first time that estimated human daily DON exposure (25 µg/kg bw) for 30 and 90 days caused low-grade inflammatory infiltration around hepatic centrilobular veins, elevated systemic IL-1ß, IL-6 and TNF-α and impaired liver function evidenced by increased serum ALT activity. At the molecular level, expressions of autophagy-related proteins as well as Cleaved Caspase-3 and Cleaved Caspase-7 were upregulated during DON exposure, which indicated the activation of autophagy and apoptosis. Importantly, AAV-mediated liver-specific overexpression of HO-1 reversed DON-induced liver damages, upregulated autophagy and attenuated apoptosis in liver, while AAV-mediated HO-1 silence aggravated DON-induced liver damages, inhibited autophagy and increased apoptosis. Furthermore, in vitro experiments demonstrated that lentivirus-mediated HO-1 overexpression in Hepa 1-6 cells prolonged the duration of autophagy and delayed the onset of apoptosis. HO-1 silence in Hepa 1-6 cells inhibited activation of autophagy and accelerated occurrence of apoptosis, and these could be recovered by CO pre-treatment. Therefore, we suppose that HO-1 might be a potential research target to protect human and animal from liver injuries induced by low dose of DON exposure.

E-vapor aerosols do not compromise bone integrity relative to cigarette smoke after 6-month inhalation in an ApoE-/- mouse model.

Cigarette smoke (CS) exposure is one of the leading risk factors for human health. Nicotine-containing inhalable products, such as e-cigarettes, can effectively support tobacco harm reduction approaches. However, there are limited comparative data on the effects of the aerosols generated from electronic vapor products (e-vapor) and CS on bone. Here, we report the effects of e-vapor aerosols and CS on bone morphology, structure, and strength in a 6-month inhalation study. Eight-week-old ApoE-/- mice were exposed to aerosols from three different e-vapor formulations-CARRIER (propylene glycol and vegetable glycerol), BASE (CARRIER and nicotine), TEST (BASE and flavor)-to CS from 3R4F reference cigarettes at matched nicotine concentrations (35 µg/L) or to fresh air (Sham) (N = 10 per group). Tibiae were analyzed for bone morphology by µCT imaging, biomechanics by three-point bending, and by histological analysis. CS inhalation caused a significant decrease in cortical and total bone volume fraction and bone density relative to e-vapor aerosols. Additionally, CS exposure caused a decrease in ultimate load and stiffness. In contrast, bone structural and biomechanical parameters were not significantly affected by e-vapor aerosol or Sham exposure. At the dissection time point, there was no significant difference in body weight or tibia bone weight or length among the groups. Histological findings revealed microcracks in cortical bone areas among all exposed groups compared to Sham control. In conclusion, because of the bone-preserving effect of e-vapor aerosols relative to CS exposure, e-vapor products could potentially constitute less harmful alternatives to cigarettes in situations in which bone health is of importance.

Bisphosphonates Reduce Smoking-Induced Osteoporotic-Like Alterations by Regulating RANKL/OPG in an Osteoblast and Osteoclast Co-Culture Model.

Co-culture models have become mandatory for obtaining better insights into bone homeostasis, which relies on the balance between osteoblasts and osteoclasts. Cigarette smoking (CS) has been proven to increase the risk of osteoporosis; however, there is currently no proven treatment for osteoporosis in smokers excluding cessation. Bisphosphonates (BPs) are classical anti-osteoclastic drugs that are commonly used in examining the suitability of bone co-culture systems in vitro as well as to verify the response to osteoporotic stimuli. In the present study, we tested the effects of BPs on cigarette smoke extract (CSE)-affected cells in the co-culture of osteoblasts and osteoclasts. Our results showed that BPs were able to reduce CSE-induced osteoporotic alterations in the co-culture of osteoblasts and osteoclasts such as decreased matrix remodeling, enhanced osteoclast activation, and an up-regulated receptor activator of nuclear factor (NF)-kB-ligand (RANKL)/osteoprotegerin (OPG) ratio. In summary, BPs may be an effective alternative therapy for reversing osteoporotic alterations in smokers, and the potential mechanism is through modulation of the RANKL/OPG ratio.