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Due to the chemical composition and structure of the target tissue, autologous bone grafting remains the gold standard for orthopedic applications worldwide. However, ongoing advancements in alternative grafting materials show that 3D-printed synthetic biomaterials offer many advantages. For instance, they provide high availability, have low clinical limitations, and can be designed with a chemical composition and structure comparable to the target tissue. This study aimed to compare the influences of particle size and sintering temperature on the mechanical properties and biocompatibility of calcium phosphate (CaP) gyroid scaffolds. CaP gyroid scaffolds were fabricated by 3D printing using powders with the same chemical composition but different particle sizes and sintering temperatures. The physicochemical characterization of the scaffolds was performed using X-ray diffractometry, scanning electron microscopy, and microtomography analyses. The immortalized human mesenchymal stem cell line SCP-1 (osteoblast-like cells) and osteoclast-like cells (THP-1 cells) were seeded on the scaffolds as mono- or co-cultures. Bone cell attachment, number of live cells, and functionality were assessed at different time points over a period of 21 days. Improvements in mechanical properties were observed for scaffolds fabricated with narrow-particle-size-distribution powder. The physicochemical analysis showed that the microstructure varied with sintering temperature and that narrow particle size distribution resulted in smaller micropores and a smoother surface. Viable osteoblast- and osteoclast-like cells were observed for all scaffolds tested, but scaffolds produced with a smaller particle size distribution showed less attachment of osteoblast-like cells. Interestingly, low attachment of osteoclast-like cells was observed for all scaffolds regardless of surface roughness. Although bone cell adhesion was lower in scaffolds made with powder containing smaller particle sizes, the long-term function of osteoblast-like and osteoclast-like cells was superior in scaffolds with improved mechanical properties.

Current treatment approaches in clinics to treat the infectious lesions have partial success thus demanding the need for development of advanced treatment modalities. In this study we fabricated an organic-inorganic composite of polypropylene fumarate (PPF) and nanohydroxyapatite (nHAP) by photo-crosslinking as a carrier of two clinically used antibiotics, ciprofloxacin (CIP) and rifampicin (RFP) for the treatment of bone infections. Carboxy terminal-PPF was first synthesized by cis-trans isomerization of maleic anhydride which was then photo-crosslinked using diethylfumarate (DEF) as crosslinker and bis-acylphosphine oxide (BAPO) as photo-initiator under UV lights (P). A composite of PPF and nHAP was fabricated by incorporating 40 % of nHAP in the polymeric matrix of PPF (PH) which was then characterized for different physicochemical parameters. CIP was added along with nHAP to fabricated CIPloaded composite scaffolds (PHC) which was then coated with RFP to synthesize RFP coated CIP-loaded scaffolds (PHCR). It was observed that there was a temporal separation in the in vitro release of two antibiotics after coating PHC with RFP with 80.48 ± 0.40 % release of CIP from PHC and 62.43 ± 0.21 % release of CIP from PHCR for a period of 60 days. Moreover, in vitro protein adsorption was also found to be maximum in PHCR (154.95 ± 0.07 μg/mL) as observed in PHC (75.42 ± 0.06 μg/mL), PH (24.47 ± 0.08 μg/mL) and P alone (4.47 ± 0.02 μg/mL). The scaffolds were also evaluated using in vivo infection model to assess their capacity in reducing the bacterial burden at the infection site. The outcome of this study suggests that RFP coated CIP-loaded PPF composite scaffolds could reduce bacterial burden and simultaneously augment bone healing during infection related fractures., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ashok Kumar reports financial support was provided by Department of Science and Technology, Ministry of Science and Technology, India. Ashok Kumar reports a relationship with Indian Institute of Technology Kanpur that includes: employment. An Indian patent on “An Antibiotic Loaded Composite Bone Active Biomaterial and Process for Synthesis Thereof” has been granted by Indian Patent Office with patent number ‘423656’., (Copyright © 2023. Published by Elsevier B.V.)

"Theory of Forms" implies that a genuine version of creatures exists beyond the shapes in this world. Stem cell, technology has adopted developmental cues to mimic real life. However, the functionality of the lab-made cells is far, from primary ones. Perhaps it is time to switch from analytical to systematic perspective in stem cell science. This, may be the way to define new horizons based on the systematic perspective and convergence of science in stem cell, biology, bridging the current gap between the shadows of real knowledge in current research and reality in future.

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 Ca 2+ -influx. However, ROS and Ca 2+ -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, Ca 2+ -influx and ROS with fluorescence probes. In contrast to mesenchymal cells, ELF-PEMF exposure did not induce ROS and Ca 2+ -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 H 2 O 2 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.

Chronic wounds affect more than 2% of the population worldwide, with a significant burden on affected individuals, healthcare systems, and societies. A key regulator of the entire wound healing cascade is transforming growth factor beta (TGF-β), which regulates not only inflammation and extracellular matrix formation but also revascularization. This present work aimed at characterizing wound tissues obtained from acute and chronic wounds regarding angiogenesis, inflammation, as well as ECM formation and degradation, to identify common disturbances in the healing process. Serum and wound tissues from 38 patients (N = 20 acute and N = 18 chronic wounds) were analyzed. The patients' sera suggested a shift from VEGF/VEGFR to ANGPT/TIE2 signaling in the chronic wounds. However, this shift was not confirmed in the wound tissues. Instead, the chronic wound tissues showed increased levels of MMP9, a known activator of TGF-β. However, regulation of TGF-β target genes, such as CTGF , COL1A1 , or IL-6, was absent in the chronic wounds. In wound tissues, all three TGF-β isoforms were expressed with increased levels of TGF-β1 and TGF-β3 and a reporter assay confirmed that the expressed TGF-β was activated. However, Western blots and immunostaining showed decreased canonical TGF-β signaling in the respective chronic wound tissues, suggesting the presence of a TGF-β inhibitor. As a potential regulatory mechanism, the TGF-β proteome profiler array suggested elevated levels of the TGF-β pseudo-receptor BAMBI. Also, tissue expression of BAMBI was significantly increased not only in chronic wounds (10.6-fold) but also in acute wounds that had become chronic (9.5-fold). In summary, our data indicate a possible regulatory role of BAMBI in the development of chronic wounds. The available few in vivo studies support our findings by postulating a therapeutic potential of BAMBI for controlling scar formation.

Diabetes is a worldwide evolving disease with many associated complications, one of which is delayed or impaired wound healing. Appropriate wound healing strongly relies on the inflammatory reaction directly after injury, which is often altered in diabetic wound healing. After an injury, neutrophils are the first cells to enter the wound site. They have a special defense mechanism, neutrophil extracellular traps (NETs), consisting of released DNA coated with antimicrobial proteins and histones. Despite being a powerful weapon against pathogens, NETs were shown to contribute to impaired wound healing in diabetic mice and are associated with amputations in diabetic foot ulcer patients. The anti-diabetic drugs metformin and liraglutide have already been shown to regulate NET formation. In this study, the effect of insulin was investigated. NET formation after stimulation with PMA (phorbol myristate acetate), LPS (lipopolysaccharide), or calcium ionophore (CI) in the presence/absence of insulin was analyzed. Insulin led to a robust delay of LPS- and PMA-induced NET formation but had no effect on CI-induced NET formation. Mechanistically, insulin induced reactive oxygen species, phosphorylated p38, and ERK, but reduced citrullination of histone H3. Instead, bacterial killing was induced. Insulin might therefore be a new tool for the regulation of NET formation during diabetic wound healing, either in a systemic or topical application.

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, belong to the most prescribed analgesic medication after traumatic injuries. However, there is accumulating evidence that NSAIDs impair fracture healing. Because bone regeneration in aged patients is subject to significant changes in cell differentiation and proliferation as well as a markedly altered pharmacological action of drugs, we herein analyzed the effects of diclofenac on bone healing in aged mice using a stable closed femoral facture model. Thirty-three mice (male n = 14, female n = 19) received a daily intraperitoneal injection of diclofenac (5 mg/kg body weight). Vehicle-treated mice (n = 29; male n = 13, female n = 16) served as controls. Fractured mice femora were analyzed by means of X-ray, biomechanics, micro computed tomography (μCT), histology and Western blotting. Biomechanical analyses revealed a significantly reduced bending stiffness in diclofenac-treated animals at 5 weeks after fracture when compared to vehicle-treated controls. Moreover, the callus tissue in diclofenac-treated aged animals exhibited a significantly reduced amount of bone tissue and higher amounts of fibrous tissue. Further histological analyses demonstrated less lamellar bone after diclofenac treatment, indicating a delay in callus remodeling. This was associated with a decreased number of osteoclasts and an increased expression of osteoprotegerin (OPG) during the early phase of fracture healing. These findings indicate that diclofenac delays fracture healing in aged mice by affecting osteogenic growth factor expression and bone formation as well as osteoclast activity and callus remodeling., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Exposure to extremely low frequency pulsed electromagnetic fields (ELF-PEMF) is supposed to simulate local EMF generated during mechanical stimulation of bone and may therefore be used to improve bone regeneration. This study aimed at optimizing the exposure strategy and investigating the underlying mechanisms of a 16 Hz ELF-PEMF, previously reported to boost osteoblast function. Comparing influences of daily continuous (30 min every 24 h) and intermittent (10 min every 8 h) exposure to the 16 Hz ELF-PEMF on osteoprogenitor cells revealed that the intermittent exposure strategy enhanced the 16 Hz ELF-PEMF effects regarding cell numbers and osteogenic function. Gene expression of piezo 1 and related Ca 2+ influx were significantly increased in SCP-1 cells with the daily intermittent exposure. Pharmacological inhibition of piezo 1 with Dooku 1 largely abolished the positive effect of the 16 Hz ELF-PEMF exposure on osteogenic maturation of SCP-1 cells. In summary, the intermittent exposure strategy enhanced the positive effects of 16 Hz continuous ELF-PEMF exposure in terms of cell viability and osteogenesis. This effect was shown to be mediated by an increased expression of piezo 1 and related Ca 2+ influx. Thus, the intermittent exposure strategy is a promising way to further optimize the therapeutic effects of the 16 Hz ELF-PEMF regarding fracture healing or osteoporosis.

Cigarette smoking promotes osteoclast activity, thus increasing the risk of secondary osteoporosis, leading to osteoporosis-associated fracture and impaired fracture healing. Heated tobacco products (HTP) are considered potential reduced-risk alternatives to cigarettes. However, their impact on bone metabolism remains to be elucidated. We developed an in vitro model that mimics in vivo bone cell interactions to comparatively evaluate the effects of HTPs and cigarette smoke on bone cell functionality and viability. We generated an in vitro coculture system with SCP-1 and THP-1 cells (1:8 ratio) cultured on a decellularized Saos-2 matrix with an optimized coculture medium. We found that, following acute or chronic exposure, particulate matter extract from the aerosol of an HTP, the Tobacco Heating System (THS), was less harmful to the bone coculture system than reference cigarette (1R6F) smoke extract. In the fracture healing model, cultures exposed to the THS extract maintained similar osteoclast activity and calcium deposits as control cultures. Conversely, smoke extract exposure promoted osteoclast activity, resulting in an osteoporotic environment, whose formation could be prevented by bisphosphonate coadministration. Thus, THS is potentially less harmful than cigarette smoke to bone cell differentiation and bone mineralization - both being crucial aspects during the reparative phase of fracture healing., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: PMI partially supported this work and David Bovard, Filippo Zanetti and Julia Hoeng were employees of PMI at the time of the study conduct., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

There is general consent that with decreasing bone mineral density the amount of marrow adipose tissue increases. While image-based techniques, claim an increase in saturated fatty acids responsible for this effect, this study shows an increase in both saturated and unsaturated fatty acids in the bone marrow. Using fatty acid methyl ester gas chromatography-mass spectrometry, characteristic fatty acid patterns for patients with normal BMD (N = 9), osteopenia (N = 12), and osteoporosis (N = 9) have been identified, which differ between plasma, red bone marrow and yellow bone marrow. Selected fatty acids, e.g. FA10:0, FA14:1, or FA16:1 n-7 in the bone marrow or FA18:0, FA18:1 n-9, FA18:1 n-7, FA20:0, FA20:1 n-9, or FA20:3 n-6 in the plasma, correlated with osteoclast activity, suggesting a possible mechanism how these fatty acids may interfere with BMD. Although several fatty acids correlated well with the osteoclast activity and BMD, there was not a single fatty acid contained in our fatty acid profile that can be claimed for controlling BMD, a fact that may be attributed to the genetic heterogeneity of the patients., (Copyright © 2023 Ehnert et al.)

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.

Cigarette smoking-induced oxidative stress has harmful effects on bone metabolism. Maqui berry extract (MBE) and ginseng extract (GE) are two naturally occurring antioxidants that have been shown to reduce oxidative stress. By using an osteoblast and osteoclast three-dimensional co-culture system, we investigated the effects of MBE and GE on bone cells exposed to cigarette smoke extract (CSE). The cell viability and function of the co-culture system were measured on day 14. Markers of bone cell differentiation and oxidative stress were evaluated at gene and protein levels on day 7. The results showed that exposure to CSE induced osteoporotic-like alterations in the co-culture system, while 1.5 µg/mL MBE and 50 µg/mL GE improved CSE-impaired osteoblast function and decreased CSE-induced osteoclast function. The molecular mechanism of MBE and GE in preventing CSE-induced bone cell damage is linked with the inhibition of the NF-κB signaling pathway and the activation of the Nrf2 signaling pathway. Therefore, MBE and GE can reduce CSE-induced detrimental effects on bone cells and, thus, prevent smoking-induced alterations in bone cell homeostasis. These two antioxidants are thus suitable supplements to support bone regeneration in smokers.

Smoking undoubtedly affects human health. Investigating 2318 representative patients at a level 1 trauma center identified delayed wound healing, tissue infections, and/or sepsis as main complications in smokers following trauma and orthopedic surgery. Therefore, smoking cessation is strongly advised to improve the clinical outcome in these patients, although smoking cessation often fails despite nicotine replacement therapy raising the need for specific interventions that may reduce the complication rate. However, the underlying mechanisms are still unknown. In diabetics, delayed wound healing and infections/sepsis are associated with increased neutrophilic PADI4 expression and formation of neutrophil extracellular traps (NETs). The aim was to investigate if similar mechanisms hold for smokers. Indeed, our results show higher PADI4 expression in active and heavy smokers than non-smokers, which is associated with an increased complication rate. However, in vitro stimulation of neutrophils with cigarette smoke extract (CSE) only moderately induced NET formation despite accumulation of reactive oxygen species (ROS). Physiological levels of nicotine and its main metabolite cotinine more effectively induced NET formation, although they did not actively induce the formation of ROS, but interfered with the activity of enzymes involved in anti-oxidative defense and NET formation. In summary, we propose increased formation of NETs as possible triggers for delayed wound healing, tissue infections, and/or sepsis in smokers after a major trauma and orthopedic surgery. Smoking cessation might reduce this effect. However, our data show that smoking cessation supported by nicotine replacement therapy should be carefully considered as nicotine and its metabolite cotinine effectively induced NET formation in vitro, even without active formation of ROS.

Background: A growing number of hepatocellular carcinoma (HCC), and recurrence frequency recently have drawn researchers' attention to alternative approaches. The concept of differentiation therapies (DT) relies on inducing differentiation in HCC cells in order to inhibit recurrence and metastasis. Hepatocyte nuclear factor 4 alpha (HNF4 α ) is the key hepatogenesis transcription factor and its upregulation may decrease the invasiveness of cancerous cells by suppressing epithelial-mesenchymal transition (EMT). This study aimed to evaluate the effect of conjugated linoleic acid (CLA) treatment, natural ligand of HNF4 α , on the proliferation, migration, and invasion capacities of HCC cells in vitro. Materials and Method. Sk-Hep-1 and Hep-3B cells were treated with different doses of CLA or BIM5078 [1-(2'-chloro-5'-nitrobenzenesulfonyl)-2-methylbenzimidazole], an HNF4 α antagonist. The expression levels of HNF4a and EMT related genes were evaluated and associated to hepatocytic functionalities, migration, and colony formation capacities, as well as to viability and proliferation rate of HCC cells., Results: In both HCC lines, CLA treatment induced HNF4α expression in parallel to significantly decreased EMT marker levels, migration, colony formation capacity, and proliferation rate, whereas BIM5078 treatment resulted in the opposite effects. Moreover, CLA supplementation also upregulated ALB, ZO1, and HNF4 α proteins as well as glycogen storage capacity in the treated HCC cells., Conclusion: CLA treatment can induce a remarkable hepatocytic differentiation in HCC cells and attenuates cancerous features. This could be as a result of HNF4a induction and EMT inhibition., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Zohre Miri-Lavasani et al.)

Cigarette smoking (CS) leads to several adverse health effects, including diseases, disabilities, and even death. Post-operative and trauma patients who smoke have an increased risk for complications, such as delayed bone or wound healing. In clinical trials, microcurrent (MC) has been shown to be a safe, non-invasive, and effective way to accelerate wound healing. Our study aimed to investigate if MC with the strength of 100 μA may be beneficial in treating CS-related healing impairment, especially in regard to angiogenesis. In this study, we investigated the effect of human keratinocyte cells (HaCaT) on angiogenesis after 72 h of cigarette smoke extract (CSE) exposure in the presence or absence of 100 μA MC. Cell viability and proliferation were evaluated by resazurin conversion, Sulforhodamine B, and Calcein-AM/Hoechst 33342 staining; the pro-angiogenic potential of HaCaT cells was evaluated by tube formation assay and angiogenesis array assay; signaling pathway alterations were investigated using Western blot. Constant exposure for 72 h to a 100 μA MC enhanced the angiogenic ability of HaCaT cells, which was mediated through the PI3K-Akt signaling pathway. In conclusion, the current data indicate that 100 μA MC may support wound healing in smoking patients by enhancing angiogenesis.

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., (© 2022. The Author(s).)

Bone mechanical properties are classically determined by biomechanical tests, which normally destroy the bones and disable further histological or molecular analyses. Thus, obtaining biomechanical data from bone usually requires an additional group of animals within the experimental setup. Finite element models (FEMs) may non-invasively and non-destructively simulate mechanical characteristics based on material properties. The present study aimed to establish and validate an FEM to predict the mechanical properties of mice tibiae. The FEM was established based on µCT (micro-Computed Tomography) data of 16 mouse tibiae. For validating the FEM, simulated parameters were compared to biomechanical data obtained from 3-point bending tests of the identical bones. The simulated and the measured parameters correlated well for bending stiffness (R2 = 0.9104, p < 0.0001) and yield displacement (R2 = 0.9003, p < 0.0001). The FEM has the advantage that it preserves the bones’ integrity, which can then be used for other analytical methods. By eliminating the need for an additional group of animals for biomechanical tests, the established FEM can contribute to reducing the number of research animals in studies focusing on bone biomechanics. This is especially true when in vivo µCT data can be utilized where multiple bone scans can be performed with the same animal at different time points. Thus, by partially replacing biomechanical experiments, FEM simulations may reduce the overall number of animals required for an experimental setup investigating bone biomechanics, which supports the 3R (replace, reduce, and refine) principle.

Chronic wounds are a serious problem in clinical work and a heavy burden for individuals and society. In order to develop novel therapies, adequate model systems for the investigation of wound healing are required. Although in past years different in vitro and in vitro wound healing models have been established, a true human-like model does still not exist. Animal models are limited in their use due to species-specific differences in the skin, a lengthy manufacturing process, experimental costs, and ethical concerns. Both 2D and 3D in vitro models are usually comprised of only one or two skin cell types and fail to capture the reaction between blood cells and skin cells. Thus, our aim was to develop an in vitro scab model to investigate early reactions in the wound healing process. The here established scab model is comprised of HaCaT cells and freshly collected blood from healthy volunteers. The generated scabs were stably cultured for more than 2 weeks. TGF-β signaling is well known to regulate the early phases of wound healing. All three TGF-β isoforms and target genes involved in extracellular matrix composition and degradation were expressed in the in vitro scabs. To validate the in vitro scab model, the effects of either additional stimulation or the inhibition of the TGF-β signaling pathway were investigated. Exogenous application of TGF-β1 stimulated matrix remodeling, which loosened the structure of the in vitro scabs with time, also induced expression of the inhibitory Smad7. Inhibition of the endogenous TGF-β signaling, on the contrary, resulted in a rapid condensation and degranulation of the in vitro scabs. In summary, the here established in vitro scab model can be used to analyze the first phases of wound healing where blood and skin cells interact, as it is viable and responsive for more than 2 weeks.

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.

Bone fracture healing is an overly complex process in which inflammation, osteogenesis, and angiogenesis are tightly coupled, and delayed fracture repair is a very common health risk. One of the major causes of delayed healing is the formation of insufficient vasculature. Precise regulation of blood vessels in bone and their interplay with especially osteogenic processes has become an emerging topic within the last years; nevertheless, regulation of angiogenesis in (early) diseased fracture repair is still widely unknown. Here, we aim to develop an in vitro model for the analysis of early fracture healing which also enables the analysis of angiogenesis as a main influencing factor. As smoking is one of the main risk factors for bone fractures and developing a delay in healing, we model smoking and non-smoking conditions in vitro to analyze diverging reactions. Human in vitro fracture hematomas mimicking smokers' and non-smokers' hematomas were produced and analyzed regarding cell viability, inflammation, osteogenic and chondrogenic differentiation, and angiogenic potential. We could show that smokers' blood hematomas were viable and comparable to non-smokers. Smokers' hematomas showed an increase in inflammation and a decrease in osteogenic and chondrogenic differentiation potential. When analyzing angiogenesis, we could show that the smokers' hematomas secrete factors that drastically reduced HUVEC proliferation and tube formation. With an angiogenesis array and gene expression analysis, we could identify the main influencing factors: Anpgt1/2, Tie2, and VEGFR2/3. In conclusion, our model is suitable to mimic smoking conditions in vitro showing that smoking negatively impacts early vascularization of newly formed tissue.

In vertebrates, bone is considered an osteoimmune system which encompasses functions of a locomotive organ, a mineral reservoir, a hormonal organ, a stem cell pool and a cradle for immune cells. This osteoimmune system is based on cooperatively acting bone and immune cells, cohabitating within the bone marrow. They are highly interdependent, a fact that is confounded by shared progenitors, mediators, and signaling pathways. Successful fracture healing requires the participation of all the precursors, immune and bone cells found in the osteoimmune system. Recent evidence demonstrated that changes of the immune cell composition and function may negatively influence bone healing. In this review, first the interplay between different immune cell types and osteoprogenitor cells will be elaborated more closely. The separate paragraphs focus on the specific cell types, starting with the cells of the innate immune response followed by cells of the adaptive immune response, and the complement system as mediator between them. Finally, a brief overview on the challenges of preclinical testing of immune-based therapeutic strategies to support fracture healing will be given., Competing Interests: Conflict-of-interest statement: The authors declare no conflict of interest for this article., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

Delayed fracture healing and fracture non-unions impose an enormous burden on individuals and society. Successful healing requires tight communication between immune cells and bone cells. Macrophages can be found in all healing phases. Due to their high plasticity and long life span, they represent good target cells for modulation. In the past, extremely low frequency pulsed electromagnet fields (ELF-PEMFs) have been shown to exert cell-specific effects depending on the field conditions. Thus, the aim was to identify the specific ELF-PEMFs able to modulate macrophage activity to indirectly promote mesenchymal stem/stromal cell (SCP-1 cells) function. After a blinded screening of 22 different ELF-PEMF, two fields (termed A and B) were further characterized as they diversely affected macrophage function. These two fields have similar fundamental frequencies (51.8 Hz and 52.3 Hz) but are emitted in different groups of pulses or rather send-pause intervals. Macrophages exposed to field A showed a pro-inflammatory function, represented by increased levels of phospho-Stat1 and CD86, the accumulation of ROS, and increased secretion of pro-inflammatory cytokines. In contrast, macrophages exposed to field B showed anti-inflammatory and pro-healing functions, represented by increased levels of Arginase I, increased secretion of anti-inflammatory cytokines, and growth factors are known to induce healing processes. The conditioned medium from macrophages exposed to both ELF-PEMFs favored the migration of SCP-1 cells, but the effect was stronger for field B. Furthermore, the conditioned medium from macrophages exposed to field B, but not to field A, stimulated the expression of extracellular matrix genes in SCP-1 cells, i.e., COL1A1 , FN1 , and BGN . In summary, our data show that specific ELF-PEMFs may affect immune cell function. Thus, knowing the specific ELF-PEMFs conditions and the underlying mechanisms bears great potential as an adjuvant treatment to modulate immune responses during pathologies, e.g., fracture healing.

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., (© 2021. The Author(s).)

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.

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-β 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-β 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., Competing Interests: The authors declare no conflict of interest. Sachtleben GmbH provided the ELF-PEMF devices (Somagen®) and the background on the physical parameters, but were not involved in the study design or the data evaluation.

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.

Background: Bone health, especially osteoporosis among ageing populations, has become an important topic for both clinical and basic researchers. The relationship between bone health and healthy lifestyles has been frequently discussed. The present study focuses on the relationship between bone health and healthy lifestyles among older adults, based on a global comparison., Methods: This narrative review was performed by collecting clinical trials, basic research and reviews on lifestyle and bone health in PubMed database., Results: Positive effects of physical activity and negative effects of malnutrition, alcohol abuse, and cigarette smoking on bone health were revealed. The relationship between bone health and drinking coffee and tea is still inconclusive. Moreover, the diversity of each region should be aware when considering healthy lifestyles to improve bone health., Conclusion: Healthy lifestyles are highly related to bone health, and different lifestyles may have different influences on regions with a high risk of bone diseases. It is practical to acknowledge the diversity of economic, religious, environmental and geological conditions in each region when providing suitable and effective recommendations for healthy lifestyles that can improve overall bone health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors have no conflicts of interests to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

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.

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.

Background: Severe acute respiratory syndrome (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), has become a global pandemic in the past months. An overall defined treatment has not yet been established. Therefore, it is important to summarize and report treatment experiences and identify patient groups that have a significantly higher risk of an adverse clinical outcome. Methods: Two hundred thirty-nine COVID-19 patients were recruited from January 25 to February 15, 2020. Demographic, clinical, laboratory, treatment management, and outcome data obtained from patients' medical records were evaluated. Results: Patients who recovered from PCR positive to negative within 2 weeks had significantly lower erythrocyte sedimentation rate (ESR) and higher C-reactive protein (CRP) levels than those recovered post 2 weeks. During antiviral treatment, COVID-19 patients with older age, comorbidities, and corticosteroid treatment required a significantly longer time to turn from PCR positive to negative COVID-19 result. Conclusion: PCR tests are of great importance to evaluate the recovery of COVID-19-positive patients, and ESR could be an indirect indicator to monitor SARS-COV-2 activity. Furthermore, our data suggest that older age, the existence of comorbidities, and corticosteroid treatment of COVID-19 patients during antiviral treatment could prolong the duration of conversion from SARS-COV-2 positive to negative., (Copyright © 2020 Zhu, Huang, Tang, Nussler and Zheng.)

Neutrophils are the most abundant immune cells in the blood. Besides common immune defense mechanisms, releasing their DNA covered with antimicrobial proteases and histones represent another strong defense mechanism: neutrophil extracellular traps. In vitro the two most common inducers of these, so called, NETs are calcium ionophores (CI) and PMA (Phorbol 12-myristate 13-acetate). Following stimulation monitoring of NET release is necessary. For now, the methods of choice are quantification of free DNA by fluorescent dyes or analysis of immunofluorescence images. As a new method we tested bio-impedance monitoring of neutrophils after stimulation with the two inducers PMA and CI in gold-electrode coated plates. Bio-impedance (cell index) was measured over time. Results were compared to the monitoring of NETs by the fluorescent DNA-binding dye Sytox Green and immunofluorescence analysis. Cell index peaked about 25 min faster following CI stimulation than following PMA stimulation. The activation in Sytox Green Assay was significantly later detectable for PMA (+ approx. 90 min) but not for CI stimulation. The earlier and faster activation by CI was also confirmed by immunofluorescence staining. Our data suggest that bio-impedance measurement allows an easy online tracking of early neutrophil activation. This offers new opportunities to monitor early phases and stimuli-dependent dynamics of NETosis., (Copyright © 2020 Linnemann et al.)

Background: Cigarette smoking (CS) is the most common method of consuming tobacco. Deleterious effects on bone integrity, increased incidence of fractures, and delayed fracture healing are all associated with CS. Over 150 of the 6500 molecular species contained in cigarette smoke and identified as toxic compounds are inhaled by CS and, via the bloodstream, reach the skeletal system. New technologies designed to develop a reduced-risk alternative for smokers are based on electronic nicotine delivery systems, such as e-cigarettes and tobacco heating systems (THS). THS are designed to heat tobacco instead of burning it, thereby reducing the levels of harmful toxic compounds released., Aim: To examine the effects of THS on osteoprogenitor cell viability and function compared to conventional CS., Methods: Human immortalized mesenchymal stem cells ( n = 3) and primary human pre-osteoblasts isolated from cancellous bone samples from BG Unfall Klinik Tübingen ( n = 5) were osteogenically differentiated in vitro with aqueous extracts generated from either the THS 2.4 "IQOS" or conventional "Marlboro" cigarettes for up to 21 d. Cell viability was analyzed using resazurin conversion assay (mitochondrial activity) and calcein-AM staining (esterase activity). Osteogenic differentiation and bone cell function were evaluated using alkaline phosphatase (AP) activity, while matrix formation was analyzed through alizarin red staining. Primary cilia structure was examined by acetylated α-tubulin immunofluorescent staining. Free radical production was evaluated with 2',7'-dichlorofluorescein-diacetate assay., Results: Our data clearly show that THS is significantly less toxic to bone cells than CS when analyzed by mitochondrial and esterase activity ( P < 0.001). No significant differences in cytotoxicity between the diverse flavors of THS were observed. Harmful effects from THS on bone cell function were observed only at very high, non-physiological concentrations. In contrast, extracts from conventional cigarettes significantly reduced the AP activity (by two-fold) and matrix mineralization (four-fold) at low concentrations. Additionally, morphologic analysis of primary cilia revealed no significant changes in the length of the organelle involved in osteogenesis of osteoprogenitor cells, nor in the number of ciliated cells following THS treatment. Assessment of free radical production demonstrated that THS induced significantly less oxidative stress than conventional CS in osteoprogenitor cells., Conclusion: THS was significantly less harmful to osteoprogenitor cells during osteogenesis than conventional CS. Additional studies are required to confirm whether THS is a better alternative for smokers to improve delays in bone healing following fracture., Competing Interests: Conflict-of-interest statement: The authors have not conflict of interest., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Cryogels represent ideal carriers for bone tissue engineering. We recently described the osteogenic potential of cryogels with different protein additives, e.g., platelet-rich plasma (PRP). However, these scaffolds raised concerns as different toxic substances are required for their preparation. Therefore, we developed another gelatin (GEL)-based cryogel. This study aimed to compare the two scaffolds regarding their physical characteristics and their influence on osteogenic and osteoclastic cells. Compared to the PRP scaffolds, GEL scaffolds had both larger pores and thicker walls, resulting in a lower connective density. PRP scaffolds, with crystalized calcium phosphates on the surface, were significantly stiffer but less mineralized than GEL scaffolds with hydroxyapatite incorporated within the matrix. The GEL scaffolds favored adherence and proliferation of the osteogenic SCP-1 and SaOS-2 cells. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin (OPG) levels seemed to be induced by GEL scaffolds. Levels of other osteoblast and osteoclast markers were comparable between the two scaffolds. After 14 days, mineral content and stiffness of the cryogels were increased by SCP-1 and SaOS-2 cells, especially of PRP scaffolds. THP-1 cell-derived osteoclastic cells only reduced mineral content and stiffness of PRP cryogels. In summary, both scaffolds present powerful advantages; however, the possibility to altered mineral content and stiffness may be decisive when it comes to using PRP or GEL scaffolds for bone tissue engineering.

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.

Hepatotoxicity is among the most frequent reasons for drug withdrawal from the market. Therefore, there is an urgent need for reliable predictive in vitro tests, which unfailingly identify hepatotoxic drug candidates, reduce drug development time, expenses and the number of test animals. Currently, human hepatocytes represent the gold standard. However, the use of hepatocytes is challenging since the cells are not constantly available and lose their metabolic activity in culture. To solve these problems many different approaches have been developed in the past decades. The aim of this review is to present these approaches and to discuss the possibilities and limitations as well as future opportunities and directions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Drug-induced liver toxicity is one of the most common reasons for the failure of drugs in clinical trials and frequent withdrawal from the market. Reasons for such failures include the low predictive power of in vivo studies, that is mainly caused by metabolic differences between humans and animals, and intraspecific variances. In addition to factors such as age and genetic background, changes in drug metabolism can also be caused by disease-related changes in the liver. Such metabolic changes have also been observed in clinical settings, for example, in association with a change in liver stiffness, a major characteristic of an altered fibrotic liver. For mimicking these changes in an in vitro model, this study aimed to develop scaffolds that represent the rigidity of healthy and fibrotic liver tissue. We observed that liver cells plated on scaffolds representing the stiffness of healthy livers showed a higher metabolic activity compared to cells plated on stiffer scaffolds. Additionally, we detected a positive effect of a scaffold pre-coated with fetal calf serum (FCS)-containing media. This pre-incubation resulted in increased cell adherence during cell seeding onto the scaffolds. In summary, we developed a scaffold-based 3D model that mimics liver stiffness-dependent changes in drug metabolism that may more easily predict drug interaction in diseased livers.

Although several researchers have attested deleterious effects of smoking to the musculoskeletal system, the association between smoking and the onset of osteoarthritis (OA) remains unclear. Here, we investigate the effect of cigarette smoke extract (CSE) on primary human chondrocytes. The present study demonstrates that physiological concentrations of CSE (0.1%-10%) inhibit the viability, proliferation, and matrix formation of chondrocytes in a dose- and time-dependent manner. Significant amounts of free radicals were generated by 10% of CSE and led to cell death. A clinical dosage (4 mg/mL) of dexamethasone (Dex) showed toxic effects on chondrocytes, and the long-time treatment by lower doses (4-400 μg/mL) induced hypertrophic changes in the chondrocytes. To substitute Dex, diclofenac (Dic, 1 μg/mL) and acetaminophen (Ace, 10 μg/mL) were tested and did not worsen the metabolic activity of CSE-exposed chondrocytes. Hyaluronic acid (HA, 5 mg/mL) combined with Dic or Ace significantly inhibited the oxidative stress and enhanced the viability and matrix formation of CSE-exposed chondrocytes. This study shows for the first time that CSE mediates the disruption of cartilage through inducing cell death by increasing oxidative stress, and that this effect is fortified by Dex. The deleterious effects of CSE on chondrocytes could be reversed by treatment with HA combined with first-line analgesic/anti-inflammatory agents.

Organ and tissue shortage are known as a crucially important public health problem as unfortunately a small percentage of patients receive transplants. In the context of emerging regenerative medicine, researchers are trying to regenerate and replace different organs and tissues such as the liver, heart, skin, and kidney. Liver tissue engineering (TE) enables us to reproduce and restore liver functions, fully or partially, which could be used in the treatment of acute or chronic liver disorders and/or generate an appropriate functional organ which can be transplanted or employed as an extracorporeal device. In this regard, a variety of techniques (e.g., fabrication technologies, cell-based technologies, microfluidic systems and, extracorporeal liver devices) could be applied in tissue engineering in liver regenerative medicine. Common TE techniques are based on allocating stem cell-derived hepatocyte-like cells or primary hepatocytes within a three-dimensional structure which leads to the improvement of their survival rate and functional phenotype. Taken together, new findings indicated that developing liver tissue engineering-based techniques could pave the way for better treatment of liver-related disorders. Herein, we summarized novel technologies used in liver regenerative medicine and their future applications in clinical settings., Competing Interests: The authors declare no conflict of interest.

In order to increase the metabolic activity of human hepatocytes and liver cancer cell lines, many approaches have been reported in recent years. The metabolic activity could be increased mainly by cultivating the cells in 3D systems or co-cultures (with other cell lines). However, if the system becomes more complex, it gets more difficult to quantify the number of cells (e.g., on a 3D matrix). Until now, it has been impossible to quantify different cell types individually in 3D co-culture systems. Therefore, we developed a PCR-based method that allows the quantification of HepG2 cells and 3T3-J2 cells separately in a 3D scaffold culture. Moreover, our results show that this method allows better comparability between 2D and 3D cultures in comparison to the often-used approaches based on metabolic activity measurements, such as the conversion of resazurin., Competing Interests: The authors declare no conflicts of interest.

The finding that alterations in electrical potential play an important role in the mechanical stimulation of the bone provoked hype that noninvasive extremely low frequency pulsed electromagnetic fields (ELF-PEMF) can be used to support healing of bone and osteochondral defects. This resulted in the development of many ELF-PEMF devices for clinical use. Due to the resulting diversity of the ELF-PEMF characteristics regarding treatment regimen, and reported results, exposure to ELF-PEMFs is generally not among the guidelines to treat bone and osteochondral defects. Notwithstanding, here we show that there is strong evidence for ELF-PEMF treatment. We give a short, confined overview of in vitro studies investigating effects of ELF-PEMF treatment on bone cells, highlighting likely mechanisms. Subsequently, we summarize prospective and blinded studies, investigating the effect of ELF-PEMF treatment on acute bone fractures and bone fracture non-unions, osteotomies, spinal fusion, osteoporosis, and osteoarthritis. Although these studies favor the use of ELF-PEMF treatment, they likewise demonstrate the need for more defined and better controlled/monitored treatment modalities. However, to establish indication-oriented treatment regimen, profound knowledge of the underlying mechanisms in the sense of cellular pathways/events triggered is required, highlighting the need for more systematic studies to unravel optimal treatment conditions., Competing Interests: The authors declare no conflict of interest.

Extremely low-frequency pulsed electromagnetic field (ELF-PEMF) therapy is proposed to support bone healing after injuries and surgical procedures, being of special interest for elderly patients. This study aimed at investigating the effect of a specific ELF-PEMF, recently identified to support osteoblast function in vitro, on bone healing after high tibial osteotomy (HTO). Patients who underwent HTO were randomized to ELF-PEMF or placebo treatment, both applied by optically identical external devices 7 min per day for 30 days following surgery. Osseous consolidation was evaluated by post-surgical X-rays (7 and 14 weeks). Serum markers were quantified by ELISA. Data were compared by a two-sided t-test (α = 0.05). Device readouts showed excellent therapy compliance. Baseline parameters, including age, sex, body mass index, wedge height and blood cell count, were comparable between both groups. X-rays revealed faster osseous consolidation for ELF-PEMF compared to placebo treatment, which was significant in patients ≥50 years (∆ mean = 0.68%/week; p = 0.003). Findings are supported by post-surgically increased bone-specific alkaline phosphatase serum levels following ELF-PEMF, compared to placebo (∆ mean = 2.2 µg/L; p = 0.029) treatment. Adverse device effects were not reported. ELF-PEMF treatment showed a tendency to accelerate osseous consolidation after HTO. This effect was stronger and more significant for patients ≥50 years. This ELF-PEMF treatment might represent a promising adjunct to conventional therapy supporting osseous consolidation in elderly patients.

Clinical and pharmaceutical applications of primary hepatocytes (PHs) are limited due to inadequate number of donated livers and potential challenges in successful maintenance of PHs in culture. Freshly isolated hepatocytes lose their specific features and rapidly de-differentiate in culture. Bipotent hepatoblasts, as liver precursor cells that can differentiate into both hepatocytes and cholangiocytes (Alb- and Ck19-positive cells, respectively), could be used as an alternative and reliable cell source to produce enough PHs for drug discovery or possible clinical applications. In this study, growth factor-free coculture systems of prenatal or postnatal murine liver stromal cells (pre-LSCs or post-LSCs, respectively) were used as feeder cells to support freshly isolated mice hepatoblasts. DLK1-positive hepatoblasts were isolated from mouse fetuses (E14.5) and cocultured with feeder cells under adherent conditions. The hepatoblasts' bipotent features, proliferation rate, and colony formation capacity were assessed on day 5 and 7 post-seeding. Immunofluorescence staining showed that the hepatoblasts remained double positive for Alb and Ck19 on both Pre- and Post-LSCs, after 5 and 7 days of coculture. Moreover, application of pre-LSCs as feeder cells significantly increased the number of DLK1-positive cells and their proliferation rate (ie, increased the number of Ki-67 positive cells) on day 7, compared to Post-LSCs group. Finally, to address our ultimate goal, which was an extension of hepatoblasts ex vivo maintenance, 3D spheres of isolated hepatoblasts were, cultured in conditioned medium (CM) derived from pre-LSCs until day 30. It was observed that the CM derived from Pre-LSCs could successfully prolong the maintenance of hepatic progenitor cells (HPCs) in 3D suspension culture., (© 2019 Wiley Periodicals, Inc.)

A variety of wound dressing are available for burns. Furthermore, although their impacts on wound healing have been studied sufficiently, their effects on blood remain unclear. Meanwhile, this aspect is extremely important, since blood interacts with the wound dressing, especially in extensive burn injuries. Therefore, the aim of this study is to evaluate the hemocompatibility and immunogenicity of different burn wound dressings. Accordingly, human whole blood (n = 5) was anticoagulated with heparin, treated with different wound dressings and incubated at 37°C for 30 minutes. Different parameters for coagulation and hemocompatibility were evaluated before and after incubation. Consequently, Jelonet, Xenoderm, and Matriderm showed higher TAT-III concentrations, Jelonet, Xenoderm, EZ Derm, and Matriderm were higher β-thromboglobulin; EZ Derm and Burntec showed higher SC5b-9 concentrations after incubation with whole blood. Our ex vivo study provided initial insights into the hemocompatibility and immunogenicity of different burn wound dressings. Moreover, Xenografts (Xenoderm and EZ Derm), Jelonet and Matriderm showed a hemostyptic effect, while EZ Derm and Burntec activated the complement system. Therefore, further studies must be conducted to analyze the possible effects in vivo., (© 2019 by the Wound Healing Society.)

Human adipose-derived mesenchymal stem/stromal cells (Ad-MSCs) have great potential for bone tissue engineering. Cryogels, mimicking the three-dimensional structure of spongy bone, represent ideal carriers for these cells. We developed poly(2-hydroxyethyl methacrylate) cryogels, containing hydroxyapatite to mimic inorganic bone matrix. Cryogels were additionally supplemented with different types of proteins, namely collagen (Coll), platelet-rich plasma (PRP), immune cells-conditioned medium (CM), and RGD peptides (RGD). The different protein components did not affect scaffolds' porosity or water-uptake capacity, but altered pore size and stiffness. Stiffness was highest in scaffolds with PRP (82.3 kPa), followed by Coll (55.3 kPa), CM (45.6 kPa), and RGD (32.8 kPa). Scaffolds with PRP, CM, and Coll had the largest pore diameters (~60 µm). Ad-MSCs were osteogenically differentiated on these scaffolds for 14 days. Cell attachment and survival rates were comparable for all four scaffolds. Runx2 and osteocalcin levels only increased in Ad-MSCs on Coll, PRP and CM cryogels. Osterix levels increased slightly in Ad-MSCs differentiated on Coll and PRP cryogels. With differentiation alkaline phosphatase activity decreased under all four conditions. In summary, besides Coll cryogel our PRP cryogel constitutes as an especially suitable carrier for bone tissue engineering. This is of special interest, as this scaffold can be generated with patients' PRP.

It is well established that smoking has detrimental effects on bone integrity and is a preventable risk factor for metabolic bone disorders. Following orthopedic surgeries, smokers frequently show delayed fracture healing associated with many complications, which results in prolonged hospital stays. One crucial factor responsible for fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs) at early stages, a mechanism mediated by transforming growth factor β (TGF-β). Although it is known that smokers frequently have decreased TGF-β levels, little is known about the actual signaling occurring in these patients. We investigated the effect of cigarette smoke on TGF-β signaling in MSCs to evaluate which step in the pathway is affected by cigarette smoke extract (CSE). Single-cell-derived human mesenchymal stem cell line (SCP-1 cells) were treated with CSE concentrations associated with smoking up to 20 cigarettes a day. TGF-β signaling was analyzed using an adenovirus-based reporter assay system. Primary cilia structure and downstream TGF-β signaling modulators (Smad2, Smad3, and Smad4) were analyzed by Western blot and immunofluorescence staining. CSE exposure significantly reduced TGF-β signaling. Intriguingly, we observed that protein levels of phospho-Smad2/3 (active forms) as well as nuclear translocation of the phospho-Smad3/4 complex decreased after CSE exposure, phenomena that affected signal propagation. CSE exposure reduced the activation of TGF-β modulators under constitutive activation of TGF-β receptor type I (ALK5), evidencing that CSE affects signaling downstream of the ALK5 receptor but not the binding of the cytokine to the receptor itself. CSE-mediated TGF-β signaling impaired MSC migration, proliferation, and differentiation and ultimately affected endochondral ossification. Thus, we conclude that CSE-mediated disruption of TGF-β signaling in MSCs is partially responsible for delayed fracture healing in smokers., Competing Interests: The authors declare no conflict of interest.

Almost all patients with chronic liver diseases (CLD) show altered bone metabolism. Depending on the etiology, this manifests in a severe osteoporosis in up to 75% of the affected patients. Due to high prevalence, the generic term hepatic osteodystrophy (HOD) evolved, describing altered bone metabolism, decreased bone mineral density, and deterioration of bone structure in patients with CLD. Once developed, HOD is difficult to treat and increases the risk of fragility fractures. Existing fractures affect the quality of life and, more importantly, long-term prognosis of these patients, which presents with increased mortality. Thus, special care is required to support the healing process. However, for early diagnosis (reduce fracture risk) and development of adequate treatment strategies (support healing of existing fractures), it is essential to understand the underlying mechanisms that link disturbed liver function with this bone phenotype. In the present review, we summarize proposed molecular mechanisms favoring the development of HOD and compromising the healing of associated fractures, including alterations in vitamin D metabolism and action, disbalances in transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) signaling with histone deacetylases (HDACs) as secondary regulators, as well as alterations in the receptor activator of nuclear factor kappa B ligand (RANKL)-osteoprotegerin (OPG) system mediated by sclerostin. Based on these mechanisms, we give an overview on the limitations of early diagnosis of HOD with established serum markers.

Background: The prevalence of diabetes mellitus (DM) or malnutrition in hospitalised patients depends on the clinical domain, but is much higher than in the normal population. In trauma surgery, this increase is frequently associated with more postoperative complications and constantly rising costs. In addition, the quality of life of this target group is decreased, but there are only limited data from departments of traumatology and/or orthopaedics. Therefore, we aim to analyse the factors influencing the postoperative complication rate as well as the quality of life of orthopaedic and trauma patients., Methods: Within this prospective trial in the period of 06/2014 to 02/2017, we analysed data of 1643 patients from traumatology - geriatric and septic traumatology - as well as endoprosthetics with regard to the clinical outcome, the complication rate and the quality of life (Short Form Health Survey 36, SF-36) associated with diabetes mellitus (DM) and the nutritional status (Nutritional Risk Screening 2002, NRS)., Results: Within our hospitalised group of trauma patients, the prevalence of diabetes mellitus was 12.4% and the risk for malnutrition (NRS ≥ 3) was 18.3%, which is much higher than in the normal population (DM 7.2%). Patients suffering from diabetes mellitus had significantly more complications than patients without diabetes mellitus. Similar results were found when comparing patients with a risk of malnutrition to the patients without. Furthermore, patients with DM evaluate their subjective quality of life lower than do patients without DM in the most domains of the SF-36, especially in the subjective-physical domains, while patients with NRS ≥ 3 assess their quality of life as being lower than do patients without a risk of malnutrition in all domains of the SF-36 (physical and mental). Additionally, we showed that reduced nutritional status has a greater influence on the decline in quality of life than did diabetes mellitus., Conclusion: Both diabetes mellitus and malnutrition seems to influence the subjective quality of life and the complication rate of hospitalised trauma patients. A nationwide data collection and targeted interventions within the frame of interdisciplinary cooperation are necessary. In this way, the postoperative complication rate as well as associated higher treatment costs could be reduced., Competing Interests: The authors declare that they have no conflict of interest./Die Autoren geben an, dass kein Interessenkonflikt besteht., (Georg Thieme Verlag KG Stuttgart · New York.)

Numerous studies have described a correlation between smoking and reduced bone mass. This not only increases fracture risk but also impedes reconstruction/fixation of bone. An increased frequency of complications following surgery is common. Here, we investigate the effect of smoking on the clinical outcome following total joint arthroplasty (TJA). 817 patients receiving primary or revision (including clinical transfers) TJA at our level-one trauma center have been randomly interviewed twice (pre- and six months post-surgery). We found that 159 patients developed complications (infections, disturbed healing, revisions, thrombosis, and/or death). Considering nutritional status, alcohol and cigarette consumption as possible risk factors, OR was highest for smoking. Notably, mean age was significantly lower in smokers (59.2 ± 1.0a) than non-smokers (64.6 ± 0.8; p < 0.001). However, the number of comorbidities was comparable between both groups. Compared to non-smokers (17.8 ± 1.9%), the complication rate increases with increasing cigarette consumption (1⁻20 pack-years (PY): 19.2 ± 2.4% and >20 PY: 30.4 ± 3.6%; p = 0.002). Consequently, mean hospital stay was longer in heavy smokers (18.4 ± 1.0 day) than non-smokers (15.3 ± 0.5 day; p = 0.009) or moderate smokers (15.9 ± 0.6 day). In line with delayed healing, bone formation markers (BAP and CICP) were significantly lower in smokers than non-smokers 2 days following TJA. Although, smoking increased serum levels of MCP-1, OPG, sRANKL, and Osteopontin as well as bone resorption markers (TRAP5b and CTX-I) were unaffected. In line with an increased infection rate, smoking reduced 25OH vitamin D3 (immune-modulatory), IL-1β, IL-6, TNF-α, and IFN-γ serum levels. Our data clearly show that smoking not only affects bone formation after TJA but also suppresses the inflammatory response in these patients. Thus, it is feasible that therapies favoring bone formation and immune responses help improve the clinical outcome in smokers following TJA.

Although human liver tumor cells have reduced metabolic functions as compared to primary human hepatocytes (PHH) they are widely used for pre-screening tests of drug metabolism and toxicity. The aim of the present study was to modify liver cancer cell lines in order to improve their drug-metabolizing activities towards PHH. It is well-known that epigenetics is strongly modified in tumor cells and that epigenetic regulators influence the expression and function of Cytochrome P450 (CYP) enzymes through altering crucial transcription factors responsible for drug-metabolizing enzymes. Therefore, we screened the epigenetic status of four different liver cancer cell lines (Huh7, HLE, HepG2 and AKN-1) which were reported to have metabolizing drug activities. Our results showed that HepG2 cells demonstrated the highest similarity compared to PHH. Thus, we modified the epigenetic status of HepG2 cells towards 'normal' liver cells by 5-Azacytidine (5-AZA) and Vitamin C exposure. Then, mRNA expression of Epithelial-mesenchymal transition (EMT) marker SNAIL and CYP enzymes were measured by PCR and determinate specific drug metabolites, associated with CYP enzymes by LC/MS. Our results demonstrated an epigenetic shift in HepG2 cells towards PHH after exposure to 5-AZA and Vitamin C which resulted in a higher expression and activity of specific drug metabolizing CYP enzymes. Finally, we observed that 5-AZA and Vitamin C led to an increased expression of Hepatocyte nuclear factor 4α (HNF4α) and E-Cadherin and a significant down regulation of Snail1 (SNAIL), the key transcriptional repressor of E-Cadherin. Our study shows, that certain phase I genes and their enzyme activities are increased by epigenetic modification in HepG2 cells with a concomitant reduction of EMT marker gene SNAIL. The enhancing of liver specific functions in hepatoma cells using epigenetic modifiers opens new opportunities for the usage of cell lines as a potential liver in vitro model for drug testing and development.

Cigarette smoking has been identified as a major risk factor for osteoporosis decades ago. Several studies have shown a direct relationship between cigarette smoking, decreased bone mineral density, and impaired fracture healing. However, the mechanisms behind impaired fracture healing and cigarette smoking are yet to be elucidated. Migration and osteogenesis of mesenchymal stem/stromal cells (MSCs) into the fracture site play a vital role in the process of fracture healing. In human nicotine, the most pharmacologically active and major addictive component present in tobacco gets rapidly metabolized to the more stable cotinine. This study demonstrates that physiological concentrations of both nicotine and cotinine do not affect the osteogenic differentiation of MSCs. However, cigarette smoke exposure induces oxidative stress by increasing superoxide radicals and reducing intracellular glutathione in MSCs, negatively affecting osteogenic differentiation. Although, not actively producing reactive oxygen species (ROS) nicotine and cotinine inhibit catalase and glutathione reductase activity, contributing to an accumulation of ROS by cigarette smoke exposure. Coincubation with N-acetylcysteine or L-ascorbate improves impaired osteogenesis caused by cigarette smoke exposure by both activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and scavenging of ROS, which thus might represent therapeutic targets to support fracture healing in smokers.