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Significance: Osteonecrosis (ON) is characterized by bone tissue death due to disturbance of the nutrient artery. The detailed process leading to the necrotic changes has not been fully elucidated....

Annual Meeting of the German Society of Biomedical Engineering, Leipzig, Germany, 29 Sep 2020 - 1 Oct 2020; Current directions in biomedical engineering 6(3), 200-204 (2020). doi:10.1515/cdbme-2020-3051 special issue: "Proceedings of the 2020 Annual Meeting of the German Society of Biomedical Engineering (29 September – 1 October 2020, Leipzig)", Published by De Gruyter, Berlin

International journal of molecular sciences 23(5), 2827 (2022). doi:10.3390/ijms23052827 special issue: "Special Issue "Recent Advances in Biological Functions of Platelet" / Special Issue Editor: Dr. Isabella Russo, Guest Editor", Published by Molecular Diversity Preservation International, Basel

Mechanosensing plays an essential role in maintaining tissue functions. Across the human body, several tissues (i.e., striated muscles, bones, tendons, ligaments, as well as cartilage) require mechanical loading to exert their physiological functions. Contrary, mechanical unloading triggers pathological remodeling of these tissues and, consequently, human body dysfunctions. At the cellular level, both mechanical loading and unloading regulate a wide spectrum of cellular pathways. Among those, pathways regulated by oxidants such as reactive oxygen species (ROS) represent an essential node critically controlling tissue organization and function. Hence, a sensitive balance between the generation and elimination of oxidants keeps them within a physiological range. Here, the Nuclear Factor-E2-related factor 2/Antioxidant response element (Nrf2/ARE) system plays an essential role as it constitutes the major cellular regulation against exogenous and endogenous oxidative stresses. Dysregulations of this system advance, i.a., liver, neurodegenerative, and cancer diseases. Herein, we extend our comprehension of the Nrf2 system to the aforementioned mechanically sensitive tissues to explore its role in their physiology and pathology. We demonstrate the relevance of it for the tissues’ functionality and highlight the imperative to further explore the Nrf2 system to understand the physiology and pathology of mechanically sensitive tissues in the context of redox biology.

Platelet-released growth factor (PRGF) is a thrombocyte concentrate lysate which, like its clinically equivalent variations (e.g., Vivostat PRF&reg, (platelet-rich fibrin)), is known to support the healing of chronic and hard-to-heal wounds. However, studies on the effect of PRGF on keratinocytes remain scarce. This study aims to identify genes in keratinocytes that are significantly influenced by PRGF. Therefore, we performed a whole transcriptome and gene ontology (GO) enrichment analysis of PRGF-stimulated human primary keratinocytes. This revealed an increased expression of genes involved in extracellular matrix (ECM) organization. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) analysis confirmed the PRGF-mediated induction of selected ECM-related factors such as transforming growth factor beta-induced protein, fibronectin 1, matrix metalloproteinase-9, transglutaminase 2, fermitin family member 1, collagen type I alpha 1 and collagen type XXII alpha 1. PRGF-induced expression of the above factors was influenced by blockade of the epidermal growth factor receptor (EGFR), a receptor playing a crucial role in wound healing. A differential induction of the investigated factors was also detected in skin explants exposed to PRGF and in experimentally generated in vivo wounds treated with Vivostat PRF&reg, Together, our study indicates that the induction of ECM-related factors may contribute to the beneficial wound-healing effects of PRGF-based formulations.

Cell & tissue research (2021). doi:10.1007/s00441-021-03416-9, Published by Springer, Berlin ; Heidelberg

It was hypothesized that strontium (Sr)-doped &beta, tricalcium phosphate (TCP)-based scaffolds have a positive effect on the regeneration of large bone defects (LBD). Readouts in our mice models were nuclear factor-kappa beta (NF-&kappa, B) activity and vascular endothelial growth factor receptor-2 (VEGFR-2) promoter activity during the healing process. A 2-mm critical-size femoral fracture was performed in transgenic NF-&kappa, B- and VEGFR-2-luciferase reporter mice. The fracture was filled with a 3D-printed &beta, TCP scaffold with or without Sr. A bioluminescence in-vivo imaging system was used to sequentially investigate NF-&kappa, B and VEGFR-2 expression for two months. After sacrifice, soft and osseous tissue formation in the fracture sites was histologically examined. NF-&kappa, B activity increased in the &beta, TCP + Sr group in the latter stage (day 40&ndash, 60). VEGFR-2 activity increased in the + Sr group from days 0&ndash, 15 but decreased and showed significantly less activity than the &beta, TCP and non-scaffold groups from days 40&ndash, 60. The new bone formation and soft tissue formation in the + Sr group were significantly higher than in the &beta, TCP group, whereas the percentage of osseous tissue formation in the &beta, TCP group was significantly higher than in the &beta, TCP + Sr group. We analyzed longitudinal VEGFR-2 promoter activity and NF-&kappa, B activity profiles, as respective agents of angiogenesis and inflammation, during LBD healing. The extended inflammation phase and eventually more rapid resorption of scaffold caused by the addition of strontium accelerates temporary bridging of the fracture gaps. This finding has the potential to inform an improved treatment strategy for patients who suffer from osteoporosis.

International journal of molecular sciences 21(8), 2925 (2020). doi:10.3390/ijms21082925 special issue: "Special Issue "Tendon/Ligament Reconstruction by Tissue Engineering" / Special Issue Editor: Prof. Dr. Gundula Schulze-Tanzil", Published by Molecular Diversity Preservation International, Basel

Background Although platelet-released growth factors (PRGF) can protect cells from inflammation or oxidative stress condition, their therapeutic efficacy for articular cartilage degeneration has been little discussed. The purpose of this study was to investigate the effect of PRGF on human articular chondrocytes under inflammatory conditions. Methods Human C-28/I2 chondrocytes were treated with PRGF, the production from liquid-preserved platelet concentrates obtained by platelet apheresis from human volunteers. Cell proliferation/viability, and collagen type (COL) II and SOX9 gene expressions for chondrogenesis were evaluated with different PRGF concentrations. Additionally, in vitro inflammatory condition was mimicked by stimulating the cells with tumor necrosis factor (TNF)-α. Under inflammation, cell viability, TNF-α gene expression, and the protein levels of cytokines including TNF-α, interleukin (IL)-1β and -6, and vascular endothelial growth factor (VEGF) angiogenesis marker, were compared with and without PRGF treatment. Results Cell proliferation/viability, and SOX9 and COL II expressions in chondrocytes stimulated with 10% PRGF were significantly higher than without treatment. Cell viability with 10% PRGF was also statistically higher than without treatment under inflammation. The TNF-α gene expression with 10% PRGF was significantly lower than without treatment under inflammation. The protein levels of endogenous TNF-α with 5% PRGF, IL-1β with 10% PRGF, and IL-6 with 5 and 10% PRGF in chondrocytes were significantly lower than untreated ones under inflammation. The VEGF-protein level in chondrocytes stimulated with 20% PRGF was significantly higher than without treatment under inflammation, while there was no significant difference between with 10% PRGF and without treatment. Conclusions Our results reveal that optimal PRGF treatment leads to the increase of chondrocyte proliferation/viability and chondrogenic markers, while it increased cell viability but reduced IL-1β and IL-6 expressions under inflammatory condition, suggesting the therapeutic role of PRGF for protection from articular cartilage degeneration through anti-inflammatory effects.

Background and objective: The use of automated systems for image recognition is highly preferred for regenerative medicine applications to evaluate stem cell differentiation early in the culturing state with non-invasive methodologies instead of invasive counterparts. Bone marrow-derived mesenchymal stem cells (BMSCs) are able to differentiate into desired cell phenotypes, and thereby promise a proper cell source for tendon regeneration. The therapeutic success of stem cell therapy requires cellular characterization prior to the implantation of cells. The foremost problem is that traditional characterization techniques require cellular material which would be more useful for cell therapy, complex laboratory procedures, and human expertise. Convolutional neural networks (CNNs), a class of deep neural networks, have recently made great improvements in image-based classifications, recognition, and detection tasks. We, therefore, aim to develop a potential CNN model in order to recognize differentiated stem cells by learning features directly from image data of unlabelled cells. Methods: The differentiation of bone marrow mesenchymal stem cells (BMSCs) into tenocytes was induced with the treatment of bone morphogenetic protein-12 (BMP-12). Following the treatment and incubation step, the phase-contrast images of cells were obtained and immunofluorescence staining has been applied to characterize the differentiated state of BMSCs. CNN models were developed and trained with the phase-contrast cell images. The comparison of CNN models was performed with respect to prediction performance and training time. Moreover, we have evaluated the effect of image enhancement method, data augmentation, and fine-tuning training strategy to increase classification accuracy of CNN models. The best model was integrated into a mobile application. Results: All the CNN models can fit the biological data extracted from immunofluorescence characterization. CNN models enable the cell classification with satisfactory accuracies. The best result in terms of accuracy and training time is achieved by the model proposed based on Inception-ResNet V2 trained from scratch using image enhancement and data augmentation strategies (96.80%, 434.55 sec). Conclusion: Our study reveals that the CNN models show good performance by identifying stem cell differentiation. Importantly this technique provides a faster and real-time tool in comparison to traditional methods enabling the adjustment of culture conditions during cultivation to improve the yield of therapeutic stem cells.

Background Chronic alcohol consumption is a known limiting factor for bone healing. One promising strategy to improve bone augmentation techniques with Bio-Oss® in oral and maxillofacial surgery might be the supportive application of platelet-concentrated biomaterials as platelet-released growth factor (PRGF). To address this matter, we performed an in vitro study investigating the protective effects of PRGF and Bio-Oss® in ethanol (EtOH) treated osteoblasts. Methods The SAOS-2 osteosarcoma cell line, with and without EtOH pretreatment was used. The cell viability, proliferation and alkali phosphatase activity (ALP) after application of 0%, 5% and 10% PRGF and Bio-Oss® were assessed. Results The application of PRGF and Bio-Oss® in EtOH impaired osteoblasts showed a significant beneficial influence increasing the viability of the osteoblasts in cell culture. The synergistic effect of Bio-Oss® and 5% PRGF on the proliferation of osteoblasts was also demonstrated. Bio-Oss® only in combination with PRGF increases the alkaline phosphatase (ALP) activity in EtOH pretreated cells. Conclusions These results indicate that the simultaneous application of PRGF and Bio-Oss® inhibits EtOH induced bone healing impairment. Furthermore, in the cells, PRGF induced a protective mechanism which might promote bone regeneration.

Die Huftkopfnekrose kann schon im jungen Alter zu einer sekundaren Coxarthrose fuhren. Mithilfe der vorliegenden Arbeit soll die Diagnose im fruhen Stadium erleichtert werden, in dem ein Gelenkerhalt beispielsweise mit der autologen Stammzelltransplantation noch moglich ist. Kurzschaftendoprothesen und neue Gleitpaarungen geben im Spatstadium Anlass zur Hoffnung auf funktionell gute Ergebnisse.

Fracture healing is a natural process that recapitulates embryonic skeletal development. In the early phase after fracture, reactive oxygen species (ROS) are produced under inflammatory and ischemic conditions due to vessel injury and soft tissue damage, leading to cell death. Usually, such damage during the course of fracture healing can be largely prevented by protective mechanisms and functions of antioxidant enzymes. However, intrinsic oxidative stress can cause excessive toxic radicals, resulting in irreversible damage to cells associated with bone repair during the fracture healing process. Clinically, patients with type-2 diabetes mellitus, osteoporosis, habitual drinkers, or heavy smokers are at risk of impaired fracture healing due to elevated oxidative stress. Although increased levels of oxidative stress markers upon fracture and effects of antioxidants on fracture healing have been reported, a detailed understanding of what causes impaired fracture healing under intrinsic conditions of oxidative stress is lacking. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been identified as a key transcriptional regulator of the expression of antioxidants and detoxifying enzymes. It further not only plays a crucial role in preventing degenerative diseases in multiple organs, but also during fracture healing. This narrative review evaluates the influence of intrinsic oxidative stress on fracture healing and sheds new light on the intriguing role of Nrf2 during bone regeneration in pathological fractures.

Platelet-released growth factors (PRGF) and its related clinically used formulations [e.g. Vivostat platelet-rich fibrin (PRF(®) )] are thrombocyte concentrate lysates that support healing of chronic, hard-to-heal and infected wounds. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound-related bacteria. In this study, we analysed the influence of PRGF on hBD-2 expression in human primary keratinocytes and the influence of Vivostat PRF(®) on hBD-2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD-2 gene and protein expressions in a concentration- and time-dependent manner. The use of blocking antibodies revealed that the PRGF-mediated hBD-2 induction was partially mediated by the epidermal growth factor receptor and the interleukin-6 receptor (IL-6R). Luciferase gene reporter assays indicated that the hBD-2 induction through PRGF required activation of the transcription factor activator protein 1 (AP-1), but not of NF-kappaB. In concordance with these cell culture data, Vivostat PRF(®) induced hBD-2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD-2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds.

Background Blunt trauma is the most frequent mechanism of injury in multiple trauma, commonly resulting from road traffic collisions or falls. Two of the most frequent injuries in patients with multiple trauma are chest trauma and extremity fracture. Several trauma mouse models combine chest trauma and head injury, but no trauma mouse model to date includes the combination of long bone fractures and chest trauma. Outcome is essentially determined by the combination of these injuries. In this study, we attempted to establish a reproducible novel multiple trauma model in mice that combines blunt trauma, major injuries and simple practicability. Methods Ninety-six male C57BL/6 N mice (n = 8/group) were subjected to trauma for isolated femur fracture and a combination of femur fracture and chest injury. Serum samples of mice were obtained by heart puncture at defined time points of 0 h (hour), 6 h, 12 h, 24 h, 3 d (days), and 7 d. Results A tendency toward reduced weight and temperature was observed at 24 h after chest trauma and femur fracture. Blood analyses revealed a decrease in hemoglobin during the first 24 h after trauma. Some animals were killed by heart puncture immediately after chest contusion; these animals showed the most severe lung contusion and hemorrhage. The extent of structural lung injury varied in different mice but was evident in all animals. Representative H&E-stained (Haematoxylin and Eosin-stained) paraffin lung sections of mice with multiple trauma revealed hemorrhage and an inflammatory immune response. Plasma samples of mice with chest trauma and femur fracture showed an up-regulation of IL-1β (Interleukin-1β), IL-6, IL-10, IL-12p70 and TNF-α (Tumor necrosis factor- α) compared with the control group. Mice with femur fracture and chest trauma showed a significant up-regulation of IL-6 compared to group with isolated femur fracture. Conclusions The multiple trauma mouse model comprising chest trauma and femur fracture enables many analogies to clinical cases of multiple trauma in humans and demonstrates associated characteristic clinical and pathophysiological changes. This model is easy to perform, is economical and can be used for further research examining specific immunological questions.

Short-stem hip arthroplasty has the potential advantage of femoral bone stock preservation, especially in view of the expected revisions in the often relatively young patients. Despite short-stem hip prosthesis are increasingly used for total hip arthroplasty, there are no sufficient mid- and long-term results especially for patients with avascular femoral head osteonecrosis. The present study investigates mid-term functional results as well as the revision rate following implantation of a short-stem prosthesis. In the period 06/2005 until 12/2013, a total of 351 short-stem hip prostheses were implanted. The study included 331 complete data sets. A retrospective analysis was performed using the Oxford Hip Score. All revisions were registered. In a total of 331 prostheses, the Oxford Hip Score was “excellent” in 66.2%, “good” in 12.7%, “fair” in 13.0%, and “poor” in 8.2% with a mean follow-up of 57.4 months (SD ± 29.8; range 24–115). In 26 cases, aseptic osteonecrosis of the hip was the indication (7.9%). The Oxford Hip Score was “excellent” in 66.7%, “good” in 0.0%, “fair” in 20.8%, and “poor" in 12.5%. The cumulated five year survival rate was 96.7%. In mid-term observation, the Metha® short-stem prosthesis shows no disadvantage in functional outcome and in survival time compared to a standard hip stem. Providing a correct indication, the Metha® short stem is a valuable option in total hip arthroplasty for younger patients with avascular osteonecrosis of the femoral head. Evaluation has shown no significant differences between aseptic osteonecrosis and other indications.

Current directions in biomedical engineering 4(1), 313-317 (2018). doi:10.1515/cdbme-2018-0076, Published by De Gruyter, Berlin

Autologous thrombocyte concentrate lysates as platelet-released growth factors (PRGF) or Vivostat Platelet Rich Fibrin (PRF®) represent important tools in modern wound therapy, especially in the treatment of chronic, hard-to-heal or infected wounds. Nevertheless, underlying cellular and molecular mechanisms of the beneficial clinical effects of a local wound therapy with autologous thrombocyte concentrate lysates are poorly understood. Recently, we have demonstrated that PRGF induces antimicrobial peptides in primary keratinocytes and accelerates keratinocytes' differentiation. In the present study we analyzed the influence of PRGF on primary human keratinocytes' proliferation. Using the molecular proliferation marker Ki-67 we observed a concentration- and time dependent inhibition of Ki-67 gene expression in PRGF treated primary keratinocytes. These effects were independent from the EGFR- and the IL-6-R pathway. Inhibition of primary keratinocytes' proliferation by PRGF treatment was confirmed in colorimetric cell proliferation assays. Together, these data indicate that the clinically observed positive effects of autologous thrombocytes concentrates in the treatment of chronic, hard-to-heal wounds are not based on an increased keratinocytes proliferation.

Chronic alcohol consumption is a known limiting factor for bone healing. One promising strategy to improve bone augmentation techniques with Bio-OssThe SAOS-2 osteosarcoma cell line, with and without EtOH pretreatment was used. The cell viability, proliferation and alkali phosphatase activity (ALP) after application of 0%, 5% and 10% PRGF and Bio-OssThe application of PRGF and Bio-OssThese results indicate that the simultaneous application of PRGF and Bio-Oss

International journal of molecular sciences 15(9), 15456-15474 (2014). doi:10.3390/ijms150915456, Published by Molecular Diversity Preservation International, Basel

Introduction Oxidative stress can impair fracture healing. To protect against oxidative damage, a system of detoxifying and antioxidative enzymes works to reduce the cellular stress. The transcription of these enzymes is regulated by antioxidant response element (ARE). The nuclear factor (erythroid-derived 2)-like2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes. Recently it has been shown that vascular endothelial growth factor (VEGF) prevents oxidative damage via activation of the Nrf2 pathway in vitro. Platelet-released growth factor (PRGF) is a mixture of autologous proteins and growth factors, prepared from a determined volume of platelet-rich plasma (PRP). It has already used to enhance fracture healing in vitro. The aim of the present study was to elucidate if platelets can lead to upregulation of VEGF and if platelets can regulate the activity of Nrf2–ARE system in primary human osteoblast (hOB) and in osteoblast-like cell line (SAOS-2). Methods Platelets and PRGF were obtained from healthy human donors. HOB and SAOS-2 osteosarcoma cell line were used. The ARE activity was analysed using a dual luciferase reporter assay system. We used Western blot to detect the nuclear accumulation of Nrf2 and the amount of cytosolic antioxidant Thioredoxin Reductase-1 (TXNRD-1), Heme Oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO1). Gene expression analysis was performed by real-time RT PCR. ELISA was used for the quantification of growth factors. Results The activity of ARE was increased in the presence of PRGF up to 50%. Western blotting demonstrated enhanced nuclear accumulation of Nrf2. This was followed by an increase in the protein expression of the aforementioned downstream targets of Nrf2. Real-time RT PCR data showed an upregulation in the gene expression of the VEGF after PRGF treatment. This was confirmed by ELISA, where the treatment with PRGF induced the protein level of VEGF in both cells. Conclusions These results provide a new insight into PRGF's mode of action in osteoblasts. PRGF not only leads to increase the endogenous VEGF, but also it may be involved in preventing oxidative damage through the Nrf2–ARE signalling. Nrf2 activation via PRGF may have great potential as an effective therapeutic drug target in fracture healing.

Background Fracture healing is a complex biological process with specific temporal expression patterns. During this process new bone tissue is formed, which is similar to the original bone in quality and structure. This occurs in four phases: inflammation, formation of a soft tissue callus, formation of a bony callus and remodelling of the bony callus. This needs the precise orchestration of each cell type involved. Objectives This article presents details of the fracture healing phases and the relevant factors. During the aging process there is an increase of reactive oxygen species and a change in expression pattern of growth factors that have a negative effect on the fracture healing process. Methods A selective review of the literature was carried out in PubMed concerning the influence of aging on fracture healing. Conclusion The healing process is regulated by systemic and local factors. An understanding of these processes and the changes during aging is necessary in order to improve the knowledge of delayed or lack of fracture healing during aging to decide when an intervention is needed.

Mediators of inflammation 2017, 5671615 (2017). doi:10.1155/2017/5671615, Published by Hindawi Publishing Corp., Sylvania, Ohio

BMC musculoskeletal disorders 18(1), 468 (2017). doi:10.1186/s12891-017-1813-9, Published by BioMed Central, London

Mediators of inflammation 2017, 6157491 (2017). doi:10.1155/2017/6157491, Published by Hindawi Publishing Corp., Sylvania, Ohio

Background Although there are many studies discussing the etiological and pathological factors leading to both, acute and chronic tendon injuries, the pathophysiology of tendon injuries is still not clearly understood. Although most lesions are uncomplicated, treatment is long and unsatisfactory due to the poor vascularity of tendon tissue. Platelet mediator concentrate (PMC) contains many growth factors derived from platelets, which can promote wound healing. In this study we investigate the effects of PMC on tenocyte proliferation and differentiation in order to provide an experimental basis for tissue regeneration strategies and to develop new treatment concepts. Methods Using enzyme linked immunosorbent assay (ELISA) we were able to quantify the several growth factors and cytokines found in PMC. Tenocytes were isolated both from human and from mouse Achilles tendons and stimulated with PMC. CyQuant® and Cell Titer Blue® assays were carried out to analyze tendon growth and viability at different concentrations of PMC. Real time RT-PCR was used to analyze tenocyte gene expression with or without PMC treatment. Immunohistochemistry was carried out to detect the tenocyte-specific antibody tenomodulin (TNMD) and scleraxis (SCX). Results We were able to detect numerous mediators such as platelet derived growth factor BB (PDGF-BB), interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF-α), transforming growth factor beta 1 (TGF-ß1), and bone morphogenetic proteins 2, 4 and 7 (BMP-4, BMP-2, BMP-7) in PMC. It was possible to show a positive effect of PMC on human tendon cell growth and viability in a dose-dependent manner. Furthermore, PMC treatment led to induction of gene expression of scleraxis (SCX), type I collagen A 1 (Col1A1) and TNMD by tenocytes. Conclusions We suggest that the use of autologous PMC may be a suitable addition to conventional tendon therapy that is capable of increasing and optimizing tendon healing and reducing the risk of recurrence.

Background Thrombocyte concentrate i.e. platelet-rich plasma (PRP) has become a popular adjunct for many surgical procedures. It is believed to improve bone and soft tissue healing. Recently antimicrobial effects of the autologous preparation were reported by several groups. In this study we investigated the antimicrobial effect of PRP against gram-negative microbes which frequently cause severe complications in orthopaedic trauma surgery. Methods Platelet-rich plasma was produced from liquid preserved thrombocyte concentrates. ELISA, Western blot and immunohistochemistry were preformed to investigate the release and content of platelet concentrates. A radial diffusion assay was used to detect antimicrobial effects of PRP. Results We detected the human beta defensin-3 in bactericidal concentrations in platelet preparations by ELISA, Western blot and immunohistochemistry. In antimicrobial testing we demonstrated effective inhibition of Escherichia coli (ATCC 11303), Bacterium megaterium (ATCC 14581), Klebsiella pneumoniae (ATCC 13883), Enterococcus faecalis (ATCC 29212) and Proteus mirabilis (ATCC21100). Conclusion With this study we demonstrate antimicrobial action of a popular adjunct for orthopaedic and trauma surgery against gram-positive and gram-negative bacteria. We have identified a possible mechanism of action via the secretion of HBD-3 as a first line defence in contaminated wounds and in elective application of PRP. This finding supports a broader spectrum of clinical indications for an autologous platelet preparation.

IL-6 gene expression is controlled by a promoter region containing multiple regulatory elements such as NF-κB, NF-IL6, CRE, GRE, and TRE. In this study, we demonstrated that TRE, found within the IL-6 promoter, is embedded in a functional antioxidant response element (ARE) matching an entire ARE consensus sequence. Further, point mutations of the ARE consensus sequence in the IL-6 promoter construct selectively eliminate ARE but not TRE activity. Nrf2 is a redox-sensitive transcription factor which provides cytoprotection against electrophilic and oxidative stress and is the most potent activator of ARE-dependent transcription. Using Nrf2 knock-out mice we demonstrate that Nrf2 is a potent activator of IL-6 gene transcription in vivo. Moreover, we show evidence that Nrf2 is the transcription factor that activates IL6 expression in a cholestatic hepatitis mouse model. Our findings suggest a possible role of IL-6 in oxidative stress defense and also give indication about an important function for Nrf2 in the regulation of hematopoietic and inflammatory processes.

Background: The healing potential in the avascular regions of the meniscus is very limited, and improving the vascularity might be a reasonable way to improve healing. Vascular endothelial growth factor (VEGF) is one of the most potent proangiogenetic factors. We hypothesized that the local application of VEGF165 would (1) improve the healing of a lesion in the avascular region of the meniscus, (2) induce angiogenesis in both the avascular and vascular regions, and (3) increase the amounts of VEGF mRNA and VEGF. Methods: In eighteen sheep, the medial menisci were cut longitudinally in the avascular region and were sutured. Three groups were established depending on the suture material: (1) uncoated Ethibond, (2) Ethibond coated with VEGF165 and its carrier Poly(D,L–Lactide) (PDLLA), and (3) Ethibond coated with PDLLA. The contralateral medial menisci served as a control group. Each of the three suture type groups included six animals. After eight weeks, the sheep were killed, and the menisci were examined macroscopically. Immunohistochemistry of Factor VIII and VEGF and real-time reverse-transcription polymerase chain reaction (RT-PCR) of VEGF mRNA were performed. Additionally, the VEGF release kinetics from the VEGF/PDLLA-coated suture were evaluated in vitro. Results: In this model, VEGF did not improve meniscal healing. It did not increase angiogenesis in the avascular or vascular region, the VEGF concentration, or the amount of VEGF mRNA. VEGF release from the coated suture peaked on Day 3 and was nearly zero on Day 9. Conclusions: The local application of VEGF165 as eluted from suture did not increase meniscal angiogenesis or improve meniscal healing. In addition, there was no effect on the amount of VEGF mRNA and VEGF. The VEGF carrier (PDLLA) may have been inadequate because of the short duration of VEGF supply. Clinical Relevance: This study shows that the application of VEGF at one time point might not be a solution to improve meniscal healing.

Gram-positive bacterial bone infections are an important cause of morbidity particularly in immunocompromised patients. Antimicrobial peptides (AP) are effectors of the innate immune system and directly kill microorganisms in the first hours after microbial infection. The aim of the present investigation was to study the expression and regulation of gram-positive specialized human beta-defensin-3 (HBD-3) in bone. Samples of healthy and osteomyelitic human bone were assessed for the expression of HBD-3. Using primary and immortalized osteoblasts (SAOS-2 cells), release and regulation of HBD-3 was evaluated after exposure to Staphylococcus aureus supernatant and/or corticosteroids using PCR, immunohistochemistry, Western blot and ELISA. To determine the role of toll-like-receptors-2 and -4 (TLR-2/-4), shRNA was used to downregulate TLRs. An osteomyelitis mouse model was created performed to investigate the release of murine beta-defensins using immunohistochemistry and RT-PCR. Cultured osteoblasts and human bone produce HBD-3 under standard conditions. The release increases within hours of bacterial supernatant exposure in cultured osteoblasts. This observation was not made in chronically infected bone samples. The shRNA-technology revealed the necessity of TLR-2 and -4 in HBD-3 induction in osteoblasts. Blocking protein synthesis with cycloheximide showed that the rapid release of HBD-3 is not dependent on a translational de novo synthesis and is not affected by glucocorticoids. The murine osteomyelitis model confirmed the in vivo release uptake of mouse beta-defensins-4 (MBD-4) in bone. This report shows the bacterial induction of HBD-3 via TLR-2 and -4 in osteoblasts and suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection. The rapid and effective induction of HBD-3 in osteoblasts incubated with conditioned media from bacteria is more likely a result of a rapid secretion of preformed HBD-3 by osteoblasts rather than a result of enhanced biosynthesis. The increased incidence of gram-positive bacterial bone infection in patients with regular intake of glucocorticoids does not seem to be caused by a deranged HBD-3 release in osteoblasts.

Background Abrasion arthroplasty (AAP) is a procedure by which intrinsic cartilage healing is believed to be stimulated. Although clinically accepted for degenerative and traumatic cartilage lesions scientific evidence at a molecular level that proves the effect of AAP is scarce. Method Mononuclear cells were extracted from postoperative joint effusions 21.5 h post AAP and simple debridement of cartilage lesions. Luminex, ELISA and FACS experiments were performed. Immunohistochemical stainings of cell cultures for cartilage markers were used to confirm the findings. Results Postoperative joint effusions after AAP showed increased contents of Mononuclear cells compared to Arthroscopic Chondroplasty (ACP). BMP-4 and IGF were increased in AAP as complared to ACP. Mononuclear cells isolated after AAP express the MSC markers CD 73, CD 105, CD 90, CD 44 and are CD34 negative. Chondrogenic differentiation was demonstrated by positive staining for Sox9, collagen II, proteoglycan, chondroitin-4-sulfate. Conclusion Our results support the clinical application of AAP as a procedure that enhances cartilage repair as an alternative to far more complex procedures that have gained popularity. Furthermore the data presented supports clinical investigations that recommend not to use suction drainage as by this procedure a considerable amount of the regeneratory potential of postoperative joint effusions might be extracted.

Mediators of inflammation 2015, 1-7 (2015). doi:10.1155/2015/132451, Published by Hindawi Publishing Corp., Sylvania, Ohio

Background Musculoskeletal injuries induce systemic inflammation which often impairs lung function contributing to morbidity. IL-10 has been shown to have a beneficial effect on immune dysfunction and organ damage after different traumatic insults. We sought to investigate the effect of inhalative IL-10 administration on the systemic and pulmonary inflammatory response in a small animal model of bilateral femoral fracture. Materials and methods Male C57/BL6 mice (6 animals per group) were subjected to bilateral femoral fracture and intramedullary nailing followed by inhalative administration of either 50 μL PBS (Fx group) or 50 μg/kg recombinant mouse IL-10 dissolved in 50 μL PBS (FxIL-10 group). All animals were sacrificed at 6, 24, or 72 h after fracture induction. Blood samples were collected and analyzed for IL-6, IL-10, KC, and MCP-1 (CCL2) plasma concentrations by Bio-Plex Pro™ assays. Pulmonary infiltration by neutrophils was assessed by myeloperoxidase (MPO) activity (ELISA) and histological analysis of lung tissue. Pulmonary ICAM-1 expression (immunohistochemistry), and pulmonary IL-6 levels (ELISA) were determined. Results Inhalative IL-10 administration showed a decrease in the pulmonary infiltration by neutrophils. A significant decrease in the expression of the adhesion molecule ICAM-1 after local IL-10 application was observed. In contrast, local IL-10 administration did not show a significant effect on the systemic inflammatory response. Conclusion Our findings suggest that inhalative IL-10 administration may beneficially modulate the pulmonary microenvironment, in which IL-10 effect on the local ICAM-1 expression seems to play a central role.

The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant ofStaphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 andS. aureusinduced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction.

To improve skin flap healing, one promising strategy in reconstructive surgery might be to optimize platelet rich plasma (PRP) bioactivity and the ischemia-altered expression of genes. We studied both the effect of PRP on ischemic flaps, and whether in vivo bioluminescence imaging (BLI) is a suitable method for the longitudinal monitoring of angiogenesis in surgical wounds. Axial murine skin flaps were created in four experimental groups. In vivo measurements of VEGFR2 expression levels were made every other day until the 14th day. The local VEGF level and microvessel density were quantified on the 14th day via ELISA and immunohistochemistry, and flap survival rates were measured. We demonstrated that PRP and induced ischemia have a beneficial influence on angiogenesis and flap healing. Combining the two resulted in a significantly robust increase in angiogenesis and flap survival rate that was corroborated by bioluminescence imaging of VEGFR2 activity. This study shows that angiogenic effects of PRP may be potentialized by the stimulus of induced ischemia during free flap harvesting, and thus the two procedures appear to have a synergistic effect on flap healing. This study further demonstrates that BLI of modulated genes in reconstructive surgery is a valuable model for longitudinal in vivo evaluation of angiogenesis.