Pharmacological effects of methysticin and L-sulforaphane through the Nrf2/ARE signaling pathway in MLO-Y4 osteocytes: in vitro study.

BACKGROUND: Oxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway. METHODS: MLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H2O2) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining. RESULTS: The experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H2O2-induced stress conditions. CONCLUSIONS: Nrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

The Contribution of the Nrf2/ARE System to Mechanotransduction in Musculoskeletal and Periodontal Tissues.

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.

Nuclear factor erythroid 2-related factor 2 (Nrf2) deficiency causes age-dependent progression of female osteoporosis.

BACKGROUND: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial transcription factor for cellular redox homeostasis. The association of Nrf2 with elderly female osteoporotic has yet to be fully described. The aim was to elucidate a potential age-dependent Nrf2 contribution to female osteoporosis in mice. METHODS: Eighteen female wild type (WT) and 16 Nrf2-knockout (KO) mice were sacrificed at different ages (12 weeks = young mature adult and 90 weeks = old) to analyze their femurs. The morphological properties (trabecular and cortical) were evaluated by micro-computed tomography (µCT) and compared to gold standard histochemistry analysis. The quasi-static compression tests were performed to calculate the mechanical properties of bones. Additionally, the population of bone resorbing cells and aromatase expression by osteocytes was immunohistochemically evaluated and empty osteocyte lacunae was counted in cortical bone. RESULTS: Old Nrf2-KO mice revealed a significantly reduced trabecular bone mineral density (BMD), cortical thickness, cortical area, and bone fraction compared to old WT mice, regardless of no significant difference in skeletally mature young adult mice between WT and KO. Specifically, while all old WT mice showed thin metaphyseal trabeculae, trabecular bone was completely absent in 60% of old KO mice. Additionally, old KO mice showed significantly more osteoclast-like cells and fewer aromatase-positive osteocytes than WT mice, whereas the occurrence of empty osteocyte lacunae did not differ between both groups. Nrf2-KO mice further showed an age-dependently reduced fracture resilience compared to age-matched WT mice. CONCLUSION: Our results suggest that chronic Nrf2 loss can lead to age-dependent progression of female osteoporosis.

Platelet-Released Growth Factors Influence Wound Healing-Associated Genes in Human Keratinocytes and Ex Vivo Skin Explants.

Platelet-released growth factors (PRGFs) or other thrombocyte concentrate products, e.g., Platelet-Rich Fibrin (PRF), have become efficient tools of regenerative medicine in many medical disciplines. In the context of wound healing, it has been demonstrated that treatment of chronic or complicated wounds with PRGF or PRF improves wound healing in the majority of treated patients. Nevertheless, the underlying cellular and molecular mechanism are still poorly understood. Therefore, we aimed to analyze if PRGF-treatment of human keratinocytes caused the induction of genes encoding paracrine factors associated with successful wound healing. The investigated genes were Semaphorin 7A (SEMA7A), Angiopoietin-like 4 (ANGPLT4), Fibroblast Growth Factor-2 (FGF-2), Interleukin-32 (IL-32), the CC-chemokine-ligand 20 (CCL20), the matrix-metalloproteinase-2 (MMP-2), the chemokine C-X-C motif chemokine ligand 10 (CXCL10) and the subunit B of the Platelet-Derived Growth Factor (PDGFB). We observed a significant gene induction of SEMA7A, ANGPLT4, FGF-2, IL-32, MMP-2 and PDGFB in human keratinocytes after PRGF treatment. The CCL20- and CXCL10 gene expressions were significantly inhibited by PRGF therapy. Signal transduction analyses revealed that the PRGF-mediated gene induction of SEMA7A, ANGPLT4, IL-32 and MMP-2 in human keratinocytes was transduced via the IL-6 receptor pathway. In contrast, EGF receptor signaling was not involved in the PRGF-mediated gene expression of analyzed genes in human keratinocytes. Additionally, treatment of ex vivo skin explants with PRGF confirmed a significant gene induction of SEMA7A, ANGPLT4, MMP-2 and PDGFB. Taken together, these results describe a new mechanism that could be responsible for the beneficial wound healing properties of PRGF or related thrombocytes concentrate products such as PRF.

Platelet-Released Growth Factors Induce Genes Involved in Extracellular Matrix Formation in Human Fibroblasts.

Platelet concentrate products are increasingly used in many medical disciplines due to their regenerative properties. As they contain a variety of chemokines, cytokines, and growth factors, they are used to support the healing of chronic or complicated wounds. To date, underlying cellular mechanisms have been insufficiently investigated. Therefore, we analyzed the influence of Platelet-Released Growth Factors (PRGF) on human dermal fibroblasts. Whole transcriptome sequencing and gene ontology (GO) enrichment analysis of PRGF-treated fibroblasts revealed an induction of several genes involved in the formation of the extracellular matrix (ECM). Real-time PCR analyses of PRGF-treated fibroblasts and skin explants confirmed the induction of ECM-related genes, in particular transforming growth factor beta-induced protein (TGFBI), fibronectin 1 (FN1), matrix metalloproteinase-9 (MMP-9), transglutaminase 2 (TGM2), fermitin family member 1 (FERMT1), collagen type I alpha 1 (COL1A1), a disintegrin and metalloproteinase 19 (ADAM19), serpin family E member 1 (SERPINE1) and lysyl oxidase-like 3 (LOXL3). The induction of these genes was time-dependent and in part influenced by the epidermal growth factor receptor (EGFR). Moreover, PRGF induced migration and proliferation of the fibroblasts. Taken together, the observed effects of PRGF on human fibroblasts may contribute to the underlying mechanisms that support the beneficial wound-healing effects of thrombocyte concentrate products.

Development of convolutional neural networks for recognition of tenogenic differentiation based on cellular morphology.

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.

Platelet-released growth factors protect articular chondrocytes from inflammatory condition.

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)-1ß 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-1ß 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-1ß and IL-6 expressions under inflammatory condition, suggesting the therapeutic role of PRGF for protection from articular cartilage degeneration through anti-inflammatory effects.

Cyclically stretched ACL fibroblasts emigrating from spheroids adapt their cytoskeleton and ligament-related expression profile.

Mechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell-cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.

Adverse Effects of Oxidative Stress on Bone and Vasculature in Corticosteroid-Associated Osteonecrosis: Potential Role of Nuclear Factor Erythroid 2-Related Factor 2 in Cytoprotection.

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. Clinically, high-dose corticosteroid therapy is one of the main culprits behind osteonecrosis of the femoral head (ONFH). Recent Advances: Numerous studies have proposed that such ischemia concerns various intravascular mechanisms. Of all reported risk factors, the involvement of oxidative stress in the irreversible damage suffered by bone-related and vascular endothelial cells during ischemia simply cannot be overlooked. Several articles also have sought to elucidate oxidative stress in relation to ON using animal models or in vitro cell cultures. Critical Issues: However, as far as we know, antioxidant monotherapy has still not succeeded in preventing ONFH in humans. To provide this desideratum, we herein summarize the current knowledge about the influence of oxidative stress on ON, together with data about the preventive effects of administering antioxidants in corticosteroid-induced ON animal models. Moreover, oxidative stress is counteracted by nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent cytoprotective network through regulating antioxidant expressions. Therefore, we also describe Nrf2 regulation and highlight its role in the pathology of ON. Future Directions: This is a review of all available literature to date aimed at developing a deeper understanding of the pathological mechanism behind ON from the perspective of oxidative stress. It may be hoped that this synthesis will spark the development of a prophylactic strategy to benefit corticosteroid-associated ONFH patients. Antioxid. Redox Signal. 35, 357-376.

Platelet-Released Growth Factors and Platelet-Rich Fibrin Induce Expression of Factors Involved in Extracellular Matrix Organization in Human Keratinocytes

Platelet-released growth factor (PRGF) is a thrombocyte concentrate lysate which, like its clinically equivalent variations (e.g., Vivostat PRF® (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®. Together, our study indicates that the induction of ECM-related factors may contribute to the beneficial wound-healing effects of PRGF-based formulations.

Effects of Strontium-Doped ß-Tricalcium Scaffold on Longitudinal Nuclear Factor-Kappa Beta and Vascular Endothelial Growth Factor Receptor-2 Promoter Activities during Healing in a Murine Critical-Size Bone Defect Model.

It was hypothesized that strontium (Sr)-doped ß-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-κB) 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-κB- and VEGFR-2-luciferase reporter mice. The fracture was filled with a 3D-printed ß-TCP scaffold with or without Sr. A bioluminescence in-vivo imaging system was used to sequentially investigate NF-κB and VEGFR-2 expression for two months. After sacrifice, soft and osseous tissue formation in the fracture sites was histologically examined. NF-κB activity increased in the ß-TCP + Sr group in the latter stage (day 40-60). VEGFR-2 activity increased in the + Sr group from days 0-15 but decreased and showed significantly less activity than the ß-TCP and non-scaffold groups from days 40-60. The new bone formation and soft tissue formation in the + Sr group were significantly higher than in the ß-TCP group, whereas the percentage of osseous tissue formation in the ß-TCP group was significantly higher than in the ß-TCP + Sr group. We analyzed longitudinal VEGFR-2 promoter activity and NF-κB 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.

Impact of Uniaxial Stretching on Both Gliding and Traction Areas of Tendon Explants in a Novel Bioreactor.

The effects of mechanical stress on cells and their extracellular matrix, especially in gliding sections of tendon, are still poorly understood. This study sought to compare the effects of uniaxial stretching on both gliding and traction areas in the same tendon. Flexor digitorum longus muscle tendons explanted from rats were subjected to stretching in a bioreactor for 6, 24, or 48 h, respectively, at 1 Hz and an amplitude of 2.5%. After stimulation, marker expression was quantified by histological and immunohistochemical staining in both gliding and traction areas. We observed a heightened intensity of scleraxis after 6 and 24 h of stimulation in both tendon types, though it had declined again 48 h after stimulation. We observed induced matrix metalloproteinase-1 and -13 protein expression in both tendon types. The bioreactor produced an increase in the mechanical structural strength of the tendon during the first half of the loading time and a decrease during the latter half. Uniaxial stretching of flexor tendon in our set-up can serve as an overloading model. A combination of mechanical and histological data allows us to improve the conditions for cultivating tendon tissues.

Different Frequency of Cyclic Tensile Strain Relates to Anabolic/Catabolic Conditions Consistent with Immunohistochemical Staining Intensity in Tenocytes.

Tenocytes are mechanosensitive cells intimately adapting their expression profile and hence, their phenotype to their respective mechanomilieu. The immunolocalization and expression intensity of tenogenic, anabolic and catabolic markers in tenocytes in response to in vitro mechanical loading have not been monitored by immunohistochemical staining (IHC). Thus, we investigated the association between IHC intensities, different stimulation frequencies, and tenogenic metabolism using a versatile mechanical stretcher. Primary tenocytes obtained from murine Achilles tendons were transferred to poly(dimethylsiloxane) (PDMS) elastomeric chamber. Chambers were cyclically stretched by 5% in uniaxial direction at a variation of tensile frequency (1 or 2 Hz) for 3 h. After stretching, cell physiology, IHC intensities of tendon-related markers, and protein level of the angiogenesis marker vascular endothelial growth factor (VEGF) were evaluated. Cell proliferation in tenocytes stimulated with 1 Hz stretch was significantly higher than with 2 Hz or without stretch, while 2 Hz stretch induced significantly reduced cell viability and proliferation with microscopically detectable apoptotic cell changes. The amount of scleraxis translocated into the nuclei and tenomodulin immunoreactivity of tenocytes treated with stretch were significantly higher than of non-stretched cells. The collagen type-1 expression level in tenocytes stretched at 1 Hz was significantly higher than in those cultivated with 2 Hz or without stretching, whereas the matrix metalloproteinase (MMP)-1 and MMP-13 immunoreactivities of cells stretched at 2 Hz were significantly higher than in those stimulated with 1 Hz or without stretching. The secreted VEGF-protein level of tenocytes stretched at 2 Hz was significantly higher than without stretching. Our IHC findings consistent with cell physiology suggest that appropriate stretching can reproduce in vitro short-term tenogenic anabolic/catabolic conditions and allow us to identify an anabolic stretching profile.

Role of Nrf2 in Fracture Healing: Clinical Aspects of Oxidative Stress.

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.

Bone-preserving total hip arthroplasty in avascular necrosis of the hip-a matched-pairs analysis.

PURPOSE: 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. METHODS: 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. RESULTS: 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%. CONCLUSION: 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.

Platelet-released growth factors inhibit proliferation of primary keratinocytes in vitro.

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.

A new multiple trauma model of the mouse.

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-1ß (Interleukin-1ß), 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.

The protective effect of platelet released growth factors and bone augmentation (Bio-Oss®) on ethanol impaired osteoblasts.

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.