RESUMEN

Fused filament fabrication (FFF) represents a straightforward additive manufacturing technique applied in the medical sector for personalized patient treatment. However, frequently processed biopolymers lack sufficient thermal stability to be used as auxiliary devices such as surgical guides. The aim of this study was to evaluate the dimensional accuracy of experimental biocopolyester blends with improved thermal characteristics after printing, annealing and sterilization. A total of 160 square specimens and 40 surgical guides for oral implant placement were printed. One subgroup of each material (n = 10) underwent thermal annealing before both subgroups were subjected to steam sterilization (134 °C; 5 min). Specimens were digitized and the deviation from the original file was calculated. The thermal behavior was analyzed using differential scanning calorimetry and thermogravimetric analysis. A one-way ANOVA and t-tests were applied for statistical analyses (p < 0.05). All biocopolyester blends showed warpage during steam sterilization. However, the material modification with mineral fillers (21-32 wt%) and nucleating agents in combination with thermal annealing showed a significantly reduced warpage of printed square specimens. Geometry of the printing object seemed to affect dimensional accuracy, as printed surgical guides showed less distortion between the groups. In summary, biocopolyesters did benefit from fillers and annealing to improve their dimensional stability.

Asunto(s)

Impresión Tridimensional , Vapor , Humanos , Esterilización

RESUMEN

Fractures of the paranasal sinuses often require surgical intervention. Persisting bone defects lead to permanent visible deformities of the facial contours. Bone substitutes for reconstruction of defects with simultaneous induction of new bone formation are not commercially available for the paranasal sinus. New materials are urgently needed and have to be tested in their future area of application. For this purpose critical size defect models for the paranasal sinus have to be developed. A ≥2.4 cm large bilateral circular defect was created in the anterior wall of the maxillary sinus in six sheep via an extraoral approach. The defect was filled with two types of an osteoconductive titanium scaffold (empty scaffold vs. scaffold filled with a calcium phosphate bone cement paste) or covered with a titanium mesh either. Sheep were euthanized after four months. All animals performed well, no postoperative complications occured. Meshes and scaffolds were safely covered with soft tissue at the end of the study. The initial defect size of ≥2.4 cm only shrunk minimally during the investigation period confirming a critical size defect. No ingrowth of bone into any of the scaffolds was observed. The anterior wall of the maxillary sinus is a region with low complication rate for performing critical size defect experiments in sheep. We recommend this region for experiments with future scaffold materials whose intended use is not only limited to the paranasal sinus, as the defect is challenging even for bone graft substitutes with proven osteoconductivity. Graphical abstract.

Asunto(s)

Sustitutos de Huesos , Ovinos , Animales , Cementos para Huesos , Titanio , Maxilar/cirugía , Fosfatos de Calcio , Regeneración Ósea , Seno Maxilar/cirugía

RESUMEN

Endogenous immune mediated reactions of inflammation and angiogenesis are components of the spinal cord injury in patients with degenerative cervical myelopathy (DCM). The aim of this study was to identify alteration of certain mediators participating in angiogenetic and inflammatory reactions in patients with DCM. A consecutive series of 42 patients with DCM and indication for surgical decompression were enrolled for the study. 28 DCM patients were included, as CSF samples were taken preoperatively. We enrolled 42 patients requiring surgery for a thoracic abdominal aortic aneurysm (TAAA) as neurologically healthy controls. In 38 TAAA patients, CSF samples were taken prior to surgery and thus included. We evaluated the neurological status of patients and controls prior to surgery including NDI and mJOA. Protein-concentrations of factors with a crucial role in inflammation and angiogenesis were measured in CSF via ELISA testing (pg/ml): Angiopoietin 2, VEGF-A and C, RANTES, IL 1 beta and IL 8. Additionally, evaluated the status of the blood-spinal cord barrier (BSCB) by Reibers´diagnostic in all participants. Groups evidently differed in their neurological status (mJOA: DCM 10.1 ± 3.3, TAAA 17.3 ± 1.2, p < .001; NDI: DCM 47.4 ± 19.7, TAAA 5.3 ± 8.6, p < .001). There were no particular differences in age and gender distribution. However, we detected statistically significant differences in concentrations of mediators between the groups: Angiopoietin 2 (DCM 267.1.4 ± 81.9, TAAA 408.6 ± 177.1, p < .001) and VEGF C (DCM 152.2 ± 96.1, TAAA 222.4 ± 140.3, p = .04). DCM patients presented a mild to moderate BSCB disruption, controls had no signs of impairment. In patients with DCM, we measured decreased concentrations of angiogenic mediators. These results correspond to findings of immune mediated secondary harm in acute spinal cord injury. Reduced angiogenic activity could be a relevant part of the pathogenesis of DCM and secondary harm to the spinal cord.

Asunto(s)

Aneurisma de la Aorta Abdominal/sangre , Citocinas/sangre , Neovascularización Fisiológica , Traumatismos de la Médula Espinal/sangre , Anciano , Aneurisma de la Aorta Abdominal/cirugía , Femenino , Humanos , Inflamación/sangre , Inflamación/patología , Inflamación/cirugía , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/cirugía

RESUMEN

The OATS (osteochondral autograft transfer system) is a standardized technique for the treatment of aseptic osteonecrosis of the knee and ankle. We show the first use and successful course of the OAT System for the treatment of a partial aseptic necrosis of a metacarpal head.

Asunto(s)

Trasplante Óseo/instrumentación , Cartílago/trasplante , Osteonecrosis/cirugía , Adolescente , Diseño de Equipo , Femenino , Humanos , Aumento de la Imagen , Metacarpo/anomalías , Metacarpo/diagnóstico por imagen , Metacarpo/cirugía , Oseointegración/fisiología , Osteonecrosis/diagnóstico por imagen , Complicaciones Posoperatorias/diagnóstico por imagen , Tomografía Computarizada por Rayos X

RESUMEN

Little is know about the pathophysiology of acute and degenerative tendon injuries. Although most lesions are uncomplicated, treatment is long and unsatisfactory in a considerable number of cases. Besides the common growth factors that were shown to be relevant for tendon integrity more recently protection against oxidative stress was shown to promote tendon healing. To improve tendon regeneration, many have advocated the use of platelet-rich plasma (PRP), a thrombocyte concentrate that can serve as an autologous source of growth factors. In this study, we investigated the effect of platelet-released growth factors (PRGF) on tenocytes. Tenocytes were isolated from the Achilles tendon of postnatal rats. Tenocyte cell cultures were stimulated with PRGF. We used a CyQuant assay and WST assay to analyse tendon cell growth and viability in different concentrations of PRGF. Migration and proliferation of cells grown in PRGF were assessed by a scratch test. A dual-luciferase assay was used to demonstrate the activation of the anti-oxidant response element (ARE) in tenocytes. A positive effect of PRGF could be shown on tendon cell growth and migratory capacity. PRGF activated the Nrf2-ARE pathway in a dose-dependent manner. Here, we provide evidence of a biological effect of PRGF on tenocytes by the promotion of tenocyte growth and activation of the Nrf2-ARE pathway. This is a novel aspect of the action of platelet concentrates on tendon growth.

Asunto(s)

Antioxidantes/metabolismo , Plaquetas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Elementos de Respuesta/genética , Tendones/citología , Tendones/efectos de los fármacos , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Masculino , Factor 2 Relacionado con NF-E2/metabolismo , Fenotipo , Ratas , Ratas Wistar , Tendones/metabolismo

RESUMEN

AIMS: Trophoblast fusion in the placenta is prerequisite to successful pregnancy and the pathological conditions related to it. The presence of syncytin-1, is not sufficient to explain the complete event and ADAM12 is a major co-player candidate. Via differential splicing, the ADAM12 gene produces a short and a long form, being the ADAM12-S and the ADAM12-L respectively. METHODS AND RESULTS: We investigated the localisation of both variants in the human placenta using whole mount in situ hybridisation, immunohistochemistry and Northern blotting in 1st (n=8) and 3rd (n=8) trimester placentae and in the case of NB in several cell lines. In Northern blotting, 1st and 3rd trimester placentae were positive for the ADAM12-S and Bewo, 293HEK, JAR, leucocytes, macrophages, 1st and 3rd trimester placentae were positive for ADAM12-L. In whole mount in situ hybridisation, the 1st and 3rd trimester placental syncytium was positive for both variants. In immunohistochemistry, ADAM12-L localised in the cytotrophoblast of both 1st and 3rd trimester placentae, while ADAM12-S localised in the complete syncytium, often including the cytotrophoblast. CONCLUSION: The different localisation of ADAM12-S and ADAM12-L indicates a possible different role making ADAM12-L a candidate for the fusion event, while the syncytial localisation of the ADAM12-S makes it a candidate for cell-cell and cell-matrix interactions between the placental syncytium and the maternal interface.

Asunto(s)

Proteínas ADAM/genética , Proteínas de la Membrana/genética , Placenta/fisiología , ARN Mensajero/genética , Proteína ADAM12 , Femenino , Humanos , Embarazo , Isoformas de Proteínas/genética , Distribución Tisular

RESUMEN

Septic arthritis is frequently observed especially in immune-compromised or chronically diseased patients and leads to functional impairment due to tissue destruction. Recently, production of antimicrobial peptides (AMP) was observed in articular cartilage after exposure to bacteria. This report examines the role of synoviocyte-derived AMPs in innate defense mechanisms of articular joints. Samples of healthy, low-grade synovialitis and septic synovial membranes were assessed for the expression of human beta-defensin-2 (HBD-2) and Toll-like receptor-2 and -4 (TLR) by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). A stable synoviocyte line (K4IM) was used for in vitro experiments and assayed for endogenous HBD-2 and TLR production after exposure to inflammatory cytokines or bacterial supernatants by reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, ELISA, and dual luciferase assay. Healthy human synovial membranes and cultured synoviocytes are able to produce HBD-2 and TLR-1-5 at basal expression levels. Samples of bacteria-colonized synovial membranes produce higher levels of HBD-2 when compared with samples of healthy tissues. K4IM synoviocytes exposed to Staphylococcus aureus, Pseudomonas aeruginosa, or proinflammatory cytokines demonstrated a clear HBD-2 transcription and protein induction. TLR-2 and -4 are known to have a critical role in the recognition of gram-positive and gram-negative bacteria in epithelia and are induced in mesenchymal synoviocytes after bacterial exposure on transcription and on protein level. This report demonstrates an unappreciated role of synovial membranes: samples of septic synovial membranes and cultured synoviocytes exposed to bacteria produce increased amounts of the AMP HBD-2 and the bacteria recognition receptors TLR-2 and -4. The induction of anti-inflammatory pathways in infected synoviocytes suggests involvement in intra-articular defense mechanisms.

Asunto(s)

Artritis Infecciosa/metabolismo , Membrana Sinovial/metabolismo , Sinovitis/metabolismo , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 4/biosíntesis , beta-Defensinas/biosíntesis , Artritis Infecciosa/microbiología , Artritis Infecciosa/patología , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , Interleucina-6/farmacología , ARN Mensajero/metabolismo , Infecciones Estafilocócicas/complicaciones , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/patología , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/fisiología , Staphylococcus epidermidis/aislamiento & purificación , Staphylococcus epidermidis/fisiología , Membrana Sinovial/microbiología , Membrana Sinovial/patología , Sinovitis/patología , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/farmacología , beta-Defensinas/genética

RESUMEN

Alpha-Synuclein (alpha-Syn) accounts, as a major component of Lewy bodies (LB), for the filamentous deposits in many cases of neurodegenerative diseases. Yet, little is known about the molecular mechanisms of neuronal loss in these diseases. The correlation between alpha-Syn oligomerization/aggregation and pathologies raises the key question of which molecular form of alpha-Syn (i.e. monomeric alpha-Syn, protofibrils or mature fibrils) represents the damage-inducing culprit in the scenario of synucleinopathies. We show that human alpha-Syn protofibrils (PFs) are potent activators of parallel proinflammatory signalling pathways (p38 and ERK1/2 MAP kinases and NF-kappaB) in microglial cells in vitro. Furthermore, stereotactic injection of alpha-Syn PFs into the substantia nigra of adult rats leads to a profound activation of microglia and adjacent neuronal cell loss, which can be attenuated by the MAP kinase inhibitor semapimod. We propose that the neurodegenerative process of alpha-synucleinopathies involves microglial activation through alpha-Syn released or extruded from cells with pathogenic alpha-Syn metabolism. Compounds that inhibit the MAPK/NF-kappaB pathways might be a promising pharmacological strategy for the treatment of the inflammatory component of synucleinopathies including PD.

Asunto(s)

Hidrazonas/farmacología , Microglía/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , alfa-Sinucleína/metabolismo , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Humanos , Masculino , Microglía/enzimología , Microglía/patología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Neuronas/enzimología , Neuronas/patología , Ratas , Ratas Wistar , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

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.

Asunto(s)

Huesos/química , Osteoblastos/metabolismo , Osteomielitis/inmunología , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 4/fisiología , beta-Defensinas/genética , Corticoesteroides/farmacología , Animales , Huesos/efectos de los fármacos , Huesos/microbiología , Regulación de la Expresión Génica , Humanos , Cinética , Ratones , Osteoblastos/química , Staphylococcus aureus/inmunología , beta-Defensinas/biosíntesis

RESUMEN

Osteomyelitis often causes functional impairment due to tissue destruction. This report demonstrates a novel previously unappreciated role of osteoblasts. Samples of osteomyelitic bone and bacterially challenged osteoblasts produce increased amounts of antimicrobial peptides in order to combat bacterial bone infection. An osteomyelitis mouse model confirmed the osseous induction of the murine homologue of human beta-defensin-2, suggesting a central role in the prevention of bacterial bone infection. Antimicrobial peptides are effectors of the innate defence system and play a key role in host protection at cellular surfaces. Some of them are produced constitutively, whereas others are induced during infection. Human beta-defensins represent a major subclass of antimicrobial peptides and act as a first line of defence through their broad spectrum of potent antimicrobial activity. The aim of the present in-vitro and in-vivo investigations was to study the expression and regulation of human beta-defensin-2 in the case of bacterial bone infection and to analyse the effects of immunosuppressive drugs on bone-derived antimicrobial peptide expression. Samples of healthy human bone, osteomyelitic bone and cultured osteoblasts (hFOB cells) were assessed for the expression of human beta-defensin-2. Regulation of human beta-defensin-2 was studied in hFOB cells after exposure to bacterial supernatants, proinflammatory cytokines and immunosuppressive drugs (glucocorticoids and methotrexate) and was assayed by enzyme-linked immunosorbent assay. An osteomyelitis mouse model was performed to demonstrate the regulation of the murine homologue of human beta-defensin-2, named murine beta-defensin-3, by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Healthy human bone and cultured osteoblasts are able to produce human beta-defensin-2 under standard conditions. Samples of infected bone produce higher levels of endogenous antibiotics, such as human beta-defensin-2, when compared with samples of healthy bone. A clear induction of human beta-defensin-2 was observed after exposure of cultured osteoblasts to gram-positive bacteria or proinflammatory cytokines. Additional treatment with glucocorticoids or methotrexate prevented bacteria-mediated antimicrobial peptide induction in cultured osteoblasts. The osteomyelitis mouse model demonstrated transcriptional upregulation of the murine homologue of human beta-defensin-2, namely murine beta-defensin-3, in bone after intraosseous contamination of the tibia. Human and murine bone have the ability to produce broad-spectrum endogenous antibiotics when challenged by micro-organisms in vitro and in vivo. Immunosuppressive drugs, such as glucocorticoids or methotrexate, may increase the susceptibility to bone infection by decreasing antimicrobial peptide expression levels in case of microbial challenge. The induction of human beta-defensin-2 following bacterial contact suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection.

Asunto(s)

Antiinfecciosos/metabolismo , Huesos/metabolismo , beta-Defensinas/metabolismo , Anciano , Animales , Estudios de Casos y Controles , Línea Celular , Dexametasona/uso terapéutico , Ensayo de Inmunoadsorción Enzimática/métodos , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Inmunosupresores/uso terapéutico , Masculino , Metotrexato/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Modelos Animales , Osteoblastos/metabolismo , Osteomielitis/tratamiento farmacológico , Osteomielitis/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus , beta-Defensinas/genética

RESUMEN

Recent studies suggest that the formyl-peptide-receptor-like-1 (FPRL1) plays an essential role in the inflammatory responses of host defense mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). We therefore analyzed whether amyloid beta1-42 (Abeta1-42) increased the activity of phospholipase D (PLD) via FPRL1, which is an enzyme involved in the secretion, endocytosis and receptor signaling. PLD activity was determined using a transphosphatidylation assay. The internalization of Abeta1-42 via FPRL1 was visualized using fluorescence microscopy and quantified by ELISA (Enzyme Linked Immunosorbent Assay). Determining receptor activity by extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement verified the Abeta1-42-induced activation of FPRL1. We were able to show that Abeta1-42 is rapidly internalized via FPRL1 in astrocytes and microglia. PLD was additionally activated by Abeta1-42 and via FPRL1 in rat glial cells. Furthermore, the ERK1/2 phosphorylation by FPRL1 agonists was dependent on the PLD product phosphatidic acid (PA). Together, these data suggest that PLD plays an important role in the regulation of Abeta1-42-induced endocytosis and FPRL1 receptor signaling.

Asunto(s)

Péptidos beta-Amiloides/metabolismo , Endocitosis/fisiología , Neuroglía/metabolismo , Fragmentos de Péptidos/metabolismo , Fosfolipasa D/metabolismo , Receptores de Formil Péptido/metabolismo , Transducción de Señal/fisiología , Péptidos beta-Amiloides/agonistas , Péptidos beta-Amiloides/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Encéfalo/citología , Células Cultivadas , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Endocitosis/efectos de los fármacos , Humanos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neuroglía/efectos de los fármacos , Oligopéptidos/farmacología , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/farmacología , Ratas , Receptores de Formil Péptido/agonistas , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Alcohol terc-Butílico/farmacología