RESUMEN
Fracture healing in the aged is associated with a reduced healing capacity, which often results in delayed healing or non-union formation. Many factors may contribute to this deterioration of bone regeneration, including a reduced 'angiogenic trauma response'. The phosphodiesterase-3 (PDE-3) inhibitor cilostazol has been shown to exert pro-angiogenic and pro-osteogenic effects in preclinical studies. Therefore, we herein analyzed in a stable closed femoral fracture model whether this compound also promotes fracture healing in aged mice. Forty-two aged CD-1 mice (age: 16-18 months) were daily treated with 30 mg/kg body weight cilostazol (n = 21) or vehicle (control, n = 21) by oral gavage. At 2 and 5 weeks after fracture, the femora were analyzed by X-ray, biomechanics, micro-computed tomography (µCT), histology, immunohistochemistry, and Western blotting. These analyses revealed a significantly increased bending stiffness at 2 weeks (2.2 ± 0.4 vs. 4.3 ± 0.7 N/mm) and an enhanced bone formation at 5 weeks (4.4 ± 0.7 vs. 9.1 ± 0.7 mm3) in cilostazol-treated mice when compared to controls. This was associated with a higher number of newly formed CD31-positive microvessels (3.3 ± 0.9 vs. 5.5 ± 0.7 microvessels/HPF) as well as an elevated expression of phosphoinositide-3-kinase (PI3K) (3.6 ± 0.8 vs. 17.4 ± 5.5-pixel intensity × 104) and runt-related transcription factor (RUNX)2 (6.4 ± 1.2 vs. 18.2 ± 2.7-pixel intensity × 104) within the callus tissue. These findings indicate that cilostazol accelerates fracture healing in aged mice by stimulating angiogenesis and the expression of PI3K and RUNX2. Hence, cilostazol may represent a promising compound to promote bone regeneration in geriatric patients.
Asunto(s)
Fracturas del Fémur , Fosfatidilinositol 3-Quinasa , Animales , Femenino , Masculino , Ratones , Angiogénesis , Cilostazol/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Curación de Fractura , Fosfatidilinositol 3-Quinasas , Inhibidores de Fosfodiesterasa 3/farmacología , Inhibidores de Fosfodiesterasa 3/uso terapéutico , Microtomografía por Rayos XRESUMEN
Fracture-healing is a highly complex and timely orchestrated process. Non-healing fractures are still a major clinical problem and treatment remains difficult. A 16 Hz extremely low-frequency pulsed electromagnetic field (ELF-PEMF) was identified as non-invasive adjunct therapy supporting bone-healing by inducing reactive oxygen species (ROS) and Ca2+-influx. However, ROS and Ca2+-influx may stimulate neutrophils, the first cells arriving at the wounded site, to excessively form neutrophil extracellular traps (NETs), which negatively affects the healing process. Thus, this study aimed to evaluate the effect of this 16 Hz ELF-PEMF on NET formation. Neutrophils were isolated from healthy volunteers and exposed to different NET-stimuli and the 16 Hz ELF-PEMF. NETs were quantified using Sytox Green Assay and immunofluorescence, Ca2+-influx and ROS with fluorescence probes. In contrast to mesenchymal cells, ELF-PEMF exposure did not induce ROS and Ca2+-influx in neutrophils. ELF-PEMF exposure did not result in basal or enhanced PMA-induced NET formation but did reduce the amount of DNA released. Similarly, NET formation induced by LPS and H2O2 was reduced through exposure to ELF-PEMF. As ELF-PEMF exposure did not induce NET release or negatively affect neutrophils, the ELF-PEMF exposure can be started immediately after fracture treatment.
Asunto(s)
Campos Electromagnéticos , Peróxido de Hidrógeno , Humanos , Especies Reactivas de Oxígeno , Campos Electromagnéticos/efectos adversos , Curación de FracturaRESUMEN
Fracture healing is characterized by an inflammatory phase directly after fracture which has a strong impact on the healing outcome. Neutrophils are strong contributors here and can release neutrophil extracellular traps (NETs). NETs are found after trauma, originally thought to capture pathogens. However, they can lead to tissue damage and impede wound healing processes. Their role in fracture healing remains unclear. In this study, the effect of isolated NETs on the function of bone-forming mesenchymal stem cells (SCP-1 cells) was examined. NETs were isolated from stimulated healthy neutrophils and viability, migration, and differentiation of SCP-1 cells were analyzed after the addition of NETs. NETs severely impaired the viability of SCP-1 cells, induced necrosis and already nontoxic concentrations reduced migration significantly. Short-term incubation with NETs had a persistent negative effect on osteogenic differentiation, as measured by AP activity and matrix formation. The addition of DNase or protease inhibitors failed to reverse the negative effect of NETs, whereas a short febrile-range temperature treatment successfully reduced the toxicity and membrane destruction. Thus, the possible modification of the negative effects of NETs in fracture hematomas could be an interesting new target to improve bone healing, particularly in patients with chronic diseases such as diabetes.
Asunto(s)
Trampas Extracelulares , Hipertermia Inducida , Células Madre Mesenquimatosas , Humanos , Osteogénesis , NeutrófilosRESUMEN
BACKGROUND AND PURPOSE: In fracture healing, ischemia caused by vascular injuries, chronic vascular diseases, and metabolic comorbidities is one of the major risk factors for delayed union and non-union formation. To gain novel insights into the molecular and cellular pathology of ischemic fracture healing, appropriate animal models are needed. Murine models are of particular interest, as they allow to study the molecular aspects of fracture healing due to the availability of both a large number of murine antibodies and gene-targeted animals. Thus, we present the development of an ischemic fracture healing model in mice. MATERIAL AND METHODS: After inducing a mild ischemia by double ligature of the deep femoral artery in CD-1 mice, the ipsilateral femur was fractured by a 3-point bending device and stabilized by screw osteosynthesis. In control animals, the femur was fractured and stabilized without the induction of ischemia. The femora were analyzed at 2 and 5 weeks after fracture healing by means of radiology, biomechanics, histology, and histomorphometry. RESULTS: The surgically induced ischemia delayed and impaired the process of fracture healing. This was indicated by a lower Goldberg score, decreased bending stiffness, and reduced bone callus formation in the ischemic animals when compared with the controls. INTERPRETATION: We introduce a novel ischemic femoral fracture healing model in mice, which is characterized by delayed bone healing. In future, the use of this model may allow both the elucidation of the molecular aspects of ischemic fracture healing and the study of novel treatment strategies.
Asunto(s)
Fracturas del Fémur , Curación de Fractura , Animales , Callo Óseo , Fracturas del Fémur/cirugía , Fijación Interna de Fracturas , Humanos , Isquemia , RatonesRESUMEN
The extracellular matrix regulates cell survival, proliferation, and differentiation. In vitro two-dimensional cell experiments are typically performed on a plastic plate or a substrate of a single extracellular matrix constituent such as collagen or calcium phosphate. As these approaches do not include extracellular matrix proteins or growth factors, they fail to mimic a complex cell microenvironment. The cell-derived matrix is an alternative platform for better representing the in vivo microenvironment in vitro. Standard decellularization of a cell-derived matrix is achieved by combining chemical and physical methods. In this study, we compared the decellularization efficacy of several methods: ammonium hydroxide, sodium dodecyl sulfate (SDS), or Triton X-100 with cold or heat treatment on a matrix of Saos-2 cells. We found that the protocols containing SDS were cytotoxic during recellularization. Heat treatment at 47 °C was not cytotoxic, removed cellular constituents, inactivated alkaline phosphatase activity, and maintained the levels of calcium deposition. Subsequently, we investigated the differentiation efficiency of a direct bone coculture system in the established decellularized Saos-2 matrix, an inorganic matrix of calcium phosphate, and a plastic plate as a control. We found that the decellularized Saos-2 cell matrix obtained by heat treatment at 47 °C enhanced osteoclast differentiation and matrix mineralization better than the inorganic matrix and the control. This simple and low-cost method allows us to create a Saos-2 decellularized matrix that can be used as an in vivo-like support for the growth and differentiation of bone cells.
Asunto(s)
Matriz Extracelular Descelularizada/síntesis química , Osteoblastos/citología , Osteoblastos/fisiología , Ingeniería de Tejidos/métodos , Huesos/citología , Huesos/efectos de los fármacos , Huesos/fisiología , Fosfatos de Calcio/química , Fosfatos de Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacología , Humanos , Osteoblastos/efectos de los fármacos , Osteocitos/citología , Osteocitos/efectos de los fármacos , Osteocitos/fisiología , Células THP-1 , Andamios del Tejido/químicaRESUMEN
Diabetes mellitus is a main risk factor for delayed fracture healing and fracture non-unions. Successful fracture healing requires stimuli from different immune cells, known to be affected in diabetics. Especially, application of mononuclear cells has been proposed to promote wound and fracture healing. Thus, aim was to investigate the effect of pre-/diabetic conditions on mononuclear cell functions essential to promote osteoprogenitor cell function. We here show that pre-/diabetic conditions suppress the expression of chemokines, e.g., CCL2 and CCL8 in osteoprogenitor cells. The associated MCP-1 and MCP-2 were significantly reduced in serum of diabetics. Both MCPs chemoattract mononuclear THP-1 cells. Migration of these cells is suppressed under hyperglycemic conditions, proposing that less mononuclear cells invade the site of fracture in diabetics. Further, we show that the composition of cytokines secreted by mononuclear cells strongly differ between diabetics and controls. Similar is seen in THP-1 cells cultured under hyperinsulinemia or hyperglycemia. The altered secretome reduces the positive effect of the THP-1 cell conditioned medium on migration of osteoprogenitor cells. In summary, our data support that factors secreted by mononuclear cells may support fracture healing by promoting migration of osteoprogenitor cells but suggest that this effect might be reduced in diabetics.
Asunto(s)
Medios de Cultivo Condicionados/metabolismo , Diabetes Mellitus/metabolismo , Curación de Fractura , Monocitos/metabolismo , Animales , Biomarcadores , Movimiento Celular , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL8/metabolismo , Quimiocinas/metabolismo , Quimiotaxis de Leucocito/inmunología , Humanos , Hiperglucemia/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Sistema de Señalización de MAP Quinasas , Monocitos/inmunología , Osteoblastos/metabolismo , Osteogénesis , Células THP-1RESUMEN
Cigarette smoking (CS) is one of the main factors related to avoidable diseases and death across the world. Cigarette smoke consists of numerous toxic compounds that contribute to the development of osteoporosis and fracture nonunion. Exposure to pulsed electromagnetic fields (PEMF) was proven to be a safe and effective therapy to support bone fracture healing. The aims of this study were to investigate if extremely low frequency (ELF-) PEMFs may be beneficial to treat CS-related bone disease, and which effect the duration of the exposure has. In this study, immortalized human mesenchymal stem cells (SCP-1 cells) impaired by 5% cigarette smoke extract (CSE) were exposed to ELF-PEMFs (16 Hz) with daily exposure ranging from 7 min to 90 min. Cell viability, adhesion, and spreading were evaluated by Sulforhodamine B, Calcein-AM staining, and Phalloidin-TRITC/Hoechst 33342 staining. A migration assay kit was used to determine cell migration. Changes in TGF-ß signaling were evaluated with an adenoviral Smad2/3 reporter assay, RT-PCR, and Western blot. The structure and distribution of primary cilia were analyzed with immunofluorescent staining. Our data indicate that 30 min daily exposure to a specific ELF-PEMF most effectively promoted cell viability, enhanced cell adhesion and spreading, accelerated migration, and protected TGF-ß signaling from CSE-induced harm. In summary, the current results provide evidence that ELF-PEMF can be used to support early bone healing in patients who smoke.
Asunto(s)
Cilios/metabolismo , Células Madre Mesenquimatosas/citología , Humo/efectos adversos , Factor de Crecimiento Transformador beta/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cilios/efectos de los fármacos , Cilios/inmunología , Campos Electromagnéticos , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Transducción de Señal/efectos de los fármacos , NicotianaRESUMEN
Peritonitis and peritonitis-associated sepsis are characterized by an increased formation of platelet-neutrophil complexes (PNCs), which contribute to an excessive migration of polymorphonuclear neutrophils (PMN) into the inflamed tissue. An important neutrophilic mechanism to capture and kill invading pathogens is the formation of neutrophil extracellular traps (NETs). Formation of PNCs and NETs are essential to eliminate pathogens, but also lead to aggravated tissue damage. The chemokine receptors CXCR4 and CXCR7 on platelets and PMNs have been shown to play a pivotal role in inflammation. Thereby, CXCR4 and CXCR7 were linked with functional adenosine A2B receptor (Adora2b) signaling. We evaluated the effects of selective CXCR4 and CXCR7 inhibition on PNCs and NETs in zymosan- and fecal-induced sepsis. We determined the formation of PNCs in the blood and, in addition, their infiltration into various organs in wild-type and Adora2b-/- mice by flow cytometry and histological methods. Further, we evaluated NET formation in both mouse lines and the impact of Adora2b signaling on it. We hypothesized that the protective effects of CXCR4 and CXCR7 antagonism on PNC and NET formation are linked with Adora2b signaling. We observed an elevated CXCR4 and CXCR7 expression in circulating platelets and PMNs during acute inflammation. Specific CXCR4 and CXCR7 inhibition reduced PNC formation in the blood, respectively, in the peritoneal, lung, and liver tissue in wild-type mice, while no protective anti-inflammatory effects were observed in Adora2b-/- animals. In vitro, CXCR4 and CXCR7 antagonism dampened PNC and NET formation with human platelets and PMNs, confirming our in vivo data. In conclusion, our study reveals new protective aspects of the pharmacological modulation of CXCR4 and CXCR7 on PNC and NET formation during acute inflammation.
Asunto(s)
Trampas Extracelulares/efectos de los fármacos , Receptor de Adenosina A2B/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Células Cultivadas , Trampas Extracelulares/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismoRESUMEN
A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.
Asunto(s)
Huesos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Redes y Vías Metabólicas/genética , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Huesos/patología , Calcificación Fisiológica/genética , Anhidrasa Carbónica II/genética , Anhidrasa Carbónica II/metabolismo , Catepsina K/genética , Catepsina K/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Osteoblastos/patología , Osteoclastos/patología , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , Células THP-1 , Fosfatasa Ácida Tartratorresistente/genética , Fosfatasa Ácida Tartratorresistente/metabolismo , Andamios del TejidoRESUMEN
Many current-generation biomedical implants are fabricated from the Ti-6Al-4V alloy because it has many attractive properties, such as low density and biocompatibility. However, the elastic modulus of this alloy is much larger than that of the surrounding bone, leading to bone resorption and, eventually, implant failure. In the present study, we synthesized and performed a detailed analysis of a novel low elastic modulus Ti-based alloy (Ti-28Nb-5Zr-2Ta-2Sn (TNZTS alloy)) using a variety of methods, including scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and tensile test. Additionally, the in vitro biocompatibility of the TNZTS alloy was evaluated using SCP-1, SaOs-2, and THP-1 cell lines and primary human osteoblasts. Compared to Ti-6Al-4V, the elastic modulus of TNZTS alloy was significantly lower, while measures of its in vitro biocompatibility are comparable. O2 plasma treatment of the surface of the alloy significantly increased its hydrophilicity and, hence, its in vitro biocompatibility. TNZTS alloy specimens did not induce the release of cytokines by macrophages, indicating that such scaffolds would not trigger inflammatory responses. The present results suggest that the TNZTS alloy may have potential as an alternative to Ti-6Al-4V.
Asunto(s)
Aleaciones/química , Aleaciones/síntesis química , Materiales Biocompatibles/química , Materiales Biocompatibles/síntesis química , Niobio/química , Tantalio/química , Estaño/química , Titanio/química , Circonio/química , Aleaciones/farmacología , Materiales Biocompatibles/farmacología , Módulo de Elasticidad , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales/métodos , Osteoblastos/efectos de los fármacos , Prótesis e Implantes , Propiedades de Superficie , Células THP-1 , Resistencia a la Tracción , Titanio/farmacologíaRESUMEN
Approx. every third hospitalized patient in Europe suffers from musculoskeletal injuries or diseases. Up to 20% of these patients need costly surgical revisions after delayed or impaired fracture healing. Reasons for this are the severity of the trauma, individual factors, e.g, the patients' age, individual lifestyle, chronic diseases, medication, and, over 70 diseases that negatively affect the bone quality. To investigate the various disease constellations and/or develop new treatment strategies, many in vivo, ex vivo, and in vitro models can be applied. Analyzing these various models more closely, it is obvious that many of them have limits and/or restrictions. Undoubtedly, in vivo models most completely represent the biological situation. Besides possible species-specific differences, ethical concerns may question the use of in vivo models especially for large screening approaches. Challenging whether ex vivo or in vitro bone models can be used as an adequate replacement for such screenings, we here summarize the advantages and challenges of frequently used ex vivo and in vitro bone models to study disturbed bone metabolism and fracture healing. Using own examples, we discuss the common challenge of cell-specific normalization of data obtained from more complex in vitro models as one example of the analytical limits which lower the full potential of these complex model systems.
Asunto(s)
Enfermedades Óseas/metabolismo , Remodelación Ósea , Huesos/metabolismo , Curación de Fractura , Animales , Enfermedades Óseas/patología , Enfermedades Óseas/fisiopatología , Huesos/patología , Huesos/fisiopatología , Comunicación Celular , Técnicas de Cultivo de Célula , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Osteocitos/metabolismo , Osteocitos/patología , Técnicas de Cultivo de TejidosRESUMEN
Cigarette smoke (CS) exposure is one of the leading risk factors for human health. Nicotine-containing inhalable products, such as e-cigarettes, can effectively support tobacco harm reduction approaches. However, there are limited comparative data on the effects of the aerosols generated from electronic vapor products (e-vapor) and CS on bone. Here, we report the effects of e-vapor aerosols and CS on bone morphology, structure, and strength in a 6-month inhalation study. Eight-week-old ApoE-/- mice were exposed to aerosols from three different e-vapor formulations-CARRIER (propylene glycol and vegetable glycerol), BASE (CARRIER and nicotine), TEST (BASE and flavor)-to CS from 3R4F reference cigarettes at matched nicotine concentrations (35 µg/L) or to fresh air (Sham) (N = 10 per group). Tibiae were analyzed for bone morphology by µCT imaging, biomechanics by three-point bending, and by histological analysis. CS inhalation caused a significant decrease in cortical and total bone volume fraction and bone density relative to e-vapor aerosols. Additionally, CS exposure caused a decrease in ultimate load and stiffness. In contrast, bone structural and biomechanical parameters were not significantly affected by e-vapor aerosol or Sham exposure. At the dissection time point, there was no significant difference in body weight or tibia bone weight or length among the groups. Histological findings revealed microcracks in cortical bone areas among all exposed groups compared to Sham control. In conclusion, because of the bone-preserving effect of e-vapor aerosols relative to CS exposure, e-vapor products could potentially constitute less harmful alternatives to cigarettes in situations in which bone health is of importance.
Asunto(s)
Huesos/efectos de los fármacos , Fumar Cigarrillos/efectos adversos , Cigarrillo Electrónico a Vapor/toxicidad , Sistemas Electrónicos de Liberación de Nicotina , Humo/efectos adversos , Vapeo/efectos adversos , Animales , Huesos/diagnóstico por imagen , Huesos/patología , Femenino , Exposición por Inhalación , Ratones Noqueados para ApoE , Factores de Tiempo , Microtomografía por Rayos XRESUMEN
Although several researchers have attested deleterious effects of smoking to the musculoskeletal system, the association between smoking and the onset of osteoarthritis (OA) remains unclear. Here, we investigate the effect of cigarette smoke extract (CSE) on primary human chondrocytes. The present study demonstrates that physiological concentrations of CSE (0.1%-10%) inhibit the viability, proliferation, and matrix formation of chondrocytes in a dose- and time-dependent manner. Significant amounts of free radicals were generated by 10% of CSE and led to cell death. A clinical dosage (4 mg/mL) of dexamethasone (Dex) showed toxic effects on chondrocytes, and the long-time treatment by lower doses (4-400 µg/mL) induced hypertrophic changes in the chondrocytes. To substitute Dex, diclofenac (Dic, 1 µg/mL) and acetaminophen (Ace, 10 µg/mL) were tested and did not worsen the metabolic activity of CSE-exposed chondrocytes. Hyaluronic acid (HA, 5 mg/mL) combined with Dic or Ace significantly inhibited the oxidative stress and enhanced the viability and matrix formation of CSE-exposed chondrocytes. This study shows for the first time that CSE mediates the disruption of cartilage through inducing cell death by increasing oxidative stress, and that this effect is fortified by Dex. The deleterious effects of CSE on chondrocytes could be reversed by treatment with HA combined with first-line analgesic/anti-inflammatory agents.
Asunto(s)
Acetaminofén/farmacología , Condrocitos/citología , Diclofenaco/farmacología , Ácido Hialurónico/farmacología , Humo/efectos adversos , Anciano , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Dexametasona/efectos adversos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Cultivo Primario de Células , Productos de TabacoRESUMEN
BACKGROUND: Club Cell protein (CC)16 correlates with lung injury and respiratory complications, which are in part triggered by polymorphonuclear leukocytes (PMNL) in severely traumatized patients (TP). CC16 exerts anti-inflammatory and immunosuppressive effects, however, its influence on PMNL functions after trauma is unknown. Here, we evaluated whether CC16 present in sera from TP could modify the biological functions of PMNL. METHODS: Sera from 16 severely injured TP without pneumonia (no P, n = 8) or with pneumonia (P, n = 8) were collected at admission to emergency department (ED) and 1 day prior pneumonia and pre-incubated with or without anti-CC16 antibody for CC16 neutralization. Samples from the equal post-injury days in the corresponding no P group were used. Neutrophils were isolated from healthy volunteers (HV, n = 5) and incubated with 20% of the serum medium from TP, respectively. In PMNL, CD62L, CD11b/CD18 and CD31 expression, migratory capacity, phagocytosis rate, oxidative burst and apoptosis were investigated. In isolated PMNL, CXCR1 and CXCR2 were neutralized before stimulation with CC16, and oxidative burst, phagocytosis and apoptosis were analyzed in neutrophils and their subsets. RESULTS: Serum from the P group enhanced significantly PMNL migration compared to no P group, while CC16-neutralization further increased the migratory rate of PMNL in both groups. CC16-neutralization increased significantly the expression of CD62L in the P group at ED. Oxidative burst was significantly increased in the P group vs. no P during the study period. CC16 seemed to have no influence on oxidative burst and phagocytosis in TP. However, in a more controlled study design, CC16 induced a significant increase of oxidative burst and a decrease of apoptosis of CD16+ granulocytes. These effects were markedly observed in mature CD16brightCD62Lbright and immune suppressive CD16brightCD62Ldim neutrophils. In mature subset, CXCR1 and CXCR2 neutralization diminished CC16-induced effects. CONCLUSIONS: CC16 in sera from multiply traumatized patients, notably of those with pneumonia, has significant effects on PMNL. The results suggest an association of CC16 with CXCR1 and CXCR2. Our data suggest that CC16 reduces the migratory capacity of PMNL and thus modulates their function in patients with respiratory complications after trauma.
Asunto(s)
Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Uteroglobina/sangre , Adulto , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Humanos , Neutrófilos/fisiología , Neumonía/metabolismo , Uteroglobina/farmacología , Heridas y Lesiones/metabolismoRESUMEN
Chronic back pain is a common disability, which is often accredited to intervertebral disc degeneration. Gold standard interventions such as spinal fusion, which are mainly designed to mechanically seal the defect, frequently fail to restore the native biomechanics. Moreover, artificial implants have limited success as a repair strategy, as they do not alter the underlying disease and fail to promote tissue integration and subsequent native biomechanics. The reported high rates of spinal fusion and artificial disc implant failure have pushed intervertebral disc degeneration research in recent years towards repair strategies. Intervertebral disc repair utilizing principles of tissue engineering should theoretically be successful, overcoming the inadequacies of artificial implants. For instance, advances in the development of scaffolds aided with cells and growth factors have opened up new possibilities for repair strategies. However, none has reached the stage of clinical trials in humans. In this review, we describe the hitches encountered in the musculoskeletal field and summarize recent advances in designing tissue-engineered constructs for promoting nucleus pulposus repair. Additionally, the review focuses on the effect of biomaterial aided with cells and growth factors on achieving effective functional reparative potency, highlighting the ways to enhance the efficacy of these treatments.
Asunto(s)
Dolor de Espalda/genética , Núcleo Celular/genética , Degeneración del Disco Intervertebral/terapia , Núcleo Pulposo/metabolismo , Dolor de Espalda/terapia , Materiales Biocompatibles/uso terapéutico , Humanos , Disco Intervertebral/fisiopatología , Degeneración del Disco Intervertebral/genética , Núcleo Pulposo/patología , Ingeniería de TejidosRESUMEN
It is well established that smoking has detrimental effects on bone integrity and is a preventable risk factor for metabolic bone disorders. Following orthopedic surgeries, smokers frequently show delayed fracture healing associated with many complications, which results in prolonged hospital stays. One crucial factor responsible for fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs) at early stages, a mechanism mediated by transforming growth factor ß (TGF-ß). Although it is known that smokers frequently have decreased TGF-ß levels, little is known about the actual signaling occurring in these patients. We investigated the effect of cigarette smoke on TGF-ß signaling in MSCs to evaluate which step in the pathway is affected by cigarette smoke extract (CSE). Single-cell-derived human mesenchymal stem cell line (SCP-1 cells) were treated with CSE concentrations associated with smoking up to 20 cigarettes a day. TGF-ß signaling was analyzed using an adenovirus-based reporter assay system. Primary cilia structure and downstream TGF-ß signaling modulators (Smad2, Smad3, and Smad4) were analyzed by Western blot and immunofluorescence staining. CSE exposure significantly reduced TGF-ß signaling. Intriguingly, we observed that protein levels of phospho-Smad2/3 (active forms) as well as nuclear translocation of the phospho-Smad3/4 complex decreased after CSE exposure, phenomena that affected signal propagation. CSE exposure reduced the activation of TGF-ß modulators under constitutive activation of TGF-ß receptor type I (ALK5), evidencing that CSE affects signaling downstream of the ALK5 receptor but not the binding of the cytokine to the receptor itself. CSE-mediated TGF-ß signaling impaired MSC migration, proliferation, and differentiation and ultimately affected endochondral ossification. Thus, we conclude that CSE-mediated disruption of TGF-ß signaling in MSCs is partially responsible for delayed fracture healing in smokers.
Asunto(s)
Enfermedades Óseas Metabólicas/etiología , Células Madre Mesenquimatosas/efectos de los fármacos , Transducción de Señal , Contaminación por Humo de Tabaco/efectos adversos , Factor de Crecimiento Transformador beta/metabolismo , Diferenciación Celular , Línea Celular , Movimiento Celular , Proliferación Celular , Cilios/efectos de los fármacos , Cilios/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Proteínas Smad/metabolismoRESUMEN
Musculoskeletal disorders, such as osteoarthritis and intervertebral disc degeneration are causes of morbidity, which concomitantly burdens the health and social care systems worldwide, with massive costs. Link N peptide has recently been described as a novel anabolic stimulator for intervertebral disc repair. In this study, we analyzed the influence on anabolic response, by delivering synthetic Link N encoding mRNA into primary human chondrocytes and mesenchymal stromal cells (SCP1 cells), Furthermore, both cell types were seeded on knitted titanium scaffolds, and the influence of Link N peptide mRNA for possible tissue engineering applications was investigated. Synthetic modified Link N mRNA was efficiently delivered into both cell types and cell transfection resulted in an enhanced expression of aggrecan, Sox 9, and type II collagen with a decreased expression of type X collagen. Interestingly, despite increased expression of BMP2 and BMP7, BMP signaling was repressed and TGFß signaling was boosted by Link N transfection in mesenchymal stromal cells, suggesting possible regulatory mechanisms. Thus, the exogenous delivery of Link N peptide mRNA into cells augmented an anabolic response and thereby increased extracellular matrix synthesis. Considering these findings, we suppose that the cultivation of cells on knitted titanium scaffolds and the exogenous delivery of Link N peptide mRNA into cells could mechanically support the stability of tissue-engineered constructs and improve the synthesis of extracellular matrix by seeded cells. This method can provide a potent strategy for articular cartilage and intervertebral disc regeneration.
Asunto(s)
Condrocitos/metabolismo , ARN Mensajero/metabolismo , Agrecanos/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 7/metabolismo , Línea Celular , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Células Cultivadas , Colágeno Tipo II/metabolismo , Colágeno Tipo X/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , ARN Mensajero/genética , Factor de Transcripción SOX9/metabolismoRESUMEN
Almost all patients with chronic liver diseases (CLD) show altered bone metabolism. Depending on the etiology, this manifests in a severe osteoporosis in up to 75% of the affected patients. Due to high prevalence, the generic term hepatic osteodystrophy (HOD) evolved, describing altered bone metabolism, decreased bone mineral density, and deterioration of bone structure in patients with CLD. Once developed, HOD is difficult to treat and increases the risk of fragility fractures. Existing fractures affect the quality of life and, more importantly, long-term prognosis of these patients, which presents with increased mortality. Thus, special care is required to support the healing process. However, for early diagnosis (reduce fracture risk) and development of adequate treatment strategies (support healing of existing fractures), it is essential to understand the underlying mechanisms that link disturbed liver function with this bone phenotype. In the present review, we summarize proposed molecular mechanisms favoring the development of HOD and compromising the healing of associated fractures, including alterations in vitamin D metabolism and action, disbalances in transforming growth factor beta (TGF-ß) and bone morphogenetic protein (BMP) signaling with histone deacetylases (HDACs) as secondary regulators, as well as alterations in the receptor activator of nuclear factor kappa B ligand (RANKL)-osteoprotegerin (OPG) system mediated by sclerostin. Based on these mechanisms, we give an overview on the limitations of early diagnosis of HOD with established serum markers.
Asunto(s)
Enfermedades Óseas Metabólicas/etiología , Hepatopatías/complicaciones , Animales , Enfermedades Óseas Metabólicas/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Calcio/metabolismo , Humanos , Hepatopatías/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Vitamina D/metabolismoRESUMEN
BACKGROUND: Transepidermal water loss (TEWL) is regarded as one of the most important parameters characterizing skin barrier integrity and has found to be higher in impaired skin barrier function. Reduced or low TEWL instead indicates skin barrier integrity or improvement. We evaluated if different mattresses/hospital beds can influence this skin barrier function by measuring TEWL before and after subjects lying in conventional and microclimate management capable mattresses/hospital beds. METHODS: We included 25 healthy subjects in our study. Measurements were made using Courage & Khazaka Multi Probe Adapter MPA with Tewameter TM300 to determine TEWL before and after the subjects were lying in conventional (Viskolastic® Plus, Wulff Med Tec GmbH, Fedderingen, Germany and Duo™ 2 mattress, Hill-Rom GmbH Essen, Germany) or microclimate management capable mattresses/hospital beds (ClinActiv + MCM™ and PEARLS AFT, Hill-Rom GmbH Essen, Germany). RESULTS: While there was no statistically significant difference in standard mattresses/hospital beds (22.19⯱â¯12.99 and 19.80⯱â¯11.48â¯g/hm2), the decrease of TEWL was statistically significant in both microclimate management capable mattresses/hospital beds we investigated (16.89⯱â¯8.586â¯g/hm2 and 17.41⯱â¯7.203â¯g/hm2) compared to baseline values (35.85⯱â¯24.51â¯g/hm2). CONCLUSION: As higher TEWL announces impaired skin barrier function these findings indicate that the choice of the mattress/hospital bed is important for skin barrier function and microclimate management systems improve skin barrier function of the skin.
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Lechos/microbiología , Epidermis/fisiopatología , Pérdida Insensible de Agua/fisiología , Agua/metabolismo , Adolescente , Adulto , Lechos/normas , Lechos/estadística & datos numéricos , Epidermis/metabolismo , Epidermis/microbiología , Femenino , Alemania , Voluntarios Sanos , Humanos , Masculino , Microclima , Persona de Mediana Edad , Agua/análisisRESUMEN
Several studies have explored the negative effects of cigarette smoke on bone healing; however, the complex pathogenesis still remains unclear. One crucial and primary factor determining effective fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs) into bone-forming cells. Recently, primary cilia, microtubule-based sensory organelles, have been shown to be critical in lineage commitment and differentiation of MSCs. Our present study indicates that exposure to cigarette smoke extract (CSE 0.1-10%) impaired osteogenic differentiation of human mesenchymal stem cell line (SCP-1) and interestingly, also affected primary cilia distribution and integrity in these cells during the differentiation. Furthermore, significant amounts of free radicals generated by CSE could be causative of primary cilia loss since treatment with 0.01% of hydrogen peroxide, a prime free radical in CSE, destroyed primary cilia in these cells. The debilitated differentiation of CSE-exposed SCP-1 cells also correlated with the significantly reduced expression of transcription factor and target genes of primary cilia-specific hedgehog signalling, a key player in osteogenic differentiation. As a treatment strategy, co-incubation of the CSE-exposed SCP-1 cells with the antioxidant resveratrol (1 µM) had a protective effect as it significantly reduced free radical production, protected the primary cilia and enhanced osteogenic differentiation. The current study shows for the first time that cigarette smoke affects primary cilia in human MSCs during osteogenic differentiation and treatment with resveratrol could reverse the effects and enhance differentiation, thus opening up potential therapeutic alternatives to treat fracture healing in smokers, in particularly, when delayed fracture healing is assumed.