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ABSTRACT: Complex regional pain syndrome (CRPS) is an inadequate local response after a limb trauma, which leads to severe pain and autonomic and trophic changes of the affected limb. Autoantibodies directed against human ß2 adrenergic and muscarinic M2 receptors (hß2AR and hM2R) have been described in CRPS patients previously. We analyzed sera from CRPS patients for autoantibodies against hß2AR, hM2R, and endothelial cells and investigated the functional effects of purified IgG, derived from 13 patients with CRPS, on endothelial cells. Eleven healthy controls, 7 radial fracture patients without CRPS, and 10 patients with peripheral arterial vascular disease served as control subjects. The CRPS-IgG, but not control IgG, bound to the surface of endothelial cells ( P < 0.001) and to hß2AR and hM2R ( P < 0.05), the latter being reversed by adding ß2AR and M2R antagonists. The CRPS-IgG led to an increased cytotoxicity and a reduced proliferation rate of endothelial cells, and by adding specific antagonists, the effect was neutralized. Regarding second messenger pathways, CRPS-IgG induced ERK1/2, p38, and STAT1 phosphorylation, whereas AKT phosphorylation was decreased at the protein level. In addition, increased expression of adhesion molecules (ICAM-1 and VCAM-1) on the mRNA level was induced by CRPS-IgG, thus inducing a pro-inflammatory condition of the endothelial cells. Our results show that patients with CRPS not only develop autoantibodies against hß2AR and hM2R, but these antibodies also interfere with endothelial cells, inducing functional effects on these in vitro, and thus might contribute to the pathophysiology of CRPS.
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Autoanticorpos , Síndromes da Dor Regional Complexa , Humanos , Células Endoteliais , Imunoglobulina G , DorRESUMO
Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.
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In the context of an aging population, unhealthy Western lifestyle, and the lack of an optimal surgical treatment, deep osteochondral defects pose a great challenge for the public health system. Biodegradable, biomimetic scaffolds seem to be a promising solution. In this study we investigated the biocompatibility of porous poly-((D,L)-lactide-ε-caprolactone)dimethacrylate (LCM) scaffolds in contrast to compact LCM scaffolds and blank cell culture plastic. Thus, morphology, cytotoxicity and metabolic activity of human mesenchymal stromal cells (MSC) seeded directly on the materials were analyzed after three and six days of culturing. Further, osteoclastogenesis and osteoclastic activity were assessed using reverse-transcriptase real-time PCR of osteoclast-specific genes, EIA and morphologic aspects after four, eight, and twelve days. LCM scaffolds did not display cytotoxic effects on MSC. After three days, metabolic activity of MSC was enhanced on 3D porous scaffolds (PS) compared to 2D compact scaffolds (CS). Osteoclast activity seemed to be reduced at PS compared to cell culture plastic at all time points, while no differences in osteoclastogenesis were detectable between the materials. These results indicate a good cytocompatibility of LCM scaffolds. Interestingly, porous 3D structure induced higher metabolic activity of MSC as well as reduced osteoclast activity.
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Células-Tronco Mesenquimais/citologia , Osteoclastos/citologia , Alicerces Teciduais/química , Materiais Biocompatíveis/química , Caproatos/química , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Lactonas/química , Masculino , Pessoa de Meia-Idade , Osteogênese/fisiologia , PorosidadeRESUMO
Investigations in male patients with fertility disorders revealed a greater risk of osteoporosis. The rodent model of experimental autoimmune-orchitis (EAO) was established to analyze the underlying mechanisms of male infertility and causes of reduced testosterone concentration. Hence, we investigated the impact of testicular dysfunction in EAO on bone status. Male mice were immunized with testicular homogenate in adjuvant to induce EAO (n = 5). Age-matched mice were treated with adjuvant alone (adjuvant, n = 6) or remained untreated (control, n = 7). Fifty days after the first immunization specimens were harvested. Real-time reverse transcription-PCR indicated decreased bone metabolism by alkaline phosphatase and Cathepsin K as well as remodeling of cell-contacts by Connexin-43. Micro computed tomography demonstrated a loss of bone mass and mineralization. These findings were supported by histomorphometric results. Additionally, biomechanical properties of femora in a three-point bending test were significantly altered. In summary, the present study illustrates the induction of osteoporosis in the investigated mouse model. However, results suggest that the major effects on bone status were mainly caused by the complete Freund's adjuvant rather than the autoimmune-orchitis itself. Therefore, the benefit of the EAO model to transfer laboratory findings regarding bone metabolism in context with orchitis into a clinical application is limited.
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Doenças Autoimunes/complicações , Osso e Ossos/metabolismo , Orquite/complicações , Osteoporose/imunologia , Animais , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Doenças Autoimunes/fisiopatologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Orquite/metabolismo , Orquite/patologia , Orquite/fisiopatologia , Osteoporose/diagnóstico por imagem , Microtomografia por Raio-XRESUMO
Bone substitute materials loaded with mediators that stimulate fracture healing are demanded in the clinical treatment in trauma surgery and orthopedics. Brain-derived neurotrophic factor (BDNF) enhances the proliferation and differentiation of mesenchymal stem cells into osteoblast. To load the implants with BDNF, a drug delivery system that allows the release of BDNF under spatiotemporal control would improve functionality. Polyelectrolyte complex nanoparticles (PECNP) have been reported as a suitable drug delivery system. The suitability of PECNP in contact with osteocytes as the main cell type of bone is not known so far. Thus, we aimed to verify that BDNF and PECNP loaded with BDNF (PECNP+BDNF) as well as pure PECNP have no negative effects on osteocytes in vitro. Therefore, the murine osteocyte cell line MLO-Y4 was treated with BDNF and PECNP+BDNF. The effects on proliferation were analyzed by the BrdU test (n = 5). The results demonstrated a significant increase in proliferation 24 h after BDNF application, whereas PECNP+BDNF did not lead to significant changes. Thus, we conclude that BDNF is an appropriate mediator to stimulate osteocytes. Since the addition of PECNP did not affect the viability of osteocytes, we conclude that PECNP are a suitable drug delivery system for bone implants.
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Fator Neurotrófico Derivado do Encéfalo/administração & dosagem , Nanopartículas/química , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Polieletrólitos/química , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismoRESUMO
Allogeneic bone derived from living donors being necessary to match demand for bone transplantation and thermodisinfection of femoral heads is an established sterilization method. During the thermodisinfection the peripheral bone is exposed to maximum 86 °C for 94 min providing 82.5 °C within the center of the femoral head for at least 15 min. This study examined the compression force of the central and representative peripheral regions of native and thermodisinfected human femoral heads to observe wether different duration and intensity of heat exposure might alter mechanic behaviour. Slices from the equatorial region of human femoral heads were taken from each 14 native and thermodisinfected human femoral heads. The central area revealed a significantly higher compression force for native (p ≤ 0.001) and for thermodisinfected bone (p = 0.002 and p = 0.005) compared with peripheral regions since no relevant differences were found between the peripheral and intermediate areas themselves. A small reduction of compression force for thermodisinfected bone was shown since this did not appear significant due to the small number of specimens. The heat exposure did not alter the pre-existing anatomical changes of the microarchitecture of the native femoral heads from the center towards the peripheral regions. The heterogeneity of microstructure of the femoral head might be of interest concerning clinical applications of bone grafts since the difference between native and thermodisinfected bone appears moderate as shown previously. The different quantity of heat exposure did not reveal any significant influence on compression force which might enable thermodisinfection of preformed bone pieces for surgical indications.
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Força Compressiva , Desinfecção , Cabeça do Fêmur/patologia , Temperatura Alta , Fenômenos Biomecânicos , Colágeno Tipo I/metabolismo , HumanosRESUMO
Fracture treatment in osteoporotic patients is still challenging. Osteoporosis emerges when there is an imbalance between bone formation and resorption in favor of resorption by osteoclasts. Thus, new implant materials for osteoporotic fracture treatment should promote bone formation and reduce bone resorption. Nanoparticles can serve as drug delivery systems for growth factors like Brain-Derived Neurotrophic Factor (BDNF), which stimulated osteoblast differentiation. Therefore, polyelectrolyte complex nanoparticles (PEC-NPs) consisting of poly(l-lysine) (PLL) and cellulose sulfate (CS), with or without addition of BDNF, were used to analyze their effect on osteoclasts in vitro. Live cell images showed that osteoclast numbers decreased after application of high PLL/CS PEC-NPs concentrations independent of whether BDNF was added or not. Real-time RT-PCR revealed that relative mRNA expression of cathepsin K and calcitonin receptor significantly declined after incubation of osteoclasts with high concentrations of PLL/CS PEC-NPs. Furthermore, Enzyme-Linked Immunosorbent Assay indicated that tartrate-resistant acidic phosphatase 5b activity was significantly reduced in the presence of high PLL/CS PEC-NPs concentrations. Consistent with these results, the pit formation analysis showed that less hydroxyapatite was resorbed by osteoclasts after incubation with high concentrations of PLL/CS PEC-NPs. BDNF had no influence on osteoclasts. We conclude that highly concentrated PLL/CS PEC-NPs dosages decreased osteoclastogenesis and osteoclasts activity. Moreover, BDNF might be a promising growth factor for osteoporotic fracture treatment since it did not increase osteoclast activity.
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Nanopartículas , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Polieletrólitos/farmacologia , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Expressão Gênica , Humanos , Nanopartículas/química , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Polieletrólitos/químicaRESUMO
Alterations in bone strength and structure were found in knockout (KO) mouse strains with deletion of several acetylcholine receptors. Interestingly, the expression of the nicotinic acetylcholine receptors (nAChR) subunit α10 was down-regulated in osteogenic differentiated mesenchymal stem cells of patients with osteoporosis whereas the expression of subunit α9 was not altered. Since nAChR subunits α9 and α10 are often combined in a functional receptor, we analyzed here the bone of adult female KO mice with single deletion of either nAChR alpha9 (α9KO) or alpha10 (α10KO). Biomechanical testing showed a significant decrease of bending stiffness and maximal breaking force in α9KO compared to their corresponding wild type mice. Furthermore, an increase in trabecular pattern factor (Tb.Pf) and structure model index (SMI) was detected by µCT in α9KO indicating reduced bone mass. On the mRNA level a decrease of Collagen 1α1 and Connexin-43 was measured by real-time RT-PCR in α9KO while no alteration of osteoclast markers was detected in either mouse strain. Using electron microcopy we observed an increase in the number of osteocytes that showed signs of degeneration and cell death in the α9KO compared to their wild type mice, while α10KO showed no differences. In conclusion, we demonstrate alterations in bone strength, structure and bio-marker expression in α9KO mice which imply the induction of osteocyte degeneration. Thus, our data suggest that nAChR containing the α9 subunit might be involved in the homeostasis of osteocytes and therefore in bone mass regulation.
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Osso e Ossos/anatomia & histologia , Deleção de Genes , Receptores Nicotínicos/genética , Fosfatase Alcalina/metabolismo , Animais , Animais Recém-Nascidos , Osso e Ossos/fisiologia , Osso Esponjoso/anatomia & histologia , Osso Cortical/anatomia & histologia , Feminino , Fêmur/anatomia & histologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteócitos/metabolismo , Osteócitos/ultraestrutura , Receptores Nicotínicos/deficiênciaRESUMO
The development of new and better implant materials adapted to osteoporotic bone is still urgently required. Therefore, osteoporotic muscarinic acetylcholine receptor M3 (M3 mAChR) knockout (KO) and corresponding wild type (WT) mice underwent osteotomy in the distal femoral metaphysis. Fracture gaps were filled with a pasty α-tricalcium phosphate (α-TCP)-based hydroxyapatite (HA)-forming bone cement containing mesoporous bioactive CaP-SiO2 glass particles (cement/MBG composite) with or without Brain-Derived Neurotrophic Factor (BDNF) and healing analyzed after 35 days. Histologically, bone formation was significantly increased in WT mice that received the BDNF-functionalized cement/MBG composite compared to control WT mice without BDNF. Cement/MBG composite without BDNF increased bone formation in M3 mAChR KO mice compared to equally treated WT mice. Mass spectrometric imaging showed that the BDNF-functionalized cement/MBG composite implanted in M3 mAChR KO mice was infiltrated by newly formed tissue. Leukocyte numbers were significantly lower in M3 mAChR KO mice treated with BDNF-functionalized cement/MBG composite compared to controls without BDNF. C-reactive protein (CRP) concentrations were significantly lower in M3 mAChR KO mice that received the cement/MBG composite without BDNF when compared to WT mice treated the same. Whereas alkaline phosphatase (ALP) concentrations in callus were significantly increased in M3 mAChR KO mice, ALP activity was significantly higher in WT mice. Due to a stronger effect of BDNF in non osteoporotic mice, higher BDNF concentrations might be needed for osteoporotic fracture healing. Nevertheless, the BDNF-functionalized cement/MBG composite promoted fracture healing in non osteoporotic bone.
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Cimentos Ósseos/uso terapêutico , Fator Neurotrófico Derivado do Encéfalo/uso terapêutico , Fêmur/patologia , Consolidação da Fratura/efeitos dos fármacos , Vidro/química , Fraturas por Osteoporose/tratamento farmacológico , Fosfatase Alcalina/metabolismo , Animais , Cimentos Ósseos/farmacologia , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/enzimologia , Calo Ósseo/patologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Proteína C-Reativa/metabolismo , Modelos Animais de Doenças , Feminino , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fraturas por Osteoporose/sangue , Fraturas por Osteoporose/diagnóstico por imagem , Fraturas por Osteoporose/patologia , Porosidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Muscarínico M3/metabolismo , Espectrometria por Raios X , Microtomografia por Raio-XRESUMO
OBJECTIVES: Donepezil inhibits the acetylcholine degradation molecule acetylcholinesterase (AChE). Clinical studies reported that Alzheimer's disease (AD) patients with hip fractures had improved bone quality and better fracture healing if they were treated with AD medication donepezil. We asked whether mesenchymal stroma cells (MSC) from an osteoporosis sheep model treated with donepezil increased their proliferation rate and mRNA expression. METHODS: Sheep were divided into 4 groups: a) untreated control group, b) sheep with bilateral ovariectomy (OVX), c) sheep with OVX and malnutrition, and d) sheep with OVX, malnutrition, and application of corticosteroid. After 8 months MSC were isolated of iliac crest biopsy, treated with donepezil, and AChE activity, proliferation rate, and mRNA expression were analyzed. RESULTS: Application of donepezil resulted in a significant decrease of AChE activity. Inhibition of AChE did not lead to a significant increase in proliferation. Expression of the osteogenic marker osteocalcin was not regulated by donepezil while the mRNA concentration of collagen was increased. CONCLUSION: AChE inhibition via donepezil resulted in an increased synthesis of osteoid which consists mainly of collagen. Thus, we suppose that increased acetylcholine levels through AChE inhibition do not support MSC proliferation but osteogenic activity probably combined with osteogenic differentiation.
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Inibidores da Colinesterase/farmacologia , Colágeno Tipo I/metabolismo , Donepezila/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoporose/metabolismo , Acetilcolinesterase/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , OvinosRESUMO
BACKGROUND: Anterior knee pain is the most common complication after intramedullary tibial nailing. The cause is often multifactorial and varies among individuals. Violation of the anterior intermeniscal ligament (AIL) during intramedullary tibial nailing might be a possible source of postsurgical anterior knee pain. Although there is a certain ambiguity regarding the importance and function of the AIL, neural structures in the AIL tissue might play a significant role with respect to functional purposes and pain perception. METHODS: We subjected 6 AIL specimens to histologic examination to identify the neural structures that are a mandatory requirement as a source of anterior knee pain. Specifically, we performed three-dimensional immunohistochemical investigation of subtyping, orientation, and detailed characterization of neural structures within the AIL tissue. RESULTS: Histologic and three-dimensional immunohistochemical examinations confirmed the presence of neural structures in all 6 AIL specimens. We identified myelinated and unmyelinated nerve fibers, as well as all types of mechanoreceptors. CONCLUSIONS: Free nerve endings are a mandatory requirement for pain perception as a result of AIL violation during tibial nailing. Our verification of all different types of mechanoreceptors in the AIL tissue makes a role of the ligament in knee joint function and proprioception highly probable. Further investigations are necessary to clarify possible correlations between neural supply and function of the AIL. Violation of the ligament during operative procedures should be avoided, although the significance of the AIL is still debated.
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Fixação Intramedular de Fraturas/efeitos adversos , Articulação do Joelho/fisiopatologia , Ligamentos Articulares/patologia , Mecanorreceptores/patologia , Dor/etiologia , Fraturas da Tíbia/cirurgia , Adulto , Biópsia por Agulha , Feminino , Fixação Intramedular de Fraturas/métodos , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal/métodos , Pessoa de Meia-Idade , Fibras Nervosas/patologia , Dor/patologia , Fraturas da Tíbia/diagnóstico por imagemRESUMO
Staphylococcus aureus can cause wide range of infections from simple soft skin infections to severe endocarditis, bacteremia, osteomyelitis and implant associated bone infections (IABI). The focus of the present investigation was to study virulence properties of S. aureus isolates from acute and chronic IABI by means of their in vivo lethality, in vitro osteoblasts invasion, biofilm formation and subsequently whole genome comparison between high and low virulent strains. Application of insect infection model Galleria mellonella revealed high, intermediate and low virulence phenotypes of these clinical isolates, which showed good correlation with osteoblast invasion and biofilm formation assays. Comparative genomics of selected high (EDCC 5458) and low (EDCC 5464) virulent strains enabled the identification of molecular factors responsible for the development of acute and chronic IABI. Accordingly, the low virulent strain EDCC 5464 harbored point mutations resulting in frame shift mutations in agrC (histidine kinase in agr system), graS (histidine kinase in graSR, a two component system) and efeB (peroxidase in efeOBU operon, an iron acquisition system) genes. Additionally, we found a mobile element (present 11 copies in EDCC 5464) inserted at the end of ß-hemolysin (hlb) and sarU genes, which are involved in the pathogenesis and regulation of virulence gene expression in coordination with quorum sensing system. All these results are in good support with the low virulence behavior of EDCC 5464. From the previous literature, it is well known that agr defective S. aureus clinical strains are isolated from the chronic infections. Similarly, low virulent EDCC 5464 was isolated from chronic implant-associated bone infections infection whereas EDCC 5458 was obtained from acute implant-associated bone infections. Laboratory based in vitro and in vivo results and insights from comparative genomic analysis could be correlated with the clinical conclusion of IABIs and allows evidence-based treatment strategies based on the pathogenesis of the strain to cure life devastating implant-associated infections.
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Osso e Ossos/microbiologia , Genoma Bacteriano/genética , Osteomielite/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Osso e Ossos/patologia , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/genética , Humanos , Sequências Repetitivas Dispersas/genética , Mariposas/microbiologia , Osteoblastos/microbiologia , Osteomielite/patologia , Peroxidase/genética , Proteínas Quinases/genética , Percepção de Quorum/genética , Staphylococcus aureus/isolamento & purificação , Virulência/genética , Sequenciamento Completo do GenomaRESUMO
INTRODUCTION: Treatment of osteoporotic fractures is still challenging and an urgent need exists for new materials, better adapted to osteoporotic bone by adjusted Young's modulus, appropriate surface modification and pharmaceuticals. MATERIALS AND METHODS: Titanium-40-niobium alloys, mechanically ground or additionally etched and titanium-6-aluminium-4-vanadium were analyzed in combination with brain-derived neurotrophic factor, acetylcholine and nicotine to determine their effects on human mesenchymal stem cells in vitro over 21 days using lactate dehydrogenase and alkaline phosphatase assays, live cell imaging and immunofluorescence microscopy. RESULTS: Cell number of human mesenchymal stem cells of osteoporotic donors was increased after 14 d in presence of ground titanium-40-niobium or titanium-6-aluminium-4-vanadium, together with brain-derived neurotrophic factor. Cell number of human mesenchymal stem cells of non osteoporotic donors increased after 21 d in presence of titanium-6-aluminium-4-vanadium without pharmaceuticals. No significant increase was measured for ground or etched titanium-40-niobium after 21 d. Osteoblast differentiation of osteoporotic donors was significantly higher than in non osteoporotic donors after 21 d in presence of etched, ground titanium-40-niobium or titanium-6-aluminium-4-vanadium accompanied by all pharmaceuticals tested. In presence of all alloys tested brain-derived neurotrophic factor, acetylcholine and nicotine increased differentiation of cells of osteoporotic donors and accelerated it in non osteoporotic donors. CONCLUSION: We conclude that ground titanium-40-niobium and brain-derived neurotrophic factor might be most suitable for subsequent in vivo testing.
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Acetilcolina/farmacologia , Ligas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Nicotina/farmacologia , Osteoporose/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Interações Medicamentosas , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade , Imagem MolecularRESUMO
Bone loss varies according to disease and age and these variations affect bone cells and extracellular matrix. Osteoporosis rat models are widely investigated to assess mechanical and structural properties of bone; however, bone matrix proteins and their discrepant regulation of diseased and aged bone are often overlooked. The current study considered the spine matrix properties of ovariectomized rats (OVX) against control rats (Sham) at 16 months of age. Diseased bone showed less compact structure with inhomogeneous distribution of type 1 collagen (Col1) and changes in osteocyte morphology. Intriguingly, demineralization patches were noticed in the vicinity of blood vessels in the OVX spine. The organic matrix structure was investigated using computational segmentation of collagen fibril properties. In contrast to the aged bone, diseased bone showed longer fibrils and smaller orientation angles. The study shows the potential of quantifying transmission electron microscopy images to predict the mechanical properties of bone tissue.
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Matriz Óssea/metabolismo , Colágeno/química , Processamento de Imagem Assistida por Computador , Ovariectomia , Coluna Vertebral/fisiologia , Animais , Densidade Óssea/fisiologia , Calcificação Fisiológica , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Desnutrição/patologia , Osteócitos/metabolismo , Ratos Sprague-DawleyRESUMO
Bone histology of decalcified or undecalcified samples depends on the investigation. However, in research each method provides different information to answer the scientific question. Decalcification is the first step after sample fixation and governs what analysis is later feasible on the sections. Besides, decalcification is favored for immunostaining and in situ hybridization. Otherwise, sample decalcification can be damaging to bone biomaterials implants that contains calcium or strontium. On the other hand, after decalcification mineralization cannot be assessed using histology or imaging mass spectrometry. The current study provides a solution to the hardship caused by material presence within the bone tissue. The protocol presents a possibility of gaining sequential and alternating decalcified and undecalcified sections from the same bone sample. In this manner, investigations using histology, protein signaling, in situ hybridization, and mass spectrometry on the same sample can better answer the intended research question. Indeed, decalcification of sections and grindings resulted in well-preserved sample and biomaterials integrity. Immunostaining was comparable to that of classically decalcified samples. The study offers a novel approach that incites correlative analysis on the same sample and reduces the number of processed samples whether clinical biopsies or experimental animals.
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Materiais Biocompatíveis/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Inclusão em Parafina , Animais , Colágeno/metabolismo , Epitopos , Feminino , Fêmur/efeitos dos fármacos , Fêmur/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Ratos Sprague-Dawley , Coloração pela Prata , Tíbia/metabolismoRESUMO
BACKGROUND: There is increasing evidence that the non-neuronal cholinergic system might be of importance for the pathology of rheumatoid arthritis. The role of M3 muscarinic acetylcholine receptor (M3R) in this regard has, however, not been investigated to date. Thus, in the present study we analyzed if M3R deficiency might have a protective effect on experimentally induced arthritis. METHODS: Collagen antibody-induced arthritis (CAIA) was evoked in M3R-deficient (M3R(-/-)) mice and wild-type (WT) littermates. Severity of arthritis was assessed by scoring of paw swelling. The joints of arthritic and nonarthritic animals were analyzed for histopathological changes regarding synovial tissue, cartilage degradation and bone destruction. Further, gene expression analysis of respective markers was performed. Systemic and local inflammatory response was determined by flow cytometry and immunohistochemistry for leukocytes as well as mRNA and protein measurements for pro-inflammatory cytokines and chemokines. RESULTS: In arthritic M3R(-/-) mice the number of leukocytes, specifically neutrophils, was enhanced even though clinical arthritis score was not significantly different between WT and M3R(-/-) mice with CAIA. In M3R(-/-) mice, levels of neutrophil chemoattractant chemokine C-X-C-motif ligand 2 (CXCL2) as well as the pro-inflammatory cytokine interleukin-6 were already strongly increased in mice with low arthritis score, whereas WT mice only showed prominent expression of these markers when reaching high arthritis scores. Furthermore, arthritic M3R(-/-) mice displayed a stronger degradation of collagen II in the articular cartilage and, most strikingly, histopathological evaluation revealed more severe bone destruction in arthritic mice with M3R deficiency compared to WT littermates. Moreover, in M3R(-/-) mice, gene expression of markers for bone degradation (matrix metalloproteinase 13, cathepsin K and receptor activator of nuclear factor-κB ligand) was already increased in mice with low arthritis score. CONCLUSIONS: Taken together, the present study shows that while M3R(-/-) mice were not protected from CAIA, they had a tendency toward a higher inflammatory response after arthritis induction than WT mice. Further, arthritis-induced joint destruction was significantly stronger in mice with M3R deficiency, indicating that stimulation of M3R might have protective effects on arthritis.
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Artrite Experimental/metabolismo , Artrite Experimental/patologia , Receptor Muscarínico M3/deficiência , Animais , Feminino , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
An improved interfacial drug delivery system (DDS) based on polyelectrolyte complex (PEC) coatings with controlled drug loading and improved release performance was elaborated. The cationic homopolypeptide poly(l-lysine) (PLL) was complexed with a mixture of two cellulose sulfates (CS) of low and high degree of substitution, so that the CS and PLL solution have around equal molar charged units. As drugs the antibiotic rifampicin (RIF) and the bisphosphonate risedronate (RIS) were integrated. As an important advantage over previous PEC systems this one can be centrifuged, the supernatant discarded, the dense pellet phase (coacervate) separated, and again redispersed in fresh water phase. This behavior has three benefits: (i) Access to the loading capacity of the drug, since the concentration of the free drug can be measured by spectroscopy; (ii) lower initial burst and higher residual amount of drug due to removal of unbound drug and (iii) complete adhesive stability due to the removal of polyelectrolytes (PEL) excess component. It was found that the pH value and ionic strength strongly affected drug content and release of RIS and RIF. At the clinically relevant implant material (Ti40Nb) similar PEC adhesive and drug release properties compared to the model substrate were found. Unloaded PEC coatings at Ti40Nb showed a similar number and morphology of above cultivated human mesenchymal stem cells (hMSC) compared to uncoated Ti40Nb and resulted in considerable production of bone mineral. RIS loaded PEC coatings showed similar effects after 24 h but resulted in reduced number and unhealthy appearance of hMSC after 48 h due to cell toxicity of RIS.
RESUMO
BACKGROUND: Use of magnesium for resorbable metal implants is a new concept in orthopaedic and dental medicine. The majority of studies on magnesium's biocompatibility in vitro have assessed the short-term effect of magnesium extract on cells. The aim of this study was to evaluate the influence of direct exposure to magnesium alloys on the bioactivity of primary human reaming debris-derived (HRD) cells. MATERIALS AND METHODS: Pure Mg, Mg2Ag, WE43 and Mg10Gd were tested for biocompatibility. The study consisted of assessment of cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, evaluation of alkaline phosphatase (ALP) content, and study of cell morphology under light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM), along with determination of calcification and pH changes induced by magnesium. RESULTS: The number of viable cells in the presence of Mg2Ag was high over the entire observation period. Inhibition of ALP content in osteogenic differentiating HRD was caused by pure Mg at day 14 and 28. All other magnesium alloys did not affect the ALP content. Exposure of HRD to magnesium increased the amount of lysosomes and endocytotic vesicles. Cellular attachment was generally the best for those crystals that formed on the surface of all materials. A decrease was observed in Ca(2+) in the medium from day 1 to day 14. CONCLUSIONS: In terms of cell morphology, cell viability and differentiation, cell density and the effect on the surrounding pH, Mg2Ag showed the most promising results. All magnesium materials induced calcification, which is beneficial for orthopaedic and dental applications.
Assuntos
Ligas , Magnésio/farmacologia , Teste de Materiais/métodos , Osteogênese/efeitos dos fármacos , Células-Tronco/ultraestrutura , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Humanos , Microscopia Eletrônica de VarreduraRESUMO
Recent studies showed that the non-neuronal cholinergic system (NNCS) is taking part in bone metabolism. Most studies investigated its role in osteoblasts, but up to now, the involvement of the NNCS in human osteoclastogenesis remains relatively unclear. Thus, aim of the present study was to determine whether the application of acetylcholine (ACh, 10(−4) M), nicotine (10(−6) M), mineralized collagen membranes or brain derived neurotrophic factor (BDNF, 40 ng/mL) influences the mRNA regulation of molecular components of the NNCS and the neurotrophin family during osteoclastogenesis. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of young healthy donors (n = 8) and incubated with bone fragments and osteoclast differentiation media for 21 days. All the results are based on the measurement of RNA. Real-time RT-PCR analysis demonstrated a down-regulation of nicotinic acetylcholine receptor (nAChR) subunit α2 and muscarinic acetylcholine receptor (mAChR) M3by osteoclastogenesis while BDNF mRNA expression was not regulated. Application of ACh, nicotine, BDNF or collagen membranes did not affect osteoclastic differentiation.No regulation was detected for nAChR subunit α7, tropomyosin-related kinase receptor B (TrkB), and cholineacetyl transferase (ChAT). Taken together, we assume that the transcriptional level of osteoclastogenesis of healthy young humans is not regulated by BDNF, ACh, and nicotine. Thus, these drugs do not seem to worsen bone degradation and might therefore be suitable as modulators of bone substitution materials if having a positive effect on bone formation.
Assuntos
Acetilcolina/farmacologia , Nicotina/farmacologia , Osteoclastos/fisiologia , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Diferenciação Celular , Colágeno , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Acetylcholinesterase (AChE) hydrolyzes acetylcholine (ACh) to acetate and choline and thereby terminates nerve impulse transmission. ACh is also expressed in bone tissue and enhances here proliferation and differentiation of osteoblasts, which makes it interesting to investigate effects of AChE deficiency on bone. To our knowledge, this is the first study that analyzed bone of heterozygous acetylcholinesterase-knockout (AChE-KO) mice. Tibia, femur, thoracic and lumbar vertebrae of 16-week-old female heterozygous AChE-KO mice and their corresponding wildtypes (WT) were analyzed using real-time RT-PCR, dual-energy X-ray absorptiometry, biomechanics, micro-computed tomography, histology and histomorphometry. Our data revealed that heterozygous AChE-KO did not cause negative effects upon bone parameters analyzed. In contrast, the number of osteoclasts per perimeter was significantly reduced in lumbar vertebrae. In addition, we found a significant decrease in trabecular perimeter of lumbar vertebrae and cortical area fraction (Ct.Ar/Tt.Ar) in the mid-diaphysis of femurs of AChE-KO mice compared to their WT. Therefore, presumably a local homozygous knockout of AChE or AChE-inhibitor administration might be beneficial for bone formation due to ACh accumulation. However, many other bone parameters analyzed did not differ statistically significantly between AChE-KO and WT mice. That might be reasoned by the compensating effect of butyrylcholinesterase (BChE).