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1.
J Bone Miner Res ; 2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33534144

RESUMO

After trauma, the formed fracture hematoma within the fracture gap contains all the important components (immune/stem cells, mediators) to initiate bone regeneration immediately. Thus, it is of great importance but also the most susceptible to negative influences. To study the interaction between bone and immune cells within the fracture gap, up-to-date in vitro systems should be capable of recapitulating cellular and humoral interactions and the physicochemical microenvironment (eg, hypoxia). Here, we first developed and characterized scaffold-free bone-like constructs (SFBCs), which were produced from bone marrow-derived mesenchymal stromal cells (MSCs) using a macroscale mesenchymal condensation approach. SFBCs revealed permeating mineralization characterized by increased bone volume (µCT, histology) and expression of osteogenic markers (RUNX2, SPP1, RANKL). Fracture hematoma (FH) models, consisting of human peripheral blood (immune cells) mixed with MSCs, were co-cultivated with SFBCs under hypoxic conditions. As a result, FH models revealed an increased expression of osteogenic (RUNX2, SPP1), angiogenic (MMP2, VEGF), HIF-related (LDHA, PGK1), and inflammatory (IL6, IL8) markers after 12 and 48 hours co-cultivation. Osteogenic and angiogenic gene expression of the FH indicate the osteoinductive potential and, thus, the biological functionality of the SFBCs. IL-6, IL-8, GM-CSF, and MIP-1ß were detectable within the supernatant after 24 and 48 hours of co-cultivation. To confirm the responsiveness of our model to modifying substances (eg, therapeutics), we used deferoxamine (DFO), which is well known to induce a cellular hypoxic adaptation response. Indeed, DFO particularly increased hypoxia-adaptive, osteogenic, and angiogenic processes within the FH models but had little effect on the SFBCs, indicating different response dynamics within the co-cultivation system. Therefore, based on our data, we have successfully modeled processes within the initial fracture healing phase in vitro and concluded that the cross-talk between bone and immune cells in the initial fracture healing phase is of particular importance for preclinical studies. © 2021 American Society for Bone and Mineral Research (ASBMR).

2.
Int J Mol Sci ; 21(23)2020 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-33256266

RESUMO

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) signaling pathway serves as an important downstream mediator for a variety of cytokines, hormones, and growth factors. Emerging evidence suggests JAK/STAT signaling pathway plays an important role in bone development, metabolism, and healing. In this light, pro-inflammatory cytokines are now clearly implicated in these processes as they can perturb normal bone remodeling through their action on osteoclasts and osteoblasts at both intra- and extra-articular skeletal sites. Here, we summarize the role of JAK/STAT pathway on development, homeostasis, and regeneration based on skeletal phenotype of individual JAK and STAT gene knockout models and selective inhibition of components of the JAK/STAT signaling including influences of JAK inhibition in osteoclasts, osteoblasts, and osteocytes.

3.
Int J Mol Sci ; 22(1)2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374446

RESUMO

Adequate tissue engineered models are required to further understand the (patho)physiological mechanism involved in the destructive processes of cartilage and subchondral bone during rheumatoid arthritis (RA). Therefore, we developed a human in vitro 3D osteochondral tissue model (OTM), mimicking cytokine-induced cellular and matrix-related changes leading to cartilage degradation and bone destruction in order to ultimately provide a preclinical drug screening tool. To this end, the OTM was engineered by co-cultivation of mesenchymal stromal cell (MSC)-derived bone and cartilage components in a 3D environment. It was comprehensively characterized on cell, protein, and mRNA level. Stimulating the OTM with pro-inflammatory cytokines, relevant in RA (tumor necrosis factor α, interleukin-6, macrophage migration inhibitory factor), caused cell- and matrix-related changes, resulting in a significantly induced gene expression of lactate dehydrogenase A, interleukin-8 and tumor necrosis factor α in both, cartilage and bone, while the matrix metalloproteases 1 and 3 were only induced in cartilage. Finally, application of target-specific drugs prevented the induction of inflammation and matrix-degradation. Thus, we here provide evidence that our human in vitro 3D OTM mimics cytokine-induced cell- and matrix-related changes-key features of RA-and may serve as a preclinical tool for the evaluation of both new targets and potential drugs in a more translational setup.

4.
Int J Mol Sci ; 21(21)2020 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-33113770

RESUMO

Rheumatoid arthritis (RA) is a chronic, inflammatory, and systemic autoimmune disease that affects the connective tissue and primarily the joints. If not treated, RA ultimately leads to progressive cartilage and bone degeneration. The etiology of the pathogenesis of RA is unknown, demonstrating heterogeneity in its clinical presentation, and is associated with autoantibodies directed against modified self-epitopes. Although many models already exist for RA for preclinical research, many current model systems of arthritis have limited predictive value because they are either based on animals of phylogenetically distant origin or suffer from overly simplified in vitro culture conditions. These limitations pose considerable challenges for preclinical research and therefore clinical translation. Thus, a sophisticated experimental human-based in vitro approach mimicking RA is essential to (i) investigate key mechanisms in the pathogenesis of human RA, (ii) identify targets for new therapeutic approaches, (iii) test these approaches, (iv) facilitate the clinical transferability of results, and (v) reduce the use of laboratory animals. Here, we summarize the most commonly used in vitro models of RA and discuss their experimental feasibility and physiological proximity to the pathophysiology of human RA to highlight new human-based avenues in RA research to increase our knowledge on human pathophysiology and develop effective targeted therapies.

5.
Biofabrication ; 12(4): 045016, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32598334

RESUMO

Understanding the pathophysiological processes of cartilage degradation requires adequate model systems to develop therapeutic strategies towards osteoarthritis (OA). Although different in vitro or in vivo models have been described, further comprehensive approaches are needed to study specific disease aspects. This study aimed to combine in vitro and in silico modeling based on a tissue-engineering approach using mesenchymal condensation to mimic cytokine-induced cellular and matrix-related changes during cartilage degradation. Thus, scaffold-free cartilage-like constructs (SFCCs) were produced based on self-organization of mesenchymal stromal cells (mesenchymal condensation) and (i) characterized regarding their cellular and matrix composition or secondly (ii) treated with interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα) for 3 weeks to simulate OA-related matrix degradation. In addition, an existing mathematical model based on partial differential equations was optimized and transferred to the underlying settings to simulate the distribution of IL-1ß, type II collagen degradation and cell number reduction. By combining in vitro and in silico methods, we aimed to develop a valid, efficient alternative approach to examine and predict disease progression and effects of new therapeutics.

6.
Int J Mol Sci ; 21(3)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-32013232

RESUMO

Both inflammatory diseases like rheumatoid arthritis (RA) and anti-inflammatory treatment of RA with glucocorticoids (GCs) or non-steroidal anti-inflammatory drugs (NSAIDs) negatively influence bone metabolism and fracture healing. Janus kinase (JAK) inhibition with tofacitinib has been demonstrated to act as a potent anti-inflammatory therapeutic agent in the treatment of RA, but its impact on the fundamental processes of bone regeneration is currently controversially discussed and at least in part elusive. Therefore, in this study, we aimed to examine the effects of tofacitinib on processes of bone healing focusing on recruitment of human mesenchymal stromal cells (hMSCs) into the inflammatory microenvironment of the fracture gap, chondrogenesis, osteogenesis and osteoclastogenesis. We performed our analyses under conditions of reduced oxygen availability in order to mimic the in vivo situation of the fracture gap most optimal. We demonstrate that tofacitinib dose-dependently promotes the recruitment of hMSCs under hypoxia but inhibits recruitment of hMSCs under normoxia. With regard to the chondrogenic differentiation of hMSCs, we demonstrate that tofacitinib does not inhibit survival at therapeutically relevant doses of 10-100 nM. Moreover, tofacitinib dose-dependently enhances osteogenic differentiation of hMSCs and reduces osteoclast differentiation and activity. We conclude from our data that tofacitinib may influence bone healing by promotion of hMSC recruitment into the hypoxic microenvironment of the fracture gap but does not interfere with the cartilaginous phase of the soft callus phase of fracture healing process. We assume that tofacitinib may promote bone formation and reduce bone resorption, which could in part explain the positive impact of tofacitinib on bone erosions in RA. Thus, we hypothesize that it will be unnecessary to stop this medication in case of fracture and suggest that positive effects on osteoporosis are likely.


Assuntos
Inibidores de Janus Quinases/farmacologia , Janus Quinases/metabolismo , Osteogênese/efeitos dos fármacos , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Janus Quinases/antagonistas & inibidores , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo
7.
Acta Biomater ; 86: 171-184, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30616076

RESUMO

Although several biomaterials for bone regeneration have been developed in the last decades, clinical application of bone morphogenetic protein 2 is clinically only approved when applied on an absorbable bovine collagen I scaffold (ACS) (Helistat; ACS-H). In research, another ACS, namely Lyostypt (ACS-L) is frequently used as a scaffold in bone-linked studies. Nevertheless, until today, the influence of ACS alone on bone healing remains unknown. Unexpectedly, in vitro studies using ASC-H revealed a suppression of osteogenic differentiation and a significant reduction of cell vitality when compared to ASC-L. In mice, we observed a significant delay in bone healing when applying ACS-L in the fracture gap during femoral osteotomy. The results of our study show for the first time a negative influence of both ACS-H and ACS-L on bone formation demonstrating a substantial need for more sophisticated delivery systems for local stimulation of bone healing in both clinical application and research. STATEMENT OF SIGNIFICANCE: Our study provides evidence-based justification to promote the development and approval of more suitable and sophisticated delivery systems in bone healing research. Additionally, we stimulate researchers of the field to consider that the application of those scaffolds as a delivery system for new substances represents a delayed healing approach rather than a normal bone healing which could greatly impact the outcome of those studies and play a pivotal role in the translation to the clinics. Moreover, we provide impulses on underlying mechanism involving the roles of small-leucine rich proteoglycans (SLRP) for further detailed investigations.


Assuntos
Colágeno Tipo I/farmacologia , Consolidação da Fratura/efeitos dos fármacos , Osteotomia , Tecidos Suporte/química , Animais , Regeneração Óssea/efeitos dos fármacos , Calo Ósseo/patologia , Calcificação Fisiológica/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/ultraestrutura , Modelos Animais de Doenças , Endotélio/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fator de Necrose Tumoral alfa/metabolismo , Microtomografia por Raio-X
8.
Genes (Basel) ; 9(5)2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748516

RESUMO

Bone is a complex tissue with a variety of functions, such as providing mechanical stability for locomotion, protection of the inner organs, mineral homeostasis and haematopoiesis. To fulfil these diverse roles in the human body, bone consists of a multitude of different cells and an extracellular matrix that is mechanically stable, yet flexible at the same time. Unlike most tissues, bone is under constant renewal facilitated by a coordinated interaction of bone-forming and bone-resorbing cells. It is thus challenging to recreate bone in its complexity in vitro and most current models rather focus on certain aspects of bone biology that are of relevance for the research question addressed. In addition, animal models are still regarded as the gold-standard in the context of bone biology and pathology, especially for the development of novel treatment strategies. However, species-specific differences impede the translation of findings from animal models to humans. The current review summarizes and discusses the latest developments in bone tissue engineering and organoid culture including suitable cell sources, extracellular matrices and microfluidic bioreactor systems. With available technology in mind, a best possible bone model will be hypothesized. Furthermore, the future need and application of such a complex model will be discussed.

9.
Mycoses ; 61(8): 600-609, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29611232

RESUMO

Aspergillosis in falcons may be associated with high mortality and difficulties in clinical and laboratory diagnosis. We previously cloned an immunogenic protein, Afmp1p, in Aspergillus fumigatus and showed that anti-Afmp1p antibodies were present in human patients with A. fumigatus infections. In this study, we hypothesise that a similar Afmp1p-based enzyme-linked immunosorbent assay (ELISA) could be applied to serodiagnose falcon aspergillosis. A specific polyclonal antibody was first generated to detect falcon serum IgY. Horseradish peroxidase-conjugate of this antibody was then used to measure anti-Afmp1p antibodies in sera collected from falcons experimentally infected with A. fumigatus, and the performance of the Afmp1p-based ELISA was evaluated using sera from healthy falcons and falcons with documented A. fumigatus infections. All four experimentally infected falcons developed culture- and histology-proven invasive aspergillosis. Anti-Afmp1p antibodies were detected in their sera. For the Afmp1p-based ELISA, the mean ± SD OD450 nm using sera from 129 healthy falcons was 0.186 ± 0.073. Receiver operating characteristics curve analysis showed an absorbance cut-off value of 0.407. One negative serum gave an absorbance outside the normal range, giving a specificity of 99.2%. For the 12 sera from falcons with confirmed aspergillosis, nine gave absorbance values ≥ cut-off, giving a sensitivity of 75%. The Afmp1p-based ELISA is useful for serodiagnosis of falcons with aspergillosis.


Assuntos
Anticorpos Antifúngicos/sangue , Antígenos de Fungos/imunologia , Aspergilose/veterinária , Doenças das Aves/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Falconiformes , Glicoproteínas de Membrana/imunologia , Testes Sorológicos/métodos , Animais , Aspergilose/diagnóstico , Curva ROC , Sensibilidade e Especificidade
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