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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.
Reprod Toxicol ; 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-33278556

RESUMO

25 years after the first Berlin Workshop on Developmental Toxicity this 10th Berlin Workshop aimed to bring together international experts from authorities, academia and industry to consider scientific, methodologic and regulatory aspects in risk assessment of developmental toxicity and to debate alternative strategies in testing developmental effects in the future. Proposals for improvement of the categorization of developmental effects were discussed as well as the update of the DevTox database as valuable tool for harmonization. The development of adverse outcome pathways relevant to developmental neurotoxicity (DNT) was debated as a fundamental improvement to guide the screening and testing for DNT using alternatives to animal methods. A further focus was the implementation of an in vitro mechanism-based battery, which can support various regulatory applications associated with the assessment of chemicals and mixtures. More interdisciplinary and translation research should be initiated to accelerate the development of new technologies to test developmental toxicity. Technologies in the pipeline are (i) high throughput imaging techniques, (ii) models for DNT screening tests, (iii) use of computer tomography for assessment of thoracolumbar supernumerary ribs in animal models, and (iv) 3D biofabrication of bone development and regeneration tissue models. In addition, increased collaboration with the medical community was suggested to improve the relevance of test results to humans and identify more clinically relevant endpoints. Finally, the participants agreed that this conference facilitated better understanding innovative approaches that can be useful for the identification of developmental health risks due to exposure to chemical substances.

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.
J Pediatric Infect Dis Soc ; 9(5): 622-625, 2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-32951037

RESUMO

The pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 infection is a severe complication of coronavirus disease 2019. Since impaired coagulation and thrombosis/endotheliitis are suspected pathomechanisms, we treated 2 patients with defibrotide, a profibrinolytic, antithrombotic, antiinflammatory oligonucleotide. Symptoms resolved during treatment. Moreover, coagulation parameters indicating hypofibrinolysis and complement activation normalized. The pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 infection is a severe complication of coronavirus disease 2019. Since impaired coagulation and thrombosis/endotheliitis are suspected pathomechanisms, 2 patients received defibrotide, a profibrinolytic, antithrombotic, antiinflammatory oligonucleotide. Symptoms resolved and hypofibrinolysis/complement activation normalized during treatment.


Assuntos
Infecções por Coronavirus/complicações , Inibidores da Agregação de Plaquetas/uso terapêutico , Pneumonia Viral/complicações , Polidesoxirribonucleotídeos/uso terapêutico , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Dor Abdominal/etiologia , Adolescente , Betacoronavirus , Fatores de Coagulação Sanguínea/análise , Criança , Infecções por Coronavirus/diagnóstico , Feminino , Febre/etiologia , Humanos , Pandemias , Pneumonia Viral/diagnóstico , Síndrome de Resposta Inflamatória Sistêmica/sangue , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Linfócitos T/imunologia
5.
ALTEX ; 37(4): 561-578, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32521037

RESUMO

The aim of the study was to establish an in vitro fracture hematoma (FH) model that mimics the in vivo situation of the human fracture gap in order to assess drug efficacy and effectiveness for the treatment of fracture healing disorders. Human peripheral blood and mesenchymal stromal cells (MSCs) were coagulated to produce in vitro FH models, which were incubated in osteogenic medium under normoxia/hypoxia and analyzed for cell composition, gene expression and cytokine/chemokine secretion. To evaluate the model, we studied the impact of dexamethasone (impairing fracture healing) and deferoxamine (promoting fracture healing). Under hypoxic conditions, MSCs represented the predominant cell population, while the frequencies of leukocyte populations decreased. Marker gene expression of osteogenesis, angiogenesis, inflammation, migration and hypoxic adaptation increased significantly over time and compared to normoxia, while cytokine/chemokine secretion remained unchanged. Dexamethasone favored the frequency of immune cells compared to MSCs, suppressed osteogenic and pro-angiogenic gene expression, and enhanced the secretion of inflammatory cytokines. Conversely, deferoxamine favored the frequency of MSCs over that of immune cells and enhanced the expression of the osteogenic marker RUNX2 and markers of hypoxic adaptation. In summary, we demonstrate that hypoxia is an important factor for modeling the initial phase of fracture healing in vitro and that both fracture-healing disrupting and promoting substances can influence the in vitro model comparable to the in vivo situation. Therefore, we conclude that our model is able to mimic in part the human FH and could reduce the number of animal experiments in early preclinical studies.

6.
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.

7.
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
8.
Bone Marrow Transplant ; 54(Suppl 2): 689-693, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31431707

RESUMO

Posttransplant relapsed B-cell precursor ALL can be cured by 2nd hematopoietic stem cell transplantation (HSCT) in 20% of patients. The major cause of death after second HSCT is leukemic relapse. One reliable predictor for survival after 2nd-HSCT are posttransplant MRD levels. Patients with detectable or increase of MRD are likely to relapse. Patients in complete molecular remission show the best leukemia-free survival and lowest cumulative incidence (CI) of relapse. As patients who undergo second or subsequent HSCT are high-risk patients, we evaluated the prophylactic use of the chimeric Fc-optimized CD19-4G7SDIE-mAb. Posttransplant relapsed CD19+ BCP-ALL patients, who underwent a second or subsequent haplo-HSCT from a T- and B-cell depleted graft received posttransplant prophylactic CD19-4G7SDIE-mAb treatment on compassionate use in complete molecular remission, to increase the antileukemic activity of the new reconstituting immune system by recruiting Fc-expressing effector cells. NK cells recovered early and robust. The 3 year overall survival in 15 evaluable patients was 56%, the 3 year event-free survival was 55% and the CI of relapse 38%. Compared to a historical control group, the CI of relapse was markedly lower and consecutively the EFS higher. Posttransplant-targeted therapy may overcome the need for unspecific GvL effect of undesired GvHD, that can cause severe morbidity and mortality. Due to a low adverse event profile the CD19-4G7SDIE-mAb may be suitable for broad administration to consolidate posttransplant MRD negativity.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Efeito Enxerto vs Leucemia/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia/terapia , Condicionamento Pré-Transplante/métodos , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Intervalo Livre de Doença , Feminino , Humanos , Resultado do Tratamento
9.
Bone Marrow Transplant ; 54(Suppl 2): 727-732, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31431711

RESUMO

Posttransplant treatment strategies are narrowed by the vulnerability of bone marrow. Building on immune cells with antitumor activity is a growing field in cancer therapy. Thus, transfer of expanded and preactivated immune cells is a promising intensification of treatment in high-risk tumor patients. We tested ex vivo expanded NK-, γδT-, and CIK cells that were generated by coincubation with irradiated K562-mb15-41BBL and Il2 and compared the expansion conditions of PBMCs versus CD3-depleted PBMCs as well as static versus semi-automated expansion. The median fold expansion was significantly higher using PBMCs and static expansion conditions. Expanded cells were preactivated with a CD56brightCD69high immunophenotype exerting excellent direct cellular cytotoxicity as well as ADCC in various tumor entities. We established a large-scale clinical-grade ex vivo expansion and activation protocol of NK-, γδT-, and CIK cells from donor-derived PBMCs of patients after haploidentical HSCT. In a patient with AML, NK/γδT/CIK cell transfer was associated with MRD response. A significant increase of direct antitumor activity and ADCC post cell transfer was documented. The results that we report here provide the rationale for clinical testing of expanded, preactivated NK/γδT/CIK cells for cancer therapy.


Assuntos
Células Matadoras Induzidas por Citocinas/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Imunofenotipagem/métodos , Células Matadoras Naturais/imunologia , Condicionamento Pré-Transplante/métodos , Transplante Haploidêntico/métodos , Humanos
10.
Sci Rep ; 9(1): 10749, 2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31341225

RESUMO

Adequate analgesia is essential whenever pain might occur in animal experiments. Unfortunately, the selection of suitable analgesics for mice in bone-linked models is limited. Here, we evaluated two analgesics - Tramadol [0.1 mg/ml (Tlow) vs. 1 mg/ml (Thigh)] and Buprenorphine (Bup; 0.009 mg/ml) - after a pre-surgical injection of Buprenorphine, in a mouse-osteotomy model. The aim of this study was to verify the efficacy of these opioids in alleviating pain-related behaviors, to provide evidence for adequate dosages and to examine potential side effects. High concentrations of Tramadol affected water intake, drinking frequency, food intake and body weight negatively in the first 2-3 days post-osteotomy, while home cage activity was comparable between all groups. General wellbeing parameters were strongly influenced by anesthesia and analgesics. Model-specific pain parameters did not indicate more effective pain relief at high concentrations of Tramadol. In addition, ex vivo high-resolution micro computed tomography (µCT) analysis and histology analyzing bone healing outcomes showed no differences between analgesic groups with respect to newly formed mineralized bone, cartilage and vessels. Our results show that high concentrations of Tramadol do not improve pain relief compared to low dosage Tramadol and Buprenorphine, but rather negatively affect animal wellbeing.


Assuntos
Analgésicos/administração & dosagem , Buprenorfina/administração & dosagem , Osteotomia , Dor Pós-Operatória/tratamento farmacológico , Tramadol/administração & dosagem , Administração Oral , Animais , Modelos Animais de Doenças , Água Potável , Feminino , Fêmur/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cuidados Pós-Operatórios/métodos
11.
PLoS One ; 14(4): e0214276, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30947253

RESUMO

Fractures in horses-whether simple fractures with just one clean break, or incomplete greenstick with stress fractures, or complications such as shattered bones can all be either minimal or even catastrophic. Thus, improvement in fracture healing is a hallmark in equine orthopedics. The fracture healing process implements a complex sequence of events including the initial inflammatory phase removing damaged tissue, re-establishment of vessels and mesenchymal stromal cells, a soft and hard callus phase closing the fracture gap as well as the remodeling phase shaping the bone to a scar-free tissue. Detailed knowledge on processes in equine fracture healing in general and on the initial phase in particular is apparently very limited. Therefore, we generated equine in vitro fracture hematoma models (FH models) to study time-dependent changes in cell composition and RNA-expression for the most prominent cells in the FH model (immune cells, mesenchymal stromal cells) under conditions most closely adapted to the in vivo situation (hypoxia) by using flow cytometry and qPCR. In order to analyze the impact of mesenchymal stromal cells in greater detail, we also incubated blood clots without the addition of mesenchymal stromal cells under the same conditions as a control. We observed a superior survival capacity of mesenchymal stromal cells over immune cells within our FH model maintained under hypoxia. Furthermore, we demonstrate an upregulation of relevant angiogenic, osteogenic and hypoxia-induced markers within 48 h, a time well-known to be crucial for proper fracture healing.


Assuntos
Consolidação da Fratura , Fraturas Ósseas/patologia , Fraturas Ósseas/terapia , Hematoma/terapia , Hipóxia/patologia , Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Animais , Biomarcadores/metabolismo , Biópsia , Sobrevivência Celular/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Hematoma/patologia , Cavalos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Oxigênio/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos
12.
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
13.
Front Immunol ; 10: 2588, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31956322

RESUMO

Macrophages are essential players in the process of fracture healing, acting by remodeling of the extracellular matrix and enabling vascularization. Whilst activated macrophages of M1-like phenotype are present in the initial pro-inflammatory phase of hours to days of fracture healing, an anti-inflammatory M2-like macrophage phenotype is supposed to be crucial for the induction of downstream cascades of healing, especially the initiation of vascularization. In a mouse-osteotomy model, we provide a comprehensive characterization of vessel (CD31+, Emcn+) and macrophage phenotypes (F4/80, CD206, CD80, Mac-2) during the process of fracture healing. To this end, we phenotype the phases of vascular regeneration-the expansion phase (d1-d7 after injury) and the remodeling phase of the endothelial network, until tissue integrity is restored (d14-d21 after injury). Vessels which appear during the bone formation process resemble type H endothelium (CD31hiEmcnhi), and are closely connected to osteoprogenitors (Runx2+, Osx+) and F4/80+ macrophages. M1-like macrophages are present in the initial phase of vascularization until day 3 post osteotomy, but they are rare during later regeneration phases. M2-like macrophages localize mainly extramedullary, and CD206+ macrophages are found to express Mac-2+ during the expansion phase. VEGFA expression is initiated by CD80+ cells, including F4/80+ macrophages, until day 3, while subsequently osteoblasts and chondrocytes are main contributors to VEGFA production at the fracture site. Using Longitudinal Intravital Microendoscopy of the Bone (LIMB) we observe changes in the motility and organization of CX3CR1+ cells, which infiltrate the injury site after an osteotomy. A transient accumulation, resulting in spatial polarization of both, endothelial cells and macrophages, in regions distal to the fracture site, is evident. Immunofluorescence histology followed by histocytometric analysis reveals that F4/80+CX3CR1+ myeloid cells precede vascularization.


Assuntos
Calo Ósseo/irrigação sanguínea , Calo Ósseo/metabolismo , Comunicação Celular , Macrófagos/metabolismo , Neovascularização Fisiológica , Animais , Biomarcadores , Regeneração Óssea , Células Endoteliais/metabolismo , Endotélio , Macrófagos/imunologia , Camundongos , Modelos Animais , Osteoblastos , Osteogênese
14.
Int J Mol Sci ; 19(8)2018 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-30087255

RESUMO

Mesenchymal stem/stromal cells (MSCs) are stem cells of the connective tissue, possess a plastic phenotype, and are able to differentiate into various tissues. Besides their role in tissue regeneration, MSCs perform additional functions as a modulator or inhibitor of immune responses. Due to their pleiotropic function, MSCs have also gained therapeutic importance for the treatment of autoimmune diseases and for improving fracture healing and cartilage regeneration. However, the therapeutic/immunomodulatory mode of action of MSCs is largely unknown. Here, we describe that MSCs express the inhibitory receptor CTLA-4 (cytotoxic T lymphocyte antigen 4). We show that depending on the environmental conditions, MSCs express different isoforms of CTLA-4 with the secreted isoform (sCTLA-4) being the most abundant under hypoxic conditions. Furthermore, we demonstrate that the immunosuppressive function of MSCs is mediated mainly by the secretion of CTLA-4. These findings open new ways for treatment when tissue regeneration/fracture healing is difficult.


Assuntos
Antígeno CTLA-4/imunologia , Células-Tronco Mesenquimais/imunologia , Adipogenia , Antígeno CTLA-4/análise , Células Cultivadas , Humanos , Tolerância Imunológica , Células-Tronco Mesenquimais/citologia , Osteogênese
15.
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.

17.
Int J Mol Sci ; 18(3)2017 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-28282868

RESUMO

Immunologically restricted patients such as those with autoimmune diseases or malignancies often suffer from delayed or insufficient fracture healing. In human fracture hematomas and the surrounding bone marrow obtained from immunologically restricted patients, we analyzed the initial inflammatory phase on cellular and humoral level via flow cytometry and multiplex suspension array. Compared with controls, we demonstrated higher numbers of immune cells like monocytes/macrophages, natural killer T (NKT) cells, and activated T helper cells within the fracture hematomas and/or the surrounding bone marrow. Also, several pro-inflammatory cytokines such as Interleukin (IL)-6 and Tumor necrosis factor α (TNFα), chemokines (e.g., Eotaxin and RANTES), pro-angiogenic factors (e.g., IL-8 and Macrophage migration inhibitory factor: MIF), and regulatory cytokines (e.g., IL-10) were found at higher levels within the fracture hematomas and/or the surrounding bone marrow of immunologically restricted patients when compared to controls. We conclude here that the inflammatory activity on cellular and humoral levels at fracture sites of immunologically restricted patients considerably exceeds that of control patients. The initial inflammatory phase profoundly differs between these patient groups and is probably one of the reasons for prolonged or insufficient fracture healing often occurring within immunologically restricted patients.


Assuntos
Consolidação da Fratura/imunologia , Hospedeiro Imunocomprometido , Inflamação/imunologia , Indutores da Angiogênese/metabolismo , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Estudos de Casos e Controles , Citocinas/metabolismo , Feminino , Fraturas Ósseas/imunologia , Fraturas Ósseas/patologia , Hematoma/imunologia , Hematoma/patologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Neovascularização Fisiológica , Fenótipo
18.
Lab Anim ; 50(6): 433-441, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27909193

RESUMO

Fracture healing is a complex regeneration process which produces new bone tissue without scar formation. However, fracture healing disorders occur in approximately 10% of human patients and cause severe pain and reduced quality of life. Recently, the development of more standardized, sophisticated and commercially available osteosynthesis techniques reflecting clinical approaches has increased the use of small rodents such as rats and mice in bone healing research dramatically. Nevertheless, there is no standard for pain assessment, especially in these species, and consequently limited information regarding the welfare aspects of osteotomy models. Moreover, the selection of analgesics is restricted for osteotomy models since non-steroidal anti-inflammatory drugs (NSAIDs) are known to affect the initial, inflammatory phase of bone healing. Therefore, opioids such as buprenorphine and tramadol are often used. However, dosage data in the literature are varied. Within this review, we clarify the background of osteotomy models, explain the current status and challenges of animal welfare assessment, and provide an example score sheet including model specific parameters. Furthermore, we summarize current refinement options and present a brief outlook on further 3R research.


Assuntos
Bem-Estar do Animal , Osteotomia/efeitos adversos , Medição da Dor/métodos , Dor Pós-Operatória/prevenção & controle , Projetos de Pesquisa , Animais , Modelos Animais de Doenças , Camundongos , Ratos
19.
Cell Mol Immunol ; 12(4): 483-92, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25382740

RESUMO

Exercise at regular intervals is assumed to have a positive effect on immune functions. Conversely, after spaceflight and under simulated weightlessness (e.g., bed rest), immune functions can be suppressed. We aimed to assess the effects of simulated weightlessness (Second Berlin BedRest Study; BBR2-2) on immunological parameters and to investigate the effect of exercise (resistive exercise with and without vibration) on these changes. Twenty-four physically and mentally healthy male volunteers (20-45 years) performed resistive vibration exercise (n=7), resistance exercise without vibration (n=8) or no exercise (n=9) within 60 days of bed rest. Blood samples were taken 2 days before bed rest, on days 19 and 60 of bed rest. Composition of immune cells was analyzed by flow cytometry. Cytokines and neuroendocrine parameters were analyzed by Luminex technology and ELISA/RIA in plasma. General changes over time were identified by paired t-test, and exercise-dependent effects by pairwise repeated measurements (analysis of variance (ANOVA)). With all subjects pooled, the number of granulocytes, natural killer T cells, hematopoietic stem cells and CD45RA and CD25 co-expressing T cells increased and the number of monocytes decreased significantly during the study; the concentration of eotaxin decreased significantly. Different impacts of exercise were seen for lymphocytes, B cells, especially the IgD(+) subpopulation of B cells and the concentrations of IP-10, RANTES and DHEA-S. We conclude that prolonged bed rest significantly impacts immune cell populations and cytokine concentrations. Exercise was able to specifically influence different immunological parameters. In summary, our data fit the hypothesis of immunoprotection by exercise and may point toward even superior effects by resistive vibration exercise.


Assuntos
Linfócitos B/imunologia , Exercício Físico , Imunidade Celular , Imunidade Humoral , Descanso , Linfócitos T/imunologia , Adulto , Citocinas/imunologia , Humanos , Masculino , Fatores de Tempo
20.
J Invest Surg ; 28(1): 1-7, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25093465

RESUMO

PURPOSE/AIM OF THE STUDY: Chlorhexidine and polyhexanide are frequently used antiseptics in clinical practice and have a broad antimicrobial range. Both antiseptics are helpful medical agents for septic wound treatment with a high potential for defeating joint infections. Their effect on human osteoblasts has, so far, not been sufficiently evaluated. The aim of this study was to investigate the activating potential of polyhexanide and chlorhexidine on inflammatory cytokines/chemokines in human osteoblasts in vitro. MATERIALS AND METHODS: Human osteoblasts were isolated and cultivated in vitro and then treated separately with 0.1% and 2% chlorhexidine and 0.04% polyhexanide as commonly applied concentrations in clinical practice. Detection of cell structure and cell morphology was performed by light microscopic inspection. Cytokine and chemokine secretion was determined by using a multiplex suspension array. RESULTS: Cell shrinking, defective cell membrane, and the loss of cell adhesion indicated cell damage of human osteoblasts after treatment with both antiseptics was evaluated by using light microscopy. Polyhexanide, but not chlorhexidine, caused human osteoblasts to secrete various interleukins (1ß, 6, and 7), interferon γ, tumor necrosis factor α, vascular endothelial growth factor, eotaxin, fibroblast growth factor basic, and granulocyte macrophage colony-stimulating factor as quantified by multiplex suspension array. CONCLUSIONS: Both antiseptics induced morphological cell damage at an optimum exposure between 1 and 10 min. But only polyhexanide mediated a pronounced secretion of inflammatory cytokines and chemokines in human osteoblasts. Therefore, we recommend a preferred usage of chlorhexidine in septic surgery to avoid the induction of an inflammatory reaction.


Assuntos
Anti-Infecciosos Locais/farmacologia , Biguanidas/farmacologia , Clorexidina/farmacologia , Citocinas/biossíntese , Osteoblastos/efeitos dos fármacos , Adulto , Anti-Infecciosos Locais/toxicidade , Biguanidas/toxicidade , Medula Óssea/patologia , Adesão Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Quimiocinas/biossíntese , Quimiocinas/metabolismo , Clorexidina/toxicidade , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Osteoartrite do Joelho/patologia , Osteoblastos/metabolismo
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