scholarly journals An Organoid for Woven Bone

2020 ◽  
Author(s):  
Anat Akiva ◽  
Johanna Melke ◽  
Sana Ansari ◽  
Nalan Liv ◽  
Robin van der Meijden ◽  
...  
Keyword(s):  
Bone Formation ◽  
Network Formation ◽  
Functional Model ◽  
Model System ◽  
Human Bone ◽  
Model Systems ◽  
Hierarchical Architecture ◽  
Woven Bone ◽  
Self Organizing

AbstractBone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel both cellular and matrix formation processes. Stem cell-based organoids are promising as functional, self-organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available.This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self-organizing co-culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in vivo osteocytes the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under influence of external triggers (mechanical stimulation, drug administration).

Effect of Oxidized Myofibrils Protein Subjected to Mutiple Freeze-Thaw Cycles on N-Nitrosamine Formation in In Vitro Model System

2012 ◽  
Vol 550-553 ◽  
pp. 1590-1594 ◽  
Author(s):  
Hua Yang ◽  
Pei Pei Meng ◽  
Rui Wang ◽  
Pei Ran Li ◽  
Peng Li ◽  
...  
Keyword(s):  
Sodium Nitrite ◽  
Protein Carbonyl ◽  
Model System ◽  
Model Systems ◽  
Secondary Amines ◽  
Freeze Thaw ◽  
Heavy Chains ◽  
Carcinogenic Substance ◽  
The Times

N-nitrosamine is a kind of carcinogenic substance, which is possibly formed in the reaction of nitrites with amino acids or secondary amines. Two in vitro model systems were designed to evaluate the influence of oxidized myofibrils protein subjected to repeated freeze-thaw cycles (0, 1, 2, 3, 4, 7, 10 times) on N-nitrosamine formation. Model system I contains diethylamine and sodium nitrite, while model system II contains only sodium nitrite as reaction solution. Oxidized myofibrils protein were added to both systems. The results revealed that as the number of freeze-thaw cycles increased, cross-linking of myosin heavy chains and the content of protein carbonyl increased, but the content of protein sulfydryl decreased, which indicates oxidization of protein occurred. The concentration of N-nitrosodiethylamine increased as the number of freeze-thaw cycles increased, especially after four cycles. Oxidized myofibrils protein promoted the formation of N-nitrosodiethylamine. The more the times of freeze-thaw cycles were subjected, the more oxidization of myofibrils protein occurred and the higher yield of the N-nitrosodiethylamine.

scholarly journals In Vitro Model Systems of Coxsackievirus B3-Induced Myocarditis: Comparison of Commonly Used Cell Lines and Characterization of CVB3-Infected iCell® Cardiomyocytes

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1835
Author(s):  
Lisa Kraft ◽  
Martina Sauter ◽  
Guiscard Seebohm ◽  
Karin Klingel
Keyword(s):  
Cell Death ◽  
Viral Load ◽  
Cell Lines ◽  
Hela Cells ◽  
Coxsackievirus B3 ◽  
Model System ◽  
Model Systems ◽  
H9c2 Cells ◽  
Cvb3 Infection

Coxsackievirus B3 (CVB3) belongs to the enteroviruses, which are a well-known cause of acute and chronic myocarditis, primarily infecting cardiac myocytes. As primary human cardiomyocytes are difficult to obtain, viral myocarditis is quite frequently studied in vitro in different non-cardiac and cardiac-like cell lines. Recently, cardiomyocytes that have been differentiated from human-induced pluripotent stem cells have been described as a new model system to study CVB3 infection. Here, we compared iCell® Cardiomyocytes with other cell lines that are commonly used to study CVB3 infection regarding their susceptibility and patterns of infection and the mode of cell death. iCell® Cardiomyocytes, HeLa cells, HL-1 cells and H9c2 cells were infected with CVB3 (Nancy strain). The viral load, CVB3 RNA genome localization, VP1 expression (including the intracellular localization), cellular morphology and the expression of cell death markers were compared. The various cell lines clearly differed in their permissiveness to CVB3 infection, patterns of infection, viral load, and mode of cell death. When studying the mode of cell death of CVB3-infected iCell® Cardiomyocytes in more detail, especially regarding the necroptosis key players RIPK1 and RIPK3, we found that RIPK1 is cleaved during CVB3 infection. iCell® Cardiomyocytes represent well the natural host of CVB3 in the heart and are thus the most appropriate model system to study molecular mechanisms of CVB3-induced myocarditis in vitro. Doubts are raised about the suitability of commonly used cell lines such as HeLa cells, HL-1 cells and H9c2 cells to evaluate molecular pathways and processes occurring in vivo in enteroviral myocarditis.

scholarly journals Nanomaterials induce different levels of oxidative stress, depending on the used model system: Comparison of in vitro and in vivo effects

2020 ◽  
Author(s):  
Isabel Karkossa ◽  
Anne Bannuscher ◽  
Bryan Hellack ◽  
Wendel Wohlleben ◽  
Julie Laloy ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Alveolar Macrophages ◽  
Model System ◽  
Alveolar Epithelial Cells ◽  
Model Systems ◽  
Alveolar Epithelial ◽  
Different Levels

Abstract Background The immense variety and constant development of nanomaterials (NMs) raise the demand for a facilitated risk assessment, for which knowledge on NMs mode of actions (MoAs) is required. For this purpose, a comprehensive data basis is of paramountcy that can be obtained using omics. Furthermore, the establishment of suitable in vitro test systems is indispensable to follow the 3R concept and to master the high number of NMs. In the present study, we aimed at comparing NM effects in vitro and in vivo using a multi-omics approach. We applied an integrated data evaluation strategy based on proteomics and metabolomics to four silica NMs and one titanium dioxide-based NM. For in vitro investigations, alveolar epithelial cells and alveolar macrophages were treated with different doses of NMs, and the results were compared to effects on rat lungs after short-term inhalations and instillations at varying doses with and without a recovery period.Results Since the production of reactive oxygen species (ROS) is described to be a critical biological effect of NMs, and enrichment analyses confirmed oxidative stress as a significant effect upon NM treatment in vitro in the present study, we focused on different levels of oxidative stress. Thus, we found opposite changes for proteins and metabolites that are related to the production of reduced glutathione in alveolar epithelial cells and alveolar macrophages, illustrating that NMs MoAs depend on the used model system. Interestingly, in vivo, pathways related to inflammation were affected to a greater extent than oxidative stress responses. Hence, the assignment of the observed effects to the levels of oxidative stress was different in vitro and in vivo as well. However, the overall classification of “active” and “passive” NMs was consistent in vitro and in vivo.Conclusions The consistent classification indicates both tested cell lines to be suitable for NM toxicity assessment even though the induced levels of oxidative stress strongly depend on the used model systems. Thus, the here presented results highlight that model systems need to be carefully revised to decipher the extent to which they can replace in vivo testing.

scholarly journals TMOD-12. THE ROLE OF INTEGRIN α V AND CD44 IN GBM MIGRATION USING HUMAN ORGANOTYPIC SLICES

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi265-vi265
Author(s):  
Zev Binder ◽  
Sarah Hyun Ji Kim ◽  
Pei-Hsun Wu ◽  
Anjil Giri ◽  
Gary Gallia ◽  
...  
Keyword(s):  
Human Brain ◽  
Cell Motility ◽  
Brain Tissue ◽  
Brain Slices ◽  
Model System ◽  
Model Systems ◽  
In Vivo Model ◽  
Human Brain Tissue

Abstract Current model systems used for GBM research include traditional in vitro cell line-based assays and in vivo animal studies. In vitro model systems offer the advantages of being easy to use, relatively inexpensive, and fast growing. However, these models lack key elements of the pathology they are attempting to model, including the biochemical and biophysical microenvironment and three-dimensional structure inherent to human brain tissue. In vivo model systems address these limitations, but have restrictions of their own. Species differences may result in non-applicable results and animal experiments are often not designed like clinical trials. Evidence of the limitations of current GBM models is found in the disparity between basic research findings and successful new treatments for GBMs in the clinic. Here we present an alternative model system for the study of human GBM cell motility and invasion, which features advantages of both in vitro and in vivo model systems. Using human organotypic brain slices as scaffolding for tumor growth, we explored the dynamic process of GBM cell invasion within human brain tissue. To demonstrate the utility of the model system, we investigated the effects of depletion of integrin α V (ITGAV) and CD44 on GBM cell motility. These two cell-surface proteins have been identified to have key functions in GBM cell motility. However, knockdown of ITGAV had little effect on tumor cell motility in organotypics while CD44 knockdown significantly reduced cell movement. Finally, we compare motility results from cells in human brain slices to those from cells growing on standard Matrigel and in mouse brain organotypics. We found significant differences in motility depending on the substrate in which the cells were moving. Our findings highlight the physiologic characteristics of human brain organotypics and demonstrate the use of real-time imaging in the ex vivo system.

The Efficiency of FK 506 in In Vitro Bone Formation by Human Bone Marrow Cells

2003 ◽  
Vol 240-242 ◽  
pp. 659-662
Author(s):  
Noriko Okumura ◽  
Takafumi Yoshikawa ◽  
Jin Iida ◽  
Kunio Ichijima ◽  
Yoshinori Takakura
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Bone Marrow Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Fk 506 ◽  
Marrow Cells

scholarly journals FRI0369 MIMICKING GLUCOCORTICOID-INDUCED OSTEOPOROSIS USING AN IN VITRO TRABECULAR HUMAN BONE MODEL

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 782.1-782
Author(s):  
A. Lang ◽  
K. Diesing ◽  
A. Damerau ◽  
S. Uzun ◽  
M. Pfeiffenberger ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Human Bone ◽  
Model Systems ◽  
Bone Homeostasis ◽  
Perfusion System ◽  
Cultivation Conditions ◽  
Bone Model ◽  
Long Term Treatment ◽  
Biomechanical Stimuli

Background:The bone matrix consists of inorganic and organic components and a variety of specialized cells such as osteoblasts, osteocytes and osteoclasts. The bone-forming osteoblasts are responsible for the production of organic matrix components; they differentiate later into osteocytes which is accompanied by matrix mineralization. Osteoclasts are multinuclear giant cells, which resorb bone. Healthy bone homeostasis is characterized by a balanced, dynamic and continuous remodeling process. Glucocorticoids (GCs) are commonly used to successfully treat patients with inflammatory rheumatic and other autoimmune diseases. However, long-term treatment with GC can potentially lead to several adverse effects such as the inhibition of osteoblast proliferation and the increase of osteoclastic activity resulting in osteoporosis.Objectives:Hence, the aim of our project is to i) develop anin vitrotrabecular human bone model, ii) integrate this bone model into a perfusion system to accelerate mineralization and provide biomechanical stimuli and iii) applying prednisolone to induce osteoporosis. Here we present our initial results describing the successful differentiation of osteoblasts and osteoclasts in a 3D environment, and the accomplished integration of the bone model into a perfusion system.Methods:In a first step, different cultivation conditions were tested to allow optimal osteogenic or osteoclastic differentiation. To this end, a) human bone marrow derived mesenchymal stromal cells (hMSCs) were treated with osteogenic medium, and b) monocytes (isolated from buffy coats) were differentiated into osteoclasts using following protocol: incubation for 3 days with 25 ng/ml M-CSF followed by an 18-day incubation with M-CSF and 50 ng/ml RANKL. Calcification of hMSCs was evaluated via Alizarin Red S staining. Osteoclasts were identified using immunofluorescence staining observing multinucleated (DAPI) giant (ß-Actin) cells with TRAP and Cathepsin K activity. Additional gene expression analyses are currently conducted using qRT-PCR and looking for osteoclast-specific genes. In parallel to the monolayer cultures, cells were transferred on β-tricalcium phosphate (βTCP) – a suitable bony-like scaffold. Furthermore, first experiments in a dynamic bioreactor platform (OSPIN GmH) were conducted to evaluate the influence of shear stress on the cells and model systems.Results:We have been able to populate the βTCP scaffold with monocytes, which were differentiated into osteoclasts (morphological changes) without any effect on cellular viability as measured by Live/Dead staining. The morphological changes of those osteoclasts such as formation of filopodia could be demonstrated by scanning electron microscopy. In addition, the cultivation of βTCP populated with hMSCs in a perfusion system showed the upregulation of osteogenic markers (RUNX2, OSX) on mRNA-level.Conclusion:These first results of our approach to develop anin vitro3D model for glucocorticoid-induced osteoporosis are promising. Our next step will be the co-cultivation of osteoblasts and osteoclasts under dynamic and optimized cultivation conditions. By combining several cell types, a suitable scaffold and biomechanical stimuli (perfusion), we aim to provide a valid testing platform to study underlying disease mechanisms and for drug development.Acknowledgments:The project has been funded by the Elsbeth Bonhoff Foundation.Disclosure of Interests:Annemarie Lang: None declared, Karoline Diesing: None declared, Alexandra Damerau: None declared, Sümeyye Uzun: None declared, Moritz Pfeiffenberger: None declared, Timo Gaber: None declared, Frank Buttgereit Grant/research support from: Amgen, BMS, Celgene, Generic Assays, GSK, Hexal, Horizon, Lilly, medac, Mundipharma, Novartis, Pfizer, Roche, and Sanofi.

scholarly journals Nanomaterials induce different levels of oxidative stress, depending on the used model system: Comparison of in vitro and in vivo effects

2021 ◽  
Author(s):  
Isabel Karkossa ◽  
Anne Bannuscher ◽  
Bryan Hellack ◽  
Wendel Wohlleben ◽  
Julie Laloy ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Alveolar Macrophages ◽  
Model System ◽  
Alveolar Epithelial Cells ◽  
Model Systems ◽  
Alveolar Epithelial ◽  
Different Levels

Abstract Background: The immense variety and constant development of nanomaterials (NMs) raise the demand for a facilitated risk assessment, for which knowledge on NMs mode of actions (MoAs) is required. For this purpose, a comprehensive data basis is of paramountcy that can be obtained using omics. Furthermore, the establishment of suitable in vitro test systems is indispensable to follow the 3R concept and to master the high number of NMs. In the present study, we aimed at comparing NM effects in vitro and in vivo using a multi-omics approach. We applied an integrated data evaluation strategy based on proteomics and metabolomics to four silica NMs and one titanium dioxide-based NM. For in vitro investigations, rat alveolar epithelial cells (RLE-6TN) and rat alveolar macrophages (NR8383) were treated with different doses of NMs, and the results were compared to effects on rat lungs after short-term inhalations and instillations at varying doses with and without a recovery period.Results: Since the production of reactive oxygen species (ROS) is described to be a critical biological effect of NMs, and enrichment analyses confirmed oxidative stress as a significant effect upon NM treatment in vitro in the present study, we focused on different levels of oxidative stress. Thus, we found opposite changes for proteins and metabolites that are related to the production of reduced glutathione in alveolar epithelial cells and alveolar macrophages, illustrating that NMs MoAs depend on the used model system. Interestingly, in vivo, pathways related to inflammation were affected to a greater extent than oxidative stress responses. Hence, the assignment of the observed effects to the levels of oxidative stress was different in vitro and in vivo as well. However, the overall classification of “active” and “passive” NMs was consistent in vitro and in vivo.Conclusions: The consistent classification indicates both tested cell lines to be suitable for NM toxicity assessment even though the induced levels of oxidative stress strongly depend on the used model systems. Thus, the here presented results highlight that model systems need to be carefully revised to decipher the extent to which they can replace in vivo testing.

Platelet-Derived Factors Allow Human Mesenchymal Stem Cells to Spontaneously Undergo Endochondral Bone Differentiation and Provide Bone Marrow Support in a Xenogenic In Vivo Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1322-1322
Author(s):  
Nathalie Etchart ◽  
Andreas Reinisch ◽  
Anna Ortner ◽  
Margareta Frühwirth ◽  
Rokhsareh Rohban ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Human Platelet ◽  
Human Bone ◽  
Mouse Bone Marrow ◽  
Bone Differentiation ◽  
Nsg Mice ◽  
Ectopic Bone

Abstract Abstract 1322 Introduction. Mesenchymal Stem/Progenitor Cells (MSPC) are regarded as the universal skeletal progenitor, theoretically capable of differentiating into cartilage, bone, tendon and muscle. Their functions as pericytes as well as key bone marrow stromal cells are also well documented. Tremendous benefits could therefore be achieved by cell therapy with MSPC. Unfortunately, the very successful research aimed at isolating, expanding and differentiating these cells in vitro has so far failed to translate into significant clinical advances. In particular, bone regeneration studies are disappointing, because application of MSPC in vivo requires their osteogenic pre-differentiation in vitro and/or their co-implantation with bone chips, and because results are highly donor-dependant. In this context, recent studies indicate that MSPC expanded from human Bone Marrow (BM) in media supplemented by Human Platelet Lysate (HPL) in lieu of Fetal Bovine Serum (FBS) have a higher rate of bone differentiation. Here, we use a new, simplified mouse model of ectopic bone formation requiring neither pre-differentiation nor bone matrix. We show that human BM-MSPC expanded in HPL-supplemented medium from all donors spontaneously form bone through an endochondral mechanism. Importantly, the ossicles generated from MSPC from almost half of the donors become the site of ectopic bone marrow development. Further experiments suggest that maintenance of MSPC stemness by platelet-derived factors during cell expansion is paramount to this effect. Methods and Results. MSPC are expanded from human bone marrow following standard protocols, with culture media supplemented either with 10% FBS or with 10% HPL. Cultured MSPC are resuspended in a non-mineral collagen/laminin matrix (Matrigel®) and injected subcutaneously into immune-deficient NSG mice. Bone formation is monitored non-invasively by osteosensitive near-infrared imaging and/or by histology on paraffin-embedded ossicles. HPL-derived MSPC from 13 out of 13 donors form bone in vivo compared to only 2 out of 10 FBS-derived MSPC. Extensive cartilage formation is observed as early as one week after implantation, while signs of ossification appear from the third week onwards. In addition, ossicles generated by HPL-MSPC from 6 out of 13 donors become colonised by mouse bone marrow, indicating that platelet-derived factors maintain the capacity of MSPC to reconstitute a functional bone marrow niche. Bone formation in vivo is considerably delayed, but not blocked, by pre-treatment of the cells with Cholera Toxin, indicating that a Gα protein-coupled receptor (GPCR-α) ligand is partly responsible for the biological effect of HPL. In vitro, HPL and FBS-derived cells display remarkably little phenotypic differences, with the notable exception of the stemness-associated surface marker SSEA-4 which is consistently more expressed on HPL- than on FBS-derived MSPC. Interestingly, serial passage in FBS medium of cells first expanded in HPL medium leads to the rapid loss of surface SSEA-4 expression, paralleled by a loss of osteogenic and bone marrow support potential in vivo. Loss of SSEA-4 expression is also observed in cells serially passaged in HPL medium containing the PDGF receptor-β inhibitor Imatinib, and ossicles derived from these cells are no longer able to attract mouse bone marrow in vivo. These data suggest that human platelet-derived factors allow MSPC to retain their stem cell potential in culture as well as in the ectopic bone microenvironment. This hypothesis is further supported by the observation that human MSPC can be re-isolated and re-expanded from bone marrow-infiltrated ossicles and, remarkably, build bone again when re-injected into NSG mice. Conclusion. Our data suggest that platelet-derived factors, including PDGF and an (as yet unidentified) GPCR-α ligand, contribute in vitro to maintain BM-MSPC stemness. In vivo, they efficiently drive BM-MSPC to differentiate along the chondrogenic and osteogenic lineages, while preserving MSPC bone marrow-support function. We conclude that therapeutic approaches using MSPC for skeletal regeneration should preferentially use early passage BM-MSPC expanded in HPL-supplemented medium. Furthermore, the observed correlation between surface SSEA-4 expression and multipotency in BM-MSPC can be exploited to monitor the quality of the cell preparations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Sialylation of Lipooligosaccharides Promotes Biofilm Formation by Nontypeable Haemophilus influenzae

2004 ◽  
Vol 72 (1) ◽  
pp. 106-113 ◽  
Author(s):  
W. Edward Swords ◽  
Miranda L. Moore ◽  
Luciana Godzicki ◽  
Gail Bukofzer ◽  
Michael J. Mitten ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Haemophilus Influenzae ◽  
Middle Ear ◽  
Biofilm Formation ◽  
Model System ◽  
Model Systems ◽  
Bacterial Persistence ◽  
Tract Infections

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis. The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community. Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi. Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions. In this study, we show that LOS containing sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo. LOS from NTHi in biofilms was sialylated, as determined by comparison of electrophoretic mobilities and immunochemical reactivities before and after neuraminidase treatment. Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in three different in vitro model systems. The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system. These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.

Sign in / Sign up

Export Citation Format

Share Document