scholarly journals Triple Culture of Primary Human Osteoblasts, Osteoclasts and Osteocytes as an In Vitro Bone Model

2021 ◽  
Vol 22 (14) ◽  
pp. 7316
Author(s):  
Anne Bernhardt ◽  
Jasmin Skottke ◽  
Max von von Witzleben ◽  
Michael Gelinsky
Keyword(s):  
Final Analysis ◽  
Cell Types ◽  
Porous Membrane ◽  
Collagen Gels ◽  
Human Osteoblasts ◽  
Culture Model ◽  
Typical Morphology ◽  
Primary Human Osteoblasts ◽  
Different Cell Types

In vitro evaluation of bone graft materials is generally performed by analyzing the interaction with osteoblasts or osteoblast precursors. In vitro bone models comprising different cell species can give specific first information on the performance of those materials. In the present study, a 3D co-culture model was established comprising primary human osteoblasts, osteoclasts and osteocytes. Osteocytes were differentiated from osteoblasts embedded in collagen gels and were cultivated with osteoblast and osteoclasts seeded in patterns on a porous membrane. This experimental setup allowed paracrine signaling as well as separation of the different cell types for final analysis. After 7 days of co-culture, the three cell species showed their typical morphology and gene expression of typical markers like ALPL, BSPII, BLGAP, E11, PHEX, MEPE, RANKL, ACP5, CAII and CTSK. Furthermore, relevant enzyme activities for osteoblasts (ALP) and osteoclasts (TRAP, CTSK, CAII) were detected. Osteoclasts in triple culture showed downregulated TRAP (ACP5) and CAII expression and decreased TRAP activity. ALP and BSPII expression of osteoblasts in triple culture were upregulated. The expression of the osteocyte marker E11 (PDPN) was unchanged; however, osteocalcin (BGLAP) expression was considerably downregulated both in osteoblasts and osteocytes in triple cultures compared to the respective single cultures.

scholarly journals In Vitro Co-culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels

2019 ◽  
Vol 20 (8) ◽  
pp. 1998 ◽  
Author(s):  
Skottke ◽  
Gelinsky ◽  
Bernhardt
Keyword(s):  
Gene Expression ◽  
Bone Tissue ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Porous Membrane ◽  
Collagen Gels ◽  
Rankl Expression ◽  
Human Osteoblasts ◽  
Apical Side

Background: Osteocytes are the key regulator cells in bone tissue, affecting activity of both osteoblasts and osteoclasts. Current in vitro studies on osteocyte-osteoblast interaction are invariably performed with rodent cells, mostly murine cell lines, which diminishes the clinical relevance of the data. Objective: The objective of the present study was to establish an in vitro co-culture system of osteoblasts and osteocytes, which is based solely on human primary cells. Methods: Three different approaches for the generation of human primary osteocytes were compared: direct isolation of osteocytes from bone tissue by multistep digestion, long-time differentiation of human pre-osteoblasts embedded in collagen gels, and short time differentiation of mature human osteoblasts in collagen gels. Co-cultivation of mature osteoblasts with osteocytes, derived from the three different approaches was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoblasts on the basal side of a porous membrane, which allowed the separate gene expression analysis for osteocytes and osteoblasts. Fluorescence microscopic imaging and gene expression analysis were performed separately for osteocytes and osteoblasts. Results: All examined approaches provided cells with typical osteocytic morphology, which expressed osteocyte markers E11, osteocalcin, phosphate regulating endopeptidase homolog, X-linked (PHEX), matrix extracellular phosphoglycoprotein (MEPE), sclerostin, and receptor activator of NF-κB Ligand (RANKL). Expression of osteocyte markers was not significantly changed in the presence of osteoblasts. In contrast, osteocalcin gene expression of osteoblasts was significantly upregulated in all examined co-cultures with differentiated osteocytes. Alkaline phosphatase (ALPL), bone sialoprotein II (BSPII), and RANKL expression of osteoblasts was not significantly changed in the co-culture. Conclusion: Interaction of osteoblasts and osteocytes can be monitored in an in vitro model, comprising solely primary human cells.

scholarly journals Therapeutic Attributes of Endocannabinoid System against Neuro-Inflammatory Autoimmune Disorders

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3389
Author(s):  
Ishtiaq Ahmed ◽  
Saif Ur Rehman ◽  
Shiva Shahmohamadnejad ◽  
Muhammad Anjum Zia ◽  
Muhammad Ahmad ◽  
...  
Keyword(s):  
Cannabinoid Receptors ◽  
Cannabinoid Receptor ◽  
Therapeutic Potential ◽  
Endocannabinoid System ◽  
Cell Types ◽  
Autoimmune Disorders ◽  
Specific Receptors ◽  
Different Cell Types

In humans, various sites like cannabinoid receptors (CBR) having a binding affinity with cannabinoids are distributed on the surface of different cell types, where endocannabinoids (ECs) and derivatives of fatty acid can bind. The binding of these substance(s) triggers the activation of specific receptors required for various physiological functions, including pain sensation, memory, and appetite. The ECs and CBR perform multiple functions via the cannabinoid receptor 1 (CB1); cannabinoid receptor 2 (CB2), having a key effect in restraining neurotransmitters and the arrangement of cytokines. The role of cannabinoids in the immune system is illustrated because of their immunosuppressive characteristics. These characteristics include inhibition of leucocyte proliferation, T cells apoptosis, and induction of macrophages along with reduced pro-inflammatory cytokines secretion. The review seeks to discuss the functional relationship between the endocannabinoid system (ECS) and anti-tumor characteristics of cannabinoids in various cancers. The therapeutic potential of cannabinoids for cancer—both in vivo and in vitro clinical trials—has also been highlighted and reported to be effective in mice models in arthritis for the inflammation reduction, neuropathic pain, positive effect in multiple sclerosis and type-1 diabetes mellitus, and found beneficial for treating in various cancers. In human models, such studies are limited; thereby, further research is indispensable in this field to get a conclusive outcome. Therefore, in autoimmune disorders, therapeutic cannabinoids can serve as promising immunosuppressive and anti-fibrotic agents.

scholarly journals Adhesion Behaviour of Primary Human Osteoblasts and Fibroblasts on Polyether Ether Ketone Compared with Titanium under In Vitro Lipopolysaccharide Incubation

Materials ◽  
2019 ◽  
Vol 12 (17) ◽  
pp. 2739 ◽  
Author(s):  
Korbinian Benz ◽  
Andreas Schöbel ◽  
Marisa Dietz ◽  
Peter Maurer ◽  
Jochen Jackowski
Keyword(s):  
Gene Expression ◽  
Protein Level ◽  
Binding Protein ◽  
Sem Images ◽  
Human Osteoblasts ◽  
Polyether Ether Ketone ◽  
Primary Human Osteoblasts ◽  
Titanium Surfaces ◽  
Ether Ketone

The aim of this in vitro pilot study was to analyse the adhesion behaviour of human osteoblasts and fibroblasts on polyether ether ketone (PEEK) when compared with titanium surfaces in an inflammatory environment under lipopolysaccharide (LPS) incubation. Scanning electron microscopy (SEM) images of primary human osteoblasts/fibroblasts on titanium/PEEK samples were created. The gene expression of the LPS-binding protein (LBP) and the LPS receptor (toll-like receptor 4; TLR4) was measured by real-time polymerase chain reaction (PCR). Immunocytochemistry was used to obtain evidence for the distribution of LBP/TLR4 at the protein level of the extra-cellular-matrix-binding protein vinculin and the actin cytoskeleton. SEM images revealed that the osteoblasts and fibroblasts on the PEEK surfaces had adhesion characteristics comparable to those of titanium. The osteoblasts contracted under LPS incubation and a significantly increased LBP gene expression were detected. This was discernible at the protein level on all the materials. Whereas no increase of TLR4 was detected with regard to mRNA concentrations, a considerable increase in the antibody reaction was detected on all the materials. As is the case with titanium, the colonisation of human osteoblasts and fibroblasts on PEEK samples is possible under pro-inflammatory environmental conditions and the cellular inflammation behaviour towards PEEK is lower than that of titanium.

scholarly journals A phenomenological density-scaling approach to lamellipodial actin dynamics

2014 ◽  
Vol 4 (6) ◽  
pp. 20140006 ◽  
Author(s):  
Alexandre Lewalle ◽  
Marco Fritzsche ◽  
Kerry Wilson ◽  
Richard Thorogate ◽  
Tom Duke ◽  
...  
Keyword(s):  
Protein Function ◽  
Cell Types ◽  
Actin Dynamics ◽  
Theoretical Modelling ◽  
Network Growth ◽  
Genetic Intervention ◽  
Regulatory Processes ◽  
Observable Behaviour ◽  
Different Cell Types

The integration of protein function studied in vitro in a dynamic system like the cell lamellipodium remains a significant challenge. One reason is the apparent contradictory effect that perturbations of some proteins can have on the overall lamellipodium dynamics, depending on exact conditions. Theoretical modelling offers one approach for understanding the balance between the mechanisms that drive and regulate actin network growth and decay. Most models use a ‘bottom-up’ approach, involving explicitly assembling biochemical components to simulate observable behaviour. Their correctness therefore relies on both the accurate characterization of all the components and the completeness of the relevant processes involved. To avoid potential pitfalls due to this uncertainty, we used an alternative ‘top-down’ approach, in which measurable features of lamellipodium behaviour, here observed in two different cell types (HL60 and B16-F1), directly inform the development of a simple phenomenological model of lamellipodium dynamics. We show that the kinetics of F-actin association and dissociation scales with the local F-actin density, with no explicit location dependence. This justifies the use of a simplified kinetic model of lamellipodium dynamics that yields predictions testable by pharmacological or genetic intervention. A length-scale parameter (the lamellipodium width) emerges from this analysis as an experimentally accessible probe of network regulatory processes.

In vitro Growth Pattern of Primary Human Osteoblasts on Calcium Phosphate- and Polymethylmethacrylate-Based Bone Cement

2017 ◽  
Vol 58 (5-6) ◽  
pp. 216-226
Author(s):  
Johannes Schauwecker ◽  
Mark Bock ◽  
Florian Pohlig ◽  
Heinz Mühlhofer ◽  
Jutta Tübel ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Calcium Phosphate ◽  
Cell Viability ◽  
Proliferation Rate ◽  
Control Analysis ◽  
Implant Loosening ◽  
Human Osteoblasts ◽  
Apoptosis Rate ◽  
Primary Human Osteoblasts

Background/Purpose: Polymethylmethacrylate (PMMA) and calcium phosphate (Ca-P) cements are widely used for arthroplasty surgery and augmentation of bone defects. However, aseptic implant loosening in absence of wear-induced osteolysis indicates an unfavourable interaction between the cement surface and human osteoblasts. Our underlying hypothesis is that cement surfaces directly modify cell viability, proliferation rate, and cell differentiation. Methods: To test this hypothesis, we examined primary human osteoblasts harvested from six individuals. These cells were pooled and subsequently seeded directly on cement pellets prepared from Palacos® R, Palacos® R+G, and Norian® Drillable cements. After incubation for 24 and 72 h, cell viability, proliferation rate, apoptosis rate, and cell differentiation were analysed. Results: Upon cultivation of human osteoblasts on cement surfaces, we observed a significantly reduced cell viability and DNA content compared to the control. Analysis of the apoptosis rate revealed an increase for cells on Palacos R and Norian Drillable, but a significant decrease on Palacos R+G compared to the control. Regarding osteogenic differentiation, significantly lower values of alkaline phosphatase enzyme activity were identified for all cement surfaces after 24 and 72 h compared to cultivation on tissue culture plastic, serving as control. Conclusions: In summary, these data suggest a limited biocompatibility of both PMMA and Ca-P cements, necessitating further research to reduce unfavourable cell-cement interactions and consequently extend implant survival.

scholarly journals Divergent functions of endotrophin on different cell populations in adipose tissue

2016 ◽  
Vol 311 (6) ◽  
pp. E952-E963 ◽  
Author(s):  
Yueshui Zhao ◽  
Xue Gu ◽  
Ningyan Zhang ◽  
Mikhail G. Kolonin ◽  
Zhiqiang An ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Lipid Accumulation ◽  
Lysyl Oxidase ◽  
Stromal Vascular Fraction ◽  
Cell Types ◽  
Marker Genes ◽  
New Perspective ◽  
Collagen Genes ◽  
Different Cell Types

Endotrophin is a cleavage product of collagen 6 (Col6) in adipose tissue (AT). Previously, we demonstrated that endotrophin serves as a costimulator to trigger fibrosis and inflammation within the unhealthy AT milieu. However, how endotrophin affects lipid storage and breakdown in AT and how different cell types in AT respond to endotrophin stimulation remain unknown. In the current study, by using a doxycycline-inducible mouse model, we observed significant upregulation of adipogenic genes in the white AT (WAT) of endotrophin transgenic mice. We further showed that the mice exhibited inhibited lipolysis and accelerated hypertrophy and hyperplasia in WAT. To investigate the effects of endotrophin in vitro, we incubated different cell types from AT with conditioned medium from endotrophin-overexpressing 293T cells. We found that endotrophin activated multiple pathological pathways in different cell types. Particularly in 3T3-L1 adipocytes, endotrophin triggered a fibrotic program by upregulating collagen genes and promoted abnormal lipid accumulation by downregulating hormone-sensitive lipolysis gene and decreasing HSL phosphorylation levels. In macrophages isolated from WAT, endotrophin stimulated higher expression of the collagen-linking enzyme lysyl oxidase and M1 proinflammatory marker genes. In the stromal vascular fraction isolated from WAT, endotrophin induced upregulation of both profibrotic and proinflammatory genes. In conclusion, our study provides a new perspective on the effect of endotrophin in abnormal lipid accumulation and a mechanistic insight into the roles played by adipocytes and a variety of other cell types in AT in shaping the unhealthy microenvironment upon endotrophin treatment.

Cultured epithelial cells derived from human foetal pancreas as a model for the study of cystic fibrosis: further analyses on the origins and nature of the cell types

1988 ◽  
Vol 90 (1) ◽  
pp. 73-77
Author(s):  
A. Harris ◽  
L. Coleman
Keyword(s):  
Cystic Fibrosis ◽  
Tissue Culture ◽  
Epithelial Cells ◽  
Culture System ◽  
Cultured Cells ◽  
Cell Types ◽  
Cultured Epithelial Cells ◽  
Different Cell Types ◽  
Foetal Pancreas

The establishment of a tissue-culture system for epithelial cells derived from human foetal pancreas has recently been reported. Further analyses have now been made on these cells in vitro, together with parallel investigation of the distribution of different cell types within the intact foetal pancreas. Results support the view that the cultured cells are ductal in origin and nature. Pancreatic epithelial cell cultures have also been established from foetuses with cystic fibrosis.

scholarly journals Bromodomain protein inhibition: a novel therapeutic strategy in rheumatic diseases

RMD Open ◽  
2018 ◽  
Vol 4 (2) ◽  
pp. e000744 ◽  
Author(s):  
Kerstin Klein
Keyword(s):  
Animal Models ◽  
Rheumatic Diseases ◽  
Transcriptional Activation ◽  
Therapeutic Strategy ◽  
Cell Types ◽  
Protein Inhibition ◽  
Different Cell Types ◽  
Novel Therapeutic

The reading of acetylation marks on histones by bromodomain (BRD) proteins is a key event in transcriptional activation. Small molecule inhibitors targeting bromodomain and extra-terminal (BET) proteins compete for binding to acetylated histones. They have strong anti-inflammatory properties and exhibit encouraging effects in different cell types in vitro and in animal models resembling rheumatic diseases in vivo. Furthermore, recent studies that focus on BRD proteins beyond BET family members are discussed.

scholarly journals Filopodyan: An open-source pipeline for the analysis of filopodia

2017 ◽  
Vol 216 (10) ◽  
pp. 3405-3422 ◽  
Author(s):  
Vasja Urbančič ◽  
Richard Butler ◽  
Benjamin Richier ◽  
Manuel Peter ◽  
Julia Mason ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Dynamic Properties ◽  
Cell Types ◽  
Molecular Heterogeneity ◽  
Interactive Editing ◽  
Motile Cells ◽  
Different Cell Types ◽  
Neuronal Growth Cones

Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.

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