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Bilthoven, Netherlands

Harkel B.T.,VU University Amsterdam | Schoenmaker T.,VU University Amsterdam | Picavet D.I.,University of Amsterdam | Davison N.L.,University of Twente | And 3 more authors.
PLoS ONE | Year: 2015

Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DCSTAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones-cytoskeletal organization that is considered to be osteoclast- specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K. © 2015 ten Harkel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source


De Haart S.J.,University Utrecht | Van De Donk N.W.C.J.,University Utrecht | Van De Donk N.W.C.J.,Dana-Farber Cancer Institute | Minnema M.C.,University Utrecht | And 12 more authors.
Clinical Cancer Research | Year: 2013

Purpose: Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells. Experimental Design: Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4+ or CD8+ CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants. Results: Bone marrow stromal cells from patients with multiple myeloma and healthy individuals, as well as vascular endothelial cells, significantly inhibited the lysis of multiple myeloma cells in a cell-cell contact-dependent manner and without substantial T-cell suppression, thus showing the induction of a cell adhesion-mediated immune resistance (CAM-IR) against CTL lysis. Further analyses revealed that adhesion to accessory cells downregulated Fas and upregulated the caspase-3 inhibitor survivin in multiple myeloma cells. Reconstitution of Fas expression with bortezomib enhanced the CTL-mediated lysis of multiple myeloma cells. Repressing survivin with the small-molecule YM155 synergized with CTLs and abrogated CAM-IR in vitro and in vivo. Conclusion: These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR. © 2013 AACR. Source


Janssen F.W.,University of Twente | Janssen F.W.,Xpand Biotechnology BV | Van Dijkhuizen-Radersma R.,Xpand Biotechnology BV | Van Oorschot A.,IsoTis SA | And 4 more authors.
Journal of Tissue Engineering and Regenerative Medicine | Year: 2010

The aim of this study was to evaluate a semi-automated perfusion bioreactor system for the production of clinically relevant amounts of human tissue-engineered bone. Human bone marrow stromal cells (hBMSCs) of eight donors were dynamically seeded and proliferated in a perfusion bioreactor system in clinically relevant volumes (10 cm3) of macroporous biphasic calcium phosphate scaffolds (BCP particles, 2-6 mm). Cell load and distribution were shown using methylene blue staining. MTT staining was used to demonstrate viability of the present cells. After 20 days of cultivation, the particles were covered with a homogeneous layer of viable cells. Online oxygen measurements confirmed the proliferation of hBMSCs in the bioreactor. After 20 days of cultivation, the hybrid constructs became interconnected and a dense layer of extracellular matrix was present, as visualized by scanning electron microscopy (SEM). Furthermore, the hBMSCs showed differentiation towards the osteogenic lineage as was indicated by collagen type I production and alkaline phosphatase (ALP) expression. We observed no significant differences in osteogenic gene expression profiles between static and dynamic conditions like ALP, BMP2, Id1, Id2, Smad6, collagen type I, osteocalcin, osteonectin and S100A4. For the donors that showed bone formation, dynamically cultured hybrid constructs showed the same amount of bone as the statically cultured hybrid constructs. Based on these results, we conclude that a semi-automated perfusion bioreactor system is capable of producing clinically relevant and viable amounts of human tissue-engineered bone that exhibit bone-forming potential after implantation in nude mice. Copyright © 2009 John Wiley & Sons, Ltd. Source


Barbieri D.,Xpand Biotechnology BV | Yuan H.,Xpand Biotechnology BV | Yuan H.,University of Twente | De Groot F.,Xpand Biotechnology BV | And 3 more authors.
Acta Biomaterialia | Year: 2011

To evaluate moldable osteoinductive putties for bone repair we combined microstructured biphasic calcium phosphate (BCP) particles with five different polymeric gels, carboxymethyl cellulose (CMC), Pluronic® F-127 (PLU), polyvinyl alcohol (PVA), chitosan (CHI) and alginate (ALG). In vitro gel dissolution showed that CMC, PLU and ALG gels dissolved rapidly (within hours), while the CHI gel took several days and the PVA gel did not dissolve within 2 weeks. Implanting the putty formulations into sheep muscle for 12 weeks demonstrated ectopic bone formation in the control BCP group as well as the putties prepared with dissolving gels (CMC, PLU, ALG and CHI). Bone was not seen in the putty comprising PVA. Quantitative data showed that the CMC and PLU gels did not significantly affect the osteoinductivity of BCP granules, while the ALG and CHI gels showed a significant decrease in bone formation. These results suggest that the dissolvability and chemistry of the gels may be factors affecting the osteoinduction of the putties. © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Source


Groen R.W.J.,University Utrecht | Noort W.A.,University Utrecht | Raymakers R.A.,University Utrecht | Prins H.-J.,University Utrecht | And 18 more authors.
Blood | Year: 2012

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with theBMmicroenvironment is amajor drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model resulted in equivalent drug responses as observed in the corresponding patients. This novel human-mouse hybrid model creates unprecedented opportunities to investigate species-specific microenvironmental influences on normal and malignant hematopoietic development, and to develop and personalize cancer treatment strategies. © 2012 by The American Society of Hematology. Source

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