Influence of metallic particles and TNF on the transcriptional regulation of NLRP3 inflammasome-associated genes in human osteoblasts.

Introduction: The release of mature interleukin (IL-) 1ß from osteoblasts in response to danger signals is tightly regulated by the nucleotide-binding oligomerization domain leucine-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome. These danger signals include wear products resulting from aseptic loosening of joint arthroplasty. However, inflammasome activation requires two different signals: a nuclear factor-kappa B (NF-κB)-activating priming signal and an actual inflammasome-activating signal. Since human osteoblasts react to wear particles via Toll-like receptors (TLR), particles may represent an inflammasome activator that can induce both signals. Methods: Temporal gene expression profiles of TLRs and associated intracellular signaling pathways were determined to investigate the period when human osteoblasts take up metallic wear particles after initial contact and initiate a molecular response. For this purpose, human osteoblasts were treated with metallic particles derived from cobalt-chromium alloy (CoCr), lipopolysaccharides (LPS), and tumor necrosis factor-alpha (TNF) alone or in combination for incubation times ranging from one hour to three days. Shortly after adding the particles, their uptake was observed by the change in cell morphology and spectral data. Results: Exposure of osteoblasts to particles alone increased NLRP3 inflammasome-associated genes. The response was not significantly enhanced when cells were treated with CoCr + LPS or CoCr + TNF, whereas inflammation markers were induced. Despite an increase in genes related to the NLRP3 inflammasome, the release of IL-1ß was unaffected after contact with CoCr particles. Discussion: Although CoCr particles affect the expression of NLRP3 inflammasome-associated genes, a single stimulus was not sufficient to prime and activate the inflammasome. TNF was able to prime the NLRP3 inflammasome of human osteoblasts.

In Vitro Analysis of the Differentiation Capacity of Postmortally Isolated Human Chondrocytes Influenced by Different Growth Factors and Oxygen Levels.

OBJECTIVE: In the present in vitro study, we analyzed the chondrogenic differentiation capacity of human chondrocytes postmortally isolated from unaffected knee cartilage by the addition of transforming growth factor-ß1 (TGF-ß1) and/or insulin-like growth factor-1 (IGF-1) and different oxygen levels. DESIGN: After 14 and 35 days, DNA concentrations and protein contents of Col1, Col2, aggrecan as well as glycosaminoglycans (GAGs) of chondrocytes cultivated as pellet cultures were analyzed. Additionally, expression rates of mesenchymal stem cell (MSC)-associated differentiation markers were assessed in monolayer cultures. RESULTS: All cultivated chondrocytes were found to be CD29+/CD44+/CD105+/CD166+. Chondrocytic pellets stimulated with TGF-ß1 showed enhanced synthesis rates of hyaline cartilage markers and reduced expression of the non-hyaline cartilage marker Col1 under hypoxic culture conditions. CONCLUSIONS: Our results underline the substantial chondrogenic potential of human chondrocytes postmortally isolated from unaffected articular knee cartilage especially in case of TGF-ß1 administration.

Inflammatory Response of Human Peripheral Blood Mononuclear Cells and Osteoblasts Incubated With Metallic and Ceramic Submicron Particles.

Inflammatory reactions associated with osteolysis and aseptic loosening are the result of wear particles generated at the articulating surfaces of implant components. The aim of the present study was to analyze the biological response of human osteoblasts and peripheral blood mononuclear cells (PBMCs) after exposure to metallic and alumina ceramic particles regarding cellular differentiation, cytokine release, and monocyte migration. Cells were exposed to particles (0.01 and 0.05 mg/ml) from an alumina matrix composite (AMC) ceramic and a CoCr28Mo6 alloy with an average size of 0.5 µm over 48 and 96 h. The expression rates of osteogenic (Col1A1, ALP) and pro-osteoclastic (RANK, Trap5b) differentiation markers as well as pro-osteolytic mediators (MMP-1, TIMP-1, IL-6, IL-8, MCP-1) were determined and soluble protein concentrations of active MMP-1, IL-6, IL-8, and pro-collagen type 1 in cell culture supernatants were evaluated. Additionally, the capacity of particle-treated osteoblasts to attract potentially pro-inflammatory cells to the site of particle exposure was investigated by migration assays using osteoblast-conditioned media. The cellular morphology and metabolism of human osteoblasts and adherent PBMCs were influenced by particle type and concentration. In human osteoblasts, Col1A1 expression rates and protein production were significantly reduced after exposing cells to the lower concentration of cobalt-chromium (CoCr) and AMC particles. Exposure to AMC particles (0.01 mg/ml) resulted in increased mRNA levels of RANK and Trap5b in adherent PBMCs. For MMP-1 gene expression, elevated levels were more prominent after incubation with CoCr compared to AMC particles in osteoblasts, which was not reflected by the protein data. Interleukin (IL)-6 and IL-8 mRNA and protein were induced in both cell types after treatment with AMC particles, whereas exposure to CoCr particles resulted in significantly upregulated IL-6 and IL-8 protein contents in PBMCs only. Exposure of osteoblasts to CoCr particles reduced the chemoattractant potential of osteoblast-conditioned medium. Our results demonstrate distinct effects of AMC and CoCr particles in human osteoblasts and PBMCs. Complex cell and animal models are required to further evaluate the impact of cellular interactions between different cell types during particle exposure.

Contribution of human osteoblasts and macrophages to bone matrix degradation and proinflammatory cytokine release after exposure to abrasive endoprosthetic wear particles.

One of the major reasons for failure after total joint arthroplasty is aseptic loosening of the implant. At articulating surfaces, defined as the interface between implant and surrounding bone cement, wear particles can be generated and released into the periprosthetic tissue, resulting in inflammation and osteolysis. The aim of the present study was to evaluate the extent to which osteoblasts and macrophages are responsible for the osteolytic and inflammatory reactions following contact with generated wear particles from Ti­6Al­7Nb and Co­28Cr­6Mo hip stems. To this end, human osteoblasts and THP­1 monocytic cells were incubated with the experimentally generated wear particles as well as reference particles (0.01 and 0.1 mg/ml) for 48 h under standard culture conditions. To evaluate the impact of these particles on the two cell types, the release of different bone matrix degrading matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), and relevant cytokines were determined by multiplex enzyme­linked immunosorbent assays. Following incubation with wear particles, human osteoblasts showed a significant upregulation of MMP1 and MMP8, whereas macrophages reacted with enhanced MMP3, MMP8 and MMP10 production. Moreover, the synthesis of TIMPs 1 and 2 was inhibited. The osteoblasts and macrophages also responded with modified expression of the inflammatory mediators interleukin (IL)­6, IL­8, monocyte chemoattractant protein­1 and vascular endothelial growth factor. These results demonstrate that the release of wear particles affects the release of proinflammatory cytokines and has a negative impact on bone matrix formation during the first 48 h of particle exposure. Human osteoblasts are directly involved in the proinflammatory cascade of bone matrix degradation. The simultaneous activation and recruitment of monocytes/macrophages boosted osteolytic processes in the periprosthetic tissue. By the downregulation of TIMP production and the concomitant upregulation of MMPs as a response to particle exposure, bone formation around implants may be suppressed, resulting in implant failure.

The potential role of human osteoblasts for periprosthetic osteolysis following exposure to wear particles.

Aseptic loosening in total hip replacement is mainly caused by wear particles inducing inflammation and osteolysis. Wear can be a consequence of micromotions at the interface between implant and bone cement. Due to complex cellular interactions, different mediators (e.g. cytokines, proteinases) are released, which can promote osteolytic processes in the periprosthetic tissue followed by loosening of the implant. Furthermore, a reduced matrix synthesis and an induced apoptosis rate can be observed. The purpose of this study was to evaluate to what extent human primary osteoblasts exposed to wear particles are involved in the osteolysis. The viability, the secretion of collagen and collagenases and the variety of released cytokines after particle exposure was examined. Therefore, human osteoblasts were incubated with particles experimentally generated in the interface between hip stems with rough and smooth surface finishings as well as different material compositions (Ti-6Al-7Nb, Co-28Cr-6Mo and 316L) and bone cement mantle made of Palacos R containing zirconium oxide particles. Commercially pure titanium particles, titanium oxide, polymethylmethacrylate and particulate zirconium oxide were used as references. The results revealed distinct effects on the cytokine release of human osteoblasts towards particulate debris. Thereby, human osteoblasts released increased levels of interleukine (IL)-6 and IL-8 after treatment with metallic wear particles. The expression of VEGF was slightly induced by all particle entities at lower concentrations. Apoptotic rates were enhanced for osteoblasts exposed to all the tested particles. Furthermore, the de novo synthesis of type 1 collagen was reduced and the expression of the matrix metalloproteinase (MMP)-1 was considerably increased. However, wear particles of Co-28Cr-6Mo stems seemed to be more aggressive, whereas particles derived from stainless steel stems caused less adverse cellular reaction. Among the reference particles, which caused less altered reactions in the metabolism of osteoblasts in general, ZrO2 can be assumed as the material with the smallest cell biological effects.

Differentiation capacity of human chondrocytes embedded in alginate matrix.

Healing capacity of cartilage is low. Thus, cartilage defects do not regenerate as hyaline but mostly as fibrous cartilage which is a major drawback since this tissue is not well adapted to the mechanical loading within the joint. During in vitro cultivation in monolayers, chondrocytes proliferate and de-differentiate to fibroblasts. In three-dimensional cell cultures, de-differentiated chondrocytes could re-differentiate toward the chondrogenic lineage and re-express the chondrogenic phenotype. The objective of this study was to characterize the mesenchymal stem cell (MSC) potential of human chondrocytes isolated from articular cartilage. Furthermore, the differentiation capacity of human chondrocytes in three-dimensional cell cultures was analyzed to target differentiation direction into hyaline cartilage. After isolation and cultivation of chondrogenic cells, the expression of the MSC-associated markers: cluster of differentiation (CD)166, CD44, CD105, and CD29 was performed by flow cytometry. The differentiation capacity of human chondrocytes was analyzed in alginate matrix cultured in Dulbecco?s modified eagle medium with (chondrogenic stimulation) and without (control) chondrogenic growth factors. Additionally, the expression of collagen type II, aggrecan, and glycosaminoglycans was determined. Cultivated chondrocytes showed an enhanced expression of the MSC-associated markers with increasing passages. After chondrogenic stimulation in alginate matrix, the chondrocytes revealed a significant increase of cell number compared with unstimulated cells. Further, a higher synthesis rate of glycosaminoglycans and a positive collagen type II and aggrecan immunostaining was detected in stimulated alginate beads. Human chondrocytes showed plasticity whilst cells were encapsulated in alginate and stimulated by growth factors. Stimulated cells demonstrated characteristics of chondrogenic re-differentiation due to collagen type II and aggrecan synthesis.