Cell viability, collagen synthesis and cytokine expression in human osteoblasts following incubation with generated wear particles using different bone cements.

In total hip arthroplasty, wear particles generated at articulating surfaces and interfaces between bone, cement and implants have a negative impact on osteoblasts, leading to osteolysis and implant loosening. The aim of this experimental study was to determine the effects of particulate wear debris generated at the interface between straight stainless steel hip stems (Exeter(®)) and three different bone cements (Palacos(®) R, Simplex™ P and Cemex(®) Genta) on cell viability, collagen synthesis and cytokine expression in human osteoblasts. Primary osteoblasts were treated with various concentrations of wear particles. The synthesis of procollagen type I and different cytokines was analysed, and markers for apoptosis and necrosis were also detected. The cytokine synthesis rates in the osteoblasts were initially increased and varied, depending on incubation time and particle concentration. Specific differences in the synthesis rates of interleukin (IL)­6, IL-8, vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) were observed with the different bone cements examined. The negative effect of the particles on the synthesis of procollagen type I and increased rates of cell apoptosis and necrosis were observed with all three cements analysed. Our present data suggest that wear particles from the interface between the total hip stem and bone cement have a significant effect on viability, cytokine expression and collagen synthesis in human osteoblasts, depending on the bone cement used.

New Coating Technique of Ceramic Implants with Different Glass Solder Matrices for Improved Osseointegration-Mechanical Investigations.

Ceramics are a very popular material in dental implant technology due to their tribological properties, their biocompatibility and their esthetic appearance. However, their natural surface structure lacks the ability of proper osseointegration, which constitutes a crucial process for the stability and, thus, the functionality of a bone implant. We investigated the application of a glass solder matrix in three configurations-consisting mainly of SiO2, Al2O3, K2O and Na2O to TZP-A ceramic specimens. The corresponding adhesive strength and surface roughness of the coatings on ceramic specimens have been analyzed. Thereby, high adhesive strength (70.3 ± 7.9 MPa) was found for the three different coatings. The obtained roughness (Rz) amounted to 18.24 ± 2.48 µm in average, with significant differences between the glass solder configurations. Furthermore, one configuration was also tested after additional etching which did not lead to significant increase of surface roughness (19.37 ± 1.04 µm) or adhesive strength (57.2 ± 5.8 MPa). In conclusion, coating with glass solder matrix seems to be a promising surface modification technique that may enable direct insertion of ceramic implants in dental and orthopaedic surgery.

TGF-ß1 and IGF-1 influence the re-differentiation capacity of human chondrocytes in 3D pellet cultures in relation to different oxygen concentrations.

To prevent de-differentiation of chondrocytes in vitro, the 3D environment, growth factors and different oxygen concentrations were considered. In this in vitro study, we quantified the influence of insulin-like growth factor (IGF)-1 and/or transforming growth factor (TGF)-ß1 under differing oxygen (5/21% O(2)) levels on the proliferation and synthesis rates of hyaline extracellular matrix (ECM) components in chondrogenic pellet cultures. Human chondrocytes isolated from articular cartilage were transferred into conical tubes to form pellets. Pellets were stimulated with TGF-ß1 and/or IGF-1. After 2 and 5 weeks of cultivation the DNA concentration and expression of pro-collagen type 1, type 2 and aggrecan were analysed. Under hypoxia the DNA content remained stable. In contrast, under normoxia, cells showed an increase of DNA concentration after stimulation with TGF-ß1/IGF-1 and TGF-ß1. Nevertheless, DNA contents under normoxia did not reach the values of hypoxic-cultivated cells. Under both culture conditions a reduced synthesis of pro-collagen type 1 could be determined. Although the expression of pro-collagen type 2 was significantly higher under normoxia, a decrease in the case of TGF-ß1/IGF-1- and IGF-1-stimulated cells was observed. Under hypoxia pro-collagen type 2 contents remained stable or increased for TGF-ß1/IGF-1-stimulated cells. Furthermore, incubation with growth factors resulted in aggrecan accumulation under hypoxia, while a reduced expression under normoxia could be determined for TGF-ß1/IGF-1- and IGF-1-stimulated cells. Our results demonstrate that the treatment with growth factors causes differences in the expression of ECM compounds within pellet cultures. While under normoxia TGF-ß1 alone leads to a positive effect of the expression of hyaline cartilage-specific ECM components, an additive effect of both growth factors was only determined under hypoxia.

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.

Oxygen consumption, acidification and migration capacity of human primary osteoblasts within a three-dimensional tantalum scaffold.

A major clinical problem within synthetic, large-scaled scaffolds is the insufficient nutrient supply resulting in inhomogeneous cell proliferation and differentiation. The aim of this study was to analyse pH value, oxygen consumption and migration of human osteoblasts within a 3D tantalum scaffold, clinically used for larger bone defects. After 24 h the oxygen concentration within the scaffold decreased significantly and remained low during incubation. Monitoring of the pH value inside the tantalum scaffold showed a slightly acidification under static culture conditions. However, cell migration within the 3D scaffold was detected. Hence, in clinical application it can be assumed that porous tantalum scaffolds can be settled by osteoblasts under critical oxygen and nutrient supply. In general, monitoring of cell migration, oxygen consumption and acidification can be a suitable instrument for creating advanced 3D bone scaffolds.

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.

Migration Capacity and Viability of Human Primary Osteoblasts in Synthetic Three-dimensional Bone Scaffolds Made of Tricalciumphosphate.

In current therapeutic strategies, bone defects are filled up by bone auto- or allografts. Since they are limited by insufficient availability and donor site morbidity, it is necessary to find an appropriate alternative of synthetic porous bone materials. Because of their osteoconductive characteristics, ceramic materials like tricalciumphosphate (TCP) are suitable to fill up bone defects. Another advantage of TCP implants is the ability of patient-specific engineering. Objective of the present in-vitro study was to analyze the migration capacity and viability of human primary osteoblasts in porous three-dimensional TCP scaffolds in a static cell culture. To obtain data of the cellular supply with nutrients and oxygen, we determined the oxygen concentration and the pH value within the 3D scaffold compared to the surrounding medium using microsensors. After eight days of cultivation we found cells on all four planes. During incubation, the oxygen concentration within the scaffold decreased by approximately 8%. Furthermore, we could not demonstrate an increasing acidification in the core of the TCP scaffold. Our results suggest that osteoblasts could migrate and survive within the macroporous TCP scaffolds. The selected size of the macropores prevents overgrowth of cells, whereby the oxygen and nutrients supply is sufficiently guaranteed.

Molecular determinants of the profibrogenic effects of endothelin-1 in pancreatic stellate cells.

AIM: To gain molecular insights into the expression and functions of endothelin-1 (ET-1) in pancreatic stellate cells (PSC). METHODS: PSCs were isolated from rat pancreas tissue, cultured, and stimulated with ET-1 or other extracellular mediators. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine into DNA and cell migration was studied in a transwell chamber assay. Gene expression at the level of mRNA was quantified by real-time polymerase chain reaction. Expression and phosphorylation of proteins were monitored by immunoblotting, applying an infrared imaging technology. ET-1 levels in cell culture supernatants were determined by an enzyme immunometric assay. To study DNA binding of individual transcription factors, electrophoretic mobility shift assays were performed. RESULTS: Among several mediators tested, transforming growth factor-beta1 and tumour necrosis factor-alpha displayed the strongest stimulatory effects on ET-1 secretion. The cytokines induced binding of Smad3 and NF-kappaB, respectively, to oligonucleotides derived from the ET-1 promoter, implicating both transcription factors in the induction of ET-1 gene expression. In accordance with previous studies, ET-1 was found to stimulate migration but not proliferation of PSC. Stimulation of ET-1 receptors led to the activation of two distinct mitogen-activated protein kinases, p38 and extracellular signal-regulated kinases (ERK)1/2, as well as the transcription factor activator protein-1. At the mRNA level, enhanced expression of the PSC activation marker, alpha-smooth muscle actin and two proinflammatory cytokines, interleukin (IL)-1beta and IL-6, was observed. CONCLUSION: This study provides novel lines of evidence for profibrogenic and proinflammatory actions of ET-1 in the pancreas, encouraging further studies with ET-1 inhibitors in chronic pancreatitis.