Costimulatory molecule expression following exposure to orthopaedic implants wear debris

Patients with long-term orthopedic implants may develop inflammatory reactions due to the accumulation of biomaterial particles both around the implant and in distant organs. The exact impact of these particles on the normal immune cell function still remain relatively unclear. Activation of T-cells following exposure to biomaterial particles is driven by macrophages and requires synergistic signals primed by both antigen presentation and costimulation. The pattern of costimulatory molecule expression (CD80,CD86) was primarily examined using immunohistochemistry on tissue specimens of bone/implant interface membranes taken from sites of bone erosion. Additionally, costimulatory molecule expression was also assessed in the monocytic leukemia cell line U937 following exposure to clinically relevant titanium aluminum vanadium (TiAlV) and stainless steel particles (FeCrNi) cultured in vitro. This study demonstrates the induction and prominent expression of CD86 on almost all macrophage subsets at the bone/implant interface, including fused forms and large multinucleated giant cells (MNGC). In vitro analysis also indicated phagocytosis of metal particles by differentiated U937 caused significant induction of both CD80 and CD86 (p < 0.01), although the expression of CD86 dominated following prolonged exposure. The data presented highlights that CD86 is the predominant costimulatory molecule ligating to the complementary CD28 molecule at the inflammatory lesion of the interface. We propose that the intracellular presence of indigestible implant material, in addition to elevated costimulatory molecule expression, may promote T-cell inflammatory reactions at sites close to and distant from the orthopedic implant.     

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.     

微小RNA调控骨髓干细胞、成骨破骨细胞在骨代谢中的影响

背景：最近研究发现微小RNA在间充质干细胞分化过程中发挥着重要的调控作用。 目的：总结探讨微小RNA在间充质干细胞中的作用,全面了解间充质干细胞分化的机制,对骨质疏松症的预防和治疗有重要意义。 方法：由第一作者检索1990/2008 PubMed数据及万方数据库有关微小RNA调控骨髓间充质干细胞从而影响骨代谢等方面的文献,英文检索词为“microRNA,mesenchymal stem cells,bone metabolism”,中文检索词为“微小RNA,骨髓间充质干细胞,骨代谢”。根据纳入标准保留30篇进一步归纳总结。 结果与结论：微小RNA通过对多种转录因子、生长因子和信号通路的调节作用,影响着间充质干细胞的自我更新和分化过程。运用高通量技术筛选出与骨髓间充质干细胞分化相关的特异性微小RNA,研究其作用的分子机制,对于骨髓间充质干细胞分化异常所致的骨代谢疾病,如骨质疏松症等发病机制的阐明具有重要作用。微小RNA在骨髓间充质干细胞的分化中作用机制的研究将为骨代谢疾病的预防和治疗提供一个新的方向。     

In vitro and in vivo activation of polymorphonuclear leukocytes in response to particulate debris

The host inflammatory response to particulate wear debris has been implicated as a principal cause of osteolysis and aseptic loosening following total joint arthroplasty. While it has long been assumed that this inflammatory response is mediated solely by a chronic process, there has been evidence to suggest that an acute response to particulate debris may be important in initiating the chronic response. We studied the in vitro and in vivo acute inflammatory responses mediated by polymorphonuclear leukocytes (PMNs) to both retrieved particulate from a catastrophically failed uncemented metal-backed acetabular component and to commercially pure particulate (polyethylene, cobalt-chrome, and titanium). Isolated, nonactivated human PMNs in vitro exhibited both a dose- and time-dependent degranulation response to opsonized particulate debris, as evidenced by release of both specific (increased lysozyme activity) and azurophilic (increased beta-glucuronidase activity) granule contents. In the rat subcutaneous pouch model in vivo, PMNs were recruited within 3-6 h after exposure to particulate debris and were noted to phagocytize particulate and subsequently degranulate, as evidenced by increased beta-glucuronidase and PMN-specific myeloperoxidase (azurophilic granule enzymes) activities. This response peaked within the first 6 h and gradually declined by 24 h. The results of this study demonstrate the presence of an acute inflammatory response mediated by PMNs both in vitro and in vivo to particulate debris, which may be important in the sequence of events that lead to the macrophage-dominated chronic inflammatory process culminating in osteolysis and aseptic loosening of total joint arthroplasties.     

Response of human osteoblasts exposed to wear particles generated at the interface of total hip stems and bone cement

Aseptic loosening of total hip replacement is mainly caused by wear particles. Abrasive wear occurs at articulating surfaces or as a consequence of micro-motions at the interface between femoral stem and bone cement. Direct impact of wear particles on osteolysis, the remodeling of the bone stock and a directly affected function of osteoblasts was described. The present study examined the response of human osteoblasts exposed to different wear particles, which were generated in a test device providing oscillating micro-motions at the interface between femoral stem and standard bone cement. Characterization of released particles was performed by quantifying the size distribution and the metal content of the wear debris. Human osteoblasts were incubated with particles obtained from hip stems with different material compositions (Ti-6Al-7Nb and Co-28Cr-6Mo) and rough and smooth surface finishings combined with standard bone cement (Palacos(R) R) containing zirconium oxide particles. Commercially pure titanium particles (cp-Ti) and particulate zirconium oxide (ZrO(2)) were used for comparative analyses. The results revealed significant (p < 0.05) reduction of the cell viability after exposure to higher concentration of metallic particles, particularly from Co-based alloys. In contrast, ZrO(2) alone showed significantly less adverse effects on the cells. When increasing metallic particle concentrations massive inhibition was also observed in the release of cytokines including interleukine-6 (IL-6) and interleukine-8 (IL-8), but the expression of Procollagen I and the cell viability showed the highest reduction after exposure to Co-based alloy particles from rough stems.     

Bisphenol A at concentrations relevant to human exposure enhances histamine and cysteinyl leukotriene release from bone marrow-derived mast cells

Abstract

Bisphenol A (BPA), a monomer of polycarbonate plastics and epoxide resin, acts as an endocrine-active compound and has been shown to enhance the inflammatory response to allergen challenge. Previous reports in rodents have demonstrated that perinatal BPA exposure alters airway inflammation following sensitization and challenge to ovalbumin in juvenile and adult offspring. Additionally, a high concentration of BPA has been shown to enhance mediator release in mast cell lines. This study aimed to determine if short-term BPA exposure, at levels relevant to human exposure, enhances mast cell release of histamine and cysteinyl leukotrienes (CysLTs). Primary murine bone marrow-derived mast cells (BMMC) produced from the femurs of female C57BL/6 mice were stimulated with BPA or estradiol (E2) in vitro. It was observed that both BPA and E2 increased BMMC histamine release over a range of nanomolar concentrations (1–1000?nM). The estrogen receptor (ER) antagonist ICI 182,780 partially blocked the ability of E2, but not BPA, to elevate histamine release. BPA also increased CysLT release, which was not abrogated by ER inhibition. It was also observed that the ability of BPA to enhance histamine and CysLT release was inhibited by blocking the extracellular signal-regulated kinase (ERK) pathway with U0126 or by chelating extracellular calcium (Ca²⁺) using EGTA. In summary, these experiments are the first to demonstrate that acute BPA exposure enhances mast cell histamine and CysLT release in vitro—an effect that is not dependent on an ER-mediated mechanism. Instead, BPA-induced mast cell histamine and CysLT release may be mediated, in part, by the ERK pathway and extracellular Ca²⁺ concentrations. These data suggest that exposure to BPA at levels relevant to human exposure may provoke an acute inflammatory response in atopic individuals via enhanced mast cell activation.     

Interaction of alveolar macrophages and airway epithelial cells following exposure to particulate matter produces mediators that stimulate the bone marrow

Exposure to ambient air pollution particles with a diameter of < 10 microm (PM(10)) has been associated with increased cardiopulmonary morbidity and mortality. We postulate that these adverse health effects are related to proinflammatory mediators produced in the lung and released into the circulation where they initiate a systemic inflammatory response. The present study was designed to determine if alveolar macrophages (AMs) and primary human bronchial epithelial cells (HBECs) interact to amplify the production of certain cytokines when exposed to ambient PM(10) (EHC-93). Candidate cytokines were measured at the mRNA level using a RNase protection assay and at the protein level by enzyme-linked immunosorbent assay (ELISA). When AM/HBEC cocultures were exposed to 100 microg/ml of PM(10), levels of tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin (IL)-1beta, IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), and IL-8 mRNA increased within 2 h (P < 0.05) and 8 h following exposure compared with control cells. GM-CSF mRNA expression was more rapidly induced in cocultured cells compared with HBECs or AMs alone. The concentrations of TNF-alpha, GM-CSF, IL-1beta, IL-6, and IL-8 in the cocultured supernatants collected after 24 h PM(10) exposure increased significantly compared with control cells. There was a significant synergistic effect between AMs and HBECs in the production of GM-CSF and of IL-6 (P < 0.05). Instillation of supernatants from HBECs cultured with PM(10) into lungs of rabbits failed to increase circulating band cell counts or stimulate the bone marrow. However, those from AM/HBEC cocultures exposed to PM(10) increased circulating band cell counts (P < 0.05) and shortened the transit time of polymorphonuclear leukocytes (PMNs) through the bone marrow compared with control co-cultures (P < 0.01). These results suggest that the interaction between AMs and HBECs during PM(10) exposure contributes to the production of mediators that induce a systemic inflammatory response.     

Immunobiology of periprosthetic inflammation and pain following ultra-high-molecular-weight-polyethylene wear debris in the lumbar spine

Introduction: Wear debris-induced osteolysis is a common cause of arthroplasty failure in several joints including the knee, hip and intervertebral disc. Debris from the prosthesis can trigger an inflammatory response that leads to aseptic loosening and prosthesis failure. In the spine, periprosthetic pain also occurs following accumulation of wear debris through neovascularization of the disc. The role of the immune system in the pathobiology of periprosthetic osteolysis of joint replacements is debatable.

Areas covered: We discussed the stimulation of pro-inflammatory and pro-protective and pro-regenerative pathways due to debris from the prosthetics. The balance between the two pathways may determine the outcome results. Also, the role of cytokines and immune cells in periprosthetic inflammation in the etiology of osteolysis is critically reviewed.

Expert commentary: Therapies targeting the inflammatory process associated with ultra-high-molecular-weight polyethylene wear debris could reduce implant failure. Additionally, therapies targeting neovascularization of discs following arthroplasty could mitigate periprosthetic pain.     

Phagocytosis of wear debris by osteoblasts affects differentiation and local factor production in a manner dependent on particle composition

Wear debris is considered to be one of the main factors responsible for aseptic loosening of orthopaedic endoprostheses. Whereas the response of cells in the monocytic lineage to foreign materials has been extensively studied, little is known about cells at the bone formation site. In the present study, we examined the hypothesis that the response of osteoblasts to wear debris depends on the chemical composition of the particles. We produced particles from commercially pure titanium (cpTi), Ti-6Al-4V (Ti-A), and cobalt-chrome (CoCr) and obtained ultrahigh molecular weight polyethylene (UHMWPE; GUR 4150) particles from a commercial source. The equivalent circle diameters of the particles were comparable: 1.0 +/- 0.96 microm for UHMWPE; 0.84 +/- 0.12 microm for cpTi; 1.35 +/- 0.09 microm for Ti-A, and 1.21 +/- 0.16 microm for CoCr. Confluent primary human osteoblasts and MG63 osteoblast-like cells were incubated in the presence of particles for 24 h. Harvested cultures were examined by transmission electron microscopy to determine if the cells had phagocytosed the particles. Particles were found intracellularly, primarily in the cytosol, in both the primary osteoblasts and MG63 cells. The chemical composition of the particles inside the cells was confirmed by energy-dispersive X-ray analysis. Morphologically, both cell types had extensive ruffled cell membranes, less-developed endoplasmic reticulum, swollen mitochondria, and vacuolic inclusions compared with untreated cells. CpTi, Ti-A, and CoCr particles were also added to cultures of MG63 cells to assess their effect on proliferation (cell number) and differentiation (alkaline phosphatase activity), and PGE2 production. All three types of particles had effects on the cells. The effect on cell number was dependent on the chemical composition of the particles; Ti-A and CoCr caused a dose-dependent increase, while cpTi particles had a biphasic effect with a maximal increase in cell number observed at the 1:10 dilution. Alkaline phosphatase specific activity was also affected and cpTi was more inhibitory than Ti-A or CoCr. PGE2 production was increased by all particles, but the magnitude of the effect was particle-dependent: CoCr > cpTi > Ti-A. This study demonstrates clearly that human osteoblast-like cells and MG63 cells can phagocytose small UHMWPE, CoCr, Ti-A, and cpTi particles. Phagocytosis of the particles is correlated with changes in morphology, and analysis of MG63 response shows that cell proliferation, differentiation, and prostanoid production are affected. This may have negative effects on bone formation adjacent to an orthopaedic implant and may initiate or contribute to the cellular events that cause aseptic loosening by inhibiting bone formation. The effects on alkaline phosphatase and PGE2 release are dependent on the chemical composition of the particles, suggesting that both the type and concentration of wear debris at an implant site may be important in determining clinical outcome.     

Bone morphogenetic protein-transduced human fibroblasts convert to osteoblasts and form bone in vivo

Experimental cell or ex vivo gene therapy for localized bone formation typically uses osteoprogenitor cells propagated from periosteum or bone marrow. Both require bone or marrow biopsies to obtain cells. We have demonstrated that implantation of gingival or dermal fibroblasts transduced with BMP ex vivo, using a recombinant adenovirus (AdCMVBMP) attached to porous biodegradable scaffolds, form bone in vivo. Here we show that BMP-7-transduced fibroblasts suspended in injectable thermoset hydrogels form complete ossicles on subcutaneous injection and repair segmental defects in rat femurs. Bone formation was preceded by an intermediate cartilage stage. To determine the fate of the implanted transduced cells, thermoset hydrogel suspensions of ex vivo BMP-7-transduced or nontransduced fibroblasts were placed in diffusion chambers and implanted to allow development in vivo without direct contact with host cells. Only the BMP-transduced fibroblasts formed bone within the diffusion chambers in vivo, revealing that BMP transduction induces osteoblastic conversion of these cells.     

Microarray-based gene expression analysis of human osteoblasts in response to different biomaterials

Several biomaterials have been widely used in bone regeneration/substitution procedures in orthopedic and oral surgery. However, how these biomaterials alter osteoblast gene expression is poorly understood. We therefore attempted to address this question by using cDNA microarray technique to identify genes that are differentially regulated in osteoblasts exposed to biomaterials comprehending the biocompatibility spectrum of bioactive (bioglass and hydroxyapatite), bioinert (Ti and stainless steel), and biotolerant (polymethylmethacrylate). By using a cDNA microarray containing 687 human IMAGE sequences, we identified in primary cultures of osteoblastic cells differentiated from the human bone marrow and exposed to these biomaterials, genes whose expression was significantly upregulated or downregulated. Among the differentially expressed genes we have found those involved with cell cycle regulation, cell differentiation and proliferation, apoptosis, cell adhesion, bone mineralization and skeletal development. These results can be relevant to a better understanding of the molecular mechanism underlying the behavior of osteoblasts in bone regenerative procedures.     

同种异基因成骨细胞对混合淋巴细胞反应体系中T细胞的免疫抑制

背景：由同种异基因骨髓间充质干细胞诱导的成骨细胞移植免疫反应各家报道不一致,差别明显。 目的：体外观察由骨髓间充质干细胞诱导而成的成骨细胞对T细胞的免疫调节作用及特点。 方法：用Ficoll-Hypaque梯度密度离心法分离出兔骨髓单个核细胞,体外扩增,获取第3代细胞,经典化学方法诱导为成骨细胞,将其按照不同的比例加入到T细胞形成双向混合淋巴细胞培养体系中,在第3,5,7天,用MTT比色法检测各组混合淋巴细胞培养体系中的T细胞增殖情况,24 h后用流式细胞仪分析各组T细胞亚群凋亡情况。 结果与结论：诱导后成骨细胞混合淋巴细胞培养体系中,成骨细胞对T细胞的增殖有抑制作用,在一定范围内,抑制作用具有量效关系。诱导后成骨细胞剂量大,时间延长,抑制程度增强;3次平均抑制率相比：1∶20组低于1∶80组(P < 0.01);1∶40组低于1∶80组(P < 0.05);诱导后成骨细胞能引起T细胞亚群凋亡,其中CD4⁺(凋亡率8.57%)亚群不如CD8⁺(凋亡率15.31%)细胞亚群凋亡显著(P < 0.01)。结果显示诱导的成骨细胞在体外能够通过细胞凋亡途径抑制T细胞的增殖,特别是CD8⁺。但这种抑制不是特别强,表明诱导的成骨细胞虽有一定的免疫性,但其免疫性较低。     

Acute systemic immune activation following conjunctival exposure to staphylococcal enterotoxin B

Conjunctival exposure to the Staphylococcus aureus superantigen staphylococcal enterotoxin B (SEB) may occur accidentally, as a result of bioterrorism, or during colonization or infection of the external eye. Using human leukocyte antigen class II transgenic mice, we show for the first time that conjunctival exposure to SEB can cause robust systemic immune activation.     

小鼠骨髓成熟与不成熟树突状细胞中RelB基因的表达

目的:探讨体外分离培养的小鼠骨髓来源的成熟与未成熟DC中核转录因子RelB(avianreticuloendotheliosisviral(v-rel)oncogenerelatedB)基因的表达。方法:无菌从C57BL/6小鼠股骨和胫骨中取出骨髓细胞,利用rmGM-CSF和rmIL-4联合诱导骨髓前体细胞产生未成熟的DC,未成熟的DC在培养结束前18h经LPS刺激获得成熟的DC,用流式细胞术分析它们的表型,用RT-PCR和免疫荧光染色法检测成熟与未成熟DC中,RelBmRNA和其蛋白的表达。结果:流式细胞术分析显示未成熟的DC中MHC-Ⅱ类分子和共刺激分子(CD86和CD40)呈低水平表达;而成熟的DC则呈高水平表达。RT-PCR和免疫荧光染色法检测结果均显示,RelB基因在未成熟的DC中呈低水平表达;而在成熟的DC中呈高水平表达,两者比较具有统计学意义(P<0.01)。结论:RelB基因的表达与小鼠骨髓来源的DC的成熟状态密切相关。抑制DC中RelB基因的表达,有可能诱导产生具有耐受原性的未成熟的DC。     

Nature of mutation in the human hprt gene following in vivo exposure to ionizing radiation of cesium-137

The current study comprises the analysis of mutations in 10 individuals accidentally exposed to cesium-137 during the 1987 radiological accident in Goiânia, Brazil. Their exposures were among the highest experienced, ranging from 1 to 7 Gy. Peripheral T-lymphocyte samples were obtained 3.3 years after the original exposure and mutation was studied at the hprt locus using the 6-thioguanine-resistance selection assay. The mutational spectrum for the exposed population is comprised of 90 independent mutants. Based on T-cell receptor analysis, only 5% (5/95) were clonally related. Mutants were initially studied using RT-PCR and directly sequenced using an automated laser fluorescent DNA sequencer. Mutants that repeatedly failed to produce cDNAs were studied using a multiplex PCR assay with genomic DNA. Missense mutations were the most frequent event recovered, comprising 40% (23/57) of the spectral sample. An excess of events involving A:T base pairs was observed, exhibiting a significant difference (χ² = 12.7, P = 0.0004) when compared to the spontaneous spectrum. This finding may reflect the effect of ionizing radiation-induced damage, suggesting a potential similarity to radiation effects in prokaryotes. At the genomic level, 36.7% (33/90) of the mutants exhibited gross structural alterations, as detected by multiplex PCR. Deletion events were over-represented in our spectral sample, displaying a twofold increase when compared to the frequency observed in the spontaneous mutation database. Environ. Mol. Mutagen. 30:385–395, 1997 © 1997 Wiley-Liss, Inc.     

Cultured human bone marrow-derived mast cells, their similarities to cultured murine E-mast cells

Homogeneous populations of human mast cells were differentiated and grown by culturing bone marrow cells in the presence of conditioned medium derived from lectin-stimulated human peripheral blood mononuclear cells. The cells obtained were similar in ultrastructure, proteoglycan type and lipid products generated upon calcium ionophore A23187, and immunological activation to the murine E-mast cells (E-MC) differentiated in culture containing IL-3. Fluorescence analysis revealed that the human E-MC expressed IgE-Fc receptors which retained bound IgE through several washes. These cells did not express cell-surface lymphoid determinants (T11, T4, T8 and B4) and myeloid determinants 'My'. However, 40% of these cells expressed monocytic surface determinants, such as M-1. The amount of histamine that was found per 10(6) cells was 525 +/- 106 ng (mean +/- SE, n = 4). These cultured mast cells possessed granular chondroitin sulphate E proteoglycan of about 180,000 MW. Following activation with either calcium ionophore A23187 or anti-hIgE challenge, these mast cells released their preformed mediators and generated mainly leukotriene C4 leukotriene B4, and platelet-activating factor. In conclusion, according to all of these criteria, these human cultured mast cells show many similarities to the murine cultured E-mast cells, and therefore could be considered as the culture analogue of the human intestinal E-mast cells identified recently.