Modulation of the phenotypic and functional properties of phagocytic macrophages by wear particles from orthopaedic implants

An attempt was made to assess the local chronic inflammatory response in patients with failed orthopaedic implant that is clinically associated with osteolysis, bone and bone marrow necrosis. The main objective was to analyse the heterogeneity of the macrophage functional subsets in the bone–implant interface membrane and to evaluate their possible role in the development of an erosive inflammatory lesion within the bone. Immunohistology was performed on 21 specimens of the bone–implant interface obtained from 17 patients during revision arthroplasty, and synovial membranes from rheumatoid (RA, n=4), and osteoarthritis (OA, n=4) patients. Three well-characterized monoclonal antibodies (MAb) recognizing antigenic determinants on specific functional subsets of macrophages (M) were used. RFD1 (interdigitating reticulum cells/antigen presenting cells, (APC), RFD7 (mature phagocytic macrophages), and RFD9 epithelioid cells and foreign body giant cells (FBGC). RFD1 was expressed on a variable number of perivascular and synovial lining M in both RA and OA synovia, at a frequency of 25%–40%. In cases with total joint replacements, the interface showed a marked increase in the expression of RFD1 (20%–90%). A considerably greater percentage of RFD1 positive M and FBGC was noted in the interfaces from cases with a high level of detectable metal particulate wear debris (mean 80%, range 60%–90%) than in cases with polyethylene wear debris (mean 30%, range 0%–50%), p 0.0001. RFD7 labelled most tissue M in each group. Immunoreactivity for RFD9 was restricted to FBGC in all cases analysed. The finding of elevated expression of RFD1 on metal-containing M and FBGC in the bone-implant interface suggests an increase in antigen-presenting phenotype and indicates that metal particles have more impact in the induction of immune-mediated responses. Such responses are characterized by sustained cellular hyperreactivity and phenotypic changes in M subsets.     

Cyclodextrin‐Derived Intrinsically Bioactive Nanoparticles for Treatment of Acute and Chronic Inflammatory Diseases

Inflammation is a common cause of many acute and chronic inflammatory diseases. A major limitation of existing anti‐inflammatory therapeutics is that they cannot simultaneously regulate pro‐inflammatory cytokine production, oxidative stress, and recruitment of neutrophils and macrophages. To overcome this limitation, nanoparticles (NPs) with multiple pharmacological activities are synthesized, using a chemically modified cyclic oligosaccharide. The manufacture of this type of bioactive, saccharide material‐based NPs (defined as LCD NP) is straightforward, cost‐effective, and scalable. Functionally, LCD NP effectively inhibits inflammatory response, oxidative stress, and cell migration for both neutrophils and macrophages, two major players of inflammation. Therapeutically, LCD NP shows desirable efficacies for the treatment of acute and chronic inflammatory diseases in mouse models of peritonitis, acute lung injury, and atherosclerosis. Mechanistically, the therapeutic benefits of LCD NP are achieved by inhibiting neutrophil‐mediated inflammatory macrophage recruitment and by preventing subsequent pro‐inflammatory events. In addition, LCD NP shows good safety profile in a mouse model. Thus, LCD NP can serve as an effective anti‐inflammatory nanotherapy for the treatment of inflammatory diseases mainly associated with neutrophil and macrophage infiltration.     

The role of newly formed vessels and cell adhesion molecules in the tissue response to wear products from orthopaedic implants

Neovascularization and activation of endothelial cells play an important role in recruitment of blood leucocytes at sites of inflammation. This study aimed to assess the pattern of vascular growth and the expression of cell adhesion molecules on vascular endothelium and inflammatory macrophages and T cells in the bone-implant interface from patients with aseptically loosened orthopaedic prostheses. ELAM-1, VCAM-1, ICAM-1 and the receptors LFA-1 and CR3 were immunolocalized on cryostat sections of the interface obtained during revision arthroplasty. The results showed that ELAM-1 was restricted to endothelium and was upregulated on different vessels in 21 cases. Its expression correlated strongly with the presence of metal wear debris. VCAM-1 was less frequently expressed (n=6 cases), and was co-expressed with ELAM-1 in three cases with metal debris. ICAM-1 was detected on a large number of vessels on the bone side in 13 cases, but was more strongly expressed on macrophage subsets and foreign body giant cells (FBGCs) on the lining layer at the implant side. This study indicates the contribution of three different pathways in the migration of inflammatory cells to the bone-implant interface in response to phagocytosis of implant degradation products. Upregulated ELAM-1 expression may suggest a role in hypersensitivity reactions. Finally the persistent expression of VCAM-1 and ICAM-1 on macrophages and FBGCs in the lining layer indicates possible cellular interactions with the extracellular matrix proteins.     

Monocyte–biomaterial interaction inducing phenotypic dynamics of monocytes: a possible role of monocyte subsets in biocompatibility

For the in vitro study of cell–biomaterial surface interactions, the choice of cell type is crucial. In vivo data indicate that during the healing of the implant in the tissues, the pivotal cell types are the macrophages. These cells, upon interaction with any foreign material, might initiate a spectrum of responses, which could lead to acute and chronic inflammatory changes affecting the biocompatibility of the implant. Whether the mechanisms governing the type of evolving inflammatory reaction could be attributed to the macrophages functional differentiation mirrored by monocyte subsets during the polymer interaction, is poorly described. This in vitro study, therefore, attempted to investigate whether different biomaterials influence monocyte cellular activity, determined by the myeloperoxidase level and mitochondrial XTT cleavage, and phenotype dynamics characterized by the presence of CD14, RM 3/1 and 27E10 antigens. It is shown that different polymers exert differential potential to influence monocytes, both in their cellular activity and their phenotypic pattern. Thus, these findings demonstrating material-induced monocyte activation and monocyte phenotype modulation, are suggestive of the monocyte role as reporter cells in evaluating the biocompatibility of a synthetic medical device.     

Monocyte, macrophage and foreign body giant cell interactions with molecularly engineered surfaces

To elucidate the mechanisms involved in monocyte/macrophage adhesion and fusion to form foreign body giant cells on molecularly engineered surfaces, we have utilized our in vitro culture system to examine surface chemistry effects, cytoskeletal reorganization and adhesive structure development, and cell receptor-ligand interactions in in vitro foreign body giant cell formation. Utilizing silane-modified surfaces, monocyte/macrophage adhesion was essentially unaffected by surface chemistry, however the density of foreign body giant cells (FBGCs) was correlated with surface carbon content. An exception to the surface-independent macrophage adhesion were the alkyl-silane modified surfaces which exhibited reduced adhesion and FBGC formation. Utilizing confocal immunofluorescent techniques, cytoskeletal reorganization and adhesive structure development in in vitro FBGC formation was studied. Podosomes were identified as the adhesive structures in macrophages and FBGCs based on the presence of characteristic cytoplasmic proteins and F-actin at the ventral cell surface. Focal adhesion kinase (FAK) and focal adhesions were not identified as the adhesive structures in macrophages and FBGCs. In studying the effect of preadsorbed proteins on FBGC formation, fibronectin or vitronectin do not play major roles in initial monocyte/macrophage adhesion, whereas polystyrene surfaces modified with RGD exhibited significant FBGC formation. These studies identify the potential importance of surface chemistry-dependent conformational alterations which may occur in proteins adsorbed to surfaces and their potential involvement in receptor-ligand interactions. Significantly, preadsorption of α₂-macroglobulin facilitated macrophage fusion and FBGC formation readily on the RGD surface in the absence of any additional serum proteins. As α₂-macroglobulin receptors are not found on blood monocytes but are expressed only with macrophage development, these results point to a potential interaction between adsorbed α₂-macroglobulin and its receptors on macrophages during macrophage development and fusion. These studies identify important surface independent and dependent effects in foreign body reaction development that may be important in the identification of biological design criteria for molecularly engineered surfaces and tissue engineered devices. © 1999 Kluwer Academic Publishers     

Dual Functional Monocytes Modulate Bactericidal and Anti‐Inflammation Process for Severe Osteomyelitis Treatment

Osteomyelitis is an inflammatory bone disease caused by infection microorganisms which leads to progressive bone destruction and loss. Drug resistance and inflammatory damage make it urgent to develop new dual‐functional therapies. Based on the powerful bactericidal effect of monocyte/macrophage cells by nature, a functional monocyte with programed anti‐inflammatory ability is promising for osteomyelitis treatment. Herein, gold nanocage (GNC)–modified monocytes are developed which contain aspirin to realize the controlled antibacterial and anti‐inflammatory process for bone infection treatment effectively. Aspirin@GNC‐laden monocytes inherit the biological functions of origin monocytes such as chemotaxis to bacteria, differentiation potential, and phagocytic ability. The controlled release of aspirin from GNC has a beneficial effect on improving the rate and amount of bone regeneration after the anti‐infection stage due to its ability to suppress the activity of natural immunity and induce osteoblast differentiation during the treatment of osteomyelitis. The present work described here is the first to utilize living monocytes to achieve a dual effect to antibacteria and anti‐inflammation in a time‐oriented and programed way, and provides an inspiration for future therapy based on this concept.     

Microscopic observations and inflammatory cytokine productions of human macrophage phagocytising submicron titanium particles

This study was performed to microscopically observe and measure inflammatory cytokine production by human macrophages phagocytosing submicron titanium (Ti) particles. Observations with secondary electron microscopy (SEM), SEM/electron probe microanalysis (EPMA) and transmission electron microscopy (TEM) indicated that macrophages [phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells] at 24 h in culture actively phagocytosed and accumulated submicron Ti particles in intracellular phagosomes, in which refinement of Ti particles occurred. The macrophages were also cultured for 24 h in four media with and without submicron Ti particles and lipopolysaccharide (LPS; components of bacteria). Whilst neither stimulus reduced cell viability, submicron Ti particles and LPS activation independently and synergistically caused the macrophages to produce three inflammatory cytokines (TNF-α, IL-1β and IL-6) at high levels in the culture supernatants. The inflammatory and osteolysis conditions caused by macrophages phagocytosing submicron Ti particles would be worsened by challenge with LPS in patients wearing Ti prostheses.     

Role of HSP70i in regulation of biomaterial-induced activation of human monocytes-derived macrophages in culture

The functioning of an implant depends on the material properties and the wound-healing process. The latter is led by an inflammatory reaction guided mainly by monocyte/macrophage activity. This in vitro study investigated human monocytes/macrophages in culture from 2 h to 10 days on silicone, polyurethane, teflon and TCPS. Analysis of cytokine release by ELISA showed that maturing macrophages have different capacities to produce cytokines TNF, IL10, IL8 and GM-CSF. The long culture-mature macrophages on all polymers produced comparable low levels of TNF, IL10 and IL8. Monocytes/macrophages on polyurethane and teflon, and those on silicone only in long culture-time produced high GM-CSF amounts, where as those on TCPS exhibited low levels of GM-CSF. FACS analysis revealed that HSP70i was highly inducible after short time culture yet this high level was maintained in long culture-mature macrophages on TCPS only, whereas on other polymers the mature macrophages showed a high reduction in HSP70i level, which demonstrated a high stress-response by cells on TCPS. Accordingly, CLSM-analysis revealed low nuclear NF-B in cells on TCPS and high nuclear NF-B in mature macrophages on silicone and polyurethane, showing a high cellular activation on the latter two polymers. This corresponded also to the high mitochondrial activity by XTT metabolism displayed by the mature macrophages on polyurethane silicone > teflon > TCPS. These data show a correlation of (1) cytokines (TNF, GM-CSF) and HSP70i, (2) NF-B and HSP70i by monocytes/macrophages after contact with polymers. Thus, HSP70i might be a useful molecular candidate for exploring biomaterial-induced inflammatory reaction.     

低压等离子喷涂MoB/CoCr涂层的组织及耐磨性

采用低压等离子喷涂技术(LPPS)制备MoB/CoCr涂层,对涂层进行磨粒磨损试验研究。采用SEM观察涂层的表面和截面形貌,显微硬度计测试涂层的力学性能,湿式橡胶轮磨粒磨损试验机测试涂层的磨粒磨损性能。结果表明,涂层组织致密,呈层状结构;涂层具有良好的力学性能,显微硬度达到930HV0.3,结合强度在71MPa以上,具有较高的耐磨性能。     

Activatable Nanoreporters for Real-Time Tracking of Macrophage Phenotypic States Associated with Disease Progression

Diagnosis of inflammatory diseases is characterized by identifying symptoms, biomarkers, and imaging. However, conventional techniques lack the sensitivities and specificities to detect disease early. Here, it is demonstrated that the detection of macrophage phenotypes, from inflammatory M1 to alternatively activated M2 macrophages, corresponding to the disease state can be used to predict the prognosis of various diseases. Activatable nanoreporters that can longitudinally detect the presence of the enzyme Arginase 1, a hallmark of M2 macrophages, and nitric oxide, a hallmark of M1 macrophages are engineered, in real-time. Specifically, an M2 nanoreporter enables the early imaging of the progression of breast cancer as predicted by selectively detecting M2 macrophages in tumors. The M1 nanoreporter enables real-time imaging of the subcutaneous inflammatory response that rises from a local lipopolysccharide (LPS) administration. Finally, the M1-M2 dual nanoreporter is evaluated in a muscle injury model, where an initial inflammatory response is monitored by imaging M1 macrophages at the site of inflammation, followed by a resolution phase monitored by the imaging of infiltrated M2 macrophages involved in matrix regeneration and wound healing. It is anticipated that this set of macrophage nanoreporters may be utilized for early diagnosis and longitudinal monitoring of inflammatory responses in various disease models.     

The effect of particle size and electrical charge on macrophage-osteoclast differentiation and bone resorption

In aseptic loosening, there is commonly periprosthetic bone loss and a heavy macrophage infiltrate in response to biomaterial wear particles generated from the implant materials. Macrophages which have phagocytosed wear particles are known to be capable of differentiation into bone resorbing osteoclasts. In this investigation we determine the role of particle size and particle charge on this process. Mouse monocytes and macrophages were co-cultured with osteoblast-like UMR106 cells and 1,25 dihydroxyvitamin D3 in the presence or the absence of (i) various sizes of latex beads (0.1, 1, 10 and 100 m) and (ii) uncharged, positively- or negatively-charged sephadex beads of uniform shape and composition. The extent of osteoclast differentiation by monocytes or foreign body macrophages was determined by the expression of the osteoclast-associated enzyme tartrate-resistant acid phosphatase and lacunar bone resorption. No significant difference in the extent of osteoclast formation and bone resorption was noted in response to particle size. Osteoclast formation was also not significantly different in the presence of positively/negatively charged and uncharged particles. These findings indicate that osteoclast formation is not significantly influenced by particle characteristics, such as particle size. They also add support to the hypothesis that macrophage involvement in periprosthetic osteolysis is not dependent on particle phagocytosis and that it may be induced by particle contact.     

Micro RNA‐30b (inhibitor) nanoparticles suppressed the lipopolysaccharide (LPS)‐induced acute kidney injury

The main aim of present study is to evaluate the effect of miR‐30b on the function of human proximal tubular epithelial cell line HK‐2 cells. For this purpose, miRNA was loaded in an ionically cross‐linked polysaccharide nanoparticle. The authors have demonstrated the influence of miR‐30b mimic and inhibitor in HK‐2 cell killing effect. Lipopolysaccharide (LPS) significantly increased the level of inflammatory cytokines of TNF‐α, IL‐1β and level was further increased with the treatment of PAg‐miR mimic consistent with the cell viability assay. Interestingly, PAg‐miR inhibitor significantly downregulated the expression of inflammatory cytokines and thereby reduced the inflammation in the body. Western blot analysis showed that LPS induced severe apoptosis of HK‐2 cells and the apoptosis was further promoted by the PAg‐miR (mimic). In contrast, PAg‐miR (inhibitor) alleviated the apoptosis of HK‐2 cells as indicated in the significantly reduced levels of Bax and c‐Caspase‐3 proteins. Overall, miR‐30b promoted LPS‐induced HK‐2 cell inflammatory injury by inducing the apoptosis and by releasing inflammatory cytokines, as well as by impairing autophagy process.Inspec keywords: biomedical materials, nanoparticles, molecular biophysics, enzymes, toxicology, injuries, nanomedicine, RNA, cellular biophysics, kidney, proteins, drugs, biochemistryOther keywords: microRNA‐30b, nanoparticles suppressed the lipopolysaccharide (LPS)‐induced, main aim, human proximal tubular epithelial cell line HK‐2 cells, polysaccharide nanoparticle, HK‐2 cell killing effect, inflammatory cytokines, IL‐1β, cell viability assay, PAg‐miR inhibitor, apoptosis, reduced levels, LPS‐induced HK‐2 cell inflammatory injury     

Human monocyte-derived macrophages and dendritic cells as targets for biomaterial cytocompatibility studies using an improved in vitro culture system

Since macrophage plays a key role in the biocompatibility process, neoplastic macrophage cell lines and human blood monocytes are commonly used as target cells for in vitro biomaterial tolerance evaluation. However, tumor cells profoundly differ from normal tissue cells and monocytes are only precursors of macrophages. It has become possible to generate recently, under adherent-free conditions, fully mature macrophages and dendritic cells from human blood monocytes in the presence of GM-CSF and GM-CSF + IL4 respectively. In the present work, we examined the effects of titanium-alloy on morphology, adhesion, cell phenotype and TNF- release activity of such differentiated cells grown in hydrophobic teflon bags. Scanning electron microscopy showed that macrophages substantially adhered and spread on titanium-alloy surface throughout the culture period, whereas only a few dendritic cells were adherent. The phenotype of both cell types remained unchanged in the presence of the tested material. However, titanium-alloy stimulated the secretion of TNF- by the macrophages of some donors. This model of culture may offer new insights into the biomaterial evaluation and may be useful for studying individual responses induced by biomaterials. © 2001 Kluwer Academic Publishers     

Neutrophil and monocyte activation in chronic kidney disease patients under hemodialysis and its relationship with resistance to recombinant human erythropoietin and to the hemodialysis procedure

The aim of the present work was to further clarify leukocyte activation due to hemodialysis (HD) procedures and to investigate its relationship with recombinant human erythropoietin resistance. Therefore, we studied the expression of CXCR1 and CD11b on neutrophils, as well as the monocyte expression of CD11b, HLA‐DR, and CD14. We studied 34 chronic kidney disease (CKD) patients under HD and recombinant human erythropoietin treatment (26 responders and 8 nonresponders to recombinant human erythropoietin therapy). All CKD patients' blood samples were collected before and immediately after the HD procedure. Eighteen healthy individuals (blood donors) were also studied as a control group. Hematological data, neutrophil (CD11b and CXCR1), and monocyte (CD11b, HLA‐DR, and CD14) cell surface markers were measured in all patients (before and after the HD procedure) and controls. When compared with the controls, CKD patients presented a significant decrease in CXCR1 neutrophil expression, and in CD14 monocyte expression, accompanied by a significant increase in HLA‐DR monocyte expression. When comparing the 2 groups of patients, we found that nonresponders showed an additional decrease in CXCR1 neutrophil expression. After the HD procedure, a statistically significant increase in CD14 and CD11b monocyte surface markers and a decrease in CXCR1 neutrophil expression and in HLA‐DR monocyte expression was found. These data further strengthen our previous studies, showing that neutrophils and monocytes are activated in CKD patients, particularly in nonresponder patients. Moreover, this activation is due, at least in part, to the HD procedure, although we should not exclude that it can also be due to the enhanced inflammatory process observed in nonresponder patients.     

Nanoparticles of a different source induce different patterns of activation in key biochemical and cellular components of the host response

Nanoparticulate materials are produced by industrial processing or engineered for specific biomedical applications. In both cases, their contact with the human body may lead to adverse reactions. Most of the published papers so far have focused on the cytotoxic effects of nanoparticles (NPs). Instead, the present in vitro study investigates the effect of different types of NP on key components of the host response such as clot formation and the inflammatory cells. The different NPs were pre-conditioned with platelet-rich human plasma for 30 min and then incubated with the blood mononuclear cells for 20 hours. The potential of the different NPs to induce clot formation, platelet activation and monocyte/macrophage differentiation was assessed by morphological analysis, immunocytochemistry and biochemical assays. The data showed that nanoparticulate materials based on antimony, silver and nickel were capable of promoting the polymerization of fibrin and the aggregation and fragmentation of platelets, leading to a moderately activated monocyte phenotype. This process was more pronounced in the case of antimony- and silver-based NPs that share a similar size and round-shaped morphology. Conversely, NPs of cobalt, titanium and iron appeared to stimulate cells to acquire a macrophage phenotype able to secrete higher levels of tumour necrosis factor α, a pro-inflammatory cytokine. Therefore, the present study provides clear indications about the subtle and adverse effects that the invasion of these materials may produce in the cardiovascular system and in vital organs.     

Screening of biomedical polymer biocompatibility in NMRI-mice peritoneal cavity: A comparison between ultra-high-molecular-weight polyethylene (UHMW-PE) and polyethyleneterephthalate (PET)

The peritoneal resident cell population is influenced by various inflammatory and immunogenic stimuli. The influence of intraperitoneal application of polyethyleneterephthalate (PET) (group A) and ultra-high-molecular-weight polyethylene (UHMW-PE) (group B) powders on peritoneal cell count and macrophage activity was investigated. Powders were tested to mimic wear particles from solid implant devices as these particles often cause chronic granulomatous inflammation. The results were compared with the inflammatory response following an abdominal midline incision (group C) and untreated animals (group D). On days 1, 7, 14 and 30 peritoneal cells were quantified and the number of active macrophages was assessed. Groups A and C mice showed a significant loss of macrophages in the peritoneal lavage at day 1 but this returned to normal values (group D) on day 7. In contrast, group B animals remained at low peritoneal cell counts but showed the highest number of active macrophages. Only in this latter group was adhesion formation and granulomatous clustering of polymer powder observed. Applying the parameters macrophage count and the number of active macrophages it can be concluded that PET elicits a weaker inflammatory reaction than UHMW-PE in mice peritoneal cavity. Thus this animal model may be used as a screening test for biomedical materials, especially their wear products.     

Quantitative Evaluation of the Cellular Uptake of Nanodiamonds by Monocytes and Macrophages

Fluorescent nanodiamonds (FNDs) with negative nitrogen-vacancy (NV⁻) defect centers are great probes for biosensing applications, with potential to act as biomarkers for cell differentiation. To explore this concept, uptake of FNDs (≈120 nm) by THP-1 monocytes and monocyte-derived M0-macrophages is studied. The time course analysis of FND uptake by monocytes confirms differing FND-cell interactions and a positive time-dependence. No effect on cell viability, proliferation, and differentiation potential into macrophages is observed, while cells saturated with FNDs, unload the FNDs completely by 25 cell divisions and subsequently take up a second dose effectively. FND uptake variations by THP-1 cells at early exposure-times indicate differing phagocytic capability. The cell fraction that exhibits relatively enhanced FND uptake is associated to a macrophage phenotype which derives from spontaneous monocyte differentiation. In accordance, chemical-differentiation of the THP-1 cells into M0-macrophages triggers increased and homogeneous FND uptake, depleting the fraction of cells that were non-responsive to FNDs. These observations imply that FND uptake allows for distinction between the two cell subtypes based on phagocytic capacity. Overall, FNDs demonstrate effective cell labeling of monocytes and macrophages, and are promising candidates for sensing biological processes that involve cell differentiation.     

Effects of adsorbed heat labile serum proteins and fibrinogen on adhesion and apoptosis of monocytes/macrophages on biomaterials

A previously established human monocyte culture protocol was used to determine the effects of varying adsorbed proteins on monocyte/macrophage adhesion and survival on dimethyl-silane (DM) or RGD modified glass coverslips. Cells were allowed to adhere for 2 h in the absence of protein or in the presence of serum, fibrinogen (Fg), heat inactivated serum (HIS), serum supplemented with Fg or HIS with Fg. Cell adhesion and apoptosis rates were determined on days 0 (2 h), 3, 7 and 10 of culture. The presence of serum alone in the initial culture was sufficient to optimize monocyte/macrophage adhesion and survival rates. Adding Fg to serum did not increase adhesion nor decrease apoptotic rates. No protein or the addition of HIS during the initial incubation period significantly decreased monocyte/macrophage adhesion and survival on both surfaces, however, the addition of Fg to HIS restored adhesion and survival rates to those seen with in the presence of serum alone on RGD surfaces. These studies demonstrate that monocyte/macrophage adhesion and survival on biomaterial surfaces are optimized by adsorbed heat labile serum proteins while adsorbed Fg plays a surface property-dependent role.     

Geometrical features of wear debris

Image analysis software was used to analyse the geometry of debris formed during the erosion of low-carbon steel by impinging solid particles. Depending on the two-dimensional aspect ratio (ratio between debris height and debris width), three different debris types could be distinguished. The most frequent type observed was a platelet-type debris as suggested by the Bellman-Levy (1981) model. This wear debris shape type covered about 60% of all acquired debris. Plain micro-machining according to Finnie’s (1959) suggestion played a negligible role only, but other processes, namely ploughing as suggested by Winter and Hutchings (1974), were more important. The statistically estimated mean debris size was about 14 μ m. About 92% of all wear debris had sizes smaller than the target material grain size. This result supports the figure that ‘secondary’ removal modes—lip or platelet, respectively, detachment from crater rims—were responsible for material removal.