Do patients with a failed metal-on-metal hip implant with a pseudotumor present differences in their peripheral blood lymphocyte subpopulations?

BACKGROUND: Early adverse tissue reactions around metal-on-metal (MoM) hip replacements, especially pseudotumors, are a major concern. Because the causes and pathomechanisms of these pseudotumors remain largely unknown, clinical monitoring of patients with MoM bearings is challenging. QUESTIONS/PURPOSES: The purpose of this study was to compare the lymphocyte subpopulations in peripheral blood from patients with a failed MoM hip implant with and without a pseudotumor and patients with a well-functioning MoM hip implant without a pseudotumor. Potential differences in the systemic immune response are expected to reflect local differences in the periprosthetic tissues. METHODS: Consenting patients who underwent a revision of a failed MoM hip implant at The Ottawa Hospital (TOH) from 2011 to 2014, or presented with a well-functioning MoM hip implant for a postoperative clinical followup at TOH from 2012 to 2013, were recruited for this study, unless they met any of the exclusion criteria (including diagnosed conditions that can affect peripheral blood lymphocyte subpopulations). Patients with a failed implant were divided into two groups: those with a pseudotumor (two hip resurfacings and five total hip arthroplasties [THAs]) and those without a pseudotumor (10 hip resurfacings and two THAs). Patients with a well-functioning MoM hip implant (nine resurfacings and three THAs) at 5 or more years postimplantation and who did not have a pseudotumor as demonstrated sonographically served as the control group. Peripheral blood subpopulations of T cells (specifically T helper [Th] and cytotoxic T [Tc]), B cells, natural killer (NK) cells, memory T and B cells as well as type 1 (expressing interferon-γ) and type 2 (expressing interleukin-4) Th and Tc cells were analyzed by flow cytometry after immunostaining. Serum concentrations of cobalt and chromium were measured by inductively coupled plasma-mass spectrometry. RESULTS: The mean percentages of total memory T cells and, specifically, memory Th and memory Tc cells were lower in patients with a failed MoM hip implant with a pseudotumor than in both patients with a failed implant without a pseudotumor and patients with a well-functioning implant without a pseudotumor (memory Th cells: 29% ± 5% [means ± SD] versus 55% ± 17%, d = 1.8, 95% confidence interval [CI] [1.2, 2.5] and versus 48% ± 14%, d = 1.6, 95% CI [1.0, 2.2], respectively; memory Tc cells: 18% ± 5% versus 45% ± 14%, d = 2.3, 95% CI [1.5, 3.1] and versus 41% ± 12%, d = 2.3, 95% CI [1.5, 3.1], respectively; p < 0.001 in all cases). The mean percentage of memory B cells was also lower in patients with a failed MoM hip implant with a pseudotumor than in patients with a well-functioning implant without a pseudotumor (12% ± 8% versus 29% ± 16%, d = 1.3, 95% CI [0.7, 1.8], p = 0.025). In addition, patients with a failed MoM hip implant with a pseudotumor had overall lower percentages of type 1 Th cells than both patients with a failed implant without a pseudotumor and patients with a well-functioning implant without a pseudotumor (5.5% [4.9%-5.8%] [median with interquartile range] versus 8.7% [6.5%-10.2%], d = 1.4, 95% CI [0.8, 2.0] and versus 9.6% [6.4%-11.1%], d = 1.6, 95% CI [1.0, 2.2], respectively; p ≤ 0.010 in both cases). Finally, serum cobalt concentrations in patients with a failed MoM hip implant with a pseudotumor were overall higher than those in patients with a well-functioning implant without a pseudotumor (5.8 µg/L [2.9-17.0 µg/L] versus 0.9 µg/L [0.6-1.3 µg/L], d = 2.2, 95% CI [1.4, 2.9], p < 0.001). CONCLUSIONS: Overall, results suggest the presence of a type IV hypersensitivity reaction, with a predominance of type 1 Th cells, in patients with a failed MoM hip implant with a pseudotumor. CLINICAL RELEVANCE: The lower percentages of memory T cells (specifically Th and Tc) as well as type 1 Th cells in peripheral blood of patients with a failed MoM hip implant with a pseudotumor could potentially become diagnostic biomarkers for the detection of pseudotumors. Although implant design (hip resurfacing or THA) did not seem to affect the results, as suggested by the scatter of the data with respect to this parameter, future studies with additional patients could include the analysis of implant design in addition to correlations with histological analyses of specific Th subsets in periprosthetic tissues.

Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo.

Plant-derived cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as its ability to effectively support osteogenic differentiation. The present study further examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and energy-dispersive spectroscopy (EDS) confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young's modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.

Development of hydrogel-based composite scaffolds containing eggshell particles for bone regeneration applications.

This study describes the development and characterization of novel composite scaffolds, made of an alginate-chitosan hydrogel matrix containing eggshell (ES) particles, for bone tissue engineering applications. Scaffolds with ES particles, either untreated or treated with phosphoric acid to create a nanotextured particle surface, were compared to scaffolds without particles. Results indicate that the nanotexturing process exposed occluded ES proteins orthologous to those in human bone extracellular matrix. Scaffolds with ES or nanotextured ES (NTES) particles had a higher porosity (81 ± 4% and 89 ± 5%, respectively) than scaffolds without particles (59 ± 5%) (p = .002 and p < .001, respectively). Scaffolds with NTES particles had a larger median pore size (113 µm [interquartile range [IQ]: 88-140 µm]) than scaffolds with ES particles (94 µm [IQ: 75-112 µm]) and scaffolds without particles (99 µm [IQ: 74-135 µm]) (p < .001 and p = .011, respectively). The compressive modulus of the scaffolds with ES or NTES particles remained low (3.69 ± 0.70 and 3.14 ± 0.62 kPa, respectively), but these scaffolds were more resistant to deformation following maximum compression than those without particles. Finally, scaffolds with ES or NTES particles allowed better retention of human mesenchymal stem cells during seeding (53 ± 12% and 57 ± 8%, respectively, vs. 17 ± 5% for scaffolds without particles; p < .001 in both cases), as well as higher cell viability up to 21 days of culture (67 ± 17% and 61 ± 11%, respectively, vs. 15 ± 7% for scaffolds without particles; p < .001 in both cases). In addition, alkaline phosphatase (ALP) activity increased up to 558 ± 164% on day 21 in the scaffolds with ES particles, and up to 567 ± 217% on day 14 in the scaffolds with NTES particles (p = .006 and p = .002, respectively, relative to day 0). Overall, this study shows that the physicochemical properties of the alginate-chitosan hydrogel scaffolds with ES or NTES particles are similar to those of cancellous bone. In addition, scaffolds with particles supported early osteogenic differentiation and therefore represent a promising new bone substitute, especially for non-load bearing applications.

PGE2 and BMP-2 in bone and cartilage metabolism: 2 intertwining pathways.

Osteoarthritis and lesions to cartilage tissue are diseases that frequently result in impaired joint function and patient disability. The treatment of osteoarthritis, along with local bone defects and systemic skeletal diseases, remains a significant clinical challenge for orthopaedic surgeons. Several bone morphogenetic proteins (BMPs) are known to have osteoinductive effects, whereof BMP-2 and BMP-7 are already approved for clinical applications. There is growing evidence that the metabolism of bone as well as the cartilage damage associated with the above disease processes are strongly inter-related with the interactions of the inflammation-related pathways (in particular prostaglandin E2 (PGE2)) and osteogenesis (in particular bone morphogenetic protein-2 (BMP-2)). There is strong evidence that the pathways of prostaglandins and bone morphogenetic proteins are intertwined, and they have recently come into focus in several experimental and clinical studies. This paper focuses on PGE2 and BMP-2 intertwining pathways in bone and cartilage metabolism, and summarizes the recent experimental and clinical data.

Do revised hip resurfacing arthroplasties lead to outcomes comparable to those of primary and revised total hip arthroplasties?

BACKGROUND: A theoretical clinical advantage of hip resurfacing (HR) is the preservation of femoral bone. HR femoral component revision reportedly yields postoperative function comparable to that of primary THA. However, few studies have looked at the outcome of both HR femoral and acetabular side revisions. QUESTIONS/PURPOSES: We determined whether (1) patients undergoing HR revision to THA have perioperative measures and outcome scores comparable to those of patients undergoing primary THA or revision of primary THA and (2) patients undergoing HR revision of both components have perioperative measures and outcome scores comparable to those of patients undergoing HR revision of the femoral component only. METHODS: We retrospectively reviewed and compared 22 patients undergoing revision HR to a THA to a matched (age, sex, BMI) group of 23 patients undergoing primary THA and 12 patients undergoing primary THA revision. Patients completed the WOMAC and SF-12 questionnaires before surgery and at latest followup (range, 24-84 months for HR revision, 28-48 months for primary THA, and 24-48 months for revision THA). Blood loss, days in hospital, complications, and outcome scores were compared among groups. RESULTS: We observed no differences in SF-12 scores but observed lower WOMAC stiffness, function, and total scores in the HR revision group than in the primary THA group. Patients undergoing HR revision of both components had comparable SF-12 and WOMAC stiffness, function, and total scores but overall lower WOMAC pain scores compared to patients undergoing HR revision of the femoral side only. The HR revision group had greater intraoperative blood loss compared to the primary THA group but not the revision THA group. CONCLUSIONS: The perioperative measures and outcome scores of HR revision are comparable to those of revision THA but not primary THA. Longer followup is required to determine whether these differences persist. Patients undergoing HR revision of one or both components can expect comparable stiffness and function. LEVEL OF EVIDENCE: Level IV, therapeutic study. See the Instructions for Authors for a complete description of levels of evidence.

Effects of cobalt and chromium ions on glycolytic flux and the stabilization of hypoxia-inducible factor-1α in macrophages in vitro.

Implant wear and corrosion have been associated with adverse tissue reactions that can lead to implant failure. Wear and corrosion products are therefore of great clinical concern. For example, Co2+ and Cr3+ originating from CoCrMo-based implants have been shown to induce a proinflammatory response in macrophages in vitro. Previous studies have also shown that the polarization of macrophages by some proinflammatory stimuli is associated with a hypoxia-inducible factor-1α (HIF-1α)-dependent metabolic shift from oxidative phosphorylation (OXPHOS) towards glycolysis. However, the potential of Co2+ and Cr3+ to induce this metabolic shift, which plays a determining role in the proinflammatory response of macrophages, remains largely unexplored. We recently demonstrated that Co2+ , but not Cr3+ , increased oxidative stress and decreased OXPHOS in RAW 264.7 murine macrophages. In the present study, we analyzed the effects of Co2+ and Cr3+ on glycolytic flux and HIF-1α stabilization in the same experimental model. Cells were exposed to 6 to 24 ppm Co2+ or 50 to 250 ppm Cr3+ . Glycolytic flux was determined by analyzing extracellular flux and lactate production, while HIF-1α stabilization was analyzed by immunoblotting. Results showed that Co2+ , and to a lesser extent Cr3+ , increased glycolytic flux; however, only Co2+ acted through HIF-1α stabilization. Overall, these results, together with our previous results showing that Co2+ increases oxidative stress and decreases OXPHOS, suggest that Co2+ (but not Cr3+ ) can induce a HIF-1α-dependent metabolic shift from OXPHOS towards glycolysis in macrophages. This metabolic shift may play an early and pivotal role in the inflammatory response induced by Co2+ in the periprosthetic environment.

Biologic activity of wear particles.

Aseptic loosening resulting from periprosthetic osteolysis continues to be an important cause of hip implant failure. Wear particles from the bearing surfaces play a major role in initiating periprosthetic osteolysis, which is also potentiated by mechanical factors such as increased synovial fluid pressure. The precise mechanisms by which wear particles induce periprosthetic osteolysis have not been fully elucidated and remain an active subject of research. Particle characteristics such as composition, size, shape, and number (especially for particles in the most biologically active, submicrometer-size range) are recognized to significantly affect the overall cell and tissue response. The production of corrosion products, especially from metal-on-metal implants, also is a clinically significant issue, and individual variability in innate and adaptive immune responses is important but not yet completely defined. Because of the increasing need to implant hip prostheses in younger and more active patients, a better understanding of the biologic activity of wear particles from bearing couples is critical in the attempt to modulate the clinical effects of these particles and to develop materials with improved wear and corrosion resistance.

Effects of metal ions on caspase-1 activation and interleukin-1ß release in murine bone marrow-derived macrophages.

Ions released from metal implants have been associated with adverse tissue reactions and are therefore a major concern. Studies with macrophages have shown that cobalt, chromium, and nickel ions can activate the NLRP3 inflammasome, a multiprotein complex responsible for the activation of caspase-1 (a proteolytic enzyme converting pro-interleukin [IL]-1ß to mature IL-1ß). However, the mechanism(s) of inflammasome activation by metal ions remain largely unknown. The objectives of the present study were to determine if, in macrophages: 1. caspase-1 activation and IL-1ß release induced by metal ions are oxidative stress-dependent; and 2. IL-1ß release induced by metal ions is NF-κB signaling pathway-dependent. Lipopolysaccharide (LPS)-primed murine bone marrow-derived macrophages (BMDM) were exposed to Co2+ (6-48 ppm), Cr3+ (100-500 ppm), or Ni2+ (12-96 ppm), in the presence or absence of a caspase-1 inhibitor (Z-WEHD-FMK), an antioxidant (L-ascorbic acid [L-AA]), or an NF-κB inhibitor (JSH-23). Culture supernatants were analyzed for caspase-1 by western blotting and/or IL-1ß release by ELISA. Immunoblotting revealed the presence of caspase-1 (p20 subunit) in supernatants of BMDM incubated with Cr3+, but not with Ni2+ or Co2+. When L-AA (2 mM) was present with Cr3+, the caspase-1 p20 subunit was undetectable and IL-1ß release decreased down to the level of the negative control, thereby demonstrating that caspase-1 activation and IL-1ß release induced by Cr3+ was oxidative stress-dependent. ELISA demonstrated that Cr3+ induced the highest release of IL-1ß, while Co2+ had no or limited effects. In the presence of Ni2+, the addition of L-AA (2 mM) also decreased IL-1ß release, below the level of the negative control, suggesting that IL-1ß release induced by Ni2+ was also oxidative stress-dependent. Finally, when present during both priming with LPS and activation with Cr3+, JSH-23 blocked IL-1ß release, demonstrating NF-κB involvement. Overall, this study showed that while both Cr3+ and Ni2+ may be inducing inflammasome activation, Cr3+ is likely a more potent activator, acting through oxidative stress and the NF-κB signaling pathway.

Effects of cobalt and chromium ions on oxidative stress and energy metabolism in macrophages in vitro.

Cobalt and chromium ions released from cobalt-chromium-molybdenum (CoCrMo)-based implants are a potential health concern, especially since both ions have been shown to induce oxidative stress in macrophages, the predominant immune cells in periprosthetic tissues. Ions of other transition metals (Cd, Ni) have been reported to inhibit the activity of mitochondrial enzymes in the electron transport chain. However, the effects of Co and Cr ions on the energy metabolism of macrophages remain largely unknown. The objective of the present study was to analyze the effects of Co2+ and Cr3+ on oxidative stress and energy metabolism in macrophages in vitro. RAW 264.7 murine macrophages were exposed to 6-18 ppm Co2+ or 50-150 ppm Cr3+ . Results showed a significant increase in two markers of oxidative stress, reactive oxygen species level and protein carbonyl content, with increasing concentrations of Co2+ , but not with Cr3+ . In addition, oxygen consumption rates (OCR; measured using an extracellular flux analyzer) showed significant decreases in both mitochondrial respiration and non-mitochondrial oxygen consumption with increasing concentrations of Co2+ , but not with Cr3+ . OCR results further showed that Co2+ , but not Cr3+ , induced mitochondrial dysfunction, including a decrease in oxidative phosphorylation capacity. Overall, this study suggests that mitochondrial dysfunction may contribute to Co2+ -induced oxidative stress in macrophages, and thereby to the inflammatory response observed in periprosthetic tissues. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3178-3187, 2018.

The quest for mechanically and biologically functional soft biomaterials via soft network composites.

Developing multifunctional soft biomaterials capable of addressing all the requirements of the complex tissue regeneration process is a multifaceted problem. In order to tackle the current challenges, recent research efforts are increasingly being directed towards biomimetic design concepts that can be translated into soft biomaterials via advanced manufacturing technologies. Among those, soft network composites consisting of a continuous hydrogel matrix and a reinforcing fibrous network closely resemble native soft biological materials in terms of design and composition as well as physicochemical properties. This article reviews soft network composite systems with a particular emphasis on the design, biomaterial and fabrication aspects within the context of soft tissue engineering and drug delivery applications.

Differential proteomic analysis of synovial fluid from hip arthroplasty patients with a pseudotumor vs. Periprosthetic osteolysis .

Adverse tissue reactions to metal implants, including pseudotumors, can compromise implant functionality and survivorship. The identification of specific proteins in the synovial fluid (SF) of hip arthroplasty patients with a pseudotumor may lead to a better understanding of the underlying pathomechanisms. The objective of the present study was to compare the protein content of SF from patients with a short-term metal-on-metal hip implant associated with a pseudotumor and patients with a long-term metal-on-polyethylene hip implant associated with periprosthetic osteolysis. Discovery proteomics was used to identify differentially abundant proteins in albumin-depleted SF. In toto, 452 distinct proteins (present in at least half of the patients in one or both groups) were identified. Thirty of these 452 proteins were differentially abundant between the two groups, including two potential biomarkers: 6-phosphogluconate dehydrogenase (which plays a major protective role against oxidative stress) for the pseudotumor group, and scavenger receptor cysteine-rich type 1 protein M130 (which is involved in low-grade inflammation) for the periprosthetic osteolysis group. Other differentially abundant proteins identified suggest the presence of an adaptive immune response (particularly a type-IV hypersensitivity reaction), necrosis, and greater oxidative stress in patients with a pseudotumor. They also suggest the presence of an innate immune response, oxidative stress, tissue remodeling, and apoptosis in both patient groups, although differences in the specific proteins identified in each group point to differences in the pathomechanisms. Overall, results provide insights into the molecular mechanisms underlying metal-related pseudotumors and periprosthetic osteolysis, and may ultimately help elucidate pseudotumor etiology and assess the risk that asymptomatic pseudotumors will develop into an aggressive lesion. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1849-1859, 2018.

Effects of cobalt and chromium ions on lymphocyte migration.

A T cell-mediated hypersensitivity reaction has been reported in some patients with CoCrMo-based implants. However, the role of cobalt and chromium ions in this reaction remains unclear. The objective of the present study was to analyze the effects of Co2+ and Cr3+ in culture medium, as well as the effects of culture supernatants of macrophages exposed to Co2+ or Cr3+ , on the migration of lymphocytes. The release of cytokines/chemokines by macrophages exposed to Co2+ and Cr3+ was also analyzed. The migration of murine lymphocytes was quantified using the Boyden chamber assay and flow cytometry, while cytokine/chemokine release by J774A.1 macrophages was measured by ELISA. Results showed an ion concentration-dependent increase in TNF-α and MIP-1α release and a decrease in MCP-1 and RANTES release. Migration analysis showed that the presence of Co2+ (8 ppm) and Cr3+ (100 ppm) in culture medium increased the migration of T lymphocytes, while it had little or no effect on the migration of B lymphocytes, suggesting that Co2+ and Cr3+ can stimulate the migration of T but not B lymphocytes. Levels of T lymphocyte migration in culture medium containing Co2+ or Cr3+ were not statistically different from those in culture supernatants of macrophages exposed to Co2+ or Cr3+ , suggesting that the effects of the ions and chemokines were not additive, possibly because of ion interference with the chemokines and/or their cognate receptors. Overall, results suggest that Co2+ and Cr3+ are capable of stimulating the migration of T (but not B) lymphocytes in the absence of cytokines/chemokines, and could thereby contribute to the accumulation of more T than B lymphocytes in periprosthetic tissues of some patients with CoCrMo-based implants. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:916-924, 2017.

Effects of hip implant modular neck material and assembly method on fatigue life and distraction force.

Hip implant neck fractures and adverse tissue reactions associated with fretting-corrosion damage at modular interfaces are a major source of concern. Therefore, there is an urgent clinical need to develop accurate in vitro test procedures to better understand, predict and prevent in vivo implant failures. This study aimed to simulate in vivo fatigue fracture and distraction of modular necks in an in vitro setting, and to assess the effects of neck material (Ti6Al4V vs. CoCrMo) and assembly method (hand vs. impact) on the fatigue life and distraction of the necks. Fatigue tests were performed on the cementless PROFEMUR® Total Hip Modular Neck System under two different loads and number of cycles: 2.3 kN for 5 million cycles, and 7.0 kN for 1.3 million cycles. The developed in vitro simulation setup successfully reproduced in vivo modular neck fracture mode and location. Neck failure occurred at the neck-stem taper and the fracture ran from the distal lateral neck surface to the proximal medial entry point of the neck into the stem. None of the necks failed under the 2.3 kN load. However, all hand-assembled Ti6Al4V necks failed under the 7.0 kN load. In contrast, none of the hand-assembled CoCrMo necks and impact-assembled necks (Ti6Al4V or CoCrMo) failed under this higher load. In conclusion, Ti6Al4V necks were more susceptible to fatigue failure than CoCrMo necks. In addition, impact assembly substantially improved the fatigue life of Ti6Al4V necks and also led to overall higher distraction forces for both neck materials. Overall, this study shows that the material and assembly method can affect the fatigue strength of modular necks. Finally, improper implant assembly during surgery may result in diminished modular neck survivability and increased failure rates. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2023-2030, 2017.

Biofabricated soft network composites for cartilage tissue engineering.

Articular cartilage from a material science point of view is a soft network composite that plays a critical role in load-bearing joints during dynamic loading. Its composite structure, consisting of a collagen fiber network and a hydrated proteoglycan matrix, gives rise to the complex mechanical properties of the tissue including viscoelasticity and stress relaxation. Melt electrospinning writing allows the design and fabrication of medical grade polycaprolactone (mPCL) fibrous networks for the reinforcement of soft hydrogel matrices for cartilage tissue engineering. However, these fiber-reinforced constructs underperformed under dynamic and prolonged loading conditions, suggesting that more targeted design approaches and material selection are required to fully exploit the potential of fibers as reinforcing agents for cartilage tissue engineering. In the present study, we emulated the proteoglycan matrix of articular cartilage by using highly negatively charged star-shaped poly(ethylene glycol)/heparin hydrogel (sPEG/Hep) as the soft matrix. These soft hydrogels combined with mPCL melt electrospun fibrous networks exhibited mechanical anisotropy, nonlinearity, viscoelasticity and morphology analogous to those of their native counterpart, and provided a suitable microenvironment for in vitro human chondrocyte culture and neocartilage formation. In addition, a numerical model using the p-version of the finite element method (p-FEM) was developed in order to gain further insights into the deformation mechanisms of the constructs in silico, as well as to predict compressive moduli. To our knowledge, this is the first study presenting cartilage tissue-engineered constructs that capture the overall transient, equilibrium and dynamic biomechanical properties of human articular cartilage.

Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro.

This study analyzed human mesenchymal stem cell (hMSC) behavior in a fibrin sealant. hMSC morphology, proliferation, and osteogenic differentiation were analyzed after up to 28 days of incubation in eight different formulations of fibrin gels (Tisseel) prepared with various concentrations of fibrinogen complex (FC) and thrombin. Cell morphology and distribution within the gels were observed by fluorescence microscopy after cell staining with calcein dye. Cell proliferation was assessed by measuring the fluorescence intensity of the cell suspension stained with calcein dye after dissolution of the gels. A standard alkaline phosphatase (ALP) assay, von Kossa staining, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze hMSC osteogenic differentiation. Cell behavior varied depending on the gel formulation. Proliferation was higher in the formulations containing a low FC concentration, but ALP activity was higher in the formulations containing a high FC concentration. Variations in thrombin concentration had a lesser effect. Small nodules of mineralization were observed at days 21 and 28 in a formulation containing a high FC concentration, in addition to a marked increase in bone sialoprotein (BSP) gene expression level as well as a lower increase in ALP and osteopontin (OPN) levels. However, there was no significant increase in osteocalcin (OCN) expression, a late marker of osteogenic differentiation, up to day 28. In conclusion, this study demonstrated that hMSC morphology, proliferation, and osteogenic differentiation in fibrin gels depended on the FC/thrombin ratio. hMSCs appeared to undergo osteogenic differentiation when seeded in Tisseel fibrin sealant containing a high FC concentration, but they did not fully differentiate into mature osteoblasts.

Quantitative analysis of macrophage apoptosis vs. necrosis induced by cobalt and chromium ions in vitro.

The potential toxicity of metal ions in tissues surrounding metal-metal hip replacements is a cause for concern. Previous studies conducted in our laboratory demonstrated that Co(2+) and Cr(3+) induce TNF-alpha secretion in macrophages, as well as cell mortality. However, the degree of apoptosis and necrosis remained to be investigated. The aim of the present study was to quantify the rate of macrophage mortality by apoptosis vs. necrosis induced by Co(2+) and Cr(3+). J774 mouse macrophages were incubated in growth medium containing 0-10 ppm Co(2+) and 0-500 ppm Cr(3+) for 24 and 48 h under conventional cell culture conditions. Transmission electron microscopy, flow cytometry (Annexin-V fluorescein isothiocyanate/propidium iodide assay) and a specific cell death detection ELISA were used to illustrate cell death and differentiate between apoptotic and necrotic cells. Cell culture exposed to low concentrations of Co(2+) (0-6 ppm) revealed a low degree of mortality. In contrast, at the highest concentrations (8-10 ppm), late apoptosis occurred within 24 h. After 48 h, however, there was a clear evidence for an increase in the rate of necrosis while apoptosis occurred at much lower rate. Macrophages exposed to Cr(3+) demonstrated a predominance of apoptosis after 24h. At concentrations lower than 250 ppm, early and late apoptosis occurred at the same rate. At higher concentrations (250-500 ppm), the number of early apoptotic cells decreased in favor of late apoptosis. After 48 h, lower concentrations of Cr(3+) (150 ppm) induced a higher degree of early apoptosis than after 24 h, and some necrosis. At higher concentrations, the percentage of early apoptotic cells decreased, while necrosis became predominant over late apoptosis. In conclusion, this study demonstrates that macrophage mortality induced by metal ions depends on the type and concentration of metal ions as well as the duration of their exposure. Overall, apoptosis was predominant after 24 h with both Co(2+) and Cr(3+) ions, but high concentrations induced mainly necrosis at 48 h. These results point to the potential for these ions of inducing tissue damage by necrosis if present in large concentrations in vivo.

Semi-quantitative analysis of cytokines in MM THR tissues and their relationship to metal particles.

Cytokines associated with osteolysis have been demonstrated in tissues surrounding failed metal-metal (MM) total hip replacements (THRs). The objective of the present study was to semi-quantify the amounts of inflammatory cytokines in tissues from 28 failed MM THRs, and determine their relationship with the quantity of metal particles. Paraffin sections were immunohistochemically stained with monoclonal antibodies: anti-IL-1-beta, anti-IL-6 and anti-TNF-alpha. Cytokines and metal particles were rated in 10 fields per tissue using standard light microscopy. Because of the use of light microscopy, only relatively large particles or agglomerations of particles were visible. Therefore, a polarized light and a semi-quantitative scheme based on the discoloration of cell cytoplasms induced by the presence of particles were used to evaluate the quantity of metal particles. Results showed an overall higher amount of IL-6 than IL-1beta while TNF-alpha remained at very low levels. For each patient, the average IL-1beta and IL-6 ratings decreased when the average particle rating increased, following a linear regression, with relatively high correlation factors (r=-0.69 for IL-1beta and r=-0.57 for IL-6). IL-1beta decreased about twice as fast as IL-6. TNF-alpha, remaining at very low levels, did not demonstrate any correlation with particle rating. When multiple tissues were available for the same patient, the correlation factors between the average cytokine and particle ratings were highly variable between samples, demonstrating the heterogeneity between the tissues from the same patient. At the cellular level, there was an even higher correlation between the quantity of metal particles and the production of IL-1beta and IL-6 (r=-0.99), while TNF-alpha did not demonstrate any correlation, remaining at very low levels. In conclusion, this study showed that tissues surrounding failed MM THRs with low to moderate quantities of metal particles can induce the production of potentially osteolytic cytokines. However, the overall number of cells producing these cytokines tended to be lower than that typically seen in tissues surrounding metal-polyethylene THRs.

Effect of cobalt and chromium ions on bcl-2, bax, caspase-3, and caspase-8 expression in human U937 macrophages.

The bcl-2 and caspase families of proteins play a central role in the modulation of apoptosis. The purpose of this study was to analyze the effect of Co(2+) and Cr(3+) ions on the expression of bcl-2, bax, caspase-3 and caspase-8 to better understand the mechanisms leading to ion-induced apoptosis in macrophages. U937 human macrophages were exposed to Co(2+) and Cr(3+) ions. The expression of proteins was measured by Western blot while caspase activities were measured by colorimetric assay. Results show that Co(2+) ions inhibited bcl-2 expression with significant effect (p<0.05) after 16 h and a maximal 52% inhibitory effect after 24 h. Co(2+) stimulated bax expression with a significant stimulation (p<0.05) after 8 h and a maximal 1.75-fold increase after 16 h. Co(2+) also stimulated the expression of the active fragment of caspase-3 as well as caspase-3 activity maximal increase after 24 h. Co(2+) ions had no effect on caspase-8 expression or activity.Cr(3+) ions inhibited bcl-2 expression with significant effect (p<0.05) after 16 h and a maximal 43% inhibitory effect after 24 h. Cr(3+) stimulated bax expression with significant stimulation (p<0.01) after 8h and a maximal 2.25-fold increase after 24 h. Cr(3+) ions also stimulated the expression of the active fragments of caspase-3 and -8, as well as the activities of both proteases. The effect of Cr(3+) ions on the expression of both caspase active fragments was maximal after 16 h incubation. In conclusion, our results suggest that the modulation of the expression of proteins from the bcl-2 and the caspase families of proteins are implicated in the induction of macrophage apoptosis by Co(2+) and Cr(3+) ions.

TNF-alpha secretion and macrophage mortality induced by cobalt and chromium ions in vitro-qualitative analysis of apoptosis.

Metal ion toxicity is a major cause for concern in metal-metal hip replacements. A previous study in our laboratory demonstrated that Co(2+) and Cr(3+) induce macrophage apoptosis in vitro at 24h, with the implication of a caspase-3 pathway. The aim of the present study was to look at the effect of a prolonged incubation time on macrophage response with regards to TNF-alpha secretion and macrophage mortality, more specifically apoptosis. J774 macrophages were exposed for up to 48 h to 0-10 ppm Co(2+) and 0-500 ppm Cr(3+). ELISA results demonstrated that Co(2+ )and Cr(3+) induced a concentration- and time-dependent increase of TNF-alpha secretion, but a decrease at the highest concentrations of Cr(3+) (350-500 ppm). This decrease was most likely due to a high toxicity of Cr(3+) at such concentrations. Higher levels of TNF-alpha were observed with Co(2+) than Cr(3+), demonstrating a higher stimulatory effect of this ion. Trypan blue and flow cytometry results demonstrated that both Co(2+) and Cr(3+) ions induce macrophage mortality in a dose- and time-dependent manner. The number of cells decreased when ion concentrations increased, especially at 48 h. In parallel with the TNF-alpha results, Co(2+) was more toxic than Cr(3+) since the maximal effects were reached with lower concentrations (8-10 ppm vs. 350-500 ppm, respectively). DNA analysis demonstrated that both Co(2+) and Cr(3+) ions induce macrophage apoptosis, with a stronger signal at 24h than at 48 h, suggesting the presence of more necrosis after 48 h. PARP cleavage, another marker of apoptosis, was observed at both 24 and 48 h, with a maximum intensity at 48 h and with the highest concentrations of ions. In conclusion, this study demonstrates that both Co(2+) and Cr(3+) ions can induce the release of TNF-alpha and macrophage mortality in a dose- and time-dependent manner. More specifically, Co(2+) and Cr(3+) ions induced apoptosis after both 24 and 48 h incubation, although DNA analysis suggested the presence of necrosis at 48 h. The relative importance of apoptosis and necrosis in the induction of macrophage mortality by these metal ions remains to be investigated.

Differential apoptotic response of J774 macrophages to alumina and ultra-high-molecular-weight polyethylene particles.

We recently identified apoptosis in in vitro wear particle-stimulated macrophages. The recent explosion of interest in apoptosis lies in the fact that it is under positive and negative regulation through evolutionary conserved biochemical pathways. It may also be possible to modulate macrophage apoptosis in the treatment of periprosthetic osteolysis. The purpose of this study was to compare the macrophage response to identically sized particles of alumina ceramic (Al2O3) and ultra-high-molecular-weight polyethylene (UHMWPE) in terms of TNF-alpha release and induction of apoptosis. J774 mouse macrophages were incubated for 0-24 h in the presence of Al2O3 and UHMWPE particles. TNF-alpha release was measured by ELISA; Poly(ADP-ribose)polymerase (PARP) and caspase-3 expression was analyzed by Western blot; DNA fragmentation (DNA laddering) was visualized on agarose gel containing ethidium bromide. Al2O3 particles induced TNF-alpha release after 4 h incubation with concentrations reaching 483 and 800 pg/ml after 24 h with 125 and 250 particles/macrophage, respectively (control = 161 pg/ml) (P < 0.05 vs. control). The same concentrations of UHMWPE particles induced a much larger and significant TNF-alpha release after only 1 h incubation, increasing up to 6250 pg/ml after 24 h (P < 0.05 vs. control). Western blot analysis demonstrated that the active caspase-3 fragment (17 kDa) and the proteolytic PARP fragment (85 kDa) were expressed after 2 h incubation with 125 and 250 Al2O3 particles/macrophage. The active caspase-3 and the PARP fragment had lower expression and appeared after a longer incubation time (8 h) with 125 and 250 UHMWPE particles/macrophage. Finally, DNA fragmentation (DNA laddering) was observed after 16 h with 125 and 250 particles of Al2O3 per macrophage whereas no laddering was induced by UHMWPE particles even after 24 h incubation. This study shows that although both Al2O3 and UHMWPE particles induce TNF-alpha release, this stimulation was much greater (8-10 times higher) with UHMWPE than Al2O3 (P < 0.05 vs. control). As well, the induction of apoptosis, as measured by activation of caspase-3, PARP cleavage and DNA laddering, is different for these two particles, being faster and more important with Al2O3 than UHMWPE. We hypothesize that the ability of Al2O3 to induce macrophage apoptosis may explain the lower TNF-alpha release observed with these particles and explain the differences seen in osteolysis patterns of ceramic-ceramic (CC) vs. metal-polyethylene (Mpe) articulations. In conclusion, apoptosis may be a major internal mechanism to decrease macrophage activity and may be a desired therapeutic endpoint. The identification of an apoptosis-related pathway in the macrophage response to ceramic particles provides crucial data for a rational approach in the treatment and/or prevention of periprosthetic osteolysis.