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.

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.

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.

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.

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.

In vitro macrophage response to nanometer-size chromium oxide particles.

An increasing number of studies have reported adverse tissue reactions around metal-on-metal (MM) hip implants. However, the origin and mechanisms of these reactions remain unclear. Moreover, the biological effects of nanometer-size chromium oxide particles, the predominant type of wear particles produced by MM implants, remain mostly unknown. The purpose of this study was to analyze the cytotoxic effects of clinically relevant nanometer-size chromium oxide particles on macrophage response in vitro. J774.A1 macrophages were cultured with either 60 nm or 700 nm commercially available Cr2 O3 particles at different concentrations. Two different particle sizes were analyzed to evaluate potential volume effects. Cell mortality was analyzed by light microscopy, flow cytometry (annexin V-fluorescein isothiocyanate and propidium iodide assay), and using a cell death detection enzyme-linked immunosorbant assay (ELISA). Tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 alpha (MIP-1α) release was measured by ELISA, and gene expression was analyzed by quantitative real-time PCR. Results showed that, at high concentrations, Cr2 O3 particles of both sizes can be cytotoxic, inducing significant decreases in total cell numbers and increases in necrosis. Results also suggested that these effects were dependent on particle volume. However, TNF-α, MCP-1, and MIP-1α cytokine release and gene expression remained low. Overall, this study demonstrates that nanometer-size particles of Cr2 O3 , a stable form of chromium oxide ceramic, have rather low cytotoxic effects on macrophages. Therefore, these particles may not be the main culprit in the initiation of the inflammatory reaction in MM periprosthetic tissues. However, other parameters (e.g., potential intracellular damage) remain to be investigated.

An assay of mammalian cell micropermeabilization based on measurements of cellular lactate production.

A method for the quantitative assay of mammalian cell micropermeabilization is described. The method is based on the permeabilization-induced loss of endogenous glycolytic cofactors and consequent discontinuation of cellular lactate production. Advantages of the method include sensitivity and precision similar to that of micropermeabilization assays based on the release of 86Rb+ from preloaded cells, avoidance of radioactivity, and simplicity of the measurements and equipment required.

Absence of evidence for metabolite-modulated association between alpha-glycerol-3-phosphate dehydrogenase and L-lactate dehydrogenase.

Evidence for the NADH-modulated formation of a complex between alpha-glycerol-3-phosphate dehydrogenase and l-lactate dehydrogenase was reported [Yong, H., Thomas, G. A., and Peticolas, W. L. (1993) Biochemistry 32, 11124-11131]. This NADH-modulated association suggested a mechanism of potentially great importance to enzyme modulation and the controversial phenomena of direct NADH channeling. In the present paper, we reproduce with additional controls the experiments described by Yong et al. ((1993) Biochemistry 32, 11124-11131). Our results conclusively demonstrate the absence of detectable association between alpha-glycol-3-phosphate dehydrogenase and l-lactate dehydrogenase.