Surface nanocavitation of titanium modulates macrophage activity.

Background: Nanoscale surface modifications are widely touted to improve the biocompatibility of medically relevant materials. Immune cells, such as macrophages, play a critical role in the initial healing events following implantation. Methods: To understand the response of macrophages to nanotopography better, we exposed U937-derived macrophages to a distinctive mesoporous titanium surface (TiNano) produced by a process of simple chemical nanocavitation, and to mechanically polished titanium (TiPolished) and glass coverslip (Glass) surfaces as controls. Cell numbers and morphology were evaluated. Osteopontin expression and that of the proinflammatory SPARC protein and its stabilin 1 receptor were analyzed. Release of inflammation-associated cytokines and chemokines was also measured. Results: Compared to the two control surfaces, there were fewer U937 cells on TiNano, and these exhibited a more rounded morphology with long filopodia. The cells showed areas of punctate actin distribution, indicating formation of podosomes. Of the three proteins examined, only osteopontin's immunofluorescence signal was clearly reduced. Irrespective of the substrate, the cytokine assay revealed important variations in expression levels of the multiple molecules analyzed and downregulation in a number of chemokines by the TiNano surface. Conclusion: These results indicate that macrophages sense and respond to the physicochemical cueing generated by the nanocavitated surface, triggering cellular and molecular changes consistent with lesser inflammatory propensity. Given the previously reported beneficial outcome of this mesoporous surface on osteogenic activity, it could be presumed that modulation of the macrophagic response it elicits may also contribute to initial bone-integration events.

A matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry-based toxicoproteomic screening method to assess in vitro particle potencies.

Knowledge of biological reactivity and underlying toxicity mechanisms of airborne particulate matter (PM) is central to the characterization of the risk associated with these pollutants. An integrated screening platform consisting of protein profiling of cellular responses and cytotoxic analysis was developed in this study for the estimation of PM potencies. Mouse macrophage (J774A.1) and human lung epithelial cells (A549) were exposed in vitro to Ottawa urban particles (EHC6802) and two reference mineral particles (TiO2 and SiO2 ). Samples from the in vitro exposure experiment were tested following an integrated classical cytotoxicity/toxicoproteomic assessment approach for cellular viability (CellTiter Blue®, lactate dehydrogenase) and proteomic analyses. Cellular proteins were pre-fractionated by molecular weight cut-off filtration, digested enzymatically and were analyzed by matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry for protein profiling and identification. Optimization of detergent removal, pre-fractionation strategies and enzymatic digestion procedures led to increased tryptic peptide (m/z) signals with reduced sample processing times, for small total protein contents. Proteomic analyses using this optimized procedure identified statistically significant (P < 0.05) PM dose-dependent changes at the molecular level. Ranking of PM potencies based on toxicoproteomic analysis were in line with classical cytotoxicity potency-based ranking. The high content toxicoproteomic approach exhibited the potential to add value to risk characterization of environmental PM exposures by complementing and validating existing cytotoxicity testing strategies.

Biodegradable chitosan microparticles induce delayed STAT-1 activation and lead to distinct cytokine responses in differentially polarized human macrophages in vitro.

Current data suggest that chitosan activates wound macrophages to release endogenous factors that guide mesenchymal stem cell (MSC) to bone fractures. We tested the hypothesis that chitosan, a polymer containing glucosamine and N-acetyl glucosamine, stimulates macrophages in different polarization states to release functional MSC chemokines and mainly anabolic factors. Low-serum conditioned medium was collected from M0, M1 and M2a U937 macrophages previously differentiated with phorbol myristate acetate (PMA) and exposed or not for 24h to chitosan microparticles (80% degree of deacetylation, DDA, 130kDa). Chitosan particles were highly phagocytosed. Chitosan enhanced anabolic factor release from M0 and M2a macrophages (MCP-1, IP-10, MIP-1beta, IL-1ra, IL-10, PDGF), and IL-1beta release, with 25- to 400-fold excess IL-1ra over IL-1beta. In M1 macrophages, chitosan enhanced IL-1beta without enhancing or suppressing inflammatory factor release (IL-6, IP-10, IL-8). M0 and M2a macrophages, with or without chitosan stimulation, produced conditioned medium that promoted 2-fold more MSC chemotaxis than low-serum control medium, while M1-conditioned medium failed to induce MSC chemotaxis. Acetylated chitosan induced U937 macrophages to release IL-1ra without STAT-6 activation, and also induced a delayed STAT-1 activation/IP-10 release response that was not observed using non-biodegradable chitosan (98% DDA, 130kDa). In primary human macrophages, acetylated chitosan enhanced IL-1ra release without inducing IL-1beta, and required PMA priming to elicit STAT-1 activation and IP-10 release. We conclude that biodegradable chitosan particles enhance M0 and M2a macrophage anabolic responses independent of the IL4/STAT-6 axis, by inducing excess IL-1ra over IL-1beta and more chemokine release, without altering their inherent capacity to attract MSCs.

Osteogenic cell cultures cannot utilize exogenous sources of synthetic polyphosphate for mineralization.

Phosphate is critical for mineralization and deficiencies in the regulation of free phosphate lead to disease. Inorganic polyphosphates (polyPs) may represent a physiological source of phosphate because they can be hydrolyzed by biological phosphatases. To investigate whether exogenous polyP could be utilized for mineral formation, mineralization was evaluated in two osteogenic cell lines, Saos-2 and MC3T3, expressing different levels of tissue non-specific alkaline phosphatase (tnALP). The role of tnALP was further explored by lentiviral-mediated overexpression in MC3T3 cells. When cells were cultured in the presence of three different phosphate sources, there was a strong mineralization response with ß-glycerophosphate (ßGP) and orthophosphate (Pi) but none of the cultures sustained mineralization in the presence of polyP (neither chain length 17-Pi nor 42-Pi). Even in the presence of mineralizing levels of phosphate, low concentrations of polyP (50 µM) were sufficient to inhibit mineral formation. Energy-dispersive X-ray spectroscopy confirmed the presence of apatite-like mineral deposits in MC3T3 cultures supplemented with ßGP, but not in those with polyP. While von Kossa staining was consistent with the presence or absence of mineral, an unusual Alizarin staining was obtained in polyP-treated MC3T3 cultures. This staining pattern combined with low Ca:P ratios suggests the persistence of Ca-polyP complexes, even with high residual ALP activity. In conclusion, under standard culture conditions, exogenous polyP does not promote mineral deposition. This is not due to a lack of active ALP, and unless conditions that favor significant processing of polyP are achieved, its mineral inhibitory capacity predominates.

Poly(ε-caprolactone) scaffolds of highly controlled porosity and interconnectivity derived from co-continuous polymer blends: model bead and cell infiltration behavior.

Porous structures destined for tissue engineering applications should ideally show controlled and narrow pore size distributions with fully interconnected pores. This study focuses on the development of novel poly(ε-caprolactone) (PCL) structures with fully connected pores of 84, 116, 141, and 162 µm average diameter, from melt blending of PCL with poly(ethylene oxide) (PEO) at the co-continuous composition, followed by static annealing and selective extraction of PEO. Our results demonstrate a low onset concentration for PEO continuity and a broad region of phase inversion. A novel in vitro assay was used to compare scaffold infiltration by 10-µm diameter polystyrene beads intended to mimic trypsinized human bone marrow stromal cells (hBMSCs). Beads showed a linear increase in the extent of scaffold infiltration with increasing pore size, whereas BMSCs infiltrated 162 and 141 µm pores, below which the cells aggregated and adhered near the seeding area with low infiltration into the porous device. While providing a baseline for non-aggregated systems, the beads closely mimic trypsinized cells at pore sizes equal to or larger than 141 µm, where optimal retention and distribution of hBMSCs are detected. A cytotoxicity assay using L929 cells showed that these scaffolds were cytocompatible and no cell necrosis was detected. This study shows that a melt blending approach produces porous PCL scaffolds of highly controlled pore size, narrow size distribution and complete interconnectivity, while the bead model system reveals the baseline potential for a homogeneous, non-aggregated distribution of hBMSCs at all penetration depths.

Interactions of U937 macrophage-like cells with decellularized pericardial matrix materials: influence of crosslinking treatment.

While macrophages have been implicated in the failure of bioprosthetic heart valves, the macrophage response to crosslinked native pericardial collagen has not been previously investigated. Using decellularized bovine pericardium (DBP) as a model for native collagen, this study investigated the response of macrophage-like cells (U937s) to DBP, either: (i) untreated, or (ii) exogenously crosslinked with glutaraldehyde or 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDC). We have previously validated the use of U937 cells as models for the response of human monocyte-derived macrophages to decellularized pericardial materials and, per our previous work, differentiated the U937 cells directly on the three material surfaces. After 72h in culture, the cells and medium were analyzed for DNA content, acid phosphatase activity, and cytokine and matrix metalloproteinase release. As well, cell/substrate samples were fixed for SEM. Fewer cells attached to or survived on the glutaraldehyde-treated substrate, and some showed an abnormal morphology compared to cells cultured on the other surfaces. Further, cells on glutaraldehyde-treated surfaces released more pro-inflammatory cytokines, more MMP-1 and less MMP-2 and MMP-9. The poor performance of the U937 macrophage-like cells on the glutaraldehyde-treated surfaces appears to be due to surface characteristics rather than to soluble aldehyde or other components leaching from the crosslinked material. These results provide evidence that crosslinking with glutaraldehyde is cytotoxic to macrophage-like cells, and that crosslinking with a zero-length crosslinker like EDC can be an acceptable alternative crosslinking treatment for biomaterials.

Biodegradable chitosan particles induce chemokine release and negligible arginase-1 activity compared to IL-4 in murine bone marrow-derived macrophages.

Alternatively activated macrophages have been implicated in the therapeutic activity of biodegradable chitosan on wound healing, however, the mechanisms of phenotypic differentiation are still unclear.In vitro, macrophages stimulated with high doses of chitosan (≥ 500 µg/mL) were reported to produce low-level markers associated with alternative activation (arginase-1) as well as classical activation (nitric oxide), and to undergo apoptosis. In this study, we tested the hypothesis that 40 kDa biodegradable chitosan (5-500 µg/mL) is sufficient to polarize mouse bone marrow-derived macrophages (BMDM) in vitro to an alternatively activated phenotype. Control cultures were stimulated with IL-4 (alternative activation), IFN-γ/LPS (classical activation), 1 µm diameter latex beads (phagocytosis), or left untreated. After 48 h of in vitro exposure, BMDM phagocytosed fluorescent chitosan particles or latex beads, and remained viable and metabolically active, although some cells detached with increasing chitosan and latex bead dosage. Arginase-1 was over 100-fold more strongly induced by IL-4 than by chitosan, which induced only sporadic and weak arginase-1 activity over untreated BMDM, and no nitric oxide. IFN-γ/LPS stimulated nitric oxide production and arginase-1 activity and high concentrations of inflammatory cytokines (IL-6, IL-1ß, TNF-α, MIP-1α/MIP-1ß), while latex beads stimulated nitric oxide and not arginase-1 activity. Chitosan or latex bead exposure, but not IL-4, tended to promote the release of several chemokines (MIP-1α/ß, GM-CSF, RANTES, IL-1ß), while all treatments promoted MCP-1 release. These data show that chitosan phagocytosis is not sufficient to polarize BMDM to the alternative or the classical pathway, suggesting that biodegradable chitosan elicits alternatively activated macrophages in vivo through indirect mechanisms.

Macrophage differentiation and polarization on a decellularized pericardial biomaterial.

The monocyte-derived macrophage (MDM), present at biomaterial implantations, can increase, decrease or redirect the inflammatory and subsequent wound healing process associated with the presence of a biomaterial. Understanding MDM responses to biomaterials is important for improved prediction and design of biomaterials for tissue engineering. This study analyzed the direct differentiation of monocytes on intact, native collagen. Human monocytes were differentiated on decellularized bovine pericardium (DBP), polydimethylsiloxane (PDMS) or polystyrene (TCPS) for 14 d. MDMs on all surfaces released high amounts of MMP-9 compared to MMP-2 and relatively little MMP-1. MDMs differentiated on DBP released more MMP-2, but less acid phosphatase activity. MDMs on all three surfaces released low amounts of cytokines, although substrate differences were found: MDMs on DBP released higher amounts of IL-6, IL-8, and MCP-1 but lower amounts of IL-10 and IL-1ra. This research provides evidence that MDMs on decellularized matrices may not be stimulated towards an activated, inflammatory phenotype, supporting the potential of decellularized matrices for tissue engineering. This study also demonstrated that the differentiation surface affects MDM phenotype and therefore study design of macrophage interactions with biomaterials should scrutinize the specific macrophage culture method utilized and its effects on macrophage phenotype.

In vitro response of monocyte-derived macrophages to a decellularized pericardial biomaterial.

Decellularized tissue-derived heart valves are an example of biomaterials derived from natural scaffolds. These types of implants are increasing in popularity although their in vivo performance is still only poorly understood and has, at times, been catastrophic. It is apparent that better understanding is required before these biomaterials can be used safely. In this study, the human monocyte-derived macrophage (MDM) response to decellularized bovine pericardium (DBP) was used as a model to predict the biological performance of these materials on implantation. Human monocytes differentiated on tissue culture polystyrene (TCPS) for 14 days were trypsinized and reseeded onto DBP, TCPS, and polydimethylsiloxane (PDMS) for 48 h. The MDMs on DBP contained less intracellular and extracellular esterase activity compared with MDMs on TCPS and PDMS, as well as less acid phosphatase activity than on TCPS. As well, morphologically, MDMs on DBP were less spread, less multinucleated and did not display many lamellipodia. Taken together, these data represent the first evidence of the MDM response to intact, native extracellular matrix, demonstrating that these cells reacted with an altered, possibly reduced foreign body response on this natural scaffold compared with the two control surfaces. This in vitro MDM cell model may provide a novel method for predicting and elucidating the biological performance of tissue-derived biomaterials, thereby directing a more rational design of biomaterials for tissue regeneration purposes.

Response of macrophage-like U937 cells to decellularized tissue heart valve materials.

BACKGROUND AND AIM OF THE STUDY: Decellularized materials, which represent a popular option for a variety of applications in regenerative medicine, including bioprosthetic heart valves, offer the opportunity to study cellular responses to extracellular matrix biochemistry and architecture. The study aim was to investigate the response of U937 macrophage-like cells (a model of the monocyte-derived macrophage, the pivotal cell to the initial and chronic cellular responses to implanted biomaterials) to decellularized bovine pericardium, to explore its expected biological performance in vivo, and to predict any adverse reactions in clinical trials. METHODS: Differentiated U937 cells were cultured on three surfaces: decellularized bovine pericardium (DBP); polydimethylsiloxane (PDMS); and tissue-culture polystyrene (TCPS). Cell lysates were analyzed for DNA (to determine cell attachment and viability), esterase (as a marker of degradative potential) and acid phosphatase activity (as a marker of the innate immune response). Cell morphology was also compared using confocal and scanning electron microscopy. RESULTS: U937 cells cultured on DBP were less spread and had less multinucleation than cells on either control polymer. No significant differences in DNA amount were observed between the substrates at each time point. In addition, cells cultured on DBP contained less acid phosphatase and esterase activity than cells on TCPS (p < 0.05). CONCLUSION: The study results suggested that U937 cells seeded onto DBP reacted with an altered, more mild, foreign body response than cells cultured on either PDMS or TCPS. This U937 cell model provides evidence that the response of macrophages to decellularized materials is not initially aggressive. The present study served as a first step in elucidating the biological mechanisms by which tissue-derived valve replacements fail in the host--an understanding that may direct a more rational design of valvular and decellularized materials.