Loren A. Matheson

Human macrophage-mediated biodegradation of polyurethanes: assessment of candidate enzyme activities

A predominant cell type associated with explanted failed devices is the monocyte-derived macrophage (MDM). However, there is still very little known about the specific cellular enzyme activities involved in interactions with these devices. The current study investigates the nature of candidate enzymes that may be involved in the degradation of polymeric biomaterials through the use of specific enzyme inhibitor agents. When MDM were incubated with a polycarbonate-based polyurethane (PCNU) synthesized with 14C-labeled hexane diisocyanate (HDI), polycarbonate diol and butanediol (BD) (referred to as 14C-HDI431), the radiolabel release (RR) measured was inhibited by phenylmethylsulfonyl fluoride, diethyl-p-nitrophenyl phosphate (serine protease/esterase inhibitors), and sodium fluoride (NaF) (a carboxyl esterase (CXE) inhibitor). Sodium taurocholate (NaT) (a cholesterol esterase (CE) stimulator) had little effect on RR. The two candidate enzymes proposed were CE and CXE, based on the fact that both were identified by immunoblotting in the releasate of MDM following 48 h incubation with 14C-HDI431. The effect of the above reagents on the RR caused by purified CE and CXE, was measured and compared to changes in their activity with p-nitrophenylbutyrate (PNB). The effect of NaF on MDM was similar to that of purified CXE (inhibitory on both RR and lysate esterase activity), suggesting the involvement of CXE. However, NaT inhibited the PNB activity of purified CXE, but had no effect on MDM-mediated RR or PNB activity, implicating another esterase in the biomaterial degradation. Since NaT stimulated CE-mediated RR and PNB activity, it may also be involved in MDM-mediated biodegradation of PCNUs. The results of these studies point to both esterases as being candidates. However, the current methods were unable to determine the relative contribution of each one to the observed biodegradation.

Changes in macrophage function and morphology due to biomedical polyurethane surfaces undergoing biodegradation

Monocytes are recruited to the material surface of an implanted biomedical device recognizing it as a foreign body. Differentiation into macrophages subsequently occurs followed by fusion to form foreign body giant cells (FBGCs). Consequently, implants can become degraded, cause chronic inflammation or become isolated by fibrous encapsulation. In this study, a relationship between material surface chemistry and the FBGC response was demonstrated by seeding mature monocyte‐derived macrophages (MDMs) on polycarbonate‐based polyurethanes that differed in their chemical structures (synthesized with poly(1,6‐hexyl 1,2‐ethyl carbonate) diol, and either 14C‐hexane diisocyanate and butanediol (BD) (referred to as HDI) or 4,4′‐methylene bisphenyl diisocyanate and 14C‐BD (referred to as MDI)) and material degradation assessed. At 48 h of cell‐material interaction, the FBGC attached to HDI were more multinucleated (73%) compared to MDI or the polystyrene (PS) control (21 and 36%, respectively). There was a fivefold increase in the synthesis and secretion of a protein with an approximate molecular weight of 48 kDa and a pI of 6.1 (determined by two‐dimensional gel electrophoresis) only from cells seeded on HDI. Immunoprecipitation confirmed that MSE and CE were synthesized and secreted de novo. Immunoblotting also showed an increase in secreted monocyte‐specific esterase (MSE) and cholesterol esterase (CE) from cells seeded on HDI relative to PS and MDI. Significantly more radiolabel (14C) release and esterase activity were elicited by MDMs on HDI than MDI (P < 0.05). The material that was more degradable (HDI), elicited greater protein synthesis and esterase secretion as well as more multinucleated MDMs than MDI, suggesting that the material surface chemistry modulates the function of MDM at the site of an inflammatory response to an implanted device. J. Cell. Physiol. 199: 8–19, 2004© 2003 Wiley‐Liss, Inc.

Effect of polyurethane chemistry and protein coating on monocyte differentiation towards a wound healing phenotype macrophage.

Tissue regeneration alternatives for peripheral vascular disease are actively being investigated; however, few studies in this area have probed the role of the wound healing monocyte-derived macrophage (MDM). Inflammatory MDMs transition to wound healing MDMs as the relative levels of tumor necrosis factor-alpha (TNF-alpha) decrease and IL-10 increase. TNF-alpha has been linked to the regulation of HMGB1 (high mobility group box 1 protein), a nuclear protein that upon macrophage stimulation can be secreted and act as a pro-inflammatory cytokine. This study investigated the influence of a degradable polar hydrophobic ionic polyurethane (D-PHI) on MDM cell expression of pro- versus anti-inflammatory markers, when the material was uncoated or pre-coated with collagen prior to cell studies. Effects were compared to similar groups on tissue culture polystyrene (TCPS). Collagen coated TCPS and D-PHI had significantly more DNA than the uncoated TCPS after 7d (p=0.001 and p=0.006 respectively); however, there was significantly less esterase activity from cells on D-PHI (+/-collagen) than for cells on TCPS after 7d (p=0.002, p=0.0003 respectively). No significant differences in esterase activity were observed between collagen coated and non-coated D-PHI surfaces. Analyses of pro-inflammatory cytokines (TNF-alpha, IL-1beta and HMGB1) secreted from differentiating monocytes adherent to D-PHI demonstrated a decrease whereas anti-inflammatory IL-10 increased over time when compared to TCPS, suggesting that D-PHI was less inflammatory than TCPS. Since D-PHI maintains cell attachment while aiding in the transition of MDM to a wound healing phenotype, this material has qualities suitable to be used in tissue engineering applications where MDM play a key role in tissue regeneration.

Polycarbonate-urethane hard segment type influences esterase substrate specificity for human-macrophage-mediated biodegradation

Previous studies have shown that esterase activity can degrade a variety of polyurethanes (PUs), including polycarbonate-based PUs (PCNUs). When cultured on PCNUs, differing in their chemistries, monocyte-derived macrophages (MDM) synthesized and secreted different amounts of both cholesterol esterase (CE) and monocyte-specific esterase (MSE). MDM were seeded on PCNUs synthesized with hexane diisocyanate (HDI) or 4,4′-methylene-bis-phenyl diisocyanate (MDI), PCN and [14C]butanediol (BD) in the ratio 3:2:1 (referred to as HDI321 or MDI321). The effect of phenylmethylsulfonyl fluoride (PMSF, a serine esterase and proteinase inhibitor), sodium fluoride (NaF, a MSE inhibitor) and sodium taurocholate (NaT, a CE stimulator) was assessed on degradation (measured by radiolabel release (RR)) and esterase activity in MDM lysate. The results were compared to the effect that these reagents had on commercially available CE and carboxyl esterase (CXE), which has a specificity similar to MSE. NaF inhibited CXE- and MDM-mediated RR to the same extent as for both PCNUs. However, the MDM-mediated RR from MDI321 was 1.8-times higher than HDI321 in the presence of NaT (P = 0.005). This study suggests that the difference in diisocyanate chemistry may dictate the relative contribution of each esterase to a specific materials degradation. This may be related to both the substrate specificity of each esterase, as well as by the relative amount of each esterase that the specific biomaterial substrates induce the cells to synthesize and secrete.

Is cell culture stressful? Effects of degradable and nondegradable culture surfaces on U937 cell function.

In vitro cell culture has become one of the most widely used techniques in biological and health sciences research, with the most common culture supports being either tissue culture grade polystyrene (TCPS) or polydimethylsiloxane (PDMS). It has previously been shown that monocyte-derived macrophages (MDMs) respond to material surface chemistry, synthesizing and releasing degradative activities that could produce products, which alter the cells response. In this study, functional parameters of differentiated U937 macrophage-like cells were compared when cultured on nondegradable standard control surfaces versus models of biomaterials (polycarbonate-based polyurethanes) used in the manufacture of medical devices previously shown to degrade and/or elicit pathways of inflammation. Although the influence of PDMS and TCPS on cell function is often underappreciated by investigators, both surfaces elicited enzyme markers of inflammation. Cells on TCPS had the highest intracellular and released esterase activities and protein levels. Cells on PDMS had the most released acid phosphatase activity and protein (P < 0.001), as well as de novo 57- and 59-kDa released proteins. The criteria for defining an activated cell phenotype become critically important when materials such as PDMS and TCPS are used as standard control surfaces whether in experiments for research in elucidating metabolic pathways or in screening drugs and materials for therapeutic uses.

Design of biodegradable polyurethanes and the interactions of the polymers and their degradation by-products within in vitro and in vivo environments

From 2005 to 2015 the development of new biodegradable polymers and the use of established biodegradable materials such as polylactic–glycolic acid have dominated the landscape of the implantable biomedical devices field, driven in large part by the need for tissue engineering (TE) and drug delivery applications. This chapter provides an overview of the contributions that degradable polyurethanes (PUs) have made over the past decade, and starts with a brief summary of PU chemistry and their mechanisms of biodegradation, condenses knowledge learned from the failure of PU devices in the 1980s, elaborates on the extensive knowledge built from the work on inflammatory processes involved in wound healing and biodegradation in the 1990s, and then provides a comprehensive analysis of new degradable PU synthesis from 2005 to 2015, with comments on their strategic uses in TE, interactions with primary and stem cells, and highlights their innovative applications in drug delivery.

The effects of phorbol ester activation and reactive oxygen species scavengers on the macrophage-mediated foreign body reaction to polyurethanes.

Phorbol myristate acetate, a protein kinase C activator, inhibited monocyte-derived macrophage (MDM)-mediated degradation of aliphatic (HDI) polycarbonate-based polyurethanes but not degradation of the aromatic polycarbonate-based polyurethane (MDI). The objectives of this study were to determine if reactive oxygen species are involved in the phorbol myristate acetate effect on esterase activity and MDM-mediated polycarbonate-based polyurethane degradation and to find a good marker of material-initiated activation of MDM. The phorbol myristate acetate-dependent effects of the material chemistry on cell activation and degradation were evaluated by adding reactive oxygen species scavengers, catalase plus superoxide dismutase to MDM and assaying possible markers of MDM activation: esterase activity, acid phosphatase activity, and high molecular weight group box 1 protein (HMGB1). All treatments reduced the esterase activity in MDM on HDI but not in MDM on MDI. Acid phosphatase was inhibited in MDM to varying degrees on all surfaces by phorbol myristate acetate or catalase plus superoxide dismutase either alone or together. Secretion of HMGB1 from MDM on HDI431 was higher than MDI; however only secretion from MDM on HDI was inhibited by phorbol myristate acetate. In MDM on HDI, catalase plus superoxide dismutase reduced intracellular HMGB1 levels +/- phorbol myristate acetate; whereas, catalase, superoxide dismutase plus phorbol myristate acetate increased intracellular HMGB1 in MDM on MDI, suggesting that esterase and HMGB1 are more specific markers of activation than acid phosphatase. Manipulation of signaling pathways may provide insight surrounding the mechanism of activation for oxidative and/or hydrolytic degradative pathways in the MDM response to material surface chemistry.