Role of protein kinase C in the monocyte-derived macrophage-mediated biodegradation of polycarbonate-based polyurethanes

Polycarbonate-polyurethanes (PCNUs) elicit a foreign body reaction during the initial tissue contact, partly mediated by the respiratory burst in monocytes, during which protein kinase C (PKC) activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase. Using an in vitro cell system, monocytes were differentiated into monocyte-derived macrophages (MDMs) and then reseeded onto three PCNUs (HDI431, HDI321, or MDI321): hexane (HDI) or 4,4-methylene bis-phenyl (MDI) diisocyanates synthesized with poly(1,6-hexyl 1,2-ethyl carbonate) diol (PCN) and 14C-labeled butanediol (BD) in the ratios 4:3:1 or 3:2:1 (diisocyanate/PCN/BD). MDM-mediated degradation was assessed by radiolabel release in the presence of a PKC activator (phorbol myristate acetate), inhibitor (H7), and a catalase/peroxidase inhibitor (NaN3). Activating PKC decreased biodegradation and esterase activity in MDMs on HDI431 and HDI321 but not MDI321, whereas H7 and NaN3 inhibited the MDM degradation of MDI321 only. Pretreatment of the PCNUs with H2O2 inhibited esterase-mediated radiolabel release from HDI431 and HDI321 but stimulated radiolabel release from MDI321. The difference in the effect of H2O2 on the HDI versus MDI PCNUs contributes to explaining the effect of PKC activation on material degradation. Understanding the mechanism by which this pathway is linked to PCNU chemistry may assist in designing materials with tailored biodegradation rates.     

Effect of Phorbol Esters on the Macrophage-Mediated Biodegradation of Polyurethanes via Protein Kinase C Activation and Other Pathways

It was previously found that re-seeding monocyte-derived macrophages (MDM) on polycarbonate-based polyurethanes (PCNUs) in the presence of the protein kinase C (PKC) activator phorbol myristate acetate (PMA) inhibited MDM-mediated degradation of PCNUs synthesized with 1,6-hexane diisocyanate (HDI), as well as esterase activity and monocyte-specific esterase (MSE) protein. However, no effect on the degradation of a 4,4′-methylene bisphenyl (MDI)-derived PCNU (MDI321) occurred. This finding suggested that oxidation, a process linked to the PKC pathway, was not activated in the same manner for all PCNUs. In the current study MDM were re-seeded onto the above PCNU surfaces with PMA, PKC-inactive 4αPMA and the PKC inhibitor bisindolylmaleimide I hydrochloride (BIM) for 48 h before assaying for PCNU degradation, esterase activity, MSE protein, DNA, cell viability and cell morphology. 4αPMA did not alter MDM-mediated HDI PCNU degradation but MDI321 degradation increased in this condition. BIM alone had no effect on any parameter; however, when BIM and PMA were added together, the PMA inhibition of biodegradation, esterase activity and MSE protein was partially reversed for MDM on HDI PCNUs only. Adding PMA to MDM on HDI PCNUs increased intercellular connections, whereas 4αPMA or BIM+PMA increased cell size. Although this study demonstrated a role for oxidation via a PKC-activated pathway in MDM-mediated PCNU degradation, phorbol esters appear to also activate non-PKC pathways that have roles in biodegradation. Moreover, the sensitivity to material surface chemistry in the MDM response to each PCNU dictates a multi-factorial degradative process involving alternate material specific oxidative and hydrolytic mechanisms.     

The human macrophage response during differentiation and biodegradation on polycarbonate-based polyurethanes: dependence on hard segment chemistry

Human monocytes, isolated from whole blood, were seeded onto tissue culture grade polystyrene (PS) and three polycarbonate-based polyurethanes (PCNUs) (synthesized with either 1,6-hexane diisocyanate (HDI) or 4,4'-methylene bis-phenyl diisocyanate (MDI), poly(1,6-hexyl 1,2-ethyl carbonate) diol (PCN) and 1,4-butanediol (BD) in different stoichiometric ratios (HDI:PCN:BD 4:3:1 or 3:2:1 and MDI:PCN:BD 3:2:1) (referred to as HDI431, HDI321 and MDI321, respectively). Following their differentiation to monocyte-derived macrophages (MDMs) the cells were trypsinized and reseeded onto each of the PCNUs synthesized with either 14C-HDI or 14C-BD and degradation was measured by radiolabel release (RR). When the differentiation surface was MDI321, there was more RR from 14C-HDI431 than from any other surface (p < 0.0001) whereas the amount of esterase (identified by immunoblotting) as well as the esterase activity was the greatest in MDM differentiated on PS, reseeded on 14C-HDI431 (p < 0.0001). The effect of potential degradation products (methylene dianiline (MDA) and BD) from the PCNUs was carried out to determine possible links between products and substrate-induced activation of MDM. MDA was found to inhibit RR 60% from MDM seeded on 14C-MDI321B (p < 0.0001), approximately 20% from 14C-HDI431 (p = 0.002) and no effect from 14C-HDI321B. MDA inhibited esterase activity 30% from MDM only on 14C-MDI321B (p = 0.003), but no effect on esterase activity was observed for the other two polymers. BD had no inhibitory effect on RR from any PCNU, but did inhibit esterase activity in MDM on 14C-HDI431 (p = 0.025). This study indicates that the degradation of a specific material is a multi-factorial process, dictated by its susceptibility to hydrolysis, the effect of specific products generated during this course of action, and perhaps not as well appreciated, the material's inherent ability to influence enzyme synthesis and release.     

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 material's 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.     

The effect of oxidation on the enzyme-catalyzed hydrolytic biodegradation of poly(urethane)s

Although the biodegradation of polyurethanes (PU) by oxidative and hydrolytic agents has been studied extensively, few investigations have reported on the combination of their effects. Since neutrophils (PMN) arrive at an implanted device first and release HOCl, followed by monocyte-derived macrophages (MDM) which have potent esterase activities and oxidants of their own, the combined effect of oxidative and hydrolytic degradation on radiolabeled polycarbonate-polyurethanes (PCNU)s was investigated and compared to that of a polyester-PU (PESU) and a polyether-PU (PEU). The PCNUs were synthesized with PCN (MW = 1,000), and butanediol (14C-BD) and one of two diisocyanates, hexane-1,6-diisocyanate (14C-HDI) or methylene bis-p-phenyl diisocyanate (MDI). The PESU and PEU were synthesized using toluene-diisocyanate (14C-TDI), with polycaprolactone and polytetramethylene oxide as soft segments respectively, and ethylene diamine as the chain extender. The effect of pre-treatment with 0.1 mM HOC1 for 1 week on the HDI-based PCNUs and both TDI-based PUs resulted in a significant inhibition of radiolabel release (RR) elicited by cholesterol esterase (CE), when compared to buffer alone, whereas the MDI-based PCNU showed a small but significant increase. When PMN were activated on the HDI-based PCNU surface with phorbol myristate acetate (PMA), HOCl was released for 3 h, and was almost completely abolished by sodium azide (AZ). Simultaneously, the PMN-elicited RR, shown previously to be due to the esterolytic cleavage by serine proteases, was inhibited approximately 75% by PMA-activation of the cells, but significantly increased relative to the latter when AZ was added. Both in vitro oxidation by HOCl and the release of HOCI by PMN were associated with the inhibition of RR and suggest perturbations between oxidative and hydrolytic mechanisms of biodegradation.     

Hydrolytic degradation of poly(carbonate)-urethanes by monocyte-derived macrophages

Polycarbonate (PCN)-based polyurethanes (PCNU) are rapidly becoming the chosen polyurethane (PU) for long-term implantation since they have shown decreased susceptibility to oxidation. However, monocyte-derived macrophages (MDM), the cell implicated in biodegradation, also contain hydrolytic activities. Hence, in this study, an activated human MDM cell system was used to assess the biostability of a PCNU, synthesized with 14C-hexane diisocyanate (HDI) and butanediol (BD), previously shown to be susceptible to hydrolysis by cholesterol esterase (CE). Monocytes, isolated from whole blood and cultured for 14 days on polystyrene (PS) to mature MDM, were gently trypsinized and seeded onto 14C-PCNU. Radiolabel release and esterase activity, as measured with p-nitrophenylbutyrate, increased for almost 2 weeks. At 1 week, the increase in radiolabel release and esterase activity were diminished by more than 50% when the protein synthesis inhibitor, cycloheximide, or the serine esterase/protease inhibitor, phenylmethylsulfonylfluoride was added to the medium. This strongly suggests that in part, it was MDM esterase activity which contributed to the PU degradation. In an effort to simulate the potential combination of oxidative and hydrolytic activities of inflammatory cells. 14C-PCNU was exposed to HOCl and then CE. Interestingly, the release of radiolabeled products by CE was significantly inhibited by the pre-treatment of PCNU with HOCl. The results of this study show that while the co-existing roles of oxidation and hydrolysis in the biodegradation of PCNUs remains to be elucidated, a clear relationship is drawn for PCNU degradation to the hydrolytic degradative activities which increase in MDM during differentiation from monocytes, and during activation in the chronic phase of the inflammatory response.     

Enzyme induced biodegradation of polycarbonate-polyurethanes: dose dependence effect of cholesterol esterase

The current study has investigated the influence of esterase activity (80-400units/ml) on the biodegradation of polycarbonate-urethanes (PCNUs) by cholesterol esterase (CE), with a particular interest in studying the influence of different hard segment structures and their contribution to sensitizing the polymer towards enzyme catalyzed hydrolysis. Polycarbonate based polyurethanes were synthesized with varying hard segment content as well as hard segment chemistry based on three different diisocyanates, 1,6-hexane diisocyanate (HDI), 4,4'-methylene bisphenyl diisocyanate (MDI) and 4,4-methylene biscyclohexyl diisocyanate (HMDI). The effect of different chemistry on surface contact angle was measured in order to define the relative chemical nature of the surfaces. The enzyme dose response was found to be lower when hard segment content in the polymer was high. There was a very strong dependence on enzyme concentration for polyurethanes with different hard segment chemistry, despite the fact that the nature of the hydrolysable polycarbonate segment remained the same. The PCNU which showed the most dramatic dependence on enzyme concentration was synthesized with HMDI. At low enzyme concentration (80units/ml) this material was the most stable of the polymers while at elevated CE concentration (400units/ml) the polymer underwent a catastrophic breakdown. The findings suggested that protein binding on the surfaces was saturated even though enzyme degradation did not achieve saturation on any of the surfaces. The role of protein binding in modulating the hydrolytic action of the enzymes at different activity levels highlights a need for further study in this area.     

The effect of oxidation on the enzyme-catalyzed hydrolytic biodegradation of poly(urethane)s

Although the biodegradation of polyurethanes (PU) by oxidative and hydrolytic agents has been studied extensively, few investigations have reported on the combination of their effects. Since neutrophils (PMN) arrive at an implanted device first and release HOCl, followed by monocytederived macrophages (MDM) which have potent esterase activities and oxidants of their own, the combined effect of oxidative and hydrolytic degradation on radiolabeled polycarbonate-polyurethanes (PCNU)s was investigated and compared to that of a polyester-PU (PESU) and a polyether-PU (PEU). The PCNUs were synthesized with PCN (MW = 1000), and butanediol (¹⁴C-BD) and one of two diisocyanates, hexane-1,6-diisocyanate (¹⁴C-HDI) or methylene bis-p-phenyl diisocyanate (MDI). The PESU and PEU were synthesized using toluene-diisocyanate (¹⁴C-TDI), with polycaprolactone and polytetramethylene oxide as soft segments respectively, and ethylene diamine as the chain extender. The effect of pre-treatment with 0.1 mM HOCl for 1 week on the HDI-based PCNUs and both TDI-based PUs resulted in a significant inhibition of radiolabel release (RR) elicited by cholesterol esterase (CE), when compared to buffer alone, whereas the MDI-based PCNU showed a small but significant increase. When PMN were activated on the HDI-based PCNU surface with phorbol myristate acetate (PMA), HOCl was released for 3 h, and was almost completely abolished by sodium azide (AZ). Simultaneously, the PMN-elicited RR, shown previously to be due to the esterolytic cleavage by serine proteases, was inhibited ~75% by PMA-activation of the cells, but significantly increased relative to the latter when AZ was added. Both in vitro oxidation by HOCl and the release of HOCl by PMN were associated with the inhibition of RR and suggest perturbations between oxidative and hydrolytic mechanisms of biodegradation.     

Isolation of methylene dianiline and aqueous-soluble biodegradation products from polycarbonate-polyurethanes

Polycarbonate-polyurethanes (PCNUs) have provided the medical device industry with practical alternatives to oxidation-sensitive polyether-urethanes (PEUs). To date, many studies have focused on PCNUs synthesized with 4,4'-methylene diphenyl-diisocyanate (MDI). The relative hydrolytic stability of this class of polyurethanes is actually quite surprising given the inherent hydrolytic potential of the aliphatic carbonate group. Yet, there has been little information reporting on the rationale for the material's demonstrated hydrolytic stability. Recent work has shown that PCNU materials have a strong sensitivity towards hydrolysis when changes are made to their hard segment content and/or chemistry. However, knowledge is specifically lacking in regards of the identification of cleavage sites and the specific nature of the biodegradation products. Using high-performance liquid chromatography, radiolabel tracers and mass spectrometry, the current study provides insight into the distribution of biodegradation products from the enzyme-catalyzed hydrolysis of five different PCNUs. The hydrolytic sensitivity of the materials is shown to be related to the distribution of products, which itself is a direct consequence of unique micro-structures formed within the different materials. While an MDI-based polymer was shown to be the most hydrolytically stable material, it was the only PCNU that produced its diamine analog, in this case 4,4'-methylene dianiline (MDA), as a degradation product. Given the concern over aromatic diamine toxicity, this finding is important and highlights the fact that relative biostability is a distinct issue from that of degradation product toxicity, and that both must be considered separately when assessing the impact of biodegradation on biomaterial in vivo compatibility.     

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.     

Material surfaces affect the protein expression patterns of human macrophages: A proteomics approach

Monocyte-derived macrophages (MDM) are key inflammatory cells and are central to the foreign body response to implant materials. MDM have been shown to exhibit changes in actin cytoskeleton, multinucleation, cell size, and function in response to small alterations in polycarbonate-urethane (PCNU) surface chemistry. Although PCNU chemistry has an influence on de novo protein synthesis, no assessments of the protein expression profiles of MDM have yet been reported. The rapid emerging field of expression proteomics facilitates the study of changes in cellular protein profiles in response to their microenvironment. The current study applied proteomic techniques, 2-dimensional electrophoresis (2-DE) combined with MALDI-ToF (matrix assisted laser desorption ionization-time of flight) mass spectrometry, to determine differences in MDM protein expression influenced by PCNU. Results indicated that MDM responded to material chemistry by modulation of structural proteins (i.e. actin, vimentin, and tubulin). Additionally, intracellular protein modulation which requires proteins responsible for trafficking (i.e. chaperone proteins) and protein structure modification (i.e. bond rearrangement and protein folding) were also altered. This study demonstrated for the first time that a proteomics approach was able to detect protein expression profile changes in MDM cultured on different material surfaces, forming the basis for utilizing further quantitative proteomics techniques that could assist in elucidation of the mechanisms involved in MDM-material interaction.     

Biodegradation of polycarbonate-based polyurethanes by the human monocytes-derived macrophage and U937 cell systems

The prominent cell type found on implanted medical devices during the chronic inflammatory response is the monocyte-derived macrophage (MDM). Using an activated in vitro cell system, it was possible to show that MDMs possess esterolytic activities that may contribute to the degradation of polyurethanes. In the present study, the U937 cell line was paralleled to the MDM cell system in order to validate the use of a cell line that could expedite studies on biomaterial biocompatibility and biostability. Using 12-o-tetradecanoylphorbol 13-acetate (PMA), the optimum differentiation time for the U937 cells was 72 h based on biodegradation, degradative potential, and (35)S-methionine uptake. After activation of the cells by resuspending from tissue culture polystyrene plates and reseeding onto a (14)C-labeled polycarbonate-based polyurethane(PCNU), both U937 cells and the MDMs elicited comparable radiolabel release (measure of polymer breakdown) and esterase activity (measure of degradative potential) at 48 h.There was no difference in the effect on radiolabel release and esterase activity elicited by both cell types with inhibitors of protein synthesis, esterase activity, and phospholipase A(2). This established that both cell types likely used similar hydrolytic activities and signaling pathways to cause degradation of the PCNU. Immunoblotting demonstrated that both cell systems secreted monocyte-specific esterase and cholesterol esterase enzymes previously shown to degrade PCNUs. The U937 cell system is more convenient and reproducible than MDMs for pursuing possible biological pathways elucidating the mechanism of polyurethane biodegradation. Once established with U937s, the pathways can then be validated with the more physiologically relevant human MDM cell system.     

Phospholipase A2 pathway association with macrophage-mediated polycarbonate-urethane biodegradation

Activation of the phospholipase A2 (PLA2) pathway is a key cell signaling event in the inflammatory response. The PLA2 family consists of a group of enzymes that hydrolyze membrane phospholipids, resulting in the liberation of arachidonic acid (AA), a precursor to pro-inflammatory molecules. Given the well-documented activating role of biomaterials in the inflammatory response to medical implants, the present study investigated the link between PLA2 and polycarbonate-based polyurethane (PCNU) biodegradation, and the effect that material surface had on PLA2 activation in the U937 cell line. PCNUs were synthesized with poly(1,6-hexyl 1,2-ethyl carbonate)diol, 1,4-butanediol and one of two diisocyanates (hexane 1,6-diisocyanate or 4,4'-methylene bisphenyl diisocyanate) in varying stoichiometries and incubated with adherent U937 cells. PLA2 inhibiting agents resulted in significantly decreased PCNU biodegradation (p < 0.05). Moreover, when activation of PLA2 was assessed (3H-AA release), significantly more 3H-AA was released from PCNU-adherent U937 cells than polystyrene-adherent U937 cells (p < 0.05) which was significantly decreased in the presence of PLA2 inhibitors. The pattern of inhibition of U937 cell-mediated biodegradation and 3H-AA release that was modulated by PCNU surface differences, suggests a role for secretory PLA2 along with cytosolic PLA2. Understanding PCNU activation of intracellular pathways, such as PLA2, will allow the design of materials optimized for their intended use.     

Enzyme-induced biodegradation of polycarbonate-polyurethanes: dependence on hard-segment chemistry

Polycarbonate urethanes (PCNUs) have been used as a replacement for traditional biomedical polyether-urethanes due to their reported resistance to oxidative biodegradation. However, relatively little is known about their hydrolytic stability in the presence of inflammatory derived enzymes. This has in part motivated the current study relating to the effect of hard segment chemistry and the microdomain structures generated by such chemistry, on the cholesterol esterase (CE) catalyzed hydrolysis of PCNUs. The bulk structures of the studied materials were characterized using gel permeation chromatography (GPC), differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), Fourier transform infrared spectroscopy (FTIR) for their bulk structures, and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) for their subsurface structures. 14C-labeled PCNUs were incubated with CE (400 units/mL), for a period of 10 weeks (pH 7.0 at 37 degrees C), and radiolabel release was used to monitor the degradation. The results showed that all of the polymers synthesized in this study were susceptible to CE-catalyzed hydrolytic degradation, and that the extent of degradation was highly dependent on the nature of hard segment interactions within the polymer and at the surface. More specifically, the degree of phase separation and soft segment crystallinity were found to be less important in comparison to the hydrogen bonding among the carbonate and urethane linkages. The rank of the different chemical groups' susceptibility to hydrolysis was as follows: nonhydrogen bonded carbonate > nonhydrogen bonded urethane > hydrogen bonded carbonate > hydrogen bonded urethane. The findings suggest that the degree of hydrogen bonding, when processed into a polyurethane material could be an important parameter to consider in the design of new biostable polyurethane products.     

Influence of surface morphology and chemistry on the enzyme catalyzed biodegradation of polycarbonate-urethanes

Abstract —Polycarbonate based polyurethanes were synthesized with varying hard segment content as well as hard segment chemistry based on three different diisocyanates,1,6-hexane diisocyanate (HDI), 4,4′-methylene bisphenyl diisocyanate (MDI) and 4,4-methylene biscyclohexyl diisocyanate (HMDI). The surface chemistry and morphology were characterized using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The polymers were incubated with cholesterol esterase (CE) in a phosphate buffer solution at 37°C over 10 weeks. XPS results showed that the surface chemistry changed as the size and chemistry of the hard segment varied within the materials. AFM images exhibited distinctive surface morphologies for all polymers, and this was particularly apparent with changes in the hard segment chemistry. The results showed that the surface of HDI polymers consisted of relatively stiff rod-like structures, which corresponded to the soft segment domains. Polymers with a higher HDI content exhibited a dense top layer containing a relatively higher hard segment component, covering the sub-surface matrix of rod like structures. The MDI based polyurethane had large aggregates on its top surface, which corresponded to the aggregation of harder components. The HMDI based polycarbonate-urethane presented a relatively homogeneous surface where no phase separation could be detected. The relative differences in hard and soft segment content in their surface structure was supported by XPS findings. The analysis of the biodegradation results, concluded that enzyme catalyzed biodegradation within these materials was initiated in amorphous soft segment regions located in the region of the interface between hard and soft segments. A higher hard segment content at the surface contributed significantly to an increase in biostability. The findings provided an enhanced understanding for the role of surface molecular structure in the enzyme catalyzed biodegradation of polyurethanes.     

Influence of surface morphology and chemistry on the enzyme catalyzed biodegradation of polycarbonate-urethanes

Polycarbonate based polyurethanes were synthesized with varying hard segment content as well as hard segment chemistry based on three different diisocyanates,1,6-hexane diisocyanate (HDI), 4.4'-methylene bisphenyl diisocyanate (MDI) and 4,4-methylene biscyclohexyl diisocyanate (HMDI). The surface chemistry and morphology were characterized using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The polymers were incubated with cholesterol esterase (CE) in a phosphate buffer solution at 37 degrees C over 10 weeks. XPS results showed that the surface chemistry changed as the size and chemistry of the hard segment varied within the materials. AFM images exhibited distinctive surface morphologies for all polymers, and this was particularly apparent with changes in the hard segment chemistry. The results showed that the surface of HDI polymers consisted of relatively stiff rod-like structures, which corresponded to the soft segment domains. Polymers with a higher HDI content exhibited a dense top layer containing a relatively higher hard segment component, covering the sub-surface matrix of rod like structures. The MDI based polyurethane had large aggregates on its top surface, which corresponded to the aggregation of harder components. The HMDI based polycarbonate-urethane presented a relatively homogeneous surface where no phase separation could be detected. The relative differences in hard and soft segment content in their surface structure was supported by XPS findings. The analysis of the biodegradation results, concluded that enzyme catalyzed biodegradation within these materials was initiated in amorphous soft segment regions located in the region of the interface between hard and soft segments. A higher hard segment content at the surface contributed significantly to an increase in biostability. The findings provided an enhanced understanding for the role of surface molecular structure in the enzyme catalyzed biodegradation of polyurethanes.     

Sulfur‐Containing Polyesters, 9. The Synthesis and Characterization of New Diphenylmethane‐Derivative Thiopolyester Diols and Their Use for the Preparation of Thermoplastic Polyurethanes

The new linear thiopolyester diols (PEs) containing sulfur in the main chain were prepared by melt polymerization of newly obtained diphenylmethane‐4,4′‐bis(methylthiopropionic acid) with excess of 1,4‐butanediol, 1,5‐pentanediol, and 1,6‐hexanediol. All these PEs (M̄_n of ≈2 000) were converted to thiopoly(ester‐urethane)s (PEUs) by addition reaction with hexamethylene diisocyanate (HDI) or 4,4′‐diphenylmethane diisocyanate (MDI) which was carried out in melt at a ratio of NCO/OH = 1 or 1.05. The resulting thermoplastic PEUs were elastomeric products, completely amorphous from MDI, with glass transition temperatures ranging from –22 to –8°C. Segmented PEUs (hard segment content ≈53–60 wt.‐%) prepared by using PEs, HDI or MDI, and bis[4‐(6′‐hydroxyhexylthio)phenyl] ether as chain extender were elastomeric materials, particularly those from MDI, with tensile strength of 9.4–12.7 MPa. All the polymers were thermally stable up to 200–270°C. The structures of the polymers were determined by Fourier transform infrared, ¹H NMR, and X‐ray analysis.     

Polyurethanes containing binaphthyl groups

Polyurethanes containing binaphthyl groups were synthesized by reaction of diisocyanates – 4,4'-diphenylmethane diisocyanate (MDI), 2,4-toluylene diisocyanate (TDI) and hexamethylene diisocyanate (HDI) – with 2,2'-(1,1'-binaphthyl-4,4'-ylenedioxy)diethanol ( 3 ). The polyurethane obtained from 3 with MDI has an intrinsic viscosity [η]⁹⁰_DMSO^90°C of 0,14 dl · g⁻¹ and a melting point of 244 – 260°C, that from 3 with TDI has an [η]^90°C_DMSO of 0,12 dl · g⁻¹ and a melting point of 181 – 191°C, and that from 3 with HDI has an [η]^90°C_DMSO of 0,11 dl · g⁻¹ and a melting point of 222 – 241°C. These polymers were found to be crystalline. Segmented polyurethanes (SPU) from 3 with prepolymers of poly(tetramethylene glycol) (PTG), (M = 1079) and MDI or HDI (mole ratio PTG:MDI (or HDI) = 1:2) were also synthesized. The SPU from MDI has an intrinsic viscosity [η]^90°C_DMSO of 0,56 dl · g⁻¹ and a melting point of 260–275°C, and that from HDI has an [η]^90°C_DMSO of 0,50 dl · g⁻¹ and a melting point of 231 – 253°C. These polymers show slight crystallinity.     

Combined alveolitis and asthma due to hexamethylene diisocyanate (HDI), with demonstration of crossed respiratory and immunologic reactivities to diphenylmethane diisocyanate (MDI)

A worker exposed intermittently to hexamethylene diisocyanate (HDI) developed episodes of dyspnea, wheezing, and fever on working days. Complete lung function tests performed when the subject was asymptomatic were normal except for increased airway responsiveness to histamine, which significantly improved after a 3 wk period off work. At that time, specific inhalation challenges with HDI were carried out. After being exposed for 5 min, the subject developed general malaise, cough, fever, and leukocytosis, together with a mixed restrictive and obstructive breathing defect. We demonstrated a subsequent increase in airway hyperexcitability, which lasted for 2 mo. The subject was also challenged with diphenylmethane diisocyanate (MDI) for 15 min. A late obstructive reaction was documented. Increased levels of specific IgG antibodies against HDI-human serum albumin (HSA) and MDI-HSA were demonstrated.     

Generation of cell adhesive substrates using peptide fluoralkyl surface modifiers

Previous studies reported on the delivery of vitamin E to the surface of a polycarbonate polyurethane (PCNU) to produce antioxidant surfaces, using a bioactive fluorinated surface modifer (BFSM). In the current report, a cell adhesive peptide sequence was coupled to the BFSM, and when blended into PCNU, generated a cell adhesive substrate. An NH2-GK*GRGD-CONH2 peptide sequence (referred to as RGD) with a dansyl label (*) on the lysine residue was coupled via the N-terminal to a BFSM precursor molecule. The resulting RGD BFSM was purified and the pmol peptide/mg BFSM value was assayed by amino acid quantification. The migration of the RGD BFSM in a PCNU blend was confirmed by X-ray photoelectron spectroscopy analysis. U937 macrophage-like cells and human monocytes were seeded onto the PCNU and blends of PCNU with non-bioactive fluorinated surface modifier or the RGD BFSM, in order to study the cell response. Both U937 cells and human monocytes adhered in greater numbers to the RGD BFSM substrate when compared to unmodified PCNU or the blend of PCNU with the non-bioactive fluorinated surface modifying macromolecule substrate. The study demonstrated a novel approach for the introduction of peptides onto the surface of polymers by modifying the surface from within the polymer as opposed to the use of cumbersome post-surface modification techniques. The generation of a peptide substrate points to the possibility of producing complex bioactive surfaces using various peptide BFSMs or pharmaceuticals simultaneously to manipulate cell functions.