Rosalind S. Labow

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.

Synthesis of surface-modifying macromolecules for use in segmented polyurethanes

Polyurethanes are one of the most important classes of thermoplastic elastomers and have been widely used in medical-device manufacturing as well as in other applications. However, their function can be limited, particularly under environmental conditions that render them susceptible to hydrolysis. Using polymeric additives that are hydrolytically stable may be one approach to modifying the surface of polyurethanes for the purpose of improving their hydrolytic resistance without compromising their structural features. In this paper, the development of a series of novel fluorine-containing polyurethane surface modifying macromolecules (SMMs) is described and their synthesis conditions are investigated. The material structure and mixing properties of the synthesized SMMs with base polyurethanes was dependent on the reactant stoichiometry and concentration for the SMM components, as well as the reaction temperature and the amount of catalyst used in the SMM synthesis. This study describes the use of low surface energy components (fluorinated tails) which showed selective migration towards the surface when added to a polyester-urea-urethane. These novel macromolecules generated a nonwettable surface while not significantly altering the apparent bulk structure of the base polymer. The advancing and receding contact angle results indicated that the surface of these modified polyurethanes showed wettability characteristics similar to that of Teflon. TM The differential scanning calorimetry thermograms for the mixtures of the SMM with the polyurethane showed that, at 5% w/w SMM in the base polyurethane, the thermal transitions were similar to that of the native base polyure-thane, indicating that the additives had no detectable effect on the polyurethane structure.

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.

Biodegradation and in vivo biocompatibility of a degradable, polar/hydrophobic/ionic polyurethane for tissue engineering applications

A degradable, polar/hydrophobic/ionic polyurethane (D-PHI) scaffold was optimized in in vitro studies to yield mechanical properties appropriate to replicate vascular graft tissue while eliciting a more wound-healing phenotype macrophage when compared to established materials. The objectives of this study were to characterize the biodegradation (in vitro and in vivo) and assess the in vivo biocompatibility of D-PHI, comparing it to a well-established, commercially-available scaffold biomaterial, polylactic glycolic acid (PLGA), recognized as being degradable, non-cytotoxic, and showing good biocompatibility. PLGA and D-PHI were formed into 6 mm diameter disk-shaped scaffolds (2 mm thick) of similar porosity (∼82%) and implanted subcutaneously in rats. Both PLGA and D-PHI scaffolds were well-tolerated at the 7 d time point in vivo. In vitro D-PHI scaffolds degraded slowly (only 12 wt% in PBS in vitro after 120 d at 37 °C). In vivo, D-PHI scaffolds degraded at a more controlled rate (7 wt% loss over the acute 7 d implant phase and subsequently a linear profile of degradation leading to a 21 wt% mass loss by 100 d (chronic period)) than PLGA scaffolds which showed an initial more rapid degradation (14 wt% over 7 d), followed by minimal change between 7 and 30 d, and then a very rapid breakdown of the scaffold over the next 60 d. Histological examination of D-PHI scaffolds showed tissue ingrowth into the pores increased with time whereas PLGA scaffolds excluded cells/tissue from its porous structure as it degraded. The results of this study suggest that D-PHI has promising qualities for use as an elastomeric scaffold material for soft TE applications yielding well integrated tissue within the scaffold and a controlled rate of degradation stabilizing the form and shape of the implant.

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.

The effect of hard segment size on the hydrolytic stability of polyether-urea-urethanes when exposed to cholesterol esterase

Previous studies have shown that both polyester and polyether-based polyurea-urethanes are susceptible to cleavage by hydrolytic enzymes. Furthermore, it has been hypothesized that the degree of hard segment micro-domain formation in polyurethane materials, as well as its structure, influences the ability of enzymes to degrade the polymers. The current study has investigated a series of segmented polyether-urea-urethanes synthesized with the same reagents but having different hard segment content. Using these materials, the relationship between the formation of hard segment domains and the hydrolysis of urea/urethane groups was specifically addressed. Both differential scanning calorimetry and X-ray photo-electron spectroscopy data indicated that the three materials differed significantly in the extent of hard segment domain formation and the nature of the chemical groups located in the top 10 nm of the surface. Biodegradation studies showed a strong dependence on hard segment domain formation and indicated that the polymer containing the highest number of hydrolytically labile urea and urethane bonds exhibited the least degradation. The ability of a polyurethane material to form hard segment micro-domains may contribute to the formation of a protective structure for the hydrolysable hard segment linkages located within the micro-domains.

Use of surface‐modifying macromolecules to enhance the biostability of segmented polyurethanes

Polyurethanes are widely used as biomaterials for medical implants because of their excellent mechanical properties and moderate biocompatibility. However, the demand for more bioresistant and biocompatible polyurethanes to meet the needs of long-term implant devices still remains an important issue. Since most biological interactions with materials occur at the interface, a significant number of studies for improving the biocompatibility of polyurethanes have concentrated on surface modification. It is well known that additives used in polymeric materials as processing aids, mold releasing agents, antioxidants, etc., migrate to the surface and change the surface properties of the material. Under certain conditions polymeric additives may also migrate toward surfaces. This study describes two fluorine-containing, surface-modifying macromolecules (SMMs) that have been evaluated for their ability to inhibit polyurethane degradation. These materials actively migrate to the upper surface of a material film when they are mixed with a base polymeric materia. Contact angle measurements for the mixture of SMM with base polyurethane indicate that the surface becomes more hydrophobic after adding the SMMs, while X-ray photoelectron spectroscopy analysis shows an enrichment of fluorine on the polymer surfaces. Differential scanning calorimetry thermograms indicate that the micro-structure, as defined by the thermal transitions of the base polymer, are not altered by the addition of SMMs. Enzyme-induced biodegradation tests exhibit a significant reduction of polyurethane degradation in the presence of these surface-resident materials. The results indicate that the SMMs have the potential to resist hydrolytic degradation mediated by lysosomal enzymes while generating a surface chemistry on the native elastomer which is similar in nature to that of a fluoropolymer, e.g., Teflon.

Model systems to assess the destructive potential of human neutrophils and monocyte-derived macrophages during the acute and chronic phases of inflammation

Isolated cell systems of human neutrophils (PMNs) and monocyte-derived macrophages (MDMs) were used to compare the destructive potential of these cells during the acute and chronic phases of inflammation, respectively. The contrast in the damage to poly(urethane)s (PUs) was monitored by measuring radiolabel release elicited from a (14)C-polyester-urea-urethane (PEUU) during incubation with both cell types. Human PMN were seeded onto polymer-coated glass slips and both radiolabel release as well as serine protease activity [assayed with N-benzyloxycarbonyl lysine thiobenzyl ester (BLT)] were measured 18 h later. Human monocytes were cultured on polystyrene tissue culture plates for 14 days, trypsinized, and seeded onto the polymer-coated glass slips; then, radiolabel release and esterase activity [assayed with p-nitrophenylbutyrate (PNB)] were measured after 18 h. Coverslips with MDM were also incubated for an additional 2 weeks. At 18 h postincubation with the PEUU, MDM elicited 25 times more radiolabel release per 10(6) cells than PMN at 18 h and continued to increase more than sevenfold over the 18-h value during the subsequent 14-day period. The BLT activity in PMN did not increase significantly during the 18-h incubation period, whereas the PNB activity in MDM increased more than fourfold. The MDM, but not the PMN elicited radiolabel release, was inhibited by the protein synthesis inhibitor cycloheximide, as was the increase in PNB activity. The data provide evidence for a hydrolytic role for MDM and, to a lesser extent PMN, in the biodegradation of implanted materials. The full implication of the release of polymer-derived chemical agents from this hydrolytic cleavage of the implanted biomaterials, on the propagation of the inflammatory response, remains to be elucidated.

Neutrophil-mediated degradation of segmented polyurethanes

The biostability of polyurethanes was evaluated using a human neutrophil cell culture. The polymers were synthesized with 14C radiolabelled components incorporated into the polyurethane chain and the amount of radiolabel released during exposure to cells and medium was used as a marker for material degradation. The effect of diisocyanate, soft segment and chain extender chemistry on the susceptibility of polymer degradation was examined. All polymers showed a release of material into the tissue culture medium which was unrelated to the cells. A significant cell-dependent release of radiolabel-containing material was found from one of the polymers (a polyester urea-urethane, TDI/PCL/ED) which increased linearly up to 96 h. The polyether-containing polyurethanes showed no significant cell-mediated degradation under similar conditions as measured by radiolabel release. Scanning electron microscopy (SEM) showed that the cells adhered to the different polyurethanes. However, no effect of neutrophils on polymer structure could be detected by this technique. The cellular response to each polymer was evaluated by measuring release of elastase-like activity (ELA) into the tissue culture media. After 24h TDI/PCL/ED showed the highest levels of ELA in the tissue culture medium. When TDI/PCL/ED was incubated with commercial elastase in vitro, a significant release of radiolabel was found which was comparable to the amount of radiolabelled material released from this polymer in contact with the neutrophils in culture. No significant amount of radiolabel was released from the corresponding polyether material (TDI/PTMO/ED) under similar conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

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.