Development of infection resistant polyurethane biomaterials using textile dyeing technology.

Infection is a major complication when using biomaterials such as polyurethane in the clinical setting. The purpose of this study was to develop a novel infection resistant polyurethane biomaterial using textile dyeing technology. This procedure results in incorporation of the antibiotic into the polymer, resulting in a slow, sustained release of antibiotic from the material over time, without the use of exogenous binder agents. Polycarbonate based urethanes were synthesized that contained either a non-ionic (bdPU) or anionic (cPU) chain extender within the polymer backbone and cast into films. The fluoroquinolone antibiotic ciprofloxacin (Cipro) was applied to bdPU and cPU using textile dyeing technology, with Cipro uptake determined by absorbance reduction of the "dyebath." These dyed bdPU/cPU samples were then evaluated for prolonged Cipro release and antimicrobial activity by means of spectrophotometric and zone of inhibition assays, respectively. Cipro release and antimicrobial activity by dyed cPU segments that were aggressively washed persisted over 9 days, compared with dyed bdPU and dipped cPU control segments that lasted < 24 hours. Dyed cPU segments, which remained in a static wash solution, maintained antimicrobial activity for 11 days (length of study), whereas controls again lost antimicrobial activity within 24 hours. Thus, application of Cipro to the cPU polymer by means of dyeing technology results in a slow sustained release of antibiotic with persistent bacteriocidal properties over extended periods of time.

Coating of Dacron vascular grafts with an ionic polyurethane: a novel sealant with protein binding properties.

The purpose of this study was to develop a novel sealant that would seal prosthetic vascular graft interstices and be accessible for protein binding. Crimped knitted Dacron vascular grafts were cleaned (CNTRL) and hydrolyzed in boiling sodium hydroxide (HYD). These HYD grafts were sealed using an 11% solids solution of a polyether-based urethane with carboxylic acid groups (PEU-D) via a novel technique that employs both trans-wall and luminal perfusion. Carboxylic acid content, determined via methylene blue dye uptake, was 2.3- and 4.2-fold greater in PEU-D segments (1.0+/-0.27 nmol/mg) as compared to HYD and CNTRL segments, respectively. Water permeation through PEU-D graft (1.1+/-2 ml/cm2 min(-1)) was comparable to collagen-impregnated Dacron (9.8+/-10 ml/cm2 min(-1)). Non-specific 125I-albumin (125I-Alb) binding to PEU-D segments (18+/-3 ng/mg) was significantly lower than HYD and CNTRL segments. 125I-Alb linkage to PEU-D using the crosslinker EDC resulted in 5.7-fold greater binding (103+/-2 ng/mg) than non-specific PEU-D controls. However, covalent linkage of 125I-Alb to PEU-D was 4.9- and 5.9-fold less than CNTRL and HYD segments with EDC, respectively. Thus, ionic polyurethane can be applied to a pre-formed vascular graft, seal the interstices and create "anchor" sites for protein attachment.

Synthesis of a novel small diameter polyurethane vascular graft with reactive binding sites.

Development of a small diameter (4 mm inner diameter [ID]) prosthetic vascular graft with functional groups accessible for covalent binding of recombinant hirudin (a potent anticoagulant) should create a more hemocompatible surface. The purpose of this study was to develop a technique for generating carboxylic acid groups on the surface of precast 4 mm ID poly-(carbonate urea)-urethane vascular grafts and to evaluate the accessibility of these groups. A polycarbonate based urethane with the chain extender 2,2-bis(hydroxymethyl)propionic acid was synthesized. A precast 4 mm ID poly(carbonate urea)-urethane vascular graft (Chronoflex [CF]; CardioTech International, Woburn, MA) was then placed into a 4% carboxylated polyurethane (cPU) solution (in 1% dimethyl acetamide) and incubated for 30 minutes (cPU graft). To determine the accessibility of the carboxylic acid groups, a standard textile technique using methylene blue dye was used. Macroscopic cross-sections, which were cut and evaluated for dye penetration, showed greatest concentration of carboxylic acid groups at the luminal and capsule surfaces, with minimal penetration into the mid-portion of the graft. Analysis of dye baths for absorbance reduction resulted in the cPU grafts having 3.7-fold and 5.4-fold more accessible carboxylic acid groups compared with untreated and dimethyl acetamide dipped CF grafts. Thus, a novel small diameter vascular graft has been developed that contains reactive carboxylic acid groups accessible for protein binding.

Bioengineering of a novel small diameter polyurethane vascular graft with covalently bound recombinant hirudin.

Development of a small diameter prosthetic vascular graft with surface based antithrombin properties should aid in maintaining early graft patency in small vessel reconstruction. The purpose of this study was to bind covalently a basecoat protein (canine serum albumin [CSAJ) and a potent antithrombin agent (recombinant hirudin [rHir]) to 4 mm inner diameter poly(carbonate urea) urethane grafts with reactive carboxylic acid groups (cPU). 125I-CSA was covalently bound to 1 cm length segments of cPU grafts using the carbodimide cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). To bind 125I-rHir covalently, CSA was modified with the heterobifunctional cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) before linkage to the cPU surface with EDC (cPU-CSA-SMCC). 125I-rHir was modified with Traut's reagent and reacted with the cPU-CSA-SMCC surface, covalently linking 125I-rHir to surface bound CSA. 125I-CSA binding to the cPU graft surface (34,235 ng/segment) was ninefold, sevenfold, and 10-fold greater than controls with nonspecifically bound 125I-CSA. Covalent linkage of 125I-rHir to the cPU-CSA-SMCC surface (9,974 ng/segment) was 172, 192, and 142-fold greater than controls with nonspecifically bound 125I-rHir. Surface antithrombin properties were characterized using a chromogenic assay to measure residual thrombin activity. Evaluation of surface antithrombin activity showed significantly greater 131I-thrombin inhibition and binding by the cPU surface with covalently bound 125I-rHir, as compared with controls. Release of 125I-rHir from the cPU surface was minimal as compared with controls. Therefore, rHir can be covalently linked to a novel small diameter polyurethane vascular graft surface while maintaining its potent antithrombin properties.

Chemical and physical characterization of a novel poly(carbonate urea) urethane surface with protein crosslinker sites.

A major complication which occurs with implantable polyurethane biomaterials is bioincompatibility between blood and the biomaterial surface. Development of a novel biodurable polyurethane surface to which biological agents, such as growth factors or anticoagulants could be covalently bound, would be beneficial. The purpose of this study was to synthesize a novel poly(carbonate urea) urethane polymer with carboxylic acid groups which would serve as "anchor" sites for protein attachment. Physical characteristics such as tensile strength, initial modulus, ultimate elongation, tear strength, water/alcohol uptake and water vapor permeation were then evaluated and compared to other biomedical-grade polyurethanes. Covalent linkage of the blood protein albumin to this novel surface was then examined. A biodurable polycarbonate-based polyurethane containing carboxylic acid groups (cPU) was synthesized using a two step procedure incorporating the chain extender 2,2-bis(hydroxymethyl)-propionic acid (DHMPA). Tensile strength of this cPU film was 2.7 and 2.6 fold greater than both a polycarbonate-based polyurethane synthesized with a 1,4-butanediol chain extender (bdPU) and Mitrathane (Mit) controls, respectively. The cPU polymer also possessed 7.8 and 31 fold greater structural rigidity upon evaluation of initial modulus as compared to the bdPU and Mit, respectively. Ultimate elongation for the bdPU films was slightly higher than the cPU and Mit films, which had comparable elongation properties. The force required to tear the bdPU film was 1.9 and 32 fold greater than the cPU and Mit films, respectively. Alcohol solution uptake by all of the polyurethane segments increased with increasing alcohol concentrations, with the cPU having the greatest uptake. Water uptake was minimal for all the polyurethanes examined and was not affected by altering pH. Water vapor permeation was lowest for the cPU films as compared to both bdPU and Mit. Swelling the cPU in 50% ethanol prior to evaluation slightly increased water vapor permeation through the films. Covalent linkage of the radiolabelled blood protein albumin (125I-BSA) to the cPU segments incubated with the heterobifunctional crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was greatest in the higher percent of ethanol as compared to controls. These results serve as foundation for developing a novel poly(carbonate urea) urethane with physical characteristics comparable to other medical-grade polyurethanes while having protein binding capabilities.

Covalent linkage of recombinant hirudin to poly(ethylene terephthalate) (Dacron): creation of a novel antithrombin surface.

Thrombus formation and intimal hyperplasia on the surface of implantable biomaterials such as poly(ethylene terepthalate) (Dacron) vascular grafts are major concerns when utilizing these materials in the clinical setting. Thrombin, a pivotal enzyme in the blood coagulation cascade primarily responsible for thrombus formation and smooth muscle cell activation, has been the target of numerous strategies to prevent this phenomenon from occurring. The purpose of this study was to covalently immobilize the potent, specific antithrombin agent recombinant hirudin (rHir) to a modified Dacron surface and characterize the in vitro efficacy of thrombin inhibition by this novel biomaterial surface. Bovine serum albumin (BSA), which was selected as the "basecoat' protein, was reacted with various molar ratios of the cross-linker sulphosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulpho-SMCC; 1:5-1:50). These BSA-SMCC complexes were then covalently linked to sodium hydroxide-hydrolysed Dacron (HD) segments via the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Covalent linkage of these complexes to HD (HD-BSA-SMCC) was not affected by any of the sulpho-SMCC cross-linker ratios assayed. rHir, which was initially reacted with 2-iminothiolane hydrochloride (Traut's reagent) in order to create sulphydryl groups, was then covalently bound to these HD-BSA-SMCC surfaces (HD-BSA-SMCC-S-rHir). The 1:50 (BSA: sulpho-SMCC) HD-BSA-SMCC-S-rHir segments bound 22-fold more rHir (111 ng per mg Dacron) compared to control segments and also possessed the greatest thrombin inhibition of the segments evaluated using a chromogenic substrate assay for thrombin. Further characterization of the HD-BSA-SMCC-S-rHir segments demonstrated that maximum thrombin inhibition was 20.43 NIHU, 14.6-fold greater inhibition than control segments (1.4 NIHU). Thrombin inhibition results were confirmed by 125I-thrombin binding experiments, which demonstrated that the 1:50 HD-BSA-SMCC-S-rHir segments had significantly greater specific thrombin adhesion compared to control segments. Non-specific 125I-thrombin binding to and release from the 1:50 HD-BSA-SMCC-S-rHir segments was also significantly less than the control segments. Thus, these results demonstrate that rHir can be covalently bound to a clinically utilized biomaterial (Dacron) while still maintaining its ability to bind and inhibit thrombin.

Modification of polyethylene terephthalate (Dacron) via denier reduction: effects on material tensile strength, weight, and protein binding capabilities.

Thrombosis remains a significant and potentially catastrophic complication of polyethylene terephthalate (Dacron) prosthetic vascular graft implantation. Numerous attempts have been made to create a novel surface that reduces the adverse effects of blood interaction with the material. The purpose of this study was to create reactive groups on Dacron without significantly altering the chemical and physical properties of the biomaterial. These groups would then serve as "anchor sites" for covalent attachment of the blood protein albumin to the surface, thus creating a more biocompatible surface. Denier reduction, an established textile chemistry procedure that creates carboxyl groups on the fiber surface via hydrolysis of the material, was performed at 100 degrees C using sodium hydroxide concentrations of 0.5, 1.0, 2.5, and 5.0% (treated materials referred to as 0.5% hydrolyzed etc.). Tensile strength determination of hydrolyzed materials revealed no statistically significant difference in material strength between control, 0.5, and 1.0% hydrolyzed materials; the 2.5 and 5.0% hydrolyzed materials had significant strength loss as compared to the controls. Significant fiber weight loss occurred in the 1.0, 2.5, and 5.0% hydrolyzed Dacron segments. The 0.5% hydrolyzed material did not have any significant weight loss. Covalent linkage of 125I-albumin to these modified materials using the crosslinker 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide hydrochloride (EDC) resulted in the 0.5% hydrolyzed material having the greatest protein binding (330 ng/mg Dacron, 2,4-fold greater than control). Incubation of the 0.5% hydrolyzed material with EDC and various concentrations of 125I-albumin resulted in the 14.80 microM solution permitting the greatest binding per milligram Dacron (330 ng/mg Dacron). Scanning electron microscopy, performed blindly, revealed no change in the 0.5% hydrolyzed Dacron as compared to untreated Dacron. The 5.0% hydrolyzed Dacron, however, had noticeable structural damage on the outer periphery of the fiber surface. Observation of the untreated Dacron with nonspecifically bound albumin showed scattered areas of albumin adherent to the fiber surface whereas covalent linkage of albumin to the 0.5% hydrolyzed Dacron via EDC crosslinking showed numerous albumin moieties on each fiber. This study demonstrates that a clinically accepted biomaterial (Dacron) can be chemically modified, without significantly altering the physical and chemical characteristics of the biomaterial, in order to covalently bind albumin to the fiber surface. Thus, these results serve as foundation for creating potential novel biomaterials without significantly altering the properties of the original biomaterial.

In vivo testing of an infection-resistant vascular graft material.

Present prosthetic arterial conduits continue to suffer the clinically and economically catastrophic complication of infection. We recently described a novel technique for binding quinolone antibiotics to Dacron based on principles of textile chemistry. This thermofixation procedure ("pad/heat") utilizes the limited fibrophilic characteristics of the quinolone antibiotic ciprofloxacin (Cipro) to permit pad/heat application and allowed controlled, sustained release from Dacron in several in vitro assays. The objective of this study was to test this infection-resistant prosthetic vascular graft material in an in vivo model. Dacron segments (1 cm2, either plain, dipped into antibiotic immediately prior to implantation, or Cipro pad/heat treated) were implanted in the dorsal subcutaneous tissue of the rabbit and directly contaminated with 10(6) Staphylococcus aureus. After 1 week, the samples were sterily harvested. Wounds were blindly graded on a scale from 1 (no evidence of infection, good tissue incorporation) to 4 (suppurative infection extending outside of the graft pocket, no gross tissue incorporation). Plain Dacron was easily infected in this model (mean grade 3.1 +/- 0.6, 92% culture positive). Notably, however, a significant (P < 0.05) wound grade difference between the dipped (2.3 +/- 1.0) and pad/heat (1.4 +/- 0.6) samples was demonstrated. Determination of adherent bacteria present on the implanted Dacron pieces by sonication and culture studies again revealed a significant difference between the dipped (56% culture positive) and pad/heat (12% culture) groups (P < 0.025). Histologic studies confirmed good tissue incorporation of the pad/heat samples. This project opens new avenues in the development of infection-resistant biomaterials.

Application of the quinolone antibiotic ciprofloxacin to Dacron utilizing textile dyeing technology.

Prosthetic arterial graft infection continues to be a significant and often devastating complication of vascular surgery. The organisms Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) are the primary pathogens causing acute and late graft infections, respectively. The objective of this study was to develop an infection-resistant prosthetic arterial graft by applying the bacteriocidal quinolone antibiotic ciprofloxacin to polyethylene terepthalate (Dacron) via thermofixation (pad/heat), a new application method founded on established textile procedures. We hypothesize that the limited fibrophilic characteristics of ciprofloxacin will permit binding to Dacron and at the same time allow persistent controlled release over an extended period of time. Using pad/heat technology, 33 micrograms (+/- 2.97 micrograms, n = 12) of ciprofloxacin was successfully bound to a 1-cm2 piece of woven Dacron. A full complement of microbiologic assays demonstrated superior, sustained antistaphylococcal activity of the pad/heat Dacron when compared to Dacron dipped into an equivalent concentration of ciprofloxacin. The sustained antimicrobial efficacy of ciprofloxacin pad/heat-treated Dacron opens new avenues in the development of infection-resistant biomaterials based on an understanding of textile chemistry.