Matthew D. Phaneuf

Evaluation of a novel hirudin-coated polyester graft to physiologic flow conditions: Hirudin bioavailability and thrombin uptake

PURPOSE Our laboratory has developed methods required to covalently bind recombinant hirudin (rHir) to the surface of polyester vascular grafts. Using alkaline hydrolysis of the polyester surface, carboxyl-binding sites are created on the outer periphery of each fiber. A series of static, in vitro experiments have demonstrated that surface-bound rHir rapidly removes and inhibits activated human alpha-thrombin from the reaction system; however, the performance of this modified graft material under physiologic flow conditions was undefined. METHODS An in vitro flow loop was used to evaluate structural stability of the 125I-rHir and 131I-albumin covalently bound to the surface of 6 mm interior diameter crimped polyester grafts exposed to either constant flow (n = 4; shear rate, 300 sec(-1)) or pulsatile flow (n = 4; maximum shear rate, 780 sec(-1)) conditions for a 7-day period. In a separate series of experiments, the kinetics of thrombin-rHir interaction were evaluated through perfusion of 125I-rHir-coated grafts (n = 6) with 131I-thrombin for a 27-hour period under constant flow conditions. Identically prepared 125I-albumin-coated grafts (n = 3) were used as controls. RESULTS Results of the stability experiments were independent of flow conditions, demonstrating moderate loss of both proteins, with rHir and albumin losses of 52.1% and 19.9% under constant flow and 49.1% and 21.6% under pulsatile flow, respectively. With results comparable with those of previous static experiments, rHir-coated grafts were significantly more effective at removing thrombin from the perfusion stream with 131I-thrombin binding densities of 3.08 +/- 0.61 and 0.64 +/- 0.04 NIHU/cm2 (p < 0.01) for rHir-coated and albumin-coated grafts, respectively. Estimates of the total amount of thrombin inactivated during the perfusion period similarly demonstrated a marked difference between the rHir-coated and control graft segments (125 +/- 8 vs. 3 +/- 14 NIHU; p < 0.005). CONCLUSIONS These in vitro flow results illustrate that polyester grafts with covalently bound rHir can provide significant reductions in local thrombin concentration under physiologic flow conditions, and can serve as a foundation with which to understand the performance of these grafts when implanted in vivo under physiologic flow and shear rates.

Isolation of genes differentially expressed at the downstream anastomosis of prosthetic arterial grafts with use of mRNA differential display

PURPOSE Downstream anastomotic intimal hyperplasia in prosthetic arterial grafts remains a major cause of delayed graft failure. The new method of messenger RNA (mRNA) differential display was used to screen numerous genes to gain insight into the molecular mechanisms of intimal hyperplasia. METHODS Fifty-centimeter-long 8 mm expanded polytetrafluoroethylene grafts were placed in four mongrel dogs from the carotid artery to the distal abdominal aorta. At 3 months the distal anastomoses and adjacent normal aortas were harvested; a portion was taken for histologic examination, and total RNA was isolated from the remainder. Differential mRNA display was used to identify candidate cDNA clones whose expression differed in anastomotic intimal hyperplasia as compared with adjacent unaffected aorta. The clones were sequenced, and national gene databases were searched. Northern blot analysis confirmed alteration of gene expression. RESULTS Approximately 5000 mRNA species were screened, and 11 candidate clones were obtained. DNA sequence revealed homology of five clones to known gene sequences. Homologous genes included an interferon-gamma-induced human gene, (IGUP I-5111), alpha-1 protease inhibitor gene, human retinoblastoma susceptibility gene, and human creatine kinase gene (two clones). Northern blot analysis revealed altered gene expression in 4 of 11, nonregulation in 1 of 11, and undetectable signals in 6 of 11. Expression of the clone representing IGUP I-5111 in the segment of intimal hyperplasia was found to be decreased over threefold to only 31% +/- 16.4% SE of the level seen in normal aorta. CONCLUSIONS The technique of mRNA differential display has identified differences in gene expression in an in vivo model of anastomotic intimal hyperplasia. Expression of RNA with homology to an interferon-gamma-induced human gene was consistently decreased within the hyperplastic region at the downstream polytetrafluoroethylene arterial anastomosis.

Gene silencing in human aortic smooth muscle cells induced by PEI-siRNA complexes released from dip-coated electrospun poly(ethylene terephthalate) grafts.

An excessive tissue response to prosthetic arterial graft material leads to intimal hyperplasia (IH), the leading cause of late graft failure. Seroma and abnormal capsule formation may also occur after prosthetic material implantation. The matricellular protein Thrombospondin-2 (TSP-2) has shown to be upregulated in response to biomaterial implantation. This study evaluates the uptake and release of small interfering RNA (siRNA) from unmodified and surface functionalized electrospun PET graft materials. ePET graft materials were synthesized using electrospinning technology. Subsets of the ePET materials were then chemically modified to create surface functional groups. Unmodified and surface-modified ePET grafts were dip-coated in siRNAs alone or siRNAs complexed with transfection reagents polyethyleneimine (PEI) or Lipofectamine RNAiMax. Further, control and TSP-2 siRNA-PEI complex treated ePET samples were placed onto a confluent layer of human aortic smooth muscle cells (AoSMCs). Complexation of all siRNAs with PEI led to a significant increase in adsorption to unmodified ePET. TSP-2 siRNA-PEI released from unmodified-ePET silenced TSP-2 in AoSMC. Regardless of the siRNA-PEI complex evaluated, AoSMC migrated into the ePET. siRNA-PEI complexes delivered to AoSMC from dip-coated ePET can result in gene knockdown. This methodology for siRNA delivery may improve the tissue response to vascular and other prosthetics.

Antibiotic Treatment of Silk to Produce Novel Infection-Resistant Biomaterials

Dye-like applications of antibiotics to silk produce infection-resistant materials for potential use in biomedical applications. Two antibiotics, doxycycline (doxy) and cipro floxacin (cipro), are applied under a variety of conditions to silk and to silk that has previously been hydrolyzed at 40°C for 20, 40, and 60 minutes. FTIR spectroscopic analyses indicate that the drastically increased sorption of antibiotics by hydrolyzed silk is attributable to both chemical and conformational changes that occur with the hydrolysis. The high sorption of doxy by hydrolyzed silk does not necessarily yield a more infection- resistant material, as determined by a zone of inhibition test. Conversely, the same hydrolysis considerably increases both the sorption of cipro and the zone of inhibition of cipro-treated silk dyed at 65 and 85°C.

Fibronectin adsorption on functionalized electrospun polycaprolactone scaffolds: Experimental and molecular dynamics studies

Designing scaffolds to modulate protein adsorption is a key to building advanced scaffolds for tissue regeneration. Protein adsorption to tissue engineering scaffolds is critical in early cell attachment, survival, and eventual proliferation. The goal of this study is to examine the effect of functionalization on fibronectin adsorption to electrospun polycaprolactone (PCL) scaffolds through experimentation using fluorescently labeled fibronectin and to couple this experimental data with analysis of interaction energies obtained through molecular dynamics (MD) simulations to develop a better understanding of the adsorption process. This study is the first to analyze and compare experimental and MD simulation results of fibronectin adsorption on functionalized electrospun PCL scaffolds. Electrospun nanofiber PCL scaffolds were treated with either 1 N NaOH (hydrolyzed) or 46% hexamethylenediamine (HMD) (aminated) solution to be compared with untreated (control) scaffolds. We found that aminated PCL scaffolds experimentally adsorbed more fibronectin than control scaffolds, whereas hydrolyzed scaffolds showed decreased adsorption. MD simulations carried out with NVT ensemble at a temperature of 310 K indicated a higher work of adhesion for both functionalized scaffolds over control. Also, the simulations revealed different conformations of fibronectin on each scaffold type after adsorption, with the arginine-glycine-aspartic acid sequence appearing most accessible on the aminated scaffolds. This suggests that functionalization affects not only the quantity of protein that will adsorb on a scaffold but how it attaches as well, which could affect subsequent cell attachment.

Glycoconjugate mediated endothelial cell adhesion to Dacron polyester film

PURPOSE The purpose of this study was to explore new strategies for enhancing specific cell type attachment to biomaterials using immobilized lectins for cell surface glycoconjugates. The lectin Ulex europaeus I (UEA I) has a high affinity for human vascular endothelial cell surface glycoconjugates. METHODS UEA I was covalently bound to polyethylene terephthalate (Dacron) with the cross-linking agent 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride to achieve oligosaccharide-mediated endothelial cell attachment to this otherwise nonadherent surface. RESULTS Experiments with radiolabeled UEA I demonstrated covalent linkage of as much as 1.35 micrograms/cm2. The lectin binding site is available after the reaction, as demonstrated in experiments a neoglycoprotein. Adhesion studies reveal a 100-fold increase in endothelial cell attachment for the UEA I/polyethylene terephthalate surface (99.7 +/- 29.6 cells/high-power field) when compared with untreated (0.7 +/- 0.5), crosslinking agent (0.4 +/- 0.3), and denatured UEA I (1.2 +/- 1.1) control groups. Five vascular endothelial cell lines adhered to the UEA I/polyethylene terephthalate surface, whereas monocytes, smooth muscle cells, and fibroblasts did not. CONCLUSION These results imply new strategies for endothelialization of prosthetic grafts and promotion of selective cell adherence to biomaterials, with emphasis on carbohydrate interactions. Moreover, this experimental system offers a model for exploring the biologic significance of the endothelial cell-UEA I ligand.

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.

Identification of multiple genes with altered expression at the distal anastomosis of healing polytetrafluoroethylene grafts

PURPOSE Anastomotic intimal hyperplasia remains a significant cause of delayed prosthetic arterial graft failure. Prior studies have identified several genes with altered expression within the hyperplastic region at the downstream polytetrafluoroethylene arterial anastomosis as compared with normal arteries. The purpose of the current study was to determine the sequence of early gene-related events at the distal anastomosis of an in vivo prosthetic arterial graft model. Messenger RNA (mRNA) differential display was used to screen for alterations in gene expression between anastomotic sites and control arterial segments. METHODS Six carotid interposition 6-mm expanded polytetrafluoroethylene grafts were placed in mongrel dogs, with the intervening carotid artery segment serving as the baseline control. Five days after graft implantation, the distal anastomotic artery segments were harvested and total RNA was isolated from both the intervening normal arteries and anastomotic segments. Differential mRNA display was used to identify candidate complementary DNA (cDNA) clones with expression that differed in anastomotic segments as compared with normal intervening arteries. Northern blot analysis confirmed alteration of gene expression. The cDNA clones were sequenced, and gene databases were searched. Novel sequences were used as probes for screening human cDNA libraries. RESULTS Approximately 7000 mRNA species were screened, and 26 candidate clones were obtained. Northern blot analysis showed altered gene expression in 10 (38%) of the clones, undetectable signals in 13 (50%), and nonregulation in 3 (12%). Seven clones with 92% homology at the nucleotide level to human alpha1 (III) procollagen gene and novel sequence were expressed only at the distal anastomosis. A clone representing apolipoprotein J and a novel sequence had increased expression at the distal anastomosis of 364% +/- 236% and 156% +/- 47%, respectively (mean percentage, control +/- standard deviation). CONCLUSIONS These studies identified genes with expressions that increased or were exclusive to the distal anastomosis of healing prosthetic arterial grafts in an in vivo prosthetic arterial graft model. Type III collagen may contribute significantly to the composition of the extracellular matrix associated with intimal hyperplasia by increasing lesion volume. Apolipoprotein J, through its association with proteases, may modulate some of the matrix changes seen early after grafting.

Development of a composite electrospun polyethylene terephthalate-polyglycolic acid material: potential use as a drug-eluting vascular graft

Intimal hyperplasia (IH), an excessive wound healing response of an injured vessel wall after bypass grafting, typically leads to prosthetic bypass graft failure. In an approach to ameliorate IH, nondegradable poly(ethylene terephthalate) or PET, which has been used in prosthetic vascular grafts for over 60 years, and biodegradable poly(glycolic acid) or PGA were electrospun using different techniques to generate a material that may serve as permanent scaffold and as a drug/biologic delivery device. PET and PGA polymers were electrospun from either a single-blended solution (ePET/ePGA-s) or two separate polymer solutions (ePET/ePGA-d). ePET/ePGA-d material revealed two distinct fibers and was significantly stronger than the single fiber ePET/ePGA-s material. After 21 days of incubation in PBS, ePET-PGA-s showed fiber strand breaks likely due to the degradation of the PGA within the ePET-ePGA-s fiber, while the ePET/ePGA-d material showed intact ePET fibers even after ePGA fiber degradation. The ePET/ePGA- material was able to release red fluorescent dye for at least 14 days. Attachment of human aortic smooth muscle cells (AoSMCs) was similar to both materials. ePET/ePGA-d materials maybe a step towards bypass graft materials that can be custom-designed to promote cellular attachment while serving as a drug delivery platform for IH prevention.

Cytotoxicity associated with electrospun polyvinyl alcohol

Polyvinyl alcohol (PVA) is a synthetic, water-soluble polymer, with applications in industries ranging from textiles to biomedical devices. Research on electrospinning of PVA has been targeted toward optimizing or finding novel applications in the biomedical field. However, the effects of electrospinning on PVA biocompatibility have not been thoroughly evaluated. In this study, the cytotoxicity of electrospun PVA (nPVA) which was not crosslinked after electrospinning was assessed. PVA polymers of several molecular weights were dissolved in distilled water and electrospun using the same parameters. Electrospun PVA materials with varying molecular weights were then dissolved in tissue culture medium and directly compared against solutions of nonelectrospun PVA polymer in human coronary artery smooth muscle cells and human coronary artery endothelial cells cultures. All nPVA solutions were cytotoxic at a threshold molar concentration that correlated with the molecular weight of the starting PVA polymer. In contrast, none of the nonelectrospun PVA solutions caused any cytotoxicity, regardless of their concentration in the cell culture. Evaluation of the nPVA material by differential scanning calorimetry confirmed that polymer degradation had occurred after electrospinning. To elucidate the identity of the nPVA component that caused cytotoxicity, nPVA materials were dissolved, fractionated using size exclusion columns, and the different fractions were added to HCASMC and human coronary artery endothelial cells cultures. These studies indicated that the cytotoxic component of the different nPVA solutions were present in the low-molecular-weight fraction. Additionally, the amount of PVA present in the 3-10 kg/mol fraction was approximately sixfold greater than that in the nonelectrospun samples. In conclusion, electrospinning of PVA resulted in small-molecular-weight fractions that were cytotoxic to cells. This result demonstrates that biocompatibility of electrospun biodegradable polymers should not be assumed on the basis of success of their nonelectrospun predecessors.