To Compare the Effects of a Standard Versus Hydrophilic Polymer Coated Airway Stent in a Porcine Model: A Randomized, Single-Blinded Study.

BACKGROUND: Stent encrustation with debris and mucostasis is a significant cause of airway injury and comorbidity, leading to ~25% of stent exchanges (1-3). Previous work from our group has shown that the experimental coating can reduce mucous adhesion in bench testing and demonstrated a signal for reducing airway injury and mucostasis in a feasibility study. OBJECTIVES: The aim of this study is to continue our inquiry in a randomized, single-blinded multi-animal trial to investigate the degree of airway injury and mucostasis using silicone stents with and without this specialized coating. METHODS: We modified commercially available silicone stents with a hydrophilic polymer from Toray Industries. We conducted an in vivo survival study in 6 mainstem airways (3 coated and 3 uncoated) of 3 pigs to compare the degree of airway injury and mucostasis between coated versus noncoated stented airways. Both stents were randomized to either left or right mainstem bronchus. The pathologist was blinded to the stent type. RESULTS: We implanted a total of six 14×15 mm silicone stents (1 per mainstem bronchi) into 3 pigs. All animals survived to termination at 4 weeks. All stents were intact; however, 1 uncoated stent migrated out. On average, all the coated stents demonstrated reduced pathology and tissue injury scores (75 vs. 68.3, respectively). The average total dried mucous weight was slightly higher in the coated stents (0.07 g vs. 0.05 g; respectively). CONCLUSION: Coated stents had lower airway injury compared with uncoated stents in this study. Of all the stents, 1 uncoated stent migrated out and was not included in the dried mucous weight totals. This could explain the slightly higher mucous weight in the coated stents. Nevertheless, this current study demonstrates promising results in lowering airway injury in stents incorporated with the hydrophilic coating, and future studies, including a larger number of subjects, would be needed to corroborate our findings.

Small caliber heparin loaded ultrafine microfiber woven graft achieved high patency rate in a preliminary study of canine carotid artery implantation.

OBJECTIVE: In the past five decades, many small caliber vascular grafts have been developed as bypasses for infrapopliteal or coronary arteries. However, reliable grafts have not been obtained owing to poor patency, mainly caused by early thrombosis or neointimal hyperplasia in the intermediate period after implantation. We developed a novel small caliber heparin-loaded polyethylene terephthalate ultrafine microfiber (HL-PET) graft and evaluated the feasibility to overcome those main causes of graft failure in canine carotid artery implantation. METHODS: The HL-PET graft with a diameter of 3 mm and length of 30 mm was made with combination of three key technologies: (1) weaving with PET ultrafine microfiber with a high biological porosity allowing for cell ingrowth, (2) heparin loading on microfiber surfaces, and (3) an outer coating with a flexible bioabsorbable polymer for prevention of blood leakage and graft kinking. Kink resistance, water permeability, and loaded heparin were assessed. One HL-PET graft each was implanted into a carotid artery of six animals. Graft patency rate and healing were assessed 24 weeks after implantation. RESULTS: Among the six grafts, five were deemed patent (patency rate of >83%), with one occluded 20 weeks after implantation. Histopathology of the patent grafts showed neointima formation with confluent endothelial cell lining (estimated mean endothelial cell coverage area, 89 ± 18%). Intimal hyperplasia at the anastomotic sites and severe chronic inflammatory responses were not observed. Immunohistochemistry with antibodies to endothelial nitric oxide synthase, alpha 2 smooth muscle actin and calponin 1 revealed luminal surface endothelial cell layer with expression of endothelial nitric oxide synthase and vascular smooth muscle cells with contractile phenotype in the subintimal layer. CONCLUSIONS: The HL-PET graft showed no early postoperative thrombosis and was able to demonstrate a high patency rate with no severe biological response observed after 24 weeks. These results strongly suggest the potential of the HL-PET graft to be used for distal bypasses.

Synthesis of poly(propylene fumarate).

This protocol describes the synthesis of 500-4,000 Da poly(propylene fumarate) (PPF) by a two-step reaction of diethyl fumarate and propylene glycol through a bis(hydroxypropyl) fumarate diester intermediate. Purified PPF can be covalently cross-linked to form degradable polymer networks, which have been widely explored for biomedical applications. The properties of cross-linked PPF networks depend upon the molecular properties of the constituent polymer, such as the molecular weight. The purity of the reactants and the exclusion of water from the reaction system are of utmost importance in the generation of high-molecular-weight PPF products. Additionally, the reaction time and temperature influence the molecular weight of the PPF product. The expected time required to complete this protocol is 3 d.

A novel approach and protocol for discovering extremely low-abundance proteins in serum.

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.

Characterization of DNA release from composites of oligo(poly(ethylene glycol) fumarate) and cationized gelatin microspheres in vitro.

This research investigates the release of plasmid DNA from novel hydrogel composites of oligo(poly(ethylene glycol) fumarate) (OPF) and cationized gelatin microspheres (CGMS), as well as the swelling and degradation of these materials in vitro. The release of total DNA and of double-stranded DNA was measured fluorescently, and the swelling properties and polymer mass loss of the hydrogels were assessed. Further, the structural integrity of the released DNA was determined through electrophoresis. It was found that plasmid DNA can be released in a sustained fashion over the course of up to 49-140 days in vitro from hydrogels of OPF synthesized from poly(ethylene glycol) of nominal molecular weights of 10 kDa and 3 kDa, respectively, with the release kinetics depending upon the material composition and the method of DNA loading. Released DNA was predominately double-stranded DNA (dsDNA) in structure and of the open-circular conformation. The results suggest that DNA release from hydrogel composites of OPF and CGMS is dominated by the degradation of the OPF component of the gels. Electrophoresis results indicate that the released DNA retains suitable conformation for potential bioactivity over the course of at least 63 days of release. Thus, these studies demonstrate the potential of composites of OPF and CGMS in controlled gene delivery applications.

Evaluation of bone regeneration by DNA release from composites of oligo(poly(ethylene glycol) fumarate) and cationized gelatin microspheres in a critical-sized calvarial defect.

This research examines the bone formation response to release of plasmid DNA encoding human Bone Morphogenetic Protein-2 from hydrogel composites consisting of cationized gelatin microspheres (CGMS) embedded within a crosslinked oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel network in a critical-sized rat cranial defect model after 30 days. Four composite groups were investigated: (1) composites with 10 microg DNA loaded into the CGMS phase, (2) composites with 10 microg DNA loaded into the OPF phase, (3) composites with 100 microg DNA loaded into the OPF phase, and (4) composites without DNA (material control). Light microscopy revealed no enhancement in bone formation for groups releasing plasmid DNA, relative to the material control group. Limited formation of new bone was observed from the defect margins and within the defect for some samples. The hydrogels swelled appreciably and fragmentation of the implants was noted to varying degrees among samples within groups, with a presence of inflammatory cells related to the degree of fragmentation. The lack of enhancement in bone formation indicates that the release of plasmid DNA from the composites was not sufficient to elicit a bone regeneration response.

Effect of hydrophilicity and agmatine modification on degradation of poly(propylene fumarate-co-ethylene glycol) hydrogels.

We synthesized positively charged biodegradable hydrogels with different poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)] block copolymers and agmatine-modified poly(ethylene glycol)-tethered fumarate [Agm-PEGF] by radical crosslinking and investigated the effect of copolymer composition and agmatine modification on their degradation. Hydrogels were incubated in phosphate-buffered saline (PBS) at 37 degrees C with periodic PBS changes. All hydrogels experienced a slight mass loss over 4 weeks, ranging from 10-20%. Hydrogels with a molar ratio of ethylene glycol repeating units to propylene fumarate repeating units (EG/PF) of 9.3 degraded faster than hydrogels with an EG/PF ratio of 0.6. Agmatine-modified hydrogels degraded faster than unmodified hydrogels. The weight swelling ratio of the hydrogels at pH 7 increased over 4 weeks while increases in the EG/PF ratio and agmatine modification showed greater swelling. After 2 weeks, degraded hydrogels swollen at pH 3 demonstrated significantly lower weight swelling ratios than at pH 5, 7, and 9. Our results suggest that the hydrophilicity of the P(PF-co-EG) copolymer and agmatine modification have a small effect on the degradation of P(PF-co-EG) hydrogels.

Protein adsorption and smooth muscle cell adhesion on biodegradable agmatine-modified poly(propylene fumarate-co-ethylene glycol) hydrogels.

We synthesized positively charged biodegradable hydrogels from poly(propylene fumarate-co-ethylene glycol) block copolymer and agmatine-modified poly(ethylene glycol)-tethered fumarate by radical crosslinking, and investigated the effect of the guanidino group of agmatine on vascular smooth muscle cell adhesion and protein adsorption to the hydrogels. In the presence of serum, the number of adherent smooth muscle cells per unit surface area increased dose-dependently from 15 to 75% of the initial seeding density at 20 h as the initial agmatine-modified monomer content increased from 0 to 200 mg/g. Cell spreading also depended on the initial monomer content. In the absence of serum, the number of adherent cells per unit surface area increased slightly from 10 to 17% of the initial seeding density as the initial monomer content increased from 0 to 200 mg/g. Cell adhesion increased significantly by adding exogenous vitronectin to serum-free medium, whereas exogenous fibronectin addition did not enhance cell adhesion. The enzyme-linked immunosorbent assay of fibronectin and vitronectin adsorbed onto the hydrogels revealed that the incorporation of positive charges into the hydrogels enhanced vitronectin, but not fibronectin, adsorption significantly. These results suggest that the guanidino group of agmatine enhanced cell adhesion by promoting the adsorption of serum components, and vitronectin may be one of the components.

Cell adhesion on poly(propylene fumarate-co-ethylene glycol) hydrogels.

We synthesized poly(propylene fumarate-co-ethylene glycol) block copolymers [P(PF-co-EG)] that were crosslinked to form hydrogels and investigated the effect of copolymer composition on cell adhesion to the hydrogels. These copolymers were water soluble when the molar ratio of ethylene glycol repeating unit to propylene fumarate repeating unit was higher than 4.4. The water content of swollen hydrogels increased from 29 to 63% and the water contact angle decreased from 38 to 21 degrees as the molar ratio increased from 0.6 to 4.4. No significant change in either property was observed for ratios higher than 4.4. In a cell adhesion assay under serum-free conditions, the number of adherent platelets and smooth muscle cells decreased from 21 to 2% and from 78 to 20% of the initial seeding density, respectively, as the molar ratio increased from 0.6 to 7.8. Adherent smooth muscle cells did not spread on the hydrogels of the compositions tested. Adherent platelets did not show any filopodia. These results suggest that the hydrophilicity of P(PF-co-EG) hydrogels is one of the factors affecting cell adhesion, and that copolymer modification may be required for enhancing cell adhesion for an application involving the copolymers as in situ crosslinkable cell carriers.

Synthesis and characterization of biodegradable cationic poly(propylene fumarate-co-ethylene glycol) copolymer hydrogels modified with agmatine for enhanced cell adhesion.

We synthesized positively charged biodegradable hydrogels by cross-linking of agmatine-modified poly(ethylene glycol)-tethered fumarate (Agm-PEGF) and poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)) to investigate the effect of the guanidino groups of the agmatine on hydrogel swelling behavior and smooth muscle cell adhesion to the hydrogels. The weight swelling ratio of these hydrogels at pH 7.0 increased from 279 +/- 4 to 306 +/- 7% as the initial Agm-PEGF content increased from 0 to 200 mg/g of P(PF-co-EG), respectively. The diffusional exponents, n, during the initial phase of water uptake were independent of the initial Agm-PEGF content and were determined to be 0.66 +/- 0.08, 0.71 +/- 0.07, and 0.60 +/- 0.05 for respective initial Agm-PEGF contents of 0, 100, and 200 mg/g. The heat of fusion of water present in the hydrogels increased from 214 +/- 11 to 254 +/- 4 J/g as the initial Agm-PEGF content increased from 0 to 200 mg/g. The number of adherent smooth muscle cells increased dose-dependently from 15 +/- 6 to 75 +/- 7% of the initial seeding density as the initial Agm-PEGF content increased from 0 to 200 mg/g. These results suggest that the incorporation of the guanidino groups of agmatine into P(PF-co-EG) hydrogels increases the hydrogel free water content and the total water content of the hydrogels and also enhances cell adhesion to the hydrogels.