Development of a universal, oriented antibody immobilization method to functionalize vascular prostheses for enhanced endothelialization for potential clinical application

Materials

Electropolished CC discs, CC stents, ePTFE grafts, and anti-human CD34 antibodies (mouse IgG2a) were provided by OrbusNeich Medical Technologies (Fort Lauderdale, FL, USA). Mouse isotype control (cat#: 015-000-002, 5 mg/ml) for CD34 antibody was obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). Mouse anti-pig CD31 antibody (cat#: MCA1746F) was purchased from Bio-Rad Canada (Mississauga, ON, Canada). FITC-conjugated rabbit anti human CD34 polyclonal antibody (cat#: 10,665-284) was provided by VWR Canada (Mississauga, ON, Canada). Recombinant human CD34 peptide containing the entire extracellular domain was procured from Abcam Inc., Toronto, ON, Canada (cat#: ab126924). Amino-(PEG)8-hydrazide-t-boc (the linker) was obtained from Quanta Biodesign (Plain City, OH, USA) and all other chemicals from Sigma-Aldrich Canada (Oakville, ON, Canada). CD34 + KG1a human leukemic progenitor cells, HEK293 human kidney epithelial cells, human umbilical vein endothelial cells (HUVECs), Iscove’s Modified Dulbecco's Medium (IMDM), Eagle's Minimum Essential Medium (EMEM), Endothelial Basal Medium (EBM) and fetal bovine serum (FBS) were purchased from ATCC (Gaithersburg, MD, USA). PD-10 Desalting Columns were from Fisher Scientific Canada (Ottawa, ON, Canada). The fluorescence dyes Calcein-AM, CMTMR and Sytox Green were obtained from Thermo Fisher Scientific Canada (Mississauga, ON, Canada).

Oriented CD34 antibody coating

Oriented CD34 antibody or isotype coating, intermediate coating, and CD34 antibody coating without t-boc removal were done as follows.

Poly-dopamine base and PEG linker construction

The material to be coated was bath sonicated in ethanol, acetone and ddH2O for 10 min each. For the deposition of poly-dopamine base and PEG linker, the cleaned material was immersed in a mixture of equal volumes of 4 mg/ml dopamine hydrochloride and 50 mg/ml Amino-(PEG)8-hydrazide-t-boc solution, both prepared with 10 mM Tris.HCl (pH 8.5), and incubated by shaking at 250 rpm at 37 °C for 24 h. This led to the generation of substrates with intermediate coating only. After 3 washes with ddH2O and 3 washes with ethanol, 10 min each by shaking at 100 rpm at room temperature, the substrates with intermediate coating were either stored in the storage buffer (SB), i.e., phosphate buffered saline (PBS, pH 7.4) containing 100 units/ml of penicillin, 100 µg/mL of streptomycin and 2.5 µg/mL of amphotericin B, in a 4 °C refrigerator until further analyzed or processed further as described below.

Removal of t-boc

The substrate with intermediate coating was dipped in dichloromethane and incubated at room temperature for 10 min followed by incubation in 2 mg/ml iodine solution prepared with dichloromethane by shaking at 100 rpm at room temperature for 5 h.

Antibody oxidization

10 mM sodium periodate solution prepared with the oxidation buffer (20 mM sodium acetate and 15 mM sodium chloride, pH 5.6) was used for antibody oxidization. CD34 antibody or isotype control was mixed with 10 mM sodium periodate solution to reach a final concentration of 10 µg/ml. The mixture was then incubated (protected from light) by shaking at 100 rpm at room temperature for 30 min. Afterwards, sodium periodate was removed using the PD-10 Desalting Column according to the manufacturer’s instructions, and CD34 antibodies were eluted with oxidation buffer.

Oriented antibody immobilization

Following t-boc removal described above, the substrate was washed 3 times with dichloromethane, 3 times with ethanol and 3 times with ddH2O (10 min each time) by shaking at 100 rpm at room temperature. Finally, the substrate was immersed in the oxidized CD34 antibodies or isotype control, and incubated at 4 °C for 24 h. After incubation, the coated material was washed one time with PBS and stored in the SB in a 4 °C refrigerator until further analyzed.

Antibody coating without t-boc removal

The substrate with intermediate coating without t-boc removal was incubated directly with oxidized CD34 antibodies at 4 °C for 24 h. After one wash with PBS, the coated substrate was stored in the SB in a 4 °C refrigerator until further analyzed.

Coating surface characterizationScanning electron microscopy (SEM)

SEM was performed as described elsewhere [48].

Immunofluorescence analysis

CD34 antibodies coated on CC discs were evaluated by immunofluorescence analysis with isotype coated discs serving as control. Coated discs were placed in a 96-well plate (1 disc/well) and blocked with PBS containing 2% bovine serum albumin (BSA) at room temperature for 1 h. After 3 washes with PBS, the disc was incubated with 100 µl of CD34 peptide (5 µg/ml) prepared with the blocking buffer at room temperature for 2 h followed by 3 washes with PBS. Subsequently, the disc was incubated with FITC-conjugated anti-CD34 polyclonal antibodies (1:50 diluted with the blocking buffer) in dark at room temperature for 1 h. After 3 washes with PBS, the disc was mounted on a glass slide and observed under fluorescence microscope.

Mechanical characterization

The integrity and deformability of the coating was assessed on a Graftmaster RX coronary stent system (Abbott Vascular, Santa Clara, California). The Graftmaster stent is configured with a layer of ePTFE sandwiched between two identical stainless steel stents. The device was coated in its unexpanded state, then expanded to its fully deployed size at nominal expansion pressure, with a size matched compliant angioplasty balloon. The expanded stent system was then analyzed by SEM with a coated unexpanded coronary stent system serving as control.

X-ray photoelectron spectroscopy (XPS)

XPS was performed as described elsewhere [48].

Contact angle measurement

Contact angle measurement was performed for bare and coated ePTFE grafts using an optical NRL Model 100–00 contact angle goniometer (NRL C.A. goniometer, Ramé-Hart, Inc., Mountain Lakes, NJ, USA) as described elsewhere [25].

Cell growth analysis

The in vitro cell compatibility of the CD34 antibody coating was tested by cell growth on coated CC discs using HUVECs maintained in EBM containing 5% FBS. A coated disc was placed in a well of a 6-well plate followed by addition of 1 × 105 HUVECs in 2 ml culture medium. The plate was transferred into a cell culture incubator. At 24 and 48 h after culture, the fluorescence dye Calcein-AM that only stains live cells was added to cells (final concentration: 1 µM) and cells were kept in the incubator for 30 min. Afterwards, the disc was removed, placed on a coverslip, and stained cells were watched and counted under fluorescence microscope. The cell number from 5 randomly chosen fields (20x) for each disc was calculated, and the average cell numbers were compared between different time points.

In vitro cell capture assay

CD34 + KG1a cells were maintained in IMDM supplemented with 20% FBS. CD34- HEK293 cells were maintained in EMEM supplemented with 10% FBS. The ability of CD34 antibody-coated substrates to bind CD34 + cells was tested with mixed CD34 + KG1a and CD34- HEK293 cells that were pre-stained with different fluorescence dyes. Briefly, KG1a and HEK293 cells were stained with Calcein-AM and CMTMR respectively, both at a final concentration of 1 µM, according to the manufacturer’s instructions. Cells were then harvested, washed once with PBS and re-suspended in PBS at a density of 2 × 106/ml for cell binding assay. The coated material was blocked with PBS containing 2% BSA at room temperature for 1 h followed by incubation with a mixture of equal volumes of KG1a and HEK293 cells in a 2 ml cryotube protected from light by shaking at 100 rpm at room temperature for 1 h. Subsequently, the substrate was thoroughly washed 6 times with PBS to remove non-specifically attached cells and bound cells were visualized under fluorescence microscope. Additionally, cell binding testing was done for coated substrates using KG1a cells with HEK293 cells serving as control. KG1a cells or HEK293 cells detached by trypsinization were washed once with PBS and re-suspended in PBS at a density of 106 cells/ml. After blocking as described above, the coated substrate was incubated with either KG1a cells or HEK293 cells by shaking at 100 rpm at room temperature for 1 h. Subsequently, the material was thoroughly washed 6 times with PBS to remove non-specifically attached cells. After fixation in 4% paraformaldehyde prepared with PBS for 15 min, bound cells were stained with Sytox Green and visualized under fluorescence microscope. To test the stability of coated CD34 antibody, the coated material was stored in the SB in a 4 °C refrigerator for 2 weeks, and then tested by cell binding assays as described above.

Pig carotid artery graft interposition

The animal protocol was approved by the Animal Care Committee of St. Michael’s Hospital, Unity Health Toronto, University of Toronto (approval number: ACC893), in accordance with the NIH Guide for the Care and Use of Laboratory Animals, 8th edition. CD34 antibody coated ePTFE grafts (5 mm in diameter) were tested for enhanced endothelialization in a carotid artery graft interposition model in a total of 9 Yorkshire male pigs weighing approximately 40 kg. Three pigs were used for each of the following grafts: CD34 antibody-coated, bare, and intermediately-coated. Before implantation, the graft was blocked in pig serum at room temperature for 30 min. Animals were fasted ~ 12 h and then anesthetized. A cervical incision was made after disinfection with alcohol and betadine to expose the unilateral carotid artery. A carotid artery interposition with the graft was performed as follows: intravenous unfractionated heparin was given and the carotid artery was clamped proximal and distal to the interposition site, and a segment of approximately 3 cm was excised and the ends of the artery were beveled. The graft was also beveled and trimmed to a length of 3 cm. Proximal and distal anastomoses were completed with a 7-0 polypropylene along a continuous suture line. Flow was restored by removing the clamps, and absence of proximal and distal anastomotic suture line leak confirmed. The cervical incision was closed in layers and the animals were allowed to recover and analgesic measures were taken by intramuscular administration of Anafen once (3 mg/kg) and oral administration of Metacam for 48 h (0.1 mg/kg). Each pig was orally administered 0.5 g of acetylsalicylic acid daily until the end-point study. At day 14 post surgery, pigs were anesthetized, and carotid ultrasound was performed using a handheld Doppler probe immediately before vessel exposure and explantation of the prosthetic material in order to confirm vessel patency. No carotid Doppler tracings were recorded. The grafts were explanted and gently flushed with PBS. Each graft was cut in two halves: one half for SEM analysis and the other half for immunostaining.

Analysis of explanted graftsSEM analysis

One half of each explanted graft was fixed with formaldehyde and glutaraldehyde 2.5% each in 0.1 M sodium cacodylate buffer (pH 7.2) at 4 °C for 24 h. The fixative was removed by washing grafts 3 times 10 min each in 0.1 M Sorensen’s Phosphate Buffer (pH 7.2). The graft was then fixed in 1% osmium tetroxide prepared with 0.1 M Sorensen’s Phosphate Buffer for 1.5 h and then washed 3 times 10 min each in 0.1 M Sorensen’s Phosphate Buffer. Afterwards, the graft was dehydrated through an ascending ethanol series of 50–100% ethanol solutions for 10 min each followed by infiltration with an ethanol:hexamethyldisilizane (HMDS) series at a ratio of 3:1, 1:1, 1:3 and 100% HMDS, all for 10 min each. After being left to dry overnight in 100% HMDS, the material was mounted on the SEM stub, sputter coated with platinum using the Bal-Tec SCD 050, and examined with a Hitachi S-2500 scanning electron microscope. The endothelium covered areas were determined in a blind manner using the Image J software as follows: the area covered or uncovered with endothelial cells that displayed a cobblestone morphology was manually selected using the free hand selection tool, and the selected area was then automatically calculated by the “Analyze > Measure” command with the “Area” being selected from the “Results > Set Measurement” menu. Each half graft was divided into 3 longitudinal sections covered by 3 consecutive SEM images (approximately 5 mm/section) for measurement, and the sum of covered or uncovered areas from the 3 sections were used to calculate the percentage of endothelialization as follows: sum of endothelialization area/(sum of endothelialization area + sum of non-endothelialization area) × 100%.

Immunostaining

The explanted grafts (one half of each graft) were fixed with 4% paraformaldehyde at room temperature for 15 min, blocked in PBS containing 2% BSA for 1 h and incubated with the mouse anti-pig CD31 antibody (1:100 dilution in PBS containing 2% BSA) at room temperature for 1 h. Following 3 washes with PBS, 5 min each, the grafts were incubated with the secondary antibodies (1:1000 dilution, DAPI for nuclear staining) under the light protection for 1 h. After 3 washes with PBS 5 min each, the graft was mounted on a coverslip and visualized under confocal microscope.

Statistical analysis

All data from in vitro study of bare and coated substrates were analyzed with Student t test. Endothelium covered areas in explanted grafts in animal study were analyzed with ANOVA with post-hoc Tukey test. A two-tailed p < 0.05 was considered statistically significant.

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