The consequence of biologic graft processing on blood interface biocompatibility and mechanics

Abstract

Processing ex vivo derived tissues to reduce immunogenicity is an effective approach to create biologically complex materials for vascular reconstruction. Due to the sensitivity of small diameter vascular grafts to occlusive events, the effect of graft processing on critical parameters for graft patency, such as peripheral cell adhesion and wall mechanics, requires detailed analysis. Isolated human umbilical vein sections were used as model allogenic vascular scaffolds that were processed with either: 1. sodium dodecyl sulfate (SDS), 2. ethanol/acetone (EtAc), or 3. glutaraldehyde (Glu). Changes in material mechanics were assessed via uniaxial tensile testing. Peripheral cell adhesion to the opaque grafting material was evaluated using an innovative flow chamber that allows direct observation of the blood-graft interface under physiological shear conditions. All treatments modified the grafts tensile strain and stiffness properties, with physiological modulus values decreasing from Glu 240±12 kPa to SDS 210±6 kPa and EtAc 140±3 kPa, P<.001. Relative to glutaraldehyde treatments, neutrophil adhesion to the decellularized grafts increased, with no statistical difference observed between SDS or EtAc treatments. Early platelet adhesion (% surface coverage) showed no statistical difference between the three treatments; however, quantification of platelet aggregates was significantly higher on SDS scaffolds compared to EtAc or Glu. Tissue processing strategies applied to the umbilical vein scaffold were shown to modify structural mechanics and cell adhesion properties, with the EtAc treatment reducing thrombotic events relative to SDS treated samples. This approach allows time and cost effective prescreening of clinically relevant grafting materials to assess initial cell reactivity.

Keywords: biocompatibility; biomaterial; decellularization; flow chamber; human umbilical vein; thrombosis; vascular tissue engineering.

Figure 1. Perfusion design

A newly designed flow chamber is used to observe platelet and leukocyte adhesion to the graft materials in real-time. Shear conditions are controlled by modulating the bulk flow rate using a high capacity syringe pump. Images are recorded via a high resolution Zeiss Axiocam monochrome Rev 3 digital camera. (A) Schematic of the perfusion circuit. (B) Photograph of the newly designed flow chamber.

Figure 2. Processing influence on the scaffold architecture

Extracted Human Umbilical Veins (HUV) were either decellularized with one of two different chemical treatments or cross-linked with glutaraldehyde. Shown in the top row (A) are representative images of HUV scaffolds immediately following treatment and after subsequent rinsing in PBS. The micro-architecture of the processed scaffolds is shown in (B), first by H&E staining (left), then by SEM imaging of the lumenal (middle), and ablumenal (right) surface. Treatments are labeled as follows: (EtAc) Ethanol/Acetone decellularization, (SDS) Sodium Dodecyl Sulfate decellularization, and (Glu) Glutaraldehyde crosslinking. represents the lumen of the HUV and the ablumen.

Figure 3. Scaffold mechanical properties

The mechanics of the HUV were tested after processing treatments followed by subsequent rinsing/sterilization. Graphs show (A) representative strain/stress curves, inset showing the complete profile with an enlargement over the physiological range, B) tensile modulus in the physiological range (from 11 to 16 kPa, represented by E in (A)), and C) tensile strain at 16 kPa. (n=3, * P<.05) Dashed lines represent the scaffold prior to chemical processing, and the grey areas surrounding the dash lines indicate the standard error.

Figure 4. Neutrophil adhesion to processed HUV

HL-60 differentiated as neutrophils were incubated with HUV for 5 hours. After incubation and subsequent exposure to a 210 s⁻¹ flow, the neutrophils adhering to the lumenal surface of the vein were (A) observed (scale bar: 100 µm) and (B) quantified (* P<.05, the dashed line represents the amount of neutrophils incubated). Neutrophil detachment as a function of shear was also observed (C). After incubation, a ramping flow was applied and the percentage of neutrophils detaching was quantified for each shear condition. (n=4).

Figure 5. Qualitative visual assessment of platelet adhesion and aggregates formation

A) Representative fluorescent images of platelet adhesion to the lumen of processed HUV over time, under a shear rate of 210 s⁻¹. White arrows represent flow direction. Scale bar: 100 µm. B) After five minutes of whole blood perfusion at a shear rate of 210 s⁻¹, platelet aggregates were imaged using scanning electron microscopy.

Figure 6. Progression of platelet adhesion and aggregation

A) Real-time analysis of the total platelet accumulation on the lumen of the HUV at a given shear rate of 210 s⁻¹. Each data point represents the mean ± S.E. for 7 separate experiments. B) After one and five minutes of perfusion, the number of platelet aggregates was quantified (Ŧ P<.05 between the three treatments, * P<.05 between 1 and 5 minutes for each treatment). C) Correlation between platelet aggregation and surface coverage was assessed at 1, 2, 3, 4, and 5 minutes. Each data point represents the mean platelet aggregates correlated to the corresponding total area covered with platelets for seven separate experiments. D) An index indicating the overall surface area covered by one platelet was calculated for each treatment (Ŧ P<.05 between the three treatments).