Flow with variable pulse frequencies accelerates vascular recellularization and remodeling of a human bioscaffold.

Despite significant advances in vascular tissue engineering, the ideal graft has not yet been developed and autologous vessels remain the gold standard substitutes for small diameter bypass procedures. Here, we explore the use of a flow field with variable pulse frequencies over the regeneration of an ex vivo-derived human scaffold as vascular graft. Briefly, human umbilical veins were decellularized and used as scaffold for cellular repopulation with human smooth muscle cells (SMC) and endothelial cells (EC). Over graft development, the variable flow, which mimics the real-time cardiac output of an individual performing daily activities (e.g., resting vs. exercising), was implemented and compared to the commonly used constant pulse frequency. Results show marked differences on SMC and EC function, with changes at the molecular level reflecting on tissue scales. First, variable frequencies significantly increased SMC proliferation rate and glycosaminoglycan production. These results can be tied with the SMC gene expression that indicates a synthetic phenotype, with a significant downregulation of myosin heavy chain. Additionally and quite remarkably, the variable flow frequencies motivated the re-endothelialization of the grafts, with a quiescent-like structure observed after 10 days of conditioning, contrasting with the low surface coverage and unaligned EC observed under constant frequency (CF). Besides, the overall biomechanics of the generated grafts (conditioned with both pulsed and CFs) evidence a significant remodeling after 55 days of culture, depicted by high burst pressure and Young's modulus. These last results demonstrate the positive recellularization and remodeling of a human-derived scaffold toward an arterial vessel.

Sequential adaptation of perfusion and transport conditions significantly improves vascular construct recellularization and biomechanics.

Recellularization of ex vivo-derived scaffolds remains a significant hurdle primarily due to the scaffolds subcellular pore size that restricts initial cell seeding to the scaffolds periphery and inhibits migration over time. With the aim to improve cell migration, repopulation, and graft mechanics, the effects of a four-step culture approach were assessed. Using an ex vivo-derived vein as a model scaffold, human smooth muscle cells were first seeded onto its ablumen (Step 1: 3 hr) and an aggressive 0-100% nutrient gradient (lumenal flow under hypotensive pressure) was created to initiate cell migration across the scaffold (Step 2: Day 0 to 19). The effects of a prolonged aggressive nutrient gradient created by this single lumenal flow was then compared with a dual flow (lumenal and ablumenal) in Step 3 (Day 20 to 30). Analyses showed that a single lumenal flow maintained for 30 days resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source), with improved distribution. In Step 4 (Day 31 to 45), the transition from hypotensive pressure (12/8 mmHg) to normotensive (arterial-like) pressure (120/80 mmHg) was assessed. It demonstrated that recellularized scaffolds exposed to arterial pressures have increased glycosaminoglycan deposition, physiological modulus, and Young's modulus. By using this stepwise conditioning, the challenging recellularization of a vein-based scaffold and its positive remodeling toward arterial biomechanics were obtained.

Impact of magnetic nanoparticle surface coating on their long-term intracellular biodegradation in stem cells.

Magnetic nanoparticles (MNPs) internalized within stem cells have paved the way for remote magnetic cell manipulation and imaging in regenerative medicine. A full understanding of their interactions with stem cells and of their fate in the intracellular environment is then required, in particular with respect to their surface coatings. Here, we investigated the biological interactions of MNPs composed of an identical magnetic core but coated with different molecules: phosphonoacetic acid, polyethylene glycol phosphonic carboxylic acid, caffeic acid, citric acid, and polyacrylic acid. These coatings vary in the nature of the chelating function, the number of binding sites, and the presence or absence of a polymer. The nanoparticle magnetism was systematically used as an indicator of their internalization within human stem cells and of their structural long-term biodegradation in a 3D stem cell spheroid model. Overall, we evidence that the coating impacts the aggregation status of the nanoparticles and subsequently their uptake within stem cells, but it has little effect on their intracellular degradation. Only a high number of chelating functions (polyacrylic acid) had a significant protective effect. Interestingly, when the nanoparticles aggregated prior to cellular internalization, less degradation was also observed. Finally, for all coatings, a robust dose-dependent intracellular degradation rate was demonstrated, with higher doses of internalized nanoparticles leading to a lower degradation extent.

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

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.

In vitro method for real-time, direct observation of cell-vascular graft interactions under simulated blood flow.

In the development of engineered vascular grafts, assessing the material's interactive properties with peripheral blood cells and its capacity to endothelialize are important for predicting in vivo graft behavior. Current in vitro techniques used for characterizing cell adhesion at the surface of engineered scaffolds under flow only facilitate a terminal quantification of cell/surface interactions. Here, we present the design of an innovative flow chamber for real-time analysis of blood-biomaterial interactions under controllable hemodynamic conditions. Decellularized human umbilical veins (dHUV) were used as model vascular allografts to characterize platelet, leukocyte, and endothelial cell (EC) adhesion dynamics. Confluent EC monolayers adhered to the lumenal surface of the grafting material were flow conditioned to resist arterial shear stress levels (up to 24 dynes/cm(2)) over a 48 h period, and shown to maintain viability over the 1 week assessment period. The basement membrane was imaged while whole blood/neutrophil suspensions were perfused across the HUV surface to quantify cell accumulation. This novel method facilitates live visualization of dynamic events, including cell adhesion, migration, and morphological adaptation at the blood-graft interface on opaque materials, and it can be used for preliminary assessment of clinically relevant biomaterials before implantation.

Preimplantation processing of ex vivo-derived vascular biomaterials: effects on peripheral cell adhesion.

The use of ex vivo-derived scaffolds as vascular conduits has shown to be a clinically valid approach to repair or bypass occluded vessels. Implantation of allogeneic tissue grafts requires careful processing to lower immunogenicity and prevent bacterial infection. However, the mechanical/chemical treatments used to prepare biological scaffolds can result in significant alterations to the native structure and surface chemistry, which can affect in vivo performance. Of particular importance for vascular grafts are binding interactions between the implanted biomaterial and host cells from the circulation and adjacent vasculature. Here we present a comparison of four strategies used to decellularize allogeneic human umbilical vein (HUV) scaffolds: ethanol/acetone, sodium chloride, sodium dodecyl sulfate (SDS), or Triton X-100. Scanning electron microscopy revealed that all four techniques achieved removal of native cells from both the lumenal and ablumenal surfaces of HUV grafts. Platelets and promyelocytic HL-60 cells showed preferential binding on the more loosely structured ablumenal surface, although low surface coverage was observed overall by peripheral blood cells. Vascular endothelial cell adhesion was highest on HUV decellularized using ethanol/acetone, and significantly higher than on SDS-processed grafts (p = 0.016). Primary cells showed high viability on the lumenal surface regardless of decellularization technique (over 95% in all cases). These results demonstrate the critical effects of various chemical processing strategies on the adhesive properties of ex vivo-derived vascular grafts. Careful application-specific consideration is warranted when selecting a processing strategy that minimizes innate responses (e.g. thrombosis, inflammation) that are often deleterious to graft survival.

The role of fibrinogen spacing and patch size on platelet adhesion under flow.

Platelet adhesion to the vessel wall during vascular injury is mediated by platelet glycoproteins binding to their respective ligands on the vascular wall. In this study we investigated the roles that ligand patch spacing and size play in regulating platelet interactions with fibrinogen under hemodynamic flow conditions. To regulate the size and distance between patches of fibrinogen we developed a photolithography-based technique to fabricate patterns of proteins surrounded by a protein-repellant layer of poly(ethylene glycol). We demonstrate that when mepacrine labeled whole blood is perfused at a shear rate of 100 s ⁻¹ over substrates patterned with micron-sized wide lines of fibrinogen, platelets selectively adhere to the areas of patterned fibrinogen. Using fluorescent and scanning electron microscopy we demonstrate that the degree of platelet coverage (3-35%) and the ability of platelet aggregates to grow laterally are dependent upon the distance (6-30 µm) between parallel lines of fibrinogen. We also report on the effects of fibrinogen patch size on platelet adhesion by varying the size of the protein patch (2-20 µm) available for adhesion, demonstrating that the downstream length of the ligand patch is a critical parameter in platelet adhesion under flow. We expect that these results and protein patterning surfaces will be useful in understanding the spatial and temporal dynamics of platelet adhesion under physiologic flow, and in the development of novel platelet adhesion assays.