Rolling the human amnion to engineer laminated vascular tissues

Abstract

The prevalence of cardiovascular disease and the limited availability of suitable autologous transplant vessels for coronary and peripheral bypass surgeries is a significant clinical problem. A great deal of progress has been made over recent years to develop biodegradable materials with the potential to remodel and regenerate vascular tissues. However, the creation of functional biological scaffolds capable of withstanding vascular stress within a clinically relevant time frame has proved to be a challenging proposition. As an alternative approach, we report the use of a multilaminate rolling approach using the human amnion to generate a tubular construct for blood vessel regeneration. The human amniotic membrane was decellularized by agitation in 0.03% (w/v) sodium dodecyl sulfate to generate an immune compliant material. The adhesion of human umbilical vein endothelial cells (EC) and human vascular smooth muscle cells (SMC) was assessed to determine initial binding and biocompatibility (monocultures). Extended cultures were either assessed as flat membranes, or rolled to form concentric multilayered conduits. Results showed positive EC adhesion and a progressive repopulation by SMC. Functional changes in SMC gene expression and the constructs' bulk mechanical properties were concomitant with vessel remodeling as assessed over a 40-day culture period. A significant advantage with this approach is the ability to rapidly produce a cell-dense construct with an extracellular matrix similar in architecture and composition to natural vessels. The capacity to control physical parameters such as vessel diameter, wall thickness, shape, and length are critical to match vessel compliance and tailor vessel specifications to distinct anatomical locations. As such, this approach opens new avenues in a range of tissue regenerative applications that may have a much wider clinical impact.

FIG. 1.

hAM preparation and rolling procedure. The hAM (A) was first decellularized, then under sterile conditions cut to 13×9 cm² sections. SMC were seeded at a concentration of 600 cells/mm² and cultured as flat sheets for 10 days before rolling (B). After a further 10 days of culture, the hAMS was rolled five times around a glass mandrel (C). A pictorial overview of construct seeding, rolling, and terminal analysis (D). Scale bar in (C)=1 cm. hAM, human amniotic membrane; hAMS, human amniotic membrane scaffold; SMC, smooth muscle cells.

FIG. 2.

Schematic of the rolling process and initial adhesion of EC and SMC monocultures. (A) Schematic on the right shows the rolling process with the layered structure, with the left image showing tunable geometries in scaffold size (lumen diameter, length, and number of membrane revolutions). Initial adhesion of EC (B) and SMC (C) monocultures was assessed 2 h after seeding using the fluorescent nuclear stain DAPI. Scale bar=50 μm. EC, endothelial cells. Color images available online at www.liebertpub.com/tec

FIG. 3.

SMC metabolic activity and cell density when cultured on and within the hAMS. Quantitative assessment of cell metabolic activity and cell proliferation for flat sheets (A) and rolled constructs (B). On flat constructs, the cells displayed an increasing cell population, with a decreasing cell metabolic activity. Conversely, cells within rolled constructs (after day 10) showed no statistical change in cell density to day 40; however, a significant increase in metabolic activity was noted (n=3).

FIG. 4.

SEM and histological image analysis. SEM of the hAMS stromal surface after sodium dodecyl sulfate decellularization (A) displayed fibers with a random orientation and no observable cellular debris. Histological sections of the decellularized hAMS show the stromal surface to have a compact fiber structure, with a relatively thick stromal layer compared with the thinner and more compact basement membrane (B). Day 10 after cell seeding, cell migration into the stromal layer was observed (C). The multilayered construct at day 20 (D), and at day 40 (E) showing areas of higher cell concentration with a more compact and remodeled ECM compared with the native ECM. Scale bar=50 μm for histological images and 5 μm for SEM images. SEM. Color images available online at www.liebertpub.com/tec

FIG. 5.

Changes in GAG concentration. GAG concentrations increased over 10 days of cell culture with the flat hAMS (A), stabilizing at day 20 to 10 mg/g of hydrated tissue. A progressive increase of GAG concentration was measured with the rolled hAMS (B) from day 20 to 40 (n=9). (n=5). *p<0.01. GAG, glycosaminoglycan.

FIG. 6.

Reverse transcription–polymerase chain reaction of rolled hAMS at day 10, 20, and 40. Variation in gene expression profiles of collagen and SMC phenotype markers α-actin and SM22 by SMC was assessed. Data were calculated using the 2^-ΔΔCt method normalized to SMC expression levels under basal culture conditions (controls), and expressed as the fold differences. At each time point, α-actin was expressed at higher levels than controls, reaching an 11-fold increase by day 40. SM22 was expressed with higher values than controls at day 40 only. Collagen expression decreased from 2.8-fold at day 20 to same values as those of the controls at day 40.

FIG. 7.

Tensile properties of the hAMS. Representative plots showing stress-strain relationships for flat (A) and rolled (B) hAMS. (C) details the ringlet test method to assess tensile properties. Single breaking points were observed on flat hAMS (A), whereas three breaking points were noted on the five times rolled hAMS. The rupture strength of the laminated hAMS was twice higher at 1.38 MPa compared with single layers of hAMS (0.77 MPa). (D) shows tabulated data of the constructs elastic modulus (over the physiological range: 80–120 mmHg) and rupture strength for both flat and rolled constructs. Color images available online at www.liebertpub.com/tec

FIG. 8.

Dynamic culture and comparative mechanics. A schematic of the dynamic culture systems process flow (A). Two peristaltic pumps drove the respective solutions media and phosphate buffered saline through the albumen and lumen flow circuits at a constant flow rate of 30 and 60 mL/min, respectively. Constructs cultured under perfusion conditions displayed a compact structure at day 40 (B). Rupture strength, elastic modulus, and mean compliance values are compared with native human carotid arteries values (C), ^#refers to theoretical calculated values. A representative load–extension profile of the rolled construct illustrates multiple failure peaks associated with each construct layer (D). Scale bar in (B)=20 μm. *refers to measured values. Color images available online at www.liebertpub.com/tec