Three-Dimensional Quantitative Micromorphology of Pre- and Post-Implanted Engineered Heart Valve Tissues

Abstract

There is a significant gap in our knowledge of engineered heart valve tissue (EHVT) development regarding detailed three-dimensional (3D) tissue formation and remodeling from the point of in vitro culturing to full in vivo function. As a step toward understanding the complexities of EHVT formation and remodeling, a novel serial confocal microscopy technique was employed to obtain 3D micro-structural information of pre-implant (PRI) and post-implant for 12 weeks (POI) EHVT fabricated from PGA: PLLA scaffolds and seeded with ovine bone-marrow-derived mesenchymal stem cells. Custom scaffold fiber tracking software was developed to quantify scaffold fiber architectural features such as length, tortuosity, and minimum scaffold fiber-fiber separation distance and scaffold fiber orientation was quantified utilizing a 3D fabric tensor. In addition, collagen and cellular density of ovine pulmonary valve leaflet tissue were also analyzed for baseline comparisons. Results indicated that in the unseeded state, scaffold fibers formed a continuous, oriented network. In the PRI state, the scaffold showed some fragmentation with a scaffold volume fraction of 7.79%. In the POI specimen, the scaffold became highly fragmented, forming a randomly distributed short fibrous network (volume fraction of 2.03%) within a contiguous, dense collagenous matrix. Both PGA and PLLA scaffold fibers were observed in the PRI and POI specimens. Collagen density remained similar in both PRI and POI specimens (74.2 and 71.5%, respectively), though the distributions in the transmural direction appeared slightly more homogenous in the POI specimen. Finally, to guide future 2D histological studies for largescale studies (since acquisition of high-resolution volumetric data is not practical for all specimens), we investigated changes in relevant collagen and scaffold metrics (collagen density and scaffold fiber orientation) with varying section spacing. It was found that a sectioning spacing up to 25 μm (for scaffold morphology) and 50 μm (for collagen density) in both PRI and POI tissues did not result in loss of information fidelity, and that sectioning in the circumferential or radial direction provides the greatest preservation of information. This is the first known work to investigate EHVT microstructure over a large volume with high resolution and to investigate time evolving in vivo EHVT morphology. The important scaffold fiber structural changes observed provide morphological information crucial for guiding future structurally based constitutive modeling efforts focused on better understanding EHVT tissue formation and remodeling.

A mathematical model for the determination of forming tissue moduli in needled-nonwoven scaffolds

“…For the present study (cf. Section 2.6), NNW scaffolds incubated in vitro for 1 month demonstrated similar tissue formation [28, 29]. …”

“…We illustrate the impact of our modeling approach by employing it to quantify the stiffness of engineered ECM after in vitro incubation and early stages of in vivo implantation obtained in a concurrent study of engineered tissue pulmonary valves in an ovine model [28, 29]. …”

Modeling the impact of scaffold architecture and mechanical loading on collagen turnover in engineered cardiovascular tissues

“…When fibroblast-like cells are seeded onto a fibrous scaffold and cultured under pulsatile conditions, they align preferentially along the fibers of the scaffold and produce collagen along the same direction (Hwang et al 2009;Niklason 2009). The neo-formed collagen fibers stay in close contact with the scaffold (Eckert et al 2011), indicating that the contact guidance by the scaffold is a crucial factor in early stages of the devel-123 opment of the collagen network. Next to collagen synthesis, also enzymatic degradation of the collagen fibers takes place.…”

Bone marrow mesenchymal stem cells for tissue engineered pulmonary valves (TEPV)

“…Replacement TEPVs developed from biodegradable scaffolds seeded with autologous cells have proven successful in large animal and human models [29,31,[59][60][61]. From a fabrication stand point, the scaffold design is critical; the 3D structure guides cellular migration, attachment and growth [62][63][64]. Ideal synthetic biodegradable scaffolds are typically porous and are designed to enable diffusion of both nutrients and waste products [29,59,65].…”

“…The ideal scaffold should be optimally biodegradable-the timing of absorption into endogenous tissue should match tissue formation. Scaffolds have been developed from polyglycolic acid (PGA), polylactic acid (PLA), combined PGA-PLA and polyhydroxyoctanoate (PHO) [29,[59][60][61][62][63][64][65]. The advantage with these materials is thermoplasticity, biodegradability and biocompatibility [29,[60][61][62][63][64][65].…”