Inflammatory and regenerative processes in bioresorbable synthetic pulmonary valves up to two years in sheep-Spatiotemporal insights augmented by Raman microspectroscopy.

In situ heart valve tissue engineering is an emerging approach in which resorbable, off-the-shelf available scaffolds are used to induce endogenous heart valve restoration. Such scaffolds are designed to recruit endogenous cells in vivo, which subsequently resorb polymer and produce and remodel new valvular tissue in situ. Recently, preclinical studies using electrospun supramolecular elastomeric valvular grafts have shown that this approach enables in situ regeneration of pulmonary valves with long-term functionality in vivo. However, the evolution and mechanisms of inflammation, polymer absorption and tissue regeneration are largely unknown, and adverse valve remodeling and intra- and inter-valvular variability have been reported. Therefore, the goal of the present study was to gain a mechanistic understanding of the in vivo regenerative processes by combining routine histology and immunohistochemistry, using a comprehensive sheep-specific antibody panel, with Raman microspectroscopy for the spatiotemporal analysis of in situ tissue-engineered pulmonary valves with follow-up to 24 months from a previous preclinical study in sheep. The analyses revealed a strong spatial heterogeneity in the influx of inflammatory cells, graft resorption, and foreign body giant cells. Collagen maturation occurred predominantly between 6 and 12 months after implantation, which was accompanied by a progressive switch to a more quiescent phenotype of infiltrating cells with properties of valvular interstitial cells. Variability among specimens in the extent of tissue remodeling was observed for follow-up times after 6 months. Taken together, these findings advance the understanding of key events and mechanisms in material-driven in situ heart valve tissue engineering. STATEMENT OF SIGNIFICANCE: This study describes for the first time the long-term in vivo inflammatory and regenerative processes that underly in situ heart valve tissue engineering using resorbable synthetic scaffolds. Using a unique combinatorial analysis of immunohistochemistry and Raman microspectroscopy, important spatiotemporal variability in graft resorption and tissue formation was pinpointed in in situ tissue-engineered heart valves, with a follow-up time of up to 24 months in sheep. This variability was correlated to heterogenous regional cellular repopulation, most likely instigated by region-specific differences in surrounding tissue and hemodynamics. The findings of this research contribute to the mechanistic understanding of in situ tissue engineering using resorbable synthetics, which is necessary to enable rational design of improved grafts, and ensure safe and robust clinical translation.

Hyaluronic acid as a macromolecular crowding agent for production of cell-derived matrices.

Cell-derived matrices (CDMs) provide an exogenous source of human extracellular matrix (ECM), with applications as cell delivery vehicles, substrate coatings for cell attachment and differentiation, and as biomaterial scaffolds. However, commercial application of CDMs has been hindered due to the prolonged culture time required for sufficient ECM accumulation. One approach to increasing matrix deposition in vitro is macromolecular crowding (MMC), which is a biophysical phenomenon that limits the diffusion of ECM precursor proteins, resulting in increased ECM accumulation at the cell layer. Hyaluronic acid (HA), a natural MMC highly expressed in vivo during fetal development, has been shown to play a role in ECM production, but has not been investigated as a macromolecule for increasing cell-mediated ECM deposition in vitro. In the current study, we hypothesized that HA can act as a MMC, and increase cell-mediated ECM production. Human dermal fibroblasts were cultured for 3, 7, or 14 days with 0%, 0.05%, or 0.5% high molecular weight HA. Ficoll 70/400 was used as a positive control. SDS-PAGE, Sircol, and hydroxyproline assays indicated that 0.05% HA-treated cultures had significantly higher mean collagen deposition at 14 days, whereas Ficoll 70/400-treated cultures had significantly lower collagen production compared to the HA and untreated controls. However, fluorescent immunostaining of ECM proteins and quantification of mean gray values did not indicate statistically significant differences in ECM production in HA or Ficoll 70/400-treated cultures compared to untreated controls. Raman imaging (a marker-free spectral imaging method) indicated that HA increased ECM deposition in human dermal fibroblasts. These results are consistent with decreases in CDM stiffness observed in Ficoll 70/400-treated cultures by atomic force microscopy. Overall, these results indicate that there are macromolecule- and cell type- dependent effects on matrix assembly, turnover, and stiffness in cell-derived matrices. STATEMENT OF SIGNIFICANCE: Cell-derived matrices (CDMs) are versatile biomaterials with many regenerative medicine applications, including as cell and drug delivery vehicles and scaffolds for wound healing and tissue regeneration. While CDMs have several advantages, their commercialization has been limited due to the prolonged culture time required to achieve CDM synthesis in vitro. In this study, we explored the use of hyaluronic acid (HA) as a macromolecular crowder in human fibroblast cell cultures to support production of CDM biomaterials. Successful application of macromolecular crowding will allow development of human cell-derived, xeno-free biomaterials that re-capitulate the native human tissue microenvironment.