Spatiotemporal assessment of pathophysiological tissue remodeling in synthetic in situ tissue-engineered heart valves in sheep


We recently developed a synthetic heart valve replacement for in situ tissue engineering (TE), based on an acellular electrospun polycarbonate-bisurea scaffold (Kluin et al. Biomaterials 2017). Upon implantation in the pulmonary position in sheep, 3/4 of the valves were functional over 12 months follow-up. However, scaffold resorption was still incomplete and the regenerated neotissue only partially resembled the native heart valve tissue. Since the underlying mechanisms of in situ TE remain incompletely understood, the overall aim of this study was to elucidate these processes by mapping the spatiotemporal expression of neomatrix and cell phenotypes in our in situ TE heart valve implants.


We performed a detailed retrospective analysis on the explanted in situ TE valves from our previous preclinical study. Paraffin-embedded longitudinal sections of explanted in situ TE heart valves, with 6 and 12 months follow-up (n=4 sheep timepoint), were analyzed using Raman microspectroscopy and comprehensive immunohistochemistry (IHC). IHC analysis was performed using a dedicated sheep specific antibody panel (>40 antibodies), which we recently developed and validated.


Overall, the Raman spectra revealed progressive development of the newly formed matrix in the fibrosa and spongiosa layers of in situ TE valves towards their native counterparts. The ventricularis- like tissue in TE valves did not fully resemble the composition of native ventricularis due to the limited development of an elastic network. Degradation of the synthetic scaffold associated with inflammatory cell infiltrates was most pronounced in the remodeling area localized in the middle portion. Of note, Raman spectrometry was able to clearly identify the 12-month malfunctioned explant that was previously diagnosed as regurgitant. IHC analysis corroborated Raman spectra data and demonstrated an extensive expression of fibrotic-like markers in this specific valve, thus differentiating it from the other functional 12-month valves.


These findings enhance our mechanistic understanding of material-driven in situ regenerative processes. Moreover, the observed differential marker expression between functional and regurgitant valves can provide us with a diagnostic set of histopathological biomarkers, which could discriminate between functional regeneration and pathologic fibrotic repair.