Tri‐Layered and Gel‐Like Nanofibrous Scaffolds with Anisotropic Features for Engineering Heart Valve Leaflets

Abstract

3D heterogeneous and anisotropic scaffolds that approximate native heart valve tissues are indispensable for the successful construction of tissue engineered heart valves (TEHVs). In this study, novel tri-layered and gel-like nanofibrous scaffolds, consisting of poly(lactic-co-glycolic) acid (PLGA) and poly(aspartic acid) (PASP), are fabricated by a combination of positive/negative conjugate electrospinning and bioactive hydrogel post-processing. The nanofibrous PLGA-PASP scaffolds present tri-layered structures, resulting in anisotropic mechanical properties that are comparable with native heart valve leaflets. Biological tests show that nanofibrous PLGA-PASP scaffolds with high PASP ratios significantly promote the proliferation and collagen and glycosaminoglycans (GAGs) secretions of human aortic valvular interstitial cells (HAVICs), compared to PLGA scaffolds. Importantly, the nanofibrous PLGA-PASP scaffolds are found to effectively inhibit the osteogenic differentiation of HAVICs. Two types of porcine VICs, from young and adult age groups, are further seeded onto the PLGA-PASP scaffolds. The adult VICs secrete higher amounts of collagens and GAGs and undergo a significantly higher level of osteogenic differentiation than young VICs. RNA sequencing analysis indicates that age has a pivotal effect on the VIC behaviors. This study provides important guidance and a reference for the design and development of 3D tri-layered, gel-like nanofibrous PLGA-PASP scaffolds for TEHV applications.

Elastomeric Trilayer Substrates with Native-like Mechanical Properties for Heart Valve Leaflet Tissue Engineering

“…Total RNA was extracted from the constructs following the manufacturer’s protocols for QIA-Shredder and RNeasy micro-kits . Subsequently, the reverse transcription of RNA into cDNA was performed with a High-Capacity cDNA Reverse Transcription Kit and the supplied instructions .…”

“…A substrate calcification study was conducted according to a published protocol. 11 PVICs (500k cells/ cm 2 ) were seeded on the PCL and PCL/PLCL substrates and then cultured in an ODM supplemented with 10% FBS, 1% penicillinstreptomycin, 1 mM dexamethasone, 10 mM β-glycerophosphate, and 50 mM ascorbic acid with for 14 days. The ODM was changed every 3 days.…”

“…Total RNA was extracted from the constructs following the manufacturer's protocols for QIA-Shredder and RNeasy micro-kits. 11 Subsequently, the reverse transcription of RNA into cDNA was performed with a High-Capacity cDNA Reverse Transcription Kit and the supplied instructions. 16 The cDNA transcripts were then probed with TaqMan assays for ACTA2, VIM, COL1A1, BGLAP, TGF-β1, TGF-β3, and ACTβ as a normalizer gene using a TaqMan Universal PCR Master Mix (Applied Biosystems).…”

“…These non-native characteristics of the substrates have led to prolonged VIC activation, which caused leaflet retraction and calcification in the tissue-engineered heart valves. VICs cultured on trilayer substrates with anisotropic structural and mechanical properties exhibit lower VIC activation and lower rates of calcification and retraction. − Further, our previous work has shown that trilayered cell-cultured constructs developed in vitro from a trilayer substrate show resistance to leaflet retraction. , Also, trilayered tissue constructs produced from trilayer leaflet substrates in the subcutaneous space of the rat model had a distinct native-like structure and protein quantities similar to that in a native leaflet. , Thus, developing substrates with both anisotropic structural and mechanical properties could be essential for successful heart valve tissue engineering …”

Control of hydrostatic pressure and osmotic stress in 3D cell culture for mechanobiological studies

“…Active biomaterials [174,[192][193][194] can be used to reproduce the chemomechanical coupling between pathophysiological mechanical loadings and OS. For example, composite biomaterials containing polyelectrolytes and collagen fibers capture the mechanism of osmotic swelling of the extracellular matrix [195], resembling both the composition and multiphasic loading function observed in several biological tissues.…”