The regeneration of complex tissues and organs remains a major clinical challenge. With a view towards bioprinting such tissues, we developed a new class of pore-forming bioink to spatially and temporally control the presentation of therapeutic genes within bioprinted tissues. By blending sacrificial and stable hydrogels, we were able to produce bioinks whose porosity increased with time following printing. When combined with amphipathic peptide-based plasmid DNA delivery, these bioinks supported enhanced non-viral gene transfer to stem cells in vitro. By modulating the porosity of these bioinks, it was possible to direct either rapid and transient (pore-forming bioinks), or slower and more sustained (solid bioinks) transfection of host or transplanted cells in vivo. To demonstrate the utility of these bioinks for the bioprinting of spatially complex tissues, they were next used to zonally position stem cells and plasmids encoding for either osteogenic (BMP2) or chondrogenic (combination of TGF-β3, BMP2 and SOX9) genes within networks of 3D printed thermoplastic fibers to produce mechanically reinforced, gene activated constructs. In vivo, these bioprinted tissues supported the development of a vascularised, bony tissue overlaid by a layer of stable cartilage. When combined with multiple-tool biofabrication strategies, these gene activated bioinks can enable the bioprinting of a wide range of spatially complex tissues.

Focal articular cartilage (AC) defects, if left untreated, can lead to debilitating diseases such as osteoarthritis. While several tissue engineering strategies have been developed to promote cartilage regeneration, it is still challenging to generate functional AC capable of sustaining high load‐bearing environments. Here, a new class of cartilage extracellular matrix (cECM)‐functionalized alginate bioink is developed for the bioprinting of cartilaginous tissues. The bioinks are 3D‐printable, support mesenchymal stem cell (MSC) viability postprinting and robust chondrogenesis in vitro, with the highest levels of COLLII and ACAN expression observed in bioinks containing the highest concentration of cECM. Enhanced chondrogenesis in cECM‐functionalized bioinks is also associated with progression along an endochondral‐like pathway, as evident by increases in RUNX2 expression and calcium deposition in vitro. The bioinks loaded with MSCs and TGF‐β3 are also found capable of supporting robust chondrogenesis, opening the possibility of using such bioinks for direct “print‐and‐implant” cartilage repair strategies. Finally, it is demonstrated that networks of 3D‐printed polycaprolactone fibers with compressive modulus comparable to native AC can be used to mechanically reinforce these bioinks, with no loss in cell viability. It is envisioned that combinations of such biomaterials can be used in multiple‐tool biofabrication strategies for the bioprinting of biomimetic cartilaginous implants.

The aortic valve (AV) experiences a complex mechanical environment, which includes tension, flexure, pressure, and shear stress forces due to blood flow during each cardiac cycle. This mechanical environment regulates AV tissue structure by constantly renewing and remodeling the phenotype. In vitro, ex vivo and in vivo studies have shown that pathological states such as hypertension and congenital defect like bicuspid AV (BAV) can potentially alter the AV’s mechanical environment, triggering a cascade of remodeling, inflammation, and calcification activities in AV tissue. Alteration in mechanical environment is first sensed by the endothelium, which in turn induces changes in the extracellular matrix, and triggers cell differentiation and activation. However, the molecular mechanism of this process is not understood very well. Understanding these mechanisms is critical for advancing the development of effective medical based therapies. Recently, there have been some interesting studies on characterizing the hemodynamics associated with AV, especially in pathologies like BAV, using different experimental and numerical methods. Here, we review the current knowledge of the local AV mechanical environment and its effect on valve biology, focusing on in vitro and ex vivo approaches.

Aortic valve (AV) calcification is an inflammation driven process that occurs preferentially in the fibrosa. To explore the underlying mechanisms, we investigated if key microRNAs (miRNA) in the AV are differentially expressed due to disturbed blood flow (oscillatory shear (OS)) experienced by the fibrosa compared to the ventricularis. To identify the miRNAs involved, endothelial-enriched RNA was isolated from either side of healthy porcine AVs for microarray analysis. Validation using qPCR confirmed significantly higher expression of 7 miRNAs (miR-100, -130a, -181a/b, -199a-3p, -199a-5p, and -214) in the fibrosa versus the ventricularis. Upon bioinformatics analysis, miR-214 was selected for further investigation using porcine AV leaflets in an ex vivo shear system. Fibrosa and ventricularis sides were exposed to either oscillatory or unidirectional pulsatile shear for 2 days and 3 & 7 days in regular and osteogenic media, respectively. Higher expression of miR-214, increased thickness of the fibrosa, and calcification was observed when the fibrosa was exposed to OS compared to the ventricularis. Silencing of miR-214 by anti-miR-214 in whole AV leaflets with the fibrosa exposed to OS significantly increased the protein expression of TGFβ1 and moderately increased collagen content but did not affect AV calcification. Thus, miR-214 is identified as a side- and shear-dependent miRNA that regulates key mechanosensitive gene in AV such as TGFβ1.

Aortic valve interstitial cells (VIC) can exhibit phenotypic characteristics of fibroblasts, myofibroblasts, and smooth muscle cells. Others have proposed that valve cells become activated and exhibit myofibroblast or fibroblast characteristics during disease initiation and progression; however, the cues that modulate this phenotypic change remain unclear. We hypothesize that the mechanical forces experienced by the valve play a role in regulating the native phenotype of the valve and that altered mechanical forces result in an activated phenotype. Using a novel ex vivo cyclic stretch and pressure bioreactor, we subjected porcine aortic valve (AV) leaflets to combinations of normal and pathological stretch and pressure magnitudes. The myofibroblast markers α-SMA and Vimentin, along with the smooth muscle markers Calponin and Caldesmon, were analyzed using immunohistochemistry and immunoblotting. Tissue structure was analyzed using Movat’s pentachrome staining. We report that pathological stretch and pressure inhibited the contractile and possibly myofibroblast phenotypes as indicated by downregulation of the proteins α-SMA, Vimentin, and Calponin. In particular, Calponin downregulation implies depolymerization of actin filaments and possible conversion to a more synthetic (non-contractile) phenotype. This agreed well with the increase in spongiosa and fibrosa thickness observed under elevated pressure and stretch that are typically indicative of increased matrix synthesis. Our study therefore demonstrates how cyclic stretch and pressure may possibly act together to modulate the AVIC phenotype.