Fiber Reinforced Cartilage ECM Functionalized Bioinks for Functional Cartilage Tissue Engineering.

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-ß3 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.

Mechanotransduction in small intestinal submucosa scaffolds: fabrication parameters potentially modulate the shear-induced expression of PECAM-1 and eNOS.

In small intestinal submucosa (SIS) scaffolds for functional tissue engineering, the impact of scaffold fabrication parameters on cellular response and tissue regeneration may relate to the mechanotransductory properties of the final arrangement of collagen fibres. We previously proved that two fabrication parameters, (a) preservation (P) or removal (R) of a dense collagen layer present in SIS, and (b) SIS in a final dehydrated (D) or hydrated (H) state, have an effect on the micromechanical environment of SIS. In a continuation of our studies, we herein hypothesized that these fabrication parameters also modulate early mechanotransduction in cells populating the scaffold. Mechanotransduction was investigated by seeding human umbilical vein endothelial cells (HUVECs) on scaffolds, exposing them to pulsatile shear stress (12 ± 4 dyne/cm2 ) for 1 h (n = 5) in a cone-and-plate shear system, and evaluating the expression of the mechanosensitive genes Pecam1 and Enos by immunofluorescence and qPCR. Expression of mechanosensitive genes was highest in PD grafts, followed by PH and RH grafts. The RD group had similar expression to that of unsheared control cells, suggesting that the RD combination potentially reduced mechanotransduction of shear to cells. We concluded that the two fabrication parameters studied, which modify SIS micromechanics, also potentially modulated the early shear-induced expression of mechanosensitive genes in seeded HUVECs. Our findings suggest that fabrication parameters influence the outcome of SIS as a therapeutic scaffold. Copyright © 2015 John Wiley & Sons, Ltd.

Identification of side- and shear-dependent microRNAs regulating porcine aortic valve pathogenesis.

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 TGFß1 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 TGFß1.

The role of inorganic pyrophosphate in aortic valve calcification.

BACKGROUND AND AIM OF THE STUDY: Aortic valve (AV) calcification is a major cause of morbidity and mortality, yet the molecular mechanisms involved are poorly understood. Hence, an ex vivo model of calcification in intact AVs was developed in order to test the role of orthophosphate and pyrophosphate (PPi), both of which factors are known to influence vascular calcification. METHODS: Porcine AV leaflets were cultured in serum-free medium under static conditions for eight days, over which time leaflet architecture and viability were preserved. Calcification was measured as the incorporation of 45Ca, with confirmation by Alizarin Red staining. RESULTS: Calcification required both a high phosphate concentration (3.8 mM) and removal of PPi with alkaline phosphatase or inorganic pyrophosphatase. Calcification occurred predominantly on the fibrosa and was arrested by the bisphosphonate etidronate, a non-hydrolyzable analog of PPi. Leaflets released PPi into the medium, and this was enhanced by MLS38949, a specific inhibitor of tissue non-specific alkaline phosphatase (TNAP). Furthermore, leaflets synthesized PPi from extracellular ATP, which was reduced by ß,γ-methylene-ATP, an inhibitor of ectonucleotide pyrophosphorylase phosphodiesterase (NPP1). CONCLUSION: The ex vivo AV calcification model developed in the present study showed that extracellular PPi, produced by valvular tissue, is a potent inhibitor of valvular calcification. In addition to synthesis, hydrolysis by TNAP also controls PPi levels and calcification. The results suggest that a decreased synthesis or increased hydrolysis of pyrophosphate may contribute to valvular calcification, and that bisphosphonates or inhibitors of TNAP are potential preventive strategies of the process. TNAP are potential preventive strategies.

Aortic valve: mechanical environment and mechanobiology.

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.

The effects of combined cyclic stretch and pressure on the aortic valve interstitial cell phenotype.

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.