Daniel S. Puperi

Integrating valve-inspired design features into poly(ethylene glycol) hydrogel scaffolds for heart valve tissue engineering.

The development of advanced scaffolds that recapitulate the anisotropic mechanical behavior and biological functions of the extracellular matrix in leaflets would be transformative for heart valve tissue engineering. In this study, anisotropic mechanical properties were established in poly(ethylene glycol) (PEG) hydrogels by crosslinking stripes of 3.4 kDa PEG diacrylate (PEGDA) within 20 kDa PEGDA base hydrogels using a photolithographic patterning method. Varying the stripe width and spacing resulted in a tensile elastic modulus parallel to the stripes that was 4.1-6.8 times greater than that in the perpendicular direction, comparable to the degree of anisotropy between the circumferential and radial orientations in native valve leaflets. Biomimetic PEG-peptide hydrogels were prepared by tethering the cell-adhesive peptide RGDS and incorporating the collagenase-degradable peptide PQ (GGGPQG↓IWGQGK) into the polymer network. The specific amounts of RGDS and PEG-PQ within the resulting hydrogels influenced the elongation, de novo extracellular matrix deposition and hydrogel degradation behavior of encapsulated valvular interstitial cells (VICs). In addition, the morphology and activation of VICs grown atop PEG hydrogels could be modulated by controlling the concentration or micro-patterning profile of PEG-RGDS. These results are promising for the fabrication of PEG-based hydrogels using anatomically and biologically inspired scaffold design features for heart valve tissue engineering.

Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde-derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma.

Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds

Advanced tissue engineered heart valves must be constructed from multiple materials to better mimic the heterogeneity found in the native valve. The trilayered structure of aortic valves provides the ability to open and close consistently over a full human lifetime, with each layer performing specific mechanical functions. The middle spongiosa layer consists primarily of proteoglycans and glycosaminoglycans, providing lubrication and dampening functions as the valve leaflet flexes open and closed. In this study, hyaluronan hydrogels were tuned to perform the mechanical functions of the spongiosa layer, provide a biomimetic scaffold in which valve cells were encapsulated in 3D for tissue engineering applications, and gain insight into how valve cells maintain hyaluronan homeostasis within heart valves. Expression of the HAS1 isoform of hyaluronan synthase was significantly higher in hyaluronan hydrogels compared to blank-slate poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Hyaluronidase and matrix metalloproteinase enzyme activity was similar between hyaluronan and PEGDA hydrogels, even though these scaffold materials were each specifically susceptible to degradation by different enzyme types. KIAA1199 was expressed by valve cells and may play a role in the regulation of hyaluronan in heart valves. Cross-linked hyaluronan hydrogels maintained healthy phenotype of valve cells in 3D culture and were tuned to approximate the mechanical properties of the valve spongiosa layer. Therefore, hyaluronan can be used as an appropriate material for the spongiosa layer of a proposed laminate tissue engineered heart valve scaffold.

Ascorbic acid promotes extracellular matrix deposition while preserving valve interstitial cell quiescence within 3D hydrogel scaffolds.

Current options for aortic valve replacements are non‐viable and thus lack the ability to grow and remodel, which can be problematic for paediatric applications. Toward the development of living valve substitutes that can grow and remodel, porcine aortic valve interstitial cells (VICs) were isolated and encapsulated within proteolytically degradable and cell‐adhesive poly(ethylene glycol) (PEG) hydrogels, in an effort to study their phenotypes and functions. The results showed that encapsulated VICs maintained high viability and proliferated within the hydrogels. The VICs actively remodelled the hydrogels via secretion of matrix metalloproteinase‐2 (MMP‐2) and deposition of new extracellular matrix (ECM) components, including collagens I and III. The soft hydrogels with compressive moduli of ~4.3 kPa quickly reverted VICs from an activated myofibroblastic phenotype to a quiescent, unactivated phenotype, evidenced by the loss of α‐smooth muscle actin expression upon encapsulation. In an effort to promote VIC‐mediated ECM production, ascorbic acid (AA) was supplemented in the medium to investigate its effects on VIC function and phenotype. AA treatment enhanced VIC spreading and proliferation, and inhibited apoptosis. AA treatment also promoted VIC‐mediated ECM remodelling by increasing MMP‐2 activity and depositing collagens I and III. AA treatment did not significantly influence the expression of α‐smooth muscle actin (myofibroblast activation marker) and alkaline phosphatase (osteogenic differentiation marker). No calcification or nodule formation was observed within the cell‐laden hydrogels, with or without AA treatment. These results suggest the potential of this system and the beneficial effect of AA in heart valve tissue engineering. Copyright

Differential cell-matrix responses in hypoxia-stimulated aortic versus mitral valves

Tissue oxygenation often plays a significant role in disease and is an essential design consideration for tissue engineering. Here, oxygen diffusion profiles of porcine aortic and mitral valve leaflets were determined using an oxygen diffusion chamber in conjunction with computational models. Results from these studies revealed the differences between aortic and mitral valve leaflet diffusion profiles and suggested that diffusion alone was insufficient for normal oxygen delivery in mitral valves. During fibrotic valve disease, leaflet thickening due to abnormal extracellular matrix is likely to reduce regional oxygen availability. To assess the impact of low oxygen levels on valve behaviour, whole leaflet organ cultures were created to induce leaflet hypoxia. These studies revealed a loss of layer stratification and elevated levels of hypoxia inducible factor 1-alpha in both aortic and mitral valve hypoxic groups. Mitral valves also exhibited altered expression of angiogenic factors in response to low oxygen environments when compared with normoxic groups. Hypoxia affected aortic and mitral valves differently, and mitral valves appeared to show a stenotic, rheumatic phenotype accompanied by significant cell death. These results indicate that hypoxia could be a factor in mid to late valve disease progression, especially with the reduction in chondromodulin-1 expression shown by hypoxic mitral valves.

Supplementary material from "Differential cell-matrix responses in hypoxia-stimulated aortic versus mitral valves"

Tissue oxygenation often plays a significant role in disease and is an essential design consideration for tissue engineering. Here, oxygen diffusion profiles of porcine aortic and mitral valve leaflets were determined using an oxygen diffusion chamber in conjunction with computational models. Results from these studies revealed the differences between aortic and mitral valve leaflet diffusion profiles and suggested that diffusion alone was insufficient for normal oxygen delivery in mitral valves. During fibrotic valve disease, leaflet thickening due to abnormal ECM likely reduces regional oxygen availability. To assess the impact of low oxygen levels on valve behaviour, whole leaflet organ cultures were created to induce leaflet hypoxia. These studies revealed a loss of layer stratification and elevated levels of hypoxia inducible factor 1-alpha in both aortic and mitral valve hypoxic groups. Mitral valves also exhibited altered expression of angiogenic factors in response to low oxygen environments when compared with normoxic groups. Hypoxia affected aortic and mitral valves differently, and mitral valves appeared to show a stenotic, rheumatic phenotype accompanied by significant cell death. These results indicate that hypoxia could be a factor in mid to late valve disease progression, especially with the reduction in chondromodulin-1 expression shown by hypoxic mitral valves.