Dmitry Belchenko

Evaluation of composition and crosslinking effects on collagen-based composite constructs

Vascular grafts are widely used for a number of medical treatments. Strength, compliance, endothelialization and availability are issues of most concern for vascular graft materials. With current approaches, these requirements are difficult to satisfy simultaneously. To explore an alternative approach, the present study has engineered the collagen gel construct by incorporating mimetic components and crosslinking the construct with different crosslinkers. The effects of component additives, such as chitosan and elastin, have been evaluated in terms of their mechanical and biological properties. Results demonstrate that the incorporation of chitosan and/or elastin alter stress-strain curves in the low stress loading region, and significantly improve the stretching ratio and ultimate stress of gel constructs compared to collagen constructs. Electron microscopy results suggest that the mechanical improvements might be due to microstructural modifications by chitosan sheets and elastin fibers. The effects of crosslinkers, such as formaldehyde, genipin and ethyl-(dimethyl aminopropyl) and carbodiimide hydrochloride (EDAC) have also been evaluated. Results demonstrate that formaldehyde, EDAC and genipin employ different mechanisms to crosslink collagen-based constructs, and use of genipin as a construct crosslinker exhibits improved elongation and endothelial coverage as compared to formaldehyde and EDAC. In addition, extending gelation time increased the elastic modulus but not the ultimate strength. Therefore, this study suggests that the mimicry of natural vessel tissues with properly crosslinked biopolymer composites could be a potential material design strategy for vascular graft materials.

Development and evaluation of microdevices for studying anisotropic biaxial cyclic stretch on cells

Mechanical effects on cells have received more and more attention in the studies of tissue engineering, cellular pathogenesis, and biomedical device design. Anisotropic biaxial cyclic stress, reminiscent of the in vivo cellular mechanical environment, may promise significant implications for biotechnology and human health. We have designed, fabricated and characterized a microdevice that imparts a variety of anisotropic biaxial cyclic strain gradients upon cells. The device is composed of an elastic membrane with microgroove patterns designed to associate cell orientation axes with biaxial strain vectors on the membrane and a Flexcell stretcher with timely controlled vacuum pressure. The stretcher generates strain profile of anisotropic biaxial microgradients on the membrane. Cell axes determined by the microgrooves are associated with the membrane strain profile to impose proper biaxial strains on cells. Using vascular smooth muscle cells as a cell model, we demonstrated that the strain anisotropy index of a cell was likely one of the determinant mechanical factors in cell structural and functional adaptations. The nuclear shape and cytoskeleton structure of smooth muscle cells were influenced by mechanical loading, but were not significantly affected by the strain anisotropy. However, cell proliferation has profound responses to strain anisotropy.

Circulating Fibrocytes Are Increased in Children and Young Adults with Pulmonary Hypertension

Chronic inflammation is an important component of the fibroproliferative changes that characterise pulmonary hypertensive vasculopathy. Fibrocytes contribute to tissue remodelling in settings of chronic inflammation, including animal models of pulmonary hypertension (PH). We sought to determine whether circulating fibrocytes were increased in children and young adults with PH. 26 individuals with PH and 10 with normal cardiac anatomy were studied. Fresh blood was analysed by flow cytometry for fibrocytes expressing CD45 and procollagen. Fibrocyte numbers were correlated to clinical and haemodynamic parameters, and circulating CC chemokine ligand (CCL)2 and CXC chemokine ligand (CXCL)12 levels. We found an enrichment of circulating fibrocytes among those with PH. No differences in fibrocytes were observed among those with idiopathic versus secondary PH. Higher fibrocytes correlated to increasing mean pulmonary artery pressure and age, but not to length or type of treatment. Immunofluorescence analysis confirmed flow sorting specificity. Differences in plasma levels of CCL2 or CXCL12, which could mobilise fibrocytes from the bone marrow, were not found. We conclude that circulating fibrocytes are significantly increased in individuals with PH compared with controls. We speculate that these cells might play important roles in vascular remodelling in children and young adults with pulmonary hypertension.

Endothelin-1, the Unfolded Protein Response, and Persistent Inflammation: Role of Pulmonary Artery Smooth Muscle Cells

Endothelin-1 is a potent vasoactive peptide that occurs in chronically high levels in humans with pulmonary hypertension and in animal models of the disease. Recently, the unfolded protein response was implicated in a variety of diseases, including pulmonary hypertension. In addition, evidence is increasing for pathological, persistent inflammation in the pathobiology of this disease. We investigated whether endothelin-1 might engage the unfolded protein response and thus link inflammation and the production of hyaluronic acid by pulmonary artery smooth muscle cells. Using immunoblot, real-time PCR, immunofluorescence, and luciferase assays, we found that endothelin-1 induces both a transcriptional and posttranslational activation of the three major arms of the unfolded protein response. The pharmacologic blockade of endothelin A receptors, but not endothelin B receptors, attenuated the observed release, as did a pharmacologic blockade of extracellular signal-regulated kinases 1 and 2 (ERK-1/2) signaling. Using short hairpin RNA and ELISA, we observed that the release by pulmonary artery smooth muscle cells of inflammatory modulators, including hyaluronic acid, is associated with endothelin-1-induced ERK-1/2 phosphorylation and the unfolded protein response. Furthermore, the synthesis of hyaluronic acid induced by endothelin-1 is permissive for persistent THP-1 monocyte binding. These results suggest that endothelin-1, in part because it induces the unfolded protein response in pulmonary artery smooth muscle cells, triggers proinflammatory processes that likely contribute to vascular remodeling in pulmonary hypertension.

Circulating Myeloid-Derived Suppressor Cells Are Increased and Activated in Pulmonary Hypertension

BACKGROUND Myeloid-derived suppressor cells (MDSCs) are increased in inflammatory and autoimmune disorders and orchestrate immune cell responses therein. Pulmonary hypertension (PH) is associated with inflammation, autoimmunity, and lung vascular remodeling. Immature myeloid cells are found in the lungs of humans and animals with PH, and we hypothesized that they would be increased in the blood of patients with PH compared with control subjects. METHODS Twenty-six children with PH and 10 undergoing cardiac catheterization for arrhythmia ablation were studied. Five milliliters of fresh blood were analyzed using flow cytometry. Results were confirmed using magnetic bead sorting and immunofluorescence, while quantitative polymerase chain reaction and intracellular urea concentration assays were used as measures of MDSC arginase-1 activation. RESULTS Flow cytometry demonstrated enrichment of circulating MDSCs among patients with PH (n = 26; mean, 0.271 × 10(6) cells/mL ± 0.17; 1.86% of CD45(+) population ± 1.51) compared with control subjects (n = 10; mean, 0.176 × 10(6) cells/mL ± 0.05; 0.57% of CD45(+) population ± 0.29; P < .05). Higher numbers of circulating MDSCs correlated to increasing mean pulmonary artery pressure (r = 0.510, P < .05). Among patients with PH, female patients had a twofold increase in MDSCs compared with male patients. Immunofluorescence analysis confirmed the results of flow cytometry. Quantitative reverse transcription polymerase chain reaction assay results for arginase-1 and measurement of intracellular urea concentration revealed increased activity of MDSCs from patients with PH compared with control subjects. CONCLUSIONS Circulating activated MDSCs are significantly increased in children with PH compared with control subjects. Further investigation of these cells is warranted, and we speculate that they might play significant immunomodulatory roles in the disease pathogenesis of PH.

Roles of genipin crosslinking and biomolecule conditioning in collagen‐based biopolymer: Potential for vascular media regeneration

The vascular media, a layer of the blood vessel wall containing smooth muscle cells (SMCs), are often the target functional tissue in the construction of artificial vessel. It contributes to mechanical properties and biological functions of vessels. The present study aimed to study effects of crosslinking and biomolecule conditions in the development of mechanically strong and stable, biologically functional constructs with potential for vascular media regeneration. Genipin was used to crosslink collagen-chitosan-elastin (CCE) constructs. Results revealed that mechanical strength, stiffness, and stability of CCE constructs significantly increased with genipin concentration, but crosslinking significantly inhibited SMC contraction of and invasion in gel constructs. No contraction or invasion was observed in those crosslinked with genipin at 5 mM or above. attenuated total reflectance Fourier transform infrared results showed crosslinking changed functional groups on CCE depending on genipin concentration. To enhance biological activities on crosslinked constructs, soluble molecule factors were incorporated, and their effects on SMC activities were evaluated. These conditions include heparin, platelet-derived transforming growth factor (PDGF), high-concentrated fetal bovine serum (h-FBS), a mixture of heparin and PDGF, and a mixture of h-FBS and PDGF. The h-FBS and PDGF mixture was found to stimulate a 3.2-fold increase in SMC contraction of the crosslinked gels. It was also found that PDGF and h-FBS, separately and in combination, induced SMC invasion in the crosslinked gels, while heparin attenuated PDGF-induced SMC invasion. Our study suggests that designing high-performance acellular constructs to encourage tissue regeneration should use a combination of crosslinking condition and biomolecule factor, striking a balance between mechanical properties and biological functions.

Anisotropic Strain Effects on Vascular Smooth Muscle Cell Physiology

Biological development is a complex and highly-regulated process, a significant part of which is controlled by mechanostimulus, or the strain imparted on a cell by its environment. Mechanostimulus is important for stem cell differentiation, from cytoskeletal assembly to cell-cell and cell-matrix adhesion [1]. The mechanics of cells and tissues play a critical role in organisms, under both physiological and pathological conditions; abnormal mechanotransduction — the mechanism by which cells sense and respond to strain — has been implicated in a wide range of clinical pathologies [2,3].Copyright