Aligned human microvessels formed in 3D fibrin gel by constraint of gel contraction.
ABSTRACT: This study aimed to form microvessels in fibrin gels, which is of interest both for studying the fundamental cell-matrix interactions as well as for tissue engineering purposes, and to align the microvessels, which would provide natural inlet and outlet sides for perfusion. The data reported here demonstrate the formation of highly interconnected microvessels in fibrin gel under defined medium conditions and the ability to align them using two methods, both of which involved anchoring the gel at both ends to constrain the cell-induced compaction. The first method used only defined medium and resulted in moderate alignment. The second method used defined and serum-containing media sequentially to achieve high levels of microvessel alignment.
Project description:The development of engineered microvessels with clinically relevant characteristics is a critical step toward the creation of engineered myocardium. Alignment is one such characteristic that must be achieved, as it both mimics native capillary beds and provides natural inlet and outlet sides for perfusion. A second characteristic that is currently deficient is cross-sectional lumen density, typically under 100 lumens/mm²; the equivalent value for human myocardium is 2000 lumens/mm². Therefore, this study examined the effects of gel compaction and interstitial flow on microvessel alignment and lumen density. Strong microvessel alignment was achieved via mechanically constrained cell-induced fibrin gel compaction following vasculogenesis, and high lumen density (650 lumens/mm²) was achieved by the subsequent application of low levels of interstitial flow. Low interstitial flow also conferred microvessel barrier function.
Project description:A major goal of tissue engineering is the creation of pre-vascularized tissues that have a high density of organized microvessels that can be rapidly perfused following implantation. This is especially critical for highly metabolic tissues like myocardium, where a thick myocardial engineered tissue would require rapid perfusion within the first several days to survive transplantation. In the present work, tissue patches containing human microvessels that were either randomly oriented or aligned were placed acutely on rat hearts post-infarction and for each case it was determined whether rapid inosculation could occur and perfusion of the patch could be maintained for 6 days in an infarct environment. Patches containing self-assembled microvessels were formed by co-entrapment of human blood outgrowth endothelial cells and human pericytes in fibrin gel. Cell-induced gel contraction was mechanically-constrained resulting in samples with high densities of microvessels that were either randomly oriented (with 420 ± 140 lumens/mm(2)) or uniaxially aligned (with 940 ± 240 lumens/mm(2)) at the time of implantation. These patches were sutured onto the epicardial surface of the hearts of athymic rats following permanent ligation of the left anterior descending artery. In both aligned and randomly oriented microvessel patches, inosculation occurred and perfusion of the transplanted human microvessels was maintained, proving the in vivo vascularization potential of these engineered tissues. No difference was found in the number of human microvessels that were perfused in the randomly oriented (111 ± 75 perfused lumens/mm(2)) and aligned (173 ± 97 perfused lumens/mm(2)) patches. Our results demonstrate that tissue patches containing a high density of either aligned or randomly oriented human pre-formed microvessels achieve rapid perfusion in the myocardial infarct environment - a necessary first-step toward the creation of a thick, perfusable heart patch.
Project description:Rat retinal microvessels (RMVs) and brain microvessels (BMVs) were mechanically isolated under ice-cold condition. Retinal and brain tissues (RT, BT) from the same animals were collected for comparisons. Total RNA were individually extracted, amplified and processed on Affymetrix rat 2.0 microarray Chips. We sought to obtain the whole gene expression profile of RMVs, RT, BMVs and BT. Differently expressed genes (DEGs) between RMVs and RT, and between RMVs and BMVs, between RT and BT were analyses. Using these DEGs, we comprehensively analyzed gene expression of the microvessels and highlighted their involvement in critical functional structures in RMVs and BMVs, such as junctional complex, transporters and signaling pathways. Overall design: Totally, we analyzed four groups: brain microvessels, brain tissue, retinal microvessels and retinal tissue. Each group contains 6 biological replicates. The tissues simples were used as controls.
Project description:Blood clots and thrombi consist primarily of a mesh of branched fibers made of the protein fibrin. We propose a molecular basis for the marked extensibility and negative compressibility of fibrin gels based on the structural and mechanical properties of clots at the network, fiber, and molecular levels. The force required to stretch a clot initially rises linearly and is accompanied by a dramatic decrease in clot volume and a peak in compressibility. These macroscopic transitions are accompanied by fiber alignment and bundling after forced protein unfolding. Constitutive models are developed to integrate observations at spatial scales that span six orders of magnitude and indicate that gel extensibility and expulsion of water are both manifestations of protein unfolding, which is not apparent in other matrix proteins such as collagen.
Project description:We demonstrate that alignment of a structured peptide or small protein solubilized in mixed phospholipid:detergent micelles or bicelles, when embedded in a compressed gel or liquid crystalline medium, can be altered by either changing the phospholipid aggregate shape, charge, or both together. For the hemagglutinin fusion peptide solubilized in bicelles, we show that bicelle shape and charge do not change its helical hairpin structure but impact its alignment relative to the alignment medium, both in charged compressed acrylamide gel and in liquid crystalline d(GpG). The method can be used to generate sets of residual dipolar couplings that correspond to orthogonal alignment tensors, and holds promise for high-resolution structural refinement and dynamic mapping of membrane proteins.
Project description:Due to the increasing needs for organ transplantation and a universal shortage of donated tissues, tissue engineering emerges as a useful approach to engineer functional tissues. Although different synthetic materials have been used to fabricate tissue engineering scaffolds, they have many limitations such as the biocompatibility concerns, the inability to support cell attachment, and undesirable degradation rate. Fibrin gel, a biopolymeric material, provides numerous advantages over synthetic materials in functioning as a tissue engineering scaffold and a cell carrier. Fibrin gel exhibits excellent biocompatibility, promotes cell attachment, and can degrade in a controllable manner. Additionally, fibrin gel mimics the natural blood-clotting process and self-assembles into a polymer network. The ability for fibrin to cure in situ has been exploited to develop injectable scaffolds for the repair of damaged cardiac and cartilage tissues. Additionally, fibrin gel has been utilized as a cell carrier to protect cells from the forces during the application and cell delivery processes while enhancing the cell viability and tissue regeneration. Here, we review the recent advancement in developing fibrin-based biomaterials for the development of injectable tissue engineering scaffold and cell carriers.
Project description:Early stages of diseases, including stroke, hypertension, angiogenesis of tumours, spinal cord injuries, etc., are closely associated with the lesions of microvasculature. Rodent models of human vascular diseases are extensively used for the preclinical investigation of the disease evolution and therapy with synchrotron radiation. Therefore, non-invasive and in vivo X-ray imaging with high sensitivity and clarity is desperately needed to visualize the microvessels in live-animal models. Contrast agent is essential for the in vivo X-ray imaging of vessels and angiomatous tissue. Because of the non-rigid motion of adjacent tissues, the short circulation time and the intermittent flow of contrast agents in vessels, it is a great challenge for the traditional X-ray imaging methods to achieve well defined images of microvessels in vivo. In this article, move contrast X-ray imaging (MCXI) based on high-brightness synchrotron radiation is developed to overcome the intrinsic defects in conventional methods. Experiments with live rodents demonstrate the practicability of the MCXI method for sensitive and intact imaging of microvessels in vivo.
Project description:Only little is known about how cells coordinately behave to establish functional tissue structure and restore microarchitecture during regeneration. Research in this field is hampered by a lack of techniques that allow quantification of tissue architecture and its development. To bridge this gap, we have established a procedure based on confocal laser scans, image processing, and three-dimensional tissue reconstruction, as well as quantitative mathematical modeling. As a proof of principle, we reconstructed and modeled liver regeneration in mice after damage by CCl(4), a prototypical inducer of pericentral liver damage. We have chosen the regenerating liver as an example because of the tight link between liver architecture and function: the complex microarchitecture formed by hepatocytes and microvessels, i.e. sinusoids, ensures optimal exchange of metabolites between blood and hepatocytes. Our model captures all hepatocytes and sinusoids of a liver lobule during a 16 days regeneration process. The model unambiguously predicted a so-far unrecognized mechanism as essential for liver regeneration, whereby daughter hepatocytes align along the orientation of the closest sinusoid, a process which we named "hepatocyte-sinusoid alignment" (HSA). The simulated tissue architecture was only in agreement with the experimentally obtained data when HSA was included into the model and, moreover, no other likely mechanism could replace it. In order to experimentally validate the model of prediction of HSA, we analyzed the three-dimensional orientation of daughter hepatocytes in relation to the sinusoids. The results of this analysis clearly confirmed the model prediction. We believe our procedure is widely applicable in the systems biology of tissues.
Project description:Tracheal tissue engineering is a promising option for the treatment of tracheal defects. In a previous study we proved the suitability of fibrin gel as a scaffold for tracheal tissue engineering. This study investigates whether the differentiation of respiratory epithelium can be increased by culturing epithelial cells in a three dimensional system containing fibroblasts embedded into fibrin gel.Respiratory epithelial cells were isolated from porcine trachea, seeded onto a fibrin gel and kept in air-liquid-interface culture for 33 days. Morphology as well as pan-cytokeratin, MUC5AC and claudin-1 expression of cells cultured on pure fibrin gel were compared to culture on gels containing fibroblasts.After two weeks, cells seeded on pure fibrin gel were multilayered, showed hyperproliferation and dedifferentiation. Co-cultured cells built up a pseudostratified epithelium. The differentiation and organization of epithelial structure improved with respect to time. After four weeks, morphology of the co-cultured respiratory epithelium resembled native tracheal epithelium. Immunohistochemistry showed that respiratory epithelium co-cultured with fibroblasts had an increasing similarity of pan-cytokeratin expression compared to native trachea. Cells cultured without fibroblasts differed in pan-cytokeratin expression from native trachea and did not show any improvement of differentiation. Immunohistochemical staining of MUC5AC and claudin-1 proved seeded cells being respiratory epithelial cells.This study indicates that adding fibroblasts to fibrin gel positively influences the differentiation of respiratory epithelium.
Project description:BACKGROUND:Recent findings have shown that the fibrin gel derived from cord blood units (CBUs) play a significant role in wound healing and tissue regeneration. The aim of this study was to standardize the fibrin gel production process in order to allow for its regular use. METHODS:CBUs (n = 200) were assigned to 4 groups according to their initial volume. Then, a two-stage centrifugation protocol was applied in order to obtain platelet rich plasma (PRP). The concentration of platelets (PLTs), white blood cells (WBCs) and red blood cells (RBCs) were determined prior to and after the production process. In addition, targeted proteomic analysis using multiple reaction monitoring was performed. Finally, an appropriate volume of calcium gluconate was used in PRP for the production of fibrin gel. RESULTS:The results of this study showed that high volume CBUs were characterized by greater recovery rates, concentration and number of PLTs compared to the low volume CBUs. Proteomic analysis revealed the presence of key proteins for regenerative medicine. Fibrin gel was successfully produced from CBUs of all groups. CONCLUSION:In this study, low volume CBUs could be an alternative source for the production of fibrin gel, which can be used in multiple regenerative medicine approaches.