Postdoc, Université Laval
Collagen-based scaffolds are cellularised with SMCs and matured in static and dynamic conditions
Coronary artery diseases are one of the leading causes of death in Western society. They develop when coronaries, the blood vessels that supply heart with blood, become damaged or diseased. The main cause is the formation of plaques, event known as atherosclerosis, that narrow the arteries thus decreasing blood flow to the heart. Although drug treatments are available to control the growth of atherosclerotic plaques, often a surgical act is necessary. Unhappily, current surgical options do not guarantee long term success and re-interventions are often required. Moreover, causes and processes responsible of the formation of atherosclerotic plaques are not yet completely understood.
To overcome these problems, vascular tissue engineering aims to (re)generate in laboratory living blood vessels. Vascular tissue engineering employs cells in combination with biomaterials such as collagen where vascular cells extracted from humans are seeded and cultured, aiming to produce structures as similar as possible to living blood vessels. The choice of the biomaterial and of the process of fabrication is pivotal, since the biomaterial itself should support the survival of the cells and stimulate them to regenerate a functional tissue. In addition, it is possible to stimulate cells with biological molecules such as growth factors and with mechanical stimuli, like the maturation in the so-called bioreactors, aiming to reproduce the physiological conditions.
The development of functional tissue engineered blood vessels would represent a terrific breakthrough in the cardiovascular field with a wide range of possible applications. In fact, in addition to their use as vascular substitutes, these systems can be profitably used in vitro models alternative to in vivo tests for the investigation of physiological and pathological processes occurring in blood vessels, the preclinical testing of drugs, biomaterials and devices, all in a controlled environment that accurately reproduces natural conditions.
Abstract: In vitro maturation of engineered vascular tissues (EVT) requires the appropriate incorporation of smooth muscle cells (SMC) and extracellular matrix (ECM) components similar to native arteries. To this end, the aim of the current study was to fabricate 4 mm inner diameter vascular tissues using mesenchymal progenitor cells seeded into tubular scaffolds. A dual-pump bioreactor operating either in perfusion or pulsatile perfusion mode was used to generate physiological-like stimuli to promote progenitor cell differentiation, extracellular elastin production, and tissue maturation. Our data demonstrated that pulsatile forces and perfusion of 3D tubular constructs from both the lumenal and ablumenal sides with culture media significantly improved tissue assembly, effectively inducing mesenchymal progenitor cell differentiation to SMCs with contemporaneous elastin production. With bioreactor cultivation, progenitor cells differentiated toward smooth muscle lineage characterized by the expression of smooth muscle (SM)-specific markers smooth muscle alpha actin (SM-α-actin) and smooth muscle myosin heavy chain (SM-MHC). More importantly, pulsatile perfusion bioreactor cultivation enhanced the synthesis of tropoelastin and its extracellular cross-linking into elastic fiber compared with static culture controls. Taken together, the current study demonstrated progenitor cell differentiation and vascular tissue assembly, and provides insights into elastin synthesis and assembly to fibers.Incorporation of elastin into engineered vascular tissues represents a critical design goal for both mechanical and biological functions. In the present study, we seeded porous tubular scaffolds with multipotent mesenchymal progenitor cells and cultured in dual-pump pulsatile perfusion bioreactor. Physiological-like stimuli generated by bioreactor not only induced mesenchymal progenitor cell differentiation to vascular smooth muscle lineage but also actively promoted elastin synthesis and fibre assembly. Gene expression and protein synthesis analyses coupled with histological and immunofluorescence staining revealed that elastin-containing vascular tissues were fabricated. More importantly, co-localization and co-immunoprecipitation experiments demonstrated that elastin and fibrillin-1 were abundant throughout the cross-section of the tissue constructs suggesting a process of elastin protein crosslinking. This study paves a way forward to engineer elastin-containing functional vascular substitutes from multipotent progenitor cells in a bioreactor.
Pub.: 12 Jul '17, Pinned: 22 Aug '17
Abstract: Blood vessels are subjected to numerous biomechanical forces that work harmoniously but, when unbalanced because of vascular smooth muscle cell (vSMC) dysfunction, can trigger a wide range of ailments such as cerebrovascular, peripheral artery, and coronary artery diseases. Human pluripotent stem cells (hPSCs) serve as useful therapeutic tools that may help provide insight on the effect that such biomechanical stimuli have on vSMC function and differentiation. In this study, we aimed to examine the effect of biomechanical strain on vSMCs derived from hPSCs. The effects of two types of tensile strain on hPSC-vSMC derivatives at different stages of differentiation were examined. The derivatives included smooth muscle-like cells (SMLCs), mature SMLCs, and contractile vSMCs. All vSMC derivatives aligned perpendicularly to the direction of cyclic uniaxial strain. Serum deprivation and short-term uniaxial strain had a synergistic effect in enhancing collagen type I, fibronectin, and elastin gene expression. Furthermore, long-term uniaxial strain deterred collagen type III gene expression, whereas long-term circumferential strain upregulated both collagen type III and elastin gene expression. Finally, long-term uniaxial strain downregulated extracellular matrix (ECM) expression in more mature vSMC derivatives while upregulating elastin in less mature vSMC derivatives. Overall, our findings suggest that in vitro application of both cyclic uniaxial and circumferential tensile strain on hPSC-vSMC derivatives induces cell alignment and affects ECM gene expression. Therefore, mechanical stimulation of hPSC-vSMC derivatives using tensile strain may be important in modulating the phenotype and thus the function of vSMCs in tissue-engineered vessels.
Pub.: 26 Jun '15, Pinned: 16 Aug '17
Abstract: Tissue engineering offers the opportunity to develop vascular scaffolds that mimic the morphology of natural arteries. We have developed a porous three-dimensional scaffold consisting of fibres of collagen and elastin interspersed together. Scaffolds were obtained by freeze-drying a suspension of insoluble type I collagen and insoluble elastin. In order to improve the stability of the obtained matrices, they were cross-linked by two different methods. A water-soluble carbodi-imide, alone or in combination with a diamine, was used for this purpose: zero- or non-zero-length cross-links were obtained. The occurrence of cross-linking was verified by monitoring the thermal behaviour and the free-amino-group contents of the scaffolds before and after cross-linking. Smooth-muscle cells (SMCs) were cultured for different periods of time and their ability to grow and proliferate was investigated. SMCs were isolated from human umbilical and saphenous veins, and the purity of the cultures obtained was verified by staining with a specific monoclonal antibody (mAb). Cultured cells were also identified by mAbs against muscle actin and vimentin. After 14 days, a confluent layer of SMCs was obtained on non-cross-linked scaffolds. As for the cross-linked samples, no differences in cell attachment and proliferation were observed between scaffolds cross-linked using the two different methods. Cells cultured on the scaffolds were identified with an anti-(alpha-smooth-muscle actin) mAb. The orientation of SMCs resembled that of the fibres of collagen and elastin. In this way, it may be possible to develop tubular porous scaffolds resembling the morphological characteristics of native blood vessels.
Pub.: 23 Mar '04, Pinned: 16 Aug '17
Abstract: Biomaterials with the capacity to innately guide cell behaviour while also displaying suitable mechanical properties remain a challenge in tissue engineering. Our approach to this has been to utilise insoluble elastin in combination with collagen as the basis of a biomimetic scaffold for cardiovascular tissue engineering. Elastin was found to markedly alter the mechanical and biological response of these collagen-based scaffolds. Specifically, during extensive mechanical assessment elastin was found to reduce the specific tensile and compressive moduli of the scaffolds in a concentration dependant manner while having minimal effect on scaffold microarchitecture with both scaffold porosity and pore size still within the ideal ranges for tissue engineering applications. However, the viscoelastic properties were significantly improved with elastin addition with a 3.5-fold decrease in induced creep strain, a 6-fold increase in cyclical strain recovery, and with a four-parameter viscoelastic model confirming the ability of elastin to confer resistance to long term deformation/creep. Furthermore, elastin was found to result in the modulation of SMC phenotype towards a contractile state which was determined via reduced proliferation and significantly enhanced expression of early (α-SMA), mid (calponin), and late stage (SM-MHC) contractile proteins. This allows the ability to utilise extracellular matrix proteins alone to modulate SMC phenotype without any exogenous factors added. Taken together, the ability of elastin to alter the mechanical and biological response of collagen scaffolds has led to the development of a biomimetic biomaterial highly suitable for cardiovascular tissue engineering.
Pub.: 04 Oct '15, Pinned: 16 Aug '17
Abstract: Nanotechnology has enabled the engineering of a variety of materials to meet the current challenges and needs in vascular tissue regeneration. In this study, four different kinds of native proteins namely collagen, gelatin, fibrinogen, and bovine serum albumin were incorporated with polyurethane (PU) and electropsun to obtain composite PU/protein nanofibers. SEM studies showed that the fiber diameters of PU/protein scaffolds ranged from 245 to 273 nm, mimicking the nanoscale dimensions of native ECM. Human aortic smooth muscle cells (SMCs) were cultured on the electrospun nanofibers, and the ability of the cells to proliferate on different scaffolds was evaluated via a cell proliferation assay. Cell proliferation on PU/Coll nanofibers was found the highest compared to other electrospun scaffolds and it was 42 % higher than the proliferation on PU/Fib nanofibers after 12 days of cell culture. The cell–biomaterial interaction studies by SEM confirmed that SMCs adhered to PU/Coll and PU/Gel nanofibers, with high cell substrate coverage, and both the scaffolds promoted cell alignment. The functionality of the cells was further demonstrated by immunocytochemical analysis, where the SMCs on PU/Coll and PU/Gel nanofibers expressed higher density of SMC proteins such as alpha smooth muscle actin and smooth muscle myosin heavy chain. Cells expressed biological markers of SMCs including aligned spindle-like morphology on both PU/Coll and PU/Gel with actin filament organizations, better than PU/Fib and PU/BSA scaffolds. Our studies demonstrate the potential of randomly oriented elastomeric composite scaffolds for engineering of vascular tissues causing cell alignment.
Pub.: 09 Apr '13, Pinned: 16 Aug '17
Abstract: Elastin, critical for its structural and regulatory functions, is a missing link in vascular tissue engineering. Several elastin-inducting compounds have previously been reported, but their relative efficiency in promoting elastogenesis by adult arterial and venous vascular smooth muscle cells (VSMCs) and fibroblasts, four main vascular and elastogenic cells, has not been described. In addition to elasto-inductive substances, microRNA-29a was recently established as a potent post-transcriptional inhibitor of elastogenesis. Here, we explored if stimulating positive regulators or blocking inhibitors of elastogenesis could maximize elastin production.We tested whether the elasto-inducing compounds IGF-1, TGF-β1 and minoxidil could indeed augment elastin production, and whether microRNA-29a antagonism could block elastin production in adult arterial and venous fibroblasts and VSMCs. The effects on elastin, lysyl oxidase and fibrillin-1 mRNA expression levels and tropoelastin protein were determined.IGF-1 and minoxidil exerted little effect on tropoelastin mRNA expression levels in all cell types, while TGF-β1 predominantly enhanced mRNA tropoelastin levels, but this mRNA increase did not impact tropoelastin protein abundance. In contrast, microRNA29a-inhibition resulted in the upregulation of tropoelastin mRNA in all cell types, but most pronounced in venous VSMCs. Importantly, microRNA-29a-antagonism also enhanced lysyl oxidase and fibrillin-1 mRNA expression, and revealed a dose-dependent increase in tropoelastin protein expression in venous VSMCs.Our studies suggest that the elastogenic potential of microRNA-29a-inhibition in vascular cells is superior to that of established elastin-stimulating compounds IGF-1, TGF-β1 and minoxidil. Thus,microRNA-29a-antagonism could serve as an attractive means of enhancing elastin synthesis in tissue engineered blood vessels.
Pub.: 09 Sep '16, Pinned: 16 Aug '17
Abstract: The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-beta and PDGF-BB). Expression of smooth muscle protein 22alpha and alpha-smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies.
Pub.: 17 Jun '08, Pinned: 16 Aug '17
Abstract: Rat smooth muscle cells (SMCs) stably transfected with the gene for the phenotype regulating protein cyclic guanosine monophosphate-dependent protein kinase (PKG) were used as a cell source in the preparation of three-dimensional (3D) collagen type I vascular constructs. PKG-transfected cells expressed severalfold higher levels of the contractile protein smooth muscle alpha-actin (SMA), relative to untransfected SMCs, both in monolayer culture and in 3D gels. The proliferation rate of PKG-transfected cells was lower than that of untransfected cells in both culture geometries. Three-dimensional collagen constructs made with PKG-transfected cells compacted to a similar degree as those made with untransfected cells, and this compaction could be augmented by biochemical stimulation with platelet-derived growth factor BB (PDGF) or transforming growth factor beta(1) (TGF). Application of cyclic mechanical strain to tubular collagen gels seeded with PKG-transfected cells resulted in a higher degree of gel compaction and circumferential matrix alignment, relative to statically grown controls, but cell proliferation and SMA expression were not affected. These results show that genetic modification of SMCs can be used as a tool to control cell function in vascular tissue engineering, and that the function of such cells can be further modulated by application of biochemical and mechanical stimulation.
Pub.: 11 Mar '04, Pinned: 16 Aug '17
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