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ABSTRACT: Controlling the structure and organization of electrospun fibers is desirable for fabricating scaffolds and materials with defined microstructures. However, the effects of microtopography on the deposition and, in turn, the organization of the electrospun fibers are not well understood. In this study, conductive polydimethylsiloxane (PDMS) templates with different micropatterns were fabricated by combining photolithography, silicon wet etching, and PDMS molding techniques. The fiber organization was varied by fine-tuning the microtopography of the electrospinning collector. Fiber conformity and alignment were influenced by the depth and the slope of microtopography features, resulting in scaffolds comprising either an array of microdomains with different porosity and fiber alignment or an array of microwells. Microtopography affected the fiber organization for hundreds of micrometers below the scaffold surface, resulting in scaffolds with distinct surface properties on each side. In addition, the fiber diameter was also affected by the fiber conformity. The effects of the fiber arrangement in the scaffolds on the morphology, migration, and infiltration of cells were examined by in vitro and in vivo experiments. Cell morphology and organization were guided by the fibers in the microdomains, and cell migration was enhanced by the aligned fibers and the three-dimensional scaffold structure. Cell infiltration was correlated with the microdomain porosity. Microscale control of the fiber organization and the porosity at the surface and through the thickness of the fibrous scaffolds, as demonstrated by the results of this study, provides a powerful means of engineering the three-dimensional structure of electrospun fibrous scaffolds for cell and tissue engineering.
Biomacromolecules 03/2013; · 5.48 Impact Factor
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ABSTRACT: Tissue-specific stem cells can be coaxed or harvested for tissue regeneration. In this study, we identified and characterized a new type of stem cells from the synovial membrane of knee joint, named neural crest cell-like synovial stem cells (NCCL-SSCs). NCCL-SSCs showed the characteristics of neural crest stem cells: they expressed markers such as Sox10, Sox17, and S100β, were clonable, and could differentiate into neural lineages as well as mesenchymal lineages, although NCCL-SSCs were not derived from neural crest during the development. When treated with transforming growth factor β 1 (TGF- β 1), NCCL-SSCs differentiated into mesenchymal stem cells (MSCs), lost the expression of Sox17 and the differentiation potential into neural lineages, but retained the potential of differentiating into mesenchymal lineages. To determine the responses of NCCL-SSCs to microfibrous scaffolds for tissue engineering, electrospun composite scaffolds with various porosities were fabricated by co-electrospinning of structural and sacrificial microfibers. The increase of the porosity in microfibrous scaffolds enhanced cell infiltration in vitro and in vivo, but did not affect the morphology and the proliferation of NCCL-SSCs. Interestingly, microfibrous scaffolds with higher porosity increased the expression of chondrogenic and osteogenic genes but suppressed smooth muscle and adipogenic genes. These results suggest that the differentiation of NCCL-SSCs can be controlled by both soluble chemical factors and biophysical factors such as the porosity of the scaffold. Engineering both NCCL-SSCs and scaffolds will have tremendous potential for tissue regeneration.
Acta biomaterialia 03/2013; · 3.98 Impact Factor
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ABSTRACT: Poly(L-lactide) (PLLA) microfibrous scaffolds produced by electrospinning were treated with mild Ar or Ar-NH3/H2 plasmas to enhance cell attachment, growth, and infiltration. Goniometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) measurements were used to evaluate the modification of the scaffold surface chemistry by plasma treatment. AFM and XPS measurements showed that both plasma treatments increased the hydrophilicity without affecting the integrity of the fibrous structure and the fiber roughness, whereas Ar-NH3/H2 plasma treatment also resulted in surface functionalization with amine groups. Culture studies of bovine aorta endothelial cells and bovine smooth muscle cells on the plasma-treated PLLA scaffolds revealed that both Ar and Ar-NH3/H2 plasma treatments promoted cell spreading during the initial stage of cell attachment and, more importantly, increased the cell growth rate, especially for Ar plasma treatment. In vitro cell infiltration studies showed that both plasma treatments effectively enhanced cell in-growth into the microfibrous scaffolds. In vivo experiments involving the subcutaneous implantation of plasma-treated PLLA scaffolds under the skin of Sprague-Dawley rats also showed increased cell infiltration. The results of this study indicate that surface treatment of PLLA microfibrous scaffolds with mild Ar or Ar-NH3/H2 plasmas may have important implications in tissue engineering. Further modifications with bioactive factors should improve the functions of the scaffolds for specific applications.
Tissue Engineering Part A 01/2013; · 4.64 Impact Factor
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ABSTRACT: The conversion of contractile vascular smooth muscle cells (SMCs) to a synthetic or proliferative phenotype is thought to play a major role in vascular diseases such as atherosclerosis and restenosis.(1-3) Our recent work presents evidence that challenges this widely accepted dogma. Our findings suggest that multipotent vascular stem cells (MVSCs) rather than SMCs are a major contributor to vascular remodeling.(4) The experimental results demonstrate that the major population of the traditionally defined "proliferative/synthetic SMCs" is derived from the differentiation of MVSCs rather than the de-differentiation of mature SMCs. Both In vitro and in vivo results suggest that vascular disease is a stem cell disease, which raises the question on the previous dogma: Is vascular disease a SMC disease?In this issue of Circulation Research, a group of leaders in the area of SMC biology wrote a commentary on this work.(5) They present evidence in the literature that seems to support the SMC de-differentiation hypothesis. However, in many previous studies, it was incorrectly assumed that the vascular cells in the primary SMC culture and in injured blood vessels were mostly derived from SMCs. Thus, the previous experimental findings on vascular cells were often attributed to SMCs, which resulted in data misinterpretation and the overstatement on the roles of SMCs. While we agree that further investigations are needed to determine the relative contribution of MVSCs and SMCs to vascular remodeling in various animal models, we respectfully disagree on some of the arguments in the commentary. [Extract].
Circulation Research 10/2012; · 9.49 Impact Factor
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ABSTRACT: Small-diameter synthetic vascular grafts have high failure rate and tissue-engineered blood vessels are limited by the scalability. Here we engineered bioactive materials for in situ vascular tissue engineering, which recruits two types of endogenous progenitor cells for the regeneration of blood vessels. Heparin was conjugated to microfibrous vascular grafts to suppress thrombogenic responses, and stromal cell-derived factor-1α (SDF-1α) was immobilized onto heparin to recruit endogenous progenitor cells. Heparin-bound SDF-1α was more stable than adsorbed SDF-1α under both static and flow conditions. Microfibrous grafts were implanted in rats by anastomosis to test the functional performance. Heparin coating improved the short-term patency, and immobilized SDF-1α further improved the long-term patency. SDF-1α effectively recruited endothelial progenitor cells (EPCs) to the luminal surface of the grafts, which differentiated into endothelial cells (ECs) and accelerated endothelialization. More interestingly, SDF-1α increased the recruitment of smooth muscle progenitor cells (SMPCs) to the grafts, and SMPCs differentiated into smooth muscle cells (SMCs) in vivo and in vitro. Consistently, SDF-1α-immobilized grafts had significantly higher elastic modulus. This work demonstrates the feasibility of simultaneously recruiting progenitor cells of ECs and SMCs for in situ blood vessel regeneration. This in situ tissue engineering approach will have broad applications in regenerative medicine.
Biomaterials 08/2012; 33(32):8062-74. · 7.40 Impact Factor
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ABSTRACT: Electrospun scaffolds are used extensively in tissue-engineering applications as they offer a cell-friendly microenvironment. However, one major limitation is the dense fibers, small pore size and consequently poor cell infiltration. Here, we employ a femtosecond (FS) laser system to ablate and create microscale features on electrospun poly(L-lactide) (PLLA) nanofibrous scaffolds. Upon determining the ablation parameters, we pattern structured holes with diameters of 50, 100 and 200 μm and spacings of 50 and 200 μm between adjacent holes on the scaffolds. The elastic moduli of ablated scaffolds decrease with the decrease in spacing and the increase in hole size. Cells seeded on the laser-ablated scaffolds exhibit different morphology but similar proliferation rate when compared with control (non-ablated) scaffold. Furthermore, animal studies indicate that ablated scaffolds facilitate endothelial cell ingrowth as well as drastically increase M2 macrophage and overall cell infiltration. These findings demonstrate that FS laser ablation can be used to increase cell infiltration into nanofibrous scaffolds. Laser ablation not only can create desired features in micrometer length scale but also presents a new approach in the fabrication of three-dimensional porous constructs for tissue engineering.
Acta biomaterialia 04/2012; 8(7):2648-58. · 3.98 Impact Factor
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Journal of clinical orthodontics: JCO 04/2012; 46(4):225-32.
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ABSTRACT: Due to high incidence of vascular bypass procedures, an unmet need for suitable vessel replacements exists, especially for small-diameter vascular grafts. Here we produced 1-mm diameter vascular grafts with nanofibrous structure via electrospinning, and successfully modified the nanofibers by the conjugation of heparin using di-amino-poly(ethylene glycol) (PEG) as a linker. Antithrombogenic activity of these heparin-modified scaffolds was confirmed in vitro. After 1 month implantation using a rat common carotid artery bypass model, heparin-modified grafts exhibited 85.7% patency, versus 57.1% patency of PEGylated grafts and 42.9% patency of untreated grafts. Post-explant analysis of patent grafts showed complete endothelialization of the lumen and neovascularization around the graft. Smooth muscle cells were found in the surrounding neo-tissue. In addition, greater cell infiltration was observed in heparin-modified grafts. These findings suggest heparin modification may play multiple roles in the function and remodeling of nanofibrous vascular grafts, by preventing thrombosis and maintaining patency, and by promoting cell infiltration into the three-dimensional nanofibrous structure for remodeling.
IEEE transactions on nanobioscience 03/2012; 11(1):22-7. · 1.71 Impact Factor
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ABSTRACT: It is generally accepted that the de-differentiation of smooth muscle cells, from the contractile to the proliferative/synthetic phenotype, has an important role during vascular remodelling and diseases. Here we provide evidence that challenges this theory. We identify a new type of stem cell in the blood vessel wall, named multipotent vascular stem cells. Multipotent vascular stem cells express markers, including Sox17, Sox10 and S100β, are cloneable, have telomerase activity, and can differentiate into neural cells and mesenchymal stem cell-like cells that subsequently differentiate into smooth muscle cells. On the other hand, we perform lineage tracing with smooth muscle myosin heavy chain as a marker and find that multipotent vascular stem cells and proliferative or synthetic smooth muscle cells do not arise from the de-differentiation of mature smooth muscle cells. In response to vascular injuries, multipotent vascular stem cells, instead of smooth muscle cells, become proliferative, and differentiate into smooth muscle cells and chondrogenic cells, thus contributing to vascular remodelling and neointimal hyperplasia. These findings support a new hypothesis that the differentiation of multipotent vascular stem cells, rather than the de-differentiation of smooth muscle cells, contributes to vascular remodelling and diseases.
Nature Communications 01/2012; 3:875. · 7.40 Impact Factor
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ABSTRACT: The heterogeneity of vascular smooth muscle cells (SMCs) is related to their different developmental origins such as the neural crest and mesoderm. Derivation of SMCs from different origins will provide valuable in vitro models for the investigation of vascular development and diseases. From the perspective of regenerative medicine and tissue engineering, an expandable cell source of SMCs is required for the construction of tissue-engineered blood vessels. In this study, we developed a robust protocol to derive neural crest stem cells (NCSCs) from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). NCSCs derived from ESCs and iPSCs were expandable with similar cell doubling times. NCSCs were capable of differentiating into neural and mesenchymal lineages. TGF-β1 induced the expression of SMC markers calponin-1, SM22α, and smooth muscle myosin heavy chain and resulted in the assembly of smooth muscle α-actin, calponin-1, and SM22α into stress fibers. This work provides a basis for using iPSCs to study SMC biology and deriving vascular cells for tissue engineering.
Cells Tissues Organs 01/2012; 195(1-2):5-14. · 2.20 Impact Factor
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ABSTRACT: Vascular smooth muscle cells (SMCs) are a major cell type involved in vascular remodeling. The various developmental origins of SMCs such as neural crest and mesoderm result in heterogeneity of SMCs, which plays an important role in the development of vascular remodeling and diseases. Upon vascular injury, SMCs are exposed to blood flow and subjected to fluid shear stress. Previous studies have shown that fluid shear stress inhibits SMC proliferation. However, the effect of shear stress on the subpopulation of SMCs from specific developmental origin and vascular bed is not well understood. Here we investigated how shear stress regulates human aortic SMCs positive for neural crest markers. DNA microarray analysis showed that shear stress modulates the expression of genes involved in cell proliferation, matrix synthesis, cell signaling, transcription and cytoskeleton organization. Further studies demonstrated that shear stress induced SMC proliferation and cyclin D1, downregulated cell cycle inhibitor p21, and activated Akt pathway. Inhibition of PI-3 kinase blocked these shear stress-induced changes. These results suggest that SMCs with neural crest characteristics may respond to shear stress in a different manner. This finding has significant implications in the remodeling and diseases of blood vessels.
Cellular and Molecular Bioengineering 12/2011; 4(4):627-636. · 1.95 Impact Factor
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ABSTRACT: Neural crest stem cells (NCSCs) are multipotent and play an important role during the development and tissue regeneration. However, the anisotropic effects of mechanical strain on NCSCs are not known. To investigate the anisotropic mechanosensing by NCSCs, NCSCs derived from induced pluripotent stem cells were cultured on micropatterned membranes, and subjected to cyclic uniaxial strain in the direction parallel or perpendicular to the microgrooves. Cell and nuclear shape were both regulated by micropatterning and mechanical strain. Among the unpatterned, parallel-patterned and perpendicular-patterned groups, mechanical strain caused an increase in histone deacetylase activity in the parallel-patterned group, accompanied by the increase of cell proliferation. In addition, mechanical strain increased the expression of contractile marker calponin-1 but not other differentiation markers in the unpatterned and parallel-patterned groups. These results demonstrated that NCSCs responded differently to the anisotropic mechanical environment. Understanding the mechanical regulation of NCSCs will reveal the role of mechanical factors in NCSC differentiation during development, and provide a basis for using NCSCs for tissue engineering.
Annals of biomedical engineering 11/2011; 40(3):598-605. · 2.41 Impact Factor
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ABSTRACT: Induced pluripotent stem cells (iPSCs) hold great promise for cell therapies and tissue engineering. Neural crest stem cells (NCSCs) are multipotent and represent a valuable system to investigate iPSC differentiation and therapeutic potential. Here we derived NCSCs from human iPSCs and embryonic stem cells (ESCs), and investigated the potential of NCSCs for neural tissue engineering. The differentiation of iPSCs and the expansion of derived NCSCs varied in different cell lines, but all NCSC lines were capable of differentiating into mesodermal and ectodermal lineages, including neural cells. Tissue-engineered nerve conduits were fabricated by seeding NCSCs into nanofibrous tubular scaffolds, and used as a bridge for transected sciatic nerves in a rat model. Electrophysiological analysis showed that only NCSC-engrafted nerve conduits resulted in an accelerated regeneration of sciatic nerves at 1 month. Histological analysis demonstrated that NCSC transplantation promoted axonal myelination. Furthermore, NCSCs differentiated into Schwann cells and were integrated into the myelin sheath around axons. No teratoma formation was observed for up to 1 year after NCSC transplantation in vivo. This study demonstrates that iPSC-derived multipotent NCSCs can be directly used for tissue engineering and that the approach that combines stem cells and scaffolds has tremendous potential for regenerative medicine applications.
Biomaterials 08/2011; 32(22):5023-32. · 7.40 Impact Factor
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ABSTRACT: Although the field of vascular tissue engineering has made tremendous advances in the past decade, several complications have
yet to be overcome in order to produce biocompatible small-diameter vascular conduits with long-term patency. Stem cells and
progenitor cells represent potential cell sources in the development of autologous (or allogeneic), nonthrombogenic vascular
grafts with mechanical properties comparable to native blood vessel. However, a better understanding of the effects of mechanical
forces on stem cells and progenitor cells is needed to properly utilize these cells for tissue engineering applications. In
this chapter, we discuss the current understanding of the effects of hemodynamic forces on the differentiation and function
of adult stem cells, embryonic stem cells, and progenitor cells. We also review the use of stem cells and progenitor cells
in vascular graft engineering.
05/2011: pages 227-244;
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ABSTRACT: Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions. However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape, HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 μm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.
Biophysical Journal 04/2011; 100(8):1902-9. · 3.65 Impact Factor
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ABSTRACT: Trauma injuries often cause peripheral nerve damage and disability. A goal in neural tissue engineering is to develop synthetic nerve conduits for peripheral nerve regeneration having therapeutic efficacy comparable to that of autografts. Nanofibrous conduits with aligned nanofibers have been shown to promote nerve regeneration, but current fabrication methods rely on rolling a fibrous sheet into the shape of a conduit, which results in a graft with inconsistent size and a discontinuous joint or seam. In addition, the long-term effects of nanofibrous nerve conduits, in comparison with autografts, are still unknown. Here we developed a novel one-step electrospinning process and, for the first time, fabricated a seamless bi-layer nanofibrous nerve conduit: the luminal layer having longitudinally aligned nanofibers to promote nerve regeneration, and the outer layer having randomly organized nanofibers for mechanical support. Long-term in vivo studies demonstrated that bi-layer aligned nanofibrous nerve conduits were superior to random nanofibrous conduits and had comparable therapeutic effects to autografts for nerve regeneration. In summary, we showed that the engineered nanostructure had a significant impact on neural tissue regeneration in situ. The results from this study will also lead to the scalable fabrication of engineered nanofibrous nerve conduits with designed nanostructure. This technology platform can be combined with drug delivery and cell therapies for tissue engineering.
Tissue Engineering Part C Methods 04/2011; 17(7):705-15. · 4.64 Impact Factor
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ABSTRACT: Surface chemical patterning of polystyrene (PS) dishes for long-term single-cell culture was accomplished by oxygen plasma treatment through the windows of a polydimethylsiloxane membrane mask that produced hydrophilic areas of different shapes and sizes, followed by overnight incubation with either Pluronic F108 solution or a mixture of Pluronic F108 solution and fibronectin. Selective cell attachment on pattern areas of PS dishes was investigated in light of cell seeding experiments and X-ray photoelectron spectroscopy measurements. Activation of the hydrophilic areas of patterned PS surfaces by serum proteins in the culture medium was conducive to cell attachment on the pattern areas of dishes incubated with only Pluronic solution. Preferential adsorption of fibronectin on hydrophilic pattern areas enhanced selective cell attachment on patterned dishes incubated with a mixture of Pluronic solution and fibronectin. Cell-culture experiments demonstrated an effect of surface patterning on both cell and nucleus shape and confirmed the long-term (>2 weeks) stability of the produced single-cell patterns in serum medium.
Journal of Biomedical Materials Research Part A 03/2011; 96(3):507-12. · 2.63 Impact Factor
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ABSTRACT: The extracellular matrix (ECM) microenvironment consists of structural and functional molecules. The ECM relays both biochemical and biophysical cues to and from the cells to modulate cell behavior and function. The biophysical cues can be engineered and applied to cells by means of spatial patterning, matrix rigidity, and matrix actuation. Tissue engineering strategies that utilize ECMs to direct stem cell organization and lineage specification show tremendous potential. This review describes the technologies for modulating ECM spatial patterning, matrix rigidity, chemical composition, and matrix actuation. The role of ECMs in vascular tissue engineering is then discussed as a model of tissue engineering and regenerative medicine.
Annals of biomedical engineering 03/2011; 39(4):1201-14. · 2.41 Impact Factor
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ABSTRACT: Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.
Biomaterials 03/2011; 32(16):3921-30. · 7.40 Impact Factor
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ABSTRACT: Trauma injuries often cause peripheral nerve damage and disability. A goal in neural tissue engineering is to develop synthetic nerve conduits for peripheral nerve regeneration having therapeutic efficacy comparable to that of autografts. Nanofibrous conduits with aligned nanofibers have been shown to promote nerve regeneration, but current fabrication methods rely on rolling a fibrous sheet into the shape of a conduit, which results in a graft with inconsistent size and a discontinuous joint or seam. In addition, the long-term effects of nanofibrous nerve conduits, in comparison with autografts, are still unknown. Here we developed a novel one-step electrospinning process, and for the first time, fabricated a seamless bi-layer nanofibrous nerve conduit: the luminal layer having longitudinally aligned nanofibers to promote nerve regeneration, and the outer layer having randomly organized nanofibers for mechanical support. Long-term in vivo studies demonstrated that bi-layer aligned nanofibrous nerve conduits were superior to random nanofibrous conduits and had comparable therapeutic effects to autografts for nerve regeneration. In summary, we showed that the engineered nanostructure had a significant impact on neural tissue regeneration in situ. The results from this study will also lead to the scalable fabrication of engineered nanofibrous nerve conduits with designed nanostructure. This technology platform can be combined with drug delivery and cell therapies for tissue engineering.
Tissue Engineering Part C Methods 03/2011; · 4.64 Impact Factor
