[Show abstract][Hide abstract] ABSTRACT: In vitro tissue engineering enables the fabrication of functional tissues for tissue replacement. In addition, it allows us to build useful physiological and pathological models for mechanistic studies. However, the translation of in vitro tissue engineering into clinical therapies presents a number of technical and regulatory challenges. It is possible to circumvent the complexity of developing functional tissues in vitro by taking an in situ tissue engineering approach that uses the body as a native bioreactor to regenerate tissues. This approach harnesses the innate regenerative potential of the body and directs the appropriate cells to the site of injury. This review surveys the biomaterial-, cell-, and chemical factor-based strategies to engineer tissue in vitro and in situ.
Annals of Biomedical Engineering 05/2014; · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Continuous flow particulate-based microfluidic processors are in critical demand for emerging applications in chemistry and biology, such as point-of-care molecular diagnostics. Challenges remain, however, for accomplishing biochemical assays in which microparticle immobilization is desired or required during intermediate stages of fluidic reaction processes. Here we present a dual-mode microfluidic reactor that functions autonomously under continuous flow conditions to: (i) execute multi-stage particulate-based fluidic mixing routines, and (ii) array select numbers of microparticles during each reaction stage (e.g., for optical detection). We employ this methodology to detect the inflammatory cytokine, interferon-gamma (IFN-γ), via a six-stage aptamer-based sandwich assay.
[Show abstract][Hide abstract] ABSTRACT: Blood vessels transport blood to deliver oxygen and nutrients. Vascular diseases such as atherosclerosis may result in obstruction of blood vessels and tissue ischemia. These conditions require blood vessel replacement to restore blood flow at the macrocirculatory level, and angiogenesis is critical for tissue regeneration and remodeling at the microcirculatory level. Vascular tissue engineering has focused on addressing these two major challenges. We provide a systematic review on various approaches for vascular graft tissue engineering. To create blood vessel substitutes, bioengineers and clinicians have explored technologies in cell engineering, materials science, stem cell biology, and medicine. The scaffolds for vascular grafts can be made from native matrix, synthetic polymers, or other biological materials. Besides endothelial cells, smooth muscle cells, and fibroblasts, expandable cells types such as adult stem cells, pluripotent stem cells, and reprogrammed cells have also been used for vascular tissue engineering. Cell-seeded functional tissue-engineered vascular grafts can be constructed in bioreactors in vitro. Alternatively, an autologous vascular graft can be generated in vivo by harvesting the capsule layer formed around a rod implanted in soft tissues. To overcome the scalability issue and make the grafts available off-the-shelf, nonthrombogenic vascular grafts have been engineered that rely on the host cells to regenerate blood vessels in situ. The rapid progress in the field of vascular tissue engineering has led to exciting preclinical and clinical trials. The advancement of micro-/nanotechnology and stem cell engineering, together with in-depth understanding of vascular regeneration mechanisms, will enable the development of new strategies for innovative therapies. For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.
Wiley Interdisciplinary Reviews Systems Biology and Medicine 10/2013; · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biochemical factors can help reprogram somatic cells into pluripotent stem cells, yet the role of biophysical factors during reprogramming is unknown. Here, we show that biophysical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically, decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)-a subunit of H3 methyltranferase-by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications.
[Show abstract][Hide abstract] ABSTRACT: Due to high incidence of vascular bypass procedures, an unmet need for suitable vessel replacements exists, especially for small-diameter (< 6 mm) vascular grafts. Here, we developed a novel, bilayered, synthetic vascular graft of 1-mm diameter that consisted of a microfibrous luminal layer and a nanofibrous outer layer, which was tailored to possess the same mechanical property as native arteries. We then chemically modified the scaffold with mucin, a glycoprotein lubricant on the surface of epithelial tissues, by either passive adsorption or covalent bonding via di-amino-poly(ethylene glycol) (PEG) linker to microfibers. Under static and physiological flow conditions, conjugated mucin was more stable than adsorbed mucin on the surfaces. Mucin could slightly inhibit blood clotting, and mucin coating suppressed platelet adhesion on microfibrous scaffolds. In the rat common carotid artery anastomosis model, grafts with conjugated mucin but not adsorbed mucin exhibited excellent patency and higher cell infiltration into the graft walls. Mucin, which can be easily obtained from autologous sources, offers a novel method for improving the hemocompatibility and surface lubrication of vascular grafts and many other implants.
Tissue Engineering Part A 08/2013; · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Induced pluripotent stem cells (iPSCs) hold great potential for cell therapy and tissue engineering. Neural crest stem cells (NCSCs) are multipotent and capable of differentiating into mesenchymal lineages. In this study, we investigated whether iPSC-derived NCSCs (iPSC-NCSCs) have potential for tendon repair. Human iPSC-NCSCs were suspended in fibrin gel and transplanted into a rat patellar tendon window defect. At 4 weeks post-transplantation, macroscopical observation showed that the repair of iPSC-NCSC-treated tendons was superior to that of non-iPSC-NCSC-treated tendons. Histological and mechanical examinations revealed that iPSC-NCSCs treatment signifcantly enhanced tendon healing as indicated by the improvement in matrix synthesis and mechanical properties. Furthermore, transplanted iPSC-NCSCs produced fetal tendon-related matrix proteins, stem cell recruitment factors, tenogenic differentiation factors, and accelerated the host endogenous repair process. This study demonstrates a potential strategy of employing iPSC-derived NCSCs for tendon tissue engineering.
Tissue Engineering Part A 07/2013; · 4.64 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Abstract Polyacrylamide gels with different stiffness and glass were employed as substrates to investigate how substrate stiffness affects the cellular stiffness of adherent hepatocellular carcinoma (HCCLM3) and hepatic (L02) cells. The interaction of how cell-substrate stiffness influences cell migration was also explored. An atom force microscope measured the stiffness of HCCLM3 and L02 cells on different substrates. Further, F-actin assembly was analyzed using immunofluorescence and Western blot. Finally, cell-surface expression of integrin β1 was quantified by flow cytometry. The results show that, while both HCCLM3 and L02 cells adjusted their cell stiffness to comply with the stiffness of the substrate they were adhered to, their tuning capabilities were different. HCCLM3 cell stiffness complied when substrate stiffness was between 1.1 and 33.7 kPa, whereas the analogous stiffness for L02 cells occurred at a higher substrate stiffness, 3.6 kPa up to glass. These ranges correlated with F-actin filament assembly and integrin β1 expression. In a migration assay, HCCLM3 cells migrated faster on a relatively soft substrate, while L02 cells migrated faster on substrates that were relatively rigid. These findings indicate that different tuning capabilities of HCCLM3 and L02 cells may influence cell migration velocity on substrates with different stiffness by regulating cy- toskeleton remodeling and integrin β1 expression.
[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] ABSTRACT: Microenvironmental biophysical stimuli influence diverse cellular functions, such as directional motility and stem cell differentiation. Previously, researchers have tuned the linear stiffness of microposts to investigate cell mechanobiological processes and direct cellular behavior; however, microposts suffer from an inherent, yet critical drawback - regulation of micropost stiffness is fundamentally limited to “biaxial” control. To overcome this issue, here we utilize three-dimensional (3D) direct-write laser lithography processes to fabricate arrays of microscale springs (μSprings). By adjusting the geometric characteristics of individual μSprings, the x-, y-, and z-axis stiffness of the cellular substrate can be customized at the microscale. COMSOL simulations were performed to characterize the theoretical “triaxial” stiffness associated with a variety of μSpring designs. Endothelial cells seeded on μSpring arrays were found to successfully deform the μSprings via cell-generated forces. By enabling user-control over the triaxial stiffness of discrete, microscale substrate features, the presented μSpring methodology could offer a powerful platform for cellular studies and applications in fields including tissue engineering, biomaterials, and regenerative medicine.
Micro Electro Mechanical Systems (MEMS), 2013 IEEE 26th International Conference on; 01/2013
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND/PURPOSE: Neurological function in patients with myelomeningocele (MMC) is limited even after prenatal repair. Neural crest stem cells (NCSCs) can improve neurological function in models of spinal cord injury. We aimed to evaluate the survival, integration, and differentiation of human NCSCs derived from induced pluripotent stem cells (iPSC-NCSCs) in the fetal lamb model of MMC. METHODS: Human iPSCs derived from skin fibroblasts were differentiated into NCSCs in vitro, mixed with hydrogel, and seeded on nanofibrous scaffolds for surgical transplantation. Fetal lambs (n=2) underwent surgical MMC creation and repair with iPSC-NCSC seeded scaffolds. Gross necropsy and immunohistochemistry were performed at term. RESULTS: IPSC-NCSCs expressed NCSC markers, maintained > 95% viability, and demonstrated neuronal differentiation in vitro. Immunohistochemical analysis of repaired spinal cords thirty days after transplantation demonstrated the co-localization of human nuclear mitotic apparatus protein (NuMA) and Neurofilament M subunit (NFM) in the area of spinal cord injury. No gross tumors were identified. CONCLUSIONS: Human iPSC-NCSCs survived, integrated, and differentiated into neuronal lineage in the fetal lamb model of MMC. This is the first description of human stem cell engraftment in a model of fetal MMC and supports the concept of using NCSCs to address spinal cord damage in MMC.
Journal of Pediatric Surgery 01/2013; 48(1):158-163. · 1.38 Impact Factor
[Show abstract][Hide abstract] 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; · 11.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Precision hydrodynamic controls of microparticles (e.g., microbeads and cells) are critical to diverse lab-on-a-chip applications. Microfluidic particulate-based arraying techniques are widely used; however, achieving full microarray resettability without sacrificing trapping performance has remained a significant challenge. Here we present a single-layer hydrodynamic methodology for releasing high numbers of microparticles after a microfluidic arraying process. Experiments with suspended streptavidin-coated polystyrene microbeads (15 μm in diameter) revealed resetting efficiencies of 100%, with trapping and loading efficiencies of 99% and 99.8%, respectively. Experiments with suspended endothelial cells (13-17 μm in diameter) revealed trapping efficiencies of 65% and 93% corresponding to arraying of one cell or at least one cell per trap, respectively, with loading efficiencies of 78%. Full cell-based resettability was also observed, with the caveat that reagents that promote cellular detachment from the substrate were required. The presented resettable microarray could be readily integrated into bead-based or cell-based microfluidic platforms to enable: (i) the retrieval of high numbers of microparticles (e.g., for subsequent analyses and/or use in additional experiments), and (ii) microarray reusability.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: "Multi-stage" fluidic reactions are integral to diverse biochemical assays; however, such processes typically require laborious and time-intensive fluidic mixing procedures in which distinct reagents and/or washes must be loaded sequentially and separately (i.e., one-at-a-time). Microfluidic processors that enable multi-stage fluidic reactions with suspended microparticles (e.g., microbeads and cells) to be performed autonomously could greatly extend the efficacy of lab-on-a-chip technologies. Here we present a single-layer microfluidic reactor that utilizes a microfluidic railing methodology to passively transport suspended microbeads and cells into distinct, adjacent laminar flow streams for rapid fluidic mixing and assaying. Four distinct molecular synthesis processes (i.e., consisting of 48 discrete fluidic mixing stages in total) were accomplished on polystyrene microbead substrates (15 μm in diameter) in parallel, without the need for external observation or regulation during device operation. Experimental results also revealed successful railing of suspended bovine aortic endothelial cells (approximately 13 to 17 μm in diameter). The presented railing system provides an effective continuous flow methodology to achieve bead-based and cell-based microfluidic reactors for applications including point-of-care (POC) molecular diagnostics, pharmacological screening, and quantitative cell biology.
Lab on a Chip 08/2012; 12(20):4168-77. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spinal cord injury (SCI) remains a major challenge for regenerative medicine. Following SCI, axon growth inhibitors and other inflammatory responses prevent functional recovery. Previous studies have demonstrated that rolipram, an anti-inflammatory and cyclic adenosine monophosphate preserving small molecule, improves spinal cord regeneration when delivered systemically. However, more recent studies showed that rolipram has some adverse effects in spinal cord repair. Here, we developed a drug-delivery platform for the local delivery of rolipram into the spinal cord. The potential of drug-eluting microfibrous patches for continuous delivery of high and low-dose rolipram concentrations was characterized in vitro. Following C5 hemisections, athymic rats were treated with patches loaded with low and high doses of rolipram. In general, animals treated with low-dose rolipram experienced greater functional and anatomical recovery relative to all other groups. Outcomes from the high-dose rolipram treatment were similar to those with no treatment. In addition, high-dose treated animals experienced reduced survival rates suggesting that systemic toxicity was reached. With the ability to control the release of drug dosage locally within the spinal cord, drug-eluting microfibrous patches demonstrate the importance of appropriate local release-kinetics of rolipram, proving their usefulness as a therapeutic platform for the study and repair of SCI.
Journal of Controlled Release 05/2012; 161(3):910-7. · 7.63 Impact Factor
[Show abstract][Hide abstract] 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.