Tissue engineering最新文献

Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium.

Cell guidance is a new tissue engineering concept aimed at total in vivo tissue engineering without the need for cell seeding. This technique aims to create a biomimetic environment through constant delivery of cytokines to different areas of an implanted scaffold, such that site-specific homing of cells can be achieved. In this study, expression of CXCR4 on mesenchymal stem cells (MSCs) was characterized by immunohistochemistry and flow cytometry, subsequent to which chemotaxis toward stromal cell-derived factor 1 (SDF-1) was demonstrated. In a subsequent three-dimensional in vitro study, MSCs were shown to migrate within a polycaprolactone scaffold in response to SDF-1, such that polarized tissue formation could be achieved. A customized cytokine microdelivery system comprising a reservoir housing system and microneedle apparatus was fabricated to ensure constant delivery of SDF-1 to the scaffold. Following on this experiment, we demonstrated in an in vivo rat bone tissue engineering model that a cytokine combination consisting of vascular endothelial growth factor, SDF-1, and bone morphogenetic protein-6 delivered at 10-day intervals through the microneedle apparatus could lead to tissue formation through migrating cell fronts, with evidence of angiogenesis and vascularization without the need for cell seeding on scaffolds prior to implantation. In summary, cell guidance offers an advancement in cellular methodology for tissue engineering, and promises a novel, minimally invasive option for tissue regeneration.

Liver tissue that is functional and viable for several weeks in vitro represents an auspicious test system for basic and applied research. In this study, a coculture system for hepatocytes (HCs) and microvascular endothelial cells (mECs) was generated applying tissue-engineering techniques, establishing the basis for a new bioartificial liver in vitro model. Porcine mECs were seeded on a decellularized porcine jejunal segment with preserved vascular structures. Porcine HCs were seeded onto this vascularized scaffold, and the resulting coculture was maintained for 3 weeks in vitro. Tissue morphology and differentiation was monitored using histology and immunohistochemistry. Tissue metabolism was monitored using daily assessment of urea and lactate production. HC monolayer cultures served as controls. The 2-stage seeding procedure resulted in a 3-dimensional coculture system harboring HC cell clusters in multiple cell layers lining the generated mEC-seeded capillary structures. It was viable for 3 weeks, and HCs maintained their morphology and differentiation. Biochemical testing revealed stable metabolic activity of the tissue culture. In contrast, HCs cultured in monolayer showed morphological dedifferentiation and an unfavorable metabolic state. Our mEC-HC coculture represents a new approach toward a functional bioartificial liver-like tissue applicable as a test system for basic and applied research.

Keratinocyte stem cell technology provides at least an adjuvant therapy to clinically close large cutaneous wounds (e.g., burn wounds). Here, the performance of keratinocyte cultures depends primarily on the quality of the bed to which they are applied. Clinical take rates for cultured keratinocyte grafts are optimal when applied to a vascularized dermal bed with minimal bacterial colonization. In the absence of autologous dermis, staged reconstruction with a dermal equivalent or dermal regeneration template is required. A novel product, Hyalomatrix, is a bilayer of an esterified hyaluronan scaffold beneath a silicone membrane. The scaffold delivers hyaluronan to the wound bed, and the silicone membrane acts as a temporary epidermal barrier. The product has been investigated in a controlled, porcine, acute full-thickness excisional wound model. Cultured autologous keratinocytes (CAKs) were delivered on Laserskin to acute full-thickness wounds treated with Hyalomatrix within chambers, and graft take rates were assessed longitudinally using image analysis. In the absence of chambers, wound contraction was assessed. Clinical CAK take rates fall sequentially with delay in application post-Hyalomatrix pre-treatment, but repeated pre-treatment removed this, with maximal take of 57.2% at 5 weeks post-wounding. In the absence of chambers, more-complete wound closure resulted from edge re-epithelialization and contraction, by a factor of 5 at 1 month, and was achieved at least 2 weeks sooner in the gold standard controls of split-thickness autograft to an acute or pre-treated wound bed. Wound contraction and late neodermal morphology (1 year) were similar in pre-treated CAKs and split-thickness autograft wounds. In this model, the Hyalomatrix wound bed pre-treatment increase in CAK take appeared to be dose dependent. The product appeared to act as a hyaluronan delivery system rather than a dermal regeneration template. The silicone membrane may limit wound bed colonization, and the combination of this temporary barrier with hyaluronan delivery and neodermis induction has been termed a barrier-delivery-induction system. The development of similar systems for serial application offers an alternative to a dermal regeneration template when CAKs are engrafted in the hostile, colonized environment of large burn wounds.

Fetal dermal fibroblasts participate in a dramatically different wound healing process compared to their adult counterparts, and it is thought that their intrinsic phenotype contributes to the unique properties of fetal repair. In particular, fibroblast migratory and contractile properties have been shown to be important in the development or lack of fibrosis/scarring. Despite extensive study to date, and multiple experimental techniques utilized by various laboratories, the precise differences between fetal and adult dermal fibroblasts remain unclear. We characterized the migratory and contractile dynamics of fetal dermal fibroblasts at the individual cell and population levels under both 2-dimensional (2D) and 3-dimensional (3D) constraints. Data indicate that (1) individual fetal fibroblasts attach and locomote quicker than adult fibroblasts, resulting in faster migration at the population level; (2) use of a 2D bioactive matrix (collagen) dramatically speeds up the transition from attachment to locomotion; and (3) fetal fibroblasts compact 2D collagen matrices faster than adult fibroblasts. These characteristics are maintained inside of a novel 3D construct, which approximates some in vivo tissue repair dynamics. Specifically, fetal fibroblasts invade this construct faster than adult fibroblasts, likely through more dynamic interactions with surrounding collagen fibers. In conclusion, the hyperactive migratory and contractile dynamics of fetal fibroblasts are qualitatively and quantitatively conserved despite transitions from individual cells to whole populations and from 2D to 3D constraints. We conclude that fetal fibroblasts display a robust phenotype, which is only partially altered by changes in substrate and geometric constraints. This phenotype likely is important in dictating the dynamics of fetal tissue repair.

Growth factors are an important tool in tissue engineering. Bone morphogenetic protein-2 and transforming growth factor-beta(1) (TGF-beta(1)) are used to provide bioactivity to surgical implants and tissue substitute materials. Mostly growth factors are used in soluble or adsorbed form. However, simple adsorption of proteins to surfaces is always accompanied by reduced stability and undefined pharmacokinetics. This study aims to prove that TGF-beta(1) can be covalently immobilized to functionalized surfaces, maintaining its ability to induce myofibroblastic differentiation of normal human dermal fibroblasts. In vivo, fibroblasts differentiate to myofibroblasts (MFs) during soft tissue healing by the action of TGF-beta(1). As surfaces for our experiments, we used slides bearing aldehyde, epoxy, or amino groups. For our in vitro cell culture experiments, we used the expression of alpha-smooth muscle actin as a marker for MFs after immunochemical staining. Using the aldehyde and the epoxy slides, we were able to demonstrate the activity of immobilized TGF-beta(1) through a significant increase in MF differentiation rate. A simple immunological test was established to detect TGF-beta(1) on the surfaces. This technology enables the creation of molecular "landscapes" consisting of several factors arranged in a distinct spatial pattern and immobilized on appropriate surfaces.

Loss of skeletal muscle profoundly affects the health and well-being of patients, and there currently is no way to replace lost muscle. We believe that a key step in the development of a prosthesis for reconstruction of dysfunctional muscular tissue is the ability to reconstitute the in vivo-like 3-dimensional (3D) organization of skeletal muscle in vitro with isolated satellite cells. In our present proof of principle studies, we have successfully constructed a multilayered culture of skeletal muscle cells, derived from neonatal satellite cells, that are distributed in a 3D pattern of organization that mimics many of the features of intact tissue. These multilayered cultures are composed of elongated multinucleated myotubes that are MyoD positive. Histological studies indicate that the multiple layers of myotubes can be distinguished. Expression of muscle-specific markers such as myosin heavy chain, dystrophin, integrin alpha-7, alpha-enolase, and beta-enolase was detected using real-time reverse transcriptase polymerase chain reaction at levels near adult values. Physiological measurements of the engineered skeletal muscle showed that they tetanize and display physiologic force length behavior, although developed force per cross-sectional area was below that of native rat skeletal muscle.

Recent studies reporting differentiation of early neural progenitors of human adipose tissue-derived stromal cells (ADSCs) has aroused interest among investigators for regenerative medicine. The aim of this study was to investigate the differentiation of ADSCs to neuron-like cells and to extend the life span of these differentiated ADSCs in vitro using our new DE-1 medium. After primary culture and expansion, ADSCs were incubated in a new long-term neuronal induction medium that maintains ADSCs in a differentiated state for 8 weeks. Neuronal differentiation was identified using immunocytochemistry, reverse-transcriptase polymerase chain reaction, and Western blotting. We found that the optimal differentiation protocol induced the ADSCs to express early neuronal markers, including nestin and neuronal nuclear antigen (NeuN), as well as the mature astrocyte marker glial fibrillary acidic protein (GFAP). Neuronal morphological characteristics were recognized in approximately 40% to 50% of the cell populations maintained over 8 weeks, and 60% to 80% of the differentiated cells expressed neuronal specific markers, including nestin, GFAP, NeuN, Trk-A, vimentin, and neuron-specific enolase. The data show that our DE-1 medium is capable of achieving a greater number of differentiated ADSCs for a longer period of time. This result bodes well for the application of ADSCs in in vivo peripheral nerve regeneration.

The objectives of this investigation were (1) to characterize the growth factor release profile of a basic fibroblast growth factor (bFGF)-coated three-dimensional (3D) polymer scaffold under static and cyclically strained conditions, and (2) to delineate the individual and collective contributions of locally released bFGF and mechanical strain on cellular morphology and gene expression in this 3D system. Scaffolds were treated with I(125)-bFGF and subjected to mechanical strain or maintained in a static environment and the media sampled for factor release over a period of 6 days. Over the first 10 hours, a burst release of 25% of the incorporated growth factor into the surrounding media was noted. At 24 hours, approximately 40% of the bFGF was released into the media, after which steady state was achieved and minimal subsequent release was noted. Mechanical stimulation had no effect on growth factor release from the scaffold in this system. To test the concerted effects of bFGF and mechanical stimulation on bone marrow stromal cells (BMSCs), scaffolds were loaded with 0, 100, or 500 ng of bFGF, seeded with cells, and subjected to mechanical strain or maintained in a static environment. Scaffolds were harvested at 1, 7, and 21 days for RT-PCR and histomorphometry. All scaffolds subjected to growth factor and/or mechanical stimulation demonstrated cellular adherence and spreading at 21 days. Conversely, in the absence of both bFGF and mechanical stimulation, cells demonstrated minimal cytoplasmic spread. Moreover, at 21 days, cells subjected to both mechanical stimulation and bFGF (500 ng) demonstrated the highest upregulation of stress-resistive (collagen I, III) and stress-responsive proteins (tenascin-C). The effect of growth factor may be dose sensitive, however, as unstrained scaffolds treated with 100 ng of bFGF demonstrated upregulation of gene expression comparable to strained scaffolds treated with lower doses of bFGF (0 or 100 ng). In conclusion, results from this study suggest that the stimulatory effects of bFGF are dose sensitive and appear to be influenced by the addition of mechanical strain. The concurrent application of biochemical and mechanical stimuli may be important in promoting the adaptation of BMSCs and driving the transcription of genes essential for synthesis of a functional ligament replacement tissue.