Tissue engineering最新文献

Objective: In this study, we describe a novel bioreactor system to deliver controlled stretch protocols to bioengineered heart muscle (BEHM) constructs. Our primary objective was to evaluate the effect of mechanical stretch on the contractile properties of three-dimensional cardiac constructs in vitro.

Methods: BEHMs were formed by culturing primary neonatal cardiac myocytes in a fibrin gel using a method previously developed in our laboratory. A custom bioreactor system was designed using SolidWorks (Concord, MA) and structural components were manufactured using fusion deposition modeling. We utilized the bioreactor to evaluate the effect of 2-, 6-, and 24-hour stretch protocols on the stretch-induced changes in contractile function of BEHMs.

Results: We were able to demonstrate compatibility of the bioreactor system with BEHMs and were able to stretch all the constructs with zero incidence of failure. We found that loading the constructs for 2, 6, and 24 hours during a 24-hour period using a stretch protocol of 1 Hz, 10% stretch did not result in any significant change in the active force, specific force, pacing characteristics, and morphological features.

Conclusions: In this study, we demonstrate compatibility of a novel bioreactor system with BEHMs and the stability of the BEHMs in response to stretch protocols.

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. Two bioresorbable nonwoven scaffolds, polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) (90/10 copolymer of L-lactide and glycolide), were seeded with human chondrocytes after initial progeny in a monolayer with a serum-free medium. Two subgroups of nontreated and plasma-treated (using low-pressure plasma technique) scaffolds were investigated. The constructs were cultivated after seeding in six-well plates with serum-free medium for 7 days and implanted subcutaneously into nude mice for 6 and 12 weeks. Chondrogenic differentiations were investigated using immunhistology and reverse transcriptase-polymerase chain reaction. Cell adhesion only differed from 50% to 65% without a significant difference between the groups. During further cultivation for 7 days, the aggrecan synthesis of the seeded constructs was always higher in the PGA groups (p < 0.05). The mRNA gene expression for collagen type II was significantly higher in the PGA groups after 6 and 12 weeks (p < 0.05). A decrease in the expression of collagen type I was investigated in all groups. The expression for collagen type X and cartilage oligomeric matrix protein (COMP) increased in all groups over time. After cell proliferation in serum-free medium, the long-term chondrogenic differentiation in PGA scaffolds in vitro is cartilage specific and may be utilized in cartilage tissue engineering applications.

Oxygen is a potent modulator of cell function and wound repair in vivo. The lack of oxygen (hypoxia) can create a potentially lethal environment and limit cellular respiration and growth or, alternatively, enhance the production of the specific extracellular matrix components and increase angiogenesis through the hypoxia-inducible factor-1 pathway. For the in vitro generation of clinically relevant tissue-engineered grafts, these divergent actions of hypoxia should be addressed. Diffusion through culture medium and tissue typically limits oxygen transport in vitro, leading to hypoxic regions and limiting the viable tissue thickness. Approaches to overcoming the transport limitations include culture with bioreactors, scaffolds with artificial microvasculature, oxygen carriers, and hyperbaric oxygen chambers. As an alternate approach, angiogenesis after implantation may be enhanced by incorporating endothelial cells, genetically modified cells, or specific factors (including vascular endothelial growth factor) into the scaffold or exposing the graft to a hypoxic environment just before implantation. Better understanding of the roles of hypoxia will help prevent common problems and exploit potential benefits of hypoxia in engineered tissues.

Innovative scaffold fabrication, angiogenesis promotion, and dynamic tissue culture techniques have been utilized to improve delivery of media into the core of large tissue constructs in tissue engineering. We have developed here an intra-tissue perfusion (ITP) system, which incorporates an array of seven micron-sized needles as a delivery conduit, to improve mass transfer into the core of thick liver tissues slices (>>300 microm mass transport limit). The ITP system improves the uniformity and distribution of media throughout the tissue, resulting in improved cell viability over the static-cultured controls. The ITP-cultured thick liver slices also exhibit improved phase I and phase II metabolic functions and albumin and urea synthetic functions after 3-day culture, which is the minimal period required by the U.S. Food and Drug Administration (FDA) for studying drug-drug interaction. This ITP system can also be used for culturing other thick tissue constructs of larger dimensions for various in vitro and in vivo applications, including bridging integration of the in vitro cultured constructs into living host tissues.

Sinusoidal endothelial cells (SECs) are notoriously difficult to culture in vitro. SECs represent a highly specialized endothelial cell (EC) population, and traditional methods of SEC isolation from the liver initiate a process of SEC dedifferentiation. Acellular extracellular matrix (ECM) scaffolds were investigated in a physiologically relevant in vitro culture model for their ability to maintain SEC phenotype. The cell culture model used SECs only or a coculture of SECs with hepatocytes on ECM substrates derived from the liver (L-ECM), bladder (UBM-ECM), or small intestine submucosa (SIS-ECM). The effect of the ECM substrate upon SEC dedifferentiation was evaluated using scanning electron microscopy (SEM) and confocal microscopy. When SECs alone were cultured on uncoated glass slides, collagen I, UBM-ECM, or SIS-ECM, SECs showed signs of dedifferentiation after 1 day. In contrast, SECs alone cultured on L-ECM maintained their differentiated phenotype for at least 3 days, indicated by the presence of many fenestrations on SEC surface, expression of anti-rat hepatic sinusoidal endothelial cells mouse IgG MoAb (SE-1), and lack of expression of CD31. When SECs were cocultured with hepatocytes on any of the ECM scaffolds, the SECs maintained a near-normal fenestrated phenotype for at least 1 day. However, SEM revealed that the shape, size, frequency, and organization of the fenestrations varied greatly depending on ECM source. At all time points, SECs cocultured with hepatocytes on L-ECM maintained the greatest degree of differentiation. The present study demonstrated that the acellular ECM scaffold derived from the liver maintained SEC differentiation in culture longer than any of the tested substrate materials. The replacement of complex tissues and 3-dimensional organs may require specialized scaffolds to support multiple, functional cell phenotypes.

To investigate the use of a scaffold seeded with genetically modified meniscal cells or mesenchymal stem cells (MSCs) for the healing of meniscal lesions, primary meniscus cells and bone marrow-derived MSCs were isolated from bovine calves and transduced with first-generation adenoviral vectors encoding green fluorescent protein, luciferase, or transforming growth factor (TGF)-beta1 complementary deoxyribonucleic acid (cDNA). The genetically modified cells were seeded in type I collagen-glycosaminoglycan (GAG) matrices and transplanted into tears of the avascular zone of bovine menisci. After 3 weeks of in vitro culture, constructs and repair tissues were analyzed histologically, biochemically, and using reverse transcriptase polymerase chain reaction. Recombinant adenovirus readily transduced meniscal cells and MSCs, and transgene expression remained high after the cells were incorporated into collagen-GAG matrices. Transfer of TGF-beta1 cDNA increased cellularitiy and the synthesis of GAG/DNA [microg/microg]. It also led to stronger staining for proteoglycans and type II collagen and enhanced expression of meniscal genes. Transplantation of the TGF-beta1 transduced constructs into meniscal lesions of the avascular zone resulted in filling of the lesions with repair tissue after 3 weeks of in vitro culture. These results indicate that TGF-beta1 cDNA delivery may affect cell-based meniscus repair approaches in vivo.

Synthetic urothelium is an important goal for the tissue-engineering field that would have great utility for treating diseases and congenital defects affecting the urinary tract. A key step in the development of synthetic tissue is optimizing the conditions for coating biomaterials with cells of interest. Initial cell attachment is an important consideration when designing tissue-engineering scaffolds. The scaffold environment must also be conducive to cell proliferation and differentiation. The most popular materials for tissue-engineering scaffold often have suboptimal properties when analyzed for cell attachment and growth. It would then be of interest to know, for urinary tract tissue-engineering applications, which extracellular matrix protein coatings can facilitate urothelial cell attachment and encourage growth. Cells grown on 96-well cycloolefin plates coated with type IV or type I collagen exhibited improved initial attachment over plates coated with fibronectin or laminin. After 20 h, deoxyribonucleic acid synthesis was found to increase in cultures grown on type IV collagen, fibronectin, and laminin. Total metabolic activity of urothelial cell cultures was also monitored, and no difference was seen between any protein-coating conditions. The development of such reliable assays will be beneficial in monitoring the fate of scaffolds seeded with human urothelial cells.

Heart valve interstitial cells (VICs) appear to have a dynamic and reversible phenotype, an attribute speculated to be necessary for valve tissue remodeling during times of development and repair. Therefore, we hypothesized that the cytoskeletal (CSK) remodeling capability of the aortic and pulmonary VICs (AVICs and PVICs, respectively), which are dominated by smooth muscle alpha-actin, would exhibit unique contractile behaviors when seeded on collagen gels. Using a porcine cell source, we observed that VIC populations did not contract the gels at early time points (2 and 4 hours) as dermal fibroblasts did, but formed a central cluster of cells prior to contraction. After clustering, VICs appeared to radiate out from the center of the gels, whereas fibroblasts did not migrate but contracted the gels locally. VIC gels treated with transforming growth factor beta1 contracted the gels rapidly, revealing similar sensitivity to the cytokine. Moreover, we evaluated the initial mechanical state of the underlying CSK by comparing AVIC and PVIC stiffness with atomic force microscopy. Not only were AVICs significantly stiffer (p < 0.001) than the PVICs, but they also contracted the gels significantly more at 24 and 48 hours (p < 0.001). Taken together, these findings suggest that the AVICs are capable of inducing greater extra cellular matrix contraction, possibly manifesting in a more pronounced ability to remodel valvular tissues. Moreover, significant mechanobiological differences between AVICs and PVICs exist, and may have implications for understanding native valvular tissue remodeling. Elucidating these differences will also define important functional endpoints in the development of tissue engineering approaches for heart valve repair and replacement.

Small pore sizes inherent to electrospun matrices can hinder efficient cellular ingrowth. To facilitate infiltration while retaining its extracellular matrix-like character, electrospinning was combined with salt leaching to produce a scaffold having deliberate, engineered delaminations. We made elegant use of a specific randomizing component of the electrospinning process, the Taylor Cone and the falling fiber beneath it, to produce a uniform, well-spread distribution of salt particles. After 3 weeks of culture, up to 4 mm of cellular infiltration was observed, along with cellular coverage of up to 70% within the delaminations. To our knowledge, this represents the first observation of extensive cellular infiltration of electrospun matrices. Infiltration appears to be driven primarily by localized proliferation rather than coordinated cellular locomotion. Cells also moved from the salt-generated porosity into the surrounding electrospun fiber matrix. Given that the details of salt deposition (amount, size, and number density) are far from optimized, the result provides a convincing illustration of the ability of mammalian cells to interact with appropriately tailored electrospun matrices. These layered structures can be precisely fabricated by varying the deposition interval and particle size conceivably to produce in vivo-like gradients in porosity such that the resulting scaffolds better resemble the desired final structure.

In this study, we delineate the sequential expression of selected growth factors associated with bone formation in vitro. Mineralization, osteocalcin, and alkaline phosphatase (ALP-2) were measured to monitor the differentiation and maturation of osteoprogenitor cells collected from C57BL mice. Bone-related growth factors, including transforming growth factor beta (TGF-beta), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor (PDGF), insulinlike growth factor (IGF)-1, vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP)-2, and BMP-7, were selected. Enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) were used to measure growth factors at the protein and messenger ribonucleic acid (mRNA) level, respectively. The results found that ALP-2 expression increased progressively over time, whereas mineralization and osteocalcin did not become evident until culture day 14. VEGF and IGF-1 were upregulated early during proliferation. PDGF and TGF-beta mRNA expression was bimodal. FGF-2 and BMP-2 mRNAs were expressed only later in differentiation. FGF-2 mRNA signal levels were highest at day 14 and remained prominent through day 28 of culture. BMP-2 showed a similar profile as FGF-2. BMP-7 was not detectable using RT-PCR or ELISA. Strong correlations existed for the expression patterns between several early-response growth factors (VEGF, TGF-beta, and IGF-1) and were also evident for several late-response growth factors (BMP-2, PDGF, and FGF-2). Differential expression for grouped sets of growth factors occurs during the temporal acquisition of bone-specific markers as osteoprogenitor cell maturation proceeds in vitro.