Tissue engineering最新文献

Photopolymerizable and degradable biomaterials are finding widespread application in the field of tissue engineering for the engineering of tissues such as bone, cartilage, and liver. The spatial and temporal control afforded by photoinitiated polymerizations has allowed for the development of injectable materials that can deliver cells and growth factors, as well as for the fabrication of scaffolding with complex structures. The materials developed for these applications range from entirely synthetic polymers (e.g., poly(ethylene glycol)) to purely natural polymers (e.g., hyaluronic acid) that are modified with photoreactive groups, with degradation based on the hydrolytic or enzymatic degradation of bonds in the polymer backbone or crosslinks. The degradation behavior also ranges from purely bulk to entirely surface degrading, based on the nature of the backbone chemistry and type of degradable units. The mechanical properties of these polymers are primarily based on factors such as the network crosslinking density and polymer concentration. As we better understand biological features necessary to control cellular behavior, smarter materials are being developed that can incorporate and mimic many of these factors.

Vascular smooth muscle cells (vSMCs) can switch between a contractile (differentiated) and a synthetic (dedifferentiated) phenotype. Synthetic, proliferative vSMCs are observed during embryogenesis, wound repair, and tissue engineering. The potential of isolated vSMCs to reverse this phenotypic modulation depends strictly on culture conditions. Previous studies have demonstrated that applied shear stress is an important signal for vSMC phenotype. The objective of this study was to determine whether applied shear stress is capable of triggering re-differentiation of vSMCs in tissue-engineered aortas. vSMCs were isolated from ovine arteries. Cells were cultured statically or exposed to two- (2D) and three-dimensional (3D) shear stress after seeding on a tubular matrix. For 3D in vivo testing, grafts were seeded additionally with endothelial cells and implanted in the descending aorta. Particular attention was paid to the expression pattern of vSMC markers, cell ultra-structure, matrix remodeling activity, and proliferative activity. Cultured vSMCs de-differentiated during static in vitro culture, but 2D and 3D in vitro shear stress promoted re-expression of vSMC markers. During in vivo culture, vSMCs progressed toward a fully differentiated phenotype. Cells were expressing markers of differentiated vSMCs and resembled a morphologically contractile vSMC phenotype. Matrix remodeling activity and proliferative activity decreased. This study demonstrates the phenotypic plasticity of vSMCs and their ability to return to a differentiated phenotype under shear stress conditions. These results are crucial for tissue engineering of blood vessels, because they indicate for the first time the in vitro potential to regain physiological functionality of isolated vSMCs.

Every tissue and organ has its own 3-dimensional (3D) extracellular matrix (ECM) organization. Cells in a 3D bioscaffold for tissue engineering typically align new ECM components according to the bioscaffold provided. Therefore, scaffolds with a specific 3D structural design resembling the actual ECM of a particular tissue may have great potential in tissue engineering. Here, we show that, using specific freezing regimes, 3D scaffolds that mimic the 3D architecture of specific tissues can be made from collagen. Three examples are given, namely, scaffolds resembling the cup-shaped parenchymal (alveolar) architecture of lung, scaffolds that mimic the parallel collagen organization of tendon, and scaffolds that mimic the 3D organization of skin. For the preparation of these tissue-specific scaffolds, we relied on simple techniques without the need for expensive or customized equipment. Freezing rate, type of suspension medium, and additives (e.g., ethanol) were found to be prime parameters in controlling scaffold morphology.

The advantage of using anatomically shaped scaffolds as compared to modeled designs was investigated and assessed in terms of cartilage formation in an artificial tracheal construct. Scaffolds were rapid prototyped with a technique named three-dimensional fiber deposition (3DF). Anatomical scaffolds were fabricated from a patient-derived computerized tomography dataset, and compared to cylindrical and toroidal tubular scaffolds. Lewis rat tracheal chondrocytes were seeded on 3DF scaffolds and cultured for 21 days. The 3-(4,5-dimethylthiazol-2yl)-2,5-dyphenyltetrazolium bromide (MTT) and sulfated glycosaminoglycan (GAG) assays were performed to measure the relative number of cells and the extracellular matrix (ECM) formed. After 3 weeks of culture, the anatomical scaffolds revealed a significant increase in ECM synthesis and a higher degree of differentiation as shown by the GAG/MTT ratio and by scanning electron microscopy analysis. Interestingly, a lower scaffold's pore volume and porosity resulted in more tissue formation and a better cell differentiation, as evidenced by GAG and GAG/MTT values. Scaffolds were compliant and did not show any signs of luminal obstruction in vitro. These results may promote anatomical scaffolds as functional matrices for tissue regeneration not only to help regain the original shape, but also for their improved capacity to support larger tissue formation.

The isolation of undifferentiated adult stem/progenitor cells remains a challenging task primarily due to the rare quantity of these cells in biological samples and the lack of unique markers. Herein, we report a relatively straightforward method for isolation of human mesenchymal stem cells (MSCs) based on their unusual resistance to osmotic lysis, which we term "osmotic selection" (OS). MSCs can remarkably withstand significant exposure to hypotonic conditions (> 30 min) with only a reversible impairment in cell proliferation and with no loss of stem cell potential after exposure. Comparison of MSCs to other circulating nonhematopoietic cells revealed a time regime, by which purification of these cells would be attainable without considerable cell loss. OS showed a 50-fold enrichment of fibroblast colony-forming units from umbilical cord blood samples when compared to commonly employed techniques. After upstream processing, isolated cells using OS were immunophenotyped to be CD14-, CD34-, CD45-, CD44+, CD105+, and CD106+, and displayed multipotent differentiation. Preliminary investigations to determine mechanisms responsible for osmolytic resistance revealed MSCs to have an ineffective volume of 59%, with the ability to double cell volume at infinite dilution. Disruption of filamentous actin polymerization by cytochalasin D sensitized MSCs to osmotic lysis, which suggests a cytoskeletal element involved in osmolytic resistance.

Bone marrow- and adipose tissue-derived stromal cells (BMSCs and ASCs, respectively) exhibit a similar capacity for osteogenic differentiation in vitro, but it is unclear whether they share a common differentiation process, because they originate from different tissues. The aim of this study was to explore BMSC and ASC osteogenic differentiation by focusing on the expression of extracellular matrix-related genes (ECMGs), which play a crucial role in osteogenesis and bone tissue regeneration in vivo. We characterized the gene expression profiles of BMSCs and ASCs using a custom complementary deoxyribonucleic acid microarray containing 55 ECMGs. Undifferentiated BMSCs and ASCs actively expressed a wide range of ECMGs. Once BMSCs and ASCs were placed in an osteogenic differentiation medium, 24 and 17 ECMGs, respectively, underwent considerable downregulation over the course of the culture period. The remaining genes were maintained at a similar expression level to corresponding uninduced cell cultures. Although the suppression phenomenon was consistent irrespective of stromal cell origin, collagen (COL)2A1, COL6A1, COL9A1, parathyroid hormone receptor, integrin (INT)-beta3, and TenascinX genes were only downregulated in osteogenic BMSCs, whereas COL1A2, COL3A1, COL4A1, COL5A2, COL15A1, osteopontin, osteonectin, and INT-beta1 genes were only downregulated in osteogenic ASCs. During this time period, cell viability was sustained, suggesting that the observed downregulation did not occur by selection and elimination of unfit cells from the whole cell population. These data suggest that osteogenically differentiating BMSCs and ASCs transition away from a diverse gene expression pattern, reflecting their multipotency toward a configuration specifically meeting the requirements of the target lineage. This change may serve to normalize gene expression in mixed populations of stem cells derived from different tissues.

This study focused on mimicking collagen structurally and biologically using various peptide sequences toward realizing an artificial collagen-like biomaterial. Collagen-mimetic peptides (CMPs) incorporating integrin-specific glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) sequence from residues 502 to 507 of collagen alpha(1)(I) were used as a bioadhesive matrix and grafted onto poly(3-hydroxybutyrate-co3-hydroxyvalerate) microspheres to optimize cell adhesion, proliferation, and functions. Cell recognition of these biomolecules appeared to be conformation dependent, with the CMP1 of higher triple helix stability being preferred. Absence of the GFOGER hexapeptide in the CMP1' and CMP2' caused an adverse effect on the level of cell adhesion (<10%). The GFOGER-containing triple-helical CMPs effectively inhibited cell adhesion to collagen in a competition assay. The cell-adhesion activity of the CMP1 was approximately 50% of that of collagen. The cell spreading on the CMP1 was comparable with that observed on collagen. The presence of the CMP1 promoted cell attachment and spreading on the microspheres and extensive cell proliferation and bridging. Slower cell proliferation was observed on the blank microspheres. Live-dead assay showed that most cells are viable after 10-day culture. The presence of CMP1 on the microspheres maintained the albumin secretion and P-450 activity levels of the liver cells for up to 14 days. Our results established the potential of CMP1 to create a collagen-like microenvironment for optimizing cellular responses for liver tissue engineering.

The derivation of definitive endoderm and in particular endocrine cell types from undifferentiated embryonic stem (ES) cells remains difficult to achieve. In this study, we investigated the potential to regulate the differentiation of ES cells into endodermal derivatives using extracellular factors previously associated with various aspects of pancreatic development. Feeder-free-cultured mouse ESD3 cells were manipulated to form embryoid bodies (EBs) in the presence of retinoic acid (RA). RA-treated EBs were subsequently exposed to sodium butyrate (SB), betacellulin (BTC) or activin A (AA). A comparative analysis was performed on these models of directed differentiation in parallel with a model of spontaneous differentiation. Lineage differentiation was determined by profiling multilineage marker transcript expression (neuronal, myogenic, exocrine and endocrine pancreas, extraembryonic and apoptotic) and subsequent protein expression within ES-derived cultures. Using a two-stage differentiation protocol developed during this study, we successfully demonstrated the derivation of an intermediate multipotential population (RA_EBs) from undifferentiated ES cells that preferentially gives rise to pancreatic endocrine insulin-expressing cell types in the presence of SB, and neuronal- and glial-like cell types in the presence of AA or BTC.

Tissue engineering is lacking inexpensive, easily applicable techniques for tissue replacement. We investigated the potential use of native veins for tissue-engineering applications in the urological field. Forty-eight porcine veins, half seeded with urothelial cells and half unseeded, were kept in vitro for 7 days. Four seeded and four unseeded scaffolds were analyzed after 3 and 7 days. The remaining 32 veins were implanted subcutaneously into 16 athymic mice. Four athymic mice were sacrificed after 2, 4, 8, and 12 weeks. Histochemistry, immunohistochemistry (anti-pancytokeratin AE1/AE3, anti-desmin), western blot analyses (CD31), and scanning electron microscopy were performed in the retrieved specimens. The histochemistry of the seeded matrices showed the presence of urothelial cells in vitro and in vivo. After 12 weeks, a multilayer of urothelial cells was present in the hemotoxylin and eosin staining, positive for anti-pancytokeratin AE1/AE3. The western blot analyses showed vascularization of the veins in vivo. The results of scanning electron microscopy revealed a cellular layer on the veins. Native venous matrices may be used as tissue-engineered constructs for reconstructing the urinary tract. The clinical relevance of this approach must be proven in a large-animal model.

Bone marrow stromal cells (BMSCs) are valuable in tissue engineering and cell therapy, but the quality of the cells is critical for the efficacy of therapy. To test the quality and identity of transplantable cells, we identified the molecular markers that were expressed at higher levels in BMSCs than in fibroblasts. Using numerous BMSC lines from tibia, femur, ilium, and jaw, together with skin and gum fibroblasts, we compared the gene expression profiles of these cells using DNA microarrays and low-density array cards. The differentiation potential of tibia and femur BMSCs was similar to that of iliac BMSCs, and different from jaw BMSCs, but all BMSC lines had many common markers that were expressed at much higher levels in BMSCs than in fibroblasts; several BMSC markers showed discrete expression patterns between jaw and other BMSCs. The common markers are probably useful in routine tests, but their efficacy may depend upon the passage number or donor age. In our study the passage number markedly altered the expression levels of several markers, while donor age had little effect on them. Considering the effects of in vivo location of BMSCs and passage, magnitude of increase in expression levels, and interindividual differences, we identified several reliable markers -- LIF, IGF1, PRG1, MGP, BMP4, CTGF, KCTD12, IGFBP7, TRIB2, and DYNC1I1 -- among many candidates. This marker set may be useful in a routine test for BMSCs in tissue engineering and cell therapy.