Tissue engineering最新文献

Because engineered tissues are designed for clinical applications in humans, a major problem is the contamination of cocultures and tissues by allogenic molecules used to grow stem cells in vitro. The protocols that are commonly applied to generate epidermal equivalents in vitro require the use of irradiated murine fibroblasts as a feeder layer for keratinocytes. In this study, we report a simple procedure for growing human keratinocytes, isolated from adult skin, to generate an epidermal construct on a collagen layer alone. In this model, no human or murine feeder layers were used to amplify cell growth, and isolated keratinocytes were seeded directly at high cell density on the collagen-coated flasks or coverslips in an epithelial growth medium containing low calcium concentration. Morphological, immunochemical, and cytokinetic features of epithelial colonies grown on the collagen layer were typical of keratinocytes and were comparable with those reported for keratinocytes grown on a feeder layer. The stratification of keratinocytes generated 3-dimensional synthetic constructs displaying a tissue architecture comparable with that of natural epidermis. Epithelial cells expressed specific markers of keratinocyte terminal differentiation, including involucrin and filaggrin. Nevertheless, the number of cell layers was lower than in natural skin, and electron microscopical analysis revealed that the overall organization of these layers was poor compared with natural epidermis, including the formation of junctional complexes, basement membrane, and keratinization. The lack of epithelial-mesenchymal interactions that occur during skin histogenesis may account for such an incomplete maturation of epidermal constructs.

Engineered functional skeletal muscle would be beneficial in reconstructive surgery. Our previous work successfully generated 3-dimensional vascularized skeletal muscle in vivo. Because neural signals direct muscle maturation, we hypothesized that neurotization of these constructs would increase their contractile force. Additionally, should neuromuscular junctions (NMJs) develop, indirect stimulation (via the nerve) would be possible, allowing for directed control. Rat myoblasts were cultured, suspended in fibrin gel, and implanted within silicone chambers around the femoral vessels and transected femoral nerve of syngeneic rats for 4 weeks. Neurotized constructs generated contractile forces 5 times as high as the non-neurotized controls. Indirect stimulation via the nerve elicited contractions of neurotized constructs. Curare administration ceased contraction in these constructs, providing physiologic evidence of NMJ formation. Histology demonstrated intact muscle fibers, and immunostaining positively identified NMJs. These results indicate that neurotization of engineered skeletal muscle significantly increases force generation and causes NMJs to develop, allowing indirect muscle stimulation.

A rotary cell culture system has been established. System quality was determined by observing the stability of the basic parameters of temperature, gas exchange, and pH, and mass transfer (time to equimolarity) between the medium circuit and the 2 cell-containing chambers was investigated. Mass transfer time for urea and several ions was approximately 30 min for the high-fiber-density chamber (HFC) and 50 min for the low-fiber-density chamber (LFC). Exchange of albumin was delayed in both chambers, highlighting the dependence of mass transfer on area of exchange and molecule size. Finally, the ability for cell growth and maintenance was tested. Densities of up to 1.2 x 10(7) immortalized cells per mL at a viability of up to 85% were obtained after 1 week of continuous, non-interfering culture of immortalized cells in the HFC. Human pancreatic islets were also cultivated in the LFC. Confocal analysis using fluorescent dyes showed that the 3-dimensional islet structure was maintained for 1 week. Promising results were obtained, which will further our ongoing efforts toward establishing a mobile cell culture system.

Exposure of human skin to solar ultraviolet (UV) light induces local and systemic immune suppression. It is known that alterations of immune functions of Langerhans cells (LCs) and dermal dendritic cells (DDCs) mediate this phenomenon. The purpose of this study was to mimic in vitro the early UV-induced skin disruption to better understand the involvement of the skin micro-environment in triggering this immunosuppressive state. We therefore developed skin equivalents (SEs) integrating LCs and DDCs derived from monocytes (mo-LCs and mo-DDCs, respectively). First, we showed that Langerin(+) mo-LC and dendritic cell (DC)-specific ICAM-3 grabbing nonintegrin (SIGN)(+) mo-DDCs were immunolocalized in situ in epidermal and dermal compartments of SEs, respectively. The SE micro-environment without immune cells displayed full cytokine profile that may ensure and maintain differentiation, localization, and immaturity of LCs and DDCs in situ, as shown by secretion of granulocyte-macrophage colony-stimulating factor, transforming growth factor beta (beta)-1, interleukin (IL)-4, IL-13, and IL-15 involved in cell differentiation; presence of complete chemokine network as macrophage inflammatory protein 3 alpha (alpha); low secretion of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), IL-1 beta, IL-6, and IL-8; and surprising secretion of immunosuppresive cytokine IL-10. Second, we demonstrated that skin micro-environment homeostasis was greatly disrupted under solar UV irradiation of SEs. In fact, we showed a pro-inflammatory state characterized by high secretion of TNF-alpha, IL-1 beta, IL-6, and IL-8 and low secretion of IL-10. This breakdown of immune homeostasis was visualized at the same time as in situ migration of mo-LCs and mo-DDCs into the dermal equivalent of SEs. Moreover, this tissue migration of mo-LCs and mo-DDCs into SEs was in accordance with the chemokine (C-C motif) receptor 7 expression and the DC-lysosome-associated membrane glycoprotein acquisition only on mo-LCs. Our results highlighted major participation of the skin micro-environment in the triggering and modulating of UV-induced skin immune responses. In addition, it could be concluded that these SEs are reliable tools for modeling biological events inaccessible in humans.

The objective of cellular cardiomyoplasty is to regenerate the myocardium using implantation of living cells. Because the extracellular myocardial matrix is deeply altered in ischemic cardiomyopathies, it could be important to create a procedure aiming at regenerating both myocardial cells and the extracellular matrix. We evaluated the potential of a collagen matrix seeded with cells and grafted onto infarcted ventricles. A myocardial infarction was created in 45 mice using coronary artery ligation. Animals were randomly assigned to 4 local myocardial treatment groups. Group I underwent sham treatment (injection of cell culture medium). Group II underwent injection of human umbilical cord blood mononuclear cells (HUCBCs). Group III underwent injection of HUCBCs and fixation onto the epicardium of a collagen matrix seeded with HUCBCs. Group IV underwent fixation of collagen matrix (without cells) onto the infarct. Echocardiography was performed on postoperative days 7 and 45, followed by histological studies. Echocardiography showed that the association between the cell-loaded matrix and the intrainfarct cell implants was the most efficient approach to limiting postischemic ventricular dilation and remodeling. Ejection fraction improved in both cell-treated groups. The collagen matrix alone did not improve left ventricular (LV) function and remodeling. Histology in Group III showed fragments of the collagen matrix thickening and protecting the infarct scars. Segments of the matrix were consistently aligned along the LV wall, and cells were assembled within the collagen fibers in large populations. Intramyocardial injection of HUCBCs preserves LV function following infarction. The use of a cell-seeded matrix combined with cell injections prevents ventricular wall thinning and limits postischemic remodeling. This tissue engineering approach seems to improve the efficiency of cellular cardiomyoplasty and could emerge as a new therapeutic tool for the prevention of adverse remodeling and progressive heart failure.

Bone marrow stromal cells (MSCs) are a promising cell source for a variety of tissue engineering applications, given their ready availability and ability to differentiate into multiple cell lineages. MSCs have been successfully used to create neotissue for cardiovascular, urological, and orthopedic reconstructive surgical procedures in preclinical studies. The ability to optimize seeding techniques of MSCs onto tissue engineering scaffolds and the ability to control neotissue formation in vitro will be important for the rational design of future tissue engineering applications using MSCs. In this study we investigated the effect of centrifugal force on seeding MSCs into a biodegradable polyester scaffold. MSCs were isolated and seeded onto porous scaffold sections composed of nonwoven polyglycolic acid mesh coated with poly(L-lactide-co-epsilon-caprolactone). Compared to standard static seeding techniques, centrifugal seeding increased the seeding efficiency by 38% (p < 0.007) and significantly improved cellular distribution throughout the scaffold. Overall, centrifugal seeding of MSCs enhances seeding efficiency and improves cellular penetration into scaffolds, making it a potentially useful technique for manipulating neotissue formation by MSCs for tissue engineering applications.

The clinical need for improved blood vessel substitutes, especially in small-diameter applications, drives the field of vascular tissue engineering. The blood vessel has a well-characterized structure and function, but it is a complex tissue, and it has proven difficult to create engineered tissues that are suitable for widespread clinical use. This review is focused on approaches to vascular tissue engineering that use proteins as the primary matrix or "scaffold" material for creating fully biological blood vessel replacements. In particular, this review covers four main approaches to vascular tissue engineering: 1) cell-populated protein hydrogels, 2) cross-linked protein scaffolds, 3) decellularized native tissues, and 4) self-assembled scaffolds. Recent advances in each of these areas are discussed, along with advantages of and drawbacks to these approaches. The first fully biological engineered blood vessels have entered clinical trials, but important challenges remain before engineered vascular tissues will have a wide clinical effect. Cell sourcing and recapitulating the biological and mechanical function of the native blood vessel continue to be important outstanding hurdles. In addition, the path to commercialization for such tissues must be better defined. Continued progress in several complementary approaches to vascular tissue engineering is necessary before blood vessel substitutes can achieve their full potential in improving patient care.

A new porous, thermoresponsive, partially biodegradable, chemically crosslinked hydrogel system was developed, characterized, and tested as a cartilage tissue-engineering scaffold for in vitro chondrocyte culture over a 4-week period. The hydrogel system was composed of poly(N-isopropylacrylamide), poly(D,L-lactic acid), and dextran segments. Pores in the hydrogels were generated using a salt leaching technique. The hydrogels showed thermoresponsive properties, with a lower critical solution temperature at approximately 32 degrees C. They continuously swelled at physiological temperature in phosphate buffered saline (pH 7.4) for at least 1 month. Chondrocytes isolated from embryonic chick sterna were seeded into the hydrogel scaffolds at room temperature and cultured at 37 degrees C for 4 weeks. Real-time reverse-transcriptase polymerase chain reaction quantification was conducted every week to study messenger ribonucleic acid levels of 3 chondrocyte phenotypic markers: type II collagen, type X collagen, and Indian hedgehog. Results suggested that chondrocytes maintained their phenotype during the 4-week in vitro culture and could mimic in vivo development. Chondrocytes were non-enzymatically harvested from the hydrogel scaffold at the end of the fourth week by simply lowering the temperature from 37 degrees C to room temperature. The harvested chondrocytes kept a round morphology, confirming the maintenance of the chondrocyte phenotype in the hydrogel scaffolds.

We tested the hypothesis that supplemental regulatory factors can improve the contractile properties and viability of cardiac tissue constructs cultured in vitro. Neonatal rat heart cells were cultured on porous collagen sponges for up to 8 days in basal medium or medium supplemented with insulin-like growth factor-I (IGF), insulin-transferrin-selenium (ITS), platelet-derived growth factor-BB (PDGF), or angiopoietin-1 (ANG). IGF and ITS enhanced contractile properties of the 8-day constructs significantly more than with unsupplemented controls according to contractile amplitude and excitation threshold, and IGF also significantly increased the amount of cardiac troponin-I and enhanced cell viability according to different assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and terminal deoxynucleotidyl transferase biotin-2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)). PDGF significantly increased the contractile amplitude of 4-day constructs and enhanced cell viability according to MTT, LDH, and TUNEL; ANG enhanced cell viability according to the LDH assay. Our results demonstrate that supplemental regulatory molecules can differentially enhance properties of cardiac tissue constructs and imply that these constructs can provide a platform for systematic in vitro studies of the effects of complex stimuli that occur in vivo to improve our basic understanding of cardiogenesis and identify underlying mechanisms that can potentially be exploited to enhance myocardial regeneration.

Tissue shortage complicates surgery of cleft lip and palate. The healing of defects on the palate impairs growth of the dentoalveolar complex because of scar tissue formation. Implantation of a matrix into the wound might overcome this adverse effect. Integra with and without a silicone top layer was implanted into standardized full-thickness wounds (Ø 6 mm) in the palatal mucoperiosteum in beagle dogs. In some wounds, the silicone layer was removed after 14 days. Control wounds did not have an implant. At 2 and 4 weeks post-surgery, the wounds were assessed for epithelialization, inflammation (hematoxylin and eosin, leucocyte protein L1), number of myofibroblasts (alpha smooth muscle actin), and general histological characteristics. Wounds filled with Integra without the silicone layer showed fewer myofibroblasts and inflammatory cells than the sham wounds. Collagen fibers were more randomly orientated in these wounds than in the sham group. Wound closure was found to be retarded, and many inflammatory cells were present when Integra with silicone was implanted. The silicone layer was lost within 4 weeks in these wounds. We conclude that, in the moist oral environment, the silicone of Integra is not required. Re-epithelialization and tissue integration proceed more favorably without it. Further research in the dentoalveolar development with Integra will be conducted in a simulated cleft palate repair in the dog model.