Preparation and characterization of human keratinocyte-fibroblast cell sheets constructed using PNIAM-co-AM grafted surfaces for burn wound healing.

Autologous skin grafting, the standard treatment for severe burns, is sometimes not possible due to the limited available skin surfaces for the procedure. With advances in tissue engineering, various cell-based skin substitutes have been developed to serve as skin replacements and to promote tissue regeneration and healing. In this work, we propose the use of cell sheet technology to fabricate keratinocyte-fibroblast tissue constructs from the temperature-responsive poly(N-isoproprylacrylamide-co-acrylamide) (PNIAM-co-AM) grafted surfaces for the treatment of burn wounds. The characteristics of the human keratinocyte and fibroblast cell sheets harvested using PNIAM-co-AM grafted surfaces were similar to those cell sheets detached from the commercially-available UpCellTM plates. Upon lowering the incubation temperature, confluent keratinocytes and fibroblasts could be detached as intact sheets, consisting of biologically active cells, as indicated by their high cell viability and their reattachment, migratory, and proliferative activities. A histological analysis of the stratified keratinocyte-fibroblast cell sheets revealed the evidence of cell migration and tissue reorganization to form two distinct epidermal and dermal layers, quite similar to the skin tissue's structure. In addition, the keratinocyte-fibroblast sheets could synthesize and release significant amounts of essential cytokines and growth factors involved in regulating the wound healing process, including IL-1α, IL-6, TNF-α, VEGF, and bFGF, implying the therapeutic effect of these cell sheets, which could be beneficial to accelerate tissue repair and regeneration, leading to faster wound healing.

Maintenance of human chondrogenic phenotype on a dendrimer-immobilized surface for an application of cell sheet engineering.

BACKGROUND: Dedifferentiation of chondrocytes during cell expansion is one of the barriers in tissue construction for cartilage repair. To understand chondrocyte behavior and improve cell expansion in monolayer culture, this study investigated the effects of morphological changes and cellular aggregation on the maintenance of chondrogenic capacity by observing the expression patterns of chondrogenic (collagen type II and aggrecan) and dedifferentiation (collagen type I) markers. Primary human chondrocytes were cultured on either a polystyrene surface (PS) or a polyamidoamine dendrimer surface with a fifth-generation (G5) dendron structure to create a one-step process of cell expansion and the maintenance of chondrogenic activities prior to the construction of cell sheets. RESULTS: During the first two passages (P0 - P2), the relative mRNA level of collagen type II decreased in all cultures, while that of collagen type I increased. Remarkably, the level of collagen type II was higher and aggrecan was retained in the chondrocytes, forming cell aggregates and showing some round-shaped cells with less production of stress fibers on the G5 surface compared to fibroblast-like chondrocytes with abundant stress fibers on the PS surface. The numbers of P2 chondrocytes on the G5 and PS surfaces were nearly the same and sufficient for construction of chondrocyte sheets using a temperature-responsive plate. Without a supporting material during cell sheet manipulation, chondrocyte sheets spontaneously detached and exhibited a honeycomb-like structure of stress fibers. Unlike the chondrocyte sheets constructed from cells on the PS surface, the chondrocyte sheets from cells on the G5 surface had higher chondrogenic activities, as evidenced by the high expression of chondrogenic markers and the low expression of dedifferentiation markers. CONCLUSIONS: The one-step process of cell expansion and maintenance of chondrogenic activity could be obtained using the G5 surface. Human chondrocyte sheets were successfully constructed with high chondrogenic activity. These findings may lead to an alternative cultivation technique for human chondrocytes that offers high clinical potential in autologous chondrocyte implantation.

Expansion of fibroblast cell sheets using a modified MEEK micrografting technique for wound healing applications.

Cell sheet engineering, a scaffold-free approach to fabricate functional tissue constructs from several cell monolayers, has shown promise in tissue regeneration and wound healing. Unfortunately, these cell sheets are often too small to provide sufficient wound area coverage. In this study, we describe a process to enlarge cell sheets using MEEK micrografting, a technique extensively used to expand skin autografts for large burn treatments. Human dermal fibroblast cell sheets were placed on MEEK's prefolded gauze without any use of adhesive, cut along the premarked lines and stretched out at various expansion ratios (1:3, 1:6 and 1:9), resulting in regular distribution of many square islands of fibroblasts at a much larger surface area. The cellular processes essential for wound healing, including reattachment, proliferation, and migration, of the fibroblasts on expanded MEEK gauze were superior to those on nylon dressing which served as a control. The optimal expansion ratio with the highest migration rate was 1:6, possibly due to the activation of chemical signals caused by mechanical stretching and an effective intercellular communication distance. Therefore, the combination of cell sheet engineering with the MEEK micrografting technique could provide high quality cells with a large coverage area, which would be particularly beneficial in wound care applications.

Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II and Stress Fiber Formation in Human Chondrocyte Sheets.

Cell sheet technology is applied to human articular chondrocytes to construct a tissue-like structure as an alternative treatment for cartilage defect. The effect of a gelatin manipulator, as a cell sheet transfer system, on the quality of the chondrocyte sheets was investigated. The changes of important chondrogenic markers and stress fibers, resulting from the cell sheet manipulation, were also studied. The chondrocyte cell sheets were constructed with patient-derived chondrocytes using a temperature-responsive polymer and a gelatin manipulator as a transfer carrier. The properties of the cell sheets, including sizes, expression levels of collagen type II and I, and the localization of the stress fibers, were assessed and compared with those of the cell sheets harvested without the gelatin manipulator. Using the gelatin manipulator, the original size of the chondrocyte cell sheets was retained with abundant stress fibers, but with a decrease in the expression of collagen type II. Without the gelatin manipulator, although the cell shrinkage occurred, the cell sheet with suppressed stress fiber formation showed significantly higher levels of collagen type II. These results support our observations that stress fiber formation in chondrocyte cell sheets affected the production of chondrogenic markers. These densely packed tissue-like structures possessed a good chondrogenic activity, indicating their potential for use in autologous chondrocyte implantation to treat cartilage defects.

Construction of a chondrocyte cell sheet using temperature-responsive poly(N-isopropylacrylamide)-co-acrylamide.

In this study, a novel temperature-responsive poly(N-isopropylacrylamide)-co-acrylamide was used to prepare a chondrocyte cell sheet. Chondrocytes were isolated from human articular cartilage and plated on the copolymer film grafted tissue culture plates. The cell attachment on the copolymer film was shown to be similar to that of the ungrafted surface. To harvest a cell sheet, the incubation temperature was reduced to 10°C for 30 minutes to allow the polymer chain to fully extend, changing the copolymer's phase from hydrophobicity to hydrophilicity. Additional incubation at 20°C for 60 minutes was necessary to activate the cellular metabolism required for cytoskeletal organization and cell detachment. A complete cell sheet recovery was achieved when a PVDF membrane was used as a cell sheet carrier. Unfortunately, the shrinkage of the cell sheet was observed. Nonetheless, the harvested cell sheet was shown to be viable and healthy.

Effects of organic solvents on the scission energy of rodlike micelles.

The linear viscoelastic response of erucyl bis(hydroxyethyl) methylammonium chloride with added KCl has been studied as a function of temperature and nonpolar solvent addition. The plateau modulus is independent of temperature from 25 to 40 degrees C, in contrast to previous studies with salicylate counterions that showed a plateau modulus increasing with temperature over this range. The average micelle length, L, predicted by the model of Cates, depends experimentally on Escis/kBT, where Escis is the scission energy of the chain and kBT is the Boltzmann constant times the absolute temperature. With ethanol addition, the calculated average contour length, L, decreases by a factor of 4 as ethanol concentration varies from 0 to 1.3 M. This corresponds to an apparent energy for scission, Escis, decrease from 81 +/- 8 to 74 +/- 7 kJ/mol. On the other hand, only 80 mM of hexane is required to cause a decrease in Escis to the same level, and for hexane addition levels above 70 mM a disruption in the plateau modulus indicates the disruption of the rodlike structure. The correspondence between the effect of temperature and the effect of solvent addition allows the development of "solvent/temperature" superposition rules to predict the rheology of these viscoelastic fluids at elevated temperatures.