Some treatments for full thickness defects of articular cartilage, such as cultured chondrocyte transplantation, have already been done. However, to overcome osteoarthritis, we must further study the partial thickness defect of articular cartilage. It is much more difficult to repair a partial thickness defect because few repairing cells can address such injured sites. We herein show that bioengineered layered chondrocyte sheets using temperature-responsive culture dishes may be a potentially useful treatment for partial thickness defects. We evaluated the property of these sheets using real-time PCR and histological findings, and allografted these sheets to evaluate the effect of treatment using a rabbit partial model. In conclusion, layered chondrocyte sheets were able to maintain the cartilageous phenotype, and could be attached to the sites of cartilage damage which acted as a barrier to prevent a loss of proteoglycan from these sites and to protect them from catabolic factors in the joint.
"Based on the above mentioned facts, they suggest that the use of bioengineered chondrocyte sheets may be potentially useful to treat partial thickness defects of articular cartilage. Because the chondrocyte sheets have good adhesion and barrier function which protect against intra-articular catabolic factors, supplying the growth factors (Kaneshiro et al., 2006). "
"The good adhesiveness of layered chondrocyte sheets onto the tissues might be related to the preservation of those molecules as well as the smooth arrangement of the basal surface, which more resembles normal cartilage surface than the top side of the cell sheet. Furthermore, while human articular chondrocytes is known to lose their chondrocyte phenotype during 2D cultivation , in the multilayered chondrocyte sheets, the expressions of chondrospecific markers, namely, type II collagen, SOX9, Aggrecan, are significantly increased in comparison to those in 2D cultured chondrocytes or in a single chondrocyte sheet, indicating that a multilayered chondrocyte sheet has a natural cartilage phenotype  . The maintenance of chondrogenic phenotype might be related to the 3D environment of layered cell sheets. "
[Show abstract][Hide abstract] ABSTRACT: Recently, regenerative medicine using engineered three-dimensional (3D) tissues has been focused. In the fields of cell therapy and regenerative medicine, mesenchymal stem cells (MSCs) are attractive autologous cell sources. While, in bioengineered tissues, a 3D environment may affect the differentiation of the stem cells, little is known regarding the effect of 3D environment on cellular differentiation. In this study, MSC differentiation in in vitro 3D tissue models was assessed by human endometrial gland-derived MSCs (hEMSCs) and cell sheet technology. hEMSC sheets were layered into cell-dense 3D tissues and were cultured on porous membranes. The tissue sections revealed that chondrocyte-like cells were found within the multilayered cell sheets even at 24 h after layering. Immunostainings of chondrospecific markers were positive within those cell sheet constructs. In addition, sulfated glycosaminoglycan accumulation within the tissues increased in proportion to the numbers of layered cell sheets. The findings suggested that a high cell density and hypoxic environment in 3D tissues by layering cell sheets might accelerate a rapid differentiation of hEMSCs into chondrocytes without the help of chondro-differentiation reagents. These tissue models using cell sheets would give new insights to stem cell differentiation in 3D environment and contribute to the future application of stem cells to cartilage regenerative therapy.
The Scientific World Journal 11/2013; 2013(4):359109. DOI:10.1155/2013/359109 · 1.73 Impact Factor
"In cell sheet therapy, cytokines and growth factors produced by the cell sheet play an important role in healing damaged tissues [11,19,20]. We found that the formation of a chondrocyte sheet structure enhanced transforming growth factor-β secretion from the cells , which implies that maintaining the membranous structure after cryopreservation is a prerequisite for function. "
[Show abstract][Hide abstract] ABSTRACT: There is considerable interest in using cell sheets for the treatment of various lesions as part of regenerative medicine therapy. Cell sheets can be prepared in temperature-responsive culture dishes and applied to injured tissue. For example, cartilage-derived cell sheets are currently under preclinical testing for use in treatment of knee cartilage injuries. The additional use of cryopreservation technology could increase the range and practicality of cell sheet therapies. To date, however, cryopreservation of cell sheets has proved impractical.
Here we have developed a novel and effective method for cryopreserving fragile chondrocyte sheets. We modified the vitrification method previously developed for cryopreservation of mammalian embryos to vitrify a cell sheet through use of a minimum volume of vitrification solution containing 20% dimethyl sulfoxide, 20% ethylene glycol, 0.5 M sucrose, and 10% carboxylated poly-L-lysine. The principal feature of our method is the coating of the cell sheet with a viscous vitrification solution containing permeable and non-permeable cryoprotectants prior to vitrification in liquid nitrogen vapor. This method prevented fracturing of the fragile cell sheet even after vitrification and rewarming. Both the macro- and microstructures of the vitrified cell sheets were maintained without damage or loss of major components. Cell survival in the vitrified sheets was comparable to that in non-vitrified samples.
We have shown here that it is feasible to vitrify chondrocyte cell sheets and that these sheets retain their normal characteristics upon thawing. The availability of a practical cryopreservation method should make a significant contribution to the effectiveness and range of applications of cell sheet therapy.
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