Cultured articular chondrocytes sheets for partial thickness cartilage defects utilizing temperature-responsive culture dishes

Abstract

The extracellular matrix (ECM) of articular cartilage has several functions that are unique to joints. Although a technique for transplanting cultured chondrocytes has already been introduced, it is difficult to collect intact ECM when using enzymes to harvest samples. Temperatureresponsive culture dishes have already been clinically applied in the fields of myocardial and corneal transplantation. Earlier studies have shown that a sheet of cultured cells with intact ECM and adhesive factors can be harvested using such culture dishes, which allow the surface properties of the dish to be reversibly altered by changing the temperature. Human chondrocytes were subjected to enzymatic digestion and then were seeded in temperatureresponsive culture dishes. A sheet of chondrocytes was harvested by only reducing the temperature after the cultured cells reached confluency. A real-time PCR analysis of the chondrocyte sheets confirmed that type II collagen, aggrecan, and fibronectin were present. These results suggested that, although chondrocytes undergo dedifferentiation in a monolayer culture, multilayer chondrocyte sheets grown in a similar environment to that of three-dimensional culture may be able to maintain a normal phenotype. A histological examination suggested that multilayer chondrocyte sheets could thus prevent the loss of proteoglycans because the area covered by the sheets was well stained by safranin-O. The present experiments suggested that temperature-responsive culture dishes are useful for obtaining cultured chondrocytes, which may then be clinically employed as a substitute for periosteal patches because such sheets can be applied without a scaffold.

Characterization of chondrocyte sheets prepared using a co-culture method with temperature-responsive culture inserts

“…() found that layered chondrocyte sheets made using UpCells adhere to damaged parts of articular cartilage and are active in tissue repair. The layered sheets were found to have chondroprotective effects (Kaneshiro et al ., ) and to restrain the catabolic factors MMP3, MMP13 and ADAMTS5 – enzymes that degrade the extracellular matrix (Mitani et al ., ). In this study, it was found that layered chondrocyte sheets fabricated using our co‐culture technique expressed genes critical to maintaining the characteristics of cartilage.…”

“…Although various techniques are available to address these issues, the focus of this study was on developing a method that did not require the use of scaffolds (Nagai et al , , ). The results indicate that the novel technique described here produces more extracellular matrix than conventional techniques (Kaneshiro et al ., , ; Mitani et al ., ) while preserving the characteristics of natural cartilage.…”

“…Experiments in animals have shown that layered chondrocyte sheets, cultured using UpCells, adhere to the injured articular cartilage and are active in tissue repair (Kaneshiro et al ., ). Compared with single‐layer cell sheets, layered sheets have a stronger chondroprotective effect (Kaneshiro et al ., ). However, because of the paucity of chondrocytes obtained from the extracellular matrix and their poor proliferative ability, it takes 4–5 weeks to prepare transplantable tissue from the harvested cells.…”

Rotator cuff repair using cell sheets derived from human rotator cuff in a rat model

“…Regarding tendon tissue engineering, Mifune et al showed tendon grafts wrapped in cell sheets composed of anterior cruciate ligament‐derived CD34‐positive cells could enhance the healing of the tendon‐bone junction by promoting increased proprioceptive recovery, graft maturation, and biomechanical strength in a rat model of anterior cruciate ligament reconstruction. Furthermore, this technique was recently used for the treatment of cartilage damage . Hamahashi et al suggested that the humoral factors produced by layered chondrocyte sheets may contribute to cartilaginous tissue repair and regeneration.…”

Development of a novel vitrification method for chondrocyte sheets

“…We have been investigating the use of chondrocyte-derived cell sheets for treatment of knee cartilage injuries [11,12,16-18]. Cell sheets can be used as autografts or allografts.…”