Background: In regenerative medicine the autologous cartilage implantation (ACI) has been used for the repair of cartilage defects. As modification of ACI, the matrix assisted ACI is used nowadays with varying results. There is a general discussion about whether supporting scaffolds should be used or whether a scaffold-free cartilage repair is the method of choice. The major problem of scaffold-free regenerates is how to keep the cells in place after transplantation. Aim of this study was to examine a new scaffold-free diffusion-culture model, which uses a mega-congregate of chondrocytes cultured at an air-medium interface. This scaffold-free high-density diffusion culture could be used to repair cartilage defects.

Material and methods: Human chondrocytes from passage 1–7 were expanded in monolayer and transferred to pellet-culture or diffusion-culture. After one week cultures were stained with toluidine blue and safranin-O and evaluated by immunohistochemical staining for type II collagen. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed for the mRNAs of cartilage markers.

Results: Positive alcian blue staining was detectable in diffusion-culture for human chondrocytes up to passage 7. Within passages the amount of proteoglycan production in relationship to the number of cells increased. There was a positive signal for Collagen type II in diffusion-cultures up to passage 7.

In qRT-PCR a redifferentiation of human chondrocytes was shown by the transfer into diffusion-culture. Within passage 1 to 3 human chondrocytes which were cultured in monolayer lost the ability to express Collagen Type II but could regain it if they were transferred to diffusion-culture. At diffusion-culture chondrocytes showed the highest expression of Collagen type II at passage 1 when compared to monolayer or to pellet-culture.

Conclusion: It could be shown that the cultivation in a scaffold-free diffusion-culture can lead to redifferentiation of human chondrocytes Chondrocytes in diffusion-cultures tend to form their own matrix and produce Collagen type II at higher amounts than in monolayer or in normal pellet-cultures. Therefore diffusion-culture congregates might be an appropriate tool to be used for a new scaffold-free cartilage regeneration approach.