15502-m-pleumeekers

DISCUSSION For successful regeneration of cartilage tissue, selection of the most appropriate cell source is crucial. This study demonstrates that cartilage matrix formation and functionality is cell source dependent; articular chondrocytes (ACs) possess the highest chondrogenic capacity in vitro , while ear chondrocytes (ECs) and nasal chondrocytes (NCs) are most potent for cartilage regeneration after subcutaneous implantation in vivo . To date, we and others have evaluated the use of chondrocytes and mesenchymal stem cells (MSCs) of several anatomical locations for their applicability in cartilage regenerative medicine. [39, 42-44, 46-59, 206-209] However, these studies often used non- expanded cells isolated from animals. Moreover, a detailed direct comparison between various chondrocyte and MSC-sources was lacking. Therefore, this study is the first to systematically compare the quality and tissue stability of engineered cartilage constructs produced by culture-expanded ACs, NCs, ECs, BMSCs and AMSCs of human origin. We have used culture-expanded human cells, to closely simulate the clinical situation. For clinical application, the use of autogeneic cells is favorable, since these cells do not elicit a tissue rejection response. However, it has been difficult to obtain appropriate numbers of cells, as donor tissue is limited and harvesting can cause large donor site morbidity. Consequently, monolayer cell-expansion has become an essential step in the process of cartilage TE. To fulfil this requirement, we culture-expanded all cells for four passages. It was obvious that different cells went through a different number of population doublings during these four passages; ACs had gone through the least number of population doublings confirming earlier findings of slow proliferation of ACs. [44, 48, 54, 57] To be able to use expanded cells for the reconstruction of cartilage defects, cells should be stimulated to regain their cartilage-matrix-forming capacity. Several research groups have shown that expanded cells can regain their chondrogenic potential under specific culture conditions: (1) the use of a 3D-culture system (2) and/or the administration of chondrogenic factors, such as TGFβ. [216] In order to generate a 3D-culture environment, we have encapsulated all cells in clinical grade alginate, since alginate enables a homogeneous cell distribution, prevents cells from floating out while permitting nutrient diffusion and oxygen transfer to the encapsulated cells and promotes the synthesis of cartilage-specific matrix components, such as sGAGs and collagen type II. [89] Surprisingly, we showed that alginate appears to have the tendency to calcify in vivo , since 20% of all constructs calcified during subcutaneous implantation. Especially, cell-free constructs and constructs encapsulating ACs suffered from this phenomenon. Also, calcification was more often seen in constructs pre- cultured in medium without TGFβ1. To our knowledge, calcium-cross-linked alginate calcifies through binding the surrounding phosphate ions to form calcium phosphate crystals. Those crystals are stable in neutral to basic environments and do not appear at pH less than 6.8. [217] We believe that the calcified constructs were possibly generated a neutral to alkaline environment prior to implantation, since these constructs were either not metabolically active (non-seeded alginate) or had a low metabolic activity due to stable cartilage formation (ACs) or due to the deficiency of TGFβ1. Obviously, in these constructs, calcification did not seem to be a consequence of instable cartilage formation, but was more likely a typical characteristic 78 CHAPTER 4