Gaissmaier C (1,2), Krackhardt T (1), Flesch I (1), Kellomäki M (3), Aicher W (4), Waris T (5), Törmälä P (3), Weise K (2), Ashammakhi N (6).

1. Natural and Medical Science Institut, Reutlingen, Germany
2. BG Trauma Center, Eberhard-Karls University, Tuebingen, Germany
3. Institute of Biomaterials, Tampere University of Technology, Tampere, Finland
4. Department of Orthopaedics, Eberhard-Karls University, Tuebingen, Germany
5. Department of Surgery, Tampere University Hospital and University of Tampere, Tampere, Finland
6. Department of Surgery, Oulu University Hospital, Oulu, Finland

Abstract
Background:
Articular cartilage is rich in collagen type II fibers, proteoglycans and characterized by a low cell density. As this tissue is avascular, chondrocytes have specific nutritional requirements and therefore can not be expanded in vitro without the risk of generating fibroblastoid cells expressing collagen type I. Therefore different growth conditions were tested for cartilage tissue engineering. Aims: To investigate the effects of human platelet supernatant (hPS) on the proliferation and metabolism of cultured human articular chondrocytes. Materials and methods: Human articular chondorcytes were obtained from three donors. Chondrocytes were isolated and culture-expanded in monolayer or seeded in three dimensional alginate beads or poly-L/D-lactide (PLDLA) knitted scaffolds.hPS was prepared and added to the cultured chondrocytes. RT-PCR was used to assess the expression of chondrocyte gene markers. Indirect immunohistochemistry was used to detect the synthesis of collagen type I and II, chondritoin sulphate, proteoglycan and agrecan. Results: hPS activated chondrocyte proliferation in monolayer cultures in a dose and time dependent manner. However, this was obtained at the cost of rapid dedifferentiation of chondrocytes towards a fibroblastoid phenotype. The expression of type II collagen, aggrecan and GDF-5 were reduced in all tested samples. Seeding the chondrocytes in PLDLA scaffolds in the presence of hPS or recultivation of chondrocytes expanded in presence of hPS into scaffolds generated a chondrocyte population capable of high type II collagen expression. Still, type I collagen production remained high. Conclusions: Human chondrocytes expanded without subcultivation in primary culture in the absence of hPS maintained a high type II collagen expression. Type II collagen expression was even enhanced in chondrocytes incubated in alginate or PLDLA scaffolds. In contrast, hPS induced type I collagen expression in all samples tested even after recultivation in scaffolds. Type II collagen and aggrecan expression were reduced indicating that such cells may not generate a proper hyaline-like cartilage.

Key words
Articular cartilage, chondrocytes, Platelet supernatant, Scaffolds, Tissue engineering.