**1. Introduction**

Collagen is one of the most frequently applied biomaterials for biomedical research as well as clinical applications [1,2]. The main industrial sources of collagen are bovine and porcine tissues; however, there is increasing demand for alternative sources. Marine collagens which can be obtained from both invertebrates and vertebrates [3,4] show promising features and have the potential to overrule mammalian collagens in biomedical applications for several reasons. Marine collagens do not bear the risk of disease translation and are not allergy-causing; they are not subjected to ethical or religious concerns, show low inflammatory response and can be obtained with high yield [1]. From the beginning of the 21st century research on marine collagens has continuously emerged [5,6]. Scaffolds for tissue engineering applications are increasingly developed from collagens of marine origin, such as from fish collagen, collagen of marine sponges, jellyfish collagen and collagen from marine gastropods [5]. In two own studies we applied marine collagens for the fabrication of porous scaffolds: first we adapted the procedure of biomimetic mineralization of bovine collagen to collagen of the Atlantic salmon *Salmo salar* and prepared mineralized porous scaffolds

from salmon collagen for the application in bone tissue engineering [7]. A second study applied collagen of the jellyfish *Rhopilema esculentum* which is structurally similar to human collagen II [8] for the preparation of porous scaffolds to be used for chondral tissue engineering [9]. In the present study, we combined fibrillized jellyfish collagen with biomimetically mineralized salmon collagen to a biphasic scaffold suitable for osteochondral defect regeneration. The applied technique was already described in 2007 for the generation of biphasic, but monolithic scaffolds from mineralized bovine tendon collagen and fibrillized collagen from calf skin/hyaluronic acid composite [10]. Joint freeze-drying and chemical crosslinking of the two different phases resulted in a scaffold material which overcame the risk of delamination of the mineralized and non-mineralized phases, since the scaffolds consisted of a unified whole [10]. Challenge in the fabrication of biphasic scaffolds for the regeneration of both cartilage and the subchondral bone layer is the mechanically stable conjunction of the different phases, which have different mechanical properties to mimic the chemical nature of elastic, water-rich chondral ECM, and the stiff, mineralized bone ECM. The main challenge, however, is the simultaneous chondrogenic and osteogenic differentiation, guided by the scaffold properties which should recapitulate the native milieu of bone and cartilage development [11–14]. Furthermore, when osteochondral tissue engineering constructs are differentiated in vitro, a suitable osteochondral medium should be developed [15,16]. The aim of the present study was to generate biphasic, but monolithic porous scaffolds from fibrillized jellyfish collagen for the chondral part and biomimetically mineralized salmon collagen for the bony part. Multipotent human mesenchymal stromal cells (hMSC) were differentiated simultaneously into chondrocytes and osteoblasts, respectively, in this biphasic scaffold in vitro. To generate a microenvironment, which guides the cells to the two differentiation lineages, we inserted the cells into the chondral part with a tenfold higher cell density compared to the bony part and, furthermore, used an alginate hydrogel for embedding the cells in the porous chondral part. Furthermore, an osteochondral medium was developed and sequential seeding of the scaffold phases with pre-differentiated MSC was performed.

### **2. Results**
