The use of allografts for the treatment of critical size cartilage defects holds disadvantages including the limited number of donors and the compromised chondrocyte viability. These limitations have prompted researchers to explore different options, such as cartilage engineering. Bioprinting, a promising tissue engineering technique, could be used to fabricate biomimetic constructs that can potentially replace allografts. The present study explores the generation of a biomimetic chondrocyte density gradient in full-thickness bioprinted Alginate/NFC scaffolds with a PCL framework and investigates the effect of this zonal distribution of cells on the production of cartilage matrix within the scaffolds. To this end, two types of scaffolds were bioprinted; one with a graded three-zone distribution of cells (bottom zone: 5×106 cells/ml, middle zone: 10×106 cells/ml, top zone: 20×106 cells/ml), and another with a homogeneous cell density (10×106 cells/ml) and cultured for 25 days. Furthermore, the mechanical properties of the multi-material scaffolds were evaluated. The results showed that a three-zone cell density gradient could be achieved using extrusion-based bioprinting. The gradient was maintained for 25 days in scaffolds cultured in non-chondrogenic media (absence of ascorbic acid) but was transformed into a two-zone gradient within the first 14 days, in cultures with chondrogenic media (presence of ascorbic acid in the medium), and maintained as such until the end of the experiment. The zonal distribution of cells led to a zonal distribution of sGAGs, after 14 days of culture, with the sGAGs deposition being increased in the areas of high cell density. However, there was no significant Collagen deposition within the scaffolds at any time point during the experiment. This study attempts to shed light into one of the gradients of the native cartilage tissue (cell density gradient), with the hope of contributing to the development of a biomimetic fully functional engineered cartilage scaffold in the future.

Objectives: With an increasing life expectancy in the western societies and more people practising extreme sports, the demand for orthopaedic implants is set to increase. Orthopaedic implant loosening is one of the main causes of revision surgeries, leading to increases in the costs of patient care and patient dissatisfaction. Better osseointegration could lead to higher success rates of primary interventions. Mesenchymal stem cells (MSC) can undergo osteogenic differentiation on the implant surfaces, promoting the implant osseointegration. Alternatively, they can commit to other differentiation paths (e.g., fibroblasts) and thus hinder the osseointegration and functionality of the implant. It is therefore fundamental to promote the osteogenic differentiation of MSC at the surface of the implant to ensure enhanced osseointegration and thus its long term success. Among the available strategies that promote osteogenic differentiation, nano and micro-scale physical patterns have proved effective but the underlying mechanisms that induce these cellular changes are not yet understood. Cellular adhesion to the surface is believed to be the key process regulating mechanotransduction (i.e., extrinsic cell signalling) and subsequently, the mesenchymal stem cell osteogenic differentiation. Nevertheless, no quantitative systematic study has been performed in order to establish the relationship between the cell adhesion and the differentiation behaviour. Furthermore, the available cellular adhesion studies are only qualitative or semi-quantitative in nature. In this work, novel fluid force microscopy (FFM) technique has been employed to characterize the adhesion properties induced by a set of osteogenic and a set of non-osteogenic submicron patterns. Methods: Two sets of substrates consisting of arrays of submicron pillars with known osteogenic potential were manufactured by means of two photon polymerization. Preosteoblast mouse cells were cultured on the patterns and on a flat control surface. The adhesion properties (i.e., the force and work of adhesion) were measured by FFM after 4 and 24 hours of cell culture. The cellular behaviour picture was completed by assessing the Young’s modulus of the living cells attached on the patterns and on the control surface. Likewise, the topographical and mechanical mapping was performed by using atomic force microscopy system. Results: In this study, the adhesion of preosteoblast cells was successfully quantified on two types of submicron pattern with known osteogenic potential, and compared to cellular adhesion data on a flat control. Therefore, the fluid force microscopy (FFM) was applied for the first time. The trend indicated that cells seeded on the osteogenic substrate adhered stronger (mean force of adhesion = 159 ± 74 nN) than cells on the non-osteogenic substrate (mean force of adhesion = 108 ± 104 nN) after 24h of incubation. Moreover, the cells adopted different morphologies and spatial distribution of the Young’s modulus depending on the culture substrate.