In the last two decades, it has been generally accepted that the interaction between bone and immune cells is critical in the process of bone healing and regeneration. Steering this immune response with the development of biomaterials could lead to implant acceptance in the long term. The understanding of osteoimmunological responses is of high importance in the development of new biomaterials. In order to validate these biomaterials, better in vitro techniques are required that could mimic this inflammatory response immediately after implantation in more detail. This study investigated the effect of a direct co-culture model on a prototype nanopattern. In addition the effect of microflows, that are normally present in vivo, induced inside a microfluidic chip, on mono and co-cultures was investigated in order to improve in vitro research. Static mono- and co-cultures with a 1:1 (CO11) and 1:2 (CO12) ratio of murine pre-osteoblasts MC3T3-E1s (OBs) and M1 stimulated murine Macrophages J774A.1 (+ LPS & IFN-gamma) were cultured under static conditions for 4 weeks to see the effect of co-culturing on inflammatory markers (TNF-alpha, PGE2, and IL-10) and osteogenic markers (ALP, Runx2 & Alizarin Red Staining). It was found that the use of a co-culture did not have a positive effect on OB differentiation. The introduction of a prototype nanopattern (500 nm height and 300 nm width) that induces a macrophage polarization shift towards an anti-inflammatory phenotype, did have a positive effect on Runx2 secretion in the co-cultures. Furthermore the effect of a dynamic flow, initiated 2 days after seeding, on osteogenic differentiation of OB and CO12 was investigated. After 7 days of dynamic culture, Runx2 levels of the mono-cultures were significantly higher than the static cultures. OBs were positively affected by forming dense cell layers throughout the entire microfluidic channel. For dynamic co-cultures, no to little cells were present inside the channel after 7 days. Finally we attempted to fabricate a microfluidic device that could "sandwich" the patterned waver inside, so the effect of both the nanopattern and microflow on mono- and co-cultures could be investigated. Cells were seeded inside the channel after 1 day, but unfortunately forces on the PMMA layer of this chip resulted in cracks and failure of this system. Our findings in the design and fabrication of the chip can help in future research towards the osteoimmunodulatory properties of biomaterials. The study on a direct co-culture model, presented in this work, opens possibilities for future research towards co-cultures inside microfluidic devices. It discusses the possibilities and the drawbacks of both the use of co-cultures as the development of microfluidic devices.

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.

Objective: Advances in the field of biomaterials have positively affected implant acceptance and nowadays nanostructured surfaces are known to have a positive effect on bone regeneration. The development of the field of osteoimmunology has contributed to a steadily increasing interest towards the investigation of the immunomodulatory effect of nanopatterned surfaces. Luckily, advances in nanofabrication methods have resulted in fabricating relatively large nanostructured areas with high resolution. This study is taking advantage of the 3D printing technique called two-photon polymerization (2PP) for the generation of 3D submicron pillar structures (patterns) to systematically investigate their effect on murine macrophages regarding cell migration, viability, cytoskeletal organisation and phenotype polarization. Methods: Six different pillar patterns were fabricated using a Photonic Professional GT (Nanoscribe, Germany) system. The pillars were characterized with scanning electron microscopy (SEM). Cell viability of J774A.1 macrophages on the nanofabricated patters was assessed by live/dead staining with calcein AM and ethidium homodimer-1 and fluorescently analyzed. Cell migration to the patterns was studied using DAPI staining. The cytoskeletal organization of the macrophages was investigated by actin staining and analyzed both through fluorescent microscopy as well as SEM. Macrophage expression of nitric oxide (NO) was tested with Griess assay. The phenotype polarization of the macrophages on the patterns was explored by dual immunofluorescent staining with iNOS/ARG-1 and CCR7/CD206. Results: It was demonstrated that different pillar heights and pillar pitches induce different cell attachment mechanisms (lamellipodia, filopodial extensions) and directly affect cytoskeletal reorganization of the macrophages after one day of seeding. Patterns with a height of 1000 nm and a pitch of 700 nm cause an increase in the number of elongated cells. Round cells are experiencing larger area growth when adherent on patterns with a pitch of 1000 nm. Elongated macrophages are strongly affected by patterns of 1000 nm height and 700 nm pitch. Moreover, patterns with a pillar height and pitch of 1000 nm are shown to be the more potent leading to a M1/M2 macrophage phenotype switch.