Biomaterial development is a long process consisting of multiple stages of design and evaluation within the context of both in vitro and in vivo testing. To streamline this process, mathematical and computational modeling displays potential as a tool for rapid biomaterial characterization, enabling the prediction of optimal physicochemical parameters. In this work, a Langmuir isotherm-based model was used to describe protein and cell adhesion on a biomimetic hydroxyapatite surface, both independently and in a one-way coupled system. The results indicated that increased protein surface coverage leads to improved cell adhesion and spread, with maximal protein coverage occurring within 48 h. In addition, the Langmuir model displayed a good fit with the experimental data. Overall, computational modeling is an exciting avenue that may lead to savings in terms of time and cost during the biomaterial development process.

Polydimethylsiloxane (PDMS) is among the most widely used materials for organ-on-chip systems. Despite itsmultiple beneficial characteristics from an engineering point of view, there is a concern about the effect of PDMSon the cells cultured in such devices. The aim of this study was to enhance the understanding of the effect of PDMSon cellular behavior in a context relevant for on-chip studies. The focus was put on an indirect effect of PDMS,namely leaching of uncrosslinked oligomers, particularly for bone regeneration applications. PDMS-based chipswere prepared and analyzed for the potential release of PDMS oligomers within the microfluidic channel whenkept at different flow rates. Leaching of uncrosslinked oligomers from PDMS was quantified as silicon concen-tration by inductively coupled plasma - optical emission spectrometry and further confirmed by mass spec-trometry. Subsequently, PDMS-leached media, with a silicon concentration matching the on-chip experiment,were prepared to study cell proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts and humanmesenchymal stem cells. The silicon concentration initially detected in the media was inversely proportional tothe tested flow rates and decreased to control levels within 52 h. In addition, by curing the material overnightinstead of 2 h, regardless of the curing temperature (65 and 80 C), a large reduction in silicon concentration wasfound, indicating the importance of the PDMS curing parameters. Furthermore, it was shown that PDMS oligo-mers enhanced the differentiation of MC3T3-E1 pre-osteoblasts, this being a cell type dependent effect as nochanges in cell differentiation were observed for human mesenchymal stem cells. Overall, this study illustrates theimportance of optimization steps when using PDMS devices for biological studies, in particular PDMS curingconditions and extensive washing steps prior to an experiment.

The reliability of conventional cell culture studies to evaluate biomaterials is often questioned, as in vitro outcomes may contradict results obtained through in vivo assays. Microfluidics technology has the potential to reproduce complex physiological conditions by allowing for fine control of microscale features such as cell confinement and flow rate. Having a continuous flow during cell culture is especially advantageous for bioactive biomaterials such as calcium-deficient hydroxyapatite (HA), which may otherwise alter medium composition and jeopardize cell viability, potentially producing false negative results in vitro. In this work, HA was integrated into a microfluidics-based platform (HA-on-chip) and the effect of varied flow rates (2, 8 and 14 µl/min, corresponding to 0.002, 0.008 and 0.014 dyn/cm², respectively) was evaluated. A HA sample placed in a well plate (HA-static) was included as a control. While substantial calcium depletion and phosphate release occurred in static conditions, the concentration of ions in HA-on-chip samples remained similar to those of fresh medium, particularly at higher flow rates. Pre-osteoblast-like cells (MC3T3-E1) exhibited a significantly higher degree of proliferation on HA-on-chip (8 μl/min flow rate) as compared to HA-static. However, cell differentiation, analysed by alkaline phosphatase (ALP) activity, showed low values in both conditions. This study indicates that cells respond differently when cultured on HA under flow compared to static conditions, which indicates the need for more physiologically relevant methods to increase the predictive value of in vitro studies used to evaluate biomaterials.

The in vitro biological characterization of biomaterials is largely based on static cell cultures. However, for highly reactive biomaterials such as calcium-deficient hydroxyapatite (CDHA), this static environment has limitations. Drastic alterations in the ionic composition of the cell culture medium can negatively affect cell behavior, which can lead to misleading results or data that is difficult to interpret. This challenge could be addressed by a microfluidics-based approach (i.e. on-chip), which offers the opportunity to provide a continuous flow of cell culture medium and a potentially more physiologically relevant microenvironment. The aim of this work was to explore microfluidic technology for its potential to characterize CDHA, particularly in the context of inflammation. Two different CDHA substrates (chemically identical, but varying in microstructure) were integrated on-chip and subsequently evaluated. We demonstrated that the on-chip environment can avoid drastic ionic alterations and increase protein sorption, which was reflected in cell studies with RAW 264.7 macrophages. The cells grown on-chip showed a high cell viability and enhanced proliferation compared to cells maintained under static conditions. Whereas no clear differences in the secretion of tumor necrosis factor alpha (TNF-α) were found, variations in cell morphology suggested a more anti-inflammatory environment on-chip. In the second part of this study, the CDHA substrates were loaded with the drug Trolox. We showed that it is possible to characterize drug release on-chip and moreover demonstrated that Trolox affects the TNF-α secretion and morphology of RAW 264.7 ​cells. Overall, these results highlight the potential of microfluidics to evaluate (bioactive) biomaterials, both in pristine form and when drug-loaded. This is of particular interest for the latter case, as it allows the biological characterization and assessment of drug release to take place under the same dynamic in vitro environment.

A requirement for clinical approval is verification of a biomaterial’s functionality and biocompatibility. However, discrepancies between in vitro and in vivo evaluations have been reported, possibly due in part to a lack of physiological relevance of typical in vitro culture set-ups. We introduce a Universal Biomaterial-on-Chip (UBoC), which is a microfluidics device that allows integration of biomaterials with varied shapes and properties and subsequent evaluation of in vitro performance under design considerations that resemble physiological conditions. In addition, UBoC operates with multifunctional modalities such as continuous perfusion, shear stress mechanostimulation and cell co-culture. The device is constructed using simple 3D printing and microfabrication techniques and its cell culture area resembles a 96-well plate (0.32 cm²). Successful cell adhesion and proliferation was observed on-chip on different materials (hydroxyapatite, titanium and fibrin) using fluorescence microscopy. Furthermore, device applicability for mechanostimulation was demonstrated through shear stimulation, where sensitivity of pre-osteoblasts to flow was captured via live Ca²⁺ imaging. Finally, the modularity of the UBoC platform for on-chip co-culture experiments was established after simple modifications of on-board fluidic arrangements. Overall, the UBoC presents a useful tool that augments existing in vitro testing strategies and enables thorough comparisons between biomaterials in tunable culture conditions.