A microfluidic-based approach to investigate the inflammatory response of macrophages to pristine and drug-loaded nanostructured hydroxyapatite.

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 â€‹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 practical guide for evaluating the osteoimmunomodulatory properties of biomaterials.

Biomaterials offer a promising approach to repair bone defects. Whereas traditional studies predominantly focused on optimizing the osteogenic capacity of biomaterials, less focus has been on the immune response elicited by them. However, the immune and skeletal systems extensively interact, a concept which is referred to as 'osteoimmunology'. This realization has fuelled the development of biomaterials with favourable osteoimmunomodulatory (OIM) properties, aiming to modulate the immune response and to support bone regeneration, thereby affecting the success of an implant. Given the plethora of in vitro assays used to evaluate the OIM properties of biomaterials, it may be challenging to select the right methods to produce conclusive results. In this review, we aim to provide a comprehensive and practical guide for researchers interested in studying the OIM properties of biomaterials in vitro. After a concise overview of the concept of osteoimmunology, emphasis is put on the methodologies that are regularly used to evaluate the OIM properties of biomaterials. First, a description of the most commonly used cell types and cell culture media is provided. Second, typical experimental set-ups and their relevant characteristics are discussed. Third, a detailed overview of the generally used methodologies and readouts, including cell type-specific markers and time points of analysis, is given. Finally, we highlight the promise of advanced approaches, namely microarrays, bioreactors and microfluidic-based systems, and the potential that these may offer to the osteoimmunology field. STATEMENT OF SIGNIFICANCE: Osteoimmunology focuses on the connection and communication between the skeletal and immune systems. This interaction has been recognized to play an important role in the clinical success of biomaterials, which has resulted in an increasing amount of research on the osteoimmunomodulatory (OIM) properties of biomaterials. However, the amount of literature makes it challenging to extract the information needed to design experiments from beginning to end, and to compare obtained results to existing work. This article intends to serve as a guide for those aiming to learn more about the commonly used experimental approaches in the field. We cover early-stage choices, such as cell types and experimental set-ups, but also discuss specific assays, including cell markers and time points of analysis.

Exploring microfluidics as a tool to evaluate the biological properties of a titanium alloy under dynamic conditions.

To bring novel biomaterials to clinical use, reliable in vitro models are imperative. The aim of this work was to develop a microfluidic tool to evaluate the biological properties of biomaterials for bone repair. Two approaches to embed medical grade titanium (Ti6Al4V) on-chip were explored. The first approach consisted of a polydimethylsiloxane microfluidic channel placed onto a titanium disc, held together by an additively manufactured fixture. In the second approach, a titanium disc was assembled onto a microscopic glass slide, using a double-sided tape microfluidic channel. Both approaches demonstrated potential for maintaining MC3T3-E1 preosteoblast-like cell cultures on-chip, as was shown by the vast majority of living cells after 1 day. In addition, the cells cultured on-chip showed a more elongated morphology compared to cells grown under static conditions and a tendency to align to the direction of the flow. For longer-term (i.e. 10 days) studies, the glass-based chip was selected. Assessment of cell viability showed a high number of living cells during the entire experimental period. Cell proliferation and differentiation studies revealed an increase in cell proliferation on-chip, suggesting that proliferation was the dominating process at the detriment of differentiation in this micrometric dynamic environment. These results illustrate the importance of optimizing in vitro cell culture conditions and how these may affect biomaterial testing outcomes. Overall, this work provides a step towards more in vivo-like microfluidic testing platforms, which are expected to provide more reliable in vitro screening of biomaterials.

Development of a Coaxial 3D Printing Platform for Biofabrication of Implantable Islet-Containing Constructs.

Over the last two decades, pancreatic islet transplantations have become a promising treatment for Type I diabetes. However, although providing a consistent and sustained exogenous insulin supply, there are a number of limitations hindering the widespread application of this approach. These include the lack of sufficient vasculature and allogeneic immune attacks after transplantation, which both contribute to poor cell survival rates. Here, these issues are addressed using a biofabrication approach. An alginate/gelatin-based bioink formulation is optimized for islet and islet-related cell encapsulation and 3D printing. In addition, a custom-designed coaxial printer is developed for 3D printing of multicellular islet-containing constructs. In this work, the ability to fabricate 3D constructs with precise control over the distribution of multiple cell types is demonstrated. In addition, it is shown that the viability of pancreatic islets is well maintained after the 3D printing process. Taken together, these results represent the first step toward an improved vehicle for islet transplantation and a potential novel strategy to treat Type I diabetes.

Additive Biomanufacturing: An Advanced Approach for Periodontal Tissue Regeneration.

Periodontitis is defined as a chronic inflammatory condition, characterized by destruction of the periodontium, composed of hard (i.e. alveolar bone and cementum) and soft tissues (i.e. gingiva and periodontal ligament) surrounding and supporting the teeth. In severe cases, reduced periodontal support can lead to tooth loss, which requires tissue augmentation or procedures that initiate a repair, yet ideally a regenerative response. However, mimicking the three-dimensional complexity and functional integration of the different tissue components via scaffold- and/or matrix-based guided tissue engineering represents a great challenge. Additive biomanufacturing, a manufacturing method in which objects are designed and fabricated in a layer-by-layer manner, has allowed a paradigm shift in the current manufacturing of medical devices and implants. This shift from design-to-manufacture to manufacture-to-design, seen from a translational research point of view, provides the biomedical engineering and periodontology communities a technology with the potential to achieve tissue regeneration instead of repair. In this review, the focus is put on additively biomanufactured scaffolds for periodontal applications. Besides a general overview of the concept of additive biomanufacturing within this field, different developed scaffold designs are described. To conclude, future directions regarding advanced biomaterials and additive biomanufacturing technologies for applications in regenerative periodontology are highlighted.