Approx. every third hospitalized patient in Europe suffers from musculoskeletal injuries or diseases. Up to 20% of these patients need costly surgical revisions after delayed or impaired fracture healing. Reasons for this are the severity of the trauma, individual factors, e.g, the patients' age, individual lifestyle, chronic diseases, medication, and, over 70 diseases that negatively affect the bone quality. To investigate the various disease constellations and/or develop new treatment strategies, many in vivo, ex vivo, and in vitro models can be applied. Analyzing these various models more closely, it is obvious that many of them have limits and/or restrictions. Undoubtedly, in vivo models most completely represent the biological situation. Besides possible species-specific differences, ethical concerns may question the use of in vivo models especially for large screening approaches. Challenging whether ex vivo or in vitro bone models can be used as an adequate replacement for such screenings, we here summarize the advantages and challenges of frequently used ex vivo and in vitro bone models to study disturbed bone metabolism and fracture healing. Using own examples, we discuss the common challenge of cell-specific normalization of data obtained from more complex in vitro models as one example of the analytical limits which lower the full potential of these complex model systems.

/pdf/use-of-in-vitro-bone-models-to-screen-for-altered-bone-1575q1uyfs.pdf

Use of in vitro bone models to screen for altered bone metabolism, osteopathies, and fracture healing: challenges of complex models

Drug-induced liver toxicity is one of the most common reasons for the failure of drugs in clinical trials and frequent withdrawal from the market. Reasons for such failures include the low predictive power of in vivo studies, that is mainly caused by metabolic differences between humans and animals, and intraspecific variances. In addition to factors such as age and genetic background, changes in drug metabolism can also be caused by disease-related changes in the liver. Such metabolic changes have also been observed in clinical settings, for example, in association with a change in liver stiffness, a major characteristic of an altered fibrotic liver. For mimicking these changes in an in vitro model, this study aimed to develop scaffolds that represent the rigidity of healthy and fibrotic liver tissue. We observed that liver cells plated on scaffolds representing the stiffness of healthy livers showed a higher metabolic activity compared to cells plated on stiffer scaffolds. Additionally, we detected a positive effect of a scaffold pre-coated with fetal calf serum (FCS)-containing media. This pre-incubation resulted in increased cell adherence during cell seeding onto the scaffolds. In summary, we developed a scaffold-based 3D model that mimics liver stiffness-dependent changes in drug metabolism that may more easily predict drug interaction in diseased livers.

https://www.mdpi.com/2079-4983/11/1/17/pdf

Development of Scaffolds with Adjusted Stiffness for Mimicking Disease-Related Alterations of Liver Rigidity.

Objective: To observe simultaneous differentiation and analyse possible interactions between co-cultured human oral mucosal stem cells (hOMSC) and mouse neural stem cells (mNSC). Materials and Methods: hOMSC and mNSC were co- cultured in mouse and in human medium, and their immunocytochemical characterization to detect survival rate and differentiation pattern was performed. Co- cultures in different media were compared to hOMSC in human medium and mNSC in mouse medium as controls. Results: Co-culture of hOMSC and mNSC in medium for human cells led to normal differentiation pattern of human cells, while mNSC were directed towards astrocytes. When the same cells were cultivated in the mouse medium, both cell types succeeded to form neurons, although mNSC showed a tendency to overgrow hOMSC. hOMSC alone in the human-specific medium differentiated towards ectodermal (Oct4, Map2) and mesodermal (Osterix) cell populations. mNSC in the mouse-specific medium differentiated towards Map2-, β3-tubulin- and NeuN-positive neurons. Conclusions: hOMSC and mNSC can form co- cultures. Different media considerably affected the differentiation pattern of co- cultures, whereas one cell population itself modestly influenced differentiation of the other cell type. The in vitro differentiation pattern of hOMSC in the mouse neural tissue environment suggested that hOMSC could be beneficial in the brain tissue affected by ischaemia.

/pdf/human-and-mouse-derived-neurons-can-be-simultaneously-nrym0ubce0.pdf

Human- and mouse-derived neurons can be simultaneously obtained by co-cultures of human oral mucosal stem cells and mouse neural stem cells.

In vitro modeling of liver fibrosis in 3D microtissues using scalable micropatterning system

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.

3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics.

In order to increase the metabolic activity of human hepatocytes and liver cancer cell lines, many approaches have been reported in recent years. The metabolic activity could be increased mainly by cultivating the cells in 3D systems or co-cultures (with other cell lines). However, if the system becomes more complex, it gets more difficult to quantify the number of cells (e.g., on a 3D matrix). Until now, it has been impossible to quantify different cell types individually in 3D co-culture systems. Therefore, we developed a PCR-based method that allows the quantification of HepG2 cells and 3T3-J2 cells separately in a 3D scaffold culture. Moreover, our results show that this method allows better comparability between 2D and 3D cultures in comparison to the often-used approaches based on metabolic activity measurements, such as the conversion of resazurin.

Cell-Type-Specific Quantification of a Scaffold-Based 3D Liver Co-Culture

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Cell-Type-Specific Quantification of a Scaffold-Based 3D Liver Co-Culture