"Development of novel bone biomaterials inevitably has a phase of in vitro testing for cytocompatibility and functional osteogenesis. This relies on defined conditions under which osteoblasts, or their precursors, are stimulated to drive a mature, matrix depositing osteoblast phenotype. Based on the in vitro results obtained, a selection is made to be further tested using in vivo studies. While this is the classically adopted route, the correlation between in vitro and in vivo results is not as strong as would be expected (1). This may not be surprising when considering the methods typically being used are decades old. This suggests there is room to optimize in vitro tests to obtain more translationally relevant data. Often overlooked is the desired route to new bone formation. Direct osteogenesis is favored when cells experience 2D environments, while embedding cells within a hydrogel leads to a 3D environment, that may be more favorable to indirect bone formation, or endochondral ossification. Which raises the question: should the culture media used in vitro be tailored to the exact bone forming mechanism desired? It has been suggested that 10 mM β-glycerophosphate typically used in osteogenesis medium can lead to spontaneous ectopic calcification, an artifact often observed when using cell free material. Classical dexamethasone containing osteogenic media also has the potential to drive adipogenesis. However, many studies typically only investigate markers associated with an osteogenic phenotype, potentially overlooking conflicting signals driving differentiation into other lineages. Taken together, this suggest that improvements are possible.
Therefore, studies testing novel biomaterials are increasing in complexity with the aim to develop more representative models and more accurately represent in vivo conditions (2).
Bioreactors can be used to improve flow and nutrient exchange, and coculture of cells can highlight interactions that may occur naturally in vivo but are lacking in monoculture studies. Increasingly, single cell sequencing is being adopted to study the differentiation of cells over time, identifying new markers and even distinct osteogenic differentiation pathways. This is key as the current range of markers may not be broad enough to make accurate predictions. Whole bone explant cultures can be combined with materials to investigate aspects such as osseointegration. As the body of knowledge increases, it is time to rethink how materials are tested
Combining bioreactors, coculture systems and improved markers and assays offers the opportunity to improve the accuracy of the in vitro results and reduce subsequent animal use as a result. While this may take some time, the outcomes will be rewarding.
- Hulsart-Billstrom G, Dawson JI, Hofmann S, Muller R, Stoddart MJ, Alini M, et al. A surprisingly poor correlation between in vitro and in vivo testing of biomaterials for bone regeneration: results of a multicentre analysis. Eur Cell Mater. 2016;31:312-22.
- Armiento AR, Hatt LP, Sanchez Rosenberg G, Thompson K, Stoddart MJ. Functional Biomaterials for Bone Regeneration: A Lesson in Complex Biology. Advanced Functional Materials.n/a(30):41.