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Review
. 2018 Oct 11:6:134.
doi: 10.3389/fbioe.2018.00134. eCollection 2018.

In vitro Models of Bone Remodelling and Associated Disorders

Robert Owen  1 , Gwendolen C Reilly  1
Affiliations
Review

In vitro Models of Bone Remodelling and Associated Disorders

Robert Owen et al. Front Bioeng Biotechnol. .

Abstract

Disruption of bone remodelling by diseases such as osteoporosis results in an imbalance between bone formation by osteoblasts and resorption by osteoclasts. Research into these metabolic bone disorders is primarily performed in vivo; however, in the last decade there has been increased interest in generating in vitro models that can reduce or replace our reliance on animal testing. With recent advances in biomaterials and tissue engineering the feasibility of laboratory-based alternatives is growing; however, to date there are no established in vitro models of bone remodelling. In vivo, remodelling is performed by organised packets of osteoblasts and osteoclasts called bone multicellular units (BMUs). The key determinant of whether osteoclasts form and remodelling occurs is the ratio between RANKL, a cytokine which stimulates osteoclastogenesis, and OPG, its inhibitor. This review initially details the different circumstances, conditions, and factors which have been found to modulate the RANKL:OPG ratio, and fundamental factors to be considered if a robust in vitro model is to be developed. Following this, an examination of what has been achieved thus far in replicating remodelling in vitro using three-dimensional co-cultures is performed, before overviewing how such systems are already being utilised in the study of associated diseases, such as metastatic cancer and dental disorders. Finally, a discussion of the most important considerations to be incorporated going forward is presented. This details the need for the use of cells capable of endogenously producing the required cytokines, application of mechanical stimulation, and the presence of appropriate hormones in order to produce a robust model of bone remodelling.

Keywords: 3D cell culture; bone remodelling; co-culture; in vitro model; osteoblast; osteoclast; osteoporosis; tissue engineering.

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Figures

Figure 1
Figure 1
Bone multicellular units in (top) trabecular and (bottom) cortical bone. In trabecular bone they initiate underneath bone remodelling canopies formed from bone lining cells and in cortical bone at points within Haversian canals.
Figure 2
Figure 2
The RANKL/RANK/OPG axis and M-CSF direct osteoclastogenesis and activation.
Figure 3
Figure 3
The five stages of bone remodelling.
Figure 4
Figure 4
Different methods of co-culturing cells. Conditioned media transfers media used in one culture to another. Well inserts culture cells in the same well but only soluble factors can exchange between cell types. Direct co-cultures can be performed in 2D or 3D and permit membrane bound and soluble factors to exert influence.
Figure 5
Figure 5
The calcium, PTH, Vitamin D3 homeostatic feedback loop.
Figure 6
Figure 6
The anabolic or catabolic effects of PTH on bone depends on application modality.
Figure 7
Figure 7
(A) Day 21 and (B) day 35 mCT scans of human osteoblast and osteoclast co-cultures registered to the original scaffold. Newly formed bone is coloured orange, resorbed areas are blue, constant/quiescent areas are grey. Adapted from (Rubert et al., 2017) under The Creative Commons Attribution–ShareAlike License (CC-BY-SA).
Figure 8
Figure 8
Osteoclasts within a 3D human osteoblast and osteoclast co-culture taken by (A) confocal laser scanning microscopy (cLSM) (actin—green, nuclei—blue) after 42 days and (B) SEM after 28 days. Osteoclast podosomes appear as dots within the cells by cLSM and thin filopodia are visible around the cell perimeter. Figure adapted from (Heinemann et al., 2011) under The Creative Commons Attribution–ShareAlike License (CC-BY-SA).
Figure 9
Figure 9
Non-invasive methods of assessing bone turnover in vitro. SVF cells can commit to osteoblastic and endothelial lineages. CD14+ cells differentiate to osteoclasts. Cell culture supernatants were analysed for (A) CICP, indicative of collagen synthesis, (B) TRAP, indicative of osteoclastic cells, (C) NTX, indicative of collagen resorption, (D) phopshate levels, where decreasing levels are indicative of mineralised matrix deposition and increasing levels are indicative of mineralised matrix resorption (*p < 0.05). Figure adapted from (Papadimitropoulos et al., 2011) under The Creative Commons Attribution-ShareAlike License (CC-BY-SA).
Figure 10
Figure 10
Requirements for a robust in vitro model of bone remodelling. A 3D co-culture of osteoblast- and osteoclast-lineage cells where the osteoblastic component are capable of endogenously producing RANKL, M-CSF and OPG. The model is cultured in a defined, serum-free medium containing physiologically relevant concentrations of important hormones, e.g., oestrogen, to permit the study of associated disorders such as postmenopausal osteoporosis. The culture can be mechanically loaded using varying force levels, for example by compression or application of fluid flow.
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