Isolation of Extracellular Vesicle Subtypes from Primary Osteoblast Cultures.

This chapter describes the isolation and characterization of extracellular vesicles (EVs) isolated from primary rodent osteoblasts and extracellular matrix. The key advantages of this assay are that it allows the isolation of EVs from the conditioned media of primary osteoblasts at varying stages of differentiation and subsequent characterization prior to downstream applications.

Isolation and Generation of Osteoclasts.

This chapter describes the isolation, expansion, staining, and imaging of osteoclasts from murine (MKS, BKD, and MMM) and human (NS, JBO, and KS) sources. We cover in detail both traditional and more modern methods of assessing osteoclast formation and function in vitro including advances in image acquisition and automated analyses. Importantly, we provide in-depth methods for human osteoclast culture systems, methods to assess human osteoclast function, and highlight potential methodological pitfalls and ways to overcome them.

A dietary intervention following incretin analog treatment restores adipose tissue functions in diet-induced obese mice.

Obesity is associated with the development of type 2 diabetes. In recent years, incretin analogs have been prescribed at a high rate for the treatment of obesity and diabetes due to their potent effects on lowering body weight and improving glucose homeostasis. However, many patients do not stay on incretin analog therapy and thereby rapidly regain body weight.

Extracellular pH is a critical regulator of osteoclast fusion, size and activation.

Osteoclast activity is regulated by extracellular pH, whereby bone resorption is near-maximally activated at pH 7.0 but limited at ≥pH 7.4.

A Machine Learning-Based Image Segmentation Method to Quantify In Vitro Osteoclast Culture Endpoints.

Quantification of in vitro osteoclast cultures (e.g. cell number) often relies on manual counting methods.

Evidence that pyrophosphate acts as an extracellular signalling molecule to exert direct functional effects in primary cultures of osteoblasts and osteoclasts.

Extracellular pyrophosphate (PP) is well known for its fundamental role as a physiochemical mineralisation inhibitor. However, information about its direct actions on bone cells remains limited. This study shows that PP decreased osteoclast formation and resorptive activity by ≤50 %.

Modulation of osteoblast differentiation and function by the P2X4 receptor.

Bone cells are known to express multiple P2 receptor subtypes, and the functional effects of receptor activation have been described for many of these. One exception is the P2X4 receptor, which despite strong expression in osteoblasts and osteoclasts, has no defined functional activity. This study used the selective P2X4 receptor antagonists, 5-BDBD and PSB-12062, to investigate the role of this receptor in bone.

Differing calcification processes in cultured vascular smooth muscle cells and osteoblasts.

Arterial medial calcification (AMC) is the deposition of calcium phosphate mineral, often as hydroxyapatite, in the medial layer of the arteries. AMC shares some similarities to skeletal mineralisation and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) towards an osteoblast-like phenotype. This study used primary mouse VSMCs and calvarial osteoblasts to directly compare the established and widely used in vitro models of AMC and bone formation.