An In Vitro Model for the Development of Mature Bone Containing an Osteocyte Network

Abstract

Bone is a dynamic tissue that remodels continuously in response to local mechanical and chemical stimuli. This process can also result in maladaptive ectopic bone in response to injury, yet pathological differences at the molecular and structural levels are poorly understood. A number of in vivo models exist but can often be too complex to allow isolation of factors which may stimulate disease progression. A self‐structuring model of bone formation is presented using a fibrin gel cast between two calcium phosphate ceramic anchors. Femoral periosteal cells, seeded into these structures, deposit an ordered matrix that closely resembles mature bone in terms of chemistry (collagen:mineral ratio) and structure, which is adapted over a period of one year in culture. Raman spectroscopy and X‐ray diffraction confirm that the mineral is hydroxyapatite associated with collagen. Second‐harmonic imaging demonstrates that collagen is organized similarly to mature mouse femora. Remarkably, cells differentiated to the osteocyte phase are linked by canaliculi (as demonstrated with nano‐computed tomography) and remained viable over the full year of culture. It is demonstrated that novel drugs can prevent ossification in constructs. This model can be employed to study bone formation in an effort to encourage or prevent ossification in a range of pathologies.

Adiponectin Reduces Bone Stiffness: Verified in a Three-Dimensional Artificial Human Bone Model In Vitro

“…Different scaffolding materials (natural and/or synthetic) have been used in the production and investigation of bone in vitro ( 4 , 6 , 10 , 11 ). Recently, a model of bone formation has been presented, where two calcium phosphate scaffolds were used as anchors and cells grown on a fibrin gel cast in between ( 12 ). However, the presence of scaffolds makes it challenging to investigate the effect of a substance on the mechanical properties of the artificial bone cell constructs.…”

A novel method for the collection of nanoscopic vesicles from an organotypic culture model

“…Organotypic bone constructs were developed by embedding periosteal osteoblastic cells into mechanically-strained fibrin scaffolds, 22 which were cultured for extended periods of time, during which, the matrix became gradually replaced with a cell-produced greater density matrix containing increasing amounts of mineral ( Fig. 2a ).…”

“…Interestingly, both the early and mature culture medium contained nanoparticles in the size range of 50 nm to 300 nm compared to controls (serum supplemented but cell-free medium), which encompasses the range in which previously described matrix mineralization vesicles were detected. 3 , 6 , 28 Mature constructs (1 year), which have been previously shown to contain approximately 70% mineral, 22 contained a higher concentration of these nanoparticles in their growth medium.…”

“…Constructs were developed using the methods described previously, 22 by seeding fibrin hydrogels with an osteoblastic cell population derived from the periosteal tissue of femoral bones. The fibrin scaffolds were produced through the interaction of the normal plasma components, thrombin and fibrinogen.…”

“…3D cultures of osteoblastic cells can provide an ideal alternative to generate a rich source of vesicles which can be collected for analysis. Recently, we have developed an organotypic model of mature bone, 22 which allows long-term culture of bone tissue, in excess of 1 year, and which shows a biochemical composition and structural organisation similar to mature bone at the centimetre scale. We present here a method for small-scale isolation of matrix vesicles from the growth medium of this bone-like system, based on surface marker immunoselection, which can provide a valuable tool for understanding the biological function of these structures.…”