Abstract
AbstractBone 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.
Highlights
The presence of the two anchors is essential for generating these structures as fibrin gels, which are seeded with cells but are not provided with anchors do not become organized by cells and contract randomly (Figures S3 and S4, Supporting Information)
The matrix starts to become mineralized from the bone-like anchor regions as early as 10 d, and these deposits grow over the subsequent days as mineral nodules form throughout the structure (Figure 1b)
Some shrinkage and a level of dispersed calcification were noted in these structures, they did not exhibit the high levels of structural organization that were notable in our cell-seeded scaffold structures, suggesting that the embedded cells are critical to enabling the reorganization and subsequent mineralization of the bone-like structures
Summary
Osseous tissue forms in various physiological circumstances, ranging from normal bone development and callusmediated fracture repair, to pathological heterotopic bone formation in extra-skeletal tissues, as seen following muscle trauma, traumatic brain and spinal cord injury, surgical procedures of the hip and knee; and in genetic conditions such as fibrodysplasia ossificans progressiva (FOP).[1,2,3,4,5,6,7,8,9] These contexts, normal or otherwise, share fundamental characteristics at many levels, including molecular (overexpression of bone morphogenic proteins, BMPs), cellular (a set of progenitor cells that commits to an osteoblastic lineage) and biomechanical (translation of the mechanical forces into structured and organized bone).[3,10,11,12,13,14,15] there are still significant gaps in our understanding of these events and in particular few models allow long-term. Periosteum cells are the main determinants in the reparative phase of bone fracture healing, when they interact with the temporary fracture callus, which serves as a scaffold for the formation of new bone.[1,22,23,24,25] they have been associated with heterotopic ossification (HO) and have the ability to give rise to endochondral bone when implanted in muscle, and may be central to this condition.[17,24,26] Following isolation, periosteum cells were seeded into fibrin hydrogels, which critically permit selfassembly and imaging of a de novo collagen matrix, and because as materials, fibrin scaffolds have structural and biochemical similarities to the microenvironment of the callus formed early in fracture healing.[27,28,29] Both cortical and trabecular bone show a similar matrix alignment at the microscale, which is essential for development and mechanical resistance To mimic this important feature, the environment of the fracture callus was further emulated by introducing two calcium phosphate (CaP) anchors at the extremities of the culture dish. We show here that antiosteogenic compounds can reverse the progression of ossification, indicating potential for interventional studies
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