Collagen Osteoid-Like Model Allows Kinetic Gene Expression Studies of Non-Collagenous Proteins in Relation with Mineral Development to Understand Bone Biomineralization

Abstract

Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental in vitro model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT- and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including BGLAP, COL1A1, and BMP2. Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of SPP1 and BGLAP, which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology.

Involvement of 3D osteoblast migration and bone apatite during in vitro early osteocytogenesis

“…The mechanical properties measured in pure dense collagen matrices (15 kPa) are close to those reported for the osteoid tissue (about 25 kPa [4,12] ) favoring a more physiological behavior of osteoblasts in our models than in looser gels [4] . The initial number of seeded cells also appears of great importance since a lower initial number of cells may slow down the migration process [13] .…”

“…Additionally, the fibrillar feature of the collagen network appears crucial since it allows the osteoblasts adhesion via integrin α1β1 and α2β1 [9,10] , favors the physiological-type shape and directs cells toward osteoblast maturation by changing the local topography [11] . At a larger scale, the collagen network density prevents cell apoptosis and promotes osteoblast differentiation [12,13] .…”

“…Thanks to their composition (type I collagen), texture (fibrils) and density (40 mg/mL), these matrices were proposed as a connective tissue model for cell culture [14] . They provide an appropriate three dimensional (3D) microenvironment for the adhesion, spreading and differentiation of osteoblasts [11,15] , for the investigation of the role of non-collagenous proteins (NCPs) in the mineralization process [13] or the study of the osteoblast behavior in a bone healing context [16] .…”

Enhanced osteoblastogenesis in three-dimensional collagen gels

“…At earlier time points, at 2, 4 or 6 h after seeding, the levels of osteoblast gene expression were similar in cells growing in 2D and 3D collagen cultures (data not shown). In a number of previous studies, 3,8,14,15 collagen gels were formed first and osteoblasts were seeded on top of the gels, allowing for the analysis of cell migration into the gels. Interestingly, while osteoblasts migrated into the gels and formed a 3D cell network, fibroblasts seeded on top of the gels migrated through them and formed a monolayer on the plastic underneath.…”

“…Transmission electron microscopy was used to demonstrate cell-cell interactions through tight junctions, but, although cytoplasmic extensions were shown to penetrate the dense collagen matrix, the cells themselves were aligned on top of the surface and did not penetrate the matrix. 14 …”