Osteoblast Differentiation and Bone Matrix FormationIn VivoandIn Vitro

Abstract

We review the characteristics of osteoblast differentiation and bone matrix synthesis. Bone in air breathing vertebrates is a specialized tissue that developmentally replaces simpler solid tissues, usually cartilage. Bone is a living organ bounded by a layer of osteoblasts that, because of transport and compartmentalization requirements, produce bone matrix exclusively as an organized tight epithelium. With matrix growth, osteoblasts are reorganized and incorporated into the matrix as living cells, osteocytes, which communicate with each other and surface epithelium by cell processes within canaliculi in the matrix. The osteoblasts secrete the organic matrix, which are dense collagen layers that alternate parallel and orthogonal to the axis of stress loading. Into this matrix is deposited extremely dense hydroxyapatite-based mineral driven by both active and passive transport and pH control. As the matrix matures, hydroxyapatite microcrystals are organized into a sophisticated composite in the collagen layer by nucleation in the protein lattice. Recent studies on differentiating osteoblast precursors revealed a sophisticated proton export network driving mineralization, a gene expression program organized with the compartmentalization of the osteoblast epithelium that produces the mature bone matrix composite, despite varying serum calcium and phosphate. Key issues not well defined include how new osteoblasts are incorporated in the epithelial layer, replacing those incorporated in the accumulating matrix. Development of bone in vitro is the subject of numerous projects using various matrices and mesenchymal stem cell-derived preparations in bioreactors. These preparations reflect the structure of bone to variable extents, and include cells at many different stages of differentiation. Major challenges are production of bone matrix approaching the in vivo density and support for trabecular bone formation. In vitro differentiation is limited by the organization and density of osteoblasts and by endogenous and exogenous inhibitors.

Adrenocorticotropic hormone and 1,25-dihydroxyvitamin D3 enhance human osteogenesis in vitro by synergistically accelerating the expression of bone-specific genes

“…ACTH 10 pM rapidly increased the expression of the cartilage link protein 1 (CRTL1) in keeping with the observation that cartilage protein expression precedes bone markers during bone differentiation in vitro . 22 ACTH also increased the expression of VDR, in keeping with the synergistic effects of ACTH and 1,25(OH2)D on rapid bone differentiation. Two-h effects of 10 nM 1,25(OH) 2 D included a large effect on the expression of VEGF-A, an expected result based on previous work.…”

“…21 Our experience is that bone formation in vitro always occurs together with cartilage markers, suggesting that differentiation of MSCs to some extent recapitulates the sequence of cartilage and bone differentiation seen in embryonic bone and in skeletal repair. 22 …”

“…This reflects that bone mineral is not a result of mass action; hydroxyapatite precipitation occurs in a closed environment and is driven to completion by outward transport of the acid produced by mineral precipitation. 22,34 This physiology is the ‘Achilles heel’ in translating in vitro effects on osteoblast differentiation into clinical results; the interesting effects of 1,25(OH) 2 D on ACTH action and, in particular, VEGF production are more likely to be physiologically important. 1 Particularly, the strong and reproducible increase in osteoblast mineral production in vitro in high calcium does not translate to bone production in response to oral calcium in the setting of adequate calcium intake.…”

“…Specifically, it supported synergistic and rapid effects of ACTH and 1,25(OH) 2 D on bone development. At 2 h, 10 pM ACTH increased TGF- β receptor expression, a major early bone or cartilage signal, 22 as well as the expression of the src homology adaptor protein Shc, which is a mediator in TGF beta signaling, 37 and of the focal adhesion kinase FAK1. ACTH 10 pM rapidly increased the expression of the cartilage link protein 1 (CRTL1) in keeping with the observation that cartilage protein expression precedes bone markers during bone differentiation in vitro .…”

The critical interplay between bone resorbing and bone forming cells

“…There are many possible signalling molecules that contribute to coupling and these include growth factors released from the bone matrix up on bone resorption, soluble and membrane products of the osteoclasts and their precursors and signals from osteocytes (Figure and further described below). In the formation phase, osteoblasts deposit new bone, unmineralized matrix called osteoid which subsequently become mineralized by the osteoblasts (Blair et al., ). As osteoblasts produce OPG, which potently suppresses osteoclastogenesis by masking RANKL, they may inhibit the initiation of additional bone remodelling cycle activity in the BMU.…”