Harry C. Blair

Bone resorption depends on the formation, by osteoclasts, of an acidic extracellular compartment wherein matrix is degraded. The mechanism by which osteoclasts transport protons into that resorptive microenvironment was identified by means of adenosine triphosphate-dependent weak base accumulation in isolated osteoclast membrane vesicles, which exhibited substrate and inhibition properties characteristic of the vacuolar, electrogenic H+-transporting adenosine triphosphatase (H+-ATPase). Identify of the proton pump was confirmed by immunoblot of osteoclast membrane proteins probed with antibody to vacuolar H+-ATPase isolated from bovine kidney. The osteoclast's H+-ATPase was immunocytochemically localized to the cell-bone attachment site. Immunoelectron microscopy showed that the H+-ATPase was present in the ruffled membrane, the resorptive organ of the cell.

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.

Bisphosphonates are useful in treatment of disorders with increased osteoclastic activity, but the mechanism by which bisphosphonates act is unknown. We used cultures of chicken osteoclasts to address this issue, and found that 1-hydroxyethylidenediphosphonic acid (EHDP), dichloromethylidenediphosphonic acid (Cl2MDP), or 3-amino-1-hydroxypropylidene-1,1-diphosphonic acid (APD) all cause direct dose-dependent suppression of osteoclastic activity. Effects are mediated by bone-bound drugs, with 50% reduction of bone degradation occurring at 500 nM to 5 microM of the different agents. Osteoclastic bone-binding capacity decreased by 30-40% after 72 h of bisphosphonate treatment, despite maintenance of cell viability. Significant inhibition of bone resorption in each case is seen only after 24-72 h of treatment. Osteoclast activity depends on ATP-dependent proton transport. Using acridine orange as an indicator, we found that EHDP reduces proton accumulation by osteoclasts. However, inside-out plasma membrane vesicles from osteoclasts transport H+ normally in response to ATP in high concentrations of EHDP, Cl2MDP, or APD. This suggests that the bisphosphonates act as metabolic inhibitors. Bisphosphonates reduce osteoclastic protein synthesis, supporting this hypothesis. Furthermore, [3H]leucine incorporation by the fibroblast, which does not resorb bone, is also diminished by EHDP, Cl2MDP and APD except when co-cultured with bisphosphonate-binding bone particles. Thus, the resorption-antagonizing capacities of EHDP, Cl2MDP and APD reflect metabolic inhibition, with selectivity for the osteoclast resulting from high affinity binding to bone mineral.