Control of Vertebrate Skeletal Mineralization by Polyphosphates

BACKGROUND:Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO(3)(-))(n)) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization. PRINCIPAL FINDINGS/METHODOLOGY:The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO(4)(3-)) concentration while permitting the accumulation of a high total PO(4)(3-) concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4',6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation. CONCLUSIONS/SIGNIFICANCE:We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled

The Consequences of Collagen Degradation on Bone Mechanical Properties

The mechanisms underlying the effect of alterations in Type I collagen on bone mechanical properties are not well defined. Clinical tools for evaluating fracture risk, such as dual energy x-ray absorptiometry (DXA) and quantitative ultrasound (QUS) focus on bone mineral and cannot detect changes in the collagen matrix. The mechanical response tissue analyzer (MRTA) is a potential tool for evaluating fracture risk. Thus, the focus of this work was to investigate the effects of collagen degradation on bone mechanical properties and examine whether clinical tools can detect these changes. Female and male emu tibiae were endocortically treated with 1 M potassium hydroxide (KOH) solution for 1-14 days and then either mechanically tested in three-point bending, fatigued to failure or fatigued to induce stiffness loss. Computed Tomography scans, DXA, QUS, MRTA and three-point bend testing in the elastic region were performed on emu tibiae before and after either KOH treatment or fatigue to induce stiffness loss. Fracture surfaces were examined to determine failure mechanisms. Bone mineral and bone collagen were characterized using appropriate techniques. Bone mineral-collagen interface was investigated using Raman spectroscopy and atomic force microscopy (AFM). Endocortical KOH treatment does not affect bone mineral however, it causes in situ collagen degradation, rather than removal and may be weakening the mineral-collagen interface. These changes result in significantly compromised mechanical properties. Emu tibiae show significant decreases in failure stress and increased failure strain and toughness, with increasing KOH treatment time. The significant increase in toughness of KOH treated bones is due to structural alterations that enhance the ability of the microstructure to dissipate energy during the failure process, thereby slowing crack propagation, as shown by fracture surface analysis. KOH treated samples exhibit a lower fatigue resistance compared to untreated samples at high stresses only for both sexes. Partial fatigue testing results in similar decreases in modulus for all groups and sexes. The MRTA detected these changes whereas DXA and QUS did not. MRTA detects changes in bone mechanical properties induced by changes in collagen quality and fatigue and could be a more effective tool for predicting fracture risk.Ph