Noncollagenous proteins in normal and pathological human bone

2004

Calcif Tissue Int

Abstract. The purpose of this study was to develop an in vitro model system for bone matrix mineralization in the absence of cells. For this model, we utilized EDTAdecalcified new-born rat tibias with the cartilaginous ends intact, allowing us to visually determine the specificity of mineralization within the bone. Our results show that supplementation of DMEM culture medium with 10mM b-glycerophosphate and 15% fetal bovine serum (FBS) results in non-physiological mineral percipitation in the tibia because of the generation of supraphysiological (5mM) levels of inorganic phosphate in the medium. The same medium supplemented only with inorganic phosphate to a final concentration of 2mM failed to mineralize a decalcified tibia matrix. However, additional supplementation of this medium with as little as 5% FBS resulted in mineralization of those regions of the type I collagen where mineral was found prior to decalcification, with no evidence for mineralization in the cartilage at the bone ends or in the periosteum. Analysis of the mineral by Fourier-transform infrared spectroscopy and powder X-ray diffraction shows that tibias that have been decalcified and then remineralized contain an apatitic mineral that is strikingly similar to the mineral in normal bone. Tendon, a type I collagen matrix not normally mineralized in vivo , also mineralizes when incubated in DMEM containing 2mM Pi and as little as 1.5% FBS, but not when incubated in DMEM without serum. These data indicate that serum contains a nucleator of type I collagen matrix mineralization, and that mineralization of type I collagen under cell culture conditions requires serum but not living cells.

Section: Discussionmentioning

confidence: 99%

Mineralization of Decalcified Bone Occurs Under Cell Culture Conditions and Requires Bovine Serum But Not Cells

Hamlin

2004

Calcif Tissue Int

“…Because fetuin is the subject of this study, it is useful to review briefly its occurrence and calcification-inhibitory activity. Fetuin is a 48-kDa glycoprotein that is synthesized in the liver and is found at high concentrations in mammalian serum (15,16) and bone (17)(18)(19)(20)(21)(22). The serum fetuin concentration in adult mammals ranges from 0.5 to 1.5 mg/ml, whereas the serum fetuin concentration in the fetus and neonate is typically far higher (16).…”

mentioning

confidence: 99%

“…The serum fetuin concentration in adult mammals ranges from 0.5 to 1.5 mg/ml, whereas the serum fetuin concentration in the fetus and neonate is typically far higher (16). Fetuin is also one of the most abundant noncollagenous proteins found in bone (17)(18)(19)(20)(21)(22), with a concentration of about 1 mg fetuin/g bone in rat (21), bovine (17), and human (19,23) bone. Despite the abundance of fetuin in bone, however, it has not been possible to demonstrate the synthesis of fetuin in calcified tissues, and it is therefore presently thought that the fetuin found in bone arises from hepatic synthesis via serum (20,22).…”

mentioning

confidence: 99%

Mineralization by Inhibitor Exclusion

Journal of Biological Chemistry

Toroian

Lim

2009

152

One of our goals is to understand the mechanisms that deposit mineral within collagen fibrils, and as a first step we recently determined the size exclusion characteristics of the fibril. This study revealed that apatite crystals up to 12 unit cells in size can access the water within the fibril, whereas molecules larger than a 40-kDa protein are excluded. Based on these observations, we proposed a novel mechanism for fibril mineralization: that macromolecular inhibitors of apatite growth favor fibril mineralization by selectively inhibiting crystal growth in the solution outside of the fibril. To test this mechanism, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentration and the only macromolecule in solution is fetuin, a 48-kDa inhibitor of apatite growth. Our experiments with this system demonstrated that fetuin determines the location of mineral growth; in the presence of fetuin mineral grows exclusively within the fibril, whereas in its absence mineral grows in solution outside the fibril. Additional experiments showed that fetuin is also able to localize calcification to the interior of synthetic matrices that have size exclusion characteristics similar to those of collagen and that it does so by selectively inhibiting mineral growth outside of these matrices. We termed this new calcification mechanism "mineralization by inhibitor exclusion," the selective mineralization of a matrix using a macromolecular inhibitor of mineral growth that is excluded from that matrix. Future studies will be needed to evaluate the possible role of this mechanism in bone mineralization.

“…Previous studies have demonstrated that fetuin is the major calcification inhibitor found in serum (3,4). Fetuin is also one of the most abundant noncollagenous proteins found in mammalian bone (5)(6)(7)(8)(9)(10), with a concentration of ϳ1 mg of fetuin/g of bone in rat (9). MGP is a small, 10-kDa vitamin K-dependent protein (11) that is secreted by a wide variety of cell types, including vascular smooth muscle cells, and is found in bone at a concentration of 0.2 mg/g in the rat (12).…”

mentioning

confidence: 99%

Biochemical Characterization of the Serum Fetuin-Mineral Complex

Journal of Biological Chemistry

Nguyen

Williamson

2003

110

The present study was carried out to characterize the fetuin-mineral complex (FMC), a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein (MGP) that was initially discovered in serum of rats treated with etidronate and appears to play a critical role in inhibiting calcification in vivo. Fetuin purified from the FMC contains 3.3 mol of protein-bound phosphate. There is 1.3 mg of FMC/ml of serum 6 h after etidronate injection, and the FMC is 46% fetuin and 53% mineral by mass. Formation of the FMC in the first 6 h after etidronate injection does not increase serum fetuin despite the fact that 50% of serum fetuin is associated with the FMC, and clearance of the FMC in the 9 -24-h interval lowers total serum fetuin by 50%. These observations suggest that the fetuin component of the FMC is derived from fetuin initially in serum and that clearance of the FMC removes the associated fetuin from circulation. One additional protein was consistently present in all preparations of the FMC, spp24 (secreted phosphoprotein 24). This 24-kDa protein is similar in domain structure to fetuin and, like fetuin and MGP, contains several residues of phosphoserine and accumulates in bone. Exogenous spp24 associated strongly with the FMC when added to serum containing it. These observations suggest that spp24 may, like fetuin and MGP, play a role in inhibiting calcification.The present experiments are a continuation of our investigations into the mechanisms by which proteins interact specifically with mineral in vivo to prevent the calcification of arteries and other soft tissues. In a previous study (1), we described the discovery of a complex of calcium, phosphate, fetuin, 1 and matrix Gla protein in the serum of rats treated with the bone active bisphosphonate etidronate and showed that the appearance of this complex in serum correlates with the inhibition of bone mineralization by etidronate. The fetuin-mineral complex reaches maximal levels 6 -9 h after etidronate injection and causes a 4-fold increase in total serum calcium without causing any increase in ionic calcium levels.The proteins associated with the fetuin-mineral complex, fetuin and matrix Gla protein (MGP), have both been shown to function as potent inhibitors of calcification in vitro. Fetuin is a 59-kDa protein that consists of two N-terminal cystatin domains and a smaller C-terminal domain. Fetuin is synthesized by the liver and secreted into blood, where it is found at a concentration of ϳ1 mg/ml in the rat (2). Previous studies have demonstrated that fetuin is the major calcification inhibitor found in serum (3, 4). Fetuin is also one of the most abundant noncollagenous proteins found in mammalian bone (5-10), with a concentration of ϳ1 mg of fetuin/g of bone in rat (9). MGP is a small, 10-kDa vitamin K-dependent protein (11) that is secreted by a wide variety of cell types, including vascular smooth muscle cells, and is found in bone at a concentration of 0.2 mg/g in the rat (12). Genetic and biochemical studies ...