The manuscript tests the hypothesis that posttranslational modification of the SIBLING family of proteins in general and osteopontin in particular modify the abilities of these proteins to regulate in vitro hydroxyapatite (HA) formation. Osteopontin has diverse effects on hydroxyapatite (HA) mineral crystallite formation and growth depending on the extent of phosphorylation. We hypothesized that different regions of full-length OPN would also have distinct effects on the mineralization process. Thrombin fragmentation of milk OPN (mOPN) was used to test this hypothesis. Three fragments were tested in a de novo HA formation assay; an N-terminal fragment (aa 1–147), a central fragment (aa 148–204) denoted SKK-fragment and a C-terminal fragment (aa 205–262). Compared to intact mOPN the C- and N-terminal fragments behaved comparably, promoting HA formation and growth, but the central SKK-fragment acted as a mineralization inhibitor. In a seeded growth experiment all fragments inhibited mineral proliferation, but the SKK-fragment was the most effective inhibitor. These effects, seen in HA-formation and seeded growth assays in a gelatin gel system and in a pH-stat experiment were lost when the protein or fragments were dephosphorylated. Effects of the fully phosphorylated protein and fragments were also altered in the presence of fibrillar collagen. The diverse effects can be explained in terms of the intrinsically disordered nature of OPN and its fragments which enable them to interact with their multiple partners.

Dentin matrix protein-1 (DMP1) is a major synthetic product of hypertrophic chondrocytes and osteocytes. Previous in vitro studies showed full-length DMP1 inhibits hydroxyapatite (HA) formation and growth, while its N-terminal fragment (37K) promotes HA formation. Since there are 3 fragments within the mineralized tissues [N-terminal, C-terminal (57K), and a chondroitinsulfate-linked N-terminal fragment (DMP1-PG)], we predicted that each would have a distinct effect on mineralization related to its interaction with HA. In a gelatin-gel system, 37K and 57K fragments were both promoters of HA formation and growth; DMP1-PG was an inhibitor. The secondary structures of the 3 fragments and the full-length protein in the presence and absence of Ca2+ and HA determined by FTIR showed that the full-length protein undergoes slight conformational changes on binding to HA, while 37K, 57K, and DMP1-PG do not change conformation. These findings indicate that distinct forms of DMP1 may work collectively in controlling the mineralization process.

Matrix extracellular phosphoglycoprotein (MEPE) is an inhibitor of mineralization in situ and in cell cultures where altered expression is associated with oncogenic osteomalacia and hypophosphatemic rickets. The purpose of this study was to determine whether the intact protein or the peptide(s) originating from this protein was responsible for the inhibition. The ability of the intact protein and the acidic, serine-and aspartate-rich MEPE-associated motif (ASARM) peptide to promote or inhibit de novo hydroxyapatite formation and growth of hydroxyapatite seed crystals, in both phosphorylated and dephosphorylated forms, was assessed at room temperature in a dynamic gel diffusion system at 3.5 and 5 days. The most effective nucleator concentration was also examined when associated with fibrillar type I collagen. The phosphorylated intact protein was an effective promoter of mineralization in the gelatin gel diffusion system, while the ASARM peptide was an effective inhibitor. When dephosphorylated both the intact protein and the ASARM peptide had no effect on mineralization. Associated with collagen fibrils, some of the effect of the intact protein was lost. This study demonstrates the importance of posttranslational modification for the site-specific activity of MEPE and its ASARM peptide. Based on studies in mice in which MEPE was ablated [5] leading to enhancement of bone formation, MEPE is believed to inhibit mineralization. This was confirmed in cell culture experiments [6,7] as well as in cell-free solution studies [8]. In the cell-free studies, it was the ASARM peptide, a substrate and ligand for PHEX, that provided the inhibition [6,8,9,10]. Similar to the other SIBLING proteins, we hypothesized that posttranslational modification of MEPE would alter its effects on mineralization and that the intact protein would have a distinct effect compared to the peptides. Thus, we tested the intact protein and the ASARM peptide in the dynamic gelatin gel system [11] to validate this hypothesis. We also investigated whether phosphorylation and dephosphorylation would alter the effects of these proteins and peptides on the mineralization process. Materials and Methods Preparation of MEPE and MEPE FragmentsMEPE protein was generated by expression in insect cells using the sf9 system, as previously described [6]. ASARM peptides (phosphorylated and without phosphate) were synthesized and purchased from Neo-MPS (now called PolyPeptide Laboratories, San Diego, CA). Dephosphorylation of the intact protein in Tris buffer was performed with alkaline phosphatase-agarose beads (Sigma, St. Louis, MO) by incubating overnight at room temperature. The Gel SystemHydroxyapatite formation and growth were monitored in the dynamic collagen gel hydroxyapatite growth system [10]. In this double diffusion system, Ca 2+ and HPO 4 2− (Pi) ions circulate at room temperature and diffuse into opposite ends of a 6-cm-long 10% gelatin gel (Bloom 275; Fisher Chemicals, Fair Lawn, NJ). The pH of the 10% gelatin solution, prepared in tris-(hyd...

Bone is a highly organized tissue in which each structural level influences the macroscopic and microscopic mechanical behavior. In particular, the quantity, quality, and distribution of the different bone components, i.e. collagen matrix and hydroxyapatite crystals, are associated with bone strength or fragility. Common spectroscopic techniques used to assess bone composition have resolutions limited to the micrometer range. In this study, our aims were two-fold: i) to develop and validate the AFM-IR methodology for skeletal tissues and ii) to apply the methodology to sheep cancellous bone with the objective to obtain novel findings on the composition and structure of trabecular packets.To develop the methodology, we assessed spatial and temporal reproducibility using a known homogeneous material (polymethylmethacrylate, PMMA). We verified that the major peak positions were similar and not shifted when compared to traditional Fourier Transform Infrared imaging (FTIRI). When AFM-IR was applied to sheep cancellous bone, the mineral-to-matrix ratio increased and the acid phosphate substitution ratio decreased as a function of tissue maturity. The resolution of the technique enabled visualization of different stages of the bone maturation process, particularly newly-formed osteoid prior to mineralization. We also observed alternating patterns of IR parameters in line and imaging measurements, suggesting the apposition of layers of alternating structure and / or composition that were not visible with traditional spectroscopic methods. In conclusion, nanoscale IR spectroscopy demonstrates novel compositional and structural changes within trabecular packets in cancellous bone. Based on these results, AFM-IR is a valuable tool to investigate cancellous bone at the nanoscale and, more generally, to analyze small dynamic areas that are invisible to traditional spectroscopic methods.