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The role of bone sialoprotein in the tendon-bone insertion.

ABSTRACT: Tendons/ligaments insert into bone via a transitional structure, the enthesis, which is susceptible to injury and difficult to repair. Fibrocartilaginous entheses contain fibrocartilage in their transitional zone, part of which is mineralized. Mineral-associated proteins within this zone have not been adequately characterized. Members of the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family are acidic phosphoproteins expressed in mineralized tissues. Here we show that two SIBLING proteins, bone sialoprotein (BSP) and osteopontin (OPN), are present in the mouse enthesis. Histological analyses indicate that the calcified zone of the quadriceps tendon enthesis is longer in Bsp(-/-) mice, however no difference is apparent in the supraspinatus tendon enthesis. In an analysis of mineral content within the calcified zone, micro-CT and Raman spectroscopy reveal that the mineral content in the calcified fibrocartilage of the quadriceps tendon enthesis are similar between wild type and Bsp(-/-) mice. Mechanical testing of the patellar tendon shows that while the tendons fail under similar loads, the Bsp(-/-) patellar tendon is 7.5% larger in cross sectional area than wild type tendons, resulting in a 16.5% reduction in failure stress. However, Picrosirius Red staining shows no difference in collagen organization. Data collected here indicate that BSP is present in the calcified fibrocartilage of murine entheses and suggest that BSP plays a regulatory role in this structure, influencing the growth of the calcified fibrocartilage in addition to the weakening of the tendon mechanical properties. Based on the phenotype of the Bsp(-/-) mouse enthesis, and the known in vitro functional properties of the protein, BSP may be a useful therapeutic molecule in the reattachment of tendons and ligaments to bone.

SUBMITTER: Marinovich R

PROVIDER: S-EPMC4875796 | biostudies-literature | 2016 May-Jul

REPOSITORIES: biostudies-literature

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The role of bone sialoprotein in the tendon-bone insertion.

Marinovich Ryan R Soenjaya Yohannes Y Wallace Gregory Q GQ Zuskov Andre A Dunkman Andrew A Foster Brian L BL Ao Min M Bartman Kevin K Lam Vida V Rizkalla Amin A Beier Frank F Somerman Martha J MJ Holdsworth David W DW Soslowsky Louis J LJ Lagugné-Labarthet François F Goldberg Harvey A HA

Matrix biology : journal of the International Society for Matrix Biology 20160128

Tendons/ligaments insert into bone via a transitional structure, the enthesis, which is susceptible to injury and difficult to repair. Fibrocartilaginous entheses contain fibrocartilage in their transitional zone, part of which is mineralized. Mineral-associated proteins within this zone have not been adequately characterized. Members of the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family are acidic phosphoproteins expressed in mineralized tissues. Here we show that two SIBL ...[more]

PMID: 26826499

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Project description:Bone sialoprotein (BSP) is an extracellular matrix (ECM) protein associated with mineralized tissues, particularly bone and cementum. BSP includes functional domains implicated in collagen binding, hydroxyapatite nucleation, and cell signaling, although its function(s) in osteoblast and osteoclast differentiation and function remain incompletely understood. Genetic ablation of BSP in Ibsp knockout (Ibsp-/-) mice results in developmental bone mineralization and remodeling defects, with alveolar bone more severely affected than the femurs and tibias of the postcranial skeleton. The role of BSP in alveolar bone healing has not been studied. We hypothesized that BSP ablation would cause defective alveolar bone healing. We employed a maxillary first molar extraction socket healing model in 42-d postnatalIbsp-/- and wild-type (WT) control mice. Tissues were collected at 0, 7, 14, 21, and 56 d postprocedure (dpp) for analysis by micro-computed tomography (microCT), histology, in situ hybridization (ISH), immunohistochemistry (IHC), and quantitative polymerase chain reaction (qPCR) array. As expected, alveolar bone healing progressed in WT mice with increasing bone volume fraction (BV/TV), bone mineral density (BMD), and tissue mineral density (TMD), transitioning from woven to mature bone from 7 to 56 dpp. Ibsp messenger RNA (mRNA) and BSP protein were strongly expressed during alveolar bone healing in parallel with other osteogenic markers. Compared to WT, Ibsp-/- mice exhibited 50% to 70% reduced BV/TV and BMD at all time points, 7% reduced TMD at 21 dpp, abnormally increased Col1a1 and Alpl mRNA expression, and persistent presence of woven bone and increased bone marrow in healing sockets. qPCR revealed substantially dysregulated gene expression in alveolar bone of Ibsp-/- versus WT mice, with significantly disrupted expression of 45% of tested genes in functional groups, including markers for osteoblasts, osteoclasts, mineralization, ECM, cell signaling, and inflammation. We conclude that BSP is a critical and nonredundant factor for alveolar bone healing, and its absence disrupts multiple major pathways involved in appropriate healing.

Project description:Bone sialoprotein (BSP) is a multifunctional extracellular matrix protein found in mineralized tissues, including bone, cartilage, tooth root cementum (both acellular and cellular types), and dentin. In order to define the role BSP plays in the process of biomineralization of these tissues, we analyzed cementogenesis, dentinogenesis, and osteogenesis (intramembranous and endochondral) in craniofacial bone in Bsp null mice and wild-type (WT) controls over a developmental period (1-60 days post natal; dpn) by histology, immunohistochemistry, undecalcified histochemistry, microcomputed tomography (microCT), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and quantitative PCR (qPCR). Regions of intramembranous ossification in the alveolus, mandible, and calvaria presented delayed mineralization and osteoid accumulation, assessed by von Kossa and Goldner's trichrome stains at 1 and 14 dpn. Moreover, Bsp(-/-) mice featured increased cranial suture size at the early time point, 1 dpn. Immunostaining and PCR demonstrated that osteoblast markers, osterix, alkaline phosphatase, and osteopontin were unchanged in Bsp null mandibles compared to WT. Bsp(-/-) mouse molars featured a lack of functional acellular cementum formation by histology, SEM, and TEM, and subsequent loss of Sharpey's collagen fiber insertion into the tooth root structure. Bsp(-/-) mouse alveolar and mandibular bone featured equivalent or fewer osteoclasts at early ages (1 and 14 dpn), however, increased RANKL immunostaining and mRNA, and significantly increased number of osteoclast-like cells (2-5 fold) were found at later ages (26 and 60 dpn), corresponding to periodontal breakdown and severe alveolar bone resorption observed following molar teeth entering occlusion. Dentin formation was unperturbed in Bsp(-/-) mouse molars, with no delay in mineralization, no alteration in dentin dimensions, and no differences in odontoblast markers analyzed. No defects were identified in endochondral ossification in the cranial base, and craniofacial morphology was unaffected in Bsp(-/-) mice. These analyses confirm a critical role for BSP in processes of cementogenesis and intramembranous ossification of craniofacial bone, whereas endochondral ossification in the cranial base was minimally affected and dentinogenesis was normal in Bsp(-/-) molar teeth. Dissimilar effects of loss of BSP on mineralization of dental and craniofacial tissues suggest local differences in the role of BSP and/or yet to be defined interactions with site-specific factors.

Project description:Orthopedic implant failure due to aseptic loosening and mechanical instability remains a major problem in total joint replacement. Improving osseointegration at the bone-implant interface may reduce micromotion and loosening. Bone sialoprotein (BSP) has been shown to enhance bone formation when coated onto titanium femoral implants and in rat calvarial defect models. However, the most appropriate method of BSP coating, the necessary level of BSP coating, and the effect of BSP coating on cell behavior remain largely unknown. In this study, BSP was covalently coupled to titanium surfaces via an aminosilane linker (APTES), and its properties were compared to BSP applied to titanium via physisorption and untreated titanium. Cell functions were examined using primary human osteoblasts (hOBs) and L929 mouse fibroblasts. Gene expression of specific bone turnover markers at the RNA level was detected at different intervals. Cell adhesion to titanium surfaces treated with BSP via physisorption was not significantly different from that of untreated titanium at any time point, whereas BSP application via covalent coupling caused reduced cell adhesion during the first few hours in culture. Cell migration was increased on titanium disks that were treated with higher concentrations of BSP solution, independent of the coating method. During the early phases of hOB proliferation, a suppressive effect of BSP was observed independent of its concentration, particularly when BSP was applied to the titanium surface via physisorption. Although alkaline phosphatase activity was reduced in the BSP-coated titanium groups after 4 days in culture, increased calcium deposition was observed after 21 days. In particular, the gene expression level of RUNX2 was upregulated by BSP. The increase in calcium deposition and the stimulation of cell differentiation induced by BSP highlight its potential as a surface modifier that could enhance the osseointegration of orthopedic implants. Both physisorption and covalent coupling of BSP are similarly effective, feasible methods, although a higher BSP concentration is recommended.

Project description:Although acellular cementum is essential for tooth attachment, factors directing its development and regeneration remain poorly understood. Inorganic pyrophosphate (PPi), a mineralization inhibitor, is a key regulator of cementum formation: tissue-nonspecific alkaline phosphatase (Alpl/TNAP) null mice (increased PPi) feature deficient cementum, while progressive ankylosis protein (Ank/ANK) null mice (decreased PPi) feature increased cementum. Bone sialoprotein (Bsp/BSP) and osteopontin (Spp1/OPN) are multifunctional extracellular matrix components of cementum proposed to have direct and indirect effects on cell activities and mineralization. Studies on dentoalveolar development of Bsp knockout (Bsp-/-) mice revealed severely reduced acellular cementum, however underlying mechanisms remain unclear. The similarity in defective cementum phenotypes between Bsp-/- mice and Alpl-/- mice (the latter featuring elevated PPi and OPN), prompted us to examine whether BSP is operating by modulating PPi-associated genes. Genetic ablation of Bsp caused a 2-fold increase in circulating PPi, altered mRNA expression of Alpl, Spp1, and Ank, and increased OPN protein in the periodontia. Generation of a Bsp knock-out (KO) cementoblast cell line revealed significantly decreased mineralization capacity, 50% increased PPi in culture media, and increased Spp1 and Ank mRNA expression. While addition of 2μg/ml recombinant BSP altered Spp1, Ank, and Enpp1 expression in cementoblasts, changes resulting from this dose were not dependent on the integrin-binding RGD motif or MAPK/ERK signaling pathway. Decreasing PPi by genetic ablation of Ank on the Bsp-/- mouse background reestablished cementum formation, allowing >3-fold increased acellular cementum volume compared to wild-type (WT). However, deleting Ank did not fully compensate for the absence of BSP. Bsp-/-; Ank-/- double-deficient mice exhibited mean 20-27% reduced cementum thickness and volume compared to Ank-/- mice. From these data, we conclude that the perturbations in PPi metabolism are not solely driving the cementum pathology in Bsp-/- mice, and that PPi is more potent than BSP as a cementum regulator, as shown by the ability to override loss of BSP by lowering PPi. We propose that BSP and PPi work in concert to direct mineralization in cementum and likely other mineralized tissues.

Dataset Information

The role of bone sialoprotein in the tendon-bone insertion.

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The role of bone sialoprotein in the tendon-bone insertion.

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