Characterization of SIBLING proteins in the mineralized tissues

• Author / Creator

  Dab, Sandeep

• Introduction: The SIBLING family of proteins is a group of non-collagenous proteins (NCPs) previously thought to be expressed only in dentin but have been demonstrated in other mineralized and non-mineralized tissues. They are believed to play vital roles in both osteogenesis and dentinogenesis. In the past, there have been random reports of their expression patterns in some mineralized tissues. Since, both osteogenesis and odontogenesis are tightly regulated lifelong processes and involve a peak of mineralization we used three different age groups. We systematically analyzed the mandibles, teeth and tibias of 1-, 3- and 6-month old WT (wild type) mice by determining immunohistochemical expression of SIBLING proteins in these mineralized tissues.
  Methods: 15 WT mice (C57BL/6J – The Jackson Laboratory; Bar Harbor, ME) were euthanized with CO2 - at ages 1, 3 and 6 months. Five hemimandibles and 5 tibias each for 1, 3 and 6-month old mice were randomly selected from dissected samples for use in the experiments. They were immediately fixed in PFA, decalcified and then embedded in paraffin. The samples were serially sectioned and mounted on slides for histological and immunohistochemical analysis. Hematoxylin and Eosin staining was done to localize various microscopic structures in the mandible and tibia of 1-, 3- and 6-month-old mice. Immunohistochemistry was done using antibodies for Dentin sialoprotein (DSP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) and osteopontin (OPN). The immunostaining pattern was observed and documented. Semi-quantitative analysis of the immunostaining intensity was done using ImageJ software [National Institutes of Health (NIH), Bethesda, MD, USA] and compared between the three age groups by 2 investigators independently.
  Results: Immunostaining of DSP in tibia showed its peak expression in the 3-month-age group and staining was especially concentrated along the calcification zone. In the molars, DSP was expressed in the dentin matrix surrounding the dentinal tubules, predentin, alveolar bone, cellular cementum, and PDL with highest expression at 1-month. A similar expression of DMP1 was seen in the tibia and dentin. BSP expression in the tibia not only increased from 1- to 3-months but also showed a broader distribution pattern in the growth plate with immunopositive staining in the resting, proliferating and hypertrophic zones. High positive staining for BSP was observed especially in the acellular cementum. BSP was also expressed in the predentin, cellular cementum, alveolar bone and periodontal ligament (PDL). OPN was mainly expressed in the bone although a lower level of OPN was observed at all age groups in the teeth. The immunostaining intensity was less for all proteins in the 6-month tibia samples.
  Conclusion: This study elaborated on the expression profile of the four SIBLING proteins at three different stages of development in the mice. Their expression showed variations in their staining intensity and temporospatial patterning concordant with skeletal and dental maturity. In the tibia, the proteins showed strong immunopositive staining in the destruction and calcification zones of growth plate. The maximum intensity of expression was found at 3-months of age corresponding to the timing of greatest conversion of osteoid into bone in the tibia. The expression in the teeth was high at 1 month corresponding to the relative time of greatest conversion of predentin into mineralized dentin. A rise in their expression at 6 months happened at the same time as increased rate of cuspal attrition and slow dentin remodeling. IHC findings for DMP1 reinforce previous studies showing increased expression with mechanical loading, and localization in regions where DSP was expressed. BSP’s broad range of expression profile in all zones of growth plate, predentin and acellular cementum which could possibly be explained by previous study showing its ability to bind with both the collagen and hydroxyapatite in an age and/or function dependent fashion. OPN’s overall expression showing a pattern consistent with the pace and extent of osteogenesis and dentinogenesis and may partake in controlled inhibition. Taken together, since the SIBLING proteins were expressed in all age groups albeit at varying levels in different tissues, these findings suggest some role in this tightly regulated mineralization process.

• Subjects / Keywords

  • DSPP
  • Osteopontin
  • DMP 1
  • Bone development
  • Bone Sialoprotein
  • SIBLING proteins
  • Dentin sialophosphoprotein
  • OPN
  • Biomineralization
  • BSP
  • DSP
  • Tooth development
  • Dentin Matrix protein
  • SIBLING

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-0m7b-f192

• License

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