1. Matrix mineralization as a trigger for osteocyte maturation Kazuharu Irie, Sadakazu Ejiri, Yasunori Sakakura, Toru Shibui, and Toshihiko Yajima Journal of Histochemistry and Cytochemistry 2008 (56):561-567

2.  Hypothesis – Mineralization of surrounding bone matrix triggers osteocytes to mature  Experiment – use bisphosphonates to prevent mineralization of deposited matrix 1-hydroxyethylidene 1,1-bisphosphonate (HEBP) Age matched rats were injected with either HEBP (40 mg/kg) or normal saline solution for 5 days. Following sacrifice, rats were fixed and mandibles dissected

3. Rat ECB Bone growth at periosteal end is tightly coupled with resorption at the EO facing surface. Periosteal surface. New bone growth. Mature bone formed during experimental period EO facing surface, mature bone being resorbed Direction of bone growth

4. Calcein labeling Calcein (10 mg/kg) injected 6 hours before first injection of HEBP. ControlHEBP Matrix formed during experimental period Periosteal surface Enamel organ facing surface Experiment shows that similar amount of bone matrix was formed during experimental period. Thicker calcein labeling in HEBP group is due to reduced osteoclastic resorption Calcein still present that in bone that would have been resorbed

5. Villaneuva and radiographic analysis Control HEBP Fixed sections were ground to thickness of 2 mm. Stained in Villaneuva solution for 10 days. Sections Were then ground to 100 µm thickness for radiography. Matrix formed during experimental period. Staining reveals it to be unmineralized in HEBP group compared to control Matrix formed during experimental period. Radiography confirms it to unmineralized in HEBP group. Experiment shows that HEBP was effective in preventing Matrix mineralization Mineralized matrix not resorbed by osteoclasts

6. Electron microscopic observation Control HEBP A – Immature osteocyte seen near periosteal surface in control group. B – Immature osteocyte in HEBP group found in unmineralized matrix C – Mature osteocyte in control group located in mineralized matrix D – Mature osteocyte within mineralized matrix from HEBP group Morphology of osteocytes. Immature osteocytes show large golgi bodies and rEr. Mature osteocytes are thinner with fewer organelles. Go= golgi body. Experiment shows morphology of immature/mature osteocytes remain consistent depending on mineralization status across control and experimental groups.

7. Histology No mention of stain used, tissue possible just counterstained with hematoxylin. Control HEBP Periosteal surface Experiment reveals that plump (immature) osteocytes present only in unmineralized areas. Mature osteocytes seen in mineralized matrix Plump osteocytes present only near the periosteal surface (unmineralized) Plump osteocytes present throughout matrix formed during experimental period.

8. Anti-SOST Immunohistochemistry ControlHEBP 5 mm sections were sliced to 80um and stained with goat anti-mouse SOST antibody at 1:100. Immunostaining shows positive signal from osteocytes throughout the mineralized area in control group. No staining present in unmineralized area of HEBP group. Electron micrograph of SOST staining in mature osteocyte Experiment shows that a marker for mature osteocytes is present only in cells within mineralized matrix.

9. Experiments  Calcein labeling shows equal amounts of bone formation between samples  Villaneuva and radiography supports the bisphosphonate preventing mineralization  Morphological analysis show that mature osteocytes appear to only be in mineralized matrix  Immunohistochemistry shows that marker for mature osteocytes only present in cells within mineralized matrix

10.  Are there any other markers for osteocyte maturation that could have been looked at?  Do we know which genes control maturation? Could this have been found out with in vitro experiments?  How many rat samples did they obtain?