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

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

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

Calcein labeling

Calcein (10 mg/kg) injected 6 hours before first injection of HEBP.

Control HEBP

Matrix formed during experimental period

Periosteal surfacePeriosteal surface

Enamel organ facing 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

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

Electron microscopic observation

Control

Control HEBP

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.

HistologyNo mention of stain used, tissue possible just counterstained with hematoxylin.

Control HEBPPeriosteal surfacePeriosteal 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.

Anti-SOST Immunohistochemistry

Control HEBP

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.

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

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?