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(99m)TC-Methylene diphosphonate uptake at injury site correlates with osteoblast differentiation and mineralization during bone healing in mice.

Zhong ZA, Peck A, Li S, VanOss J, Snider J, Droscha CJ, Chang TA, Williams BO - Bone Res (2015)

Bottom Line: In an ex vivo culture system, calvarial cells were differentiated into osteoblasts with osteogenic medium, pulsed with (99m)Tc-MDP at different time points, and quantitated for (99m)Tc-MDP uptake with a gamma counter.We also found that (99m)Tc-MDP uptake was associated with osteoblast maturation and mineralization in vitro.This study provides direct and biological evidence for (99m)Tc-MDP uptake in osteoblasts during bone healing in vivo and in vitro.

View Article: PubMed Central - PubMed

Affiliation: Center for Skeletal Disease Research, Van Andel Research Institute , Grand Rapids, MI, USA ; Department of Internal Medicine, Center for Musculoskeletal Health, UC Davis Medical Center , Davis, CA, USA.

ABSTRACT
(99m)Tc-Methylene diphosphonate ((99m)Tc-MDP) is widely used in clinical settings to detect bone abnormalities. However, the mechanism of (99m)Tc-MDP uptake in bone is not well elucidated. In this study, we utilized a mouse tibia injury model, single-photon emission computed tomography (gamma scintigraphy or SPECT), ex vivo micro-computed tomography, and histology to monitor (99m)Tc-MDP uptake in injury sites during skeletal healing. In an ex vivo culture system, calvarial cells were differentiated into osteoblasts with osteogenic medium, pulsed with (99m)Tc-MDP at different time points, and quantitated for (99m)Tc-MDP uptake with a gamma counter. We demonstrated that (99m)Tc-MDP uptake in the injury sites corresponded to osteoblast generation in those sites throughout the healing process. The (99m)Tc-MDP uptake within the injury sites peaked on day 7 post-injury, while the injury sites were occupied by mature osteoblasts also starting from day 7. (99m)Tc-MDP uptake started to decrease 14 days post-surgery, when we observed the highest level of bony tissue in the injury sites. We also found that (99m)Tc-MDP uptake was associated with osteoblast maturation and mineralization in vitro. This study provides direct and biological evidence for (99m)Tc-MDP uptake in osteoblasts during bone healing in vivo and in vitro.

No MeSH data available.


Related in: MedlinePlus

99mTc-MDP uptake correlated with osteoblast differentiation in vitro. (a) Calvarial cells were isolated from Ocn-Cre;mT/mG neonates, differentiated into osteoblasts with osteogenic medium, and observed under a fluorescent microscopy. Green fluorescence indicates differentiated mature osteoblasts, and red fluorescence indicates all other non-osteoblast cells; (b) RNA was collected from differentiating osteoblasts at the indicated time points. Real-time PCR was performed to detect osteocalcin mRNA level normalized to the endogenous β-actin level; (c and d) Calvarial cells were plated on 30-cm plates and differentiated with osteogenic media for the indicated time. Then cells were pulsed with 99mTc-MDP solution and visualized with a SPECT scanner. The actual images were taken with all the plates placed at the same layer and scanned at the same time (c). The quantitated 99mTc-MDP signal from each individual plate was normalized to total 99mTc-MDP signal from all plates (d).
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fig5: 99mTc-MDP uptake correlated with osteoblast differentiation in vitro. (a) Calvarial cells were isolated from Ocn-Cre;mT/mG neonates, differentiated into osteoblasts with osteogenic medium, and observed under a fluorescent microscopy. Green fluorescence indicates differentiated mature osteoblasts, and red fluorescence indicates all other non-osteoblast cells; (b) RNA was collected from differentiating osteoblasts at the indicated time points. Real-time PCR was performed to detect osteocalcin mRNA level normalized to the endogenous β-actin level; (c and d) Calvarial cells were plated on 30-cm plates and differentiated with osteogenic media for the indicated time. Then cells were pulsed with 99mTc-MDP solution and visualized with a SPECT scanner. The actual images were taken with all the plates placed at the same layer and scanned at the same time (c). The quantitated 99mTc-MDP signal from each individual plate was normalized to total 99mTc-MDP signal from all plates (d).

Mentions: We next isolated primary calvarial cells from 3-day-old Ocn-Cre;mT/mG neonates and differentiated the cells into osteoblasts with osteogenic medium. Using a fluorescent microscope, we observed that calvarial cells were differentiating into GFP+ mature osteoblasts (Figure 5a), which was confirmed by real-time PCR of Ocn expression (Figure 5b). The calvarial cells were pulsed with 99mTc-MDP at different time points (day 0, 4, 7, 14, or 21 post-differentiation), and scanned by a planar gamma imager together with 99mTc standards. We found that the 99mTc-MDP uptake increased with osteoblast marker Ocn expression (Figure 5c and d). We further measured the 99mTc-MDP from the cell fraction collected by trypsinization and the residual mineral fraction dissolved in diluted hydrochloride. The 99mTc-MDP correlated with osteoblast maturation both in the cell fraction and in residual mineral fraction (Figure S2). We detected a small amount of calcium (by Alizarin red staining) and phosphate (by Von Kossa staining) in calvarial cells after 14 days of differentiation, and much higher levels of calcium and phosphate after 21 days of differentiation (Figure S3).


(99m)TC-Methylene diphosphonate uptake at injury site correlates with osteoblast differentiation and mineralization during bone healing in mice.

Zhong ZA, Peck A, Li S, VanOss J, Snider J, Droscha CJ, Chang TA, Williams BO - Bone Res (2015)

99mTc-MDP uptake correlated with osteoblast differentiation in vitro. (a) Calvarial cells were isolated from Ocn-Cre;mT/mG neonates, differentiated into osteoblasts with osteogenic medium, and observed under a fluorescent microscopy. Green fluorescence indicates differentiated mature osteoblasts, and red fluorescence indicates all other non-osteoblast cells; (b) RNA was collected from differentiating osteoblasts at the indicated time points. Real-time PCR was performed to detect osteocalcin mRNA level normalized to the endogenous β-actin level; (c and d) Calvarial cells were plated on 30-cm plates and differentiated with osteogenic media for the indicated time. Then cells were pulsed with 99mTc-MDP solution and visualized with a SPECT scanner. The actual images were taken with all the plates placed at the same layer and scanned at the same time (c). The quantitated 99mTc-MDP signal from each individual plate was normalized to total 99mTc-MDP signal from all plates (d).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472149&req=5

fig5: 99mTc-MDP uptake correlated with osteoblast differentiation in vitro. (a) Calvarial cells were isolated from Ocn-Cre;mT/mG neonates, differentiated into osteoblasts with osteogenic medium, and observed under a fluorescent microscopy. Green fluorescence indicates differentiated mature osteoblasts, and red fluorescence indicates all other non-osteoblast cells; (b) RNA was collected from differentiating osteoblasts at the indicated time points. Real-time PCR was performed to detect osteocalcin mRNA level normalized to the endogenous β-actin level; (c and d) Calvarial cells were plated on 30-cm plates and differentiated with osteogenic media for the indicated time. Then cells were pulsed with 99mTc-MDP solution and visualized with a SPECT scanner. The actual images were taken with all the plates placed at the same layer and scanned at the same time (c). The quantitated 99mTc-MDP signal from each individual plate was normalized to total 99mTc-MDP signal from all plates (d).
Mentions: We next isolated primary calvarial cells from 3-day-old Ocn-Cre;mT/mG neonates and differentiated the cells into osteoblasts with osteogenic medium. Using a fluorescent microscope, we observed that calvarial cells were differentiating into GFP+ mature osteoblasts (Figure 5a), which was confirmed by real-time PCR of Ocn expression (Figure 5b). The calvarial cells were pulsed with 99mTc-MDP at different time points (day 0, 4, 7, 14, or 21 post-differentiation), and scanned by a planar gamma imager together with 99mTc standards. We found that the 99mTc-MDP uptake increased with osteoblast marker Ocn expression (Figure 5c and d). We further measured the 99mTc-MDP from the cell fraction collected by trypsinization and the residual mineral fraction dissolved in diluted hydrochloride. The 99mTc-MDP correlated with osteoblast maturation both in the cell fraction and in residual mineral fraction (Figure S2). We detected a small amount of calcium (by Alizarin red staining) and phosphate (by Von Kossa staining) in calvarial cells after 14 days of differentiation, and much higher levels of calcium and phosphate after 21 days of differentiation (Figure S3).

Bottom Line: In an ex vivo culture system, calvarial cells were differentiated into osteoblasts with osteogenic medium, pulsed with (99m)Tc-MDP at different time points, and quantitated for (99m)Tc-MDP uptake with a gamma counter.We also found that (99m)Tc-MDP uptake was associated with osteoblast maturation and mineralization in vitro.This study provides direct and biological evidence for (99m)Tc-MDP uptake in osteoblasts during bone healing in vivo and in vitro.

View Article: PubMed Central - PubMed

Affiliation: Center for Skeletal Disease Research, Van Andel Research Institute , Grand Rapids, MI, USA ; Department of Internal Medicine, Center for Musculoskeletal Health, UC Davis Medical Center , Davis, CA, USA.

ABSTRACT
(99m)Tc-Methylene diphosphonate ((99m)Tc-MDP) is widely used in clinical settings to detect bone abnormalities. However, the mechanism of (99m)Tc-MDP uptake in bone is not well elucidated. In this study, we utilized a mouse tibia injury model, single-photon emission computed tomography (gamma scintigraphy or SPECT), ex vivo micro-computed tomography, and histology to monitor (99m)Tc-MDP uptake in injury sites during skeletal healing. In an ex vivo culture system, calvarial cells were differentiated into osteoblasts with osteogenic medium, pulsed with (99m)Tc-MDP at different time points, and quantitated for (99m)Tc-MDP uptake with a gamma counter. We demonstrated that (99m)Tc-MDP uptake in the injury sites corresponded to osteoblast generation in those sites throughout the healing process. The (99m)Tc-MDP uptake within the injury sites peaked on day 7 post-injury, while the injury sites were occupied by mature osteoblasts also starting from day 7. (99m)Tc-MDP uptake started to decrease 14 days post-surgery, when we observed the highest level of bony tissue in the injury sites. We also found that (99m)Tc-MDP uptake was associated with osteoblast maturation and mineralization in vitro. This study provides direct and biological evidence for (99m)Tc-MDP uptake in osteoblasts during bone healing in vivo and in vitro.

No MeSH data available.


Related in: MedlinePlus