Increase, Cell death leads to Altered Bone Cell Homeostasis

Reference 

Experiment Description 

Result 

Aguirre et al., 2006 

In vivo. Female swiss Webster mice (C57BL/6 genetic background) were suspended via their tail to stimulate microgravity conditions. Bone resorption was determined by evaluating osteoclast number. Osteocyte and osteoblast apoptosis were detected. 

Following tail suspension of mice, significant increases in osteocyte and osteoblast apoptosis were observed by day 3. There was a maximum increase of ~2.3-fold and ~1.8-fold in cortical and cancellous osteocyte apoptosis, respectively, on day 7. A ~2.6-fold increase in osteoblast apoptosis was measured at day 3 and sustained until day 7. This was associated with a significant 0.53-fold decrease in osteoblast number on day 3, which was restored to above controls on day 18 as it increased by 1.9-fold compared to the group without tail suspension. A 4.6-fold increase was observed in osteoclast number on day 18 relative to controls. 

Yang et al., 2020 

In vitro. Male 14-week-old wildtype and transgenic mice (CD1 background) were unloaded using tail suspension. Apoptosis was measured by TUNEL staining. Bone blood serum markers were measured via enzyme-linked immunosorbent assay (ELISA) for osteocalcin (OCN) as an indicator for bone formation, and TRAP-5b as an indicator for bone resorption. In bone sections, osteoclasts and osteoblasts were identified by hematoxylin, eosin and TRAP staining. 

Hindlimb unloaded wildtype mice had an overall ~2.7-fold increase in osteocyte apoptosis, as well as a 3-fold increase in osteoblast apoptosis after 7 days of unloading. 

At day 7 and 28, significantly reduced number of osteoblasts (~0.3-fold and ~0.7-fold) was found in conjunction with reduced ALP (~0.4-fold and ~0.6-fold) gene expression. Further, serum marker OCN was significantly reduced (~0.5-fold and ~0.6-fold) at both time points indicating impaired bone formation. In contrast, at day 7 and 28, significantly increased number of osteoclasts (~13-fold and ~2.1-fold) was found in conjunction with increased cathepsin K (~8-fold and ~4.3-fold) gene expression. Further, serum marker TRAP5b was significantly increased (~3.5-fold and ~2-fold, respectively) at day 7 and 28 indicating increased bone resorption. 

Wright et al., 2015 

In vivo. The right hindlimbs of 20-week-old male C57BI/6 mice were irradiated with 2 Gy of X-rays at a rate of 1.6 Gy/min. Apoptotic osteocytes were measured by TUNEL. Osteoclast number was determined by TRAP stain. 

In vitro. Osteocyte-like cells (MLO-Y4) and osteoblast cells (MC3T3) were irradiated with 0-20 Gy X-rays. Annexin V was used as a marker of cellular apoptosis. 

In vivo. 2 Gy X-ray exposure resulted in a 2.5-fold increase in percentage of apoptotic osteocytes in trabecular bone. Osteoclast number increased significantly by ~1.8-fold after 2 Gy irradiation in the right hindlimb. 

In vitro, exposure to increasing doses of radiation from 0-20 Gy led to a linear dose-dependent increase in osteocyte apoptosis (MLO-Y4 cell culture) up to ~13.7-fold above controls at 20 Gy. Osteoblast apoptosis (MC3T3 cell culture) similarly increased in a dose-dependent fashion from 4-20 Gy, with a maximum increase of ~2.5-fold at 20 Gy (only significant increase). Osteoclasts increased significantly in MLO-Y4 coculture at 8 Gy, and calvarial osteoblasts decreased by ~0.5-fold at 10 Gy. 

Chandra et al., 2014 

In vivo. 4-month-old female rats were irradiated with 16 Gy of small animal radiation research platform (SARRP) X-rays, fractionated into two 8 Gy doses at a rate of 1.65 Gy/min. TUNEL staining in tibial trabecular bone was performed to determine osteoblast apoptosis. Osteoblast number was determined using static histomorphometry. 

Exposure to 16 Gy X-rays increases osteoblast apoptosis by ~7-fold and resulted in a ~0.25-fold decrease in osteoblast number. A significant decrease in osteoclast surface was also observed and is inconsistent with other radiation studies. The authors suggest the imbalance of radiation effects may lead to relatively higher osteoclast activity compared to osteoblast activity, leading to overall bone resorption. 

Chandra et al., 2017 

In vivo. Male C57BL/6 mice (8–10 weeks) were exposed to 8 Gy X-ray radiation at a rate of 1.65 Gy/min. Apoptosis was determined with a TUNEL assay. Osteoblast number was determined by static histomorphometry. 

8 Gy radiation exposure led to a ~3.9-fold increase in the number of TUNEL-positive osteoblasts and a ~0.5-fold decrease in osteoblast number.   

Liu et al., 2018 

In vitro. hBMMSCs were irradiated with 8 Gy of X-rays at a rate of 1.24 Gy/min. Apoptosis was measured with using an Annexin V-FITC staining kit. ALP activity was determined with a kit, and bone deposition was determined by Alizarin red staining. 

Apoptosis rate of osteoblast precursor cells (hBMMSCs) exposed to 8 Gy X-ray radiation increased ~3-fold, resulting in a ~0.5-fold decrease in ALP activity and bone deposition, as measured by optical density of calcium nodules. 

Huang et al., 2019 

Ex vivo. bmMSCs from the tibiae and femur of rats were irradiated with 2 Gy of ⁶⁰Co gamma rays at a rate of 0.83 Gy/min. Apoptosis was determined with Annexin V staining. bmMSCs were analyzed for changes in bone cell function following irradiation through measuring levels of ALP. 

Exposure to 2 Gy gamma radiation resulted in a ~4-fold increase in osteoblast apoptosis and led to a significant ~0.3-fold decrease in ALP activity. 

Li et al., 2020 

In vitro. Osteoblastic MC3T3-E1 cells of mice were irradiated with 0.25, 0.5, 1, 2, or 4 Gy of X-ray radiation. Apoptosis was determined by the Bcl-2/Bax ratio through western blot as well as caspase-3 activity with an assay kit. ALP activity was determined with an assay kit. 

X-ray radiation exposure resulted in a significant, dose-dependent decrease in the Bcl-2/Bax ratio at 0-4 Gy with a maximum decrease of ~0.6-fold below controls at 4 Gy, indicating a significant shift of osteoblasts towards apoptosis. There was also a dose-dependent increase in caspase-3 activity at 0.5-4 Gy with significant increases at 0.5 Gy and greater and a maximum increase of 1.6-fold above controls at 4 Gy. This was accompanied by a dose dependent linear decrease in ALP activity with significant decreases at 0.5 Gy and greater, and a maximum decrease of ~0.3-fold below controls at 4 Gy. 

Li et al., 2015 

In vitro. Calvarial osteoblasts of Male rats were irradiated using 0, 1, 2, 5, 10 Gy of ¹³⁷Cs gamma rays at a rate of 0.76 Gy/min. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to determine caspase-3 levels and apoptosis was measured by Annexin v fluorescence. ALP activity was determined to measure osteoblastogenesis. 

Osteoblasts exposed to 1-10 Gy radiation observed an exponential dose-dependent increase in caspase-3 with significant increases at 5 and 10 Gy and a maximum increase of 3-fold above controls at 10 Gy. A maximum increase in osteoblast apoptosis was observed under 2 Gy at ~1.6-fold above control, with the first significant increase at 1 Gy. This resulted in a roughly inverse-exponential dose-dependent decrease in ALP activity down to ~0.7-fold below controls at 10 Gy, with the first significant increase at 5 Gy. 

Huang et al., 2018 

In vitro. Murine RAW264.7 macrophage cells were irradiated with 2 Gy of gamma rays at a rate of 0.83 Gy/min. Annexin V-FITC/PI was used as a measure for apoptosis. TRAP staining was used to determine osteoclast differentiation. 

Exposure of RAW264.7 osteoclast cells to 2 Gy gamma radiation had a 5.26-fold increase in apoptosis percentage, from 1.86% to 9.78%. This resulted in a 2-fold increase in TRAP-stained cell number and 2.4-fold increase in total resorption area. 

Reference 

Experiment Description 

Result 

Li et al., 2020 

In vitro. Osteoblastic MC3T3-E1 cells of mice were irradiated with 0.25, 0.5,1,2, and 4 Gy of X-ray radiation. Autophagy markers were determined by western blot. ALP activity was determined by an assay kit. 

X-ray irradiation of osteoblasts linearly and dose-dependently increased LC3II/LC3I protein expression up to ~2.5-fold above controls under 1 Gy, after which it remained consistently elevated under 2 and 4 Gy. There were also dose-dependent increases in ATG5 and Beclin-1 up to ~1.75- and 3-fold above controls under 4 Gy, respectively. These increases in markers of autophagy induction were accompanied by substantial, dose-dependent inverse-exponential decrease in ALP activity down to 0.3-fold below control levels under 2 Gy 

Reference 

Experiment Description 

Result 

Aguirre et al., 2009 

In vivo. Female swiss Webster mice (C57BL/6 genetic background) were suspended via their tail to stimulate microgravity conditions. Bone homeostasis (biomechanical testing, bone histomorphometry) was assessed in lumbar vertebra (L1-L5). Bone resorption was determined by evaluating osteoclast number. Osteocyte and osteoblast apoptosis were detected by ISEL.

Hindlimb unloading of mice led to significant increase in cortical and trabecular osteocyte apoptosis and osteoblast apoptosis on day 3 of unloading, which remained increased up to day 18. Control mice had an increase in osteoblast apoptosis on day 18 such that the increased apoptosis under unloading conditions was non-significant on that day. Osteoblast number was significantly decreased by day 3 of unloading, returned to control levels by day 7, and surpassed controls by 2-fold on day 18. Significantly increased osteoclast number was not observed until day 18 of unloading. 

Yang et al., 2020 

In vitro. Male 14-week-old wildtype and transgenic mice (CD1 background) were unloaded using tail suspension. The tibia were scanned via micro-CT at 28 days after un-loading. Apoptosis was measured by TUNEL staining. Bone blood serum markers were measured via ELISA for OCN as indicator for bone formation, and TRAP-5b as indicator for bone resorption. In bone sections osteoclasts and osteoblasts were identified by hematoxylin, eosin and TRAP staining. 

On day 7 of unloading, ALP decreased ~0.4-fold and OCN decreased ~0.5-fold, while TRAP-5b increased ~3.5-fold, indicating enhanced osteoclast activity and decreased osteoblast activity. This was further shown by a ~13-fold increase in osteoclast number and 3.7-fold decrease in osteoblast number on day 7 of unloading. 

On day 28 of unloading, there were further decreases in osteoblastogenesis markers (~0.6-fold decrease in ALP activity and 0.6-fold decrease in OCN expression), and an overall 3-fold decrease in osteoblast number. 

Osteocyte and osteoblast apoptosis under in vitro simulated microgravity was increased by ~2-3-fold by day 7 of unloading. Significant decreases in several markers of osteoblastogenesis were observed on day 7, which were attenuated relative to contemporaneous controls on day 28. A similar trend was observed for osteoclastogenesis. 

Wright et al., 2015 

In vivo. The right hindlimbs of 20-week-old male C57BI/6 mice were irradiated with 2 Gy of X-rays at a rate of 1.6 Gy/min. Apoptotic osteocytes were measured by TUNEL. Osteoclasts and osteoblasts as measures of altered bone cell homeostasis were determined by TRAP. 

In vitro. Osteocyte-like cells (MLO-Y4) and osteoblast cells (MC3T3) were irradiated with 2-20 Gy X-rays. Annexin V was used as a marker of cellular apoptosis. 

In vitro radiation exposure of osteocytes (MLO-Y4) resulted in significant increases in apoptosis by 24 hours post-exposure, which increased several-fold by 48 hours. Osteoblast (MC3T3) apoptosis was also increased by 24 hours post-irradiation and remained increased up to 48 hours. Calvarial osteocyte apoptosis was not increased until 10 days post-irradiation. In vivo radiation exposure resulted in significant increase in hindlimb trabecular osteocyte apoptosis at 7 days post-irradiation. Significantly increased osteoclast number was observed at around the same time at 1 week post-irradiation, however, no significant changes in osteoblast number were observed. 

Li et al., 2020 

In vitro. Osteoblastic MC3T3-E1 cells of mice were irradiated with 0.25, 0.5, 1, 2, or 4 Gy of X-ray radiation. Apoptosis was determined by the Bcl-2/Bax ratio through western blot as well as caspase-3 activity with an assay kit. ALP activity was determined with an assay kit. All endpoints were measured 72h post-irradiation. 

X-ray radiation exposure from 0.25-4 Gy led to a dose-dependent decrease in the Bcl-2/Bax ratio down to 40% below controls, indicating a significant shift of osteoblasts towards apoptosis. There was also a dose-dependent increase in caspase-3 activity from 0.5-4 Gy up to 1.6-fold above controls. This was accompanied by a dose dependent linear decrease in ALP activity down to 0.3-fold below controls under 4 Gy. 

Chandra et al., 2014 

In vivo. 3-month-old female rats were irradiated with 16 Gy of SARRP X-rays, fractionated into two 8 Gy doses at a rate of 1.65 Gy/min. TUNEL staining in tibial trabecular bone was performed to determine osteoblast apoptosis. Osteoblast number was determined using static histomorphometry. 

Exposure to 16 Gy X-rays increases osteoblast apoptosis by ~7-fold at 2 weeks post-irradiation and resulted in a ~0.25-fold decrease in osteoblast number by day 28 post-irradiation. A significant decrease in osteoclast surface was also observed on day 28 post-irradiation and is inconsistent with other radiation studies. The authors suggest the imbalance of radiation effects may lead to relatively higher osteoclast activity compared to osteoblast activity, leading to overall bone resorption. 

Chandra et al., 2017 

In vivo. An experiment was conducted on male C57BL/6 mice (8–10 weeks) exposed to 8 Gy X-ray radiation at a rate of 1.65 Gy/min. Apoptosis was determined with a TUNEL assay. Osteoblast number was determined by static histomorphometry. 

8 Gy radiation exposure led to a ~3.9-fold increase in the number of TUNEL-positive osteoblasts 2 weeks after irradiation and a 0.5-fold decrease in osteoblast number 4 weeks after irradiation.   

Liu et al., 2018 

In vitro. hBMMSCs were irradiated with 12 Gy of X-rays at a rate of 1.24 Gy/min. Apoptosis was measured using an Annexin V-fluorescein isothiocyanate staining kit. ALP activity was determined with a kit, and bone deposition was determined by Alizarin red staining. 

Apoptosis rate of osteoblast precursor cells (human bone marrow mesenchymal stem cells) exposed to 12 Gy X-ray radiation increased 3-fold after 24h, resulting in a 0.5-fold decrease in ALP activity after 1 week and bone deposition after 3 weeks, as measured by optical density of calcium nodules.