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1,25(OH)2D3 alters growth plate maturation and bone architecture in young rats with normal renal function.

Idelevich A, Kerschnitzki M, Shahar R, Monsonego-Ornan E - PLoS ONE (2011)

Bottom Line: Concomitant with altered chondrocyte maturation, 1,25(OH)(2)D(3) increased chondrocyte proliferation and apoptosis in terminal hypertrophic cells.In vitro treatment of the chondrocytic cell line ATDC5 with 1,25(OH)(2)D(3) lowered differentiation and increased proliferation dose and time-dependently.Micro-CT analysis of femurs from 1-week 1,25(OH)(2)D(3)-treated group revealed reduced cortical thickness, elevated cortical porosity, and higher trabecular number and thickness. 1-month administration resulted in a similar cortical phenotype but without effect on trabecular bone.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Food Science and Nutrition, Hebrew University of Jerusalem, Rehovot, Israel.

ABSTRACT
Whereas detrimental effects of vitamin D deficiency are known over century, the effects of vitamin D receptor activation by 1,25(OH)(2)D(3), the principal hormonal form of vitamin D, on the growing bone and its growth plate are less clear. Currently, 1,25(OH)(2)D(3) is used in pediatric patients with chronic kidney disease and mineral and bone disorder (CKD-MBD) and is strongly associated with growth retardation. Here, we investigate the effect of 1,25(OH)(2)D(3) treatment on bone development in normal young rats, unrelated to renal insufficiency. Young rats received daily i.p. injections of 1 µg/kg 1,25(OH)(2)D(3) for one week, or intermittent 3 µg/kg 1,25(OH)(2)D(3) for one month. Histological analysis revealed narrower tibial growth plates, predominantly in the hypertrophic zone of 1,25(OH)(2)D(3)-treated animals in both experimental protocols. This phenotype was supported by narrower distribution of aggrecan, collagens II and X mRNA, shown by in situ hybridization. Concomitant with altered chondrocyte maturation, 1,25(OH)(2)D(3) increased chondrocyte proliferation and apoptosis in terminal hypertrophic cells. In vitro treatment of the chondrocytic cell line ATDC5 with 1,25(OH)(2)D(3) lowered differentiation and increased proliferation dose and time-dependently. Micro-CT analysis of femurs from 1-week 1,25(OH)(2)D(3)-treated group revealed reduced cortical thickness, elevated cortical porosity, and higher trabecular number and thickness. 1-month administration resulted in a similar cortical phenotype but without effect on trabecular bone. Evaluation of fluorochrome binding with confocal microscopy revealed inhibiting effects of 1,25(OH)(2)D(3) on intracortical bone formation. This study shows negative effects of 1,25(OH)(2)D(3) on growth plate and bone which may contribute to the exacerbation of MBD in the CKD pediatric patients.

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1-week administration of 1,25(OH)2D3 suppresses intracortical mineral deposition in young rats.4 weeks old male Spague Dawley rats received daily i.p. injections of 1 µg/kg 1,25(OH)2D3 (n = 6, VD) or vehicle (n = 6, Ctr) for a period of 7 days. Both groups also received i.p. injections of mineral binding fluorochromes, 40 mg/kg calcein (green) on day 2 and 20 mg/kg alizarin complexon (red) on day 5. Femurs were removed, mid-diaphysis segments transversally cut into 200 µm sections, embedded acrylate and polished until a final thickness of 80 µm. Samples were mounted on glass slides and subjected to LSCM. (A) 10× magnification. White arrows point to the fluorochrome binding surrounding cortical pores. Note the markedly impaired mineral deposition in the 1,25(OH)2D3-treated group compared to the control group. (B) 43× magnification. Representative images from n = 3 in each group are shown.
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pone-0020772-g006: 1-week administration of 1,25(OH)2D3 suppresses intracortical mineral deposition in young rats.4 weeks old male Spague Dawley rats received daily i.p. injections of 1 µg/kg 1,25(OH)2D3 (n = 6, VD) or vehicle (n = 6, Ctr) for a period of 7 days. Both groups also received i.p. injections of mineral binding fluorochromes, 40 mg/kg calcein (green) on day 2 and 20 mg/kg alizarin complexon (red) on day 5. Femurs were removed, mid-diaphysis segments transversally cut into 200 µm sections, embedded acrylate and polished until a final thickness of 80 µm. Samples were mounted on glass slides and subjected to LSCM. (A) 10× magnification. White arrows point to the fluorochrome binding surrounding cortical pores. Note the markedly impaired mineral deposition in the 1,25(OH)2D3-treated group compared to the control group. (B) 43× magnification. Representative images from n = 3 in each group are shown.

Mentions: Micro-CT analysis of cortical mineral density (BMD) demonstrated significant 10±2% and 22±2% reduction during 1-week and 1-month 1,25(OH)2D3 treatments respectively. In attempt to elucidate the reasons for 1,25(OH)2D3-increased cortical porosity and reduced BMD, we monitored the dynamics of bone formation by injecting two fluorochromes, calcein and alizarin complexone at different time points, known to bind precipitated mineral. Confocal microscopy images showed progressive time dependent bone deposition (first green calcein and subsequently red alizarine complexone) leading to pore closure in the control group (Fig. 6A,B). 1-week treatment with 1,25(OH)2D3 demonstrated markedly reduced fluorochrome staining, particularly surrounding pore area indicating hampered bone formation and thus reduced pore closure. Bone apposition at the periosteal side does not appear to be affected, supported by the unchanged T.Ar. values (Fig. 4C, 5C). These data suggest that larger percentage of pores observed during 1,25(OH)2D3 administration results from repressed bone formation in the interior and the endosteal side of the cortical bone of young growing rats.


1,25(OH)2D3 alters growth plate maturation and bone architecture in young rats with normal renal function.

Idelevich A, Kerschnitzki M, Shahar R, Monsonego-Ornan E - PLoS ONE (2011)

1-week administration of 1,25(OH)2D3 suppresses intracortical mineral deposition in young rats.4 weeks old male Spague Dawley rats received daily i.p. injections of 1 µg/kg 1,25(OH)2D3 (n = 6, VD) or vehicle (n = 6, Ctr) for a period of 7 days. Both groups also received i.p. injections of mineral binding fluorochromes, 40 mg/kg calcein (green) on day 2 and 20 mg/kg alizarin complexon (red) on day 5. Femurs were removed, mid-diaphysis segments transversally cut into 200 µm sections, embedded acrylate and polished until a final thickness of 80 µm. Samples were mounted on glass slides and subjected to LSCM. (A) 10× magnification. White arrows point to the fluorochrome binding surrounding cortical pores. Note the markedly impaired mineral deposition in the 1,25(OH)2D3-treated group compared to the control group. (B) 43× magnification. Representative images from n = 3 in each group are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113808&req=5

pone-0020772-g006: 1-week administration of 1,25(OH)2D3 suppresses intracortical mineral deposition in young rats.4 weeks old male Spague Dawley rats received daily i.p. injections of 1 µg/kg 1,25(OH)2D3 (n = 6, VD) or vehicle (n = 6, Ctr) for a period of 7 days. Both groups also received i.p. injections of mineral binding fluorochromes, 40 mg/kg calcein (green) on day 2 and 20 mg/kg alizarin complexon (red) on day 5. Femurs were removed, mid-diaphysis segments transversally cut into 200 µm sections, embedded acrylate and polished until a final thickness of 80 µm. Samples were mounted on glass slides and subjected to LSCM. (A) 10× magnification. White arrows point to the fluorochrome binding surrounding cortical pores. Note the markedly impaired mineral deposition in the 1,25(OH)2D3-treated group compared to the control group. (B) 43× magnification. Representative images from n = 3 in each group are shown.
Mentions: Micro-CT analysis of cortical mineral density (BMD) demonstrated significant 10±2% and 22±2% reduction during 1-week and 1-month 1,25(OH)2D3 treatments respectively. In attempt to elucidate the reasons for 1,25(OH)2D3-increased cortical porosity and reduced BMD, we monitored the dynamics of bone formation by injecting two fluorochromes, calcein and alizarin complexone at different time points, known to bind precipitated mineral. Confocal microscopy images showed progressive time dependent bone deposition (first green calcein and subsequently red alizarine complexone) leading to pore closure in the control group (Fig. 6A,B). 1-week treatment with 1,25(OH)2D3 demonstrated markedly reduced fluorochrome staining, particularly surrounding pore area indicating hampered bone formation and thus reduced pore closure. Bone apposition at the periosteal side does not appear to be affected, supported by the unchanged T.Ar. values (Fig. 4C, 5C). These data suggest that larger percentage of pores observed during 1,25(OH)2D3 administration results from repressed bone formation in the interior and the endosteal side of the cortical bone of young growing rats.

Bottom Line: Concomitant with altered chondrocyte maturation, 1,25(OH)(2)D(3) increased chondrocyte proliferation and apoptosis in terminal hypertrophic cells.In vitro treatment of the chondrocytic cell line ATDC5 with 1,25(OH)(2)D(3) lowered differentiation and increased proliferation dose and time-dependently.Micro-CT analysis of femurs from 1-week 1,25(OH)(2)D(3)-treated group revealed reduced cortical thickness, elevated cortical porosity, and higher trabecular number and thickness. 1-month administration resulted in a similar cortical phenotype but without effect on trabecular bone.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Food Science and Nutrition, Hebrew University of Jerusalem, Rehovot, Israel.

ABSTRACT
Whereas detrimental effects of vitamin D deficiency are known over century, the effects of vitamin D receptor activation by 1,25(OH)(2)D(3), the principal hormonal form of vitamin D, on the growing bone and its growth plate are less clear. Currently, 1,25(OH)(2)D(3) is used in pediatric patients with chronic kidney disease and mineral and bone disorder (CKD-MBD) and is strongly associated with growth retardation. Here, we investigate the effect of 1,25(OH)(2)D(3) treatment on bone development in normal young rats, unrelated to renal insufficiency. Young rats received daily i.p. injections of 1 µg/kg 1,25(OH)(2)D(3) for one week, or intermittent 3 µg/kg 1,25(OH)(2)D(3) for one month. Histological analysis revealed narrower tibial growth plates, predominantly in the hypertrophic zone of 1,25(OH)(2)D(3)-treated animals in both experimental protocols. This phenotype was supported by narrower distribution of aggrecan, collagens II and X mRNA, shown by in situ hybridization. Concomitant with altered chondrocyte maturation, 1,25(OH)(2)D(3) increased chondrocyte proliferation and apoptosis in terminal hypertrophic cells. In vitro treatment of the chondrocytic cell line ATDC5 with 1,25(OH)(2)D(3) lowered differentiation and increased proliferation dose and time-dependently. Micro-CT analysis of femurs from 1-week 1,25(OH)(2)D(3)-treated group revealed reduced cortical thickness, elevated cortical porosity, and higher trabecular number and thickness. 1-month administration resulted in a similar cortical phenotype but without effect on trabecular bone. Evaluation of fluorochrome binding with confocal microscopy revealed inhibiting effects of 1,25(OH)(2)D(3) on intracortical bone formation. This study shows negative effects of 1,25(OH)(2)D(3) on growth plate and bone which may contribute to the exacerbation of MBD in the CKD pediatric patients.

Show MeSH
Related in: MedlinePlus