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The effects of 1α, 25-dihydroxyvitamin D3 and transforming growth factor-β3 on bone development in an ex vivo organotypic culture system of embryonic chick femora.

Smith EL, Rashidi H, Kanczler JM, Shakesheff KM, Oreffo RO - PLoS ONE (2015)

Bottom Line: In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis.We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation.Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

View Article: PubMed Central - PubMed

Affiliation: Bone & Joint Research Group, Human Development and Health, University of Southampton, Southampton, United Kingdom.

ABSTRACT
Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

No MeSH data available.


Related in: MedlinePlus

μCT analysis of organotypic cultured embryonic femurs (E11) in basal and TGF-β3 conditions.A, μCT images (whole femur tissue; saggital sections; segmented mineralized bone (green); and cross sectional sections of the central diaphysis region) of the embryonic chick femurs organotypic cultured in basal and TGF-β3 (5ng/ml and 15ng/ml) for 10 days. B, Femur lengths. ***P < 0.001 increase in basal cultured femur lengths compared to non-cultured (NC) femurs; #P < 0.05 decrease in TGF-β3 cultured femur length compared to basal cultured femurs. C, μCT morphometric indices of the structure of the E11 embryonic chick femurs either non-cultured (NC) or organotypic cultured in basal and TGF-β3 media for 10 days. Values are means ± s.d. (n = 4 femurs per group) ***P < 0.01 increase μCT bone morphometric indices of non-cultured embryonic chick femurs compared to basal cultured femurs. ***P < 0.01 increase in μCT bone morphometric indices of basal cultured femurs compared to non-cultured femurs #P < 0.05, ###P < 0.001 increase/decrease in μCT bone morphometric indices of TGF-β3 (5 ng/mL and 15 ng/ml) cultured embryonic femurs compared to basal cultured embryonic femurs. (Scale bar = 1mm).
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pone.0121653.g004: μCT analysis of organotypic cultured embryonic femurs (E11) in basal and TGF-β3 conditions.A, μCT images (whole femur tissue; saggital sections; segmented mineralized bone (green); and cross sectional sections of the central diaphysis region) of the embryonic chick femurs organotypic cultured in basal and TGF-β3 (5ng/ml and 15ng/ml) for 10 days. B, Femur lengths. ***P < 0.001 increase in basal cultured femur lengths compared to non-cultured (NC) femurs; #P < 0.05 decrease in TGF-β3 cultured femur length compared to basal cultured femurs. C, μCT morphometric indices of the structure of the E11 embryonic chick femurs either non-cultured (NC) or organotypic cultured in basal and TGF-β3 media for 10 days. Values are means ± s.d. (n = 4 femurs per group) ***P < 0.01 increase μCT bone morphometric indices of non-cultured embryonic chick femurs compared to basal cultured femurs. ***P < 0.01 increase in μCT bone morphometric indices of basal cultured femurs compared to non-cultured femurs #P < 0.05, ###P < 0.001 increase/decrease in μCT bone morphometric indices of TGF-β3 (5 ng/mL and 15 ng/ml) cultured embryonic femurs compared to basal cultured embryonic femurs. (Scale bar = 1mm).

Mentions: The addition of the chondrogenic factor TGF-β3 (5 ng/mL and 15 ng/mL) resulted in a statistically significant effect on the bone development of the organotypic cultured femurs. TGF-β3 significantly reduced the growth of the organotypic cultured femurs (E11) compared to control basal femurs (Figs. 4A and B). In addition, μCT analysis demonstrated that TGF-β3 reduced bone morphometric indices of BV, BV/TV, Tb.No, and increased Tb.Sp within these femurs compared to the basal control femurs (Fig. 4C). These results correlated with the data obtained from the histological sections which demonstrated a reduction in Sirius red staining, and expression of Type I collagen and STRO-1+ in femurs cultured with TGF-β3 (Fig. 5 and S2 Fig) (-ve controls S4 Fig). Furthermore, TGF-β3 increased alcian blue staining, and expression of Type II collagen and PCNA in the cultured femurs (Fig. 5 and S2 Fig) (-ve controls S4 Fig). TGF-β3 enhanced chondrocyte proliferation within the diaphyseal (Fig. 6A) and the epiphyseal (Fig. 6B) regions of the femur as evidenced by increased PCNA positive cells compared to the total number of cells. The addition of TGF-β3 to the cultured femurs significantly increased the GAG content compared to the basal cultured femurs (Fig. 6C).


The effects of 1α, 25-dihydroxyvitamin D3 and transforming growth factor-β3 on bone development in an ex vivo organotypic culture system of embryonic chick femora.

Smith EL, Rashidi H, Kanczler JM, Shakesheff KM, Oreffo RO - PLoS ONE (2015)

μCT analysis of organotypic cultured embryonic femurs (E11) in basal and TGF-β3 conditions.A, μCT images (whole femur tissue; saggital sections; segmented mineralized bone (green); and cross sectional sections of the central diaphysis region) of the embryonic chick femurs organotypic cultured in basal and TGF-β3 (5ng/ml and 15ng/ml) for 10 days. B, Femur lengths. ***P < 0.001 increase in basal cultured femur lengths compared to non-cultured (NC) femurs; #P < 0.05 decrease in TGF-β3 cultured femur length compared to basal cultured femurs. C, μCT morphometric indices of the structure of the E11 embryonic chick femurs either non-cultured (NC) or organotypic cultured in basal and TGF-β3 media for 10 days. Values are means ± s.d. (n = 4 femurs per group) ***P < 0.01 increase μCT bone morphometric indices of non-cultured embryonic chick femurs compared to basal cultured femurs. ***P < 0.01 increase in μCT bone morphometric indices of basal cultured femurs compared to non-cultured femurs #P < 0.05, ###P < 0.001 increase/decrease in μCT bone morphometric indices of TGF-β3 (5 ng/mL and 15 ng/ml) cultured embryonic femurs compared to basal cultured embryonic femurs. (Scale bar = 1mm).
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pone.0121653.g004: μCT analysis of organotypic cultured embryonic femurs (E11) in basal and TGF-β3 conditions.A, μCT images (whole femur tissue; saggital sections; segmented mineralized bone (green); and cross sectional sections of the central diaphysis region) of the embryonic chick femurs organotypic cultured in basal and TGF-β3 (5ng/ml and 15ng/ml) for 10 days. B, Femur lengths. ***P < 0.001 increase in basal cultured femur lengths compared to non-cultured (NC) femurs; #P < 0.05 decrease in TGF-β3 cultured femur length compared to basal cultured femurs. C, μCT morphometric indices of the structure of the E11 embryonic chick femurs either non-cultured (NC) or organotypic cultured in basal and TGF-β3 media for 10 days. Values are means ± s.d. (n = 4 femurs per group) ***P < 0.01 increase μCT bone morphometric indices of non-cultured embryonic chick femurs compared to basal cultured femurs. ***P < 0.01 increase in μCT bone morphometric indices of basal cultured femurs compared to non-cultured femurs #P < 0.05, ###P < 0.001 increase/decrease in μCT bone morphometric indices of TGF-β3 (5 ng/mL and 15 ng/ml) cultured embryonic femurs compared to basal cultured embryonic femurs. (Scale bar = 1mm).
Mentions: The addition of the chondrogenic factor TGF-β3 (5 ng/mL and 15 ng/mL) resulted in a statistically significant effect on the bone development of the organotypic cultured femurs. TGF-β3 significantly reduced the growth of the organotypic cultured femurs (E11) compared to control basal femurs (Figs. 4A and B). In addition, μCT analysis demonstrated that TGF-β3 reduced bone morphometric indices of BV, BV/TV, Tb.No, and increased Tb.Sp within these femurs compared to the basal control femurs (Fig. 4C). These results correlated with the data obtained from the histological sections which demonstrated a reduction in Sirius red staining, and expression of Type I collagen and STRO-1+ in femurs cultured with TGF-β3 (Fig. 5 and S2 Fig) (-ve controls S4 Fig). Furthermore, TGF-β3 increased alcian blue staining, and expression of Type II collagen and PCNA in the cultured femurs (Fig. 5 and S2 Fig) (-ve controls S4 Fig). TGF-β3 enhanced chondrocyte proliferation within the diaphyseal (Fig. 6A) and the epiphyseal (Fig. 6B) regions of the femur as evidenced by increased PCNA positive cells compared to the total number of cells. The addition of TGF-β3 to the cultured femurs significantly increased the GAG content compared to the basal cultured femurs (Fig. 6C).

Bottom Line: In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis.We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation.Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

View Article: PubMed Central - PubMed

Affiliation: Bone & Joint Research Group, Human Development and Health, University of Southampton, Southampton, United Kingdom.

ABSTRACT
Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

No MeSH data available.


Related in: MedlinePlus