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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

Analysis of diaphyseal and epiphyseal cell proliferation and glycosaminoglycan content of E13 organotypic cultured femurs.A, diaphyseal and B, epiphyseal cell proliferation data for TGF-β3 treated femurs (E13); *P < 0.05; **P < 0.01. C, Glycosaminoglycan (GAG) content (expressed as percentage of tissue weight) of embryonic chick femurs, either non-cultured (NC) or cultured for 10 days in basal media, alone or supplemented with either 5 ng/mL or 15 ng/mL of TGF-β3. GAG content was significantly reduced in both groups of organotypic cultured femurs with TGF-β3 compared to basal cultured femurs (*P < 0.05). Values are mean ± s.d. (n = 4 femurs per group).
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pone.0121653.g009: Analysis of diaphyseal and epiphyseal cell proliferation and glycosaminoglycan content of E13 organotypic cultured femurs.A, diaphyseal and B, epiphyseal cell proliferation data for TGF-β3 treated femurs (E13); *P < 0.05; **P < 0.01. C, Glycosaminoglycan (GAG) content (expressed as percentage of tissue weight) of embryonic chick femurs, either non-cultured (NC) or cultured for 10 days in basal media, alone or supplemented with either 5 ng/mL or 15 ng/mL of TGF-β3. GAG content was significantly reduced in both groups of organotypic cultured femurs with TGF-β3 compared to basal cultured femurs (*P < 0.05). Values are mean ± s.d. (n = 4 femurs per group).

Mentions: In studies using the more developed E13 embryonic femurs, addition of TGF-β3 resulted in a modest effect on growth (Fig. 7A and B) and bone structure indices with reduced levels of BV, BV/TV, Tb.No, and increased Tb.Sp within these femurs compared to the basal control femurs. Interestingly, 15 ng/mL TGF-β3 significantly reduced the levels of Tb.Th compared to the basal cultured femurs which was not observed in the E11 femurs (Fig. 7C). Histological examination of sections from the femurs demonstrated reduced collagen as determined by Sirius red staining, reduced Type I and II collagen expression, reduced mineralization, and reduced cell proliferation within the hypertrophic chondrocytes (Fig. 8 and S3 Fig) (-ve controls S4 Fig). No observable differences were detected in the expression of STRO-1+ between the groups except for the femurs cultured in the presence of 15 ng/mL TGF-β3 which displayed a thicker bone collar containing cells positive for the expression of STRO-1+ (Fig. 8 and S3 Fig). TGF-β3 decreased the chondrocyte proliferation of the diaphyseal (Fig. 9A) and the epiphyseal (Fig. 9B) regions of the femur as indicated by reduced numbers of PCNA positive cells compared to the total number of cells. In contrast to the E11 embryonic cultured femurs the addition of TGF-β3 to E13 embryonic cultured femurs significantly reduced the levels of GAG compared to the femurs cultured in basal conditions (Fig. 9C).


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)

Analysis of diaphyseal and epiphyseal cell proliferation and glycosaminoglycan content of E13 organotypic cultured femurs.A, diaphyseal and B, epiphyseal cell proliferation data for TGF-β3 treated femurs (E13); *P < 0.05; **P < 0.01. C, Glycosaminoglycan (GAG) content (expressed as percentage of tissue weight) of embryonic chick femurs, either non-cultured (NC) or cultured for 10 days in basal media, alone or supplemented with either 5 ng/mL or 15 ng/mL of TGF-β3. GAG content was significantly reduced in both groups of organotypic cultured femurs with TGF-β3 compared to basal cultured femurs (*P < 0.05). Values are mean ± s.d. (n = 4 femurs per group).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383569&req=5

pone.0121653.g009: Analysis of diaphyseal and epiphyseal cell proliferation and glycosaminoglycan content of E13 organotypic cultured femurs.A, diaphyseal and B, epiphyseal cell proliferation data for TGF-β3 treated femurs (E13); *P < 0.05; **P < 0.01. C, Glycosaminoglycan (GAG) content (expressed as percentage of tissue weight) of embryonic chick femurs, either non-cultured (NC) or cultured for 10 days in basal media, alone or supplemented with either 5 ng/mL or 15 ng/mL of TGF-β3. GAG content was significantly reduced in both groups of organotypic cultured femurs with TGF-β3 compared to basal cultured femurs (*P < 0.05). Values are mean ± s.d. (n = 4 femurs per group).
Mentions: In studies using the more developed E13 embryonic femurs, addition of TGF-β3 resulted in a modest effect on growth (Fig. 7A and B) and bone structure indices with reduced levels of BV, BV/TV, Tb.No, and increased Tb.Sp within these femurs compared to the basal control femurs. Interestingly, 15 ng/mL TGF-β3 significantly reduced the levels of Tb.Th compared to the basal cultured femurs which was not observed in the E11 femurs (Fig. 7C). Histological examination of sections from the femurs demonstrated reduced collagen as determined by Sirius red staining, reduced Type I and II collagen expression, reduced mineralization, and reduced cell proliferation within the hypertrophic chondrocytes (Fig. 8 and S3 Fig) (-ve controls S4 Fig). No observable differences were detected in the expression of STRO-1+ between the groups except for the femurs cultured in the presence of 15 ng/mL TGF-β3 which displayed a thicker bone collar containing cells positive for the expression of STRO-1+ (Fig. 8 and S3 Fig). TGF-β3 decreased the chondrocyte proliferation of the diaphyseal (Fig. 9A) and the epiphyseal (Fig. 9B) regions of the femur as indicated by reduced numbers of PCNA positive cells compared to the total number of cells. In contrast to the E11 embryonic cultured femurs the addition of TGF-β3 to E13 embryonic cultured femurs significantly reduced the levels of GAG compared to the femurs cultured in basal conditions (Fig. 9C).

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