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.
Highlights
ΜCT analysis demonstrated that Transforming Growth Factor-β3 (TGF-β3) reduced bone morphometric indices of Bone Volume (BV), Bone Volume/Total (tissue) Volume (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)
E11 femurs cultured in basal and 1α,25(OH)2D3 supplemented media were analyzed for alcian blue/Sirius red, von Kossa-mineralization, expression of collagen Type I & II, the proliferation marker proliferating cell nuclear antigen (PCNA) and STRO-1+. (TIF)
Summary
Materials6 well tissue culture plates were obtained from Greiner BioOne, UK. Millicell culture inserts (0.4μm pore size) (Cat No PICM03050) were purchased from Millipore (30 mm diameter). Bones were transferred to six well plates and positioned resting on 0.4 μm filter well inserts at the interface between the air and the basal culture medium (1 mL of basal tissue culture medium (TCM) consisting of α-MEM, penicillin (100 U/ mL), streptomycin (100 μg/mL), and ascorbic acid 2-phosphate (100 μM)). E11 femurs were organotypic cultured in basal TCM (1 mL) and basal TCM supplemented with 1α,25(OH)2D3 (25 nM). E11 and E13 femurs were organotypic cultured in basal TCM (1 mL) and basal TCM supplemented with TGF-β3 (5 ng/mL & 15 ng/mL). At the end of the experiments, organotypic cultured femurs were washed in 1x PBS and fixed in 4% paraformaldehyde (PFA). The non-cultured and organotypic cultured femurs were scanned using μCT and either assessed for quantification of glycosaminoglycan (GAG) content using a dimethylmethylene blue (DMMB) colorimetric assay, or processed for histology
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