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. 2017 Mar;100(3):298-310.
doi: 10.1007/s00223-016-0215-6. Epub 2016 Dec 2.

Theobromine Upregulates Osteogenesis by Human Mesenchymal Stem Cells In Vitro and Accelerates Bone Development in Rats

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Free PMC article

Theobromine Upregulates Osteogenesis by Human Mesenchymal Stem Cells In Vitro and Accelerates Bone Development in Rats

Bret H Clough et al. Calcif Tissue Int. .
Free PMC article

Abstract

Theobromine (THB) is one of the major xanthine-like alkaloids found in cacao plant and a variety of other foodstuffs such as tea leaves, guarana and cola nuts. Historically, THB and its derivatives have been utilized to treat cardiac and circulatory disorders, drug-induced nephrotoxicity, proteinuria and as an immune-modulator. Our previous work demonstrated that THB has the capacity to improve the formation of hydroxyl-apatite during tooth development, suggesting that it may also enhance skeletal development. With its excellent safety profile and resistance to pharmacokinetic elimination, we reasoned that it might be an excellent natural osteoanabolic supplement during pregnancy, lactation and early postnatal growth. To determine whether THB had an effect on human osteoprogenitors, we subjected primary human bone marrow mesenchymal stem cells (hMSCs) to osteogenic assays after exposure to THB in vitro and observed that THB exposure increased the rate of osteogenesis and mineralization by hMSCs. Moreover, THB exposure resulted in a list of upregulated mRNA transcripts that best matched an osteogenic tissue expression signature as compared to other tissue expression signatures archived in several databases. To determine whether oral administration of THB resulted in improved skeletal growth, we provided pregnant rats with chow supplemented with THB during pregnancy and lactation. After weaning, offspring received THB continuously until postnatal day 50 (approximately 10 mg kg-1 day-1). Administration of THB resulted in neonates with larger bones, and 50-day-old offspring accumulated greater body mass, longer and thicker femora and superior tibial trabecular parameters. The accelerated growth did not adversely affect the strength and resilience of the bones. These results indicate that THB increases the osteogenic potential of bone marrow osteoprogenitors, and dietary supplementation of a safe dose of THB to expectant mothers and during the postnatal period could accelerate skeletal development in their offspring.

Keywords: Anabolics; Nutrition; Preclinical studies; Stromal/stem cells; Theobromine.

Figures

Figure 1
Figure 1. Effect of THB on osteogenic capacity of hMSCs
Panel A: Flow cytometric profiling of surface markers on one of the hMSC preparations used in the study. The horizontal line on the plots represents gating of positively stained cells as defined by isotype controls. Panel B–D: In vitro osteogenic (panel B), adipogenic (panel C) and chondrogenic (panel D) differentiation of a representative hMSC preparation using classical culture conditions. Osteogenic monolayers are stained with the calcium binding dye alizarin red S, adipogenic monolayers are stained with the lipid binding dye oil red O and histological sections of chondrogenic micromasses are stained with toluidine blue, resulting in purple coloration in the presence of cartilage. For the osteogenic and adipogenic assays, control experiments in the absence of differentiation factors (control media) are presented.
Figure 2
Figure 2. Effect of THB on osteogenic capacity of hMSCs
Panel A: Colorimetric ALP assays on intact monolayers demonstrate a dose-dependent upregulation of activity in the presence of THB. Panel B: ELISA for OPG in culture media demonstrates a dose-dependent upregulation of OPG secretion in the presence of THB. Panel C: Cell recoveries from THB treated cultures. Panel D: Monolayers were treated for 8 days in the presence of OBM containing THB, then transferred to THB-containing OMM for 14 additional days. High doses of THB accelerate mineralization when detected by ARS staining at 7 and 14 days. Panel E: Densitometry profiles of the stained monolayers are presented below each plate, internally normalized to intensity of untreated controls. Panel F: Micrographs of control (above) and 50 µM THB-treated monolayers (below) after 7 days after ARS staining. Fifty-µM THB treatment results in generation of raised, calcium rich-nodules. Statistics for panel B–E: Data presented as means and standard deviations (n=3) tested by one-sided ANOVA and Dunnett’s or Tukey’s post-test. Comparisons are between the vehicle and THB doses, p<0.05 *, p<0.01 **, p<0.005 ***.
Figure 3
Figure 3. Effect of exposure to THB on cranial growth at birth and on body-masses of offspring throughout lactation
Panel A: Scheme describing the experimental timeline. Panel B: The volume of skulls from new-born male pups as measured by µCT. Panel C: The cranial diameter at the midpoint between the anterior and posterior cranial extremity in new-born male pups as measured by µCT. Panel D: Mean humerus length. Panel E: Neonatal body mass. For box and whisker plots in Panels B –E, boxes represent 25–75 percentile, horizontal line represents median, plus sign represents mean and error bars represent range of data. Panel F: Post-natal body mass of offspring. Data presented as means and standard deviations (n=20–25) with Student’s t-tests between treated and appropriate control group, p<0.005 ***.
Figure 4
Figure 4. Effect of exposure to THB on trabecular parameters of the proximal tibia in 50 day-old offspring
Panel A: Trabecular thickness. Panel B: Trabecular spacing. Panel C: Trabecular number. Panel D: Trabecular surface area. Panel E: Representative axial image of trabecular structures in control and THB treated tibiae. While the thickness of trabecular structures are equivalent, THB treated tibiae possess a greater frequency. Panel F: Depth of trabecular structures presented as a fraction of the entire length of the bone. Panel G: Representative longitudinal image of trabecular structures in control and THB treated tibiae. For Panel A–D and F, data are presented as box and whisker plots (n=5–6). P values are calculated by Student’s t-test (D–F) or Mann-Whitney test (B,C).
Figure 5
Figure 5. Effect of exposure to THB on femoral length, cortical thickness and biomechanical strength in 50 day old offspring
Panel A: Femoral length. Panel B: Average cortical thickness at midpoint of diaphysis. Panel C: Polar moment of inertia at midpoint of diaphysis. Panel D: Energy required to break. Panel E: Energy required until yield. Data are presented as box and whisker plots (n=10). P values are calculated by Student’s t-test.

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