Purpose: Prolonged use of proton pump inhibitors may cause bone loss, and limited therapeutic agents are available to prevent this skeletal side effect. The combination of annatto tocotrienol, a bone anabolic agent, with calcium presents a novel strategy to prevent bone loss caused by proton pump inhibitors. This study aims to compare the effects of calcium alone and in combination with annatto tocotrienol or vitamin D₃ (Caltrate Plus) in preventing bone loss caused by pantoprazole.

Methods: Three-month-old Sprague Dawley male rats (n=30) were randomised into five groups (n=6/group). Bone loss was induced by pantoprazole (3 mg/kg p.o.) in four groups, and they were treated concurrently with either calcium carbonate (77 mg p.o.), calcium carbonate (77 mg p.o.) plus annatto tocotrienol (60 mg/kg p.o.) or Caltrate Plus (31 mg p.o.) for 60 days. The rats were euthanised at the end of the experiment, and their femurs were harvested for X-ray micro-computed tomography, bone cellular histomorphometry and bone mechanical strength analysis.

Results: Pantoprazole caused significant deterioration of trabecular bone microstructures but did not affect other skeletal indices. Calcium supplementation with or without annatto tocotrienol prevented the deterioration of trabecular microstructures at the femur but did not improve other skeletal indices. Annatto tocotrienol did not enhance the skeletal actions of calcium, whereas Caltrate Plus did not affect the bone health indices in these rats.

Conclusion: Calcium supplementation per se can prevent the deterioration of bone trabecular microstructures in rats receiving long-term treatment of pantoprazole.

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Introduction: The aim of the present study was to determine the effects of vitamins D and E on the proliferation, morphology, and differentiation of human dental pulp stem cells (hDPSCs).

Methods: In this in vitro experimental study, hDPSCs were isolated, characterized, and treated with vitamins D and E, individually and in combination, utilizing different doses and treatment periods. Changes in morphology and cell proliferation were evaluated using light microscopy and the resazurin assay, respectively. Osteoblast differentiation was evaluated with alizarin red S staining and expression of RUNX2, Osterix, and Osteocalcin genes using real-time RT-PCR.

Results: Compared with untreated cells, the number of cells significantly reduced following treatment with vitamin D (49%), vitamin E (35%), and vitamins D + E (61%) after 144 h. Compared with cell cultures treated with individual vitamins, cells treated with vitamins D + E demonstrated decreased cell confluence, with more extensive and flatter cytoplasm that initiated the formation of a significantly large number of calcified nodules after 7 days of treatment. After 14 days, treatment with vitamins D, E, and D + E increased the transcription of RUNX2, Osterix, and Osteocalcin genes.

Conclusions: Vitamins D and E induced osteoblastic differentiation of hDPSCs, as evidenced by the decrease in cell proliferation, morphological changes, and the formation of calcified nodules, increasing the expression of differentiation genes. Concurrent treatment with vitamins D + E induces a synergistic effect in differentiation toward an osteoblastic lineage.

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Bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, indicating that stimulation of osteoblast migration could be a promising osteoanabolic strategy. We showed that purified δ-tocotrienol (δ-TT, 10 μg/mL), isolated from commercial palm oil (Elaeis guineensis) fraction, stimulates the migration of both MC3T3-E1 osteoblast-like cells and primary human bone marrow mesenchymal stem cells (BMSC) as detected by wound healing assay or Boyden chamber assay respectively. The ability of δ-TT to promote MC3T3-E1 cells migration is dependent on Akt phosphorylation detected by Western blotting and involves Wnt/β-catenin signalling pathway activation. In fact, δ-TT increased β-catenin transcriptional activity, measured using a Nano luciferase assay and pretreatment with procaine (2 µM), an inhibitor of the Wnt/β-catenin signalling pathway, reducing the wound healing activity of δ-TT on MC3T3-E1 cells. Moreover, δ-TT treatment increased the expression of β-catenin specific target genes, such as Osteocalcin and Bone Morphogenetic Protein-2, involved in osteoblast differentiation and migration, and increased alkaline phosphatase and collagen content, osteoblast differentiation markers. The ability of δ-TT to enhance the recruitment of BMSC, and to promote MC3T3-E1 differentiation and migratory behavior, indicates that δ-TT could be considered a promising natural anabolic compound.

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