Enhanced Osteogenesis of Adipose Derived Stem Cells with Noggin Suppression and Delivery of BMP-2

Bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors. However, BMPs are highly pleiotropic molecules and their supra-physiological high dose requirement leads to adverse side effects and inefficient bone formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity. In this study, we intrinsically stimulated BMP signaling in adipose derived stem cells (ASCs) by downregulating noggin, a potent BMP antagonist, using an RNAi strategy. ASCs transduced with noggin shRNA significantly enhanced osteogenic differentiation of cells. The potency of endogenous BMPs was subsequently enhanced by stimulating ASCs with exogenous BMPs at a significantly reduced dose. The level of mineralization in noggin shRNA treated ASCs when treated with BMP-2 was comparable to that of control shRNA treated cell treated with 10-fold more BMP-2. The complementary strategy of noggin suppression + BMP-2 to enhance osteogenesis was further confirmed in 3D in vitro environments using scaffolds consisting of chitosan (CH), chondroitin sulfate (CS), and apatite layer on their surfaces designed to slowly release BMP-2. This finding supports the novel therapeutic potential of this complementary strategy in bone regeneration.     

Vitamin D Promotes Odontogenic Differentiation of Human Dental Pulp Cells via ERK Activation

The active metabolite of vitamin D such as 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) is a well-known key regulatory factor in bone metabolism. However, little is known about the potential of vitamin D as an odontogenic inducer in human dental pulp cells (HDPCs) in vitro. The purpose of this study was to evaluate the effect of vitamin D₃ metabolite, 1α,25(OH)₂D₃, on odontoblastic differentiation in HDPCs. HDPCs extracted from maxillary supernumerary incisors and third molars were directly cultured with 1α,25(OH)₂D₃ in the absence of differentiation-inducing factors. Treatment of HDPCs with 1α,25(OH)₂D₃ at a concentration of 10 nM or 100 nM significantly upregulated the expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein1 (DMP1), the odontogenesis-related genes. Also, 1α,25(OH)₂D₃ enhanced the alkaline phosphatase (ALP) activity and mineralization in HDPCs. In addition, 1α,25(OH)₂D₃ induced activation of extracellular signal-regulated kinases (ERKs), whereas the ERK inhibitor U0126 ameliorated the upregulation of DSPP and DMP1 and reduced the mineralization enhanced by 1α,25(OH)₂D₃. These results demonstrated that 1α,25(OH)₂D₃ promoted odontoblastic differentiation of HDPCs via modulating ERK activation.     

Tanshinone IIA enhances BMP-2-stimulated commitment of C2C12 cells into osteoblasts via p38 activation

In this study, we demonstrate a stimulatory effect of tanshinone IIA isolated from the root of Salvia miltiorrhiza on the commitment of bi-potential mesenchymal precursor C2C12 cells into osteoblasts in the presence of bone morphogenetic protein (BMP)-2. At low concentrations, tanshinone IIA enhanced BMP-2-stimulated induction of alkaline phosphatase (ALP), an early phase biomarker of osteoblast differentiation, and mRNA expression of BMPs. ALP induction was inhibited by the BMP antagonist noggin, suggesting that tanshinone IIA enhances the osteogenic activity of BMP signaling. Furthermore, considering the tanshinone IIA-mediated enhancement of BMP-2-stimulated Smad-Runx2 activities, tanshinone IIA could enhance the osteogenic activity of BMP-2 via acceleration of Smad-Runx2 activation. Additionally, pharmacologic inhibition studies suggest the possible involvement of p38 in the action of tanshinone IIA. The p38 inhibitor SB202190 strongly and dose-dependently inhibited tanshinone IIA-enhanced ALP induction. SB202190 also dose-dependently inhibited the tanshinone IIA-induced p38 activation and combined tanshinone IIA-BMP-2-induced Smad activation. In conclusion, tanshinone IIA enhances the commitment of C2C12 cells into osteoblasts and their differentiation through synergistic cross talk between tanshinone IIA-induced p38 activation and BMP-2-induced Smad activation. These activations could subsequently induce the activation of Runx2, which induces osteogenesis via regulation of the osteogenic factors BMP and ALP expression.     

A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue

To evaluate the potential of three stem cells for cell therapy and tissue engineering applications, the biological behavior and osteogenic capacity of the newly introduced cord-blood-derived, unrestricted somatic stem cells (USSC) were compared with those of mesenchymal stem cells isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). There was no significant difference between the rates of proliferation of the three stem cells. During osteogenic differentiation, alkaline phosphatase (ALP) activity peaked on day 7 in USSC compared to BM-MSC which showed the maximum value of ALP activity on day 14. However, BM-MSC had the highest ALP activity and mineralization during osteogenic induction. In addition, AT-MSC showed the lowest capacity for mineralization during differentiation and had the lowest ALP activity on days 7 and 14. Although AT-MSC expressed higher levels of collagen type I, osteonectin and BMP-2 in undifferentiated state, but these genes were expressed higher in BM-MSC during differentiation. BM-MSC also expressed higher levels of ALP, osteocalcin and Runx2 during induction. Taking together, BM-MSC showed the highest capacity for osteogenic differentiation and hold promising potential for bone tissue engineering and cell therapy applications.     

Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-κB ligand expression in osteoblasts

1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] induces osteoclast formation via induction of receptor activator of NF-κB ligand (RANKL, also called TNF-related activation-induced cytokine: TRANCE) in osteoblasts. In cocultures of mouse bone marrow cells and osteoblasts, 1,25(OH)₂D₃ induced osteoclast formation in a dose-dependent manner, with maximum osteoclast formation observed at concentrations greater than 10^?9 M of 1,25(OH)₂D₃. In the presence of bone morphogenetic protein 2 (BMP-2), the maximum formation of osteoclasts was seen with lower concentrations of 1,25(OH)₂D₃ (greater than 10^?11 M), suggesting that BMP-2 enhances osteoclast formation induced by 1,25(OH)₂D₃. In addition, the expressions of RANKL mRNA and proteins were induced by 1,25(OH)₂D₃ in osteoblasts, and further upregulated by BMP-2. In mouse bone marrow cell cultures without 1,25(OH)₂D₃, BMP-2 did not enhance osteoclast differentiation induced by recombinant RANKL and macrophage colony-stimulating factor (M-CSF), indicating that BMP-2 does not target osteoclast precursors. Furthermore, BMP-2 up-regulated the expression level of vitamin D receptor (VDR) in osteoblasts. These results suggest that BMP-2 regulates mouse osteoclast differentiation via upregulation of RANKL in osteoblasts induced by 1,25(OH)₂D₃.     

BMP-13 Emerges as a Potential Inhibitor of Bone Formation

Bone morphogenetic protein-13 (BMP-13) plays an important role in skeletal development. In the light of a recent report that mutations in the BMP-13 gene are associated with spine vertebral fusion in Klippel-Feil syndrome, we hypothesized that BMP-13 signaling is crucial for regulating embryonic endochondral ossification. In this study, we found that BMP-13 inhibited the osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. The endogenous BMP-13 gene expression in MSCs was examined under expansion conditions. The MSCs were then induced to differentiate into osteoblasts in osteo-inductive medium containing exogenous BMP-13. Gene expression was analysed by real-time PCR. Alkaline phosphatase (ALP) expression and activity, proteoglycan (PG) synthesis and matrix mineralization were assessed by cytological staining or ALP assay. Results showed that endogenous BMP-13 mRNA expression was higher than BMP-2 or -7 during MSC growth. BMP-13 supplementation strongly inhibited matrix mineralization and ALP activity of osteogenic differentiated MSCs, yet increased PG synthesis under the same conditions. In conclusion, BMP-13 inhibited osteogenic differentiation of MSCs, implying that functional mutations or deficiency of BMP-13 may allow excess bone formation. Our finding provides an insight into the molecular mechanisms and the therapeutic potential of BMP-13 in restricting pathological bone formation.     

Co-stimulation with bone morphogenetic protein-9 and FK506 induces remarkable osteoblastic differentiation in rat dedifferentiated fat cells

Dedifferentiated fat (DFAT) cells, which are isolated from mature adipocytes using the ceiling culture method, exhibit similar characteristics to mesenchymal stem cells, and possess adipogenic, osteogenic, chondrogenic, and myogenic potentials. Bone morphogenetic protein (BMP)-2 and -9, members of the transforming growth factor-β superfamily, exhibit the most potent osteogenic activity of this growth factor family. However, the effects of BMP-2 and BMP-9 on the osteogenic differentiation of DFAT remain unknown. Here, we examined the effects of BMP-2 and BMP-9 on osteoblastic differentiation of rat DFAT (rDFAT) cells in the presence or absence of FK506, an immunosuppressive agent. Co-stimulation with BMP-9 and FK506 induced gene expression of runx2, osterix, and bone sialoprotein, and ALP activity compared with BMP-9 alone, BMP-2 alone and BMP-2 + FK506 in rDFAT cells. Furthermore, it caused mineralization of cultures and phosphorylation of smad1/5/8, compared with BMP-9 alone. The ALP activity induced by BMP-9 + FK506 was not influenced by addition of noggin, a BMP antagonist. Our data suggest that the combination of BMP-9 and FK506 potently induces osteoblastic differentiation of rDFAT cells.     

Direct effects of vitamin D3 analogues on G-protein mediated signalling systems in rat osteosarcoma cells and rat pituitary adenoma cells

In normal rats treated with 1,25(OH)₂D₃ or 24,25(OH)₂D₃, serum Ca²⁺, ALP, PRL and GH are significantly altered. In order to study the primary effect of vitamin D₃ analogues on target organ function, rat UMR 106 osteosarcoma and GH₃ pituitary adenoma cells in monolayer culture were exposed accordingly.Surprisingly, prolonged exposure of these cell lines to physiological levels of either 1,25(OH)₂D₃ or 24,25(OH)₂D₃ did not significantly affect the secretory parameters (ALP, PRL or GH) tested. However, 1,25(OH)₂D₃ exposure significantly reduced PTH- and Gpp(NH)p-elicited AC as well as Gpp(NH)p-stimulated PLC activities in the UMR 106 cells. These changes were accompanied by an increase and decrease in the membrane contents of the G-protein subunits G₃₆ and G_q/11, respectively. In contrast, 24,25(OH)₂D₃ remained without significant biological effect on these signalling systems despite concomitantly augmented levels of G₃₆. TRH- and Gpp(NH)p-elicited PLC activities in the GH₃ cells were significantly reduced by 1,25(OH)₂D₃ with a concurrent reduction in cellular amounts of G_q/11, however, 24,25(OH)₂D₃ did not significantly alter any signalling systems nor G-proteins analyzed.It is concluded that the osteoblastic and pituitary cell secretion of ALP, PRL and GH remain unaffected by the presence of 1,25(OH)₂D₃ and 24,25(OH)₂D₃, despite distinct alterations in components of G-protein mediated signalling pathways. Hence, other factors like ambient Ca²⁺ may be responsible for the perturbed secretory patterns of ALP and PRL seen in vitamin D₃ treated rats.Abbreviations AC adenylate cyclase - ALP alkaline phosphatase - BGP osteocalcin - BSA bovine serum albumin - DA dopamine - DAG diacylglycerol - GH growth hormone - GHRH growth hormone releasing hormone - Gpp(NH)p guanosine 5-[-imido]triphosphate - G-protein guanine nucleotide-binding regulatory protein - G_s etc. G_s protein -subunit - IP₃ inositol 1,4,5 trisphosphate - OAF osteoclast activating factor - PGE₂ prostaglandin E₂ - PKA & PKC protein kinase A & C - PLC phospholipase C - PRL prolactin - PTH parathyroid hormone - SRIF somatostatin - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide - 25(OH)D₃ 25 hydroxy vitamin D₃ - 1,25(OH)₂D₃ 1·25 dihydroxy vitamin D₃ - 24,25(OH)₂D₃ 24,25 dihydroxy vitamin D₃     

Individual and combined effects of calciotropic hormones and growth factors on mineral metabolism in embryonic chick tibiae

Summary We have investigated single and combined effects of calciotropic hormones and growth factors on the regulation of alkaline phosphatase (ALP) activity and calcium metabolism in an optimized serum-free bone organ culture system of embryonic chick tibiae. Parathyroid hormone PTH(1–34) alone mobilized calcium from bone tissue time- and dose-dependently and inhibited ALP activity. Both the bisphosphonate (BM 21.0955) and to a lesser extent salmon calcitonin alone slightly increased calcium uptake and inhibited the stimulation of bone resorption by PTH(1–34). 1,25(OH)₂D₃ mobilized calcium and inhibited ALP activity in contrast to 24,25(OH)₂D₃ which inhibited ALP activity but had no significant effect on calcium metabolism. Interestingly the combination of PTH(1–34) with 1,25(OH)₂D₃ but not 24,25(OH)₂D₃ reduced calcium mobilization. The combination of the midregional fragment PTH(28–48), which by itself has no effect on calcium metabolism, with 1,25(OH)₂D₃ reduced calcium mobilization more efficiently. Several PTH-regulated mediators have been assayed in this system. Of the tested growth factors, IGF-I at high concentrations caused bone resorption with no effect on ALP activity. TGF-β₁ (transforming growth factor β) and BMP-2 had no significant effect on calcium metabolism; however, ALP activity was inhibited by TGF-β₁ and induced dose dependently by BMP-2. Of the other factors known to be present in bone, platelet-derived growth factor (PDGF_A/B) and epidermal growth factor (EGF) had a small effect on calcium mobilization but had no effect on ALP activity. bFGF reduced ALP activity slightly without an effect on calcium metabolism. Our results show that this in vitro system can mimic some interactions of calciotropic hormones in vivo and allows the assaying of mediators in terms of regulation of ALP activity and of calcium metabolism.     

Bone morphogenetic proteins 4 and 2/7 induce osteogenic differentiation of mouse skin derived fibroblast and dermal papilla cells

Heterotopic ossification is a pathological condition in which bone forms outside the skeletal system. It can also occur in skin, which is the case in some genetic disorders. In addition to precursor cells and the appropriate tissue environment, heterotopic ossification requires inductive signals such as bone morphogenetic proteins (BMP). BMPs are growth and differentiation factors that have the ability to induce cartilage and bone formation in ectopic sites. The objective of this study is to explore the effect of the BMP-4 homodimer and BMP-2/7 heterodimer on the osteogenic differentiation of primary mouse skin fibroblasts and hair follicle dermal papilla (DP) cells. Osteogenic differentiation was induced by osteogenic induction medium (OS) containing 10 nM dexamethasone. The effect of BMP-4 and BMP-2/7 was studied using alkaline phosphatase (ALP) and calcium assays after 1.5, 3 and 5 weeks of differentiation. Fibroblasts and DP cells were able to differentiate into osteoblast-like matrix mineralizing cells. The first visible sign of differentiation was the change of morphology from rounded to more spindle-shaped cells. BMP-4 and BMP-2/7 exposure elevated ALP activity and calcium production significantly more than OS alone. The osteogenic response to BMP-4 and BMP-2/7 was similar in fibroblasts, whereas, in DP cells, BMP-2/7 was more potent than BMP-4. OS alone could not induce osteogenic differentiation in DP cells. Clear and consistent results show that dermal fibroblasts and stem cells from the dermal papilla were capable of osteogenic differentiation. The BMP-2/7 heterodimer was significantly more effective on hair follicular dermal stem cell differentiation.     

PTH(7-84) inhibits PTH(1-34)-induced 1,25-(OH)2D3 production in murine renal tubules

To elucidate whether PTH(7-84), a degradation product of PTH(1-84), which inhibits PTH(1-84)-induced bone resorption, also exerts an antagonistic effect on the kidney, we studied the effect of PTH(7-84) on PTH(1-34)-induced production of 1,25-(OH)₂D₃ in primary cultured murine renal tubules.Neonatal mouse renal tubules cultured in serum-free MEM for 7 days were treated with PTH(1-34) and/or PTH(7-84). Three hours after addition of 25-OHD₃ (10⁻⁶ M), 1,25-(OH)₂D₃ was determined. PTH(1-34) stimulated the conversion of 25-OHD₃ to 1,25-(OH)₂D₃, and PTH(7-84) dose-dependently inhibited this process. Real-time PCR revealed that PTH(1-34) increased the expression level of 1α-hydroxylase mRNA, whereas PTH(7-84) did not affect the expression level 1α or 24-hydroxylase mRNA.These in vitro data suggest that PTH(7-84) elicits an antagonistic effect in renal tubules through receptors different from the type I PTH/PTHrP receptor. This may at least partly account for the decreased serum level of 1,25-(OH)₂D in patients with severe primary hyperparathyroidism with renal failure.     

Role of 1α,25(OH)2 vitamin D3 on α-[1-C]MeAIB accumulation in immature rat testis

1,25D₃ is critical for the maintenance of normal reproduction since reduced fertility is observed in male rats on a vitamin D-deficient diet. Vitamin D-deficient male rats have incomplete spermatogenesis and degenerative testicular changes. In the present study we have examined the ionic involvement and intracellular messengers of the stimulatory effect of 1,25D₃ on amino acid accumulation in immature rat testis. 1,25D₃ stimulates amino acid accumulation from 10⁻¹² to 10⁻⁶ M by increasing the slope to reach a maximum value at 10⁻¹⁰ M, as compared to the control group. No effect was observed at a lower dose (10⁻¹³ M). Time-course showed an increase on amino acid accumulation after 15, 30, and 60 min of incubation with 1,25D₃ (10⁻¹⁰ M). 1,25D₃ stimulated amino acid accumulation in 11-day-old rat testis but not in testis that were 20 days old. Cycloheximide totally blocked the 1,25D₃ action on amino acid accumulation. Furthermore, a localized elevation of cAMP increased the stimulatory effect of 1,25D₃ and the blockage of PKA nullified the action of the hormone. In addition, 1,25D₃ action on amino acid accumulation was also mediated by ionic pathways, since verapamil and apamine diminished the hormone effect. The stimulatory effect of 1,25D₃ on amino acid accumulation is age-dependent and specific to this steroidal hormone since testosterone was not able to change amino acid accumulation in both ages studied. This study provides evidence for a dual effect for 1,25D₃, pointing to a genomic effect that can be triggered by PKA, as well as to a rapid response involving Ca²⁺/K⁺ channels on the plasma membrane.     

BMP-2, VEGF and bFGF synergistically promote the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) were treated with bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) dose-dependently and time-dependently. Together they caused a strong synergistic effect on the osteogenic differentiation of MSCs, with lower concentrations of each factor being enough to show the synergistic promotion (50 ng BMP-2/ml, 1 ng VEGF/ml and 10 ng bFGF/ml). When both VEGF and bFGF were added in the early proliferating stage (the first 7 days) and BMP-2 was added in the late differentiation stage (the last 7 days), osteogenic differentiation of MSCs could be enhanced more effectively.     

Platelet-rich plasma and 1,25(OH)2 vitamin D3 synergistically stimulate osteogenic differentiation of adult human mesenchymal stem cells

Mesenchymal stem cell proliferation and differentiation are regulated by growth factors and systemic hormones in a concerted manner. We have examined the effects of platelet-rich plasma (PRP) and its interaction with 1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] on the differentiation and proliferation of human mesenchymal stem cells (hMSCs). PRP stimulated cell proliferation in a manner inversely proportional to the dose. Exposure to PRP inhibited hMSC osteogenic differentiation. Co-treatment with PRP and 1,25(OH)₂D₃, however, synergistically stimulated osteogenic differentiation. Serial co-treatment with PRP and 1,25(OH)₂D₃ showed that this synergistic effect was due to the modulation of a 1,25(OH)₂D₃-induced differentiation by PRP rather than vice versa.     

Autoradiographic studies with 3H 1,25 (OH)2 vitamin D3 and 3H 25 (OH) vitamin D3 in rat parathyroid glands

Summary After injection of radiolabeled 1,25 (OH)₂ vitamin D₃, nuclear concentration of radioactivity is observed in parenchymal cells of the parathyroid gland in pregnant, adult male, and 10-day male neonatal rats. In competition studies with unlabeled 1,25 (OH)₂ vitamin D₃, but not with 25 (OH) vitamin D₃, nuclear uptake is prevented. Experiments with ³H 25 (OH) vitamin D₃, in contrast to ³H 1,25 (OH)₂ vitamin D₃, do not show nuclear concentration in cells of the parathyroid. The results of the autoradiographic studies suggest the presence of receptors for a direct effect of 1,25 (OH)₂ vitamin D₃ on the parathyroid gland for modulation of parathyroid hormone secretion.     

Extracellular calcium stimulates osteogenic differentiation of human adipose-derived stem cells by enhancing bone morphogenetic protein-2 expression

Bone morphogenetic protein-2 (BMP-2) promotes the differentiation of non-osteogenic mesenchymal cells to osteogenic cells. In this study, we isolated human adipose-derived stem cells (hASCs) and investigated the effects of recombinant human BMP-2 (rhBMP-2) and extracellular Ca²⁺ concentration ([Ca²⁺]_out) on the osteogenic differentiation of hASCs. rhBMP-2 promoted calcium deposition in hASCs and stimulated the mRNA expressions of six proteins known to be involved in the osteogenic differentiation of hASCs: Runx2, osterix, alkaline phosphatase, osteonectin, bone sialoprotein and osteocalcin. Elevation of [Ca²⁺]_out enhanced the level of alkaline phosphatase enzyme, increased the mRNA expressions of Runx2 and osteocalcin and induced the expressions of BMP-2 mRNA and protein in hASCs. Elevation of [Ca²⁺]_out transiently increased the intracellular Ca²⁺ concentration ([Ca²⁺]_in) due to activation of the calcium-sensing receptor (CaSR). The Ca²⁺-induced expressions of BMP-2 mRNA and protein were inhibited by the calmodulin antagonist, W-7. Furthermore, elevation of [Ca²⁺]_out decreased the cytoplasmic level of phosphorylated nuclear factor of activated T-cell-2 (NFAT-2) and increased the nuclear level of dephosphorylated NFAT2. Taken together, these results suggest that rhBMP-2 promotes the osteogenic differentiation of hASCs. Furthermore, an increase in [Ca²⁺]_out enhances the expression of BMP-2 via activation of the CaSR, elevation of [Ca²⁺]_in and stimulation of Ca²⁺/calmodulin-dependent NFAT-signaling pathways.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.