Background
Caffeine is a pharmacologically active xanthine (1,3,7-trimethylxanthine) that is present in coffees, teas, soft drinks, food, and drugs [
1].
Despite the beneficial effects of caffeine on the central nervous system and the respiratory system, characterized by reduced fatigue and bronchodilation, respectively, the adverse effects of caffeine on the body are more widely documented. Caffeine is known to cause insomnia and anxiety as well as increased blood pressure and pulse. In addition, caffeine is considered a risk factor for osteoporosis and periodontal disease and a causative agent of fetal malformations, reductions in body weight, reduced length of long bones and reduced length of vertebrae in newborns [
1-
7].
Caffeine crosses the placenta leading to substantial amounts in amniotic fluid, umbilical cord blood, newborn plasma and newborn urine and is also carried by the mother’s milk in both mice and humans [
8-
11]. Pregnant women in the third trimester of pregnancy with low caffeine consumption had mean serum levels of caffeine in their umbilical cord blood of 0.48 μg/ml (0 to 10.49 μg/ml). Pregnant women with caffeine intake greater than 300 mg/day averaged 2.1 μg/ml of caffeine in their cord blood [
9]. Even in humans, maternal ingestion of 36–335 mg of caffeine has been shown to be transmitted in the milk, which contained between 2.09 to 7.17 μg/ml caffeine [
11]. Previous studies have shown that caffeine causes changes in the bones of fetuses and young rats. In rat fetuses born to mothers treated with high doses of caffeine, teratogenic changes such as cleft palate, limb malformations, and ectrodactyly [
12-
14] have been observed, as well as reduced bone mass [
15] and reduced bone mineral content [
4]. In the offspring of rats treated with caffeine at doses of 25, 50 and 100 mg/kg, osteopenia in the long bones and vertebrae was observed [
15]. High doses of caffeine inhibit the endochondral bone growth of offspring when administered to rats during pregnancy and lactation. The effects of caffeine are more damaging to cartilage growth in newborns than to pups at weaning [
15]. In humans, caffeine intake above 540 mg/day during pregnancy is associated with decreased weight and fetal growth, and combining caffeine intake with smoking and alcohol consumption may further increase risk [
16,
17].
The mechanism that underlies the changes caused by caffeine, especially in the bones, requires further study. The effects of caffeine on bone metabolism are controversial. Some studies suggest that the consumption of caffeine has been associated with low bone mass and increased fracture risk and that caffeine directly enhances differentiation and maturation of osteoclasts [
18,
19].
Additionally, it has been postulated that one mechanism by which caffeine alters bone formation and growth is by affecting mesenchymal stem cells and osteoblasts [
20,
21].
In vitro studies have concluded that caffeine can potentially harm osteoblasts, causing a reduction in viability and synthetic activity [
20-
23]. The dosage of caffeine can influence the osteogenic differentiation of mesenchymal stem cells [
24]. Low doses of caffeine (0.1 mM) in culture medium can increase the mineralization, alkaline phosphatase activity and differentiation genes such as osteocalcin, osteoprotegerin, and Runx-2, whereas higher doses (≥0.3 mM) can suppress differentiation [
24]. However, little is known about the effects, associated factors and molecular mechanisms of caffeine action during skeletogenesis. Most previous studies that have addressed this issue have investigated the effects of adding caffeine to cultured mesenchymal stem cells and osteoblasts [
20-
23]. This, however, is the first study to examine the osteogenic potential of osteoblasts extracted from neonatal rats born to mothers treated with caffeine throughout pregnancy.
The osteogenic potential of the cells was evaluated using parameters such as alkaline phosphatase activity; collagen synthesis; gene expression of Runx-2, collagenous proteins and non-collagenous proteins (osteocalcin, osteopontin, sialoprotein); and the synthesis and mineralization of the extracellular matrix. Runx-2 regulates collagenous and non-collagenous protein factors that play a key role in the various stages of differentiation in the extracellular matrix [
25-
29], while alkaline phosphatase is an early marker of osteogenic differentiation [
30,
31]; these factors are thus important to evaluate.
Therefore, with the aim of elucidating the mechanism of action of caffeine in osteoblasts, this study analyzed the effects of caffeine on the synthetic activity of cells extracted from the calvaria of newborn rats whose mothers received different doses of caffeine during pregnancy.
Discussion
Previous studies with cultured osteoblasts showed that caffeine can be harmful [
20-
23] and may be considered a risk factor for osteoporosis [
18,
29] and a causative agent of fetal malformations [
5,
12,
14,
15]. The mechanisms that underlie these changes in bone tissue remain poorly understood [
5,
12,
15,
38-
40]. To the best of our knowledge, this work describes for the first time the effects of caffeine administration during pregnancy on the osteogenic potential of osteoblasts derived from pups.
The majority of cells extracted from calvaria are osteoblasts, although the derived cell population also includes pre-osteoblasts [
41]. In the present study, these cells were evaluated after passaging three times in osteogenic medium to stimulate the differentiation of all extracted cells. To characterize and compare the osteogenic potential of the osteoblasts extracted from each experimental group, several parameters were evaluated: alkaline phosphatase activity, collagen synthesis, and the synthesis and mineralization of the extracellular matrix. During the process of cell differentiation, osteoblasts produce the bone extracellular matrix, which is composed of collagenous and non-collagenous proteins. Many of these proteins play a key role in the various stages of differentiation and mineralization of the bone matrix; here we highlight osteocalcin, sialoprotein and osteopontin [
20,
41,
42], which were evaluated in this study.
Our results were surprising and interesting. In contrast with previously reported results from
in vitro studies [
20,
21], we found that caffeine, predominantly at 50 mg/kg, increased the osteogenic potential of osteoblasts, as characterized by increased alkaline phosphatase activity, collagen synthesis, mineralization of nodules and expression of osteogenic genes including osteocalcin, osteopontin, Runx-2, sialoprotein, alkaline phosphatase and type I collagen.
The methodology used and the results obtained in this study were different from those reported by other researchers; previous studies showed that the viability and activity of osteoblasts decreased significantly when exposed to increasing doses of caffeine (0.4, 0, 5, 1.0 and 10 mM) in the culture medium [
20-
23]. The most important aspect of our method of caffeine administration is that the drug was not added to the culture medium as in most of the previous studies; we administered caffeine to mothers during pregnancy, and the drug passed through the placenta to the fetus. The doses of caffeine used in this study were chosen based on previously observed effects on endochondral ossification in the offspring of rats treated with caffeine [
15]. When added directly to the culture medium, caffeine inactivates cell survival signaling and promotes programmed cell death by a mitochondria-dependent cascade; cell death thus occurs by apoptosis and necrosis [
20-
23]. Furthermore, there is decreased expression of genes, enzymes and proteins expressed during osteogenesis, such as Runx-2, alkaline phosphatase, type I collagen, osteocalcin, osteopontin, and histones [
21-
23,
43]. The results obtained with the addition of caffeine to osteoblast cultures demonstrate that in addition to reducing cell viability, caffeine also inhibits the synthesis of the extracellular matrix [
22,
23].
However,
in vitro assays may be different from
in vivo and
ex vivo assays because the cellular microenvironment of the organism is difficult to reproduce
in vitro due to numerous interdependent intrinsic and extrinsic factors. The control of proliferation, differentiation and cell maintenance is carried out by genes, cytokines, developmental and growth factors and cellular interactions [
44-
46]. Thus, even with our knowledge of the regulation of the cellular microenvironment, it is difficult to develop
in vitro models that can simulate drug effects in the body [
44-
46]. Although
in vitro assays with cultured osteoblasts reproduce the cellular sequences that occur before and during the formation of bone matrix
in vivo [
41], the drugs used in in vitro studies are added directly to the culture medium and not metabolized by the body. This lack of drug metabolism may potentiate the effects of drugs on cells [
21,
43]. The aim of this study was to observe the effects of caffeine on the metabolism of osteoblasts in pups whose mothers were exposed to caffeine during pregnancy, taking into consideration the metabolism of the drug by the mother and its passage to the fetus through the placenta.
At 7 and 14 days, alkaline phosphatase activity and collagen synthesis were significantly higher in the group that received 50 mg/kg caffeine than in controls, based on both colorimetric assays and RT-PCR. Alkaline phosphatase is an early marker of osteogenic differentiation [
47,
48]; therefore, the increase in alkaline phosphatase suggests an increase in differentiation and early osteogenesis in the treated group compared with the control group. However, the increase in the gene transcript does not necessarily mean that the protein was also increased. In tests of the conversion of MTT into formazan crystals, osteoblasts from rat neonates that received a dose of 50 mg/kg caffeine showed a lower conversion of MTT into formazan at 7 and 14 days of culture. We cannot infer that these results are due to a reduction in cell viability. We postulate that this observation may be due to an increase in differentiation, as evidenced by collagen synthesis, MTT, and the synthesis of non-collagenous proteins and mineralization nodules, which are discussed below. The MTT assay is a cell viability test; the formation of formazan crystals depends on the mitochondrial activity of viable cells. However, mitochondrial activity also varies with cell maturity, and the decrease in mitochondrial activity during maturation means that differentiated cells frequently show a reduction in formazan crystal formation [
49,
50]. At caffeine doses of 25 mg/kg and 50 mg/kg, the osteoblasts showed decreased conversion of MTT into formazan crystals after 7 and 14 days of culture. At 21 days, the group treated with 100 mg/kg caffeine showed higher conversion of MTT into formazan crystals than the controls. Except for the 50 mg/kg group, it is difficult to explain the changes observed in the MTT assays of the groups that were treated with caffeine. Because the MTT assay is dependent on mitochondrial activity, more studies should be conducted to study the effects of caffeine on these cellular organelles.
Of the three doses of caffeine studied, the 50 mg/kg dose resulted in elevated type I collagen and alkaline phosphatase expression, enhanced synthesis and mineralization of nodules and increased expression of non-collagenous proteins such as osteocalcin, collagen, sialoprotein, Runx-2 and osteopontin compared with the control group at the studied time points. These proteins are important indicators of late osteogenic differentiation [
41,
51-
57]. Therefore, it can be inferred that the osteoblasts cultured from the offspring of rats treated with caffeine at a dose of 50 mg/kg showed increased osteogenic potential in the early and late stages of differentiation. The increased osteogenic potential of osteoblasts in this study should be interpreted with caution, and further studies are needed to understand the relationship between these results and the bone changes observed
in vivo. This increase should not necessarily be interpreted as beneficial for bone growth because any imbalance in cellular function, whether an increase or decrease, may impair skeletogenesis. Caffeine binds to adenosine receptors, and modulate several others receptors including glucocorticoid receptors, insulin, estrogen, androgen, vitamin D, cannabinoid, glutamate and adrenergic receptors, all of which are expressed in osteoblasts or osteoprogenitor cells and have important functions during osteoblast differentiation [
23,
58-
72]. However, little is known about the action of caffeine on these receptors or the resultant effects on endochondral bone formation and growth. Therefore, unfortunately, it is too early to determine the implications for humans or to suggest a mechanism of action for the observed increase in osteogenic factors. Numerous factors in the cellular microenvironment could also be responsible for the observed effects, although more studies are needed to identify these factors and elucidate their mechanisms of action.
Although 25 mg/kg of caffeine also increased the expression of osteocalcin, sialoprotein and osteopontin at two of the time points studied, the synthesis of mineralization nodules was not affected at this dose. This result is likely due to the reduction of collagen expression observed in this group. The formation of mineralization nodules depends on the synthesis of non-collagenous proteins and collagen prior to the mineralization process [
41]. A dose of 100 mg/kg caffeine also promoted changes in the transcript levels of osteocalcin, osteopontin and sialoprotein at 7 days but did not alter the synthesis of mineralization nodules compared with the controls. However, it is important to note that with the exception of the MTT assay at 21 days, the 25 mg/kg and 100 mg/kg doses were similar to the controls in all parameters evaluated.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors contributed to the data collection and manuscript preparation. AMS, LG, AMG, NM and RS contributed to the cell culture and other in vitro assays. AMS, NM and RS also participated in all statistical analyses and interpretation of the data. All authors approved the final version of the manuscript.