Vitamin D and Calcium in Osteoporosis, and the Role of Bone Turnover Markers: A Narrative Review of Recent Data from RCTs

Osteoporosis is a common disease, defined primarily by a low measured bone density, which is associated with an increased risk of fragility fractures. Low calcium intake and vitamin D deficiency seem to be positively correlated with the prevalence of osteoporosis. Although they are not suitable for the diagnosis of osteoporosis, the biochemical markers of bone turnover can be measured in serum and/or urine, enabling the assessment of the dynamic bone activity and the short-term effectiveness of the osteoporosis treatment. Calcium and vitamin D are essential for maintaining bone health. The aim of this narrative review is to summarize the effects of vitamin D and calcium supplementation separately and in combination, on bone density and circulating serum and blood plasma vitamin D, calcium, parathyroid hormone levels, markers of bone metabolism concentrations, and clinical outcomes, such as falls and osteoporotic fractures. We searched the PubMed online database to find clinical trials from the last five years (2016–April 2022). A total of 26 randomized clinical trials (RCTs) were included in this review. The present reviewed evidence suggests that vitamin D alone or in combination with calcium increases circulating 25(OH)D. Calcium with concomitant vitamin D supplementation, but not vitamin D alone, leads to an increase in BMD. In addition, most studies did not detect significant changes in circulating levels of plasma bone metabolism markers, nor in the incidence of falls. Instead, there was a decrease in blood serum PTH levels in the groups receiving vitamin D and/or Ca supplementation. The plasma vitamin D levels at the beginning of the intervention, and the dosing regimen followed, may play a role in the observed parameters. However, further study is needed to determine an appropriate dosing regimen for the treatment of osteoporosis and the role of bone metabolism markers.


Introduction
Osteoporosis is a common condition, defined primarily on the basis of bone density assessment, and it is associated with an increased risk of fragility fractures. There are two types of osteoporosis, depending on the etiology of its development: primary and secondary. Primary osteoporosis is defined as osteoporosis that occurs after menopause (postmenopausal osteoporosis), or with increasing age (senile osteoporosis). Secondary osteoporosis is caused by various disorders and drug side effects [1]. Secondary causes of bone loss, which include adverse effects of drug therapy, endocrine disorders, eating osteoporosis is defined as BMD that is 2.5 standard deviations (SD) or more below the mean value for young, healthy women (T-score < −2.5 SD), while severe osteoporosis is defined as a T-score below −2.5 SD with one or more fragility fractures [15].
In addition, computed tomography (CT) and peripheral quantitative computed tomography (pQCT) are 3D techniques that use X-ray attenuation to construct images of bone [16]. For the measurement of the spine and other areas, a full-size CT scanner is mainly used, while special pQCT scanners are used to measure the radius, tibia, or the anus of the femur [16].

Biomarkers
Although biochemical markers of bone turnover cannot be used to diagnose osteoporosis, they can be measured in serum and/or urine, allowing a dynamic assessment of bone activity and health [17,18]. Moreover, they have been shown to reflect the effectiveness of osteoporosis treatment in a relatively short period of time [19]. In addition, alterations in bone turnover indices may indicate accelerated bone loss or other primary or secondary bone disorders [1]. The available biochemical markers are classified into bone formation markers, which include bone alkaline phosphatase (bALP), osteocalcin (OC), procollagen type I aminotellarpropeptide (P1NP), and bone resorption markers, which include deoxypyridoline (DPD), pyridinoline, N-and C-terminal telopeptides of type I collagen (NTx and CTx, respectively), and which have some prognostic significance for fracture [1,14]. As has already been underlined by the European Food Safety Authority (EFSA) in 2016, more research is needed to delineate the relationship between bone turnover markers and vitamin D status [20]. Moreover, due to the large analytical variation in measuring methods and biological variability, which affects their use in individual patients, they cannot be used for routine clinical evaluations yet [1].
Calcium phosphate homeostasis is mainly regulated by vitamin D, parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) [21]. Vitamin D deficiency results in a reduced absorption of calcium and phosphate from the gut, which then increases the absorption of these ions by the bones, increasing PTH production [22]. An inverse association of circulating PTH levels with calcium intake is generally observed, implying that PTH decreases with increasing dietary calcium intake [13].

Treatment
Several effective treatments, along with pharmacological interventions, can lead to a reduction in fracture risk in postmenopausal women [14]. Diet and physical activity are the primary modifiable factors associated with bone health, although other lifestyle and environmental factors may also play an important role [16]. Optimal vitamin D status promotes skeletal health and is recommended as a specific treatment in people at high risk for fragility fractures [23]. Adequate amounts of calcium are also required for the mineralization of fractures, and the reduced bone recovery often observed in osteoporotic patients can be attributed to calcium and vitamin D deficiencies. Therefore, calcium and vitamin D supplementation is widely recommended for the prevention of osteoporosis and subsequent fractures in osteoporotic patients [7]. More specifically, recommendations for postmenopausal women for osteoporosis management include a daily calcium intake of 800-1200 mg from diet and/or supplements (if dietary intake is insufficient), along with dietary protein ingestion [14]. A daily dose of 800 IU vitamin D is recommended for postmenopausal women who are at an increased risk of fracture, and those having vitamin D insufficiency [14].
However, the recent data are quite contradictory. Some studies showed that calcium supplementation combined with vitamin D reduced the risk of fractures [24][25][26], while others showed no effect [21,[27][28][29][30]. A meta-analysis suggested that concomitant supplementation with calcium and vitamin D led to a significantly reduced risk of total fractures by 15% (summary relative risk estimates, SRRE: 0.85; 95% CI, 0.73-0.98) and a 30% reduced risk of hip fractures (SRRE: 0.70; 95% CI, 0.56-0.87) [31]. In addition, the dosage regimen Diseases 2023, 11, 29 4 of 26 seems to play a role. A meta-analysis concluded that the combination of calcium and vitamin D significantly increased femoral neck BMD only when the dose of vitamin D intake was less than 400 IU/day (standard mean differences, SMD = 0.335; 95% CI: 0.113 to 0.558), compared to higher doses (SMD = −0.098; 95% CI: −0.109 to 0.305), whereas calcium had no effect on femoral neck BMD [12]. In addition, it was observed that only dairy products fortified with calcium and vitamin D, and not the combination of calcium and vitamin D supplements, had a significant effect on total BMD (SMD = 0.784; 95% CI: 0.322 to 1.247) and lumbar spine BMD (SMD = 0.320; 95% CI: 0.146 to 0.494) [12].
The aim of this review was to summarize recent data from the supplementation of vitamin D and calcium as sole compounds or a combination, regarding their effects on bone density, circulating serum and blood plasma levels of vitamin D, calcium, parathyroid hormone, and bone metabolism markers, as well as clinical outcomes, such as falls and OF.

Methods
The The inclusion criteria for studies were (1) that they would be randomized clinical trials (RCTs); (2) that the interventions involved only vitamin D supplementation, calcium supplementation, or a combination of the above; and (3) that they were conducted in an adult population. Studies with only per os supplementation in supplement form were included. More specifically, studies with supplemented/fortified foods were excluded from the present analysis, since foods contain other nutrients that may affect bone density. Studies in people with disorders that may affect bone and calcium metabolism (e.g., chronic kidney disease, HIV, pregnancy, thyroid, glucocorticoid use, and the use of antiepileptic and antidepressant drugs) and those that were not in English were also excluded. In addition, studies in which the intervention and comparison groups did not allow for the assessment of the independent contribution of vitamin D or calcium to outcome were excluded. For example, when these supplements were taken in a multivitamin or were used as part of a multimodal intervention that included other pharmacological agents, or environmental or behavioral interventions.
A search on vitamin D and osteoporosis resulted in 259 studies, 17 of which met the inclusion criteria. A search strategy on calcium and osteoporosis identified 114 studies, but none of them met the inclusion criteria. Finally, from the search strategy on vitamin D, calcium, and osteoporosis, 67 studies were detected, from which 9 were included in this review as they met the inclusion criteria. A total of 26 studies were finally included in this review.

Vitamin D and BMD
Vitamin D supplementation has been generally considered beneficial for the prevention and treatment of osteoporosis. However, the results are controversial. Of the 17 clinical studies, most found no difference [38,40,46], two found a decrease in BMD [13,37], and only two documented an increase in bone density or related variables, such as the bone mineral balance between the supplementation and control group [39,43]. More specifically, in the study of Rangarajan et al., 16 young males (18-35 years) with vitamin D deficiency participated. They were treated with 60,000 IU of vitamin D weekly for 3 years, and the bone mineral changes were assessed with the use of isotopes [39]. The study concluded that the mean increase in the bone mineral balance of subjects receiving the intervention was 0.04 ± 0.05, compared to a net negative value for the control group (−0.03 ± 0.01) [39]. Within the supplemented group, the subgroup with low dietary Ca intake showed a higher Ca isotope enrichment, resulting in a significant change in BMD compared to the control group [39]. A randomized clinical trial (48.5% women) of 8 week supplementation with vitamin D (50,000 IU weekly) in healthy adults of 20-60 years old showed that the changes in T-score in the intervention group were significantly greater than those that were observed in the control group (0.81 vs. 0.30, respectively, p < 0.001) [43]. In addition, the Z-score changes in the intervention group were significantly greater than in the control group [43].
The dosing regimen of vitamin D appears to influence the overall outcome and markers of osteoporosis. In the study of Burt et al., mean serum 25(OH)D measurements did not change for the 400 IU group from baseline to 36 months (76.3-77.4 nmol/L), whereas a significant increase was observed in the 4000 IU group in the first 3 months (81.3-115.3 nmol/L), with a further increase to 132.2 nmol/L by month 36 [41]. An increase was also observed from baseline to 3 and 18 months in the 10000 IU group (78.4-188.0-200.4 nmol/L), but then, 25(OH)D was decreased to 144.4 nmol/L at 36 months [41]. However, such a high dose of vitamin D administration was associated with significantly greater bone loss, possibly due to a reduction in plasma PTH [41].

Vitamin D and PTH
Of the eight studies that also measured plasma PTH levels [18,[33][34][35][38][39][40][41], one detected no significant change in PTH between the supplementation and placebo group [34], while all others observed a decrease in PTH concentration. Indeed, an RCT in 160 postmenopausal women identified a significant decrease in PTH levels by 21.3% in the supplementation group, before and after the intervention (p < 0.001), while a small increase of 8.5% was found in the placebo group before and after the intervention (p = 0.493), with a significant difference between the two groups (p = 0.002) at the end of the study [18]. Similarly, an RCT in men concluded that plasma PTH concentration changed from 64.2 ± 23.9 at baseline, to 43.1 ± 13.7 pg/mL in the intervention group [39]. In addition, based on seasonality, higher levels were observed in the January-March period, compared to the April-June period (p = 0.006) [38].

Vitamin D and Falls
Regarding the incidents of falls, five clinical trials were detected, which included this parameter in the assessment outcomes of vitamin D intervention. All [37,40,41,48] but one [34] reported no significant changes in falls. One study reported a higher incidence of falls in the intervention group [34]. More particularly, in that study, the adjusted risk ratio of the rate of falls and recurrent falls was 1.95-fold (95%CI, 1.23-3.08) and 2.8-fold (95%CI, 1.43-5.50) higher, respectively, in women in the placebo group, compared with the vitamin D supplementation group (p = 0.003) [34].
Bone turnover markers appear to be associated with factors other than vitamin D supplementation. Interestingly, body mass index (BMI) may play a role in the concentration of these markers, since individuals with BMI < 26.4 kg/m2 (n = 100) had significantly higher values of CTX and OC [38]. Furthermore, the levels of vitamin D in the blood serum may also play an important role. For example, in vitamin D deficiency, bone alkaline phosphatase (ALP) is considered as a good index, since its increment is related to rickets [50] and osteomalacia [51]. Among the participants treated with cholecalciferol, subjects who achieved serum 25(OH)D ≥ 75 nmol/L had greater increases in serum β-CTX (224% vs. 146%, p = 0.02) and TRAP5b (22.2% vs. 9.1%, p = 0.007), but smaller decreases in serum calcium (−1.3% vs. −1. 9%; p = 0.005) and calcium-phosphorus product (−2.6% vs. −3.3%; p = 0.02), compared with subjects with serum 25(OH)D < 75 nmol/L [44].

Combined Vitamin D, Ca Supplementation, and BMD
All [21,[24][25][26][27]29,30,52] but one study [28] measured BMD before and after the intervention. Increases in total BMD were observed in several studies [26,27,29,52], which were more significant in high-dose supplemented groups [26,52]. More specifically, 3750 IU vitamin D/day and 1000 mg Ca/day were compared to 600 IU vitamin D/day and 1000 Ca/day, and the high dose was more effective in increasing BMD [26,52]. However, some studies showed no effect of vitamin D and calcium supplementation on total BMD [21,25,30], even in high doses of 4800 IU vitamin D per day and 1200 mg Ca per day [30]. Site-specific BMD was also assessed. Increases were observed in lumbar spine BMD [24][25][26][27], while in some studies, no difference in lumbar spine was documented after vitamin D and Ca intervention [30,52]. Hip BMD was increased in one study [26], and remained unchanged in the rest of the studies [24,30,52]. It is noted that in the study of Rahme et al., there were significant increases in the percentage change in total hip and lumbar spine BMD, but not in the femoral neck at 12 months of intervention within groups, while the percentage change in the subset of BMD increased significantly only in the high-dose group [26]. Interestingly, in the study of Pop et al., a differentiation of results was documented, depending on the analysis followed [28]. Specifically, in the as-treated analysis, no differences were observed between BMD in the vitamin D3-treated groups for femoral neck, rostrum, lumbar spine, total hip, and total-body BMD, in the as-treated analysis [28]. On the other hand, the intention-to-treat (ITT) analysis yielded a statistically significant interaction between treatment and time for cortical thickness (p < 0.03), while there was a loss of total and trabecular IP over time in all groups (p < 0.05) [28]. Both ITT and treatment analyses showed a decrease over time in trabecular volumetric BMD in all women (p < 0.05), but between groups, the changes were not significant [28]. These differences were not associated with higher levels of vitamin D3 intake (2000 and 4000 IU) [28]. Furthermore, no ethnic difference has been found in the percentage change in BMD (African American vs. Caucasian) [30], or in the correlation between the absolute change in serum 1,25(OH)2D level and the change in BMD [30].
The observed differences may be differentiated according to baseline serum 25(OH)D and/or PTH levels. Indeed, there was a significant difference in total body BMD by dose, in subjects with baseline serum 25OHD ≤50 nmol/L, while the percentage change in BMD was not significant [30]. It is noted that in the adjusted analysis for multiple comparisons, the pairwise differences were not significant [30]. Subjects with serum 25(OH)D concentrations of below 20 ng/mL, and a PTH level of above 76 pg/mL showed a trend for a higher percentage change in BMD at 12 months in all skeletal regions in the high-dose group compared to the low dose group, which reached significance only in the total hip at 12 months [26].

Combined Vitamin D and Ca Supplementation, and Circulating 25(OH)D
All RCTs evaluated the change in 25(OH)D in blood serum and documented increases either within the intervention groups, or between the intervention and control groups [21,[24][25][26][27][28][29][30]52]. In a one-year intervention, subjects receiving the highest dose of vitamin D (3750 IU) +1200 mg Ca had a change in serum levels from 20.65 ± 7.89 to 36 ± 9.73 ng/mL compared to subjects supplemented with a low dose of vitamin D (600 IU) + 1200 mg Ca, in which levels changed from 20.06 ± 6.92 ng/mL to 25.96 ± 6.88 ng/mL (p < 0.001) [30]. In addition, free 25(OH)D increased from 5.33 ± 1.87 to 6.86 ± 2.07 ng/L after a low dose, and from 5.38 ± 2.14 to 8.92 ± 3.15 ng/L after high dose supplementation [30]. In a 6-month intervention in two groups (one low dose 400 IU vitamin D +0.5 µg calcitriol+ 1000 mg Ca and one high-dose 1000 IU vitamin D+ 1000 mg Ca) per protocol analysis gave the following results: after 6 months of treatment, serum 25(OH)D levels increased from 11.8 ± 8.8 to 59 ± 35 ng/mL in the cholecalciferol and Ca group, and from 8.7 ± 6.2 to 28 ± 29 ng/mL in the calcitriol, cholecalciferol, and Ca groups, and a significant difference was also observed between the groups (p = 0.05) [25]. In another intervention, only the group receiving inactive vitamin D and the group receiving inactive vitamin D along with exercise had a significant increase at 12 months [24]. In obese patients, the increase in serum 25(OH)D in response to 600, 2000, or 4000 IU vitamin D3/day was 3.8 ± 4.1, 7.4 ± 6.5 and 14.1 ± 8.1 ng/mL, respectively, with the interaction between time and treatment being significant.

Combined Vitamin D and Ca Supplementation, and Circulating Calcium
Differentiated effects were found in serum calcium concentrations in blood serum after the intervention. One RCT identified no significant differences in serum calcium concentrations between groups at baseline or after intervention, and no subjects had hypercalcemia (>10.5 mg/dL) at 12 months [28]. Another study observed a decrease in mean serum calcium concentrations after 16 weeks within the placebo group, but with no differences in mean calcium change compared to the vitamin D group [21]. Feng et al. showed that there was a significant increase in blood calcium levels in all groups [24], while Tenekol et al. documented increases only in the group receiving calcitriol, cholecalciferol, and Ca [25]. Finally, Munshi et al. recorded an increase in serum calcium-although this was not statistically significant-in the supplementation group, while a decrease was observed in the placebo group at the end of 6 and 12 months [27].

Combined Vitamin D, Ca Supplementation, and PTH
Six studies [21,25,26,[28][29][30] assessed PTH levels in blood serum, and all documented a decrease in the intervention group. After 16 weeks of vitamin D supplementation, a decrease in the mean iPTH concentrations was observed within and between the two groups, with the difference only within the treatment group being significant (4.89 ± 1.86 pmol/L vs. 3.71 ± 1.31 pmol/L, p = 0.002) [21]. Similarly, the mean serum PTH level decreased from 131 ± 62 to 71 ± 51 pg/mL in the cholecalciferol and Ca group, and from 235 ± 193 to 58 ± 38 pg/mL in the calcitriol, cholecalciferol, and Ca groups (p = 0.003, between groups) [25]. In addition, serum PTH was significantly reduced only in participants in the supplementation group, and not between the three groups (placebo, dairy, vitamin D, and calcium supplements) [29]. Pop et al., after 12 months of treatment, concluded that PTH decreased by 10.6 ± 16.6% (p < 0.01); however, the change did not differ significantly between groups of different vitamin D dosing (500 IU, 2000 IU, and 4000 IU) [28], while Rahme et al. (2017) recorded a gradual decrease in PTH levels at 6 and 12 months, only in the low supplemental dose group (600 IU) vs. a higher dose (3750 IU) [26]. In terms of ethnic groups, serum PTH decreased as circulating vitamin D was increasing, in both Caucasian and African American women [30]. There was no correlation between baseline serum PTH level and the percentage change in spine BMD, femoral neck BMD, or serum NTx, but there was a significant positive correlation between the PTH level and the percentage change in total body BMD (r = 0.18, p = 0.020) [30].

Combined Vitamin D and Ca Supplementation, and Falls/Fractures
One study recorded a significant increase in OF [26], while the others observed no difference between the groups in the OF [21,24,25,[27][28][29][30]. Only one study with combined vitamin D and Ca supplementation included in the review evaluated falls in participants, but the differences were not significant [24].

Combined Vitamin D and Ca Supplementation, and Bone Turnover Biomarkers
A total of six studies measured the concentration of bone turnover markers in the plasma of the participants [21,[25][26][27][28][29]. Two showed an increase in bone metabolism indicators [25,28], while three observed a decrease in concentration [26,27,29], and one reported non-significant changes [21].
More particularly, OC increased by 43 ± 104% in the high-dose vitamin D group, and by 111 ± 276% in the low dose vitamin D group (p > 0.05 between groups) [25]. In contrast to these findings, Rahme et al. observed a 20% to 22% decrease in the OC and CTx levels in both groups (low and high dose), with no significant differences between the two groups [26]. Decreases in both the OC and serum TRAP-5b levels, with the decline being significantly higher in the treatment group compared to the standard care group, were documented, indicating reduced bone turnover [27]. As far as CTx is concerned, in the study of Ilich et al., it increased in the control group and decreased in the vitamin D+ Ca supplementation group [29], while in the study of Tanakol, both CTx and NTx increased [25]. PGF-23 was measured in only one study, and no differences were recorded after vitamin D + Ca supplementation [21].

Discussion
A total of 26 randomized clinical trials were included in this review to summarize the recent data from vitamin D and calcium supplementation, both solely and in combination, and to study their effects on bone density, circulating vitamin D, calcium, parathyroid hormone, markers of bone turnover, and clinical outcomes, i.e., falls and osteoporotic fractures.
The ingested vitamin D is hydroxylated in the liver and kidneys, and is converted to its active form, i.e., 1,25-dihydroxyvitamin D (1,25(OH)2D) [53]. This active circulating form of the vitamin binds to vitamin D receptor (VDR), exerts several actions, and regulates gene expression [53]. It increases intestinal calcium and phosphorus absorption, it stimulates calcium renal reabsorption, and it interacts with PTH and activates osteoclasts that are responsible for bone resorption [20]. In this context, vitamin D promotes calcium absorption and helps to maintain adequate serum calcium concentrations to enable the normal mineralization of the bone. In parallel, vitamin D is needed for bone growth and bone remodeling by osteoblasts and osteoclasts [54].
Consistently with clinical studies on bone density, only few studies detected significant differences in BMD after sole vitamin D supplementation [26,39,43], which is in line with a meta-analysis regarding vitamin D effects [55]. The co-administration of calcium and vitamin D led to increases in total BMD in most studies [26,27,29,52], which were more significant in high-dose supplemented groups [26,52], while in others, it had no effect [21,25,30]. A meta-analysis of 23 trials prescribing vitamin D plus calcium (19 double-blinded trials, and 18 placebo controlled trials) with a mean duration of 23.5 months, including 4083 participants (92% women, mean age 59 years) with variable ingested vitamin D doses (<800 IU/day in 10 trials), reported a small increase in BMD at the femoral neck, but not at other sites, that the authors attributed to chance [47]. A high dose-and long-term supplementation of vitamin D may be also associated with bone loss, probably due to a decrease in plasma PTH [41], which is observed in both men [39] and women [18]. Changes in PTH with vitamin D supplementation are likely to directly affect parathyroid cells through an effect on osteoclast (CTx) activity leading to a dose-dependent accelerated decrease in bone formation and observed volumetric BMD [33,41].
Moreover, several factors may affect the effectiveness of vitamin D intervention, such as sex, baseline BMD, baseline vitamin D status, and the duration of the treatment. For example, Burt et al. reported that during the menopausal and postmenopausal period, a high dose of sole vitamin D supplementation is required in women closer to age 55 and after age 65, due to greater bone loss and accelerated bone loss, respectively [41]. A clinical trial at the bone density clinic at Kowsar Hospital in Semnan showed that the prevalence of vitamin D deficiency in osteopenic and osteoporotic patients was higher than in normal subjects, an observation which was associated with age and sex [43]. Furthermore, it was shown that the incidence of osteoporosis decreased through vitamin D treatment [43]. In these studies, the duration of the treatment was~3 years [26,39,43], while studies showing no effect had a duration of~1 y or less [36][37][38]40] suggesting a significant role of treatment duration to achieve a beneficial effect on BMD. However, a longer duration of treatment in healthy subjects may not have a positive change on BMD [48], while lessened or null effects have been observed in men [38]. Serum 25(OH)D has been positively associated with BMD [56], but the effects of baseline 25(OH)D on the effectiveness of supplementation are less clear. Some studies show no effect of baseline serum 25(OH)D on the outcomes of supplementation [30], while a potential effect in total hip BMD may be present, if low serum 25(OH)D is combined with high PTH (>76 pg/mL) [26]. Moreover, a beneficial effect was observed in subjects with low baseline 25 OHD levels, and at doses < 800, as compared to > 800 IU/day [47].
All [13,18,33,34,[36][37][38][39][40]46,48] but one [35] study included in this narrative review showed that circulating 25(OH)D levels increased after sole vitamin D supplementation, even in cases of low baseline 25(OH)D concentration [34]. Similarly, all included RCTs reported an increase in circulating 25(OH)D after supplementation with both vitamin D and calcium [21,[24][25][26]28,29,52], although according to a meta-analyses, circulating 1,25(OH)2D may be suppressed through calcium co-administration (vitamin D alone increased circulating 25(OH)D by 18.6 pmol/L; 95% CI = 12.7-24.4 pmol/L, while vitamin D plus calcium increased circulating 25(OH)D by 4.9 pmol/L; 95% CI = 0.4 −10.2 pmol/L; p 0.001) [57]. In addition, a dose-dependent and significant increase in serum 25(OH)D was observed after the intervention with sole vitamin D [21,24,25], or its combination with calcium [41]. Our results are in line with the findings of a meta-analysis in overweight and obese adults that concluded that a daily dose of 1000 IU vitamin D supplement can increase serum 25(OH)D levels and reduce PTH levels [58]. Along with the interpretation of the results regarding circulating vitamin D, it should be also noted that higher plasma 25(OH)D levels were observed during the period of July-September, compared to the period of January-March [38].
Differentiated results were present in the case of circulating calcium pre-and postsupplementation, since some studies reported a non-significant effect [21,27,28] or an increase [24] after the intervention with vitamin D and calcium, or with a combination of calcitriol, vitamin D, and calcium [25].
All studies that measured serum PTH pre-and post-intervention with vitamin D reported a significant decrease in supplementation groups [21,25,26,[28][29][30]. The percentage of PTH decrease was dependent on the duration, dose, and ethnicity [26,30]. Indeed, in Caucasian and African American women, serum PTH decreased as circulating 25(OH)D increased over the range of serum 25(OH)D 50-175 nmol/L, suggesting a vitamin D-PTH interaction [59,60]. Indeed, vitamin D reduces PTH gene expression, and it has an inhibitory effect on the proliferation of parathyroid cells [20]. However, the combined effects of calcium and vitamin D supplementation on PTH are less clear-cut. Evidence from RCTs suggests that bone benefits are evident with vitamin D supplementation at adequate calcium intakes and at normal levels of PTH [61]. In a previous review, vitamin D was found to lower PTH, but this effect was not apparent in the presence of calcium supplementation [62]. Interestingly, Lerchbaum et al. (2019), reported that vitamin D supplementation reduced PTH levels only in normal-weight men, whereas in men with higher BMIs, there was no significant effect [38]. It was thus hypothesized that overweight/obese subjects might require higher vitamin D doses in order for beneficial effects to be exerted on PTH levels [38]. It is well known that BMI has a unidirectional negative effect on vitamin D metabolism. Fillipo et al. has also previously described the possible intrinsic beneficial metabolic effects of vitamin D itself in overweight and obese patients, which influence adiposity and metabolic outcomes [63].
It is not clear whether the improvement in circulating vitamin D through supplementation can reduce the frequency of falls and fractures. Indeed, most studies reported no significant effects of vitamin D [37,40,41,48] or combinations of vitamin D with calcium [24] on falls. Other studies have reported that vitamin D and calcium supplementation (not only calcium) leads to an increase in BMD and reduces the risk of OF [12,31,64,65], while others have shown no effect, supporting our results [66,67]. Our results are similar to a meta-analysis that concluded that vitamin D had no effect on the total fractures, hip fractures, or falls [55], while several meta-analysis have shown that the combination of calcium and vitamin D significantly reduced the fractures risk [12,65,68]. This reduction was more pronounced among women with low plasma 25(OH)D concentrations and calcium co-administration [34]. Interestingly, it has been noted that an intermittent high dose supplementation of vitamin D was associated with an increased risk of falls and fractures [69]. The exoskeletal effects of vitamin D are strongly correlated with the development of sarcopenia disease [63]. Moreover, it is possible that the total diet and/or dietary protein may be important in addressing this issue, since sarcopenia often co-exists with osteoporosis (osteosarcopenia) [70]. In this case, animal protein may be a key factor for modifying body composition and sarcopenia, such as in the case of cancer, which is also related to sarcopenia [71,72]. In addition, inflammatory indices, such as platelet activating factor, are related to OF [73], and an antioxidant-rich diet and/or fatty acid profile may lessen its deleterious actions [74,75].
Regarding the effects of vitamin D supplementation on bone turnover markers, such as CTx, bALP, OC, TRAP-5b, and P1NP, most studies included in the present review found a no significant effect of vitamin D [18,35,42,44,46,48,49]. Interventions with supplements of vitamin D and calcium have shown increases [25,28], decreases [26,27,29], or no significant changes [21]. A significant decrease in the serum levels of bone turnover markers may possibly result after large doses of calcium (1000 mg elemental) taken in combination with vitamin D [26], or in a combination of calcium and vitamin D supplements with other substances, such as collagen peptides [76]. The effects of vitamin D on bone turnover markers may be limited to subjects with low 25(OH)D levels [77],while no effects on CTx and OC in men with low 25(OHD levels (<50 nmol/L, n = 85) were documented [38]. The effects of vitamin D on bone turnover may also depend on the body's calcium status, since in cases of hypocalcemia, active vitamin D may also activate osteoclasts to achieve calcium homeostasis [54]. In general, these small effects of vitamin D supplementation on bone turnover biomarkers are in line with previous studies performed in hypertensive patients [49], healthy postmenopausal women [33], and healthy young and elderly adults [78], as well as healthy obese men and women [79]. Moreover, several inter-correlations between them may affect the observed results [49,80,81], while glucose and HbA1c may also affect their levels [82,83].
Besides environmental factors, the genetic background may also influence the effects of supplementation [71]. For example, 35 genes and several single-nucleotide polymorphisms (SNPs) have been connected to vitamin D status, which could potentially alter individual 25(OH)D responses to supplementation [84]. In parallel, dietary habits may influence vitamin D status [71,85],while gene-diet interactions may also take place [86], although they may not at last largely affect BMD [87,88]. In addition, concomitant calcium supplementation may reduce compliance with vitamin D, due to calcium-induced digestive problems, which probably supported the decision in some trials to try vitamin D supplementation alone [89]. Increased vitamin D doses may also be related to hypercalcemia and hypercalciuria [41,90], with the risk being higher in subjects with calcium co-administration [32]. However, not all studies documented a high risk of hypercalcemia [36,40].
Limitations of several studies were the short duration, the low sample size, [38,40,46], and no placebo comparator during the study period [40], while other factors such as exercise, muscle, and weight loss may affect BMD more than vitamin D alone [46,70]. Gender bias may be also inherent, since most studies have been conducted in women, and the age distribution is quite narrow and shifted to older ages [91]. Moreover, no much data on ethnic-related differences are reported [91]. Last but not least, several vitamin D-drug interactions should be taken into account. Statins increase vitamin D [92] and anti-epileptic treatment, and are often used as co-analgesic treatment in the elderly [93], and they induce vitamin D catabolism [94]. In real life, other medicines for osteoporosis may be prescribed [14], and potential additional diet-drug interactions should be taken into account.

Conclusions
Osteoporosis is a systemic skeletal disorder with significant negative effects on the general health and quality of life after menopause and in old age. In addition to specific pharmacological and/or hormonal treatments, supplementation strategies seem to be very important, and they are generally recommended [14]. Vitamin D supplementation, alone or in combination with Ca, is considered as being fundamental to enhancing the positive effects of any particular therapy in patients, who are more fragile and at higher risk of vertebral and non-vertebral fragility fractures due to disorders related to bone metabolism (e.g., osteoporosis or vitamin D deficiency). The present reviewed evidence suggests that vitamin D alone or in combination with calcium increases circulating 25(OH)D. Calcium with concomitant vitamin D supplementation, but not vitamin D alone, leads to an increase in BMD, while no significant differences were documented for the reduction in the risk of total fractures. Figure 1 presents a summary of the conclusions of the review. Further research is needed to investigate the possible role of vitamin D and calcium on bone turnover markers.
supplementation alone [89]. Increased vitamin D doses may also be related to hypercalcemia and hypercalciuria [41,90], with the risk being higher in subjects with calcium coadministration [32]. However, not all studies documented a high risk of hypercalcemia [36,40].
Limitations of several studies were the short duration, the low sample size, [38,40,46], and no placebo comparator during the study period [40], while other factors such as exercise, muscle, and weight loss may affect BMD more than vitamin D alone [46,70]. Gender bias may be also inherent, since most studies have been conducted in women, and the age distribution is quite narrow and shifted to older ages [91]. Moreover, no much data on ethnic-related differences are reported [91]. Last but not least, several vitamin D-drug interactions should be taken into account. Statins increase vitamin D [92] and anti-epileptic treatment, and are often used as co-analgesic treatment in the elderly [93], and they induce vitamin D catabolism [94]. In real life, other medicines for osteoporosis may be prescribed [14], and potential additional diet-drug interactions should be taken into account.

Conclusions
Osteoporosis is a systemic skeletal disorder with significant negative effects on the general health and quality of life after menopause and in old age. In addition to specific pharmacological and/or hormonal treatments, supplementation strategies seem to be very important, and they are generally recommended [14]. Vitamin D supplementation, alone or in combination with Ca, is considered as being fundamental to enhancing the positive effects of any particular therapy in patients, who are more fragile and at higher risk of vertebral and non-vertebral fragility fractures due to disorders related to bone metabolism (e.g., osteoporosis or vitamin D deficiency). The present reviewed evidence suggests that vitamin D alone or in combination with calcium increases circulating 25(OH)D. Calcium with concomitant vitamin D supplementation, but not vitamin D alone, leads to an increase in BMD, while no significant differences were documented for the reduction in the risk of total fractures. Figure 1 presents a summary of the conclusions of the review. Further research is needed to investigate the possible role of vitamin D and calcium on bone turnover markers.  Author Contributions: Conceptualization, G.V., S.K.P. and A.P.; methodology, G.V. and P.D.; writingoriginal draft preparation: G.V., S.K.P., P.D., F.S.K., C.G. and E.L.; writing-review and editing, G.V., S.K.P., P.D., D.T., C.G., E.L. and A.P.; supervision, A.P. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.