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Efficacy of Vitamin D3 Buccal Spray Supplementation Compared to Other Delivery Methods: A Systematic Review of Superiority Randomized Controlled Trials

(1) Background: Vitamin D deficiency is an important public health concern and supplementation is common for this deficiency. Many different modes of delivering supplementation have been proposed in order to enhance absorption and utilization. The present review compared the efficacy of vitamin D3 buccal spray against other forms of supplementation delivery. (2) Methods: The protocol was registered at PROSPERO (CRD42019136146). Medline/PubMed, CENTRAL and clinicaltrials.gov were searched from their inception until September 2019, for randomized controlled trials (RCTs) that compare vitamin D3 delivery via sublingual spray against other delivery methods. Eligible RCTs involved humans, of any age and health status, published in any language that evaluated changes in plasma 25(OH)D concentrations. Three reviewers independently extracted data, assessed risk of bias (RoB) and the quality of the trials. (3) Results: Out of 9759 RCTs, four matched the predefined criteria. Intervention duration ranged from 30 days to 3 months whereas vitamin D3 dosage ranged between 800 and 3000 IU/day. One RCT advocated for the superiority of buccal spray in increasing plasma 25(OH)D concentrations, although several limitations were recorded in that trial. The rest failed to report differences in post-intervention 25(OH)D concentrations between delivery methods. Considerable clinical heterogeneity was observed due to study design, intervention duration and dosage, assays and labs used to perform the assays, population age and health status, not allowing for synthesis of the results. (4) Conclusions: Based on the available evidence, delivery of vitamin D3 via buccal spray does not appear superior to the other modes of delivery. Future RCTs avoiding the existing methodological shortcomings are warranted.


Introduction
Vitamin D 3 is an essential fat-soluble nutrient involved in a plethora of metabolic pathways [1][2][3][4][5]. When consumed within the dietary reference level limits, vitamin D exerts multiple health

Selection of Studies and Interventions of Interest
Initially, three independent reviewers (M.G.G., K.G. and M.P.N.) identified studies from their titles and abstracts. Full-text articles were retrieved to assist decision-making in cases when deemed necessary. Any disagreement between reviewers was resolved by a senior researcher (D.G.G.).
Data were extracted using a predefined Microsoft Excel data extraction form, including study (design, funding, allocation concealment, protocol registry, country, recruitment site) and participant characteristics (age, health conditions, discontinued/dropouts), intervention details (form, duration, dosage, adverse events), comparators, and clinical outcomes to produce an overview table of all eligible studies.
Characteristics of the retrieved RCTs were evaluated with the Cochrane risk of bias (RoB) 2.0 tool [54] by two reviewers (M.P.N. and M.G.G.) independently, in order to present bias comprehensively. A more experienced author (D.P.B.) assessed between-reviewer differences. The RoB results classified studies as being of "high", "unclear" or "low" risk of bias. Additionally, the Oxford quality scoring system (Jadad score) [55] was applied on each RCT to assess trial quality.

Study Selection
A total of 9759 studies were screened by title and abstract and 13 were assessed for eligibility criteria (full-text screening), out of which nine were excluded for having a different mode of supplementation delivery, comparing against placebo, or lacking a RCT design. The PRISMA flowchart [39] was applied to illustrate the step-by-step exclusion of unrelated/duplicate retrieved records, leading to the final selection of four RCTs that met the predefined inclusion criteria ( Figure 2).  [56] of the studies selection process. Figure 2. PRISMA flowchart [56] of the studies selection process.       ; ‡ Total n was 20 in each group, but the second comparator (placebo) was omitted from the present analyses; ¥ The second comparator (placebo), was omitted from the present analyses (n = 25); § At initial allocation, as this was a cross-over study;ˆThe mmol/l reported appears to be a typo and should probably be nmol/L. Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D 3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a design, and the remaining two [58,59] used parallel interventions. One RCT was multicent single-blinded. The rest were single-center, two of which used open-label [57,58] and double-blind masking [59]. Intervention duration ranged between 30 days to 3 month mainly performed during winter time. Only one RCT [59] evaluated participants' skin to the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as p are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Pe recruited children with neuro-disabilities. On the other hand, Satia [51] included two arms, one consisting of healthy subjects, and the other comprising patients with mal syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these compari omitted from the present analyses for not fulfilling the "superiority" comparison criterion

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58 assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomiz funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed tha [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT by Penagini [58] demonstrated the most quality issues, including bias in the randomizatio deviations from the intended interventions, overall bias and unclear risk outcomes measur selective reporting. In contrast, the study conducted by Williams [59] received the high score among all of the studies. Additionally, Todd [57] failed to separate the intention-to the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the C [60] components, including a flow diagram or details concerning dropouts and the participants at each stage. None of the RCTs reported any post-intervention adverse ev Williams et al. [59] who reported small blisters on the cheek and tongue of two participan Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal sp the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining t [57][58][59] did not report any difference between intervention and comparator groups, and in similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW Table 2 summarizes the characteristics of the retrieved RCTs. Tw design, and the remaining two [58,59] used parallel interventions. One single-blinded. The rest were single-center, two of which used op double-blind masking [59]. Intervention duration ranged between 3 mainly performed during winter time. Only one RCT [59] evaluated the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU are concerned, Satia [51], Todd [57] and Williams [59] used adult sa recruited children with neuro-disabilities. On the other hand, Satia arms, one consisting of healthy subjects, and the other comprising syndrome. The Todd [57] trial was restricted to the recruitment of hea Satia [51] and Williams [59] also compared against a placebo omitted from the present analyses for not fulfilling the "superiority" c

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table  assessed as having a high-risk of overall bias, for lacking a predefined funding disclosure. Williams and associates [59] also conducted a trial the predefined intervention duration was not kept. The RCT by Satia substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (T [51] and Todd [57] trials exhibited several bias-related issues. On the o by Penagini [58] demonstrated the most quality issues, including bias deviations from the intended interventions, overall bias and unclear ri selective reporting. In contrast, the study conducted by Williams [59 score among all of the studies. Additionally, Todd [57] failed to sepa the per-protocol analyses, whereas Penagini [58] and Williams [59] l [60] components, including a flow diagram or details concerning participants at each stage. None of the RCTs reported any post-inter Williams et al. [59] who reported small blisters on the cheek and tong Satia [51] was the only one who advocated for the superiority of v the other modes of delivery in increasing plasma 25(OH)D concentrati [57][58][59] did not report any difference between intervention and compa similarity and equal efficacy between different modes of vitamin D3 d Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Todd [57] Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a design, and the remaining two [58,59] used parallel interventions. One RCT was multicent single-blinded. The rest were single-center, two of which used open-label [57,58] and double-blind masking [59]. Intervention duration ranged between 30 days to 3 month mainly performed during winter time. Only one RCT [59] evaluated participants' skin to the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as p are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Pe recruited children with neuro-disabilities. On the other hand, Satia [51] included two arms, one consisting of healthy subjects, and the other comprising patients with mal syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these compari omitted from the present analyses for not fulfilling the "superiority" comparison criterion

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58 assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomiz funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed tha [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT by Penagini [58] demonstrated the most quality issues, including bias in the randomizatio deviations from the intended interventions, overall bias and unclear risk outcomes measur selective reporting. In contrast, the study conducted by Williams [59] received the high score among all of the studies. Additionally, Todd [57] failed to separate the intention-to the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the C [60] components, including a flow diagram or details concerning dropouts and the participants at each stage. None of the RCTs reported any post-intervention adverse ev Williams et al. [59] who reported small blisters on the cheek and tongue of two participan Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal sp the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining t [57][58][59] did not report any difference between intervention and comparator groups, and in similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW Table 2 summarizes the characteristics of the retrieved RCTs. Tw design, and the remaining two [58,59] used parallel interventions. One single-blinded. The rest were single-center, two of which used op double-blind masking [59]. Intervention duration ranged between 3 mainly performed during winter time. Only one RCT [59] evaluated the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU are concerned, Satia [51], Todd [57] and Williams [59] used adult sa recruited children with neuro-disabilities. On the other hand, Satia arms, one consisting of healthy subjects, and the other comprising syndrome. The Todd [57] trial was restricted to the recruitment of hea Satia [51] and Williams [59] also compared against a placebo omitted from the present analyses for not fulfilling the "superiority" c

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table  assessed as having a high-risk of overall bias, for lacking a predefined funding disclosure. Williams and associates [59] also conducted a trial the predefined intervention duration was not kept. The RCT by Satia substantial deviations from the reported intended interventions.  [58] demonstrated the most quality issues, including bias deviations from the intended interventions, overall bias and unclear ri selective reporting. In contrast, the study conducted by Williams [59 score among all of the studies. Additionally, Todd [57] failed to sepa the per-protocol analyses, whereas Penagini [58] and Williams [59] l [60] components, including a flow diagram or details concerning participants at each stage. None of the RCTs reported any post-inter Williams et al. [59] who reported small blisters on the cheek and tong Satia [51] was the only one who advocated for the superiority of v the other modes of delivery in increasing plasma 25(OH)D concentrati [57][58][59] did not report any difference between intervention and compa similarity and equal efficacy between different modes of vitamin D3 d Nutrients 2020, 12, x FOR PEER REVIEW Table 2 summarizes the characteristics of the retr design, and the remaining two [58,59] used parallel int single-blinded. The rest were single-center, two of double-blind masking [59]. Intervention duration ra mainly performed during winter time. Only one RCT the study. Vitamin D3 dosage ranged from 800 [58] to are concerned, Satia [51], Todd [57] and Williams [5 recruited children with neuro-disabilities. On the ot arms, one consisting of healthy subjects, and the ot syndrome. The Todd [57] trial was restricted to the rec Satia [51] and Williams [59] also compared ag omitted from the present analyses for not fulfilling the

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illust assessed as having a high-risk of overall bias, for lack funding disclosure. Williams and associates [59] also c the predefined intervention duration was not kept. Th substantial deviations from the reported intended inte  [59] Quality assessment of the RCTs based on the Jad [51] and Todd [57] trials exhibited several bias-related by Penagini [58] demonstrated the most quality issues deviations from the intended interventions, overall bia selective reporting. In contrast, the study conducted score among all of the studies. Additionally, Todd [57 the per-protocol analyses, whereas Penagini [58] and [60] components, including a flow diagram or deta participants at each stage. None of the RCTs reporte Williams et al. [59] who reported small blisters on the Satia [51] was the only one who advocated for the the other modes of delivery in increasing plasma 25(OH [57][58][59] did not report any difference between interven similarity and equal efficacy between different modes

Penagini [58]
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW Table 2 summarizes the characteristics of the retrieved RCTs. Two t design, and the remaining two [58,59] used parallel interventions. One RC single-blinded. The rest were single-center, two of which used opendouble-blind masking [59]. Intervention duration ranged between 30 mainly performed during winter time. Only one RCT [59] evaluated pa the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU pe are concerned, Satia [51], Todd [57] and Williams [59] used adult sam recruited children with neuro-disabilities. On the other hand, Satia [51 arms, one consisting of healthy subjects, and the other comprising p syndrome. The Todd [57] trial was restricted to the recruitment of health Satia [51] and Williams [59] also compared against a placebo, bu omitted from the present analyses for not fulfilling the "superiority" com

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. assessed as having a high-risk of overall bias, for lacking a predefined p funding disclosure. Williams and associates [59] also conducted a trial of the predefined intervention duration was not kept. The RCT by Satia [5 substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Tab [51] and Todd [57] trials exhibited several bias-related issues. On the othe by Penagini [58] demonstrated the most quality issues, including bias in deviations from the intended interventions, overall bias and unclear risk o selective reporting. In contrast, the study conducted by Williams [59] r score among all of the studies. Additionally, Todd [57] failed to separate the per-protocol analyses, whereas Penagini [58] and Williams [59] lack [60] components, including a flow diagram or details concerning dro participants at each stage. None of the RCTs reported any post-interve Williams et al. [59] who reported small blisters on the cheek and tongue Satia [51] was the only one who advocated for the superiority of vita the other modes of delivery in increasing plasma 25(OH)D concentrations [57][58][59] did not report any difference between intervention and comparat similarity and equal efficacy between different modes of vitamin D3 deliv

Williams [59]
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions. Table 3. Summary risk of bias [54] assessment of the included randomized controlled trials.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.

Deviations from intended interventions
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery. marizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover emaining two [58,59] used parallel interventions. One RCT was multicenter [51] and The rest were single-center, two of which used open-label [57,58] and one used sking [59]. Intervention duration ranged between 30 days to 3 months and was d during winter time. Only one RCT [59] evaluated participants' skin tone during in D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants atia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] n with neuro-disabilities. On the other hand, Satia [51] included two participant sting of healthy subjects, and the other comprising patients with malabsorption odd [57] trial was restricted to the recruitment of healthy adults. nd Williams [59] also compared against a placebo, but these comparisons were present analyses for not fulfilling the "superiority" comparison criterion.
nd Quality Assessment of Studies bias of the included studies is illustrated in Table 3. The Penagini [58] trial was ng a high-risk of overall bias, for lacking a predefined protocol, randomization, and re. Williams and associates [59] also conducted a trial of high overall bias, given that ntervention duration was not kept. The RCT by Satia [51] was of unclear bias, with tions from the reported intended interventions.
Summary risk of bias [54] assessment of the included randomized controlled trials.

Deviations from intended interventions
Missing outcome data

Selection of the reported result
Overall bias essment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia 7] trials exhibited several bias-related issues. On the other hand, the RCT performed demonstrated the most quality issues, including bias in the randomization process, the intended interventions, overall bias and unclear risk outcomes measurement and ng. In contrast, the study conducted by Williams [59] received the highest quality of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT s, including a flow diagram or details concerning dropouts and the number of ach stage. None of the RCTs reported any post-intervention adverse event, except 9] who reported small blisters on the cheek and tongue of two participants. as the only one who advocated for the superiority of vitamin D3 buccal spray against of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs eport any difference between intervention and comparator groups, and indicated the ual efficacy between different modes of vitamin D3 delivery. Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.  Table 2 summarizes the characteristics of the retrieved RCTs. Two trials [51,57] had a crossover design, and the remaining two [58,59] used parallel interventions. One RCT was multicenter [51] and single-blinded. The rest were single-center, two of which used open-label [57,58] and one used double-blind masking [59]. Intervention duration ranged between 30 days to 3 months and was mainly performed during winter time. Only one RCT [59] evaluated participants' skin tone during the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU per day. As far as participants are concerned, Satia [51], Todd [57] and Williams [59] used adult samples, whereas Penagini [58] recruited children with neuro-disabilities. On the other hand, Satia [51] included two participant arms, one consisting of healthy subjects, and the other comprising patients with malabsorption syndrome. The Todd [57] trial was restricted to the recruitment of healthy adults.
Satia [51] and Williams [59] also compared against a placebo, but these comparisons were omitted from the present analyses for not fulfilling the "superiority" comparison criterion.

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. The Penagini [58] trial was assessed as having a high-risk of overall bias, for lacking a predefined protocol, randomization, and funding disclosure. Williams and associates [59] also conducted a trial of high overall bias, given that the predefined intervention duration was not kept. The RCT by Satia [51] was of unclear bias, with substantial deviations from the reported intended interventions.  [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D3 delivery.
Nutrients 2020, 12, x FOR PEER REVIEW Table 2 summarizes the characteristics of the retrieved RCTs. Two t design, and the remaining two [58,59] used parallel interventions. One RC single-blinded. The rest were single-center, two of which used opendouble-blind masking [59]. Intervention duration ranged between 30 mainly performed during winter time. Only one RCT [59] evaluated pa the study. Vitamin D3 dosage ranged from 800 [58] to 3000 [57,59] IU pe are concerned, Satia [51], Todd [57] and Williams [59] used adult sam recruited children with neuro-disabilities. On the other hand, Satia [51 arms, one consisting of healthy subjects, and the other comprising p syndrome. The Todd [57] trial was restricted to the recruitment of health Satia [51] and Williams [59] also compared against a placebo, bu omitted from the present analyses for not fulfilling the "superiority" com

Risk of Bias and Quality Assessment of Studies
The risk of bias of the included studies is illustrated in Table 3. assessed as having a high-risk of overall bias, for lacking a predefined p funding disclosure. Williams and associates [59] also conducted a trial of the predefined intervention duration was not kept. The RCT by Satia [5 substantial deviations from the reported intended interventions.  [59] Quality assessment of the RCTs based on the Jadad [55] scale (Tab [51] and Todd [57] trials exhibited several bias-related issues. On the othe by Penagini [58] demonstrated the most quality issues, including bias in deviations from the intended interventions, overall bias and unclear risk o selective reporting. In contrast, the study conducted by Williams [59] r score among all of the studies. Additionally, Todd [57] failed to separate the per-protocol analyses, whereas Penagini [58] and Williams [59] lack [60] components, including a flow diagram or details concerning dro participants at each stage. None of the RCTs reported any post-interve Williams et al. [59] who reported small blisters on the cheek and tongue Satia [51] was the only one who advocated for the superiority of vita the other modes of delivery in increasing plasma 25(OH)D concentrations [57][58][59] did not report any difference between intervention and comparat similarity and equal efficacy between different modes of vitamin D3 deliv Quality assessment of the RCTs based on the Jadad [55] scale (Table 2) revealed that the Satia [51] and Todd [57] trials exhibited several bias-related issues. On the other hand, the RCT performed by Penagini [58] demonstrated the most quality issues, including bias in the randomization process, deviations from the intended interventions, overall bias and unclear risk outcomes measurement and selective reporting. In contrast, the study conducted by Williams [59] received the highest quality score among all of the studies. Additionally, Todd [57] failed to separate the intention-to-treat from the per-protocol analyses, whereas Penagini [58] and Williams [59] lacked many of the CONSORT [60] components, including a flow diagram or details concerning dropouts and the number of participants at each stage. None of the RCTs reported any post-intervention adverse event, except Williams et al. [59] who reported small blisters on the cheek and tongue of two participants.
Satia [51] was the only one who advocated for the superiority of vitamin D 3 buccal spray against the other modes of delivery in increasing plasma 25(OH)D concentrations. The remaining three RCTs [57][58][59] did not report any difference between intervention and comparator groups, and indicated the similarity and equal efficacy between different modes of vitamin D 3 delivery.

Discussion
Although a variety of delivery methods exist for most dietary supplements, systematic reviews and meta-analyses on the efficacy of each mode are lacking. The present systematic review indicates that vitamin D 3 delivery via buccal spray does not differ from other supplementation methods in increasing plasma 25(OH)D levels. In parallel, the small number of retrieved RCTs and the high degree of clinical heterogeneity among them did not allow for a safe synthesis of the results as initially intended.
The Satia [51] trial was the only one that reported positive findings regarding the superiority of vitamin D 3 delivery via buccal spray compared to capsules. However, the trial has limitations regarding the washout duration. According to Senn [61], if the duration of the washout is reasonable, substantial carry-over effects are unlikely to occur. On the other hand, as Todd and associates [57] note, the washout duration must be based on the US Food and Drug Administration (FDA) rule of thumb [62,63], which is five times the plasma half-life of the measured substance, herein 25(OH)D, is needed to achieve elimination of more than 95% of the substance from the body. Given that the plasma half-life of total 25(OH)D is approximately 15 days [64], ten weeks are needed to wash out any supplementation effect. Thus, based on the FDA guidelines, the duration of washout carried out by Satia [51] (10 days) appears inadequate.
According to the literature, interpretation of vitamin D assay results should be performed with caution, as not all methods are equal [65]. Farrell [66] revealed that automated immunoassays tend to demonstrate variable performance, and often fail to meet specific performance goals. On the other hand, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method used by Todd [57] tends to exhibit greater accuracy, lower variability and less bias [65,66]. Apart from the distinct assays, all three trials used independent laboratories and this has been shown to produce further variations in the results [67], as most laboratories fail to adhere to quality assurance standards and comply with international standardization processes.
Apart from the low Jadad [55] score and high risk of bias, the Penagini [58] trial demonstrated several additional shortcomings. Except for supplements, two known physiological pathways exist for increasing 25(OH)D concentrations, with the first being epidermal synthesis via sun exposure and the second through dietary intake. Concerning the latter, several studies suggest that vitamin D absorption is enhanced with concomitant fat intake or other oily vehicles [68]. Penagini [58] did not report controlling for these factors, failed to state the season in which the intervention was implemented and to include the assessment of usual dietary vitamin D intake, which introduces possible bias in the trial results. According to Rees [69], lifestyle variations account for one half of the variability in vitamin D supplementation response; thus, all trials should adjust for these factors in advance.
An additional limitation of the included RCTs is the lack of vitamin D genetic variants assay. As with most procedures in the human body, vitamin D absorption and utilization are also epiphenomena related to hereditary susceptibility, which suggests a personalized response [10,70]. Hence, genetic variations in 25-hydroxylase and vitamin D-binding protein have been shown to alter supplementation response [71,72], although the produced effect appears small compared to that of lifestyle components [69]. However, none of the included RCTs reported assessing vitamin D genetic variants or controlling for them during sample recruitment and group allocation.
Taking into account all of the above issues, the clinical heterogeneity of the retrieved RCTs appears to be multifactorial, which stems from the different study design, assays and laboratories used to perform the assays, intervention dosage, duration and season, washout duration, participant age and health status, allocation concealment and usual dietary intake. Although individually these factors are often encountered in meta-analyses, when only four trials are concerned, the coexistence of all these factors exacerbates heterogeneity and does not allow for a safe synthesis of the results. Indeed, in an attempt to pool findings (K.G.), considerable statistical heterogeneity was observed; thus, we considered that based on the currently available evidence at this time, a systematic review would be more robust compared to a meta-analysis.
Secondary analyses and synthesis of the findings of trials assessing the efficacy of vitamin D supplementation are required to produce robust results [73]. To this point, there are no other published systematic reviews that evaluate different modes of delivering dietary supplements. The present review was structured to assess the efficacy of vitamin D 3 supplementation from a different point of view: the superiority of buccal spray mode of delivery. Of note, one protocol for a systematic review with some similar features was published approximately a year ago (CRD42018118580) [74], although no preliminary or final findings have been reported until now. Distinct differences exist between the two protocols, with the present one focusing solely on vitamin D 3 , using a RCT design as an inclusion criterion, while assessing any form of vitamin D 3 oral spray supplementation delivery. On the other hand, the other protocol [74] reported the inclusion of any quasi-experimental study, focuses on both vitamin D 2 and D 3 intervention studies, while excluding spray interventions applied to the buccal mucosa, as performed in the Williams [59] trial included herein (use of sublingual spray). Additionally, a variety of methodological differences can be observed, including the search strategy, databases, search strings and keyword combinations applied, the tools used for assessing the quality and bias of studies (with the Jadad [55] and RoB 2.0 [54] being used herein, compared to the Joanna Briggs Institute (JBI) and GRADE [75] applied in the other protocol), and distinct data extraction protocols. In comparison to the aforementioned protocol [74], the present review has more restrictions with regard to the search strategy, as well as concerning the eligibility criteria, narrowing down the results to a great extent, while differentiating primary outcomes synthesis. Subsequently, based on the distinct methodological designs, inclusion/exclusion criteria, search strategy and vitamin D form based on the reported PICOs, the two studies would be expected to retrieve different primary studies, resulting in distinctive findings overall. Saldanha [76] noted that even when similar interventions are compared in trials or systematic reviews, differences in perspectives, goals, and constraints between trialists and reviewers explain differences in the outcomes. Nevertheless, as in primary research, also in meta-research, studies addressing similar research questions are required to inform practice and produce more robust recommendations. Given that 67% of the published meta-analyses tend to have at least one other overlapping meta-analysis, with a median of two meta-analyses per topic [77], and the fact that many differences exist between the two protocols, the two systematic reviews are expected to yield different findings based on a distinct qualitative synthesis of primary studies and are both required.

Conclusions
Thorough examination and critical appraisal of the current evidence reveals that despite the higher economic cost of the buccal spray, it does not appear to be superior to the other modes of vitamin D 3 delivery. More RCTs are required to investigate its efficacy in distinct populations, including patients with malabsorption problems. The limitations of the existing trials highlighted herein could serve as a primer for the design of future, relevant RCTs in order to reduce heterogeneity, increase trial comparability, and increase the validity of individual RCT results. Nevertheless, vitamin D 3 delivery via buccal spray might be preferred by populations with swallowing problems, or those receiving a great variety of supplements and/or medications, who wish to limit their intake of pills and capsules.