Comparative evaluation of salivary alkaline phosphatase levels and cephalometric changes during single-step versus two-step mandibular advancement with twin block in growing children – A randomized clinical trial

Trial design, ethical approval, and registry

The study design was a randomized controlled trial. The study design was approved by the Institutional Ethics Committee (Ref. no. MUHS/PG-T/E2/09/23/2023dated on 23.03.2023), and consent from the participating subjects was obtained in advance. The trial was registered in the Clinical Trial Registry – India (Ref. no. CTRI/2024/04/066030).

Sample size calculation

The sample size of this study was calculated based on the study conducted by Al-Khatieeb et al.^[10] considering a first-type error (a) level of 5% and a second-type error (b) level of 20%. Determination of sample size was done using the OpenEpi Version 3 software and it yielded an approximate sample size of 40 samples. However, considering 10% dropouts, the final sample determined was 44 patients.

Participants, setting, and eligibility criteria

The total sample consisted of 44 participants with Class II division I malocclusion in the age group of 10–14 years (22 males and 22 females). Inclusion criteria were as follows: No previous history of any orthodontic treatment, each subject with a full cusp class II relationship bilaterally with convex facial profile, participants with an overjet more than 7 mm, Virtual Treatment objective (VTO) improved with anterior positioning of mandible, ANB angle more than 4° with favorable growth pattern, and subjects with cervical vertebral maturation indicators stage-3 and MP3-FG and G. Exclusion criteria included syndromic patients, children with disorders of bone metabolism, hepatobiliary disorders and systemic inflammatory diseases, children with compromised oral hygiene, and children taking drugs that can alter salivary flow. Once the participant had agreed to the study protocol, a written consent that had been approved by the ethics committee was recorded.

Randomization

A universal sampling technique till achieving desired sample size was conducted. Randomization was done using a computer-generated sequence. Participants who met the criteria were included sequentially and allocated randomly to one of two groups: (1) Group 1 – single-step advancement with twin block (22 patients [8 males and 14 females]) and (2) Group 2 – two-step advancement with twin block (22 patients [14 males and 8 females]).

Blinding

The study participants were not informed of the treatment allocation. Data analysis was performed by assessors who were blinded and unaware of participants’ group allocation.

Intervention

Orthodontic treatment protocol

Both the groups received a conventional twin block design. Retention was by Adams clasps in 0.8-mm stainless steel on the maxillary first molars and the mandibular first premolars. Additional retention was provided by ball-end clasps. A maxillary labial bow in 0.8-mm stainless steel was used passively for retention. When maxillary arch expansion was required, a midline screw was used and turned once per week.

In group 1, single-step advancement was done with sagittal advancement up to 70% of maximum protrusive movement till achieving a class I molar relationship and vertical opening of 2–3 mm beyond the postural rest position of the mandible which opened the mandible 5–7 mm in the molar region [Figure 1].

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Figure 1:

Twin block with single-step advancement.

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In group 2, participants underwent a two-step advancement: the initial bite registration was performed with a sagittal advancement of approximately 50% of the total intended mandibular advancement. After 3 months of treatment, a second bite registration was recorded, achieving full mandibular advancement to establish a Class I molar relationship, while maintaining a vertical opening of 2–3 mm beyond the mandibular postural rest position [Figures 2 and 3].

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Figure 2:

Twin block with two-step advancement–1^st Step.

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Figure 3:

Twin block with two-step advancement–2^nd Step.

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All of the appliances were made with a high-quality, self-curing acrylic. With both approaches, selective trimming was done as and when required.

Salivary Alkaline assessment

For the assessment of salivary ALP, all the subjects were instructed to refrain from drinking, eating, chewing gum, and brushing for an hour before saliva collection. A sample volume of 3 mL unstimulated saliva was collected in a sterile, graduated, leak-proof test tube with a screw cap, and subjects were sitting in the upright position using the passive drool method. To avoid the circadian rhythm in salivary enzymes, all the samples were collected in the morning from 9 am to 11 am. Salivary samples were sealed and labelled for identification.

In the Biochemistry Laboratory, the samples were centrifuged for 3 min at 3000 RPM (relative centrifugal force dependent on rotor radius). This protocol was sufficient to obtain a clear supernatant, and samples were visually inspected to confirm the absence of cellular debris before analysis. Although longer centrifugation durations have been reported for saliva processing, all samples in the present study were processed using the same protocol across groups and time points, and the clarified supernatant was suitable for automated kinetic ALP measurement.

Following centrifugation, saliva supernatants were analyzed immediately or stored at −20°C until analysis. Samples underwent no more than one freeze–thaw cycle, and all analyses were completed within three months of sample collection.

All the samples were analyzed using an Autoanalyzer (AU-5800 system) working on the principle of spectrophotometry with di-nitrophenyl phosphate as substrate. The rate of absorbance change was measured at 405 nm at a reaction temperature of 37°C, and enzyme activity was expressed in international units per liter (IU/L). All assays were performed using manufacturer-supplied reagent kits and calibration standards, with instrument parameters automatically controlled by the analyzer software.

According to manufacturer specifications and routine laboratory quality control data, the assay demonstrated acceptable analytical performance, with intra-assay and inter-assay coefficients of variation below 5% and 8%, respectively. The analytical sensitivity of the method was adequate to reliably quantify the salivary ALP concentrations observed in the present study [Figures 4-6].

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Figure 4:

Collected and labeled samples.

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Figure 5:

Centrifugation of samples.

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Figure 6:

Autoanalyzer (Beckman Coulter-AU 5800 system).

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Outcomes (primary and secondary) and any changes after trial commencement

The primary outcome was to assess and compare the changes in salivary ALP level in single-step versus two-step advancement of the mandible using the twin block in growing skeletal class II children. Baseline salivary ALP levels before twin block delivery (M0) were recorded, followed by monthly assessment of salivary ALP levels after twin block delivery for 6 months (M1, M2, M3, M4, M5, M6).

The secondary outcome was to assess and compare the changes in skeletal and dentoalveolar effects of twin block therapy between group 1 and group 2. Lateral cephalograms were taken in centric occlusion pre-treatment (M0) and after 6 months of treatment (M6). All the cephalograms were taken using a single machine, PLANMECA (PM-2002), with an anode to midsubject distance of 5 feet. To evaluate and compare the skeletal and dentoalveolar effects, Pancherz’s cephalometric measurements were used in this study [Figure 7].

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Figure 7:

Modified Pancherz analysis.

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Statistical analysis

Data obtained was compiled on an MS Office Excel Sheet (v 2019, Microsoft Redmond Campus, Redmond, Washington, United States). Data were subjected to statistical analysis using the Statistical Package for the Social Sciences (SPSS version 26.0, IBM). Descriptive statistics such as frequencies and percentages for categorical data and mean and SD for numerical data have been depicted.

Normality of numerical data for salivary ALP was checked using the Shapiro–Wilk test and was found that the data did not follow a normal curve; hence, non-parametric tests have been used for comparisons. Inter-group comparison (2 groups) was done using the Mann–Whitney U-test. Intra-group comparison was done using Friedman’s test (for >2 observations), followed by pairwise comparison using the Wilcoxon signed-rank test.

Baseline comparisons between groups were performed using Mann–Whitney U tests for continuous variables and Chi-square tests for categorical variables, including sex distribution. Fisher’s exact test was considered where appropriate; however, all categorical comparisons satisfied chi-square assumptions.

To account for potential confounding due to sex and age, adjusted analyses were performed for the primary outcome. Longitudinal salivary ALP levels were analyzed using a mixed-effects model with subject as a random intercept and group, time, group × time interaction, sex, and age as fixed effects. Secondary skeletal and dental outcomes were analyzed using unadjusted between-group comparisons, as baseline values did not differ significantly between groups.

For all the statistical tests, P < 0.05 was considered to be statistically significant, keeping a error at 5% and b error at 20%, thus with 80% power of the study.

Participant flow

[Figure 8] shows the flow of participants through the trial. Forty-four participants who fulfilled the inclusion criteria were enrolled in the trial and continued to undergo follow-up appointments as per protocol. Later, four participants dropped out from study because they failed to attend follow-up.

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Figure 8:

Prisma flow chart.

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Baseline data

[Table 1] and [Graph 1] present the demographic details. The mean of the subjects in the one-step and two-step advancement groups was 12.55 years and 12.95 years, respectively. There were 7 males and 13 females in the one-step group and 14 males and 6 females in the two-step advancement group.

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Graph 1:

Demographic details.

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Baseline demographic characteristics are summarized in [Table 2]. There were no statistically significant differences between the one-step and two-step groups with respect to age or baseline salivary ALP levels (P > 0.05). Although sex distribution differed numerically between groups, this difference did not reach statistical significance on Chi-square testing (P = 0.057).

Numbers analyzed for each outcome, estimation, precision, and subgroup analyses

[Table 3] and [Graph 2] compare the salivary ALP levels between the two groups. While the two-step group generally showed higher ALP levels, none of the P-values fell below 0.05, although M4 (P = 0.053) is marginally close to statistical significance.

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Graph 2:

Comparison of salivary alkaline phosphatase levels between the two groups.

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After adjustment for sex and age, the mixed-effects model demonstrated a significant effect of time and a significant group × time interaction, while the overall group main effect remained non-significant [Supplementary Table S1].

[Tables 4 and 5] present a comparison of pre-operative skeletal and dental parameters between two groups: “One-step” and “Two-step.” The table shows that for all the listed skeletal parameters, the P-values are all >0.05. This means that there are no statistically significant differences between the “One-step” and “Two-step” groups in terms of their pre-operative skeletal and dental parameters.

Comparison of post-treatment skeletal changes between the two groups presented in [Table 6] and [Graph 3] shows that the change in SNB, ANB, PgOlp, and PgOlp+CoOlp was significantly greater in the Group 2 patient with two-step advancements as compared to the single-step advancement group. A statistically significant difference was observed in the change of SNB angle (P = 0.027*). The “One-step” group experienced a mean change of 2.20°, whereas the “Two-step” group showed a larger mean change of 2.95°. The difference in the change of ANB angle between the groups was statistically significant (P = 0.017*); the mean change in group 1 was 2.85 and in group 2 was 3.85. There was a statistically significant difference in the change of PgOlpCoOlp (P = 0.014*). Group 1 had a mean change of 2.68, compared to Group 2, in which a mean change of 4.85 was present.

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Graph 3:

Comparison of change in skeletal parameters between the two groups. ANB: A point Nasion B point angle, AngleNAPog: Nasion A point Pogonion angle, AngleSNGoGn Angle between Sella Nasion plane and mandibular plane, ANSMe: Anterior Nasal Spine Menton, CoANS: Condylion Anterior Nasal Spine, CoGn: Condylion Gnathion, CoGo: Condylion Gonion, CoOlp: Condylion to occlusal line perpendicular, GoGn: Gonion Gnathion, NaMe: Nasion Menton, PgOlp: Pogonion to occlusal line perpendicular, PgOlpCoOlp: Pogonion to Condylion on occlusal line perpendicular, PointAOLp: A point to occlusal line perpendicular, SGo: Sella Gonion, SNA: Sella Nasion A point angle, SNB: Sella Nasion B point angle, Beta angle: Sagittal skeletal discrepancy angle W angle: Sagittal jaw relationship angle.

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Both groups showed post-treatment increases in vertical linear and angular dimensions (e.g., Na–Me, ANS–Me, S-Go, Co–Gn). However, the magnitude of vertical change did not differ significantly between the groups. (P > 0.58).

Comparison of post-treatment dental changes between the two groups presented in [Table 7] and [Graph 4] revealed that none of the anteroposterior dental measurements, except iiOLpminuspgOlp, showed a statistically significant difference in the change between the two groups with a greater difference in Group 1 with a P = 0.045.

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Graph 4:

Comparison of change in dental parameters between the two groups. is: Maxillary incisor; ii: Mandibular incisor; ms: Maxillary first molar; mi: Mandibular first molar; Olp: Occlusal line perpendicular; A point: Subspinale; Pg (Pogonion): Most anterior point on the chin; U1PP: Upper incisor to palatal plane; U6PP: Upper first molar to palatal plane; L1MP: Lower incisor to mandibular plane; L6MP: Lower first molar to mandibular plane; S-line: Steiner’s line.

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Salivary ALP Assessment in Group 1

Salivary ALP level depicts the underlying bone depository phenomenon. Flórez-Moreno et al.^[16] and Abdul Wahab et al.^[17] evaluated the salivary ALP changes during fixed orthodontic treatment. Orthodontic tooth movement occurs in a series of alternate cycles of bone deposition and resorption. All formative cells, such as fibroblasts, osteoblasts, and cementoblasts, are associated with ALP activity. Salivary ALP level depicts the underlying bone depository phenomenon.

The results of the present study showed a gradual rise in mean salivary ALP level from M0 (836.85 IU/L) to M3 (875.60 IU/L) interval. Peak mean salivary ALP level was seen during the M3 interval. Later on, the values declined from M3 to M6 (847.45 IU/L). The salivary ALP level at M6 was higher than at M0.

Mandibular condylar cartilage (MCC) is a type of secondary cartilage; it is covered by a mesenchymal tissue layer rather than the perichondrium found on primary cartilage. It has the tendency to react to forces of mechanical loading of the condyle by functional appliances. As explained by Shen and Darendeliler, under mechanical loading, mesenchymal cells in the MCC differentiate into chondrocytes, followed by hypertrophy, matrix mineralization, and subsequent vascular invasion with osteoblast recruitment, reflecting the transition from chondrogenesis to osteogenesis.^[18,19] The orthodontic tooth movement due to mesialization of mandibular molars and distalization of maxillary molars^[20] also adds up to total salivary ALP. Immediately beneath the condylar articular surface is a quiescent layer of reserve chondrocytes that proliferate only under mechanical loading, followed in depth by the proliferative, hypertrophic, and erosive zones.^[21]

In the first group, bite registration for the twin block appliance was done with single-step mandibular advancement; the mechanical condylar loading was done only once. A bell-shaped curve of salivary ALP levels observed in this study is because of increased activity of hypertrophic chondrocytes and osteoblasts under mechanical loading. Once the chondrogenesis in proliferative and hypertrophic zones and osteogenesis in the erosive zone are completed, there is no further mechanical stimulus to resting zone cells to divide and proliferate. Hence, after reaching peak activity, salivary ALP levels gradually decline. The peak mean salivary ALP levels at M3 interval and gradual decline from M3 to M6 can be because of average life span of osteoblasts to be 90–120 days.^[22]

Salivary ALP assessment in group 2

Group 2 (two-step advancement group) demonstrated a progressive increase in mean salivary ALP levels from M0 (862.15 IU/L) to M4 (919.30 IU/L), with the highest mean value recorded at M4. A slight decline was noted thereafter, reaching 879.85 IU/L at M6. Importantly, similar to Group 1, the ALP level at M6 was still higher than the baseline, indicating sustained osteogenic activity induced by the twin block appliance in both advancement protocols.

The two-step protocol produced marginally higher and more prolonged ALP levels at every time point, yet none of the gains were statistically significant; the closest to significance was at M4, where the P-value approached significance (P = 0.053), implying only a tentative biological edge over the single-step approach.

Leung et al.^[14] supported by Rabie and Al-Kalaly^[23] showed that SWA sustains vascular endothelial growth factor (VEGF) and thus angiogenesis and bone formation after each incremental step, whereas in single-step advancement, VEGF returns toward baseline in few days. Similar to this, our results reveal salivary ALP, an osteogenic marker, declining in the single-step group after the 3^rd month but rising steadily in the two-step group, peaking at the 4^th month following the second activation. This second mechanical stimulus appears to re-stimulate condylar remodeling, prolonging biological activity. Although the intergroup difference was not statistically significant, the trend underscores the biological advantage of two-step advancement in sustaining remodeling processes.

Based on the findings of Rabie and Al-Kalaly^[23] and Turner et al.,^[24] it can be concluded that the initial mandibular shifts in stepwise protocols must exceed a biological threshold to stimulate bone growth in the condyle and fossa. If too small, enough load would not be generated to start the cellular and vascular cascade. Animal studies show that larger moves (over ~2 mm) yield more bone, but the ideal stress level for human mandibular growth is still unknown.

In this study, mandibular advancement was performed in two phases, with approximately half of the total advancement achieved in the first step, aligning with the findings of the above study which emphasizes the importance of an adequately sized initial advancement to effectively trigger condylar remodeling and osteogenesis.

Our results suggest that SWA with a twin-block appliance elicits a longer-lasting, more favorable remodeling response in the condyle likely by extending the period of chondrocyte proliferation and endochondral ossification. While intergroup differences never reached statistical significance (P > 0.05), the near-significant P = 0.053 at 4^th month shows that the two-step protocol keeps the condylar cartilage under continuous stimulation, similar to Leung et al.,^[14] results. After adjustment for sex and age using a mixed-effects model, the two-step protocol demonstrated a more sustained salivary ALP trajectory over time. Although sex was not a significant predictor in the adjusted model, the potential influence of sexual dimorphism during pubertal growth should be considered when interpreting the results. To clarify the underlying remodeling dynamics, future studies should track additional biomarkers such as insulin-like growth factor-1, transforming growth factor, and fibroblast growth factor and by longer follow-ups to see whether elevated ALP translates into durable skeletal improvements.

Salivary ALP levels may be influenced by local periodontal inflammation, oral hygiene status, and transient inflammatory conditions. Although participants were clinically examined to exclude overt periodontal disease and acute oral infections, formal periodontal indices such as plaque index or gingival index were not recorded at baseline. Consequently, the potential influence of subclinical gingivitis on salivary ALP levels cannot be entirely excluded. However, all participants were orthodontic patients with comparable oral hygiene instructions and monitoring throughout the study period, and baseline ALP levels did not differ significantly between groups, suggesting that systematic periodontal bias was unlikely to account for the observed longitudinal trends.

Skeletal anteroposterior cephalometric measurements

SNB angle increased significantly more in the two-step group than in the one-step group. A greater increase in SNB angle reflects a larger forward advancement of the mandibular base, confirming that two-step advancement stimulates more effective mandibular adaptation.

Our findings contradict Pancherz^[25] study, who argued that pushing the mandible in a single step into an incisal edge-to-edge position maximizes skeletal change. However, his conclusion was based solely on correlating mandibular growth with the size of a single “bite jump” and did not test gradual, stepwise advances. Conversely, Chen et al.^[26] conducted a meta-analysis of five studies and showed that stepwise advancement produced a significantly greater increase in SNB angle than a single-step approach an outcome similar to our results.

ANB angle decreased in both groups, but the reduction was significantly greater with two-step advancement. This indicates superior sagittal correction of maxilla–mandible relation with the two-step approach.

This result is similar to the meta-analysis conducted by Chen et al.^[26] which showed that functional appliances with stepwise appliances produced more favorable effects on improving the relationship of maxilla and mandible, which is in support of our study.

Our result is in accordance with the study conducted by Aras et al.^[27] where they compared two groups of subjects at the peak of the pubertal growth period treated with the Functional Mandibular Advancer appliance using either single-step or SWA. There were statistically significant changes in SNB, Pg horizontal, ANB, Co-Gn, and Co-Go measurements in both groups; these changes were greater in the stepwise advancement with the exception of Co-Go.

Linear pogonion measures – Pg–OLp and Pg–Olp+Co-OLp – both showed significantly larger posterior-to-anterior shifts in the two-step group, underlining a more pronounced chin advancement.

This parameter is in contrast to the study conducted by Banks et al.^[28] where they concluded that the use of incremental advancement of the Twin-block did not influence the outcome of treatment, in terms of process or morphological effects. As a result, they did not support the suggestion that step-wise advancement produces a better orthopedic response in relation to the change in mandibular length.

Our study results are similar to the conclusions made in the meta-analysis by Chen et al.^[26] They concluded a significantly greater increase in the sagittal displacement of point Pog in patients treated with functional appliances with stepwise advancement compared with single-step advancement.

Vertical skeletal changes

Both groups showed post-treatment increases in vertical linear and angular dimensions. However, the magnitude of vertical change did not differ significantly between the groups.

When Class II patients use functional appliances, a clockwise rotation of the mandible is unwanted because it aggravates their mandibular retrognathism. Our outcome differs from what was reported by Doshi and Mahindra^[29] who compared effective temporomandibular joint growth changes after stepwise and maximum advancement with twin block appliance and concluded that overall, the stepwise advancement group showed a markedly greater vertical condylar increase and mild anterior mandibular rotation, confirming a more favorable vertical growth pattern than the single-step advancement group.

Our findings indicate that the change in mandibular plane angle showed a similar effect in both the groups. Similar to our result, Chen et al.^[26] concluded that no significant difference between the step-wise advancement and single-step advancement groups was observed in vertical changes of the mandible.

Dental cephalometric measurements

Limitation

• Although the sample size was adequate to detect clinically relevant differences, larger multicenter studies are required to improve generalizability. Imaging constraints – twodimensional lateral cephalograms cannot capture transverse or threedimensional skeletal adaptations.

• Different methods of salivary enzyme assessment give different results regarding the activity of the enzymes. Again, the sexual dimorphism and ethnic variation also play an important role in total salivary ALP. Hence, there is a need for further research in this field with a standardized protocol at various geographic locations. The salivary levels of different enzymes are comparatively lesser than serum levels.

• Although sex distribution differed numerically between groups, the difference did not reach statistical significance. Nevertheless, residual confounding related to sexual dimorphism during pubertal growth cannot be entirely excluded.

• Baseline periodontal indices (plaque index and gingival index) were not recorded; therefore, the influence of subclinical gingival inflammation on salivary ALP levels cannot be completely ruled out.