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. 2024 Aug 6;35(12):1546–1559. doi: 10.1111/clr.14342

A 3D micro‐CT assessment of composition and structure of bone tissue after vertical and horizontal alveolar ridge augmentation using CAD/CAM‐customized titanium mesh

Alessandro Cucchi 1, Gregorio Marchiori 2,✉, Maria Sartori 2, Milena Fini 3, Antonino Fiorino 4, Raffaele Donati 5, Giuseppe Corinaldesi 6, Melania Maglio 2
PMCID: PMC11629439  PMID: 39106169

Abstract

Objectives

To date, no studies have exploited micro‐CT in humans to evaluate bone morphology and structure after bone augmentation with CAD/CAM‐customized titanium mesh, in mandible and maxilla. The aim of this study was to assess the composition and microstructure of bone biopsy through micro‐CT analysis.

Materials and Methods

Bone augmentation at both maxillary and mandible sites was performed on 30 patients randomly treated with customized mesh, either alone (M−) or covered with resorbable membrane (M+), in both cases filled 50:50 with autogenous bone and xenograft. After 6 months, biopsies were taken and micro‐CT was performed on consecutive 1‐mm‐thick VOIs from coronal to apical side, measuring tissue volumes, trabecular thickness, spacing, and number.

Results

In both groups, irrespective of membrane use, bone tissue (M−: 29.76% vs. M+: 30.84%) and residual graft material (M−: 14.87% vs. M+: 13.11%) values were similar. Differences were site‐related (maxillary vs. mandibular) with higher percentage of bone tissue and trabecular density of low‐mineralized bone and overall bone in the mandible.

Conclusions

The composition and structure of bone tissue, as assessed by micro‐CT after alveolar ridge augmentation using CAD/CAM‐customized titanium meshes, showed similar features regardless of whether a collagen membrane was applied.

Keywords: bone biopsies, bone regeneration, CAD/CAM mesh, micro‐CT, randomized clinical trial, resorbable membrane

1. INTRODUCTION

One of the most innovative techniques for alveolar ridge augmentation involves the use of titanium mesh, custom‐developed by means of computer‐aided design/computer‐aided manufacturing (CAD/CAM) and direct metal laser sintering (DMLS) technologies. Various papers have confirmed the clinical and radiographic efficacy of customized titanium meshes, showing how the use of titanium mesh represented a predictable method for the rehabilitation of complex atrophic sites (Briguglio et al., 2019; Chiapasco et al., 2021; Cucchi, Vignudelli, Franceschi, et al., 2021).

Over the years, titanium mesh has been used alone to overcome inadequacies impinging on the preservation of cell occlusiveness due to the presence of macropores (Kubo et al., 1998; Pineda et al., 1996; Rakhmatia et al., 2013); some authors have suggested covering the mesh with resorbable membranes to improve cell occlusiveness, increase bone gain, and reduce mesh exposure rates (Assenza et al., 2001; Cucchi et al., 2017; Cucchi, Chierico, Fontana, et al., 2019; Degidi et al., 2003).

Few in vivo studies in the literature have evaluated the effect of resorbable collagen membranes in combination with titanium mesh on the promotion of exophytic bone formation in guided bone regeneration (GBR). The results have been mixed. A first study performed on rabbit parietal bone showed how using a commercially available collagen membrane to cover a titanium mesh could promote a greater amount of newly formed bone than titanium mesh alone (Shin et al., 2013). More recently, by contrast, two in vivo studies have demonstrated how the association between collagen membrane and titanium mesh apparently conferred no additional benefits in terms of the quality of the newly formed bone (Borges et al., 2020; Paeng et al., 2022).

In those in vivo studies, together with standard histology, micro‐computed tomography (micro‐CT) emerged as a promising solution for advancement in tissue characterization. Micro‐CT is a consolidated imaging technology for bone and widely employed in orthopedics. Excellent spatial resolution, detail detectability, and intrinsic 3D are characteristics, which provide better diagnostic accuracy and have extended the applications of micro‐CT to the dental field.

In particular, micro‐CT analyses on bone biopsies provide the unique possibility of performing a non‐destructive micro‐structural investigation of tissue of heterogeneous density, morphology, and structure, and open the way to further complementary analyses, such as histology. Integrating conventional analyses with microtomographic investigation facilitates an in‐depth and transversal evaluation of the structural, architectural, and biological parameters that can give an indication of the effect of the mesh on dental ridge augmentation.

According to the authors' current knowledge, no clinical studies have yet exploited micro‐CT analyses to investigate the role of the resorbable membrane covering the titanium mesh, either concerning conventional mesh or customized CAD/CAM meshes. The aims of the present study are: (i) to investigate the effect of the resorbable membrane over CAD/CAM‐customized titanium meshes in terms of bone, soft tissue, and void formation by means of 3D micro‐CT in humans bone biopsies after vertical and horizontal ridge augmentation both in the mandible and in the maxilla; (ii) to set up a microtomographic approach for the study of the progression and morphological and structural characteristics of bone tissue regeneration in the presence of biomaterials for applications in regenerative medicine and orthopedics.

2. MATERIALS AND METHODS

2.1. Study design

This study represents the third part of the parallel‐group, double‐blinded (study subjects, research investigators, outcome assessor, and data analysts), monocentric, independent, randomized, non‐inferiority clinical trial conducted at the Unit of Oral and Maxillofacial Surgery (DIBINEM, Alma Mater Studiorum—University of Bologna, Italy) and in accordance with all the relevant requirements of the Declaration of Helsinki and the CONSORT guidelines. This clinical trial was approved by the inter‐company Ethical Committee of Bologna and Imola (protocol CMF 01/2017, study code: CE 17139) and was registered on ClinicalTrials.gov website (ClinicalTrials.gov ID: NCT04286334).

Since the primary aim of the randomized clinical trial was to compare complication rates using custom‐made titanium mesh alone (group M−) and custom‐made titanium mesh covered by resorbable collagen membranes (group M+), the calculated sample size was fixed at 30 patients in order to compensate possible drops‐outs, as previously reported (Cucchi, Vignudelli, Franceschi, et al., 2021).

The inclusion criteria for the study were: (1) age > 18 years; (2) partial edentulism of maxilla or mandible; (3) vertical and/or horizontal bone defect requiring a 3D‐bone augmentation for prosthetically guided implant placement; (4) informed consent to experimental treatment; and consent to processing of personal data.

All patients who met the inclusion criteria received written information about the study and provided written informed consent before participation in any study‐related procedure.

After enrollment, each patient received a unique identification number and was randomly assigned to a study group, according to a previously computer‐generated sequence.

Of the 30 patients, 15 were randomly assigned to group M− (titanium mesh alone) and treated using customized titanium mesh (Figure 1a) filled with 50:50 autogenous bone and high‐porosity xenograft; the other 15 were assigned to group M+ (titanium mesh plus membrane) and received the same customized titanium mesh and the same bone grafting material, but all covered with resorbable collagen membranes.

FIGURE 1.

FIGURE 1

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Measurements of maximum vertical bone defect on 3D stereolitographic model and CBCT scan section (a). Digital planning of the bone augmentation: Lateral and occlusal views of the 3D model, graft volume, and customized mesh (b).

In this randomized clinical trial, patients' blindness was maintained by not disclosing their group assignment. All surgical procedures at T0 were performed by blinded surgeons (A.C.; E.R.), until the revealing of the assigned group just before the flaps closure (not‐complete blindness). Similarly, the clinical data examiner at T1 (E.V.) was blinded. For the micro‐CT analysis, all investigators (G.M.; M.S.; M.M.) were blinded to the study groups, because they received the biopsy samples coded with unique identification numbers. Similarly, the statistician (A.F.), who was not part of the study group, received the data without any knowledge of the group assignments.

2.2. Clinical procedures

The clinical procedures have been described previously (Cucchi, Vignudelli, Franceschi, et al., 2021). Briefly, the digital planning of GBR included the evaluation of bone defects on pre‐operative cone beam computed tomography (CBCT) (.DICOM files) (Figure 1a) and the following 3D projects of the bone augmentation and the customized mesh on virtual stereolitographic models (.STL files) using a dedicated software (Figure 1b).

The bone augmentation surgery (T0 protocol) comprised the following steps: flap elevation and passivation; cortical perforation in order to promote the migration of osteogenic and osteoprogenitor cells and to ensure revascularization of the bone graft; bone harvesting using a bone scraper (Safescraper, Meta, Reggio Emilia, Italy); creation of a 50:50 mixture of autogenous bone and high‐porosity xenograft (Z‐core, Osteogenics, Lubbock, Texas, USA); filling and fixation of the custom‐made DMLS titanium mesh (3D Mesh, BTK Biotec, Dueville, Italy) using osteosynthesis screws (Profix, Osteogenics); application of a cross‐linked collagen membrane (Cytoplast RTM, Osteogenics), if patient allocation was group M+; and finally, primary closure of the surgical flaps.

After 6 months (T1 protocol), soft tissue healing was assessed in order to confirm the absence of healing complications, such as mesh exposure or infection (Figure 2a,b), and post‐healing CBCT was obtained to evaluate the bone formation under the mesh (Figure 2c). Then, all the treated sites were reopened for the following steps: mesh removal (Figure 3a–c), implant placement (Figure 3d–f), and bone tissue biopsy harvesting using a 4‐mm‐diameter trephine burr (Figure 3g). Pseudoperiosteum type (types 1, 2, 3) and bone density were clinically measured using a UNC‐15 periodontal probe (Cucchi, Sartori, Aldini, et al., 2019). A unique identification number was assigned to each biopsy specimen to blind the two operators who performed all micro‐CT analyses.

FIGURE 2.

FIGURE 2

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Soft tissue healing 6 months after surgery, without exposure and/or infection: Lateral (a) and occlusal views (b). CBCT scan section of the maxilla after the bone augmentation using customized mesh (c).

FIGURE 3.

FIGURE 3

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Augmented site showing the regenerated bone tissue after mesh removal (a); the occlusal part of the mesh removed with its pseudo‐periosteum (b); the osseointegrated borders of the mesh fractured during removal (c). Implant design and surface (d); augmented site showing the implants placed in the regenerated bone (lateral view) (e); augmented site showing the implants placed in the regenerated bone (occlusal view) (f). Biopsy of regenerated bone tissue taken from implant site (g).

2.3. Micro‐CT analyses

After fixation, the biopsies were removed from alcohol and mounted within Eppendorf plastic tubes to prevent movement during micro‐CT scanning. The biopsies were scanned in the SkyScan 1172 (Bruker, Belgium) at a nominal resolution (pixel size) of 5 μm employing an 0.5‐mm‐thick aluminum filter and an applied X‐ray tube voltage of 70 kV. Camera pixel binning was not applied. The scan orbit was 180° with a rotation step of 0.35°.

Reconstruction was carried out with a modified Feldkamp algorithm using the SkyScan™ NRecon software (version 1.7.4.6, Bruker) accelerated by GPU. Gaussian smoothing (value: 1), ring artifact reduction (value: 5), and beam hardening correction (value: 30%) were applied.

Volume of interest (VOI) selection, segmentation to binary, and morphometric analysis were all performed using SkyScan CT‐Analyser software (“CTAn” 1.20.3.0 version, Bruker).

The micro‐CT evaluation of bone biopsy was subdivided into three different phases to assess every aspect of the regenerated bone tissue:

2.3.1. Overview and VOI identification

Biopsy reconstructions were oriented corono‐apically from top to bottom, where generally bone was prevalent on the apical side and graft was prevalent on the coronal side. VOIs of 1 mm thickness were defined from coronal to apical side and wrapping the sample (Helder et al., 2018; Schulten et al., 2013). Up to seven VOIs could be identified on some micro‐CT imaged biopsies, despite only five VOIs being considered to compare groups M− and M+, in order to obtain sufficiently large samples for statistical analysis. Furthermore, since mean values of vertical bone gain (VBG) are 4.7 mm in group M− and 6.4 mm in group M+ (Cucchi, Vignudelli, Franceschi, et al., 2021), micro‐CT analysis of regenerated bone are more reliable between VOI‐1 and VOI‐5.

2.3.2. Assignment of gray levels to the heterogeneous tissues: analysis of biopsy composition

Grayscale global thresholds on micro‐CT images were selected by averaging the thresholds determined on three slices for three bone biopsies by two independent observers: the thresholds were set at 73–123 for “all” bone (AB) and at 124–255 for graft (MAT). “Non‐mineralized” tissue (NMT) was defined with 30–72 thresholds, while “void” represented the rest of the VOI, with gray levels less than 30. Distinguishing between bone, graft material, and connective tissue/bone marrow (i.e., “non‐mineralized” tissue) was common with reference studies (Helder et al., 2018; Schulten et al., 2013). Areas occupied by those materials were clearly visible and recognizable on micro‐CT images, and thus, their grayscale thresholds were easily identified and computer‐supported analysis was set as well. Moreover, bone was subdivided into “low‐mineralized” bone (L‐B) with 73–106 thresholds and “high‐mineralized” bone (H‐B) with 107–123 thresholds; to this end, histological slices from the same samples analyzed in micro‐CT were used (Cucchi et al., 2024), exploiting the observation of the biological structures and the different intensities of histological staining to relate them to the identifiable thresholds.

2.3.3. Quantitative analysis of 3D parameters: analysis of biopsy morphometry (trabecular architecture)

3D morphometric parameters were calculated for the selected VOIs. Noise objects were removed from the binarized image by despeckle and closing morphological operations in CTAn. Then, the percentage of tissue volume/total volume was calculated for: non‐mineralized tissue (NMT/TV = NMV%), low‐mineralized bone (L‐B/TV = L‐B%), high‐mineralized bone (H‐B/TV = H‐B%), all bone (AB/TV = AB%), residual biomaterial (MAT/TV = MAT%), and voids (%). In addition, the proportion of bone surface to total volume was also assessed in each VOI, in relation to “low‐mineralized” bone (L‐B_BS/TV), “high‐mineralized” bone (H‐B_BS/TV), and the sum of those (AB_BS/TV). Finally, trabecular thickness (TbTh), number of traversals across the trabecular bone per unit length (TbN), and 3D space between trabeculae (TbSp) were evaluated in “low‐mineralized” bone, “high‐mineralized” bone, and all bone in each VOI. The definitions, symbols, and units for bone morphometric parameters follow the ASBMR standardized nomenclature (Dempster et al., 2013).

2.4. Statistical analyses

An Excel data collection form and data management system were used (Microsoft Excel 2011; Windows, ver. 14.0.0; Microsoft Corp., Redmond, WA, USA). All data were entered by a single‐blinded operator (G.M.). Prior to entry, all data were evaluated in terms of accuracy and completeness. For each continuous variable the mean, median (where necessary), standard deviation (SD), and the 95% Confidence Interval (95% CI) were reported. This statistical analysis was based on the hypothesis that group M− would not be inferior to group M+ in relation to All Bone (AB) VOI (primary outcome). The threshold for non‐inferiority of group M− compared with group M+ was decided at less than 10% difference in the mean change. The non‐inferiority test was performed for All Bone VOI, NMT VOI, MAT VOI, and Void VOI (one‐sided 95% confidence interval approach), specifying the largest difference that was clinically acceptable for each outcome (delta = D). Since the sample size was calculated for the first part of the study, in which primary outcome was the complication rates (Cucchi, Vignudelli, Franceschi, et al., 2021), a post hoc power analysis for the third part was carried out, considering one‐sided significance level of .05 and non‐inferiority margin of 10%.

In addition, to evaluate significant differences in terms of intervention groups and jaw of interest, a superiority analysis was carried out. The null hypothesis (H0) was that there is no true difference in All Bone VOI between groups and anatomical sites, while the alternative hypothesis (H1) suggests that there is a difference between the intervention groups and jaws. The comparison of means to evaluate statistically significant differences was performed by t test, Wilcoxon rank sum test, and Wilcoxon matched‐pairs signed rank test where necessary. Where a non‐parametric test was used, the median is always reported.

Furthermore, all the micro‐CT variables were evaluated based on bone density detected (3 categories) and pseudoperiosteum type observed (3 categories) using the one‐way ANOVA and Tukey's post hoc test.

Possible linear correlation was investigated by applying the Pearson's or, when the variables did not have a normal distribution or a nonlinear relationship, Spearman's correlation test. Correlations were reported specifying the p‐value and the Pearson (r) or Spearman (rs) correlation coefficient.

Testing of normality was carried out with the Skewness/Kurtosis tests (normal distribution if p‐value > .05). The threshold value decided for determining the statistical significance corresponds to a p‐value of .05 (5%). The statistician was blinded and external to the working group (A.F.). Data analysis was performed with Stata/IC software (StataCorp LLC, College Station, TX, USA).

3. RESULTS

3.1. Study population

The study included 30 patients with vertical and/or horizontal bone defects (83.3% vertical and horizontal and 16.7% horizontal), both at mandibular (53.3%) and maxillary sites (46.7%), in both the anterior (23.3%) and posterior regions (76.7%).

Five biopsies/patients (2 in group M−; 3 in group M+) were excluded from micro‐CT analysis/bone tissue biopsy because healing complications occurred (3 early exposure and 2 infections without exposure). No other patients dropped out or were excluded from micro‐CT analyses. Therefore, a total of 25 samples underwent micro‐CT analyses before histological processing and analysis: 13 belonging to group M− and 12 belonging to group M+. Using 25 biopsies, considering the ALL BONE values in the two intervention groups (M−: 27.68% and M+: 30.81%), the statistical power from post hoc analysis resulted to be 21%.

3.2. Overview of micro‐CT images

The three‐dimensional and bi‐dimensional observations of the samples after micro‐CT reconstruction show the presence of bone regeneration in association with remaining graft materials in all the investigated biopsies, with a clear display of graft and bones (Figure 4). A visual observation of micro‐CT images detected no evident differences between the two study groups in terms of regenerated bone. The evidence of bone compact structure attributable to the morphology of the native bone is detected in few cases. Biopsy dimensions appeared different between cases. The distribution and dimension of the residual biomaterial appeared considerably heterogeneous among the samples, in some cases distributed along the entire length of the biopsy, in others concentrated in the coronal part rather than the apical part.

FIGURE 4.

FIGURE 4

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Renderings of the micro‐CT reconstructions of two different biopsies (a and b), in virtual cross‐section on the coronal plane (frame with broken line). In (c), coronal projection of biopsy B; in (d), comparative hematoxylin/eosin digitalized histological image of the same biopsy (scale bar 2 mm), showing the main structures for the selection of specific thresholding (high‐mineralized bone (H‐M); low‐mineralized bone (L‐B); non‐mineralized tissue (NMT); material (MAT)).

3.2.1. Analysis of biopsy composition

In the entire population, without distinguishing between M− and M+ and without considering the VOIs, All Bone tissue (AB) obtained with CAD/CAM mesh and 50% of autogenous bone and 50% of high‐porosity xenograft represented 29.22%, where 5.31% is classified as high‐mineralized bone (H‐B) and 23.35% as low‐mineralized bone (L‐B); the residual particles of grafting material volume (MAT) accounted for 14.18%, while the non‐mineralized tissue volume (NMT) accounted for 25.33%, the remaining 31.28% is represented by voids.

Looking at the group values, distinguishing between M− and M+, the distribution of tissue composition was similar in the two study groups: 27.68% of bone tissue (5.41% H‐B vs. 21.59% L‐B), 15.58% of biomaterial volume (MAT), and 25.24% of non‐mineralized tissue volume (NMT), in group M−; and 30.81% (5.21% vs. 25.19%), 12.73%, and 25.42% in group M+, respectively. Complete data including mean/median, SD, 95% CIs, and estimated differences are shown in Table S1a (Data S1) and Figure 5.

FIGURE 5.

FIGURE 5

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A Multiple Pie Chart. Graph shows the percentage distribution of All Bone (AB), residual particles of grafting material (MATV), Non‐Mineralized Tissue (NMT), and Void (VOID) in Group M− (without collagen membrane) and in group M+ (with collagen membrane).

The non‐inferiority analysis confirmed the null hypothesis that the group without membrane is not inferior to the group with membrane in terms of bone tissue (Graph 1).

GRAPH 1.

GRAPH 1

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Total Bone Volume: Non‐inferiority analysis. Error bars indicated one‐sided 95% confidence intervals for the difference in the All Bone Volume mean values between the M− and M+ groups (M− minus M+). The red broken line delineating the difference in the score (Δ = 7.80) shows the non‐inferiority margin (delta); tinted area indicates zone of non‐inferiority. The CI does not include Δ and the data prove non‐inferiority of group M− compared with group M+. Although there is no statistically significant difference between the two treatments, group M+ tends to be better than group M− in terms of All Bone Volume.

Additional non‐inferiority analyses regarding MAT, NMT and voids volume were also performed to investigate all variables of the regenerated tissue. Although group M− was proven not to be inferior to group M+ regarding non‐mineralized tissue (Graph 2), a biomaterial‐related trend in favor of group M+ was observed and the null hypothesis of non‐inferiority was not confirmed (Graph 3).

GRAPH 2.

GRAPH 2

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Non‐mineralized tissue volume: non‐inferiority analysis. Error bars indicated one‐sided 95% confidence intervals for the difference in NMT mean values between the M− and M+ groups (M− minus M+). The red broken line delineating the difference in the score (Δ = 4.23) shows the non‐inferiority margin (delta); tinted area indicates zone of non‐inferiority. The CI does not include Δ, and the data prove non‐inferiority of group M− compared with group M+. Although there is no statistically significant difference between the two treatments, group M− tends to be better than group M+ in terms of NMT Volume.

GRAPH 3.

GRAPH 3

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Residual graft material volume: non‐inferiority analysis. Error bars indicated one‐sided 95% confidence intervals for the difference in MAT Volume mean values between the M− and M+ groups (M− minus M+). The red broken line delineating the difference in the score (Δ = 4.48) shows the non‐inferiority margin (delta); tinted area indicates zone of non‐inferiority. The CI includes Δ, and the data do not prove non‐inferiority of group M− compared with group M+. Although there is no statistically significant difference between the two treatments, group M+ tends to be better than group M− in terms of graft volume.

Nevertheless, no statistically significant differences were observed between the two study groups for any of the investigated parameters according to the superiority analyses (p > .05) (Graph 4).

GRAPH 4.

GRAPH 4

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Void volume: non‐inferiority analysis. Error bars indicated one‐sided 95% confidence intervals of the difference in Void Volume mean values between the M− and M+ groups (M− minus M+). The red broken line delineating the difference in the score (Δ = 7.40) shows the non‐inferiority margin (delta); tinted area indicates zone of non‐inferiority. The CI does not include Δ, and the data prove non‐inferiority of group M− compared with group M+. Although there is no statistically significant difference between the two treatments, group M+ tends to be better than group M− in terms of Void Volume.

The analysis performed in each VOI for the different parameters did not show any significant difference between the two study groups, although the percentages of AB in group M+ were slightly higher than those in group M−. The difference between M+ and M− was mainly due to the difference in low‐mineralized bone rather than in high‐mineralized bone, and this trend was observed in all VOIs. An opposite trend was observed in the percentage of MAT, which was slightly superior in group M− compared to group M+ along the whole depth of the biopsies, even if without statistical significance (p > .05).

Observing the values of MAT along the different VOIs, a trend of progressive reduction from the most coronal VOI (VOI‐1) to the most apical one (VOI‐5) was distinctly observed in both groups; by contrast, the NMT remained steady along the biopsies. Complete data for all variables in each VOI, including mean/median SD, 95% CIs, and estimated differences, are reported in Table S1b (Data S1) and Figures 6 and 7.

FIGURE 6.

FIGURE 6

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Bone tissue by groups. Referred to the entire biopsy, it shows the percentage (mean value and SD) of All Bone, High‐Mineralized Bone (H‐B), and Low‐Mineralized Bone (L‐B) between the two study groups (M− and M+).

FIGURE 7.

FIGURE 7

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MAT, MNT, and Void volume by groups. Referred to the entire biopsy, it shows the percentage (mean value and SD) of residual particles of grafting material and Non‐Mineralized Tissue (NMT) and Void between the two study groups (M− and M+).

Mean values were also evaluated comparing sites in the maxilla and in the mandible. Considering the entire population, a statistically significant difference was found between mandible and maxilla in regard to the bone tissue. In particular, bone samples showed significantly higher rates of AB and L‐B in the mandible compared to the maxilla, with mean estimated differences of 12.77% and 14.99%, respectively (p = .009 and p = .006, respectively). Other parameters related to AB and L‐B, such as AB and L‐B surfaces in relation to total volume, were superior in mandibular than in maxillary sites (p = .011 and p = .031, respectively). No differences in terms of NMT and MAT were observed according to the jaw of interest. All values are reported in detail in Table S2a (Data S1).

A comparison between the bone density clinically assessed during implant site preparation and the bone density based on micro‐CT values (AB + MAT/TV) was performed. In sites classified as hard density, medium density, and soft density, the values of micro‐CT density were 47.57%, 55.84%, and 36.64%, respectively. Comparing all the measured data for the abovementioned subgroups, statistical differences were found for AB and L‐B because soft density showed significantly lower values compared to medium density (p = .0001) and compared to hard density too (p = .044). A slightly significant difference was also observed in the total bone surface in relation to total volume (p = .0459). All mean values are shown in Table S2b (Data S1).

3.2.2. Analysis of biopsy morphometry (trabecular architecture)

In the entire population, parameters related to tissue morphometry (TbTh, TbN, and TbSp) for the bone tissue were 0.04 mm, 7.01 mm−1, and 0.15 mm, respectively. Comparing these parameters in high‐mineralized bone and in low‐mineralized bone, the values showed significant differences for all parameters: 0.013 vs. 0.03 mm (p = .0001), 3.98 vs. 8.05 mm−1 (p = .0001), and 0.21 vs. 0.16 mm (p = .0001).

The values of TbTh, TbN, and TbSp were similar in the two study groups, observing any statistical difference between them. Similarly, H‐B and L‐B showed significant differences in relation to the trabecular parameters in both groups. Complete data are reported in Table S3a (Data S1) and Figures 8, 9, 10. A three‐dimensional gray‐scale rendering for each harvesting site and treatment, together with relative Hounsfield Units highlighting the different tissues and biomaterial, and with TbTh mapping, is showed in Figure 11.

FIGURE 8.

FIGURE 8

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Trabecular thickness. Referred to the entire biopsy, it shows the mean values and SD of trabecular thickness between the two groups (M− and M+) in All Bone, High‐Mineralized Bone (H‐B), and Low‐Mineralized Bone (L‐B).

FIGURE 9.

FIGURE 9

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Trabecular space. Referred to the entire biopsy, it shows the mean values and SD of trabecular space between the two groups (M− and M+) in All Bone, High‐Mineralized Bone (H‐B), and Low‐Mineralized Bone (L‐B).

FIGURE 10.

FIGURE 10

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Trabecular number. Referred to the entire biopsy, it shows the mean values and SD of trabecular number between the two groups (M− and M+) in All Bone, High‐Mineralized Bone (H‐B), and Low‐Mineralized Bone (L‐B).

FIGURE 11.

FIGURE 11

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Micro‐CT gray‐scale rendering (left) of representative bone biopsies—(a) from mandible, M−, (b) from mandible, M+, (c) from maxilla, M+, (d) from maxilla, M−—together with Hounsfield Units (HU) mapping (center; frontal cut; default calibration: 0 gray value = −1000 HU, 255 gray value = 8830 HU) and trabecular thickness (TbTh) mapping of apical and coronal sides (right).

The analysis performed in each VOI for the different parameters did not show any significant difference either between the two study groups or between the progressive VOIs, confirming that tissue morphometry of regenerated bone is similar along the biopsies. The same differences between H‐B and L‐B were observed in all VOIs. Complete data including mean/median, SD, 95% CIs, and estimated differences are reported in Table S3b (Data S1).

Regarding the jaw of interest, mandibular sites showed significantly higher values of TbN (p = .028) and significantly lower values of TbSp (p = .029), compared to maxillary sites. Similar results were observed for low‐mineralized bone (p = .010 and p = .018), but not for high‐mineralized bone where the same trend was not confirmed statistically. Complete data are reported in Table S4a (Data S1).

Finally, in regard to clinical bone density, in soft density, the values of TbN were lower and the values of TbSp were higher, compared to medium and hard density, and these significant differences were observed both in low‐mineralized bone and high‐mineralized bone. Values are fully reported in Table S4b (Data S1).

4. DISCUSSION

The improvement in CAD/CAM technology, with the possibility of modeling the prosthesis according to the material used and the site of implant, optimizing geometry and implant characteristics, has made a huge contribution to optimizing osteointegrative processes in orthopedics. The evaluation of the effectiveness of these new tools greatly benefits from advanced technologies such as micro‐CT. The relevance of the technique in both orthopedic and dental fields is now evident, as it enables the integration and advancement of studies ranging from bone tissue engineering to restorative dentistry, including research on the mineral density of hard tissues and bone growth. Most micro‐CT analysis data relating to customized meshes currently recorded in the literature were evaluated with pre‐clinical studies. Recently, Paeng et al. (2022) analyzed the added effect of collagen membrane in the treatment of peri‐implant defects with titanium meshes on dogs, using both micro‐CT and histomorphometric analysis after 16 weeks of healing. Although no statistically significant differences were observed because of the small sample size, the authors concluded that comparable results were observed both with and without the use of CM and/or BS. Li et al. (2018) in a comparative study, showed how the combination of collagen membrane and titanium mesh could repair peri‐implant bone defects in dogs, with a major bone regeneration rate compared to titanium mesh or collagen alone.

The present clinical study has been developed in continuity with a previous one (Cucchi, Vignudelli, Sartori, et al., 2021) where micro‐CT analyses on clinical samples were employed to compare guided bone regeneration using PTFE membranes versus titanium meshes plus collagen membranes.

To have a comprehensive investigation of the influence of collagen membrane on bone regeneration, the authors in the present study decided to perform a standardized micro‐CT analysis subdividing the biopsies for every millimeter of depth (VOI‐1, VOI‐2, VOI‐3, VOI‐4, VOI‐5) in order to detect any progression in the bone growth/graft material resorption. The image was segmented by applying thresholding, which made it possible to differentiate not only the material graft from the bone tissue, but also to distinguish low‐mineralized and high‐mineralized bone, in order to provide an effective estimate of the bone remodeling, which had occurred. This is analogous to what is done in histological analyses where, by means of specific staining techniques, it is possible to discriminate among bone, graft, and connective tissue.

The hypothesis of the study was confirmed, and the two groups showed similar percentages of bone tissue (29.8% M− and 30.8% M+). The percentages of non‐mineralized tissue (NMT) and grafting material (MATV) were also similar in the two study groups (25.1% in M− and 25.5% in M+) and for residual graft (14.9% in M− and 13.1% in M+).

The results obtained from the histomorphometric study (Cucchi et al., 2024) are consistent with those of the present paper, showing comparable values between the two methods.

In group M−, bone tissue and grafting material were 34.3% and 11.5% in histological analysis, similar to micro‐CT results (29.8% and 14.9%, respectively); correspondingly, in group M+, the same parameters were comparable (35.3% and 14.6% in histological analysis vs. 30.8% and 13.1% in micro‐CT, respectively).

The consistency between the results obtained in the two studies provides important feedback on the validity of the chosen methodology, that is, on the possibility of applying image thresholds corresponding to different levels of mineralization of the bone tissue. Moreover, performing a 3D analysis by micro‐CT, with respect to what usually carried out by histology that is limited to 2D, makes the evaluation of the experimental outcomes more exhaustive.

The evaluation of all the data collected, with the relative significant differences, has led to some considerations that will require in‐depth analysis in further research: regardless of the use of collagen membrane, new bone formation was obtained in both groups, even if the combination TM + CM showed some advantages over the regeneration process. In particular, distinguishing between M− and M+ and without considering the VOIs, the distribution of tissue composition was similar in the two study groups but M+ showed a trend favorable in terms of slightly more bone formation and less residual graft material.

Then, the analysis performed in each VOI showed a slightly higher percentage of bone tissue (AB) in group M+, mainly due to the difference in low‐mineralized bone rather than in high‐mineralized bone, while the residual biomaterial (MATV) was slightly superior in group M−; the typical trend of progressive reduction from the most coronal VOI (VOI‐1) to the most apical one (VOI‐5) was observed in both groups.

It is to note that these results coming from the thresholding between “low‐mineralized” and “high‐mineralized” tissues should be considered for relative comparisons between the two study groups more than as absolute values. To enhance data description and analysis, these values were always coupled with “all bone” analysis.

Since micro‐CT image high grayscale values do not necessarily indicate the presence of new bone tissue, as these values obtained from micro‐CT scans can be influenced by various factors, including bone density, bone quality, and image processing, the aim of micro‐CT analysis was not to distinguish “new bone” and “old bone,” but to just subdivide the vital bone tissue into “high‐mineralized bone” and “low‐mineralized bone.” Then, it is to remember that sites augmented using GBR approach usually show newly formed bone in the space under membranes/meshes; this bone formation can be considered completely different respect to sites augmented using onlay/inlay graft approach that show the revascularization of grafted bone and the remodeling of old bone into new bone (Chiapasco et al., 2013; Laino et al., 2014; Silva et al., 2017; Spinneto et al., 2013).

The correlation between bone formation and bone density was also evaluated. In the case of soft density, the formation of new bone showed lower mean values, both regarding bone volume, bone surface, and number of traversals, while 3D space between trabeculae was higher in soft‐density bone.

The present study represents the first study reporting micro‐CT analysis data after GBR both in the mandible and in the maxilla. Consequently, an interesting comparison between maxillary and mandibular surgical sites was performed.

A statistically significant difference was found in terms of higher percentage of bone tissue (AB and L‐B) in mandibular sites when compared to maxillary sites after 6 months of healing. This finding may support and influence surgeons' decisions in clinical practice, establishing a difference in regenerated bone quality in the maxilla and in the mandible.

The trabecular analysis performed in each VOI did not show any significant difference either between the two study groups or between the progressive VOIs. Thus, tissue morphometry of regenerated bone is similar along the biopsies.

The trabecular analysis performed in the jaw of interest revealed that the trabecular density (TbN) was larger in the mandible than in the maxilla, both in low‐mineralized bone (L‐B) and in all bone (AB); on the other hand, 3D space between trabeculae (TbSp) was more widely represented in the maxilla, both in low‐mineralized bone and in all bone.

The present work is part of an activity of the research group that is consolidating the use of micro‐CT to investigate outcomes of different regenerative approaches, making the analysis that is conducted increasingly detailed and sophisticated, exploiting tools that can be easily applied in analogous studies of bone regeneration.

When compared to our previous study about micro‐CT analysis of GBR procedures (Cucchi, Vignudelli, Franceschi, et al., 2021), the present work showed some changes. In more detail, both mandibular and maxilla, either anterior or posterior surgical sites were included. Moreover, in the 50:50 bone mixture created to fill the titanium mesh, allograft was replaced with xenograft: this biomaterial has longer reabsorption times but is not osteoinductive. Finally, bony tissue biopsies were performed 3 months earlier (6 instead of 9 months after bone regeneration).

The main peculiarity of the present publication is the aim of the study. In fact, the present study was the first human study involving a micro‐CT investigation of the role of resorbable membrane over titanium mesh for the improvement of bone regeneration, comparing the extent of regeneration with and without the presence of membranes. However, some drawbacks make it difficult to draw firm conclusions. A higher number of specimens should be collected and analyzed to obtain a statistical significance for the trends described above. Biopsies showed great variability attributable to different surgical sites (mandibula/maxilla, anterior/posterior) and varying widths of the defects (large/small). Furthermore, the sample size used was based on the primary outcome of the randomized clinical trial. Lastly, it was not possible to determine whether the differences obtained between the two study groups were influenced by the potential healing of each patient.

The present study also has some strengths: specimens were examined investigating bone morphology and microstructure, on precise 3D images with high, micrometric, spatial resolution. Micro‐CT provides fundamental information, given that the mechanical properties of bone largely depend on its 3D architecture (Karim & Vashishth, 2011), and, moreover, is not operator‐dependent, because values are based on a gray scale (González‐García & Monje, 2013a, 2013b; Monje et al., 2014; Rebaudi et al., 2004; Zou et al., 2011).

The study was designed as a randomized triple‐blinded comparative clinical trial. Treated sites included both maxillary and mandibular defects: the presence of sites with differences in bone density and in osteogenetic potential allowed evaluation of the bone regeneration under different conditions. Finally, the analysis included numerous parameters. It was conducted for the whole specimen and for subdivided VOIs, giving a comprehensive evaluation of the regenerated bone.

5. CONCLUSIONS

The results of this non‐inferiority randomized clinical trial showed that customized titanium meshes are effective for new bone formation both with and without resorbable membranes; these results have been confirmed by both biopsy composition and morphometry using micro‐CT. The application of resorbable membrane over a customized CAD/CAM titanium mesh did not significantly influence the amount of bone regeneration; however, a trend toward more favorable results was observed when a membrane was applied (more bone tissue and fewer residual particles of graft material). Furthermore, clinical investigations with a higher number of patients are needed to assess the role of resorbable membranes applied over titanium mesh and draw firm conclusions.

AUTHOR CONTRIBUTIONS

Alessandro Cucchi: Conceptualization; investigation; validation; resources. Gregorio Marchiori: Investigation; methodology; validation; writing – review and editing; visualization. Maria Sartori: Methodology; writing – review and editing. Milena Fini: Resources; writing – review and editing. Antonino Fiorino: Formal analysis; writing – original draft; software; visualization. Raffaele Donati: Writing – original draft; writing – review and editing. Giuseppe Corinaldesi: Methodology; data curation; writing – review and editing. Melania Maglio: Investigation; writing – original draft; methodology; validation; visualization; data curation.

CONFLICT OF INTEREST STATEMENT

The authors declare that no potential conflict of interest exists.

INFORMED CONSENT STATEMENT

Informed consent was obtained from all subjects involved in the study.

Supporting information

Data S1:

CLR-35-1546-s001.zip (454.5KB, zip)

ACKNOWLEDGMENTS

The authors would like to express sincere gratitude to Dr. Elisabetta Vignudelli (E.V.) and Dr. Emanuele Randellini (E.R.) for their invaluable contributions to the initial phase of this study protocol (T0 and T1). Osteogenics Biomedical donated the bone xenograft and collagen membrane and Biotec BTK donated the meshes used in this study. Data belonged to the authors; the Osteogenics Biomedical and Biotec BTK were not involved in the trial and in the analysis execution or publications of results. Open access funding provided by BIBLIOSAN.

Cucchi, A. , Marchiori, G. , Sartori, M. , Fini, M. , Fiorino, A. , Donati, R. , Corinaldesi, G. , & Maglio, M. (2024). A 3D micro‐CT assessment of composition and structure of bone tissue after vertical and horizontal alveolar ridge augmentation using CAD/CAM‐customized titanium mesh. Clinical Oral Implants Research, 35, 1546–1559. 10.1111/clr.14342

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1:

CLR-35-1546-s001.zip (454.5KB, zip)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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