Detailed information about the study protocol and the procedures was recently published [12, 13]. The a priori sample size calculation was performed applying a one-way ANOVA considering the data in Ru et al. [27].

The animal trials were authorised by the Governmental Animal Care and Use Committee (Reference No.: 81–02.04.2018.A342; Landesamt für Natur, Umwelt und Verbraucherschutz Recklinghausen, Nordrhein-Westfalen, Germany; date: January 11, 2019) and were conducted in agreement with the German Animal Welfare Law (Tierschutzgesetz, TSchG) and the European Union Directive 2010/63/EU. The study was performed according to the ARRIVE Guidelines [26] and the Guide for the Care and Use of Laboratory Animals.

In this follow-up investigation, the rats were retrospectively analysed after alveolar cleft repair with either autograft or human xenograft (N = 6 per group). In one group, the cleft repair was performed with autologous bone from the hip, while in the other group, human xenogeneic bone substitute (maxgraft, Botiss Biomaterials, Zossen, Germany) was used.

At the time of the artificial maxillary cleft creation, the rats were 8 weeks old and had a mean average weight of 465 ± 34 g, while after another 4 weeks the cleft repair was performed. Finally, four weeks after the maxillary reconstruction, the orthodontic tooth movement intervention started and lasted for 8 weeks (Fig. 1A–C). At the end of the experimental procedures, the animals were 16 weeks old with an average body weight of 542 ± 32 g. Subsequently, the animals were euthanised by cervical dislocation under general anaesthesia.

View of the surgical site on the left maxilla and palate. Top: tongue base; bottom: mouth tip. A Preparation of the artificial alveolar cleft in the front of the first molar on the left side of the rat’s maxilla with an ultrasonic device. B Maxillary cleft repair was performed with an autograft from the ischial tuberosity of the hip or (C) a human xenograft. D Orthodontic appliance based on a 0.14 N nickel/titanium closed coil tension spring fixed between the first molar and the incisors

Cleft creation, maxillary reconstruction and orthodontic device placement were performed under general anaesthesia with a cocktail of ketamine (80–100 mg/kg, i.p.), medetomidine hydrochloride (0.15–0.25 mg/kg, i.p.) and buprenorphine (0.03–0.05 mg/kg, s.c.). Antibiotic administration (cefuroxime: 15 mg/kg, s.c.) started after the operation at a 24-hour interval for seven consecutive days. Atipamezole hydrochloride (0.75 mg/kg, i.p.) was administered to support the recovery process, and buprenorphine (0.03–0.05 mg/kg, s.c.) was given for maximum 5 days when necessary.

The required radiological examinations were performed in vivo using a μCT system (U-CT OI, MILabs, Utrecht, the Netherlands) at three time points: immediately after the jaw reconstruction (T0), 4 weeks after cleft repair and before the initiation of the orthodontic tooth movement (T1), and 12 weeks after cleft repair or 8 weeks of orthodontic tooth movement (T2).

The radiology parameters were the following: ultra-focus magnification and rotation of 360° at an increment of 0.75° with 0.3 s/degree. The data were reconstructed at an isotropic voxel size of 40 μm. The Micro-CT data were down-sampled to a voxel size of 80 μm. The images of cross-sectional slices were rendered to 3D iso-surfaces. For the analysis of the reconstructed maxillae and the root resorption of the first molars, both regions were segmented in micro-CT images using all the anatomical planes.

For the bone analysis, a coat with a fixed 10-voxel thickness was calculated around the segment using the morphological operation [28]. Then, the bone tissue was segmented within the coat’s volume via thresholding. The reconstructed maxilla and the surrounding bone were then analysed for BMD, BV/TV, Tb.Th and Tb.Sp (Fig. 2A–D). Radiological changes (Δ = T2-T0) within the reconstructed part of the maxilla were defined by the difference among these measurements.

Radiological imaging after cleft repair. A The autologous bone (in green) in three-dimensional (3D) micro-computed tomography (micro-CT) volume rendering (B) in the transverse, (C) coronal and (D) sagittal planes. (E) Three-dimensional reconstruction of the extracted teeth before (in purple) and (F) after (in yellow) the orthodontic treatment. The white arrows point to root resorption signs

For the root analysis, all roots were individually delineated, and their volumes were calculated for all three measurements (T0–T2). The root resorption was analysed by subtracting the root volume at T2 from the root volume at T1 (Fig. 2E–H).

After resection of the affected part of the maxilla including the orthodontically moved first molar, the samples were stored in 4% formalin (Otto Fischar GmbH & Co. KG, Saarbrücken, Germany), followed by decalcification in a 20-fold volume of ethylenediaminetetraacetic acid (EDTA, MolDecalcifer, Menarini, Florence, Italy) for 4 weeks at 37 °C. Afterwards, the samples were deposited in 5% sucrose/phosphate-buffered saline for 24 h, followed by shock freezing in liquid nitrogen and finally embedded (TissueTek, Sakura, Alphen, Netherlands).

Subsequently, longitudinal sections through the tooth and the surrounding hard and soft tissue or cross-sections from the area immediately in front of the first molar (all sections were 7 μm thick) were collected and fixed on super frost slides for drying. Then, the samples were submerged in acetone for 10 min and stained with toluidine blue, according to a standard protocol. The specimens were observed under digital microscopy with software support (OLYMPUS digital microscope DSX-1000, Olympus Hamburg, Germany).

The region of the augmented bone, the newly formed bone, and the interior and exterior of the augmented substitutes were observed to evaluate the osseous build-up or the bone substitutes that were still present (Fig. 3). The amount of root resorption was defined as the area between the intact parts of the root surfaces (Fig. 4).

Toluidine blue staining of the reconstructed jaw after cleft repair and orthodontic tooth movement. Microscopy imaging of (A) the autologous bone and (B) the xenogeneic/human bone. Representative radiological slices on the transverse plane for (C) the autologous and (D) the xenogeneic/human bone

Toluidine blue staining of longitudinal sections of the first molar and the surrounding hard and soft tissue after cleft repair and orthodontic tooth movement. A The autologous and (B) xenogeneic/human bones. Representative radiological slices on the transverse plane for (C) the autologous and (D) the xenogeneic/human bone

The Shapiro-Wilk test was applied to confirm the normal distribution of the data. Statistical comparisons between the groups were performed with the unpaired nonparametric Mann-Whitney test with compared ranks. The correlations between the radiological (BMD, BV/TV, Tb.Sp and Tb.Th) and the histological measurements to determine the newly formed bone (mm2) and the resorption lacunae (mm2) were analysed with the Pearson correlation coefficient (r). The latter was also used to study the correlation between the radiological and histological determination of root resorptions. All analyses were performed on Prism (version 8, GraphPad Software Inc., La Jolla, CA, USA). The level of significance was set at p ≤ 0.05. All results are expressed as mean ± standard deviation (SD).