The study was approved by the Clinical Research Ethics Committees of the Academic Center for Dentistry of Amsterdam (code ACTA 202061), Vrije Universiteit Amsterdam, The Netherlands, and Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine (code SH9H-2019-T231-4), China. This trial was conducted following the international standard for clinical investigations with medical devices (ISO 14155:2020). Written informed consent was obtained from all participants.

A total of 40 patients were recruited in this study (15 in rhBMP-2/BioCaP/β-TCP group, 15 in β-TCP group, and 10 in the natural healing group). All patients met the selection criteria shown in Tables 1 and 2. The patients were randomly divided into the following three groups: rhBMP-2/BioCaP/β-TCP, β-TCP, and natural healing (kept unfilled) (controls). After tooth extraction, the socket was filled with either rhBMP-2/BioCaP/β-TCP (particle size 0.25–1 mm, made by Shanghai Rebone Biomaterials Co., Ltd.) or β-TCP (particle size 0.25–1 mm) β-TCP group, while the natural healing group was not filled with any CaP material. Two layers of collagen membrane (Geistlich Bio-Gide® bilayer collagen membrane, 25 × 25 mm) were used to cover the filling materials in the alveolar sockets of all patients. The soft tissue was sutured. Immediately after surgery, the first CBCT scan was taken and the GVs were recorded. Six weeks after the procedure, a second CBCT scan was obtained before placement of the implant. In addition, a biopsy of 2.3 mm in diameter and 6 mm height was taken using a trephine drill (3 and 2 mm, outer and inner diameters, respectively) at the same point of the implant insertion (Figs. 1, 2). The operation was completed when the implant was inserted and the sutures were removed after 2 weeks.

Schematic of clinical trial procedure. A The patients were randomly divided into rhBMP-2/BioCaP/β-TCP, β-TCP, and natural healing groups; B tooth extraction, C the tooth socket filled with either rhBMP-2/BioCaP/β-TCP or β-TCP in relative groups, D the first layer of collagen membrane covered the alveolar sockets and 2-3 mm over the socket edge), E the second collagen membranes, F sutured, G 2 weeks later, took out the suture, H after 6 weeks, soft tissue flap releasing, I a trephine drill (outer diameter 3 mm, inter 2.3 mm diameter) is used to obtain the biopsy, J collected biopsy (2.3 mm in diameter × 6 mm in height)

Intra-oral photographs of socket preservation and dental implant surgery. A The upper left first premolar needed to be removed. B The gingival biotype was a thick tissue biotype. C Tooth extraction. D The tooth socket was filled with rhBMP-2/BioCaP/β-TCP. E Two layers of collagen membrane covered the alveolar socket. F Sutured. G 6 weeks later. H Soft tissue flap releasing. I Obtained the biopsy and inserted an implant. J, K Guided bone regeneration (GBR) for the horizontal bone gain. L Sutured

The CBCT images were collected using the Planmeca 3D Imaging System (field-of-view of 8 cm (D) × 8 cm (H), resolution 0.16 mm, Planmeca, Finland). After being exported as digital imaging and communications in medicine (DICOM) files, the data were analyzed using planning software (Nobel Clinician, Nobel Biocare, Sweden) for GV measurement (Fig. 3). Three-dimensional images focused on the socket site to identify the central point of the pulp cavity at the enamel–cementum junction of the mesial and distal adjacent teeth were obtained. The connection of the two points helps to determine the coronal plane (Fig. 4A). The horizontal reference line was taken through the highest alveolar ridge of extraction fossae (crest of the alveolar ridge) (Fig. 4B). The buccal-lingual section followed the center line of the root of the tooth. Then the tooth-long axis was followed to identify the 3 mm point (GV measurement point) from the baseline, and the GV was measured based on the software function (Fig. 4C). A 3D model showed the 3D information (Fig. 4D), and the schematic diagram of the GV point is shown in Fig. 5. All measurements were performed by three independent investigators, and the GV change was calculated as the difference between baseline (GV1) and final GV obtained at 6 weeks after material filling (GV change = GV1 – GV2).

Data import. A Panoramic tomography. B Buccal-lingual section of the surgery area. C 3D model

The measurement point of GV. A Look for the central point of the pulp cavity at the enamel-cementum junction of the mesial and distal adjacent teeth. The connection of the two points helps determine the coronal plane. B The horizontal reference line was taken through the highest alveolar ridge of extraction fossae (crest of the alveolar ridge). C The buccal-lingual section followed the center line of the root of the tooth. Then the tooth-long axis was followed to find the 3 mm point (GV measurement point) from the baseline and measured the GV based on the software function. D 3D view

Schematic diagram of the identification of measurement point for GV. The GV measurement baseline is on the alveolar ridge’s crest. Following the long axis of the tooth, the red dot is a point for measuring bone density underneath 3 mm of baseline. The center part of the biopsy is the same as the GV-measured point of the CBCT image

Tissue biopsies were immersed in 10% neutral formalin solution for 24 h with the trephines.

Thereafter, samples were dehydrated with alcohol gradients after flushing, and embedded with polymethyl methacrylate. The samples were sectioned opposite to the long axis of the biopsy, and five tissue sections of 600 µm thicknesses (1 mm spacing) were collected for each piece, polished to a final thickness of 50–100 µm, and stained with McNeal’s tetrachrome staining. The Image Pro Plus program (version 6.0, Media Cybernetics) was used to calculate the area of new bone, the residual material, and the unmineralized tissue at the center slice of the biopsy (Fig. 6) The center part of the biopsy as the same as the GV-measured point of the CBCT image area was measured. Measurements were performed by three independent pathologists, and the mean value of these measurements was considered.

Light micrographs of biopsy slice (diameter 1.1 mm) in the natural healing group (A), rhBMP-2/BioCaP/β-TCPgroup (B), and β-TCP group (C) 6 weeks after implantation. Stained with McNeil’s Tetra chrome basic fuchsine and toluidine blue O. a Residual material, b new bone, c unmineralized tissue

Categorical data are expressed as frequencies and percentages, and continuous data as mean (± standard deviation, SD) or median and interquartile range (IQR) (25th–75th percentile). Analysis was performed in the per-protocol (PP) dataset, that is, all patients who were randomized, received the intervention, and completed the study procedures. A single-factor analysis of variance (ANOVA) was used to compare CVs changes among the three study groups. The Mann–Whitney U test was used to compare the area of residual material between rhBMP-2/BioCaP/β-TCP and β-TCP groups, and the Kruskal–Wallis test for the comparison of the new bone area and unmineralized tissue area among the three study groups. Interrater reliability was assessed with the Cronbach’s alpha (α) coefficient. Statistical significance was set at P ≤ 0.05. The IBM Statistical Package for the Social Sciences software (SPSS) (version 23.0) was used for the analysis of data.