Ridge preservation using octacalcium phosphate collagen to induce new bone containing a vascular network of mainly Type H vessels | Scientific Reports

Scientific Reports volume 14, Article number: 25335 (2024) Cite this article

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Many studies have shown that it is important to use bone grafts that are easy to mold, bioabsorbable, and stable over time. We focused on Type H blood vessels, which were discovered by Kusumbe et al. in 2014 to be responsible for the interaction between angiogenesis and osteogenesis. The aim of this study was to assess the effect of octacalcium phosphate collagen (OCP/Col), on the healing processes of the extraction socket and the alveolar bone surrounding the extraction socket. Ridge preservation of rat lower first molars was conducted using OCP/Col, and a series of experiments involving micro-CT scanning, observations of new bone, bone morphometry measurements, histological and immunohistochemical analyses, and second harmonic generation imaging were conducted to analyze bone mass, bone quality, angiogenesis, and mechanical properties. The results demonstrate that the calcification level was not very high when using OCP/Col for RP. Moreover, the newly formed bone is rich in vascular components and collagen fibers that are essential for bone tissue remodeling. These characteristics of OCP/Col in RP could contribute significantly to the construction of a rich vascular network around dental implants immediately after implant placement and the subsequent acquisition of osseointegration and reconstruction of the surrounding tissue.

Maintaining appropriate alveolar bone height and width is crucial to the smooth progress of prosthetic and orthodontic treatment 1,2,3,4,5,6. However, tooth extraction results in alveolar bone resorption in both the vertical and horizontal directions 7,8. “Ridge preservation” (RP) is a surgical treatment with the goal of protecting the alveolar ridge that is conducted at the same time as tooth extraction to prevent post-extraction alveolar bone resorption and maintain the width and height of the alveolar ridge 9,10,11. The efficacy of RP has already been demonstrated in numerous systematic reviews and meta-analyses, and it is used extensively in clinical practice worldwide. Many reports have described its value for maintaining bone prior to implant insertion, as pretreatment prior to cosmetic prosthetic treatment, and in orthodontic treatment involving tooth extractions 12,13,14,15,16,17,18. The graft material used for RP may be autologous bone, either an allograft or xenograft, or artificial bone 14. The bone graft materials come in the form of blocks, small pieces, granules, and others. Autologous bone possesses the three factors required for bone regeneration (osteogenic potential, osteoinductivity, and osteoconductivity), and is thus still the gold standard for bone graft materials 17. However, Wu et al. previously reported that autologous bone graft was limited by factors including the surgical invasiveness of harvesting, limitations on the amount that can be harvested, and post-grafting bone resorption 19.

For this reason, in recent years many clinicians have been using artificial bone grafts such as Bio-Oss (bovine sintered bone: Geistlich Sons Ltd., Wolhusen, Switzerland), hydroxyapatite (HA), and beta-tricalcium phosphate (β-TCP) as alternatives to autologous bone 20,21. Bio-Oss has excellent osteoconductivity and morphological stability, but its nature as bovine inorganic bone granules means that its replacement by bone tissue is extremely slow 22. HA bone graft materials are also only replaced by bone tissue very slowly, like Bio-Oss. However, both Bio-Oss and HA bone graft materials have the advantage that the bone that is eventually formed is maintained long-term. The distinctive features of β-TCP bone graft materials include their osteoconductivity and bioresorbability, low osteogenic potential, and gradual replacement by bone 23. Artificial bone grafting materials suitable for use in RP must be easily shaped, have good bioaffinity with the surrounding tissues, and have high bioresorbability 24,25. Kouketsu et al. reported that the rapid acquisition of both bone mass and bone quality by the new bone forming around the bone graft material is important for post-RP healing of the extraction socket. They also suggested that the capacity to induce abundant blood vessels within the new bone may be important for subsequent bone remodeling 26. Kusumbe et al. discovered Type H vessels in 2014, where the H stands for high expression of CD31 and Endomucin, and Type H vessels are known to express bone markers such as Runx2 and OSX around Type H vessels 27,28. On the other hand, Yan et al. demonstrated that Type H vessels regulate bone remodelling in extraction socket healing 29. Type H vessels have been reported to play a role in the interaction between osteogenesis and angiogenesis. Therefore, it is possible to determine the effect of graft materials on bone formation by examining the percentage of Type H blood vessels.

In 2019, Toyobo Co., Ltd. combined octacalcium phosphate (OCP) with medical-grade collagen to develop OCP/collagen (OCP/Col) as a new composite bone graft material 24,25. OCP is a precursor of bioapatite that can be synthesized artificially. This OCP has been shown to promote differentiation into osteoprogenitor cells and to possess excellent bioresorbability. Kouketsu et al. have reported that, in the rat calvarium, OCP/Col possesses osteoinductivity, as well as osteoconductivity, and that angiogenesis during the early healing period and replacement with bone tissue in the later healing period were both faster than those for other bone graft materials 26. Kibe et al. and Matsui et al. used OCP/Col for cleft jaw bone grafting, and they reported that good bone regeneration was achieved 30,31. However, much remains unknown about the effect of the use of OCP/Col on the healing process in the extraction socket after RP.

We therefore hypothesise that OCP/Col-applied RP promotes Type H vessels formation and better healing. The objective of this study was to investigate the effect of OCP/Col on the healing processes of the extraction socket and the alveolar bone surrounding the extraction socket. Therefore, we conducted RP of the rat lower first molar using OCP/Col, analyzed bone mass and bone quality, and conducted quantitative assessments of angiogenesis and mechanical properties.

The animals used in this study were male Wistar rats (4 weeks old, mean weight 80 g, n = 180). The rats were housed under a 12-h light/dark cycle with ad libitum access to food and water. Animal care and maintenance and experimental handling were approved by the Ethics Committee of Tokyo Dental College (approval no. 223101) and conducted in accordance with the animal care guidelines of the institution. The authors have complied with the ARRIVE guidelines for the reporting of research involving animals.

At each age in days, rats were randomly selected for use in a control group (CTL group: n = 6) and four experimental groups, an OCP group (n = 6) and a Collagen group (n = 6) and an OCP/Col group (n = 6) and a β-TCP group (n = 6). The rats in the CTL group underwent lower first molar extraction only, and those in the experimental groups underwent lower first molar extraction followed by RP. The bone graft material used were OCP (Toyobo Co., Ltd., Tokyo, Japan), Collagen (Toyobo Co., Ltd., Tokyo, Japan) and OCP/Col (Bonarc®, artificial bone using collagen, Toyobo Co., Ltd., Tokyo, Japan) and β-TCP (Osferion, Olympus, Tokyo, Japan) (Fig. 1a).

(a) Experimental protocol. The CTL group, in which only lower first molar extraction was conducted, is compared with the experimental groups (OCP/Col and β-TCP groups) that underwent RP. Animals were euthanized on Days 0, 1, 4, 7, 14, and 28. (b) Designation of axes and regions of interest. (c) Method of calculating the extraction socket alveolar bone width and height: The maximum width of the extraction socket is measured, and the height is measured along a line perpendicular to the midpoint of the maximum width of the extraction socket. (d) Method of calculating the percentage of the surface area occupied by positive cells: The area of a specific part of the lower or upper part of the lower first molar mesiodistal root extraction socket is taken as the total area. The area occupied by cells staining positive for each antibody is visualized using Image J. The percentage of the surface area occupied by positive cells is calculated by dividing the total area by the area occupied by cells staining positive for each antibody.

The surgical procedure was conducted under general anesthesia induced by the intraperitoneal administration of a mixture of three anesthetics (medetomidine hydrochloride 0.75 mg/kg, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan); midazolam 4.0 mg/kg, Sandoz K.K., Tokyo, Japan; and butorphanol tartrate 5.0 mg/kg, Meiji Seika Pharma Co., Ltd., Tokyo, Japan). Lower first molar extraction was performed using a dental explorer. As RP, after the lower first molar had been extracted, the mesiodistal root extraction socket was filled with one of the bone graft materials (OCP, Collagen, OCP/Col, β-TCP) and covered with a wafer sheet. After the completion of the procedure, the rats were immediately administered a medetomidine antagonist (atipamezole hydrochloride 0.75 mg/kg, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) intraperitoneally to maintain their body temperature. On Days 0, 1, 4, 7, 14, and 28 after the procedure, 6 rats in each group were euthanized, and their mandibles were harvested. After mandibular harvesting was complete, bone specimens including the lower first molar socket were harvested and fixed by immersion in 4% phosphate-buffered paraformaldehyde for 2 days at 4 °C.

As reference axes, the mesiodistal axis of each specimen was designated the X-axis, the direction perpendicular to the inferior border of mandibular plane the Y-axis, and the buccolingual direction the Z-axis. ROIs were designated in the lower first molar mesial root and distal root extraction sockets (Fig. 1b). Microscopic computed tomography (micro-CT) images and slices were prepared in the YZ plane.

All samples were scanned by micro-CT (μCT-50, Scanco Medical AG, Wangen-Brüttisellen, Switzerland). Scanning was performed under the following conditions: tube voltage 90 kV; tube current 155 μA; image matrix 3400 × 3400; and slice thickness 2 μm. Three-dimensional (3D) images were reconstructed by the volume-rendering technique using 3D reconstruction software (TRI/3D-BON, Ratoc System Engineering, Tokyo Japan).

The width and height of the buccal-side and lingual-side alveolar bone in each group on post-extraction Days 7, 14, and 28 were measured using micro-CT analysis software (μCT Analysis Software, ScancoMedical AG), and the resorption rate (%) for the alveolar bone width and height at the extraction socket at each time point was calculated according to the following formulae (Fig. 1c).

Extraction socket alveolar bone width resorption rate (%) = (1 − extraction socket alveolar bone width (mm)/dentulous mandible alveolar bone width (mm)) × 100.

Extraction socket alveolar bone height resorption rate (%) = (1 − extraction socket alveolar bone height (mm)/dentulous mandible alveolar bone height (mm)) × 100.

The new bone formation process in the mesial and distal extraction sockets on post-extraction Days 0, 4, 7, 14, and 28 was observed on micro-CT images, and bone mineral density (BMD) measurements and morphological measurements (BV/TV, Tb.Th, Tb.N, and Tb.Sp) on ROIs were made using the built-in software. β-TCP and OCP granules are excluded in the measurement.

The specimens were fixed in 4% phosphate-buffered paraformaldehyde at 4 °C and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) at 4 °C for 3 weeks. They were then embedded in paraffin by the usual method, and after 4-μm-thick slices had been prepared, hematoxylin–eosin (H-E) staining was conducted.

The immunohistochemical evaluation was conducted using the consecutive thin slices also used for histological examination. Runx2 immunohistochemical staining was conducted to evaluate bone lineage factors and vascular endothelial growth factor (VEGF), endomucin, and CD31 for the assessment of blood vessel lineages. Anti-VEGF antibody (dilution 1:200, Proteintech, Illinois, USA: 19003-1-AP), anti-endomucin antibody (dilution 1:100, Novus Biologicals, Colorado, USA: NBP3-03543), anti-CD31 antibody (dilution 1:500,Abcam, Cambridge, GBR: ab182981), and anti-Runx2 antibody (dilution 1:100,GeneTex, California, USA: GTX00792) were used as primary antibodies. The paraffin slices were immersed in xylene and ethanol in that order, washed with phosphate-buffered saline (PBS) to remove paraffin, and then autoclaved (95 °C, 10 min) as a heat-induced epitope retrieval and washed again with PBS. Next, blocking treatment was performed using 0.3% hydrogen peroxide in methanol, followed by washing with PBS. After blocking nonspecific reactions with skim milk, the primary antibody was added and incubated at 4 °C over night. The cells were then washed with PBS and Histofine Simple Stain Rat MAX-PO (Rabbit) (Nichirei Bioscience Co., Ltd., Tokyo, Japan), colored with DAB (Funakoshi Co, Ltd., Tokyo, Japan), and hematoxylin was used for contrast staining. After washing with water and dehydration with alcohol, the specimens were permeabilized with xylene and treated for encapsulation.

The ROIs were then further subdivided into the upper and lower parts of the extraction socket, and the percentages of the surface area occupied by VEGF-positive cells, endomucin and CD31-positive cells, and Runx2-positive cells were calculated using an analytical tool (Image J, National Institutes of Health, Bethesda, MD, USA) for quantitative evaluation (Fig. 1d).

For image analysis of the cryosections, the dissected mandibular was decalcified using 20% Morse solution (Fujifilm Wako PureChemical, Osaka, Japan) for 24 h at 4 °C (or used without decalcification) and subsequently incubated with 10%, 20%, and 30% sucrose solutions for more than 2 h at 4 °C for cryoprotection. The samples were then embedded in super cryo-embedding medium (Section-Lab, Hiroshima, Japan). The cryosections of 14 µm thickness were cut according to Kawamoto’s film method using a Cryofilm type IIIC and a tungsten carbide knife (Section-Lab, Hiroshima, Japan) .The samples were pre-treated with 0.25% Triton X-100 at 24 ± 2 °C for 15 min and incubated with primary antibodies overnight at 24 ± 2 °C. Anti-CD31 antibody (dilution 1:100, R&D Systems, USA: FAB3628G) and anti-Endomucin antibody (dilution 1:400, Thermo Fisher Scientific, USA:PA5-115178) was used for primary antibodies. It was then incubated with secondary antibodies for 2 h at 24 ± 2 °C, followed by a drop of DAPI, mounted in 30% glycerol, covered with a coverslip and sealed with nail polish. Alexa Fluor 555 conjugated-anti-rabbit IgG (dilution 1:500, CST, USA:4413S) was used as secondary antibody and Hoechst 33342 (dilution 1:1000, Thermo Fisher Scientific, USA:H3570) as DAPI. Z-stacks of images were obtained at 1 µm intervals between sections of 14 µm thickness. Fluorescence images were acquired using laser scanning confocal microscopes (LSM 510 and LSM 880, Axio Observer, Oberkochen, Germany) equipped with Plan-APOCHROMAT (20 × /0.8) and ZEN 2.3 black edition (Carl Zeiss) 32.

Second harmonic generation (SHG) images were acquired with a multiphoton confocal microscopy system (LSM 880 Airy NLO, Carl Zeiss, Oberkochen, Germany) with an excitation laser (Chameleon Vision II, wavelengths 680–1080 nm, repetition rate 80 MHz pulse width 140 fs; Coherent Inc., Santa Clara, CA, USA) and an objective lens (Plan-Apochromat 10 × /0.8 M27; Carl Zeiss). The excitation wavelength for collagen fiber observations was 880 nm. ZEN software (Carl Zeiss) was used for SHG image acquisition. After SHG image acquisition, the collagen fiber bundles in the ROIs were traced by using Imaris 8.4 (Bitplane AG, Zurich, Switzerland), and their diameters were measured.

For the statistical analysis, Tukey’s test was used to test the significance of differences between the mean values of each group, with p < 0.05 considered significant.

Micro-CT observations showed that, from post-extraction Day 7, regions exhibiting radiopacity increased in the extraction sockets in all groups (Fig. 2). On post-extraction Day 28, the radiopacity of the new bone was equivalent to that of the bone surrounding the extraction socket. In terms of radiopacity, in the OCP/Col group and OCP group, Collagen group, it was impossible to distinguish between the bone graft material and the new bone at any time point. In the β-TCP group, however, even on post-RP Day 28, it was possible to distinguish between new bone and β-TCP granules.

Micro-CT images of the lower first molar extraction sockets in each group and measurement results. (a) Micro-CT images of the lower first molar distal root extraction sockets. (b) Extraction socket alveolar bone width and height resorption rate (%) (c) BMD measurement results (*p < 0.05) (d) BV/TV, Tb.Th, Tb.N, and Tb.Sp measurement results (*p < 0.05).

The width resorption rate at each time point was significantly lower in both groups that underwent RP compared with the CTL group. BMD measurements showed that, on post-RP Day 7, BMD was significantly higher in the β-TCP group and OCP group than in the OCP/Col group, and on post-RP Days 14, it was significantly higher in the β-TCP group than in the control and OCP/Col and Collagen groups. BMD measurements of post RP Day28 showed significantly higher in the β-TCP group than in the other groups. Bone morphometry measurements showed that, on post-extraction Days 4 and 7, there was no significant difference between any of the groups. BV/TV was significantly higher in the OCP/Col group than in the other groups on post-extraction Days 14 and 28. Tb.Th was significantly higher in the OCP/Col group than in the other groups on post-extraction Days 14. On post-extraction Day 28, Tb.Th was significantly higher and Tb.Sp was significantly lower in the OCP/Col group than in the β-TCP group and OCP group. There was no significant difference in Tb.N between any of the groups.

The results of H-E staining showed that, on post-extraction Day 1, the extraction sockets in all the groups were filled with clotted blood. On post-extraction Day 4, the clotted blood had been replaced by granulation tissue. On post-extraction Day 7, new bone connected to the lower part and the lateral walls of the extraction socket was evident. On post-extraction Day 14, both the amount and thickness of new bone trabeculae in the extraction socket had increased. On post-extraction Day 28, the boundary between the new bone in the extraction socket and the surrounding alveolar bone became unclear (Fig. 3). Observations of the surroundings of the graft material on post-extraction Day 1 showed the presence of large amounts of blood cell components and inflammatory cell infiltration, mainly by neutrophils, surrounding the graft material in the OCP/Col and β-TCP groups (Fig. 4).

H–E staining of the lower first molar distal root extraction socket on Days 1, 4, 7, 14, and 28 (5 × magnification, scale bar 500 μm).

Magnified images of H-E-stained specimens of lower first molar distal root extraction sockets around the graft material on post-extraction Day 1 (40 × magnification, scale bar 50 μm. *OCP. → : Aterocollagen. β: β-TCP granules).

On post-extraction Day 4, abundant collagen fibers and large amounts of blood cell components were apparent, particularly in the OCP/Col group (Fig. 5). In the OCP/Col group, there was a large amount of glanulation tissues (GT) consisting of new blood vessels, undifferentiated mesenchymal cells, leukocytes, and collagen fibers.

Magnified images of H-E-stained specimens of glanulation tissues on post-extraction Day 4 (40 × magnification, scale bar 50 μm. → : Glanulation tissues).

The percentage of the surface area occupied by VEGF-positive cells was significantly higher in the OCP/Col group than in the control and β-TCP groups in both the upper and lower parts of the extraction socket (Fig. 6). The percentage of the surface area occupied by endomucin and CD31-double positive cells was also significantly higher in the OCP/Col group than in the β-TCP group.

Percentages of surface area occupied by positive cells in the upper and lower parts of the lower first molar mesiodistal root extraction socket (*p < 0.05) (a) Upper part of extraction socket (b) Lower part of extraction socket (c) Immunohistochemical staining image of each antibody in OCP/Col group (d) Confocal images of Endomucin (red), CD31 (green) and DAPI (blue) immunostained.

The percentage of the surface area of the upper part of the extraction socket occupied by Runx2-positive cells was significantly higher in the OCP/Col group than in the control and β-TCP groups.

Cryosections on day 4 post-extraction showed more positive Endomucin and CD31 findings in the OCP, Collagen, and OCP/Col groups than in the CTL and β-TCP groups.

On post-extraction Day 7, compared with the OCP/Col group, there was less new bone formation from the bundle bone in both the CTL group and the β-TCP group, and GT was still present (Fig. 7). A comparison of H-E staining of the extraction socket lateral walls on post-extraction Days 14 and 28 showed that, in the OCP/Col group, new bone with small intertrabecular gaps that contained abundant blood vessels was being created. In the OCP/Col group, almost all the GT had been replaced by new bone, and the majority of this new bone exhibited a lamellar bone structure with many intervening blood vessels. In the control and β-TCP groups, the replacement of GT by bone tissue was still incomplete on post-extraction Day 14, and new bone formation from the bundle bone was evident.

Magnified images of H–E-stained specimens of the lateral wall of the lower first molar distal root extraction socket from bundle bone (40 × magnification, scale bar 50 μm. BB Bundle bone, GT Glanulation tissues, NB New bone, BC blood cell, β β-TCP granules).

Figure 8 shows SHG images and the results of collagen fiber bundle diameter measurements. Collagen fiber bundles of thickness ≥ 375 nm are shown in light blue. Almost no thick collagen fiber bundles were observed in the control and β-TCP groups, but in the OCP/Col group, collagen fibers running alongside the trabecular bone were evident. A comparison of collagen fiber bundle diameters showed that they were significantly thicker in the OCP/Col group than in the control and β-TCP groups on Days 7, 14, and 28.

SHG imaging of the lower first molar mesiodistal root extraction socket. (a) SHG image of a lower first molar distal root extraction socket from the CTL group on post-extraction Day 28 (5 × magnification, scale bar 500 μm) (b) SHG image of a lower first molar distal root extraction socket from the OCP/Col group on post-extraction Day 28 (5 × magnification, scale bar 500 μm) (c) SHG image of a lower first molar distal root extraction socket from the β-TCP group on post-extraction Day 28 (5 × magnification, scale bar 500 μm) (d) Collagen fiber bundles diameters in the lower first molar mesiodistal root extraction socket on Days 7, 14, and 28 (*p < 0.05).

Schropp et al. reported that, in most cases in humans, decreases in alveolar bone height and width both occurred immediately after extraction 7. According to Arioka et al., alveolar bone resorption also occurs immediately after tooth extraction in rodents 33, and RP is considered to be effective for protection of the alveolar ridge after tooth extraction. In the present study, the post-extraction alveolar bone resorption rate was lower in both groups that underwent RP than in the CTL group, with the reduction in width in particular being significantly lower, demonstrating the effectiveness of RP. The resorbability of the different bone graft materials and their replaceability by bone were also investigated. OCP/Col and OCP, Collagen itself has low radiopacity, and after its use in the extraction socket, it was not detected at all by micro-CT analysis. However, β-TCP granules were clearly detected at every time point until post-extraction Day 28. This result is consistent with Kamakura et al.’s report that β-TCP persists at 12 weeks post-grafting, suggesting that long-term follow-up is therefore required after β-TCP use 34. On histological evaluation, the regular array of collagen fibers in the OCP/Col and the OCP were observable until Day 4, but from Day 7, both the OCP and collagen fibers were difficult to distinguish in the GT and new bone. OCP and collagen groups are difficult to identify even with micro-CT and are consistent with histological findings. These results suggested that the high replaceability of the OCP/Col stems from the fact that its collagen fibers quickly blend into the GT and disappear. Tanuma et al. and Jose et al. also used OCP/Col in canine calvarial defects, and they reported that, though the replacement rate was not 100%, it provided better replaceability than β-TCP or existing HA bone graft materials 35,36. Taken together with the present results, this supports the idea that the risk of inflammation due to residual foreign matter within the tissue is low.

In both the OCP/Col and β-TCP groups that underwent RP, a significantly greater amount of bone was evident on post-extraction Day 14 than in the CTL group. In particular, the BV/TV and Tb.Th values were significantly higher in the OCP/Col group than in the β-TCP group, whereas Tb.Sp was significantly lower, indicating that the bone was thicker, and the intertrabecular gaps were smaller. A comparison of H-E-stained images from the same sites also showed that, on Day 7, replacement of the GM with new bone had begun, and the extraction socket in the OCP/Col group was forming plexiform-like bone containing a large amount of vascular components. These results suggest that alveolar bone resorption was suppressed in the OCP/Col and β-TCP groups that underwent RP, and that new bone formation occurred earlier in the OCP/Col group than in the β-TCP group, indicating that rapid extraction socket healing can be expected. The increase in BMD in the OCP/Col group was more gradual than that in the β-TCP group, indicating that calcification occurred more rapidly in the latter group. Collagen fiber diameter was also significantly thicker in the OCP/Col group than in the other two groups. This indicated that an environment favorable to collagen calcification was created in the OCP/Col group. It can accordingly be inferred from the present results that, although a large amount of calcified new bone is formed as a result of RP using OCP/Col, its level of calcification is low, and the bone is rich in organic components.

Cardaropoli et al. conducted long-term histological observations of the healing process of canine extraction sockets, and they investigated each stage in detail 37. They reported that both the formation of sufficient GT and immediate replacement with new bone are essential to extraction socket healing, and they suggested that angiogenesis within the extraction socket may play an important role. Yan et al. showed that Type H vessels appear during the extraction socket healing process, and they reported that the new bone is generated around Type H blood vessels 27. The immunohistochemical evaluation conducted in the present study also showed that, in all groups, which underwent normal extraction, a vascular network including Type H vessels was formed within the GT of the extraction socket on post-extraction Day 4. On post-RP Day 4, the percentage of the surface area occupied by VEGF-positive cells was significantly higher in both the lower and upper parts of the extraction socket in the OCP/Col group, indicating the creation of an environment conducive to angiogenesis. The percentage of the surface area occupied by cells co-expressing endomucin and CD31 was also significantly higher, suggesting that the development of a Type H vascular network had started right at the beginning of the healing process, earlier than in the CTL group and the β-TCP group. Numerous Runx2-positive cells were also present close to these Type H vessels in the OCP/Col group, a result consistent with that reported by Kusumbe et al. 26. In addition, Collagen group strongly expressed Type H vessels in cryosection images taken 4 days after tooth extraction, indicating that Collagen is primarily responsible for vascular guidance in OCP/Col, and OCP promotes new bone formation.

In conclusion, this suggested that RP using OCP/Col may promote new bone formation by inducing a vascular network consisting mainly of Type H vessels in the extraction socket at an early stage.

The present results suggest that, though OCP/Col is somewhat inferior to other bone graft materials from the perspective of achieving initial fixation through the rapid formation of calcified bone immediately post-extraction, it induces the rich vascular network that is essential for bone tissue remodeling, giving it the great advantages of achieving rapid osseointegration after dental implant insertion and promoting the reconstruction of the surrounding tissue. Chung et al. reported that in orthodontic treatment involving tooth extraction, tooth movement is delayed due to new cortical bone formation in the healing of the extraction socket 38. Gölz et al. reported that gingival recession due to alveolar bone resorption during orthodontic treatment of extraction sockets can cause delay tooth movement 4. In other words, although we want to avoid alveolar bone resorption after tooth extraction in orthodontic treatment involving tooth extraction, we really do not want the new bone generated by RP to interfere with tooth movement. A characteristic of RP using OCP/Col is that the level of calcification is not very high, and the newly formed bone is rich in vascular components and collagen fibers. Ridge preservation using OCP/Col after tooth extraction for orthodontic treatment may thus suppress extraction-induced alveolar bone resorption and delay tooth movement.

All data generated and analyzed during this study are included in this published article.

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The authors thank Toyobo Co., Ltd. for providing the specimens used in the present study.

This study received research funding from Toyobo Co., Ltd. The study sponsor had no role in data collection, management, analysis; study design, conduct, or interpretation of study findings; or the preparation, review, or approval of the manuscript submitted for publication.

Oral Health Science Center, Tokyo Dental College, 2-9-18, Kandamisaki-Cho, Chiyoda-Ku, Tokyo, 101-0061, Japan

Naoki Kaida, Satoru Matsunaga, Keisuke Sugahara, Shinichi Abe & Yasushi Nishii

Department of Orthodontics, Tokyo Dental College, 2-9-18, Kandamisaki-Cho, Chiyoda-Ku, Tokyo, 101-0061, Japan

Naoki Kaida, Chie Tachiki & Yasushi Nishii

Department of Anatomy, Tokyo Dental College, 2-9-18, Kandamisaki-Cho, Chiyoda- u, Tokyo, 101-0061, Japan

Satoru Matsunaga & Shinichi Abe

Department of Oral and Maxillofacial Implantology, Tokyo Dental College, 2-9-18, Kandamisaki-Cho, Chiyoda-Ku, Tokyo, 101-0061, Japan

Yuto Otsu

Department of Oral Pathobiological Science and Surgery, Tokyo Dental College, 2-9-18, Kandamisaki-Cho, Chiyoda-Ku, Tokyo, 101-0061, Japan

Keisuke Sugahara & Akira Katakura

Department of Oral Ultrastructural Science, Tokyo Dental College, 2-9-18, Kandamisaki-Cho, Chiyoda-Ku, Tokyo, 101-0061, Japan

Norio Kasahara & Hitoshi Yamamoto

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Conceptualization, N.K., S.M. and Y.N.; methodology, N.K., C.T., S.M., K.S., Y.O., and Y. N.; data curation, N.K., C.T., K.S.,S.A., A.K. and Y.N.; writing—original draft prep-aration, N.K and C.T.; writing—review and editing, S.M., K.S., A.K. and H.Y.; visualization, N.K., Y.O., S.M. and N.Y.; supervision, C.T., H.Y., S.M., A.K. and Y.N.; project administration, C.T., S.M. and Y.N. All authors have read and agreed to the published version of the manuscript.

Correspondence to Satoru Matsunaga.

The authors declare no competing interests.

The experimental protocol of this study was approved by the Animal Welfare Committee of Tokyo Dental College based on the Animal Care Standards of this institution (approval no. 233101). This article does not contain any studies with human participants performed by any of the authors, so informed consent was not required.

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Kaida, N., Matsunaga, S., Tachiki, C. et al. Ridge preservation using octacalcium phosphate collagen to induce new bone containing a vascular network of mainly Type H vessels. Sci Rep 14, 25335 (2024). https://doi.org/10.1038/s41598-024-75931-y

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Received: 04 December 2023

Accepted: 09 October 2024

Published: 25 October 2024

DOI: https://doi.org/10.1038/s41598-024-75931-y

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