Ridge Preservation with an Octacalcium Phosphate Collagen Complex (OCP/Col) Promotes New Bone Formation and Type H Vessel Distribution with Osterix Expression in Extraction Sockets in Mice

Ridge Preservation with an Octacalcium Phosphate Collagen Complex (OCP/Col) Promotes New Bone Formation and Type H Vessel Distribution with Osterix Expression in Extraction Sockets in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ridge Preservation with an Octacalcium Phosphate Collagen Complex (OCP/Col) Promotes New Bone Formation and Type H Vessel Distribution with Osterix Expression in Extraction Sockets in Mice Yojiro Koizumi, Satoru Matsunaga, Chie Tachiki, Toshihide Mizoguchi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9091395/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Purpose Alveolar bone resorption after tooth extraction leads to gingival recession and invagination, which may result in the stalling of tooth movement and difficulties in orthodontic tooth movement. Bone substitutes used for alveolar ridge preservation should exhibit bioaffinity, appropriate initial calcification, ease of handling, replacement by bone, long-term stability, and formation of an abundant vascular network. Therefore, this study focused on ridge preservation (RP) using an octacalcium phosphate collagen composite (OCP/Col). Type H vessels are known to appear at sites of active bone formation, and osteoblast markers are observed in their vicinity, creating a microenvironment conducive to osteogenesis. The objective of this study was to investigate the distribution of newly formed bone and Type H vessels in extraction sockets following ridge preservation with OCP/Col, as well as the expression of Osterix. Methods RP of the lower first molars in mice was performed using OCP/Col. Bone mass, bone quality, and angiogenesis were analyzed by micro-computed tomography, bone morphometry, and histological and fluorescence immunohistochemical staining. Results In RP with OCP/Col, the granularity of the bone substitute tended not to be preserved, and immature woven bone formation was observed, which prevented alveolar bone resorption. A microcapillary vascular network including Type H vessels was formed, and osteoblast markers were induced in the surrounding area, promoting new bone formation. Conclusions These findings suggest that RP with OCP/Col promotes early new bone formation associated with Type H vessels and may be beneficial for orthodontic treatment. Type H vessels ridge preservation bone substitute material bone quality analysis orthodontic tooth movement Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Maintaining the width and height of the alveolar bone after tooth extraction is vital for the smooth progress of prosthetic or orthodontic treatment [ 1 – 4 ]. In the normal healing process of the extraction socket, it is initially filled with a blood clot that is replaced with granulation tissue and the direct formation of new bone [ 5 ]. Because alveolar bone is the supporting bone for teeth, extractions may cause disuse atrophy, leading to alveolar bone resorption and gingival invagination [ 6 , 7 ]. In particular, the periodontal tissue changes of gingival recession and invagination associated with orthodontic extractions are known to have direct adverse effects on both cosmetic appearance and jaw function [ 8 – 11 ]. Ridge preservation (RP) is a surgical therapy used to maintain the alveolar bone post-extraction [ 12 ]. RP is also performed prior to dental implant treatment to enable early, strong bone formation in the extraction socket and its surroundings [ 13 ]. The bone substitutes used for RP include both xenogenic bone substitutes made from ground dried animal bone [ 14 – 15 ] and synthetic bone substitutes made from hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) [ 16 – 18 ]. Xenogenic bone substitutes derived from animal bone provide a scaffold that preserves space for new bone formation [ 19 ]. However, their replacement by newly formed bone is extremely slow, which may compromise the rate and quality of bone regeneration. Synthetic bone substitutes are highly osteoconductive and allow adjustment of their physicochemical properties [ 20 – 24 ]. Nevertheless, they exhibit variability in osteogenic potential and may require a prolonged period to integrate with newly formed bone, potentially delaying bone remodeling [ 24 ]. The octacalcium phosphate collagen complex (OCP/Col), developed by Kamakura et al., is a synthetic bone substitute with high osteoregenerative potential [ 25 ]. Kamakura et al. reported favorable bone formation following the use of OCP/Col in cranial defects and jaw clefts [ 26 ]. OCP/Col consists of octacalcium phosphate (OCP) combined with collagen (Col) [ 27 ]. OCP is a precursor of biological apatite and has been reported to promote differentiation of osteoprogenitor cells into osteogenic cells [ 27 ]. Animal studies by Kaida et al. suggested enhanced capillary formation in alveolar bone following RP with OCP/Col [ 28 ]. However, the mechanisms underlying the effects of RP with OCP/Col remain incompletely understood and require further investigation. Capillary vessels are more than just a constituent of granulation tissue in wound healing; they also play other important roles, including the removal of foreign substances and the supply of oxygen and nutrients to tissues [ 29 ]. Sun et al. reported that CD31 (PECAM-1) is a surface marker of vascular endothelial cells that is expressed at high levels at the junctions with neighboring endothelial cells [ 30 ]. CD31 is an adhesive stress-response protein that reportedly contributes to maintaining endothelial cell junctions, angiogenesis, and the rapid restoration of the vascular permeability barrier following inflammation [ 31 ]. Furthermore, endomucin, which is a mucin-like glycoprotein expressed in vascular endothelial cells, is also expressed in vascular endothelial cells of capillary vessels and veins, but it is not expressed in arteries [ 32 , 33 ]. Several capillary vessel subtypes within bone that differ both morphologically and functionally have been reported [ 34 , 36 ]. In the bone modeling process in the growth plates of mouse long bones, Type H vessels characterized by high expression levels of both CD31 and endomucin are abundant beneath the hypertrophic cartilage of the growth plate. Osterix-positive osteoprogenitor cells are observed in the vicinity of endothelial cells of Type H vessels, and these reportedly interact with each other via the exchange of growth factors to promote angiogenesis-osteogenesis coupling [ 34 , 35 ]. Yan et al. reported a significant increase in Type H vessels with concomitant perivascular accumulation of Runx2-positive osteoblasts and augmentation of bone mass [ 37 ]. We hypothesized that Type H vessels are formed within newly generated bone following RP with OCP/Col and that post-extraction bone formation is promoted by these vessels and surrounding osteoprogenitor cells. The aim of this study was to clarify how OCP/Col used as a bone substitute to fill extraction sockets promotes new bone formation during the early healing phase by investigating the distribution of Type H vessels generated in extraction sockets after RP with OCP/Col and the expression of osteoinductive factors around them. Materials and Methods Materials The experimental animals used were six-week-old male mice (mean weight 21.0 g). These mice were reared in a 12-h light/dark cycle with unlimited access to chow and water. The animal experiments were conducted with the authorization of the Tokyo Dental College Animal Experimentation Ethics Committee (approval no. 253102). Experimental procedure The surgical procedure was conducted under general anesthesia induced by the intraperitoneal administration of a mixture of three anesthetics (0.75 mg/kg medetomidine hydrochloride, Nippon Zenyaku Kogyo, Fukushima, Japan; 4.0 mg/kg midazolam, Sandoz, Tokyo, Japan; and 5.0 mg/kg butorphanol tartrate, Meiji Seika Pharma, Tokyo, Japan). In all mice, the lower right first molar was extracted with a dental probe. The CTL group underwent only lower right first molar extraction, and the experimental groups underwent RP after lower right first molar extraction. OCP/Col (Bonarc artificial bone with collagen, Toyobo, Tokyo, Japan), β-TCP (Osferion, Olympus, Tokyo, Japan), and Bio-Oss (Geistlich Sons Ltd, Wolhusen, Switzerland) were used as bone substitutes. The mice were randomly assigned to a CTL group (n = 12 per time point) and the different experimental groups (OCP/Col group: n = 12 per time point; β-TCP group: n = 12 per time point; and Bio-Oss group: n = 12 per time point). After lower right first molar extraction, the mesiodistal root extraction socket was filled with one of the bone substitutes for RP and covered with a wafer sheet. To reverse medetomidine-induced anesthesia, a medetomidine antagonist (0.75 mg/kg atipamezole hydrochloride, Nippon Zenyaku Kogyo, Fukushima, Japan) was administered intraperitoneally. The mice in each group were then sacrificed on Days 1, 4, 7, and 14, respectively (Fig. 1 A). Bone samples including the lower right first molar extraction socket were immediately collected and fixed by immersion in 4% paraformaldehyde phosphate buffer solution at 4°C for 2 days. Designation of reference axes and regions of interest (ROIs) The samples consisted of the portion of the mandible containing the extraction sockets of the first molar mesiodistal root at the front and the first molar distal root at the back, excised as a single block and trimmed. ROIs were designated in the area surrounding the lower right first molar mesial and distal root extraction sockets. The reference axes of the samples were designated as follows: the X-axis as the line joining the midpoints of the lines joining the left and right mandibles at the lowest points of the anterior and posterior thickened portions of the mandibles in the mesiodistal direction; the Z-axis as the buccolingual orientation with respect to the X-Y plane (Fig. 1 B); and the Y-axis as the direction perpendicular to the mandibular plane, which contains the line joining the lowest points of the anterior mandibles (a) and the lowest points of the posterior mandibles (p) (Fig. 1 C). Micro-CT scanning All samples were scanned by micro-CT (µCT-50, Scanco Medical AG, Wangen-Brüttisellen, Switzerland). The scanning conditions were as follows: tube voltage, 90 kV; tube current, 155 µA; image matrix, 3400 × 3400 and slice thickness, 20 µm. Two-dimensional slice images in the XZ, XY, and YZ planes were acquired by volume rendering using three-dimensional reconstruction analysis software (TRI/3D-BON, Ratoc System Engineering, Tokyo, Japan). Alveolar bone width and height Three-dimensional reconstruction and measurement software (µCT Analysis Software, Scanco Medical AG) was used to measure the alveolar bone width and height on the buccal and lingual sides of the lower first molar distal root extraction socket on Days 1, 4, 7, and 14 (Fig. 1 D). The alveolar bone width and height in the extraction sockets of each group were measured in the YZ plane on micro-CT images, and the resorption rates (%) were calculated as follows: Extraction socket alveolar bone width resorption rate (%) = [1 – extraction socket alveolar bone width (mm)/dentulous mandible (left) alveolar bone width (mm)] ⋅ 100 Extraction socket alveolar bone height resorption rate (%) = [1 – extraction socket alveolar bone height (mm)/dentulous mandible (left) alveolar bone height (mm)] ⋅ 100 Bone mineral density measurement and bone morphometry Bone mineral density (BMD) measurements and bone morphometry in the lower first molar mesial and distal root extraction sockets were conducted on post-extraction Days 1, 4, 7, and 14 using TRI/3D-BON (bone morphometric software). The following parameters were measured: bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Granular radiopaque areas with CT values ≥ 1500 were regarded as Bio-Oss or β-TCP and excluded from the ROIs. Histological assessments After fixation by immersion in 4% paraformaldehyde phosphate buffer solution at 4°C for 2 days, specimens were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 3 weeks. They were then embedded in paraffin by the usual method, and 4-µm-thick sections were prepared in the mesiodistal direction at the buccolingual center, followed by hematoxylin–eosin (HE) staining and histological observation. Fluorescence immunohistochemical staining assessments Following general anesthesia, the specimens underwent perfusion fixation with 4% paraformaldehyde, after which they were subjected to immersion fixation. Specimens were decalcified in 20% Morse solution (Fujifilm Wako Pure Chemical, Osaka, Japan) at 4°C for 24 h. They were then incrementally immersed at 4°C for ≥ 2 h in 10%, 20%, and 30% sucrose solutions. The samples were then embedded in mounting medium for ultra-low-temperature embedding (Section-Lab, Hiroshima, Japan). Following Kawamoto’s film technique, 14-µm-thick frozen sections were prepared in the mesiodistal direction at the buccolingual center using Cryofilm type IIIC and a tungsten carbide knife (Section-Lab, Hiroshima, Japan). Samples were pretreated at 24 ± 2°C for 15 min in 0.25% Triton X-100 (1:100, A16046-AE, Thermo Fisher Scientific, Waltham, MA, USA), and then incubated at 24 ± 2°C overnight with the primary antibody. The primary antibodies used were Mouse/Rat CD31/PECAM-1 Alexa Fluor 488-conjugated Antibody (1:100 dilution, FAB3628G, R&D Systems, Minneapolis, MN, USA), Anti Endomucin (V7 C7) Mouse (1:200 dilution, sc-65495, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and Anti-Sp7/Osterix Antibody (1:800, ab22552, Abcam, Cambridge, UK). As a negative control, sections were incubated with the antibody diluent without primary antibodies, followed by identical secondary antibody incubation. The secondary antibodies used were Donkey Anti-Rat IgG (H&L), Alexa Fluor 555 (1:1000 dilution, ab150154, Thermo Fisher Scientific) and Goat Anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (1:1000 dilution, A21245, Thermo Fisher Scientific). Sections were incubated for 2 h at room temperature with the secondary antibodies. Nuclear counterstaining was performed with DAPI. The specimens were covered with a cover slip and sealed with nail varnish. Fluorescence images were acquired using a laser scanning confocal microscope (LSM 880, Carl Zeiss, Oberkochen, Germany) equipped with a Plan-Apochromat objective (20×/0.8) and ZEN 2.3 black edition software. From each 14-µm-thick section, 20 optical slices were obtained at 0.5-µm intervals along the Z-axis. Quantitative histological analysis The distributions of CD31-positive (CD31+) and endomucin-positive (endomucin+) vessels within the first molar extraction sockets were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). Areas positive for CD31 alone, endomucin alone, or both markers were measured. Merged images were separated into green (CD31) and red (endomucin) channels, and threshold values distinguishing positive signals from background were set (CD31: 0–115; endomucin: 0–160). The areas of CD31-positive (A) and endomucin-positive (B) vessels were measured individually. Regions showing merged signals for both markers (C) were identified using the Image Calculator function. The proportion of vascular surface area positive for both CD31 and endomucin was calculated as follows: C / (A + B − C). Statistical analysis Data are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test. A value of P < 0.05 was considered statistically significant. All statistical analyses were conducted using SPSS version 27 (IBM Corp., Chicago, IL, USA). Results Micro-CT observations On Day 1, no radiopacity was evident in the first molar distal root extraction socket of the CTL group, and the same was true for the OCP/Col group. In the Bio-Oss and β-TCP groups, high-density bone substitute was evident. On Day 4, there was still no visible radiopacity in the CTL group, but in the OCP/Col group, a radiopaque area was evident in the bottom of the extraction socket. On Day 7, progression of the radiopacity in the first molar distal root extraction socket was evident in both the CTL and OCP/Col groups in comparison with the other groups, with no radiopaque areas suggestive of new bone formation observed in either the Bio-Oss or the β-TCP group. On Day 14, the radiopacity at the bottom of the extraction socket had extended upward in the CTL group, and in the OCP/Col group, the radiopacity filled the entire extraction socket. Radiopaque regions considered to be granules were still present in both the Bio-Oss and β-TCP groups (Fig. 2 A). Alveolar bone width and height On Day 7, the extraction socket width resorption rate was significantly lower in the OCP/Col group than in the CTL group. The extraction socket width resorption rates were also significantly lower in the Bio-Oss and β-TCP groups than in the CTL group. The extraction socket height resorption rates were significantly lower in the OCP/Col, Bio-Oss, and β-TCP groups than in the CTL group from Day 7 onward (Fig. 2 B, C). BMD and bone morphometry On Days 7 and 14, BMD was significantly lower in the OCP/Col group than in the Bio-Oss and β-TCP groups (Fig. 3 A). On Day 14, BV/TV was higher compared with Day 7 in the OCP/Col group, and on Day 14, its value was significantly higher in the OCP/Col group than in any of the other groups (Fig. 3 B). On Days 7 and 14, the value of Tb.Th was significantly higher in the OCP/Col group than in all other groups (Fig. 3 D). On Day 14, the value of Tb.Sp was significantly lower in the OCP/Col group than in the Bio-Oss and β-TCP groups (Fig. 3 E). Histological observations On Day 4, blood clots were observed in the extraction sockets of all groups (Fig. 4 ). On Day 7, granulation tissue composed predominantly of neutrophils, fibroblasts, and capillary vessels was evident in all groups. New bone formation connecting the bottom and side walls of the extraction socket was observed in the CTL and OCP/Col groups, whereas residual granules remained in the extraction sockets of the Bio-Oss and β-TCP groups (Fig. 4 ). On Day 14, markedly increased new bone formation was evident in the OCP/Col group. Residual granules persisted in the Bio-Oss and β-TCP groups, although new bone formation was observed at the bottom of the extraction sockets (Fig. 4 ). Fluorescence immunohistochemical staining of the first molar distal root extraction socket On Day 4, CD31- and endomucin-expressing cells were evident in the bottom and walls of the extraction socket in the OCP/Col group (Fig. 5 ). On Day 7, vascular endothelial cells expressing both CD31 and endomucin were observed in the OCP/Col group, and a newly formed capillary vascular network expressing both markers extended upward from the bottom and walls of the extraction socket. Numerous Osterix-positive cells were also observed in the vicinity of vascular endothelial cells expressing both CD31 and endomucin (Fig. 5 ). On Day 14, increased numbers of vascular endothelial cells expressing both CD31 and endomucin were observed in the OCP/Col and β-TCP groups, and Osterix-positive cells were also evident around these vessels. In the OCP/Col group, vascular endothelial cells expressing both CD31 and endomucin were particularly prominent. Bone substitute granules remained in the Bio-Oss and β-TCP groups, with newly formed capillary vessels observed between the granules (Fig. 5 ). The proportion of vascular surface area positive for both CD31 and endomucin in the first molar extraction socket was significantly higher in the OCP/Col group than in the other groups on Days 4, 7, and 14 (Fig. 6 ). Discussion Schropp et al. reported that most of the decrease in the width and height of alveolar bone due to bone resorption occurs immediately after extraction [ 1 ]. Araujo et al. also reported that buccal and lingual bone resorption was evident in dogs after tooth extraction [ 7 ]. Because post-extraction alveolar bone resorption adversely affects subsequent prosthetic treatment, RP has been introduced in dental implant treatment [ 38 ]. RP with a bone substitute has been shown to be effective in suppressing physiological post-extraction bone resorption [ 39 ]. Orthodontic extractions are often performed to secure space for tooth movement prior to treatment [ 40 ]. Ideally, after orthodontic extraction, the extraction socket should heal rapidly without vertical or horizontal alveolar bone resorption. Klein et al. found that RP performed after orthodontic extraction promoted extraction-socket healing with abundant new bone formation [ 41 ]. In the present study, alveolar bone height and width were significantly greater in all RP groups than in the CTL group. Suppression of the immediate post-extraction alveolar bone resorption is essential for subsequent dental treatment, and these findings further support the necessity of RP, particularly in orthodontic extractions. Extraction socket healing and new bone formation were compared among the different bone substitutes used for RP. On Day 14, BV/TV and Tb.Th were significantly higher in the OCP/Col group than in the other groups, indicating thicker and denser trabeculae. HE-stained observations showed that replacement with new bone began on Day 7, with plexiform-like bone containing abundant vascular components in the OCP/Col group. These findings suggest that new bone formation progressed more rapidly in the OCP/Col group than in the Bio-Oss and β-TCP groups, potentially enabling faster extraction-socket healing. However, BMD increased more slowly in the OCP/Col group than in the Bio-Oss and β-TCP groups, suggesting greater mineralization in the Bio-Oss and β-TCP groups. This indicates that although a large amount of new bone is formed after RP with OCP/Col, its calcification progresses gradually and the resulting bone contains a higher organic component. The tissue absorbability and bone replacement capacity of the bone substitutes were also examined. OCP/Col showed low radiodensity and was not visible on post-extraction micro-CT images, whereas Bio-Oss and β-TCP remained as distinct radiopaque granules up to Day 14. This finding was consistent with the report by Kamakura et al. that residual β-TCP was still present 12 weeks after use, indicating the need for long-term follow-up [ 26 ]. On histological assessment, the regular arrangement of collagen fibers and OCP in OCP/Col was visible until Day 4, but from Day 7 it became indistinguishable within the newly formed bone. These findings suggest that OCP/Col is readily replaced by new bone. In a study of OCP/Col used for skull defects in dogs, Tanuma et al. also reported that although the bone replacement rate did not reach 100%, it was superior to that of β-TCP and hydroxyapatite-based bone substitutes [ 42 ]. Taken together with the present results, the risk of OCP/Col persisting in tissue as a foreign body appears low, and the likelihood of inducing inflammation is minimal. Cardaropoli et al. studied extraction sockets in dogs and reported that rapid replacement with new bone is essential for extraction-socket healing and that angiogenesis plays an important role in this process [ 5 ]. Yan et al. further demonstrated that osteoid formation occurs around Type H vessels during extraction-socket healing [ 37 ]. The immunohistochemical assessments in this study showed that, on Day 4, a vascular network containing vessels with characteristics of Type H vessels was observed in the extraction socket in the CTL group. In the OCP/Col group, an angiogenic environment had already been established by Day 4 after RP, and the proportion of CD31- and endomucin-positive surface area was significantly higher, suggesting that a Type H–like vascular network developed at an earlier stage of healing than in the CTL group. In addition, numerous Osterix-positive cells were evident around these vessels in the OCP/Col group, consistent with the findings reported by Zhang et al. [ 43 ]. These findings suggest that RP with OCP/Col may promote the early formation of a vascular network enriched in Type H–like vessels within the extraction socket, thereby facilitating new bone formation. Although OCP/Col showed slower mineralization than other bone substitutes, it appears to have the advantage of promoting rapid bone formation and reconstruction of surrounding bone tissue, potentially through induction of a Type H–associated vascular network involved in bone remodeling. RP is reportedly effective in suppressing alveolar bone resorption and contributes to maintaining bone mass at the defect site [ 44 ]. However, granules may persist for a long period, and this might affect tooth movement in orthodontic treatment, potentially delaying tooth movement or raising the risk of root resorption by an abnormal concentration of stress. These findings suggest that RP with OCP/Col after orthodontic extraction may suppress extraction-related alveolar bone resorption and help prevent delayed tooth movement, indicating its potential usefulness in orthodontic treatment. Several limitations should be considered when interpreting these results. This study was conducted in a murine model and the evaluation was limited to the early healing phase. Nevertheless, our findings suggest that OCP/Col creates a favorable microenvironment during early extraction-socket healing, which may contribute to improved orthodontic outcomes by maintaining an adequate alveolar bone environment. Further long-term studies are needed to clarify the effects of OCP/Col on bone maturation and remodeling. Conclusion Residual bone substitute granules were unlikely to persist after RP with OCP/Col, which prevented alveolar bone resorption while maintaining early bone mass and forming new bone with abundant vasculature. Early extraction socket healing was associated with the development of microvasculature showing features consistent with Type H vessels, together with induction of osteogenic markers. Declarations Acknowledgements The authors thank Toyobo Co., Ltd. for providing the materials used in this study. Contributions The authors confirm their contribution to the paper as follows: Conceptualization: N.K., S.M., Y.N.; Methodology: N.K., C.T., S.M., K.S., N.K., Y.N.; Data curation: N.K., C.T., K.S., S.A., A.K., Y.N.; Writing - original draft: N.K., C.T.; Writing - review & editing: S.M., K.S., A.K., H.Y.; Visualization: N.K., N.K., S.M., N.Y.; Supervision: C.T., H.Y., S.M., A.K., Y.N.; Project administration: C.T., S.M., Y.N. All authors read and approved the final manuscript. Funding This study was supported in part by a KAKENHI grant (#24K12985) from the Japan Society for the Promotion of Science. The study also received research funding from Toyobo Co., Ltd. The sponsor had no role in the study design, data collection, analysis, or interpretation, or in writing the manuscript. Data availability The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The animal experiments were approved by the Tokyo Dental College Animal Experimentation Ethics Committee (approval no. 253102). Consent for publication Not applicable. Competing interests The authors declare that this study was conducted under a joint research agreement with Toyobo Co., Ltd., which provided research funding. The authors declare no competing interests other than the funding described above. References Schropp L, Wenzel A, Kostopoulos L, Karring T. Bone healing and soft tissue contour changes following single-tooth extraction: a clinical and radiographic 12-month prospective study. Int J Periodontics Restorative Dent. 2003;23(4):313-23. Darby I, Chen ST, Buser D. 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Clinical and histologic evaluation of granular Beta-tricalcium phosphate for the treatment of human intrabony periodontal defects: a report on five cases. J Periodontol. 2010;81(2):325-34. https://doi.org/10.1902/jop.2009.090386. Kamakura S, Sasaki K, Honda Y, Anada T, Suzuki O. Octacalcium phosphate combined with collagen orthotopically enhances bone regeneration. J Biomed Mater Res B Appl Biomater. 2006;79(2):210-17. https://doi.org/10.1002/jbm.b.30531. Kamakura S, Sasaki K, Homma T, Honda Y, Anada T, Echigo S, Suzuki O. The primacy of octacalcium phosphate collagen composites in bone regeneration. J Biomed Mater Res A. 2007;83(3):725-33. https://doi.org/10.1002/jbm.a.31332. Kamakura S, Sasano Y, Shimizu T, Hatori K, Suzuki O, Kagayama M, Motegi K. Implanted octacalcium phosphate is more resorbable than beta-tricalcium phosphate and hydroxyapatite. J Biomed Mater Res. 2002;59(1):29-34. https://doi.org/10.1002/jbm.1213. Kaida N, Matsunaga S, Tachiki C, Otsu Y, Sugahara K, Kasahara N, Abe S, Katakura A, Yamamoto H, Nishii Y. Ridge preservation using octacalcium phosphate collagen to induce new bone containing a vascular network of mainly Type H vessels. Sci Rep. 2024;14(1):25335. https://doi.org/10.1038/s41598-024-75931-y. Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell. 2011;146(6):873-87. https://doi.org/10.1016/j.cell.2011.08.039. Sun J, Paddock C, Shubert J, Zhang HB, Amin K, Newman PJ, Albelda SM. Contributions of the extracellular and cytoplasmic domains of platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) in regulating cell-cell localization. J Cell Sci. 2000;113(Pt 8):1459-69. https://doi.org/10.1242/jcs.113.8.1459. Privratsky JR, Paddock CM, Florey O, Newman DK, Muller WA, Newman PJ. Relative contribution of PECAM-1 adhesion and signaling to the maintenance of vascular integrity. J Cell Sci. 2011;124(Pt 9):1477-85. https://doi.org/10.1242/jcs.082271. Kuhn A, Brachtendorf G, Kurth F, Sonntag M, Samulowitz U, Metze D, Vestweber D. Expression of endomucin, a novel endothelial sialomucin, in normal and diseased human skin. J Invest Dermatol. 2002;119(6):1388-93. https://doi.org/10.1046/j.1523-1747.2002.19647.x. Morgan SM, Samulowitz U, Darley L, Simmons DL, Vestweber D. Biochemical characterization and molecular cloning of a novel endothelial-specific sialomucin. Blood. 1999;93(1):165-75. Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature. 2014;507(7492):323-28. https://doi.org/10.1038/nature13145. Ramasamy SK, Kusumbe AP, Schiller M, Zeuschner D, Bixel MG, Milia C, Gamrekelashvili J, Limbourg A, Medvinsky A, Santoro MM, Limbourg FP, Adams RH. Blood flow controls bone vascular function and osteogenesis. Nat Commun. 2016;7:13601. https://doi.org/10.1038/ncomms13601. Langen UH, Pitulescu ME, Kim JM, Enriquez-Gasca R, Sivaraj KK, Kusumbe AP, Singh A, Di Russo J, Bixel MG, Zhou B, Sorokin L, Vaquerizas JM, Adams RH. Cell-matrix signals specify bone endothelial cells during developmental osteogenesis. Nat Cell Biol. 2017;19(3):189-201. https://doi.org/10.1038/ncb3476. Yan ZQ, Wang XK, Zhou Y, Wang ZG, Wang ZX, Jin L, Yin H, Xia K, Tan YJ, Feng SK, Xie PL, Tang SY, Fang CY, Cao J, Xie H. H-type blood vessels participate in alveolar bone remodeling during murine tooth extraction healing. Oral Dis. 2020;26(5):998-1009. https://doi.org/10.1111/odi.13321. Avila-Ortiz G, Elangovan S, Kramer KW, Blanchette D, Dawson DV. Effect of alveolar ridge preservation after tooth extraction: a systematic review and meta-analysis. J Dent Res. 2014;93(10):950-58. https://doi.org/10.1177/0022034514541127. Majzoub J, Ravida A, Starch-Jensen T, Tattan M, Suárez-López Del Amo F. The influence of different grafting materials on alveolar ridge preservation: a systematic review. J Oral Maxillofac Res. 2019;10(3):6. https://doi.org/10.5037/jomr.2019.10306. Lundström AF. Malocclusion of the teeth regarded as a problem in connection with the apical base. Int Orthod Oral Surg Radiogr. 1925;11:1109-33. Klein Y, Fleissig O, Stabholz A, Chaushu S, Polak D. Bone regeneration with bovine bone impairs orthodontic tooth movement despite proper osseous wound healing in a novel mouse model. J Periodontol. 2019;90(2):189-99. https://doi.org/10.1002/jper.17-0550. Tanuma Y, Matsui K, Kawai T, Matsui A, Suzuki O, Kamakura S, Echigo S. Comparison of bone regeneration between octacalcium phosphate/collagen composite and β-tricalcium phosphate in canine calvarial defect. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;115(1):9-17. https://doi.org/10.1016/j.oooo.2011.12.029. Zhang J, Pan J, Jing W. Motivating role of type H vessels in bone regeneration. Cell Prolif. 2020;53(9):e12874. https://doi.org/10.1111/cpr.12874. Artzi Z, Kozlovsky A, Nemcovsky CE, Weinreb M. The amount of newly formed bone in sinus grafting procedures depends on tissue depth as well as the type and residual amount of the grafted material. J Clin Periodontol. 2005;32(2):193-99. https://doi.org/10.1111/j.1600-051x.2005.00656.x. Additional Declarations Competing interest reported. The study also received research funding from Toyobo Co., Ltd. The sponsor had no role in the study design, data collection, analysis, or interpretation, or in writing the manuscript. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 17 Apr, 2026 Reviews received at journal 16 Apr, 2026 Reviews received at journal 12 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers invited by journal 06 Apr, 2026 Editor assigned by journal 27 Mar, 2026 Submission checks completed at journal 11 Mar, 2026 First submitted to journal 11 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9091395","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":619924583,"identity":"5e61ff1b-1e73-4de9-84a3-5d87de2def7e","order_by":0,"name":"Yojiro Koizumi","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Yojiro","middleName":"","lastName":"Koizumi","suffix":""},{"id":619924584,"identity":"21fdd939-eb1c-4a38-a9d6-8d8cdee53bfb","order_by":1,"name":"Satoru Matsunaga","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYBADOQaGBDCDmQjFEDXGPCRrSeyBaiEMdNvPH/7Mu8MufT978gOGHzUM7OaEtJidSWaT5j2TnNvD88yAsecYA7NlAyEtB5LZmHnbmHN7JHIYGHgbGJgNDhDScv4x82fetvp0HqAWxr9EabmRzCDN23Y4AaSFmThbbjw2k5zbdtyw58wzg8MyxySI8Mv5xMcf3rZVy7O3Jz98+KbGJplgiKEAoJMkkg1I0gICdqRrGQWjYBSMguEOAIgUOLVQahvNAAAAAElFTkSuQmCC","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":true,"prefix":"","firstName":"Satoru","middleName":"","lastName":"Matsunaga","suffix":""},{"id":619924585,"identity":"faa93402-b443-4d93-a10d-27191312f90f","order_by":2,"name":"Chie Tachiki","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Chie","middleName":"","lastName":"Tachiki","suffix":""},{"id":619924586,"identity":"12400257-0df6-42e0-9971-12729ea47363","order_by":3,"name":"Toshihide Mizoguchi","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Toshihide","middleName":"","lastName":"Mizoguchi","suffix":""},{"id":619924587,"identity":"a3fd3315-3132-4a50-86ae-102d165e8753","order_by":4,"name":"Keisuke Sugahara","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Keisuke","middleName":"","lastName":"Sugahara","suffix":""},{"id":619924588,"identity":"eb700ca8-eb98-482b-9397-05779ac74415","order_by":5,"name":"Norio Kasahara","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Norio","middleName":"","lastName":"Kasahara","suffix":""},{"id":619924589,"identity":"c07e5070-cbb3-4d47-bbd5-c4ba4e5c0f78","order_by":6,"name":"Satoshi Ishizuka","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Ishizuka","suffix":""},{"id":619924590,"identity":"282d1d8d-6048-45e2-8004-6e7aba7e089e","order_by":7,"name":"Naoki Kaida","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Kaida","suffix":""},{"id":619924592,"identity":"e7a72017-c7da-48e1-a17d-4e4dc7e7caa5","order_by":8,"name":"Hitoshi Yamamoto","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Hitoshi","middleName":"","lastName":"Yamamoto","suffix":""},{"id":619924596,"identity":"e0de5683-5b69-4a8b-8d2f-20bd385b40a9","order_by":9,"name":"Akira Katakura","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Katakura","suffix":""},{"id":619924598,"identity":"bd44e095-2509-4968-b957-0ed2f1f1f4b0","order_by":10,"name":"Yasushi Nishii","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Yasushi","middleName":"","lastName":"Nishii","suffix":""}],"badges":[],"createdAt":"2026-03-11 07:39:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9091395/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9091395/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106725324,"identity":"f5ab3e53-b04d-4411-a79e-f772347db0e5","added_by":"auto","created_at":"2026-04-12 18:32:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6182814,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental protocol and definition of reference axes. \u003cstrong\u003eA\u003c/strong\u003e Experimental protocol. A CTL group that underwent only lower first molar extraction was compared with experimental groups for which RP was conducted (the OCP/Col, Bio-Oss, and β-TCP groups). Mice were sacrificed on Days 1, 4, 7, and 14. \u003cstrong\u003eB\u003c/strong\u003e Designation of reference axes and regions of interest. \u003cstrong\u003eC\u003c/strong\u003e Definition of anatomical reference points. Point a indicates the lowest point of the anterior mandible, and point p the lowest point of the posterior mandible. The Y-axis is perpendicular to the mandibular plane passing through point p. \u003cstrong\u003eD\u003c/strong\u003e Measurement of extraction socket alveolar bone width and height. The maximum socket width was measured, and height was determined along a perpendicular line from the midpoint.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-9091395/v1/66f2076c7e92ee6ef646658d.png"},{"id":106603783,"identity":"cf63b6d7-ddf5-444c-ac6c-9dfa0323041c","added_by":"auto","created_at":"2026-04-10 10:48:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9442668,"visible":true,"origin":"","legend":"\u003cp\u003eMicro-CT images of the lower first molar extraction sockets and measurement results.\u003cstrong\u003e A\u003c/strong\u003e Micro-CT images of the lower first molar distal root extraction socket in the YZ plane. † Bio-Oss granules; ▲ β-TCP granules. Scale bar = 200 μm.\u003cstrong\u003e B, C\u003c/strong\u003e Extraction socket alveolar bone width and height resorption rates (%). On Days 7 and 14, the height and width resorption rates were significantly lower in the OCP/Col, Bio-Oss, and β-TCP groups than in the CTL group. Data are shown as mean ± SD. *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-9091395/v1/da445d6df6e2f26a80e5b95c.png"},{"id":106603781,"identity":"b66cf01e-e234-41a2-ac05-e90c61049277","added_by":"auto","created_at":"2026-04-10 10:48:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2033731,"visible":true,"origin":"","legend":"\u003cp\u003eBone morphometric analysis of extraction sockets.\u003cstrong\u003e A\u003c/strong\u003e Bone mineral density (BMD), \u003cstrong\u003eB\u003c/strong\u003e Bone volume fraction (BV/TV), \u003cstrong\u003eC\u003c/strong\u003etrabecular number (Tb.N), \u003cstrong\u003eD\u003c/strong\u003etrabecular thickness (Tb.Th), \u003cstrong\u003eE\u003c/strong\u003etrabecular separation (Tb.Sp).\u003c/p\u003e\n\u003cp\u003eData are shown as mean ± SD. *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-9091395/v1/9d9d1a01fef31e6e701ade97.png"},{"id":106726670,"identity":"2edf9f0b-521d-42eb-b99b-33ef0609ae70","added_by":"auto","created_at":"2026-04-12 18:36:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22828208,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin–eosin (HE)–stained sections of lower first molar extraction sockets from each group on Days 4, 7, and 14. Black dotted lines indicate extraction sockets. # blood clot; ◆OCP/Col; † Bio-Oss granules; ▲ β-TCP granules. Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-9091395/v1/a31e796c1ad5f5f98789f534.png"},{"id":106726485,"identity":"cfb41768-02d1-442d-a216-640032ae9272","added_by":"auto","created_at":"2026-04-12 18:36:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28697145,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal images of specimens stained by fluorescence immunohistochemistry of lower first molar extraction sockets from each group on Days 4, 7, and 14. Red indicates endomucin, green CD31, and blue DAPI. White arrowheads indicate Type H vessels. White dotted lines indicate extraction sockets. † Bio-Oss granules; ▲ β-TCP granules. Scale bar = 200 μm. \u003cstrong\u003eb\u003c/strong\u003eCD31- and endomucin-positive vessels are observed in the OCP/Col group at the bottom and along the walls of the extraction socket. \u003cstrong\u003ef\u003c/strong\u003eCD31- and endomucin-positive vessels are observed in the OCP/Col group, with adjacent Osterix expression (light blue). \u003cstrong\u003eg, h\u003c/strong\u003e Residual bone substitute granules remain in the Bio-Oss and β-TCP groups. \u003cstrong\u003ej\u003c/strong\u003eA vascular network of CD31- and endomucin-positive vessels extends toward the upper part of the extraction socket in the OCP/Col group. \u003cstrong\u003el\u003c/strong\u003e Similar vessels are also observed in the β-TCP group.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-9091395/v1/afcc4f528197e36088d27db5.png"},{"id":106725693,"identity":"90a020f9-3602-42de-8106-77a9549e4f8d","added_by":"auto","created_at":"2026-04-12 18:33:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1343312,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of CD31- and endomucin-positive vascular surface area. Schematic illustration of the method used to calculate the proportion of vascular surface area positive for both CD31 and endomucin using ImageJ. The proportion of CD31⁺/endomucin⁺ vessels was calculated as: C / (A + B − C), where A represents the CD31-positive area, B the endomucin-positive area, and C the double-positive (merged) area indicating Type H vessels.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD. *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-9091395/v1/69710dd3a266c75d3f408326.png"},{"id":107704840,"identity":"902a0958-b7fa-4ab9-bb10-2cedff7b7abd","added_by":"auto","created_at":"2026-04-24 09:00:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":94651384,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9091395/v1/6805207f-db52-417e-84cf-83a31adba5f0.pdf"}],"financialInterests":"Competing interest reported. The study also received research funding from Toyobo Co., Ltd. The sponsor had no role in the study design, data collection, analysis, or interpretation, or in writing the manuscript.","formattedTitle":"Ridge Preservation with an Octacalcium Phosphate Collagen Complex (OCP/Col) Promotes New Bone Formation and Type H Vessel Distribution with Osterix Expression in Extraction Sockets in Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMaintaining the width and height of the alveolar bone after tooth extraction is vital for the smooth progress of prosthetic or orthodontic treatment [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the normal healing process of the extraction socket, it is initially filled with a blood clot that is replaced with granulation tissue and the direct formation of new bone [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Because alveolar bone is the supporting bone for teeth, extractions may cause disuse atrophy, leading to alveolar bone resorption and gingival invagination [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In particular, the periodontal tissue changes of gingival recession and invagination associated with orthodontic extractions are known to have direct adverse effects on both cosmetic appearance and jaw function [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRidge preservation (RP) is a surgical therapy used to maintain the alveolar bone post-extraction [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. RP is also performed prior to dental implant treatment to enable early, strong bone formation in the extraction socket and its surroundings [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The bone substitutes used for RP include both xenogenic bone substitutes made from ground dried animal bone [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and synthetic bone substitutes made from hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Xenogenic bone substitutes derived from animal bone provide a scaffold that preserves space for new bone formation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, their replacement by newly formed bone is extremely slow, which may compromise the rate and quality of bone regeneration. Synthetic bone substitutes are highly osteoconductive and allow adjustment of their physicochemical properties [\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Nevertheless, they exhibit variability in osteogenic potential and may require a prolonged period to integrate with newly formed bone, potentially delaying bone remodeling [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The octacalcium phosphate collagen complex (OCP/Col), developed by Kamakura et al., is a synthetic bone substitute with high osteoregenerative potential [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Kamakura et al. reported favorable bone formation following the use of OCP/Col in cranial defects and jaw clefts [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. OCP/Col consists of octacalcium phosphate (OCP) combined with collagen (Col) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. OCP is a precursor of biological apatite and has been reported to promote differentiation of osteoprogenitor cells into osteogenic cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Animal studies by Kaida et al. suggested enhanced capillary formation in alveolar bone following RP with OCP/Col [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the mechanisms underlying the effects of RP with OCP/Col remain incompletely understood and require further investigation.\u003c/p\u003e \u003cp\u003eCapillary vessels are more than just a constituent of granulation tissue in wound healing; they also play other important roles, including the removal of foreign substances and the supply of oxygen and nutrients to tissues [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Sun et al. reported that CD31 (PECAM-1) is a surface marker of vascular endothelial cells that is expressed at high levels at the junctions with neighboring endothelial cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. CD31 is an adhesive stress-response protein that reportedly contributes to maintaining endothelial cell junctions, angiogenesis, and the rapid restoration of the vascular permeability barrier following inflammation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, endomucin, which is a mucin-like glycoprotein expressed in vascular endothelial cells, is also expressed in vascular endothelial cells of capillary vessels and veins, but it is not expressed in arteries [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral capillary vessel subtypes within bone that differ both morphologically and functionally have been reported [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the bone modeling process in the growth plates of mouse long bones, Type H vessels characterized by high expression levels of both CD31 and endomucin are abundant beneath the hypertrophic cartilage of the growth plate. Osterix-positive osteoprogenitor cells are observed in the vicinity of endothelial cells of Type H vessels, and these reportedly interact with each other via the exchange of growth factors to promote angiogenesis-osteogenesis coupling [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Yan et al. reported a significant increase in Type H vessels with concomitant perivascular accumulation of Runx2-positive osteoblasts and augmentation of bone mass [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe hypothesized that Type H vessels are formed within newly generated bone following RP with OCP/Col and that post-extraction bone formation is promoted by these vessels and surrounding osteoprogenitor cells.\u003c/p\u003e \u003cp\u003eThe aim of this study was to clarify how OCP/Col used as a bone substitute to fill extraction sockets promotes new bone formation during the early healing phase by investigating the distribution of Type H vessels generated in extraction sockets after RP with OCP/Col and the expression of osteoinductive factors around them.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe experimental animals used were six-week-old male mice (mean weight 21.0 g). These mice were reared in a 12-h light/dark cycle with unlimited access to chow and water. The animal experiments were conducted with the authorization of the Tokyo Dental College Animal Experimentation Ethics Committee (approval no. 253102).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental procedure\u003c/h3\u003e\n\u003cp\u003eThe surgical procedure was conducted under general anesthesia induced by the intraperitoneal administration of a mixture of three anesthetics (0.75 mg/kg medetomidine hydrochloride, Nippon Zenyaku Kogyo, Fukushima, Japan; 4.0 mg/kg midazolam, Sandoz, Tokyo, Japan; and 5.0 mg/kg butorphanol tartrate, Meiji Seika Pharma, Tokyo, Japan). In all mice, the lower right first molar was extracted with a dental probe. The CTL group underwent only lower right first molar extraction, and the experimental groups underwent RP after lower right first molar extraction. OCP/Col (Bonarc artificial bone with collagen, Toyobo, Tokyo, Japan), β-TCP (Osferion, Olympus, Tokyo, Japan), and Bio-Oss (Geistlich Sons Ltd, Wolhusen, Switzerland) were used as bone substitutes. The mice were randomly assigned to a CTL group (n\u0026thinsp;=\u0026thinsp;12 per time point) and the different experimental groups (OCP/Col group: n\u0026thinsp;=\u0026thinsp;12 per time point; β-TCP group: n\u0026thinsp;=\u0026thinsp;12 per time point; and Bio-Oss group: n\u0026thinsp;=\u0026thinsp;12 per time point). After lower right first molar extraction, the mesiodistal root extraction socket was filled with one of the bone substitutes for RP and covered with a wafer sheet. To reverse medetomidine-induced anesthesia, a medetomidine antagonist (0.75 mg/kg atipamezole hydrochloride, Nippon Zenyaku Kogyo, Fukushima, Japan) was administered intraperitoneally.\u003c/p\u003e \u003cp\u003eThe mice in each group were then sacrificed on Days 1, 4, 7, and 14, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Bone samples including the lower right first molar extraction socket were immediately collected and fixed by immersion in 4% paraformaldehyde phosphate buffer solution at 4\u0026deg;C for 2 days.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDesignation of reference axes and regions of interest (ROIs)\u003c/h3\u003e\n\u003cp\u003eThe samples consisted of the portion of the mandible containing the extraction sockets of the first molar mesiodistal root at the front and the first molar distal root at the back, excised as a single block and trimmed. ROIs were designated in the area surrounding the lower right first molar mesial and distal root extraction sockets.\u003c/p\u003e \u003cp\u003eThe reference axes of the samples were designated as follows: the X-axis as the line joining the midpoints of the lines joining the left and right mandibles at the lowest points of the anterior and posterior thickened portions of the mandibles in the mesiodistal direction; the Z-axis as the buccolingual orientation with respect to the X-Y plane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB); and the Y-axis as the direction perpendicular to the mandibular plane, which contains the line joining the lowest points of the anterior mandibles (a) and the lowest points of the posterior mandibles (p) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\n\u003ch3\u003eMicro-CT scanning\u003c/h3\u003e\n\u003cp\u003eAll samples were scanned by micro-CT (\u0026micro;CT-50, Scanco Medical AG, Wangen-Br\u0026uuml;ttisellen, Switzerland). The scanning conditions were as follows: tube voltage, 90 kV; tube current, 155 \u0026micro;A; image matrix, 3400 \u0026times; 3400 and slice thickness, 20 \u0026micro;m. Two-dimensional slice images in the XZ, XY, and YZ planes were acquired by volume rendering using three-dimensional reconstruction analysis software (TRI/3D-BON, Ratoc System Engineering, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eAlveolar bone width and height\u003c/h3\u003e\n\u003cp\u003eThree-dimensional reconstruction and measurement software (\u0026micro;CT Analysis Software, Scanco Medical AG) was used to measure the alveolar bone width and height on the buccal and lingual sides of the lower first molar distal root extraction socket on Days 1, 4, 7, and 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The alveolar bone width and height in the extraction sockets of each group were measured in the YZ plane on micro-CT images, and the resorption rates (%) were calculated as follows:\u003c/p\u003e \u003cp\u003eExtraction socket alveolar bone width resorption rate (%) = [1 \u0026ndash; extraction socket alveolar bone width (mm)/dentulous mandible (left) alveolar bone width (mm)] \u0026sdot; 100\u003c/p\u003e \u003cp\u003eExtraction socket alveolar bone height resorption rate (%) = [1 \u0026ndash; extraction socket alveolar bone height (mm)/dentulous mandible (left) alveolar bone height (mm)] \u0026sdot; 100\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBone mineral density measurement and bone morphometry\u003c/h2\u003e \u003cp\u003eBone mineral density (BMD) measurements and bone morphometry in the lower first molar mesial and distal root extraction sockets were conducted on post-extraction Days 1, 4, 7, and 14 using TRI/3D-BON (bone morphometric software). The following parameters were measured: bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Granular radiopaque areas with CT values\u0026thinsp;\u0026ge;\u0026thinsp;1500 were regarded as Bio-Oss or β-TCP and excluded from the ROIs.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistological assessments\u003c/h3\u003e\n\u003cp\u003eAfter fixation by immersion in 4% paraformaldehyde phosphate buffer solution at 4\u0026deg;C for 2 days, specimens were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 3 weeks. They were then embedded in paraffin by the usual method, and 4-\u0026micro;m-thick sections were prepared in the mesiodistal direction at the buccolingual center, followed by hematoxylin\u0026ndash;eosin (HE) staining and histological observation.\u003c/p\u003e\n\u003ch3\u003eFluorescence immunohistochemical staining assessments\u003c/h3\u003e\n\u003cp\u003eFollowing general anesthesia, the specimens underwent perfusion fixation with 4% paraformaldehyde, after which they were subjected to immersion fixation. Specimens were decalcified in 20% Morse solution (Fujifilm Wako Pure Chemical, Osaka, Japan) at 4\u0026deg;C for 24 h. They were then incrementally immersed at 4\u0026deg;C for \u0026ge;\u0026thinsp;2 h in 10%, 20%, and 30% sucrose solutions. The samples were then embedded in mounting medium for ultra-low-temperature embedding (Section-Lab, Hiroshima, Japan). Following Kawamoto\u0026rsquo;s film technique, 14-\u0026micro;m-thick frozen sections were prepared in the mesiodistal direction at the buccolingual center using Cryofilm type IIIC and a tungsten carbide knife (Section-Lab, Hiroshima, Japan). Samples were pretreated at 24 \u0026plusmn; 2\u0026deg;C for 15 min in 0.25% Triton X-100 (1:100, A16046-AE, Thermo Fisher Scientific, Waltham, MA, USA), and then incubated at 24 \u0026plusmn; 2\u0026deg;C overnight with the primary antibody. The primary antibodies used were Mouse/Rat CD31/PECAM-1 Alexa Fluor 488-conjugated Antibody (1:100 dilution, FAB3628G, R\u0026amp;D Systems, Minneapolis, MN, USA), Anti Endomucin (V7 C7) Mouse (1:200 dilution, sc-65495, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and Anti-Sp7/Osterix Antibody (1:800, ab22552, Abcam, Cambridge, UK). As a negative control, sections were incubated with the antibody diluent without primary antibodies, followed by identical secondary antibody incubation. The secondary antibodies used were Donkey Anti-Rat IgG (H\u0026amp;L), Alexa Fluor 555 (1:1000 dilution, ab150154, Thermo Fisher Scientific) and Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (1:1000 dilution, A21245, Thermo Fisher Scientific). Sections were incubated for 2 h at room temperature with the secondary antibodies. Nuclear counterstaining was performed with DAPI. The specimens were covered with a cover slip and sealed with nail varnish.\u003c/p\u003e \u003cp\u003eFluorescence images were acquired using a laser scanning confocal microscope (LSM 880, Carl Zeiss, Oberkochen, Germany) equipped with a Plan-Apochromat objective (20\u0026times;/0.8) and ZEN 2.3 black edition software. From each 14-\u0026micro;m-thick section, 20 optical slices were obtained at 0.5-\u0026micro;m intervals along the Z-axis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative histological analysis\u003c/h2\u003e \u003cp\u003eThe distributions of CD31-positive (CD31+) and endomucin-positive (endomucin+) vessels within the first molar extraction sockets were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). Areas positive for CD31 alone, endomucin alone, or both markers were measured. Merged images were separated into green (CD31) and red (endomucin) channels, and threshold values distinguishing positive signals from background were set (CD31: 0\u0026ndash;115; endomucin: 0\u0026ndash;160). The areas of CD31-positive (A) and endomucin-positive (B) vessels were measured individually. Regions showing merged signals for both markers (C) were identified using the Image Calculator function. The proportion of vascular surface area positive for both CD31 and endomucin was calculated as follows: C / (A\u0026thinsp;+\u0026thinsp;B \u0026minus; C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). One-way analysis of variance (ANOVA) was performed, followed by Tukey\u0026rsquo;s post hoc test. A value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were conducted using SPSS version 27 (IBM Corp., Chicago, IL, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMicro-CT observations\u003c/h2\u003e \u003cp\u003eOn Day 1, no radiopacity was evident in the first molar distal root extraction socket of the CTL group, and the same was true for the OCP/Col group. In the Bio-Oss and β-TCP groups, high-density bone substitute was evident. On Day 4, there was still no visible radiopacity in the CTL group, but in the OCP/Col group, a radiopaque area was evident in the bottom of the extraction socket. On Day 7, progression of the radiopacity in the first molar distal root extraction socket was evident in both the CTL and OCP/Col groups in comparison with the other groups, with no radiopaque areas suggestive of new bone formation observed in either the Bio-Oss or the β-TCP group. On Day 14, the radiopacity at the bottom of the extraction socket had extended upward in the CTL group, and in the OCP/Col group, the radiopacity filled the entire extraction socket. Radiopaque regions considered to be granules were still present in both the Bio-Oss and β-TCP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAlveolar bone width and height\u003c/h2\u003e \u003cp\u003eOn Day 7, the extraction socket width resorption rate was significantly lower in the OCP/Col group than in the CTL group. The extraction socket width resorption rates were also significantly lower in the Bio-Oss and β-TCP groups than in the CTL group. The extraction socket height resorption rates were significantly lower in the OCP/Col, Bio-Oss, and β-TCP groups than in the CTL group from Day 7 onward (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBMD and bone morphometry\u003c/h2\u003e \u003cp\u003eOn Days 7 and 14, BMD was significantly lower in the OCP/Col group than in the Bio-Oss and β-TCP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). On Day 14, BV/TV was higher compared with Day 7 in the OCP/Col group, and on Day 14, its value was significantly higher in the OCP/Col group than in any of the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). On Days 7 and 14, the value of Tb.Th was significantly higher in the OCP/Col group than in all other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). On Day 14, the value of Tb.Sp was significantly lower in the OCP/Col group than in the Bio-Oss and β-TCP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHistological observations\u003c/h2\u003e \u003cp\u003eOn Day 4, blood clots were observed in the extraction sockets of all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). On Day 7, granulation tissue composed predominantly of neutrophils, fibroblasts, and capillary vessels was evident in all groups. New bone formation connecting the bottom and side walls of the extraction socket was observed in the CTL and OCP/Col groups, whereas residual granules remained in the extraction sockets of the Bio-Oss and β-TCP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). On Day 14, markedly increased new bone formation was evident in the OCP/Col group. Residual granules persisted in the Bio-Oss and β-TCP groups, although new bone formation was observed at the bottom of the extraction sockets (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence immunohistochemical staining of the first molar distal root extraction socket\u003c/h2\u003e \u003cp\u003eOn Day 4, CD31- and endomucin-expressing cells were evident in the bottom and walls of the extraction socket in the OCP/Col group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). On Day 7, vascular endothelial cells expressing both CD31 and endomucin were observed in the OCP/Col group, and a newly formed capillary vascular network expressing both markers extended upward from the bottom and walls of the extraction socket. Numerous Osterix-positive cells were also observed in the vicinity of vascular endothelial cells expressing both CD31 and endomucin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). On Day 14, increased numbers of vascular endothelial cells expressing both CD31 and endomucin were observed in the OCP/Col and β-TCP groups, and Osterix-positive cells were also evident around these vessels. In the OCP/Col group, vascular endothelial cells expressing both CD31 and endomucin were particularly prominent. Bone substitute granules remained in the Bio-Oss and β-TCP groups, with newly formed capillary vessels observed between the granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe proportion of vascular surface area positive for both CD31 and endomucin in the first molar extraction socket was significantly higher in the OCP/Col group than in the other groups on Days 4, 7, and 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSchropp et al. reported that most of the decrease in the width and height of alveolar bone due to bone resorption occurs immediately after extraction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Araujo et al. also reported that buccal and lingual bone resorption was evident in dogs after tooth extraction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Because post-extraction alveolar bone resorption adversely affects subsequent prosthetic treatment, RP has been introduced in dental implant treatment [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. RP with a bone substitute has been shown to be effective in suppressing physiological post-extraction bone resorption [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Orthodontic extractions are often performed to secure space for tooth movement prior to treatment [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Ideally, after orthodontic extraction, the extraction socket should heal rapidly without vertical or horizontal alveolar bone resorption. Klein et al. found that RP performed after orthodontic extraction promoted extraction-socket healing with abundant new bone formation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the present study, alveolar bone height and width were significantly greater in all RP groups than in the CTL group. Suppression of the immediate post-extraction alveolar bone resorption is essential for subsequent dental treatment, and these findings further support the necessity of RP, particularly in orthodontic extractions.\u003c/p\u003e \u003cp\u003eExtraction socket healing and new bone formation were compared among the different bone substitutes used for RP. On Day 14, BV/TV and Tb.Th were significantly higher in the OCP/Col group than in the other groups, indicating thicker and denser trabeculae. HE-stained observations showed that replacement with new bone began on Day 7, with plexiform-like bone containing abundant vascular components in the OCP/Col group. These findings suggest that new bone formation progressed more rapidly in the OCP/Col group than in the Bio-Oss and β-TCP groups, potentially enabling faster extraction-socket healing. However, BMD increased more slowly in the OCP/Col group than in the Bio-Oss and β-TCP groups, suggesting greater mineralization in the Bio-Oss and β-TCP groups. This indicates that although a large amount of new bone is formed after RP with OCP/Col, its calcification progresses gradually and the resulting bone contains a higher organic component. The tissue absorbability and bone replacement capacity of the bone substitutes were also examined. OCP/Col showed low radiodensity and was not visible on post-extraction micro-CT images, whereas Bio-Oss and β-TCP remained as distinct radiopaque granules up to Day 14. This finding was consistent with the report by Kamakura et al. that residual β-TCP was still present 12 weeks after use, indicating the need for long-term follow-up [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn histological assessment, the regular arrangement of collagen fibers and OCP in OCP/Col was visible until Day 4, but from Day 7 it became indistinguishable within the newly formed bone. These findings suggest that OCP/Col is readily replaced by new bone. In a study of OCP/Col used for skull defects in dogs, Tanuma et al. also reported that although the bone replacement rate did not reach 100%, it was superior to that of β-TCP and hydroxyapatite-based bone substitutes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Taken together with the present results, the risk of OCP/Col persisting in tissue as a foreign body appears low, and the likelihood of inducing inflammation is minimal. Cardaropoli et al. studied extraction sockets in dogs and reported that rapid replacement with new bone is essential for extraction-socket healing and that angiogenesis plays an important role in this process [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Yan et al. further demonstrated that osteoid formation occurs around Type H vessels during extraction-socket healing [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe immunohistochemical assessments in this study showed that, on Day 4, a vascular network containing vessels with characteristics of Type H vessels was observed in the extraction socket in the CTL group. In the OCP/Col group, an angiogenic environment had already been established by Day 4 after RP, and the proportion of CD31- and endomucin-positive surface area was significantly higher, suggesting that a Type H\u0026ndash;like vascular network developed at an earlier stage of healing than in the CTL group. In addition, numerous Osterix-positive cells were evident around these vessels in the OCP/Col group, consistent with the findings reported by Zhang et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These findings suggest that RP with OCP/Col may promote the early formation of a vascular network enriched in Type H\u0026ndash;like vessels within the extraction socket, thereby facilitating new bone formation. Although OCP/Col showed slower mineralization than other bone substitutes, it appears to have the advantage of promoting rapid bone formation and reconstruction of surrounding bone tissue, potentially through induction of a Type H\u0026ndash;associated vascular network involved in bone remodeling.\u003c/p\u003e \u003cp\u003eRP is reportedly effective in suppressing alveolar bone resorption and contributes to maintaining bone mass at the defect site [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, granules may persist for a long period, and this might affect tooth movement in orthodontic treatment, potentially delaying tooth movement or raising the risk of root resorption by an abnormal concentration of stress. These findings suggest that RP with OCP/Col after orthodontic extraction may suppress extraction-related alveolar bone resorption and help prevent delayed tooth movement, indicating its potential usefulness in orthodontic treatment.\u003c/p\u003e \u003cp\u003eSeveral limitations should be considered when interpreting these results. This study was conducted in a murine model and the evaluation was limited to the early healing phase. Nevertheless, our findings suggest that OCP/Col creates a favorable microenvironment during early extraction-socket healing, which may contribute to improved orthodontic outcomes by maintaining an adequate alveolar bone environment. Further long-term studies are needed to clarify the effects of OCP/Col on bone maturation and remodeling.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eResidual bone substitute granules were unlikely to persist after RP with OCP/Col, which prevented alveolar bone resorption while maintaining early bone mass and forming new bone with abundant vasculature. Early extraction socket healing was associated with the development of microvasculature showing features consistent with Type H vessels, together with induction of osteogenic markers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Toyobo Co., Ltd. for providing the materials used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm their contribution to the paper as follows:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eConceptualization: N.K., S.M., Y.N.; Methodology: N.K., C.T., S.M., K.S., N.K., Y.N.; Data curation: N.K., C.T., K.S., S.A., A.K., Y.N.; Writing - original draft: N.K., C.T.; Writing - review \u0026amp; editing: S.M., K.S., A.K., H.Y.; Visualization: N.K., N.K., S.M., N.Y.; Supervision: C.T., H.Y., S.M., A.K., Y.N.; Project administration: C.T., S.M., Y.N.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported in part by a KAKENHI grant (#24K12985) from the Japan Society for the Promotion of Science. The study also received research funding from Toyobo Co., Ltd. The sponsor had no role in the study design, data collection, analysis, or interpretation, or in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments were approved by the Tokyo Dental College Animal Experimentation Ethics Committee (approval no. 253102).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this study was conducted under a joint research agreement with Toyobo Co., Ltd., which provided research funding. The authors declare no competing interests other than the funding described above.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSchropp L, Wenzel A, Kostopoulos L, Karring T. Bone healing and soft tissue contour changes following single-tooth extraction: a clinical and radiographic 12-month prospective study. Int J Periodontics Restorative Dent. 2003;23(4):313-23.\u003c/li\u003e\n\u003cli\u003eDarby I, Chen ST, Buser D. Ridge preservation techniques for implant therapy. Int J Oral Maxillofac Implants. 2009;24:260-71.\u003c/li\u003e\n\u003cli\u003eChappuis V, Engel O, Reyes M, Shahim K, Nolte LP, Buser D. Ridge alterations post-extraction in the esthetic zone: a 3D analysis with CBCT. J Dent Res. 2013;92:195S-201S. https://doi.org/10.1177/0022034513506713.\u003c/li\u003e\n\u003cli\u003eAra\u0026uacute;jo MG, Silva CO, Misawa M, Sukekava F. Alveolar socket healing: what can we learn? 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J Clin Periodontol. 2005;32(2):193-99. https://doi.org/10.1111/j.1600-051x.2005.00656.x.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-implant-dentistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"IJID","sideBox":"Learn more about [International Journal of Implant Dentistry](https://journalimplantdent.springeropen.com/)","snPcode":"40729","submissionUrl":"https://submission.nature.com/new-submission/40729/3","title":"International Journal of Implant Dentistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Type H vessels, ridge preservation, bone substitute material, bone quality analysis, orthodontic tooth movement","lastPublishedDoi":"10.21203/rs.3.rs-9091395/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9091395/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose \u003c/strong\u003eAlveolar bone resorption after tooth extraction leads to gingival recession and invagination, which may result in the stalling of tooth movement and difficulties in orthodontic tooth movement. Bone substitutes used for alveolar ridge preservation should exhibit bioaffinity, appropriate initial calcification, ease of handling, replacement by bone, long-term stability, and formation of an abundant vascular network. Therefore, this study focused on ridge preservation (RP) using an octacalcium phosphate collagen composite (OCP/Col). Type H vessels are known to appear at sites of active bone formation, and osteoblast markers are observed in their vicinity, creating a microenvironment conducive to osteogenesis. The objective of this study was to investigate the distribution of newly formed bone and Type H vessels in extraction sockets following ridge preservation with OCP/Col, as well as the expression of Osterix.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eRP of the lower first molars in mice was performed using OCP/Col. Bone mass, bone quality, and angiogenesis were analyzed by micro-computed tomography, bone morphometry, and histological and fluorescence immunohistochemical staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eIn RP with OCP/Col, the granularity of the bone substitute tended not to be preserved, and immature woven bone formation was observed, which prevented alveolar bone resorption. A microcapillary vascular network including Type H vessels was formed, and osteoblast markers were induced in the surrounding area, promoting new bone formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e These findings suggest that RP with OCP/Col promotes early new bone formation associated with Type H vessels and may be beneficial for orthodontic treatment.\u003c/p\u003e","manuscriptTitle":"Ridge Preservation with an Octacalcium Phosphate Collagen Complex (OCP/Col) Promotes New Bone Formation and Type H Vessel Distribution with Osterix Expression in Extraction Sockets in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-10 10:48:02","doi":"10.21203/rs.3.rs-9091395/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-18T00:24:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-16T23:22:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T00:54:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257501606202141318053388616352254924456","date":"2026-04-08T23:14:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187738765683695241666874840665496628648","date":"2026-04-08T07:55:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-06T07:33:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-27T11:51:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-12T01:58:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Implant Dentistry","date":"2026-03-11T07:23:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-implant-dentistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"IJID","sideBox":"Learn more about [International Journal of Implant Dentistry](https://journalimplantdent.springeropen.com/)","snPcode":"40729","submissionUrl":"https://submission.nature.com/new-submission/40729/3","title":"International Journal of Implant Dentistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c0ad546-eb56-47c1-816f-df2f7206fda7","owner":[],"postedDate":"April 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-18T00:38:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-10 10:48:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9091395","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9091395","identity":"rs-9091395","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}