Abstract
Background/Aim: Maxillary sinus floor elevation is a well-established procedure for increasing bone volume in the posterior maxilla, yet the regenerative outcome depends strongly on the choice of grafting material. This clinical study compared a high-temperature sintered xenograft (Bio-Oss®) and a low-temperature processed xenograft (Ti-Oss®) with regard to their regenerative and immunological profiles.
Patients and Methods: Eight patients underwent split-mouth sinus augmentation with both materials, and biopsies were retrieved at 6 months after implant placement. Histological, histomorphometrical, and immunohistochemical analyses were performed to assess bone formation, material resorption, and tissue compatibility. Immunohistochemistry was applied to evaluate the local immune response, focusing on macrophage polarization and multinucleated giant cell activity. Quantitative histomorphometry determined the relative areas of newly formed bone, residual graft material, and connective tissue.
Results: Histopathological and histomorphometrical analyses demonstrated comparable levels of new bone formation in both groups, confirming reliable osteoconduction. Immunohistochemical evaluation revealed tartrate-resistant acid phosphatase isoform 5a (TRAP5A) expression in multinucleated giant cells adherent to both materials. Interestingly, Bio-Oss® induced a higher proportion of anti-inflammatory (CD163+) macrophages, whereas Ti-Oss® triggered a significantly greater number of pro-inflammatory multinucleated giant cells (CD11c+).
Conclusion: These findings indicate that although both xenogeneic substitutes (Bio-Oss® and Ti-Oss®) achieve bone regeneration, they elicit distinct immune responses, which may influence long-term remodeling and graft integration. Consideration of osteoimmunological properties is therefore essential when selecting biomaterials for clinical sinus augmentation.
- Maxillary sinus floor elevation
- xenogeneic bone substitute
- osteoimmunology
- macrophage polarization
- bone regeneration
Introduction
Maxillary sinus floor elevation is a well-established surgical approach for increasing alveolar bone height in the posterior maxilla, allowing stable placement of dental implants. The biological success of this procedure depends not only on mechanical space maintenance but also on the regenerative capacity of the grafting material, including its ability to support osteoconduction, vascularization, and balanced immune modulation. Autologous bone remains the gold standard, yet its limitations –including donor-site morbidity, limited availability, and increased operative time– have driven the clinical adoption of xenogeneic bone substitute materials (BSMs) (1, 2).
Among these, Bio-Oss® (Geistlich Pharma AG, Wolhusen, Switzerland) has become the most frequently used xenograft in sinus augmentation. Bio-Oss® is processed through high-temperature sintering (>700°C), which ensures the elimination of organic material and provides long-term scaffold stability. However, this thermal treatment alters the mineral phase and surface architecture by increasing hydroxyapatite crystallinity, reducing microporosity, and sometimes causing vitrification (3-5). While this produces a slow-resorbing scaffold that can maintain volume for extended periods, it might limit its remodeling capacity (6-8). Moreover, the resulting dense crystalline surfaces are less conducive to protein adsorption and macrophage adhesion, which can favor pro-inflammatory (M1) macrophage polarization, prolonging the inflammatory phase, and might thus reduce regenerative potential from an immunological standpoint (9). By contrast, Ti-Oss® (Osstem Implant Co., Ltd., Busan, Republic of Korea) is produced by a low- to mid-temperature biomimetic process, which preserves the natural trabecular morphology and multiporous architecture of cancellous bone. Importantly, this approach prevents the complete transformation of calcium phosphates into stoichiometric hydroxyapatite and instead maintains intermediate phases, especially octacalcium phosphate (OCP) (10, 11).
OCP has been described as a metastable precursor of biological apatite and is considered a critical contributor to biomaterial osteogenicity. Unlike dense hydroxyapatite, OCP is more soluble and resorbable under physiological conditions, allowing controlled release of calcium and phosphate ions during remodeling (12, 13). These physicochemical properties can enable gradual replacement of graft particles by newly mineralized bone. OCP-containing surfaces can also exhibit superior wettability and protein adsorption profiles (14, 15), which should facilitate the binding of osteogenic growth factors and enhance osteoblast attachment, proliferation, and differentiation. In preclinical models, OCP has been shown to accelerate matrix mineralization and support more homogeneous and rapid new bone formation compared to high-temperature sintered xenografts (16).
Beyond these direct osteogenic effects, OCP also plays a crucial role in modulating the immune response. The field of osteoimmunology highlights the central role of macrophages in orchestrating tissue repair (17). Biomaterials that preserve OCP and maintain micro-/nano-topography promote a shift toward M2-like (pro-regenerative) macrophage polarization, accompanied by the release of anti-inflammatory cytokines (interleukin-10, transforming growth factor-β), thereby establishing a favorable microenvironment for angiogenesis and osteogenesis (12, 13). Conversely, surfaces dominated by highly crystalline hydroxyapatite often sustain M1 macrophage activity, characterized by elevated pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β), which may hinder bone regeneration and increase the risk of fibrous encapsulation (18, 19).
In the unique environment of the sinus cavity, characterized by relatively poor vascularity and limited osteogenic potential, the regenerative potency of the graft becomes especially critical. Low-temperature xenografts such as Ti-Oss, with their preserved OCP phases and multiporosity, may therefore confer distinct advantages by enabling faster vascular penetration, stimulating osteoblast differentiation, and supporting balanced immune responses (20). Early evidence suggests that these materials provide improved bone-to-graft contact, enhanced angiogenesis, and more rapid graft substitution when compared to conventional high-temperature xenografts (20).
Despite these theoretical and experimental advantages, comparative clinical data directly evaluating Bio-Oss and Ti-Oss in sinus lift procedures are limited. Few studies have examined not only the volumetric and histological outcomes of graft integration but also the immune response associated with sintering-dependent mineral phases (21). Understanding the interaction between sintering temperature, OCP preservation, and host immune modulation may provide essential insights for selecting biomaterials for this indication.
The aim of the present study was to compare the regenerative and immunological outcomes of Bio-Oss and Ti-Oss in maxillary sinus augmentation. Therefore, established clinical, histological and histomorphometrical methodologies were used to analyse the tissue distribution, with focus on new bone formation and the host immune profile induced by both materials (18, 22-25).
Patients and Methods
Clinical procedure and biopsy collection. The sinus tissue samples analyzed in this study were obtained from eight patients who underwent follow-up surgery for dental implant placement at the private dental practice Leader Stom, Astana, Kazakhstan. The research received approval from the local Ethics Committee at meeting number 01/2025. Six months prior, all patients had undergone surgery with both of the xenogeneic BSMs in split-mouth sinus floor augmentation in preparation for dental implants.
Patients included in this study underwent cone beam computed tomography (CBCT) prior to surgery for surgical planning. Before surgery, patients were given perioperative antibiotics (875/125 mg amoxiclav) and oral disinfection via rinsing with chlorhexidine solution (0.2% for 60 s). Sinus augmentation was performed under local anesthesia using 1.7 ml Ultracain® DS Forte (4% articaine hydrochloride with epinephrine 1:100,000; Sanofi-Aventis, Frankfurt, Germany) per infiltration site–and conscious sedation. Following mucoperiosteal flap elevation, a lateral bone window was created using diamond burs. Then, the Schneiderian membrane was elevated using hand instruments. In the case of perforation, a porcine pericardium-based collagen membrane was positioned beneath the Schneiderian membrane. The BSMs were rehydrated in sterile saline solution and randomly placed into the cavity, covered with another collagen membrane. The mucoperiosteal flap was re-adapted and fixed using polytetrafluoroethylene sutures. A low-dose CBCT to check surgery was performed in order to exclude the presence of perforations with material penetration to the sinus cavity. The postoperative medication included 875/125 mg amoxiclav twice daily for 7 days; 400 mg ibuprofen three times daily for 2 days; Traumeel® tablets (Biologische Heilmittel Heel GmbH, Baden-Baden, Germany) at a dosage of one tablet three times daily for 7 days; Otriven nasal spray twice daily for 5 days, 0.2% chlorhexidine mouth rinse three times daily for 7 days.
After a healing period of 6 months, CBCT was performed for implant planning. At the time of implant placement, biopsies were exclusively taken from the grafted bone area. An initial preparation for the implant was made with a hollow trephine burr of 2.8 mm outer diameter with copious irrigation. In this way, a biopsy from the inner core of the trephine burr of approximately 2.5 mm diameter and 8-10 mm long was obtained. Two biopsies were obtained from each sinus. The biopsies were placed instantly in buffered 4% formalin solution. Bone level tapered (BLT) implants (Institut Straumann AG, Basel, Switzerland) were placed according to prior planning. Wound closure and postoperative medication were analogues to those of the augmentation procedure. Postoperative panoramic radiographs (orthopantomograms) were obtained for evaluation of the implant placement. The implants were uncovered and loaded 4 months after placement. At 1 and 2 years after placement, orthopantomogram imaging and clinical evaluation of the implants was performed.
Histological preparation, histopathological analysis and histomorphometrical measurements. The procedures were conducted based on methodologies established by Barbeck and colleagues (18, 22-25). In brief, the biopsies were initially decalcified in 10% Tris-buffered ethylenediaminetetraacetic acid (Carl Roth, Karlsruhe, Germany) at 37°C for 10 days followed by dehydration in a series of increasing alcohol concentrations and final xylol exposure. Then the biopsies were embedded in paraffin followed by sectioning by means of a rotation microtome (SLEE, Mainz, Germany) to a thickness of 3-5 μm. For histopathological and histomorphometrical analyses, hematoxylin-eosin, toluidine and Masson-Goldner staining were prepared.
Serial sections were used for immunohistochemical staining using antibodies to CD11c (ab219799; [EPR21826]) to identify pro-inflammatory cells and CD163 (ab182422; [EPR19518]) to identify anti-inflammatory cells. Furthermore, TRAP5A immunostaining (ab216025; [ACP5/1070]) was conducted to identify inflammatory phagocytes. A biotinylated goat anti-rabbit IgG secondary antibody (ab64256) was also used. All antibodies were sourced from Abcam (Cambridge, UK). The immunohistochemical staining protocol followed the method described by Lindner et al. (22). Briefly, sections were treated with citrate buffer and proteinase K at pH 8 for 20 minutes in a water-bath at 96°C, then equilibrated using TBS-Tween buffer. Slides underwent treatment with H2O2 and avidin and biotin blocking solutions (Avidin/Biotin Blocking Kit; Vector Laboratories, Burlingame, CA, USA), followed by incubation with the respective primary antibody for 30 minutes, then with the secondary antibody. The avidin-biotin-peroxidase complex (ThermoFisher Scientific, Dreeich, Germany) was applied for 30 min, and counterstaining was performed by bluing.
Histological and histomorphometrical analyses adhered to protocols established by Barbeck et al. (22, 26). Microphotographs were taken with a light microscope (Axio Scope A1; Zeiss, Oberkochen, Germany) connected to a digital camera (Axiocam 105; Zeiss). For histomorphometrical analysis of the relative areas of newly formed bone, residual graft material, and connective tissue, as well as the occurrence of the different phagocytes, slides were digitized using a scanning microscope setup consisting of an Axio Scope A1 microscope, scanning table, and ZEN Core software V3 (Zeiss). Occurrence of pro- and anti-inflammatory cells (CD11c- or CD163-positive cells, respectively) was assessed using an ImageJ plugin (National Institutes of Health, Bethesda, MA, USA) developed by Lindner and colleagues (22), which measured the number of positively stained cells per unit area (mm2).
Radiographic evaluation. The postoperative CBCT and the pre implant placement CBCT images were analysed using a OsiriX Lite DICOM viewer (Pixmeo SARL, Bernex, Switzerland). The volume (mm3) of the implanted BSM was calculated for each situs. Additionally, orthopantomography was used to evaluate peri-implant bone stability.
Statistical analyses. Statistical analysis included the normality test (Shapiro-Wilk test), analysis of variance, and a subsequent least significant difference post-hoc test using GraphPad Prism 9 software (GraphPad Software Inc., La Jolla, CA, USA). Values were considered statistically significant when p-values were less than 0.05. Graphs were then generated based on the means and standard deviations.
Results
Histopathological analysis. The histopathological analysis revealed that the implantation areas of both BSMs were completely covered with new bone tissue up to the apical regions, which were identified almost exclusively on the granular surfaces (Figure 1) of both materials. Thus, this growth pattern indicated purely osteoconductive bone growth. Analysis of bone integration further showed that the surfaces of both types of BSM were covered by new bone matrix over large areas (Figure 2A). Furthermore, in the areas of implantation of both BSMs, ongoing bone formation with active osteoblast fringes was still evident in some places (Figure 2B). The analysis of the tissue reactions to the BSM granules revealed that both materials produced a moderate inflammatory tissue reaction, which included both macrophages and multinucleated giant cells (MNGCs) on the material surfaces (Figure 2C). The overall reaction appeared to be slightly more pronounced in the case of Ti-Oss biopsies as a higher frequency of MNGCs was observed.
Overview of exemplary biopsies from areas implanted with Bio-Oss and (B) Ti-Oss analyzed in the present study. In both areas, newly formed bone (BT) was identifiable along the entire length of the biopsies on the bone substitute (BS) surfaces. RB: Residual bone tissue. Masson-Goldner staining, 100× magnification.
Characterization of bone substitute integration and cellular response. (A) Osseointegration of the granules of bone substitute (BS) materials was comparable in the areas implanted with Bio-Oss and Ti-Oss. Masson-Goldner staining. (B) Bone formation with active osteoblast fringes (green arrowheads) for both Bio-Oss and Ti-Oss. Toluidine staining. (C) Tissue reactions to the material granules in Bio-Oss and Ti-Oss involved macrophages (black arrowheads) and multinucleated giant cells (yellow arrowheads). Toluidine staining. BT: Bone tissue; CT: connective tissue. All images were acquired at 100× magnification, scale bars: 50 μm.
The analysis of the immunological response to materials via immunohistochemistry revealed that all MGNCs adherent to surfaces of both BSMs expressed TRAP5A (Figure 3A). Moreover, it was clearly evident that the CD163-positive, and thus anti-inflammatory, macrophages were located a short distance from the granules or in the surrounding connective tissue using both BSMs, while the phagocytes at the surface of materials showed no signs of expression of this molecule (Figure 3B). In contrast, the material-adherent cells showed clear CD11c expression using both BSMs and was almost exclusively limited to the cells on the surface of granules (Figure 3C).
Immunohistochemical visualization of the cellular immune response at the bone substitute interfaces. (A) Tartrate-resistant acid phosphatase 5a (TRAP5A) expression of multinucleated giant cells (yellow arrowheads) at the surface of Bio-Oss and Ti-Oss bone substitute (BS) granules. (B) Expression of CD163 within macrophages (yellow arrows) located a short distance from BS granules or in the surrounding connective tissue (CT) using Bio-Oss and Ti-Oss. CD163-negative macrophages (black arrowheads) and multinucleated giant cells (white arrowheads) at the granule surfaces. (C) CD11c expression by material-adherent macrophages (yellow arrows) and multinucleated giant cells (yellow arrowheads) was limited to areas with Bio-Oss and Ti-Oss. BT: Bone tissue. All images were acquired at 400× magnification, scale bars: 10 μm.
Histomorphometrical analysis. The histomorphometrical analysis of the tissue distribution (areas of newly formed bone, residual graft material, and connective tissue) showed no significant interindividual differences between all three measurement values in both groups indicating comparable regenerative outcomes at this level (Figure 4). However, the intraindividual analysis revealed a highly significantly higher percentage of connective tissue (p< 0.01) compared to the newly formed bone and remaining BSM when using Bio-Oss (Figure 4). Furthermore, the percentage of remaining BSM was significantly higher (p<0.05) when using Ti-Oss (Figure 4).
Results of the histomorphometrical measurements of tissue distribution after implantation of bone substitute materials (BSM). B: Bio-Oss; CT: connective tissue; T: Ti-Oss. Data are expressed as the mean±standard deviation. Each study group consisted of eight replicates. Data were analyzed by analysis of variance with least significant difference post hoc test (Shapiro–Wilk for normality). Significantly different at: *p<0.05, **p<0.01 and ***p<0.001.
The histomorphometrical analysis of the macrophage response also showed no significant interindividual differences between the values by BSM, indicating comparable immunological outcomes at this level (Figure 5A). However, significantly higher numbers of anti-inflammatory M2-macrophages (p<0.05) were detected in areas implanted with Bio-Oss (Figure 5A).
Results of the histomorphometrical measurements of macrophage subtype (A) and the occurrence of M1- and M2-multinucleated giant cells (MNGCs) (B) after implantation of bone substitute materials. B: Bio-Oss; T: Ti-Oss. Data are expressed as the mean ± standard deviation. Each study group consisted of eight replicates. Data were analyzed by analysis of variance with least significant difference post hoc test (Shapiro-Wilk for normality). Significantly different at: *p<0.05 and **p<0.01.
In contrast, the histomorphometrical analysis of the response revealed a significantly higher number of pro-inflammatory MNGCs (p<0.05) in the areas implanted with Ti-Oss (Figure 5B). Moreover, a significantly higher number of pro-inflammatory (M1) MNGCs compared to anti-inflammatory (M2) MNGCs (p<0.01) was found in areas implanted with Ti-Oss (Figure 5B).
Radiographic evaluation. Radiographic evaluation demonstrated that there were no significant differences between the postoperative volume of the implanted BSM and the volume observed at 6 months, either within or between the two BSMs (Figure 6).
Results of the orthopantomography measurements to evaluate the volume of the implanted bone substitute materials, immediately and 6 months after implantation. B: Bio-Oss; T: Ti-Oss. Data are expressed as the mean±standard deviation. Each study group consisted of eight replicates. Data were analyzed by analysis of variance with least significant difference post hoc test (Shapiro-Wilk for normality).
Discussion
The histomorphometrical analysis demonstrated that both Bio-Oss and Ti-Oss achieved comparable regenerative outcomes, as reflected by the absence of significant differences in newly formed bone, connective tissue, and remaining biomaterial fractions. This observation is in line with previous studies reporting the predictable osteoconductive properties of both xenogeneic BSMs (27-29). Despite these similarities, the intraindividual statistical analysis revealed distinct tissue distribution patterns, highlighting material-specific differences in the regenerative process.
In areas implanted with Bio-Oss, the proportion of connective tissue was significantly higher compared to both the fractions of newly formed bone and the remaining biomaterial. This finding might suggest a higher fibrotic tissue response to the xenogeneic material at this time point. Such patterns have been described before, with Bio-Oss being characterized by its long-term volume stability but also by induction of slight fibrosis post-implantation (27, 30, 31). In contrast, the significantly higher percentage of remaining BSM in areas implanted with Ti-Oss may indicate enhanced material persistence, which, while potentially beneficial for space maintenance, might also delay remodeling into mature bone. This aligns with earlier reports on the reduced biodegradability of certain synthetic hydroxyapatite-based substitutes (32).
The immunological analysis further underlined material-dependent differences. Both BSMs elicited overall comparable macrophage responses, suggesting that neither Bio-Oss nor Ti-Oss provokes excessive inflammatory reactions. Nevertheless, in areas implanted with Bio-Oss, the analysis revealed a shift toward anti-inflammatory M2 macrophages, which has been associated with a pro-regenerative microenvironment and improved healing outcomes (33, 34). By contrast, Ti-Oss exhibited a significantly higher number of MNGCs, which were predominantly of a pro-inflammatory phenotype. This pro-inflammatory polarization may represent a foreign-body response, possibly reflecting the physicochemical characteristics of this BSM (25). Importantly, the predominance of pro-inflammatory MNGCs over anti-inflammatory ones in areas implanted with Ti-Oss may negatively influence tissue integration, as sustained inflammation has been linked to impaired bone regeneration and fibrous encapsulation (25, 35, 36). This immunological response might also be influenced by the presence of OCP, as it was described to elicit a dual immunological profile, initially triggering inflammation via interleukin-1 release and macrophage activation, yet also promoting a regenerative immune environment by skewing macrophage polarization toward the anti-inflammatory M2 phenotype (37). However, its pro-inflammatory activity in the context of cartilage repair was also shown (37). Thereby, OCP crystals stimulated significant nitrous oxide release from chondrocytes in a dose-dependent manner by upregulating inducible nitric oxide synthase through p38 and c-JUN N-terminal kinase signaling pathways (37). Moreover, it was also described by Barbeck et al. that material degradation is mainly carried out by pro-inflammatory cells of the macrophage and MNGC lines (38). Thus, the described OCP component might trigger material degradation and thus a local pro-inflammatory milieu. In this case, the material-induced immune response does not seem to lead to a regenerative failure as the foreign-body response to a bone substitute can end in its fibrous encapsulation (25). As bone regeneration and osseoconduction were observed, the higher pro-inflammatory MNGC response might only be linked to material degradation via phagocytosis.
The expression of TRAP5A in biomaterial-adherent macrophages and MNGCs is increasingly recognized as a marker of chronic foreign-body response (39). TRAP5A, the macrophage-associated isoform of acid phosphatase 5, is upregulated in inflammatory macrophages and MNGCs adhering to implants (39). Its activity promotes cell migration and organization on biomaterial surfaces by dephosphorylating osteopontin, thereby reducing integrin-mediated adhesion (40). In addition, TRAP5A contains a redox-active di-iron center that can catalyze reactive oxygen species generation, which is known to contribute to material degradation but may also exacerbate local tissue damage and fibrosis (41). Thus, TRAP5A expression in biomaterial-adherent macrophages and MNGCs reflects a pro-inflammatory phenotype that supports persistence of the foreign-body reaction, which might be necessary for degradation of bioresorbable biomaterials (42).
Radiographic evaluation demonstrated that there were no significant differences between the postoperative volume of the defect area of the implanted BSM and its volume at 6 months, nor between BSMs. This finding aligns with previous investigations reporting stable graft volumes over similar time frames; for instance, a randomized split-mouth sinus augmentation study found that xenogeneic and synthetic BSMs showed no statistically significant volumetric reduction at 6 months, despite an increase in bone density (43). Additionally, long-term follow-up analyses of maxillary sinus floor augmentations using xenografts, alone or combined with autologous bone, reported negligible bone volume changes (approximately 2%) over an average of 6.6 years (44). These results collectively suggest that both BSMs employed in this study exhibit favorable volumetric stability in both the short and longer term, supporting their utility in clinical bone-augmentation procedures.
The present study has limitations that should be acknowledged. In particular, the fact that immunohistochemical assessment was restricted to selected phagocyte markers, providing only a partial view of the complex cellular interactions underlying material degradation and tissue regeneration. Finally, although the split-mouth design minimizes interindividual variability, localized anatomical or vascular differences might still have influenced the regenerative outcomes.
In conclusion, our findings suggest that while both materials support bone regeneration and volume stability at a comparable level, their distinct interactions with the host immune system and remodeling processes result in different qualitative tissue responses. Bio-Oss appears to favor an anti-inflammatory milieu and connective tissue integration, whereas Ti-Oss induces a stronger pro-inflammatory MNGC reaction. These differences underscore the importance of considering not only osteoconductive potential but also immunomodulatory properties when selecting BSMs for clinical application. Future studies should address the long-term implications of these material-specific responses, particularly regarding functional bone regeneration, stability, and the quality of newly formed bone.
Footnotes
Authors’ Contributions
Conceptualization: T.K.; resources: T.K., D.R., B.M., B.D., D.B., O.J. and M.B.; data curation: T.K. and M.B.; writing – original draft preparation: T.K. and M.B.; writing – review and editing: T.K., R.S., D.R., B. M., B.D., D.B., O.J. and M.B.; visualization: M.B.; funding acquisition: T.K. and M.B. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
All the Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All the Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine-learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received September 9, 2025.
- Revision received October 8, 2025.
- Accepted November 14, 2025.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).












