Bovine amnion membrane-hydroxyapatite composite enhances alveolar bone regeneration by modulating osteoclast activity and woven bone formationAbstract
Tooth extraction without proper socket prevention will lead to bone loss. Applying bovine amnion membrane-hydroxyapatite (BAM-HA composite) can maintain bone structure through osteoclast and woven bone formation. The purpose of the study to evaluate the different compositions of BAM-HA composite as socket preservation by analyzing osteoclast and woven bone formation. Thirty male Sprague-Dawley rats underwent lower anterior teeth extraction. Each socket treated by BAM-HA composite with different ratio or composition 30:70, 35:65, 40:60 and control group treated with BioOss collagen. Fourteen and twent-eight-days after treatment, the mandible was collected for histological analysis for osteoclast and woven bone formation using hematoxyline-eosine staining. Statistical analysis was performed using two-way ANOVA and Tukey’s post-hoc test with p<0.05 consider as statistical differences. BAM-HA composite treated groups showed a reduction in osteoclast numbers compared to the control and BioOss groups (p<0.05), indicating a suppression of excessive bone resorption. Concurrently, BAM-HA composite-treated groups, particularly composition 30:70 and 35:65, exhibited the highest levels of woven bone formation (p<0.05), suggesting enhanced bone regeneration. The application of the BAM-HA composite enhances alveolar bone regeneration in Sprague-Dawley rats tooth extraction sockets by reducing osteoclast number and promoting woven bone formation, suggesting its potential as an effective bone graft material to maintain the alveolar bone height.
Keywords:
composite; bovine amnion membrane; hydroxyapatite; woven bone; osteoclasts
Resumo
A extração dentária sem a prevenção adequada do alvéolo leva à perda óssea. A aplicação de membrana de âmnio bovino com hidroxiapatita (compósito BAM-HA) pode manter a estrutura óssea por meio da formação de osteoclastos e osso neoformado. O objetivo do estudo foi avaliar as diferentes composições do compósito BAM-HA como elemento de preservação do alvéolo, analisando a formação de osteoclastos e osso neoformado. Trinta ratos machos Sprague-Dawley foram submetidos à extração de dentes anteriores inferiores. Cada alvéolo foi tratado com compósito BAM-HA com diferentes proporções ou composições: 30:70, 35:65 e 40:60, e o grupo controle foi tratado com colágeno Bio-Oss. Após 14 e 28 dias do tratamento, a mandibula foi coletada para análise histológica da formação de osteoclastos e osso neoformado, utilizando coloração com hematoxilina-eosina. A análise estatística foi realizada utilizando ANOVA bidirecional e teste post-hoc de Tukey com p<0,05, considerados como diferenças estatísticas. Os grupos tratados com o compósito BAM-HA apresentaram redução no número de osteoclastos em comparação aos grupos controle e Bio-Oss (p<0,05), indicando supressão da reabsorção óssea excessiva. Concomitantemente, os grupos tratados com o compósito BAM-HA, particularmente as composições 30:70 e 35:65, exibiram os maiores níveis de formação de osso reticular (p<0,05), sugerindo maior regeneração óssea. A aplicação do compósito BAM-HA potencializa a regeneração óssea alveolar em alvéolos de extração dentária de ratos Sprague-Dawley, reduzindo o número de osteoclastos e promovendo a formação de osso reticular, sugerindo assim seu potencial como um material de enxerto ósseo eficaz para manter a altura do osso alveolar.
Palavras-chave:
compósito; membrana de âmnio bovino; hidroxiapatita; tecido ósseo; osteoclastos
1. Introduction
Tooth extraction without socket preservation can lead to significant alveolar bone resorption, compromising future prosthetic rehabilitation such as dental implants (Quisiguiña Salem et al., 2023). Various xenografts have been used as composite scaffolds for socket preservation, demonstrating their ability to maintain alveolar bone structure (Minetti et al., 2023). To address this, various xenografts and synthetic scaffolds have been used to preserve socket volume and structure (Minetti et al., 2023). However, these materials present several limitations, including low resorption rates, limited osteoinductive properties, risk of immune reaction, and high cost. Moreover, many commercially available materials primarily act as passive fillers and lack the biological cues necessary to actively promote bone regeneration (Jiang et al., 2020). One promising biomaterial for socket preservation is bovine amniotic membrane (BAM), which contains amide A, amide B, amide I, amide II, and triple-helical collagen structures. These protein structures, particularly the amide bands, represent characteristic vibrational modes of collagen molecules that indicate a high content of structurally intact proteins. The triple-helical collagen structure is essential for providing mechanical strength and stability to the scaffold, while also supporting cell adhesion, migration, and differentiation—key processes in wound healing and bone regeneration. Together, these features contribute to the biological activity and structural integrity of BAM, making it a suitable matrix for socket preservation. BAM has a low porosity size, higher porosity proportion of 64%, and a swelling capacity of 1320 times its weight (Octarina et al., 2022b). The application of BAM in extraction sockets has been shown to promote new bone regeneration by enhancing fibroblast proliferation, angiogenesis, and the expression of bone-related growth factors such as interleukin 6 (IL-6) and bone morphogenetic protein (BMP-2) (Indrawati et al., 2019). While fibroblasts do not directly induce bone formation, they contribute to the formation of extracellular matrix and cytokine signaling that facilitate subsequent osteogenesis.
However, BAM alone has limitations, including low compressive strength, larger pore size, and suboptimal swelling and biodegradation rates. To enhance its mechanical properties and bioactivity, BAM has been combined with hydroxyapatite (HA) to form a BAM-HA composite. Studies have shown that different BAM-to-HA compositions (30:70, 35:65, and 40:60 w/w) improve the physical characteristics of the composite. Specifically, the 35:65 composition has been reported to exhibit favorable structural and biological properties, including high porosity, excellent swelling capacity, slow degradation rate, and strong fibroblast viability, making it a promising candidate for tissue regeneration applications (Agustantina et al., 2023; Octarina et al., 2022a). Enhancing mechanical properties in socket preservation materials is essential to provide temporary structural support, maintain space for bone ingrowth, and resist collapse under masticatory forces. While these materials do not need to match the exact strength of native alveolar bone, sufficient mechanical stability is crucial during the early healing phase to facilitate effective bone regeneration. Additionally, incorporation of hydroxyapatite into the composite increases its bioactivity by mimicking the mineral phase of natural bone, promoting osteoconduction, and enhancing cell adhesion, proliferation, and differentiation, thereby supporting new bone formation.
The clinical application of BAM composites has shown increased collagen density, osteoblast proliferation, and upregulation of key osteogenic markers such as bone morphogenic protein 2 (BMP-2), RUNX2, and osteocalcin in the alveolar socket (Octarina et al., 2024). However, limited studies have explored the role of BAM in modulating osteoclast activity and promoting woven bone formation, two critical processes in early alveolar bone remodeling after tooth extraction. While osteoclasts facilitate the removal of necrotic bone to shape the socket, woven bone serves as the initial scaffold for regeneration. In the context of socket preservation, osteoclasts and woven bone play essential roles in bone remodeling and regeneration (Omi and Mishina, 2022; Shah et al., 2019). Osteoclasts facilitate bone resorption by removing necrotic bone and shaping the socket to accommodate new bone formation (Udeabor et al., 2023). This regulated resorption is crucial for maintaining alveolar ridge integrity and preventing excessive bone loss, which could otherwise compromise implant placement. Meanwhile, woven bone serves as the initial bone matrix formed during the healing process. It provides a temporary scaffold that stabilizes the extraction site and undergoes remodeling into mature lamellar bone with enhanced mechanical properties.
The purpose of this study is to evaluate the effectiveness of BAM-HA composite in socket preservation by analyzing osteoclast activity and woven bone formation. Understanding the balance between osteoclast-mediated resorption and woven bone deposition will provide insights into the regenerative potential of these composites. Furthermore, optimizing the composition of BAM and HA could enhance bone remodeling, ultimately improving clinical outcomes in socket preservation and implant dentistry.
2. Materials and Methods
2.1. Design of the study
The present study was conducted as post-test only control group design. All the protocol of this study was registered and approved by Ethic committee in Faculty of Dental Medicine Universitas Airlangga under registration number 842/HRECC.FODM/XI?2022.
2.2. Animal
This study used Sprague Dawley rats aged 2-3 months with a body weight of 250-300 g. Each rat has cages individually, with free access to water and food. The light in room cages was maintained 12-hour dark and 12-hours light.
2.3. Alveolar bone regeneration model
An alveolar bone regeneration model was utilized to assess the effects of a BAM-HA composite. To induce alveolar bone regeneration, the first mandibular molar of rats was extracted. Following extraction, the sockets were filled with BAM-HA composite in varying ratios (30:70, 35:65, and 40:60). The positive control group received Bio-Oss® (Geistlich Bio-Oss® Collagen, Geistlich Pharma AG, Bahnhofstrasse, Wolhusen, Switzerland), while the negative control group was left untreated.
During the procedure, rats were anesthetized via intraperitoneal administration of a ketamine-xylazine mixture (2:1, v/v). At 14- and 28-days post-treatment, the rats were sacrificed, and mandibular specimens were collected for further tissue analysis.
2.4. Preparation of BAM-HA composite
BAM samples weighing 3 g, 3.5 g, and 4 g were prepared. Each sample was cut into pieces, immersed in 40 mL of 0.9% NaCl solution, and soaked for 5 minutes. The soaked samples were then homogenized into an amniotic slurry using a blender for 10 minutes. Subsequently, 7 g, 6.5 g, and 6 g of hydroxyapatite were added to the slurry, mixed thoroughly until a homogeneous mixture was obtained, and poured into a square acrylic mold (5 × 5 cm, 0.5 cm thickness). The samples were then frozen at -80 °C for 24 hours, followed by freeze-drying for 48 hours. The BAM and hydroxyapatite used in this study were prepared following the protocol described by Octarina et al. (2022b).
2.5. Osteoclast and woven bone
Histological sections were stained with hematoxylin and eosin to visualize osteoclasts and woven bone cells. The stained sections were examined using a NIKON Eclipse SI microscope equipped with a Nikon digital camera, and images were captured with NIS Elements D software at 400× magnification. For each section, five different areas were analyzed.
2.6. Statistical analysis
The data were tested for normality and homogeneity before statistical analysis. A two-way ANOVA followed by Tukey’s post-hoc test was performed to evaluate the osteoclast cell count and differences in woven bone formation between days 14 and 28 following the application of BAM-HA composite in sockets of Sprague-Dawley rats. Comparisons were made among the treatment, positive control, and negative control groups. Data analysis was conducted using the SPSS software.
3. Results
Extraction sockets were treated with various BAM-HA composite. Osteoclast numbers were assessed at 14- and 28-days post-treatment to evaluate bone resorption and remodeling dynamics.
At 14 days, the control group (untreated sockets) exhibited the highest osteoclast count (6.00 ± 0.82), indicating active bone resorption during the early healing phase. The BioOss-treated group showed a moderate reduction in osteoclast numbers (4.75 ± 1.26), whereas all composites from BAM-HA composite-treated groups exhibited a further decline in osteoclast activity, suggesting an initial modulation of bone resorption (Figures 11B).
Histological and quantitative analysis of bone regeneration following treatment with BAM-Hydroxyapatite composites. (A) Representative H&E stained sections of bone defects treated with BAM-Hydroxyapatite composites (30:70, 35:65, and 40:60), Bio-Oss, and control at 14 and 28 days post-implantation; (B) Quantification of osteoclast numbers across different treatment groups at 14 and 28 days, demonstrating a significant reduction in osteoclast presence in BAM-Hydroxyapatite-treated groups compared to the BioOss; (C) Quantification of woven bone formation, indicating a significant increase in new bone formation in BAM-Hydroxyapatite-treated groups relative to the control (*p<0.05, **p<0.01, ***p<0.001, ****p< 0.0001). Data are presented as mean ± standard deviation.
By 28 days, osteoclast numbers significantly decreased across all groups. The BAM-HA composite (30:70) showed the lowest osteoclast count (1.00 ± 0.10), followed by BAM-HA composite (35:65) and (40:60) (1.33 ± 0.58; 3.00 ± 1.00; respectively). These findings indicate that BAM-HA composite reduces osteoclast activity over time, which may help to limit excessive bone resorption during healing. The reduction in osteoclast numbers further indicates that BAM-HA composite may provide a favorable bone-healing environment by minimizing excessive bone resorption while supporting bone regeneration.
At 14 days, the control group exhibited the lowest presence of woven bone structures (1.25 ± 0.50), indicating minimal early bone formation. The xenograft and BAM-HA composite (40:60) groups showed moderate improvement (2.25 ± 0.96; 2.50 ± 1.29; respectively), while the BAM-HA composite (30:70) (5.67 ± 0.58), and (35:65) (5.00 ± 1.00) groups showed the highest levels of woven bone formation, suggesting accelerated early osteogenesis (Figure 1C).
By 28 days, all BAM-HA composite -treated groups showed a significant increase in woven bone formation compared to the control and Bio-Oss groups. The BAM-HA composite (30:70) and (35:65) composites exhibited the highest levels of woven bone, indicating their potential to enhance bone regeneration. Statistical analysis revealed highly significant differences between the BAM-HA composite groups and the control, further supporting the osteogenic potential of these formulations.
These findings suggest that BAM-HA composite, particularly the 30:70 and 35:65 formulations, effectively promote bone regeneration by accelerating woven bone formation, highlighting their potential for improving post-extraction socket healing.
4. Discussion
The findings of this study underscore the pivotal roles of osteoclasts and woven bone in the regeneration of alveolar bone following tooth extraction. Osteoclasts are essential for bone remodeling, as they resorb necrotic bone and shape the socket to accommodate new bone formation (Singh et al., 2023). However, excessive osteoclast activity can lead to detrimental bone loss (Bi et al., 2017). Conversely, woven bone serves as the initial scaffold for bone regeneration (Shapiro and Wu, 2019), providing a temporary but highly cellular and vascularized structure that supports early stabilization of the extraction site (Bixel et al., 2024). Over time, woven bone undergoes remodeling into mature lamellar bone, which possesses superior mechanical properties necessary for functional load-bearing (Hart et al., 2020).
The application of bioactive composites, such as BAM-HA, has been shown to modulate osteoclast activity and enhance woven bone formation, thereby creating an optimal microenvironment for bone regeneration (Octarina et al., 2022a). The observed modulation of osteoclast activity and enhancement of woven bone formation aligns with previous studies that have demonstrated the osteoinductive properties of BAM composites (Agustantina et al., 2023). For instance, the increased of osteoblast proliferation and upregulation of osteogenic markers such as BMP-2, RUNX2, and osteocalcin following the application of BAM composites (Octarina et al., 2024).
In our study, the BAM-HA composites, particularly at the 30:70 and 35:65 ratios, effectively reduced osteoclast numbers and promoted woven bone formation. These results are consistent with the findings of Wibowo et al. (2023), who observed that BAM application led to increased osteoblast and osteocyte activity, as well as enhanced collagen formation in the alveolar bone socket of Sprague-Dawley rats (Wibowo et al., 2023).
Furthermore, the increased hydroxyapatite content in the BAM-HA composite formulations was associated with enhanced bone regeneration (Agustantina et al., 2023; Octarina et al., 2022b). This is in line with the previous study, which found that higher hydroxyapatite content in BAM-HA composites led to increased collagen thickness, osteoblast proliferation, and expression of osteogenic-related factors such as BMP2, RUNX2, and osteocalcin (Octarina et al., 2024).
The clinical application of BAM-HA composites has been associated with bone regeneration. These findings suggest a strong connection between composite application and bone regeneration mechanisms. BMP-2 plays a key role in promoting osteoblast differentiation and bone matrix production (Zhou et al., 2023), while RUNX2 is a crucial transcription factor for osteoblast maturation (Komori, 2017). Osteocalcin, a late-stage bone marker, indicates ongoing mineralization and bone tissue formation (Zoch et al., 2016). The upregulation of these markers in BAM-HA-treated sockets corresponds with the enhanced woven bone formation observed in this study, confirming that the composite provides a favorable environment for bone healing.
The present findings highlight the potential of BAM-HA composites as bioactive grafting materials capable of modulating osteoclast activity, enhancing woven bone formation, and upregulating key osteogenic markers such as BMP-2, RUNX2, and osteocalcin. Translationally, this provides a mechanistic basis for the rational design of composite formulations optimized for alveolar bone regeneration. Clinically, the use of BAM-HA composites may accelerate socket healing, preserve alveolar ridge dimensions, and provide a more favorable foundation for subsequent implant placement. By reducing excessive bone resorption and promoting the transition from woven to lamellar bone, BAM-HA composites hold promise as cost-effective and biocompatible alternatives to conventional grafting materials, with potential applications extending beyond oral surgery to broader orthopedic indications.
This study has several limitations. It was conducted in an animal model, which may not fully represent human bone healing, and the observation period was limited to the early healing phase (14 and 28 days), without assessing long-term outcomes or lamellar bone maturation. Additionally, molecular mechanisms underlying the observed effects were not explored, and only selected BAM-HA ratios were tested, warranting further investigation with broader formulations and clinical validation.
5. Conclusion
Overall, the study suggests that BAM-HA composites, especially the 30:70 and 35:65 formulations, are effective biomaterials for promoting bone regeneration in post-extraction sockets. Their ability to suppressing excessive osteoclast activity and promoting woven bone formation through osteoblast activation and extracellular matrix deposition. This dual mechanism contributes to improved socket preservation outcomes, making BAM-HA a promising material for clinical applications in alveolar bone regeneration.
Data Availability Statement
The research data are only available upon request to the corresponding author elly-m@fkg.unair.ac.id
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Editor:
Takako Matsumura Tundisi
