Abstract
Background
After an extraction, the alveolar bone may undergo shrinkage, making it more challenging to place dental implants and compromising the appearance of prosthetic rehabilitation. Studies show that alveolar bone resorption could reach 50% of the original ridge width within the first six months after extraction. Xenografts have shown promising outcomes because they are biocompatible, resemble human bone, and facilitate the growth of new bone. Xenografts, on the other hand, are mostly used as a scaffold for new bone to grow on and don't cause bone growth on their own. Because of this, their integration and remodeling often take a long time and don't always happen completely. This has led researchers to look into other biological agents that can speed up healing and bone growth. Quercetin has been shown to be bioactive, meaning it can fight inflammation and act as an antioxidant. Using a bone xenograft or another suitable carrier or scaffold with quercetin may make it more stable and concentrated at the defect site. This combination may provide the osteoconductive framework necessary for new bone deposition, as well as the osteopromotive qualities of quercetin, to accelerate the cellular and molecular processes related to healing.
Methods
This study involved 64 healthy Wistar rats. The animals were randomly divided into four groups. The animals were euthanized seven to twenty-one days after extraction.
Combining quercetin with a xenogenic bone graft greatly speeds up the repair of the alveolar socket. The combination encourages the production of new bone early on.
Conclusion
The combination of quercetin and bone xenograft significantly improved the repair of alveolar bone sockets in Wistar rats.
Keywords: Quercetin, Bone xenograft, Tooth socket, Collagen matrix, Osteocalcin
1. Introduction
Tooth extraction is a common dental procedure that is often needed because of cavities, gum disease, or injury. It is always followed by a series of physiological bone remodeling mechanisms that affect the size of the alveolar ridge. The alveolar bone may lose height and width after an extraction, which makes it harder to put in dental implants and makes the aesthetic results of prosthetic rehabilitation worse. Studies show that alveolar bone resorption can reach 50% of the initial ridge width in the first six months after extraction. This shows how important it is to use good socket preservation methods [[1], [2], [3]]. People often use bone grafting materials to keep the socket the right size and help the bone grow back. Xenografts, especially those from cows or pigs, have worked well because they are biocompatible, look like human bone, and help new bone grow. Xenografts mainly serve as a framework for new bone to grow, but they don't have the ability to do so on their own. As a result, their integration and remodeling often proceed slowly and incompletely, leading researchers to explore other biological agents that can promote bone production and accelerate healing [[4], [5], [6]].
Flavonoids are a group of polyphenolic compounds found in plants that have bioactive properties that have made them interesting in regenerative medicine. Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is one of the most studied compounds in this group because it is an antioxidant, anti-inflammatory, and osteogenic. Quercetin has been shown to eliminate free radicals, reduce lipid peroxidation, and inhibit the activation of pro-inflammatory transcription factors such as NF-κB. It also stops osteoclasts from changing into other cells and controls the production of cytokines, which is good for bone repair [[7], [8], [9]]. Numerous laboratory and animal studies have demonstrated that quercetin enhances osteoblastic activity by upregulating alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and bone morphogenetic protein-2 (BMP-2). These chemicals are very important for the processes of matrix mineralization and osteogenic differentiation. Quercetin also helps new blood vessels grow by turning on pathways that make VEGF work. This is important for getting nutrients to the bone and making new tissue while it heals [[10], [11], [12]]. Quercetin has been shown to have bioactivity, but it can't help bones heal because it doesn't dissolve well or get into the body easily. If you mix quercetin with a good carrier or scaffold, like a bone xenograft, it may become more stable and concentrated at the problem site.
This combination may provide the osteoconductive framework necessary for new bone deposition, along with the osteopromotive properties of quercetin, to expedite the cellular and molecular processes involved in healing. Prior research has investigated the amalgamation of natural chemicals with graft materials to enhance bone regeneration outcomes, demonstrating synergistic benefits in facilitating osteogenesis and mitigating inflammatory responses [13,14].
In alveolar bone healing, an optimal regenerative strategy should stimulate the recruitment and differentiation of osteoprogenitor cells, augment neovascularization, and increase the deposition of mineralized matrix within the extraction socket. Histological and immunohistochemical analyses are essential methodologies for assessing biological processes, yielding insights into tissue architecture, cellular activity, and the expression of pivotal osteogenic markers, including osteocalcin (OCN) and BMP-2 [15]. These indicators denote bone development and remodeling during the healing phase. Currently, there is insufficient evidence regarding the concurrent use of quercetin and bone xenograft in alveolar bone healing models. The possible synergistic relationship between the antioxidant and osteoinductive characteristics of quercetin and the structural and osteoconductive properties of xenografts has not been thoroughly investigated in the realm of post-extraction socket healing. Thus, the current investigation sought to assess the healing efficacy of a novel combination of quercetin and bone xenograft in the alveolar bone sockets of Wistar rats. The study utilized histological and immunohistochemical techniques to evaluate new bone production, regulation of the inflammatory response, and the osteogenic markers during the healing process. The combination of quercetin with xenograft is posited to augment bone regeneration relative to xenograft alone by expediting osteoblast development and enhancing matrix mineralization. The results of this investigation are anticipated to yield significant insights regarding the prospective implementation of this innovative combination in clinical bone regeneration and socket preservation methodologies.
2. Materials and methods
2.1. Study design
This study was designed as an experimental laboratory study employing a post-test-only control group design to evaluate the effects of a new combination of quercetin and bone xenograft on alveolar bone socket healing in Wistar rats.
2.2. Experimental animals
This study used 64 healthy Wistar rats, 12 weeks old and weighing between 200 and 250 g. There were 64 rats in all, and they were randomly put into four experimental groups, with eight rats in each group. Each group was split into two separate groups of animals, one for each evaluation time point (Day 7 and Day 21), with 8 animals in each group at each time point.
We chose Wistar rats because they consistently respond physiologically in tests of bone regeneration, are easy to handle, and have a well-documented pattern of alveolar bone healing that closely resembles the remodeling of human bones. The animals were acclimatized for one week before the experiment in a controlled setting (temperature 29 ± 2 °C, 12-h light/dark cycle) and were given a standard pellet meal and unlimited access to water [16].
2.3. Experimental groups
The animals were randomly put into four experimental groups. Following initial group allocation, animals were randomly assigned to independent cohorts corresponding to each evaluation time point (Day 7 and Day 21), with eight animals in each group. After the teeth were extracted, the animals were randomly put into four groups for the experiment.
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Control group (C) was the baseline for comparison. In this group, the extraction sockets were permitted to heal on their own without any biomaterials or drugs.
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Quercetin group (Q) (Sigma-Aldrich, St. Louis, MO, USA) the sockets received a topical administration of quercetin solution directly into the extraction socket.
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Xenograft group (X) (Osstem bone graft), the sockets were filled only with bone xenograft material.
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Quercetin + Xenograft group (QX) the sockets received a treatment of both quercetin and bone xenograft. Quercetin gel and bone xenograft were mixed in a 1:1 (w/v) ratio.
All components were utilized immediately after tooth extraction in aseptic settings. After that, the sockets were sutured to keep the materials in place, and all of the animals received routine postoperative care during the experiment. Because histological evaluation required tissue harvesting, animals were sacrificed at each evaluation time point. Therefore, Day 7 and Day 21 represent independent cohorts rather than longitudinal measurements from the same animals. The bone graft material was an anorganic bovine bone graft commercially available (Osstem, South Korea). The graft is made of deproteinized bovine bone mineral that has undergone high temperature treatment to remove organic substances and leaves behind a more crystalline structure made of hydroxyapatite. The dimension of the particles was between about 0.25 and 1.0 mm, which was an osteoconductive scaffold capable of being used to retain alveolar sockets.
Quercetin was incorporated into a 2% (w/v) carboxymethylcellulose (CMC) gel at a dose of 25 μg per socket, and the quercetin gel was mixed with bone xenograft material in a 1:1 (w/v) ratio before being applied directly into the extraction socket immediately after tooth extraction. A 1:1 (w/v) ratio of quercetin-loaded gel to xenograft was used to achieve a homogeneous mixture, facilitating even drug distribution around the graft particles without compromising the osteoconductive scaffold properties of the xenograft.
2.4. Surgical procedure
All surgical procedures were performed under aseptic conditions. Rats were anesthetized using an intramuscular injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). A standardized extraction of the left mandibular incisor was performed using small dental forceps with gentle rotational movements to avoid fracture of the alveolar socket (Fig. 1). Hemostasis was achieved with sterile gauze compression. Immediately after extraction, the respective treatments were applied to the socket according to group allocation. The mucosa was repositioned and sutured using resorbable 5-0 vicryl sutures to maintain the material in place and protect the wound. The rats were maintained on a soft diet for 48 h and monitored daily for signs of distress or infection.
Fig. 1.
Wistar before tooth extraction (1a), The protocol for the extraction of anterior teeth is illustrated in figure (1b), Wistar incisor teeth after extraction (1c).
2.5. Histological processing
At 7 and 21 days post extraction, the mandibular were carefully dissected, and specimens containing the extraction socket were collected and fixed in 10% neutral-buffered formalin for 48 h. After fixation, specimens were decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for three weeks at room temperature, with the solution being changed every three days. After decalcification, the samples were dried in different amounts of alcohol, cleaned in xylene, and then put in paraffin. We used a rotary microtome to make serial sections that were 4–5 μm thick. Then we put them on glass slides so they could be stained.
2.6. Histology staining
H&E staining was performed to evaluate the histological features of the healing socket, including inflammatory cell infiltration, fibroblast proliferation, vascularization. Masson's trichrome staining was used to differentiate collagen fibers and assess the maturation of connective tissue.
2.7. Immunohistochemical analysis
We used immunohistochemistry (IHC) to assess the levels of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN). After deparaffinization and rehydration, antigen retrieval was done using citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, followed by incubation with 5% bovine serum albumin (BSA) to prevent nonspecific binding. The sections were incubated overnight at 4 °C with primary antibodies anti-BMP-2 (1:200, Abcam, UK) and anti-OCN (1:200, Santa Cruz Biotechnology, USA). Detection was achieved using a biotin-streptavidin-peroxidase system with diaminobenzidine (DAB) as the chromogen. The expression of BMP-2 and OCN was analyzed using a light microscope at 400 × magnification.
2.8. Statistical analysis
We used SPSS version 27.0 (IBM, USA) to do the statistical analyses. The data were presented as mean ± standard deviation (SD). The Shapiro–Wilk test was used to see if the data was normally distributed. We first used ANOVA to find differences between groups and time points. Then we used Tukey's post hoc test, Kruskal-Wallis, and Mann-Whitney. A P-value below 0.05 was deemed statistically significant.
3. Results
3.1. Histological evaluation of bone healing
3.1.1. Control group
At 7 days post-extraction, the control sockets exhibited typical early healing characteristics by abundant inflammatory cell infiltration, fibroblast proliferation (Fig. 2), and minimal osteoid tissue and collagen formation. By day 21, most of the socket area has early woven bone formation along the socket wall. Partial bone trabeculae filled the alveolus, but bone maturation remained incomplete, with persistent marrow spaces and fibrous tissue remnants. These findings correspond with normal post-extraction bone healing dynamics described by Cardaropoli et al. (2003), where spontaneous bone fill occurs gradually but incompletely [6].
Fig. 2.
Fibroblast Cells and Inflammatory cells were apparent in the socket area on day 7 and 21. Control Group (2a day 7, 2b day 21), Quercetin (2c day 7, 2d day 21), Xenograft (2e day 7, 2f day 21), Quercetin + Xenograft (2g day 7, 2h day 21), Images captured at × 400 magnification.
3.2. Xenograft group
In sockets filled with xenograft, day 7 showed the integration of graft particles surrounded by inflammatory cells, immature osteoid tissue, and a collagen matrix. By day 21, the xenograft particles were embedded in a dense collagen matrix with active osteoblasts at the periphery, indicating osteoconduction. The presence of residual particles surrounded by new bone was consistent with the known slow resorption profile of xenografts [4,5]. Histomorphometrically, the xenograft group demonstrated moderate new bone area and higher bone–graft contact compared to the control, but lower than the QX combination group.
3.3. Quercetin group
On day 7, sockets that had been treated with quercetin had fewer inflammatory cells and more fibroblasts and osteoblasts growing, as well as more collagen being made. By day 21, the osteoid formation was more organized and the trabeculae were thicker than in the control group. Quercetin's antioxidant and anti-inflammatory properties may have facilitated osteoblastic differentiation and inhibited osteoclastic resorption, as evidenced by both in vitro and in vivo research [9,10]. The early maturation of the bone matrix in this group indicates that quercetin may facilitate the initial phases of bone remodeling, possibly by influencing BMP-2.
3.4. Quercetin + Xenograft combination group (QX)
On day 7, there was only a little inflammatory infiltration, but there were a lot of blood vessels around the graft particles and a thick collagen matrix (Fig. 4). By day 21, new woven bone had formed, filling in the space between the xenograft particles (Fig. 3). This showed that the osteoconductive and osteoinductive activity had increased. Histomorphometric quantification showed that the QBX group had the most new bone area (p < 0.05) compared to all other groups. This supports the idea that quercetin and xenograft work together. As was shown in osteogenic models, quercetin may boost the expression of VEGF and BMP-2, which could lead to more angiogenesis and bone matrix formation [12,13].
Fig. 4.
Collagen fibers were apparent in the socket on day 7 and 21. Control Group (4a day 7, 4b day 21), Quercetin (4c day 7, 4d day 21), Xenograft (4e day 7, 4f day 21), Quercetin + Xenograft (4g day 7, 4h day 21), Images captured at × 400 magnification.
Fig. 3.
Osteoblast Cells and New bone were apparent in the socket on day 7 and 21. Control Group (3a day 7, 3b day 21), Quercetin (3c day 7, 3d day 21), Xenograft (3e day 7, 3f day 21), Quercetin + Xenograft (3g day 7, 3h day 21), Images captured at × 400 magnification.
4. Immunohistochemical evaluation
4.1. BMP-2 expression
BMP-2 immunoreactivity was predominantly situated in osteoblasts and preosteoblasts adjacent to the nascent bone trabeculae. On day 7, the QX group exhibited the highest BMP-2 expression, succeeded by the quercetin group, the xenograft group, and the control group (Fig. 5). This finding corroborates the evidence that quercetin can augment BMP-2 transcription through the ERK and PI3K/Akt pathways, consequently expediting osteoblast differentiation [11,14].
Fig. 5.
BMP-2 expressing cells were apparent in the socket on day 7 and 21. Control Group (5a day 7, 5b day 21), Quercetin (5c day 7, 5d day 21), Xenograft (5e day 7, 5f day 21), Quercetin + Xenograft (5g day 7, 5h day 21), Images captured at × 400 magnification.
4.2. Osteocalcin (OCN) expression
The QX group had strong cytoplasmic staining for osteocalcin on days 7 and 21. Quantitatively, OCN expression was significantly elevated compared to all other groups, indicating enhanced bone maturation (Fig. 6).
Fig. 6.
OC expressing cells were apparent in the socket on day 7 and 21. Control Group (6a day 7, 6b day 21), Quercetin (6c day 7, 6d day 21), Xenograft (6e day 7, 6f day 21), Quercetin + Xenograft (6g day 7, 6h day 21), Images captured at × 400 magnification.
The current study demonstrates that the combination of quercetin with a xenogenic bone graft markedly improves alveolar socket healing compared with individual applications or the control (Fig. 7). The combination facilitates the early resolution of inflammation, angiogenesis, and the formation of new bone, as demonstrated by histological maturity and elevated expression of BMP-2 and OCN. These results support previous studies that suggest bioflavonoids may enhance bone formation and that xenograft scaffolds may be stable enough to replace bone [1,17]. The synergistic effects indicate a promising biomaterial combination for forthcoming translational applications in alveolar ridge preservation and implant dentistry.
Fig. 7.
A diagram of the results for the fibroblast, Osteoblast Cell, Collagen Matrix, BMP-2 and OC expression on days 7 and 21.
Histological and immunohistochemical quantification was performed on standardized digital images captured at × 400 magnification from predefined regions of interest within the extraction socket. For each specimen, quantitative analysis was performed on three non-overlapping microscopic fields selected from standardized regions of interest. Fibroblasts and osteoblasts were quantified by manual cell counting in selected high-power fields and expressed as the mean number of cells per field in the apical region post-extraction socket. Collagen fiber deposition was quantified using ImageJ analysis software by calculating the percentage of positively stained area relative to the total tissue area. The expression of the BMP-2 and OCN was done by counting the immunopositive cells of the osteoblast and preosteoblasts manually. Two histologists carried out all the analyses, having no idea of the groups or time point to minimize observer bias.
4.3. Statistical results
The statistical results of the present study are shown in the following tables.
Comparative analysis of fibroblast, osteoblast, collagen fibers, BMP-2 and Osteocalcin among all treatment groups on the 7th day and 21st day (Table 1, Table 2, Table 3, Table 4, Table 5). This table demonstrates significant gaps between all treatment groups (95% CI), The large treatment effects were evidenced by the analysis of effect sizes (η2) in all measured histological and immunohistochemical outcomes that indicates that the biological relevance of the applied interventions is very high.
Table 1.
Mean and SD of Fibroblast cells on day 7 and 21. Different superscripts revealed significant variation (95% CI).Treatment effects are large at both time points, which suggests that the explanation of the variance by group differences is high.
Table 2.
Mean and SD of Osteoblast cells on day 7 and 21. Different superscripts revealed significant variation (95% CI).Treatment effects are large at both time points, which suggests that the explanation of the variance by group differences is high.
Table 3.
Mean and SD of Collagen Fibers on day 7 and 21. Different superscripts revealed significant variation (95% CI). Treatment effects are large at both time points, which suggests that the explanation of the variance by group differences is high.
Table 4.
Mean and SD of BMP-2 on day 7 and 21. Different superscripts revealed significant variation (95% CI).Treatment effects are large at both time points, which suggests that the explanation of the variance by group differences is high.
Table 5.
Mean and SD of OC on day 7 and 21. Different superscripts revealed significant variation (95% CI).Treatment effects are large at both time points, which suggests that the explanation of the variance by group differences is high.
5. Discussion
The present study evaluated the healing potential of a novel combination of quercetin and bone xenograft on the alveolar bone socket of Wistar rats through histological and immunohistochemical analyses. The results demonstrated that the combination treatment (group III) enhanced new bone formation, improved collagen maturation, and increased expression of osteogenic markers, including bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN), compared with groups treated with quercetin or xenograft alone. These results show that the bioactive characteristics of quercetin and the osteoconductive scaffold of the xenograft work together to speed up socket healing and make bones stronger. After a tooth is pulled, the alveolar bone heals in a complicated way that includes inflammation, the creation of granulation tissue, the deposition of osteoid, and the remodeling of bone. In untreated sockets (control group), early healing was marked by inflammatory cell infiltration and restricted bone trabeculae development, aligning with the natural progression of socket repair. Xenograft-treated sockets, on the other hand, revealed early stability of the blood clot and a well-organized matrix that let osteoblasts connect, which demonstrated that xenografts help bones grow [4,6]. Xenografts are biologically inert and lack osteoinductive molecules, leading to delayed remodeling and the persistence of residual graft particles even months after implantation [3,5,18]. This limitation justifies the need for biologically active adjuncts that can enhance osteoblastic differentiation and matrix mineralization a role quercetin appears to fulfill in the present study.
Onions, apples, and berries all have quercetin, which is a flavonoid. It is well known for its strong anti-inflammatory effects. It gets rid of reactive oxygen species (ROS), stops lipid peroxidation, and changes the amounts of inflammatory cytokines like IL-1β, IL-6, and TNF-α. Reducing oxidative stress in the healing socket environment helps fibroblasts and osteoblasts stay alive, stops tissue necrosis, and speeds up the growth of granulation tissue [12,19]. Quercetin increases osteogenesis at the molecular level by turning on the BMP-2/Runx2 signaling axis. This axis is important for the growth of osteoblasts and the formation of bone matrix. BMP-2 increases the levels of osteogenic markers such as ALP, osteopontin, and OCN. Runx2, on the other hand, controls the transcription of genes that are specific to osteoblasts. The increased levels of BMP-2 and OCN positivity in the QX group demonstrate that quercetin not only stimulated local osteogenic signaling but also enhanced the biological response to the xenograft scaffold [10,11]. Quercetin also alters macrophage polarization by enhancing the M2 phenotype, which secretes growth factors and anti-inflammatory cytokines that facilitate tissue repair. This immunomodulatory effect may explain the less inflammatory infiltration and better tissue organization seen in the combination group when looking at histology.
The integration of quercetin with xenograft material signifies a viable biofunctional composite strategy. The xenograft gives the three-dimensional structure that osteoconduction needs, while quercetin is a bioactive enhancer that changes the cellular milieu and encourages osteoinduction. Similar synergistic effects have been shown with additional natural substances, including curcumin, resveratrol, and alendronate, when integrated into graft or scaffold systems [13,14]. The increased bone development seen in the QX group is in line with studies that suggest that giving quercetin locally can boost angiogenesis and osteoblastic differentiation, which leads to more new bone formation and collagen deposition. Also, quercetin's antioxidant qualities probably helped keep redox equilibrium at the graft site, which kept osteogenic cells from being damaged by oxidative stress and dying [20]. From a biomaterials standpoint, the incorporation of quercetin in the xenograft matrix may have affected ion exchange kinetics and protein adsorption, promoting enhanced recruitment of osteoprogenitor cells and the deposition of extracellular matrix proteins. All of these actions speed up the change from woven bone to lamellar bone during the remodeling phase.
Histologically, the QX-treated sockets displayed more extensive new bone trabeculae characterized by well-organized collagen fibers, as seen by Masson's trichrome staining. Collagen maturation is an essential factor in determining bone quality, as type I collagen acts as a framework for mineral deposition and establishes mechanical strength. Quercetin has been shown to increase collagen production and cross-linking by increasing the activity of lysyl oxidase and decreasing the breakdown of matrix metalloproteinases (MMPs). This mechanism may elucidate the higher developed collagen matrix observed in the QX group relative to the other groups [21,22]. Also, the group had a lower inflammatory response and better blood flow, which suggests that quercetin helped angiogenesis, which is important for getting nutrients to cells and bringing in osteoblasts.
Quercetin can induce angiogenic activity by activating VEGF-initiated signaling pathways. Thus, such mechanisms might be in line with the increased vascularization that was found in the current study. Quercetin could cause angiogenic effect and this effect is mediated by the alterations of the major intracellular signaling cascades, particularly, PI3K/Akt and ERK1/2. Activation of these pathways has broadly been associated with endothelial cell survival, growth and migration each which is essential in neovascularization and consequential repair of the tissues. Quercetin has been proposed in helping a microenvironment in which regeneration is able to occur. However, since the analysis of these molecular events has not been done in the current study, the given explanation can be regarded as a speculation including references to the literature that has already been published and not the direct result of our findings. Further studies using molecular studies would be necessary in the future in order to comprehend to what extent quercetin influences the angiogenic signaling in bone repair [8,12,23].
Immunohistochemical investigation validated the augmented osteogenic activity within the combo group. At early time points (7 days), BMP-2 expression was significantly higher, indicating that osteoblast differentiation was actively occurring. This discovery supports earlier studies showing that quercetin increases BMP-2 expression in osteoblast-like cells and helps mineralized nodules form. Osteocalcin (OCN), a late-stage marker of bone formation, showed higher levels of expression in the QX group at 21 days. This means that new bone was becoming more mineralized and mature. These results are consistent with earlier animal studies demonstrating that quercetin enhances mineral apposition rates and bone density when used in conjunction with graft materials [22,24].
The results of this study have important effects on how to keep alveolar bone healthy and how to treat it with regenerative medicine. It is very important to heal the socket after an extraction in order to place an implant correctly. Instead of using expensive recombinant growth factors like rhBMP-2, which can have bad side effects, you can use biologically active compounds like quercetin to do this [25]. The combination of quercetin and xenograft may serve as a biomimetic and safer option that improves bone quality while reducing inflammation and oxidative damage. The quercetin–xenograft combination was associated with enhanced osteogenic-related activity, as evidenced by histological and immunohistochemical findings. Nevertheless, despite the encouraging current findings, additional concerns require further examination prior to clinical application. These encompass the optimization of quercetin dosage, the design of the delivery system, and the long-term assessment of graft remodeling and biomechanical strength. Additionally, molecular investigations into the downstream pathways of BMP-2 and Runx2 activation may yield enhanced understanding of the signaling mechanisms underlying the observed effects.
5.1. Limitations of the study
The sample size was limited to a small number of animals, which may not have fully represented how human bones work. There was no study of the biomechanical properties of the new bone, and there was also no study of the systemic pharmacokinetic data for quercetin. Also, only two immunohistochemical markers were evaluated. Future studies should include a wider range of markers, such as ALP, VEGF, and osteopontin, to get a full picture of osteogenesis.
6. Future perspectives
Further research employing larger animal models and controlled release methodologies for quercetin may validate its therapeutic effectiveness in clinical bone regeneration. Furthermore, examining the synergistic effects of quercetin in conjunction with other bioactive compounds, such as chitosan, platelet-rich fibrin, or stem cell-derived exosomes, may yield improved restorative outcomes. Subsequent investigations, including functional evaluations and pathway-specific analyses, are necessary to definitively ascertain whether these osteogenic-related effects fulfill the criteria for authentic osteoinduction.
7. Conclusion
The combination of quercetin and bone xenograft significantly enhanced alveolar bone socket healing in Wistar rats by promoting fibroblast, osteoblast differentiation, increasing collagen maturation, and upregulating the expression of BMP-2 and Osteocalcin.
Author contributions
The manuscript was written, revised, and edited with input from CK and IRK. All authors have approved the manuscript after reading the published version.
Ethics
The Scientific Research Ethics Committee of the Faculty of Dentistry, Airlangga University, Indonesia, has approved this research procedure.
Funding
This research received no external funding
Conflicts of interest
None.
Footnotes
Peer review under responsibility of the Japanese Society for Regenerative Medicine.
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