Evaluation of Xenograft-Enhanced Osteogenesis and Modulation of Systemic Inflammation in Post-Extraction Alveolar Bone Defects (An Experimental Rat Study)

Evaluation of Xenograft-Enhanced Osteogenesis and Modulation of Systemic Inflammation in Post-Extraction Alveolar Bone Defects (An Experimental Rat Study) | 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 Evaluation of Xenograft-Enhanced Osteogenesis and Modulation of Systemic Inflammation in Post-Extraction Alveolar Bone Defects (An Experimental Rat Study) Aliaa A. Habib, Eman M. Salem, Ghadir Elnawawy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8941374/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Introduction: While tooth extraction is a routine dental procedure, it often leads to alveolar bone resorption, which changes the shape and size of the ridge. Over the past years, various bone graft materials have been investigated, with demineralized freeze-dried bovine bone xenograft (DFDBBX) being among the most widely used in dentistry. DFDBBX exhibits both osteoinductive and osteoconductive properties, thereby enhancing new bone formation. Objectives This study aimed to evaluate bone healing and systemic biological responses following tooth extraction and socket preservation with a xenograft in an animal model simulating clinical conditions. Histological analysis assessed local bone regeneration, while hematological measurements of pro-inflammatory (CRP, TNF-α, IL-6, IL-1β), anti-inflammatory (IL-10), and bone metabolism markers (Osteocalcin, BMP-1) provided a comprehensive evaluation of local and systemic effects. Materials and methods Forty-five white rats, animals were divided into three groups: Control (no extraction), Extraction without bone grafting, and Extraction with bone grafting. For histological analysis, tissues were fixed in 10% formalin, decalcified using an EDTA-based solution, and stained with H&E and Masson's trichrome to assess bone healing and osteogenic markers. Blood samples were collected and analyzed for inflammatory markers (CRP, Procalcitonin, TNF-α, IL-6, IL-1β, IL-10) and bone formation markers (BMP-1, Osteocalcin) using commercially available ELISA kits. Results CRP, TNF-α, Procalcitonin, and IL-6 differed significantly among groups at 4 and 6 weeks ( p<. 001) and decreased over time in both extraction groups. Histologically, the Extraction-only group showed granulation tissue and incomplete trabecular bone by 6 weeks, whereas the Extraction + Bone Graft group exhibited early osteoid at 2 weeks, osteoblast-lined new bone at 4 weeks, and mature trabecular bone with organized collagen and normal osteocytes by 6 weeks. Masson's Trichrome confirmed enhanced collagen deposition and matrix organization in the grafted group. Conclusion Xenografts enhance post-extraction bone healing by promoting organized trabecular and collagen matrix formation while modulating systemic inflammation, supporting ridge preservation, and creating optimal conditions for future restorative procedures. Alveolar bone preservation Xenograft Osteogenesis Tooth extraction Inflammatory markers Bone regeneration Rat model Figures Figure 1 INTRODUCTION Tooth extraction is a common procedure in dentistry; however, it is frequently followed by alveolar bone resorption that alters both the morphology and dimensions of the alveolar ridge. ( 1 ) This resorptive process primarily affects the horizontal dimension and is more pronounced on the buccal aspect than on the lingual or palatal surfaces. ( 2 ) The most rapid bone loss occurs within the first six weeks to two years after extraction, with significant and irreversible dimensional changes observed as early as six months, resulting in up to 40% loss of bone height and 60% loss of ridge width. ( 3 , 4 ) . It is well recognized that extraction leads to a reduction in both buccolingual and apico-coronal dimensions of the alveolar ridge. ( 5 ) This resorptive process can compromise future dental rehabilitation, particularly implant placement; however, it can be minimized through minimally traumatic extraction techniques combined with socket preservation using regenerative graft materials to maintain ridge dimensions and promote bone healing. ( 6 – 10 ) Adequate preservation of bone volume is essential, as insufficient support compromises the placement and long-term stability of dental implants and fixed prostheses, thereby negatively affecting function, retention, esthetics, and patient comfort. ( 11 ) Graft options include autografts from the patient, allografts from human donors, xenografts from other species, and synthetic substitutes such as calcium phosphate, hydroxyapatite, or bioactive glass. ( 12 – 14 ) These materials promote bone repair through three main biological processes: osteogenesis, osteoconduction, and osteoinduction. ( 15 ) Osteogenesis refers to new bone formation by osteoblasts contained within the graft; osteoconduction provides a scaffold that supports bone growth across its surface; while osteoinduction triggers undifferentiated stem cells in surrounding tissues to transform into bone-forming cells. ( 16 ) Autogenous bone grafts are often regarded as the “gold standard”; however, their use is limited by the need for a second surgical site and restricted availability. ( 17 , 18 ) Allografts sourced from bone banks are more accessible but carry the drawback of potential disease transmission. ( 19 , 20 ) Consequently, xenografts and synthetic substitutes have become the most widely adopted alternatives in dental practice. ( 20 ) Over the past years, various bone graft materials have been investigated, with demineralized freeze-dried bovine bone xenograft (DFDBBX) being among the most widely used in dentistry. DFDBBX exhibits both osteoinductive and osteoconductive properties, thereby enhancing new bone formation. ( 21 ) As a xenograft derived from bovine bone, it shares key structural characteristics with human bone, including crystalline composition, porosity, and carbonate content, which support natural osteoconduction. By stabilizing blood clots and providing a mineral- and collagen-rich scaffold, xenografts facilitate angiogenesis, revascularization, and osteoblast migration from the base of the socket, thereby reducing alveolar bone resorption and promoting effective bone regeneration. ( 9 , 22 ) Optimizing the quality of the existing bone grafting materials and looking for novel and better bone-substitute materials is crucial in improving the clinical outcome. Experimental testing of various grafting materials requires establishing an appropriate biological model to conduct studies and evaluate their clinical effects on osteogenesis and healing. Animal models with simulated bone defects are considered appropriate as experimental models for testing clinical interventions. ( 23 – 25 ) Next to congenitally induced models, surgically created bone defects in animals also seem suitable for experimental studies regarding bone grafting material's histologic and biomechanical properties. Moreover, it is essential to allow proper timing for the healing of the defect and establish an alveolar cleft of appropriate width that mimics the human scenario of a skeletal defect extending to the nasal mucosa and the adjacent teeth, and is suitable for clinical testing. ( 26 , 27 ) The present study aims to assess bone healing histologically using an animal model developed to create bone defects that closely replicate clinical conditions for testing tissue-engineered bone substitute materials without compromising animal health. MATERIAL AND METHODS Forty-five rats, aged 8–10 weeks, weighing approximately 250–300 grams, were obtained from the animal house of Pharos University in Alexandria (PUA). The ethical approval was obtained from the Unit of Research Ethics Approval Committee (UREAC) of PUA (02202410273282), complying with the ARRIVE reporting guidelines. Rats were housed in standard experimental conditions and kept in well-ventilated plastic cages with wood shavings as bedding with a 12-hour light/dark cycle and ad libitum access to food and water for at least 7 days before the experiment. Animals are fasted for 6 hours before the surgery to reduce the risk of aspiration during anesthesia. They were allocated to three equal groups: Control (No-extraction) group: Fifteen rats without intervention. Fifteen rats underwent tooth extraction without a bone graft. Fifteen rats underwent tooth extraction followed by Xenograft bone graft placement. - Time points for evaluation: 2, 4, and 6 weeks post-extraction. - Ethical Considerations: All procedures are carried out in accordance with the ethical guidelines for animal research and approved by Pharos University (PUA 02202410273282). Materials - Anesthetics and Analgesics: Ketamine (60 mg/kg) for general anesthesia. Xylazine (10 mg/kg) to enhance sedation by intraperitoneal injection for general anesthesia. - Bone Graft Materials: Xenograft bone graft (OneGraft) (lab-made). - Histological Analysis : 10% Formalin solution for tissue fixation. Decalcifying solution (e.g., EDTA-based solution) for bone decalcification. Hematoxylin and Eosin (H&E) staining kit for histological assessment of bone healing. Immunohistochemically (Masson's trichrome stain) reagents for markers of osteogenesis (e.g., Osteocalcin, Runx2). Methods: I. Surgical Procedure: · The aseptic technique is followed throughout the procedure. · Tooth Extraction: The tooth is extracted using forceps. Care is taken to avoid damage to the surrounding bone. The Extraction aimed to create a bone defect. · Bone Graft Placement: After Extraction, the alveolar socket is debrided to remove any remaining soft tissue and bone debris. A bone graft is prepared and inserted into the socket to fill the defect using xenograft and finely minced before graft placement. The graft is compacted to ensure stable placement within the socket. The surgical site is irrigated with sterile saline to maintain cleanliness · Closure: The mucosal incision is sutured using absorbable sutures (3-0 Vicryl) in a simple interrupted pattern - Postoperative Care: Animals are kept in a warm recovery area until they are fully awake from anesthesia. Postoperative analgesia is administered (Carprofen 5 mg/kg) for 3 days. An antibiotic (Enrofloxacin 10 mg/kg) was given for 5 days to prevent infection. Rats are monitored daily for signs of infection, pain, or complications. II. Histological procedure (28) - Fixation: The left half of the mandibles was fixed immediately in 10% neutral buffered formalin solution for 48 hours: - Preparation of the 10% neutral buffered formalin solution: 40% formalin (100 ml) Tap water (900ml) Sodium phosphate monobasic (4.0gm) Sodium phosphate dibasic anhydrous (6.5gm) - Washing: After fixation, the specimens were washed in running water to remove the formalin. - Decalcification: The specimens were decalcified in 8% trichloroacetic acid and were tested for complete decalcification by piercing the hard tissue with a needle far from the target socket. The tissue was ready for further treatment when the needle easily entered the bone. Then, the mandibles were washed in running water for at least 24 hours to remove all the acid. - Dehydration: The specimens were gradually dehydrated by passing through increasing percentages of ethyl alcohol (40%, 60%, 80%, 95%, and absolute alcohol), remaining in each dish for several hours. - Clearance: The specimen is passed from alcohol through two changes of xylene, a clearing agent that is miscible with both alcohol and paraffin. - Infiltration: The specimens were removed from the xylene to be infiltrated with paraffin. They were placed in a dish of melted embedding paraffin, and the dish was put into a constant-temperature oven regulated to about 60° C. The specimens were then left in the oven for 2 hours. - Embedding: When the specimen is completely infiltrated with paraffin, it is embedded in a small paper box filled with melted paraffin, and with warm forceps, the specimen is removed from the dish of melted paraffin and placed in the center of the box of paraffin in a direction to obtain a longitudinal bucco-lingual section. - Cutting: The hardened paraffin block was mounted on a paraffin-coated wooden cube and then clamped on a precision rotary microtome, which was adjusted to cut serial sections of 5 μm in thickness. - Mounting: The wax ribbons were mounted on the glass slide and then placed in a constant-temperature oven. - Staining: The stains used in this study were H&E (Hematoxylin and Eosin) and trichrome stains. · Haematoxylin and Eosin stain (29) · Erlish's hematoxylin Staining of the specimen · Sections were deparaffinized in xylene and then transferred into descending grades of alcohol. (100%,90%,70%,50%) · Sections were brought down to the water for 3 minutes. · Then, sections were stained with hematoxylin for 5 minutes. · After that, sections were washed in running tap water for 3 minutes. · The excess stain was removed by decolorizing (differentiating) in 0.5-1% hydrochloric acid · in 70% alcohol for a few seconds. Thus, the blue staining of the hematoxylin was changed to red by the action of the acid. · The decolorization was stopped, and the blue color was regained by washing it in alkaline and running tap water for at least 5 minutes. · The sections were stained in a 1% solution of Eosin for 1 minute and then washed in water. · Dehydration in ascending grades of alcohol, clearing in xylene, and mounting with Canada Balsam were performed after staining and washing. Then, the sections were ready for examination with the light microscope. Following mandibular dissection, the left hemimandible of each specimen was processed for light microscopic (LM) analysis to assess histological alterations across experimental groups. Specimens were fixed in 10% neutral-buffered formalin and subsequently decalcified in 10% ethylenediaminetetraacetic acid (EDTA) solution (pH 7.0–7.4) at room temperature, with frequent solution replacement to ensure effective decalcification. After thorough rinsing under running water, the tissues were processed routinely and embedded in paraffin blocks according to standard histological procedures⁽¹⁾. Serial sections of 5 μm thickness were obtained and stained with hematoxylin and eosin (H&E) and mason's trichrome stains for general morphological assessment⁽ 1 ⁾. Histological examination was performed using a light microscope equipped with a digital imaging system (Leica ICC50 HD), and representative fields were photographed and appropriately labeled. III. Blood sample examination (3,28,30) Blood samples were collected from the tail vein of the rats in glass tubes without anticoagulant and left at room temperature for 30 min for spontaneous clotting. Clotted blood was centrifuged at 2000 rpm for 5 min at 4°C, pipette off the top yellow serum layer without disturbing the white buffy layer. The serum was frozen at -80°C for analysis, and the serum was used for the determination of the following parameters: Some inflammatory markers and interleukins such as C-reactive protein (CRP), Procalcitonin, Tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-1β (IL-1β). Also, some Bone formation markers, such as Bone morphogenetic protein 1 (BMP1) and Osteocalcin. Determination of inflammatory markers and interleukins in serum CRP was measured using ELISA kits (Cat. No. CYT294) purchased from Millipore Laboratory Corporation (USA and Canada). Procalcitonin was measured using an ELISA kit (Cat. No. RK03873) purchased from Abclonal Technologies (Wuhan, China). TNF-α, IL-6, IL-1β, and IL-10 were measured using ELISA kits (Cat. No. RK00029) for TNF-α, (Cat. No. ER0042) for IL-6, (Cat. No. ER1094) for IL-1β, (Cat. No. ER0033) for IL-10, purchased from Finetest biotech Inc. (USA). Determination of some bone formation markers in serum BMP was measured using an ELISA kit (Cat. No. RE2765R) purchased from Reed Biotech Ltd (Wuhan, China). Osteocalcin was measured using an ELISA kit (Cat. No. RK09279) purchased from Abclonal technologies (Wuhan, China). RESULTS This study included 45 rats and was divided equally into three groups: Control, Extraction without bone graft, and Extraction with bone graft. I. Histological Results: Figure (1) A. Hematoxylin and Eosin (H&E) Stainin g : Control Group (No extraction): The alveolar crest was intact and positioned normally just below the cemento-enamel junction. The alveolar bone exhibited well-organized lamellar architecture from the crest to the apical region, including the interradicular areas adjacent to the periodontal ligament. Osteocytes were evenly distributed within the mature bone matrix, and no evidence of inflammation or tissue disruption was observed throughout the study period. Extraction-Only Group : At 2 weeks, the extraction socket exhibited a loose connective tissue matrix with marked inflammatory infiltrate. Early granulation tissue was evident, and minimal new bone formation was observed at the periphery of the socket. By 4 weeks, a moderate degree of trabecular bone extended from the socket walls, while central fibrous connective tissue persisted. The inflammatory response had decreased compared with the earlier time point. At 6 weeks, bone trabeculae were more prominent, with areas of woven bone forming within the socket; however, healing remained incomplete, as residual fibrous tissue continued to occupy the central defect. Extraction + Bone Graft Group: At 2 weeks, graft particles were clearly identifiable and surrounded by early connective tissue infiltration. Mild inflammatory cell presence was noted, and early osteoid formation was observed adjacent to the graft surfaces. By 4 weeks, osteoblasts were lining the newly forming bone around the graft particles, and the amount of woven bone was greater than in the Extraction-only group. Inflammatory cells were markedly reduced, indicating progression of healing. At 6 weeks, the defect exhibited abundant newly formed bone with mature trabecular structures. The graft particles were partially resorbed and integrated with host bone. Bony trabeculae were dense, containing cellular, normally sized bone marrow spaces lined by endosteal cells with visible reversal lines. Osteocyte lacunae were of normal size and distribution, and the tissue architecture closely resembled native bone, indicating advanced bone healing. B. Masson's Trichrome Staining : Masson's Trichrome confirms (H&E) results and highlights improved collagen deposition and tissue organization in the grafted group. The maturation of bone matrix is more advanced at each interval compared to the extraction-only group. Control Group: Staining revealed dense collagen-rich connective tissue, and mature lamellar bone stained deep blue. The organization of collagen bundles was uniform and compact. Extraction-Only Group: In this group, collagen deposition progressed over time. At 2 weeks, predominantly red-stained areas indicated immature connective tissue and early granulation tissue, with sparse collagen. By 4 weeks, collagen content increased (light blue areas) and showed early organization around forming trabeculae. At 6 weeks, moderate blue staining suggested further collagen deposition, but the bone matrix remained immature compared with controls, with fibrous tissue still present within the socket. Extraction + Bone Graft Group: The group showed accelerated collagen maturation. At 2 weeks, blue-stained collagen fibers surrounded graft particles, and the extracellular matrix showed early organization. By 4 weeks, staining intensified and was more widespread, indicating progressive collagen maturation, particularly at bone-graft interfaces. At 6 weeks, a well-organized, collagen-rich bone matrix was observed, with dense blue staining of newly formed trabeculae, complete resorption of graft remnants, evident reversal lines, and mature, parallel collagen fibers, indicating advanced remodeling and restoration of native bone architecture. II. Hematological Results (data are presented as mean ± SD) - CRP (mg/L) Table (1) CRP levels differed significantly among the three groups at 4 and 6 weeks ( p<. 001). CRP decreased from 4 to 6 weeks in both Extraction-only ( p=. 009) and Extraction + Bone Graft groups ( p=. 024), with no change in the Control group ( p=. 417). Pairwise comparisons showed CRP was lower in the grafted group than in the Extraction-only group ( p=. 001), and lowest in the Control group ( p<. 001). - BMP1 (pg/mL) Table (2) BMP-1 levels differed significantly among the three groups at 4 and 6 weeks ( p<. 001). Levels increased from 4 to 6 weeks in the Extraction-only group ( p=. 037), while no significant changes were observed in the Control ( p=1. 000) or Extraction + Bone Graft groups ( p=. 471). - Osteocalcin (ng/mL) Table (3) Osteocalcin levels differed significantly among the three groups at 4 and 6 weeks ( p<. 001). Levels increased from 4 to 6 weeks in the Extraction + Bone Graft group ( p=. 040), while no significant changes were observed in the Control ( p=. 800) or Extraction-only groups ( p=. 106). - TNF-alpha (ng/mL) Table (4) TNF-α levels differed significantly among the three groups at 4 and 6 weeks ( p<. 001). Levels decreased from 4 to 6 weeks in both the Extraction-only ( p<. 001) and Extraction + Bone Graft groups ( p=. 001), while no significant change was observed in the Control group ( p=. 715). - Procalcitonin (pg/mL)Table (5) Procalcitonin levels differed significantly among the three groups at 4 and 6 weeks ( p<. 001). Levels decreased from 4 to 6 weeks in both the Extraction-only ( p=. 017) and Extraction + Bone Graft groups ( p=. 001). IL-6 (pg/mL)Table (6) IL-6 levels differed significantly among the three groups at 4 and 6 weeks ( p<. 001). Levels decreased from 4 to 6 weeks in both the Extraction-only and Extraction + Bone Graft groups ( p<. 001 for both), while no significant change was observed in the Control group ( p=. 905). - IL-1B (pg/mL) Table (7) IL-1β levels differed significantly among the three groups at 4 and 6 weeks ( p<. 001). Levels decreased from 4 to 6 weeks in the Extraction-only group ( p<. 001), while no significant change was observed in the Control ( p=. 197) or Extraction + Bone Graft groups ( p=. 052). - IL-10 (pg/mL)Table (8) IL-10 levels differed significantly among the three groups at 4 and 6 weeks ( p<. 001). Levels remained unchanged in the Control group ( p=. 850), decreased in the Extraction-only group ( p=. 001), and increased in the Extraction + Bone Graft group ( p<. 001) from 4 to 6 weeks. DISCUSSION The present study aimed to establish a reliable animal model simulating a clinical environment for evaluating bone regeneration following tooth extraction, and to assess the effect of bone grafting on the healing process through histological and histochemical analysis at various time intervals. Our findings demonstrate that bone grafting substantially accelerates and enhances alveolar bone regeneration compared to ungrafted defects. Histological evaluation using H&E staining revealed that the extraction-only group exhibited a typical healing response characterized by initial granulation tissue formation, followed by progressive but incomplete bone regeneration over six weeks. This is in accordance with previous studies describing the natural sequence of socket healing, where inflammatory cell infiltration and fibrous tissue predominated in early stages, followed by delayed woven bone formation. (31) In contrast, defects treated with bone grafts showed significantly improved healing outcomes. As early as two weeks, the presence of osteoid formation and mild inflammatory response indicated early osteogenic activity around graft particles. By six weeks, grafted sockets demonstrated mature trabecular bone formation with clear evidence of graft integration and resorption. This suggests that the graft served as an osteoconductive scaffold, facilitating cellular migration and bone matrix deposition, consistent with the established biological role of bone substitutes in enhancing osteogenesis. (3,32) . The results were further substantiated by Masson’s Trichrome staining, which provided insight into the extracellular matrix and collagen maturation. Collagen deposition was sparse and disorganized in the early stages of healing in the extraction-only group. Although gradual organization was noted by six weeks, the extent of matrix maturation remained inferior to that observed in the grafted group. In grafted sockets, collagen fibers were more abundant and better organized throughout the healing period, with robust matrix formation clearly supporting advanced bone tissue development. The control group, as expected, showed normal histological architecture with mature lamellar bone and dense collagen matrix, providing a baseline for comparison. The striking differences in the healing trajectory between the grafted and non-grafted groups underscore the clinical importance of biomaterial application in enhancing socket healing, particularly in cases where natural healing is compromised or slow. Our findings align with multiple studies reporting improved bone formation with various grafting materials, including xenografts and composite bioactive scaffolds. (33–35) The accelerated healing observed in our grafted group may be attributed not only to the osteoconductive properties of the graft material but also to its potential to stabilize the clot and maintain space for tissue ingrowth. Trauma from tooth extraction can trigger inflammation by activating immunocompetent cells such as macrophages and mast cells, which stimulate the production of pro-inflammatory cytokines, particularly TNF-α and IL-1. ( 15 ) Even after the extraction wound has healed, mechanical stimuli from mastication may reactivate these cytokines via neurogenic pathways, leading to continued alveolar bone resorption. ( 16 ) Proinflammatory cytokines such as IL-1, IL-2, IL-6, IL-8, and TNF-α are highly sensitive diagnostic markers since their levels can rise both systemically in the blood and locally in oral fluids. ( 17 ) Acting as antigen-nonspecific defense factors, they play a central role in immune and inflammatory responses. In the context of dental implant surgery, an imbalance between pro- and anti-inflammatory cytokines may impair osseointegration and promote peri-implant tissue destruction. ( 18 ) Moreover, uncontrolled cytokine alterations reflect heightened inflammatory and tissue-destructive activity in the oral cavity and maxillofacial region, as these mediators are released by lymphocytes, macrophages, serum transudate, and salivary gland secretions during inflammatory events or surgical interventions. ( 19 ) A thorough evaluation of inflammatory markers is crucial for planning dental implant and maxillofacial treatments. Markers such as IL-1, IL-2, IL-6, IL-8, TNF-α, and C-reactive protein are linked to systemic and oral disease pathogenesis and help predict treatment outcomes. Measuring these markers in blood or saliva is especially useful for patients with systemic conditions, as oral and systemic inflammation can mutually influence overall health. The present study revealed that all inflammatory markers (CRP, TNF-α, Procalcitonin, IL-6, and IL-1β) were elevated in both experimental groups compared with the control, consistent with the acute inflammatory response to surgical trauma and tissue injury. ( 20 , 21 ) At four weeks, the extraction without bone graft group showed persistently higher levels of systemic inflammation compared to the extraction with bone graft group. By six weeks, both groups demonstrated a reduction, but inflammatory markers in the grafted group approached control values, whereas the ungrafted group remained elevated. These findings suggest that bone grafting not only preserves the alveolar ridge but also helps modulate the host inflammatory response, promoting a faster return to baseline systemic conditions. These findings suggest that bone grafting after tooth extraction not only preserves the alveolar bone but also modulates the post-extraction inflammatory response, promoting a faster return to baseline systemic inflammation. By stabilizing the extraction site and reducing tissue trauma, grafting may create a more controlled healing environment that limits the release of inflammatory mediators such as CRP, TNF-α, IL-6, IL-1β, and Procalcitonin. Consequently, patients receiving grafts tend to show a quicker reduction of systemic inflammation compared with those left ungrafted, whose inflammatory markers remain elevated for a longer period. CRP, IL-6, and TNF-α are well-established markers of systemic inflammation and have been widely studied in periodontal and peri-implant healing, while elevated Procalcitonin—typically linked with systemic infection—may also reflect the acute inflammatory burden of surgical trauma. ( 22 ) The earlier reduction of these mediators in the grafted group highlights the potential systemic benefits of socket preservation, particularly in medically compromised patients where prolonged inflammation may impair healing or exacerbate comorbidities. These results suggest that bone grafting not only aids in ridge preservation but also stabilizes the wound environment, dampening prolonged cytokine release and facilitating faster normalization of systemic inflammation. Such modulation may be particularly beneficial in medically compromised patients, where persistent inflammation could impair healing or exacerbate comorbidities. The reduction in inflammatory markers observed in the bone-grafted group may be attributed to several mechanisms. Placement of a graft stabilizes the extraction socket, minimizes collapse of the alveolar walls, and reduces secondary trauma, thereby attenuating the release of pro-inflammatory cytokines. Avila-Ortiz et al. ( 23 ) , in a systematic review and meta-analysis, confirmed that alveolar ridge preservation (ARP) through socket grafting effectively limits post-extraction bone loss in nonmolar sites, both horizontally and vertically. Furthermore, xenografts and demineralized freeze-dried bone matrices provide a scaffold for cell migration and angiogenesis, facilitating tissue repair with complete osseous integration. ( 24 ) This controlled environment may explain the quicker normalization of systemic inflammation in grafted sockets compared to prolonged cytokine release in ungrafted sites. Similarly, Canullo et al. (2021) ( 25 ) reported that patients receiving bone grafts after extraction showed reduced inflammatory levels and fewer soft tissue healing complications, although the differences did not reach statistical significance. Together, these findings suggest that bone grafting not only preserves alveolar bone but also contributes to the modulation of the systemic inflammatory response. Inflammation is primarily driven by activated macrophages, which release pro-osteolytic mediators such as TNF-α, IL-1β, IL-6, and IL-8. These cytokines promote monocyte recruitment, inhibit osteoblastic differentiation, and induce osteoclastogenesis, making macrophage activation a central factor in peri-implant osteolysis. ( 26 ) The production of inflammatory cytokines by peripheral blood monocyte-derived macrophages (PBMMs) in response to biomaterials is therefore critical, as it significantly influences tissue integration and regeneration. Rani et al. (2024) ( 27 ) investigated the immune response to demineralized and decellularized bovine bone substitutes and found that these materials did not trigger significant inflammatory responses in PBMMs, suggesting their potential to reduce inflammation during bone healing. In contrast, treatment with a demineralized bone (DMB) substitute resulted in markedly higher proinflammatory cytokine expression, with TNF-α increasing 3.4-fold (p < 0.05) and IL-1β mRNA rising 21.75-fold (p < 0.0001) compared to controls and decellularized bone (DCC, 5.01-fold). Similarly, studies using murine air pouch models have demonstrated that different commercially available demineralized bone matrices (DBM) elicit variable inflammatory reactions, underscoring the importance of considering both the efficacy and immunological safety of graft processing methods. ( 28 ) Moreover, modifications in the physico-chemical properties of biomaterials can promote macrophage polarization favorable for tissue regeneration. ( 29 ) For example, an in vitro pilot study by Panahipour et al. ( 30 ) demonstrated that DBM reduced IL-1β and IL-6 expression in macrophage cells, suggesting that certain DBM preparations can modulate inflammatory responses. These conflicting results likely stem from differences in graft processing techniques, which alter the physico-chemical and topographical characteristics of the materials, as well as the potential presence of residual native cells capable of provoking inflammation. Inflammation triggered by tooth extraction is characterized by the release of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α, which impair osteoblastic differentiation, stimulate osteoclastogenesis, and contribute to alveolar bone resorption. Beyond localized bone changes, these cytokines may also influence systemic metabolism, contributing to hyperlipidemia and vascular dysfunction. Elevated levels of CRP and fibrinogen further amplify this systemic response, with CRP synthesized by the liver in reaction to inflammatory signals from oral infections such as periodontitis. (31, 32) Esteves-Lima et al. (32) reported that individuals with periodontitis exhibited significantly higher CRP levels, supporting the link between oral inflammation and systemic disease. Clinical data reinforce this connection: D’Aiuto et al. (33) observed a rise in CRP 24 hours after periodontal therapy or extractions, while Ide et al. (34) found rapid IL-6 and TNF-α elevations within two hours of subgingival scaling. However, Bahrani-Mougeot et al. (35) found no clear association between inflammation severity and the 12 cytokines assessed, particularly IL-6 and TNF-α. Interestingly, individuals with healthier periodontal status tended to show higher circulating cytokine levels compared to those with poorer periodontal health, though the differences were not statistically significant. Consistent with these findings, our results also suggest that TNF-α shows little correlation with inflammatory status. Given that cytokines are often present at very low concentrations in plasma, the possibility of false-negative results must be considered. Using a xenograft for socket preservation helps maintain alveolar ridge volume, provides a scaffold for bone regeneration, and stabilizes healing by moderating inflammation. Ungrafted sockets, in contrast, show faster resorption, unpredictable bone healing, and potential complications for prosthetic rehabilitation. IL-1β and IL-6 amplify inflammation by promoting chemokine release, prostaglandin production, and connective tissue degradation. They recruit neutrophils, monocytes, and fibroblasts, supporting normal healing when regulated, but sustained elevations can delay wound repair. IL-6 in gingival tissues also aids healing by protecting open wounds from bacterial invasion. (36) Karnes et al. (37) demonstrated that TNF-α enhances mesenchymal cell recruitment by upregulating intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), thereby improving intrinsic migration capacity. It also promotes the proliferation and differentiation of mesenchymal precursor cells into chondrogenic and osteogenic phenotypes, partly through the induction of early BMP-2 secretion in neighboring osteoblasts. This dual role—supporting mesenchymal stem cell repopulation at physiological levels while driving osteoclastogenesis when elevated—positions TNF-α as both a critical regulator of bone remodeling and a potential therapeutic target. However, Freitas et al. (38) reported that blocking TNF-α, while suppressing acute inflammation, impaired healing by delaying cell migration, reducing fibroblast activity and collagen maturation, and limiting angiogenesis. Similarly, Saim et al. (39) highlighted TNF-α’s role in post-odontectomy swelling through increased vascular permeability, with IL-1 and IL-6 further enhancing immune cell recruitment and fluid accumulation at the injury site. When a socket is left ungrafted, extraction trauma leads to a pronounced and prolonged inflammatory response, reflected by elevated systemic and local markers such as CRP, TNF-α, Procalcitonin, IL-6, and IL-1β. These cytokines and proteins drive osteoclastic activity, suppress osteoblastic differentiation, and prolong tissue breakdown, resulting in accelerated bone resorption and delayed healing. Bone formation markers (BMPI, Osteocalcin, and IL-10) were initially lower in both experimental groups compared with the control. Over time, the extraction with bone graft group demonstrated a significant increase in all three markers, particularly at six weeks, whereas the extraction without bone graft group showed either minimal improvement (BMPI, Osteocalcin) or a decline (IL-10). These findings suggest that bone grafting positively influences the expression of bone formation markers. The significant increase in BMPI, Osteocalcin, and IL-10 in the grafted group indicates enhanced osteogenic activity and a more favorable anti-inflammatory environment, which supports faster and more effective bone regeneration. In contrast, the minimal improvement or decline observed in the non-grafted group highlights the limited regenerative potential of extraction sites without grafting, emphasizing the benefits of bone grafts in promoting both bone formation and immunomodulation during healing. Inflammation, on the other hand, can increase osteoclast numbers through the action of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β), as well as via the receptor activator of nuclear factor-κB ligand (RANKL) and its receptor RANK. (40, 41) The RANK–RANKL system is a central regulator of bone remodeling, with TNF and the TNF receptor family further influencing cell proliferation and apoptosis. Elevated osteoclast activity driven by this pathway ultimately contributes to alveolar bone resorption. (42) Kresnoadi et al. ( 16 ) reported that the DFDBBX + PEG group showed an increase in osteoblasts and a decrease in osteoclasts at 7 and 30 days compared with the PEG (control) group, indicating enhanced bone regeneration. A significant difference in RANKL expression (P = 0.002 < 0.05) was also observed between the groups, suggesting reduced inflammation due to socket preservation with DFDBBX. Additionally, osteocalcin levels in the DFDBBX-only group were higher than in the control at both time points, reflecting its osteogenic potential, as osteoblasts synthesize non-collagenous proteins such as osteocalcin. Consistently, Khan (43) reported that placing graft material into the extraction socket can promote osteoinduction and stimulate new bone formation. Motamedian et al. (44) demonstrated that demineralized bovine bone matrix (DBBM) stimulates the expression of both early and late osteogenic genes in dental pulp stem cells (DPSCs) after one week in osteogenic medium and two weeks in standard medium, whereas Seebach et al. (45) reported no early osteogenic gene expression when mesenchymal stem cells were cocultured with DBBM for 10 days. In terms of bone formation, markers such as BMP1, osteocalcin, and IL-10 initially showed lower levels than the control, likely due to the immediate catabolic response following extraction. Over time, however, the grafted group exhibited significant increases in all three markers, particularly by week six. Osteocalcin, a non-collagenous protein secreted by osteoblasts during bone matrix formation, and BMP1, a critical regulator of extracellular matrix assembly, were markedly higher in grafted sockets, reflecting enhanced osteoblastic activity and accelerated bone remodeling. (46) Interestingly, IL-10, an anti-inflammatory cytokine with immunomodulatory and pro-healing properties, increased significantly only in the grafted group, whereas the non-grafted group showed a decline. (47) This supports the notion that bone grafting fosters a balanced inflammatory response, promoting a faster transition from a pro-inflammatory to a regenerative phase. Short et al. (48) further highlighted IL-10’s role in regulating inflammation while preserving the integrity of the healing process. Socket preservation with a xenograft stabilizes the clot and protects the wound from excessive collapse, reducing the release of pro-inflammatory mediators and creating a more controlled, less destructive healing environment compared with ungrafted sockets. Socket preservation with a xenograft moderates the post-extraction inflammatory response by stabilizing the clot and preventing excessive wound collapse, thereby reducing the release of pro-inflammatory mediators. Consequently, inflammatory markers tend to normalize faster in grafted sockets compared with ungrafted ones, creating a more controlled and less destructive healing environment. Simultaneously, bone formation markers such as BMP1, osteocalcin, and IL-10 show a more favorable trend in grafted sites. Osteocalcin, secreted by active osteoblasts, reflects enhanced osteogenic activity, while BMP1 indicates early bone matrix synthesis. IL-10, an anti-inflammatory cytokine, supports tissue regeneration by downregulating excessive inflammation. In contrast, ungrafted sockets exhibit lower or declining levels of these markers, consistent with impaired bone regeneration. Overall, xenograft use in socket preservation not only maintains alveolar ridge volume mechanically but also optimizes the biological environment by limiting inflammation and promoting bone formation and remodeling, highlighting its advantage over extraction without grafting. Sivolella et al. (49) found that xenografts promoted new bone formation and provided sufficient support for implant placement compared with extraction alone, with histologic outcomes indicating more favorable tissue repair and less connective tissue infiltration. Barbecket al. (50) demonstrated that the physical properties of bone substitute materials, including xenografts and synthetic grafts, influence immune cell responses, with macrophages and monocytes modulating the expression of inflammatory and regulatory cytokines such as IL-10, IL-6, IL-1β, IL-4, and TNF-α, ultimately affecting healing outcomes. Supporting this, Lyu et al., (51) showed that IL-10 treatment drives macrophage polarization toward a reparative phenotype, decreasing inflammation and enhancing osteogenesis in mesenchymal stem cells, highlighting the critical role of IL-10 and regulatory pathways in bone regeneration. Taken together, the findings indicate that Graft placement after tooth extraction preserves the alveolar ridge and modulates systemic inflammation, promoting faster bone formation and improved healing. This creates optimal conditions for future implants or prosthetics, especially in patients with systemic conditions. The accelerated healing likely reflects both the graft’s osteoconductive properties and its ability to stabilize the clot and maintain space for tissue ingrowth. CONCLUSIONS Xenograft socket preservation enhances bone regeneration and maintains alveolar ridge dimensions by promoting osteogenic activity and a balanced inflammatory response. Grafted sites showed organized bone and collagen formation with reduced systemic inflammation, whereas ungrafted sockets exhibited delayed healing and greater ridge resorption. These findings support the clinical potential of xenografts to improve post-extraction healing and optimize conditions for future restorative procedures. RECOMMENDATIONS Xenografts are recommended for socket preservation to enhance bone regeneration, maintain ridge dimensions, and modulate inflammation, especially in patients planning implants, with systemic risk factors, or in esthetically critical areas. Monitoring biomarkers such as CRP, TNF-α, IL-6, IL-1β, procalcitonin, BMP-1, osteocalcin, and IL-10 can guide individualized post-extraction care. Further clinical trials are needed to confirm these benefits and assess long-term implant and prosthetic outcomes. LIMITATIONS This study has several limitations. The follow-up period of 2, 4, and 6 weeks provides only early insights into bone healing and may not reflect long-term maturation and stability. Evaluation was limited to histological analysis without assessing functional or mechanical properties of the regenerated bone. As an animal study, the findings may not fully translate to human clinical conditions. Only one type of graft material was tested, limiting comparison with other alternatives. Additionally, the sample size may affect the statistical power and generalizability of the results. Abbreviations DFDBBX Demineralized Freeze-Dried Bovine Bone Xenograft BMP Bone Morphogenetic Protein CRP C-reactive protein TNF Tumor Necrosis Factor IL Interleukin Declarations Ethics approval: We declare and confirm that the work covered in this manuscript has been conducted only after receiving relevant institutional ethical approvals, PUA02202410273282, Unit of Research Ethics Approval Committee [UREAC], Pharos University in Alexandria. I declare that the submitted manuscript is original, has not been published before, and is not being considered for publication elsewhere. We understand that the Corresponding Author is the contact for the Editorial process. Consent for publication: Not Applicable. Data availability: The data that support the findings of this study are available within the article. Conflict of Interest: The authors declare no conflicts of interest. Funding Statement: The authors received no specific funding for this study. Authors' contributions: Aliaa A. Habib; surgical procedure, acquisition of data, analysis, and interpretation of data collected. Eman M. Salem; histological procedure and guarantor of the manuscript for final approval. Ghadir Elnawawy; hematological results. Acknowledgments: The authors acknowledge Prof. ElSayed Amr Basma for his effort in the statistical analysis of the work. References Van der Weijden F, Dell'Acqua F, Slot DE. Alveolar bone dimensional changes of post-extraction sockets in humans: a systematic review. J Clin Periodontol. 2009;36(12):1048–58. Vittorini Orgeas G, Clementini M, De Risi V, de Sanctis M. Surgical techniques for alveolar socket preservation: a systematic review. Int J Oral Maxillofac Implants. 2013;28(4):1049–61. Allegrini S Jr., Koening B Jr., Allegrini MR, Yoshimoto M, Gedrange T, Fanghaenel J, et al. Alveolar ridge sockets preservation with bone grafting–review. Ann Acad Med Stetin. 2008;54(1):70–81. Pagni G, Pellegrini G, Giannobile WV, Rasperini G. Postextraction alveolar ridge preservation: biological basis and treatments. Int J Dent. 2012;2012:151030. Kim S, Kim S-G. Advancements in alveolar bone grafting and ridge preservation: a narrative review on materials, techniques, and clinical outcomes. Maxillofacial Plast Reconstr Surg. 2024;46(1):14. Deo V, Bhongade M. Pathogenesis of periodontitis: role of cytokines in host response. Dent Today. 2010;29(9):60–2. 4. Lawlor F, Camp R, Greaves M. Epidermal interleukin 1 alpha functional activity and interleukin 8 immunoreactivity are increased in patients with cutaneous T-cell lymphoma. J Invest Dermatol. 1992;99(4):514–5. Yoshimura A, Hara Y, Kaneko T, Kato I. Secretion of IL-1β, TNF‐α, IL‐8 and IL‐1ra by human polymorphonuclear leukocytes in response to lipopolysaccharides from periodontopathic bacteria. J Periodontal Res. 1997;32(3):279–86. Horváth A, Mardas N, Mezzomo LA, Needleman IG, Donos N. Alveolar ridge preservation. A systematic review. Clin Oral Investig. 2013;17(2):341–63. Bartee BK. Extraction site reconstruction for alveolar ridge preservation. Part 1: rationale and materials selection. J Oral Implantol. 2001;27(4):187–93. Irinakis T. Rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. J Can Dent Assoc. 2006;72(10):917–22. Khojasteh A, Kheiri L, Motamedian SR, Nadjmi N. Regenerative medicine in the treatment of alveolar cleft defect: A systematic review of the literature. J Cranio-Maxillofacial Surg. 2015;43(8):1608–13. Caballero M, Morse JC, Halevi AE, Emodi O, Pharaon MR, Wood JS, et al. Juvenile swine surgical alveolar cleft model to test novel autologous stem cell therapies. Tissue Eng Part C: Methods. 2015;21(9):898–908. Ad De R, Meijer G, Dormaar T, Janssen N, Van Der Bilt A, Slootweg P, et al. β-TCP versus autologous bone for repair of alveolar clefts in a goat model. Cleft Palate-Craniofacial J. 2011;48(6):654–62. Oryan A, Alidadi S, Moshiri A, Maffulli N. Bone regenerative medicine: classic options, novel strategies, and future directions. J Orthop Surg Res. 2014;9(1):18. Cypher TJ, Grossman JP. Biological principles of bone graft healing. J foot ankle Surg. 1996;35(5):413–7. Goulet JA, Senunas LE, DeSilva GL, Greenfield MLV. Autogenous iliac crest bone graft: complications and functional assessment. Clin Orthop Relat Research®. 1997;339:76–81. Brunsvold MA, Mellonig JT. Bone grafts and periodontal regeneration. Periodontology 2000 1993;1(1):80–91. Nasr HF, Aichelmann-Reidy ME, Yukna RA. Bone and bone substitutes. Periodontol 2000. 1999;19:74–86. Allegrini S Jr, Koening B Jr, Allegrini M, Yoshimoto M, Gedrange T, Fanghaenel J, et al. editors. Alveolar ridge sockets preservation with bone grafting–review. Annales Academiae Medicae Stetinensis; 2008. Kresnoadi U, Rahmania PN, Caesar HU, Djulaeha E, Agustono B, Ari MDA. The role of the combination of Moringa oleifera leaf extract and demineralized freeze-dried bovine bone xenograft (xenograft) as tooth extraction socket preservation materials on osteocalcin and transforming growth factor-beta 1 expressions in alveolar bone of Cavia cobaya. J Indian Prosthodont Soc. 2019;19(2):120–5. Cohen RE, Alsuwaiyan A, Wang B-Y. Xenografts and periodontal regeneration. J Orthod Endod. 2015;1(1):1–6. Pilanci O, Cinar C, Kuvat S, Altintas M, Guzel Z, Kilic A. Effects of hydroxyapatite on bone graft resorption in an experimental model of maxillary alveolar arch defects. Arch Clin Exp Surg (ACES). 2013;2:170–5. Raposo-Amaral CE, Kobayashi GS, Almeida AB, Bueno DF, Freitas FRS, Vulcano LC, et al. Alveolar osseous defect in rat for cell therapy: preliminary report. Acta Cirurgica Brasileira. 2010;25:313–7. Sawada Y, Hokugo A, Nishiura A, Hokugo R, Matsumoto N, Morita S et al. A trial of alveolar cleft bone regeneration by controlled release of bone morphogenetic protein: an experimental study in rabbits. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2009;108(6):812 – 20. Dalia E-B, Smith SJ, Germane N, Sharawy M. New technique for creating permanent experimental alveolar clefts in a rabbit model. Cleft Palate-Craniofacial J. 1993;30(6):542–7. El Deeb M, Horswell B, Waite DE. A primate model for producing experimental alveolar cleft defects. J Oral Maxillofac Surg. 1985;43(7):523–7. Sikorska E, Wołosz D, Kasarełło K, Koperski Ł, Górnicka B, Cudnoch-Jędrzejewska A. Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina. J Visualized Experiments (JoVE) 2024, (210):e66663. Sullivan-Brown J, Bisher ME, Burdine RD. Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin. Nat Protoc. 2011;6(1):46–55. Salem EM, Abdelfatah OM, Hanafy RA, El-Sharkawy RM, Elnawawy G, Alghonemy WYJBOH. Comparative study of pulpal response following direct pulp capping using synthesized fluorapatite and hydroxyapatite nanoparticles. 2025;25(1):17. Tables Tables 1 to 8 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables1.docx Tables2.docx Tables3.docx Tables4.docx Tables5.docx Tables6.docx Tables7.docx Tables8.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 01 May, 2026 Reviewers agreed at journal 18 Apr, 2026 Reviewers invited by journal 17 Apr, 2026 Editor invited by journal 20 Mar, 2026 Editor assigned by journal 26 Feb, 2026 Submission checks completed at journal 25 Feb, 2026 First submitted to journal 25 Feb, 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8941374","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":628015382,"identity":"6b8ff02a-19cd-4e8c-969a-864bf352d74c","order_by":0,"name":"Aliaa A. Habib","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYDACdsYGIHmAwYCZh/EBRCiBgBZmhBZmAyK1gEmgFgYeNgmitPA3Mzd++MFwR96cnfdYdWGbHQM/e44Bw4dfuLVIHGZsluxheGa4s5kv7fbMtmQGyZ43Bowz+/BYc5ixQYIHSG44zGN2m7eNmcHgRo4BM28Pbh3yQFt+/mE4bA/SUszbVs9gD9LyF48Wg8OMbdJAWxJBWph52w4zGEgAtTD8wK3FEKjFWsbgcDLQL8nSM84d55E486zgYG8Dbi1yx9sf33xTcdh2O//Zg58Lyqrl+NuTNz748QeP9yHOg1CgOOIBMQ4wthHSwoDQAgUEbRkFo2AUjIIRBACxtVBj+LflRQAAAABJRU5ErkJggg==","orcid":"","institution":"Pharos University in Alexandria","correspondingAuthor":true,"prefix":"","firstName":"Aliaa","middleName":"A.","lastName":"Habib","suffix":""},{"id":628015383,"identity":"a2df7874-11cf-4411-abdc-a6b6a35c515d","order_by":1,"name":"Eman M. Salem","email":"","orcid":"","institution":"Pharos University in Alexandria","correspondingAuthor":false,"prefix":"","firstName":"Eman","middleName":"M.","lastName":"Salem","suffix":""},{"id":628015385,"identity":"07a5c725-bd5a-461a-b12f-67e35abab901","order_by":2,"name":"Ghadir Elnawawy","email":"","orcid":"","institution":"Pharos University in Alexandria","correspondingAuthor":false,"prefix":"","firstName":"Ghadir","middleName":"","lastName":"Elnawawy","suffix":""}],"badges":[],"createdAt":"2026-02-22 20:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8941374/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8941374/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107741398,"identity":"4e5995ea-613b-4294-a186-ca15e4a592bf","added_by":"auto","created_at":"2026-04-24 15:03:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1828951,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative photomicrographs of H\u0026amp;E and trichrome-stained sections showing histological changes in the extraction socket over time. Two weeks after extraction, a light photomicrograph (A1\u0026amp; C1) (Extraction-Only Group) showing the socket filled with granulation tissue (GT) characterized by high density of fibers, blood vessels, and inflammatory cells. While LMs of the healing socket (B1\u0026amp;D1)(Graft group) showing newly formed bone trabeculae extending from the lateral walls of the healing socket, forming a bony bridge filling the center of the socket (black arrows). Note the graft material(black star). (H\u0026amp;E,trichrome Χ100).\u003c/p\u003e\n\u003cp\u003eAfter four weeks from extraction, LMs of the healing socket (A2\u0026amp; C2) (Extraction-Only Group) showing newly formed bone trabeculae extending from the lateral walls of the healing socket, forming a starting bony bridge filling the center of the socket. (H\u0026amp;E,trichrome Χ100) . While LMs of the healing socket (B2\u0026amp;D2)(Graft group), Light photomicrograph showing great amount of bone spicules occupy large part of the socket, starting from the border to the center (black arrows). Note: great density of bone spicules and their continuous osseointegration between the native (N) and newly formed bone(NB). (H\u0026amp;E,trichrome Χ100).\u003c/p\u003e\n\u003cp\u003eAfter 6 weeks, LM of the healing socket (A3\u0026amp; C3) (Extraction-Only Group) showing the newly formed woven bone trabeculae of variable thickness with large marrow spaces and numerous osteocytes confined to the base of the socket. (H\u0026amp;E,trichrome Χ100). While LMs of the healing socket (B3\u0026amp;D3)(Graft group) LM revealing well-organized mature compact bone with Haversian system showing central haversian canal (arrow heads) surrounded by numerous osteocytes (short arrows). Several remodeling lines are also seen (yellow arrows),\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8941374/v1/aa55935e58daf17caf51a512.png"},{"id":108008673,"identity":"b86ec6fd-e6f9-4d0d-bac1-4cfe81ea6529","added_by":"auto","created_at":"2026-04-28 13:07:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2456003,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8941374/v1/eb32cbc4-d9e5-4706-bb34-dd30f1e95474.pdf"},{"id":107741404,"identity":"fe3855e7-390d-45d1-b66a-0aa35311ab3b","added_by":"auto","created_at":"2026-04-24 15:03:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20064,"visible":true,"origin":"","legend":"","description":"","filename":"Tables1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8941374/v1/c75a6d905726336d63d67568.docx"},{"id":108006434,"identity":"6149cb3d-75d0-4c7b-94c6-79a9dcc7b00b","added_by":"auto","created_at":"2026-04-28 12:55:34","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19804,"visible":true,"origin":"","legend":"","description":"","filename":"Tables2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8941374/v1/2842ecf2010b3afadc66cccc.docx"},{"id":107869686,"identity":"7dcd11dc-59d7-4405-a587-e91ec12c5437","added_by":"auto","created_at":"2026-04-27 07:37:51","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19750,"visible":true,"origin":"","legend":"","description":"","filename":"Tables3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8941374/v1/8c6b9ac5e3d132dc236a0c7c.docx"},{"id":107869455,"identity":"40fdcf92-962a-4ff3-b086-1216e42d8828","added_by":"auto","created_at":"2026-04-27 07:37:04","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":19663,"visible":true,"origin":"","legend":"","description":"","filename":"Tables4.docx","url":"https://assets-eu.researchsquare.com/files/rs-8941374/v1/a5301fad60120a41957cf47d.docx"},{"id":107741400,"identity":"742cff5c-d550-43a9-a666-ac8273003f24","added_by":"auto","created_at":"2026-04-24 15:03:02","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":19705,"visible":true,"origin":"","legend":"","description":"","filename":"Tables5.docx","url":"https://assets-eu.researchsquare.com/files/rs-8941374/v1/3fde7c37916430a7dace5d6d.docx"},{"id":107868903,"identity":"1107eff7-677c-48cf-8d4f-d4a84e166ba8","added_by":"auto","created_at":"2026-04-27 07:34:47","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":19619,"visible":true,"origin":"","legend":"","description":"","filename":"Tables6.docx","url":"https://assets-eu.researchsquare.com/files/rs-8941374/v1/23c87ed0417741caead70089.docx"},{"id":107868972,"identity":"5a845102-1a49-427b-a86e-665b1c7e6181","added_by":"auto","created_at":"2026-04-27 07:35:25","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":19734,"visible":true,"origin":"","legend":"","description":"","filename":"Tables7.docx","url":"https://assets-eu.researchsquare.com/files/rs-8941374/v1/8f4e6dad54909ebd0c43b5b6.docx"},{"id":107741406,"identity":"55e7fab9-03be-42df-859c-ffa8f5b7181d","added_by":"auto","created_at":"2026-04-24 15:03:02","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":19919,"visible":true,"origin":"","legend":"","description":"","filename":"Tables8.docx","url":"https://assets-eu.researchsquare.com/files/rs-8941374/v1/02d5a9694c23e223c32c2a1c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of Xenograft-Enhanced Osteogenesis and Modulation of Systemic Inflammation in Post-Extraction Alveolar Bone Defects (An Experimental Rat Study)","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTooth extraction is a common procedure in dentistry; however, it is frequently followed by alveolar bone resorption that alters both the morphology and dimensions of the alveolar ridge.\u003csup\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/sup\u003e This resorptive process primarily affects the horizontal dimension and is more pronounced on the buccal aspect than on the lingual or palatal surfaces.\u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/sup\u003e The most rapid bone loss occurs within the first six weeks to two years after extraction, with significant and irreversible dimensional changes observed as early as six months, resulting in up to 40% loss of bone height and 60% loss of ridge width.\u003csup\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/sup\u003e. It is well recognized that extraction leads to a reduction in both buccolingual and apico-coronal dimensions of the alveolar ridge.\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis resorptive process can compromise future dental rehabilitation, particularly implant placement; however, it can be minimized through minimally traumatic extraction techniques combined with socket preservation using regenerative graft materials to maintain ridge dimensions and promote bone healing.\u003csup\u003e(\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAdequate preservation of bone volume is essential, as insufficient support compromises the placement and long-term stability of dental implants and fixed prostheses, thereby negatively affecting function, retention, esthetics, and patient comfort.\u003csup\u003e(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eGraft options include autografts from the patient, allografts from human donors, xenografts from other species, and synthetic substitutes such as calcium phosphate, hydroxyapatite, or bioactive glass.\u003csup\u003e(\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/sup\u003e These materials promote bone repair through three main biological processes: osteogenesis, osteoconduction, and osteoinduction.\u003csup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e)\u003c/sup\u003e Osteogenesis refers to new bone formation by osteoblasts contained within the graft; osteoconduction provides a scaffold that supports bone growth across its surface; while osteoinduction triggers undifferentiated stem cells in surrounding tissues to transform into bone-forming cells.\u003csup\u003e(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAutogenous bone grafts are often regarded as the \u0026ldquo;gold standard\u0026rdquo;; however, their use is limited by the need for a second surgical site and restricted availability.\u003csup\u003e(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/sup\u003e Allografts sourced from bone banks are more accessible but carry the drawback of potential disease transmission.\u003csup\u003e(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/sup\u003e Consequently, xenografts and synthetic substitutes have become the most widely adopted alternatives in dental practice.\u003csup\u003e(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOver the past years, various bone graft materials have been investigated, with demineralized freeze-dried bovine bone xenograft (DFDBBX) being among the most widely used in dentistry. DFDBBX exhibits both osteoinductive and osteoconductive properties, thereby enhancing new bone formation. \u003csup\u003e(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e As a xenograft derived from bovine bone, it shares key structural characteristics with human bone, including crystalline composition, porosity, and carbonate content, which support natural osteoconduction. By stabilizing blood clots and providing a mineral- and collagen-rich scaffold, xenografts facilitate angiogenesis, revascularization, and osteoblast migration from the base of the socket, thereby reducing alveolar bone resorption and promoting effective bone regeneration. \u003csup\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOptimizing the \u003cb\u003equality of the existing bone grafting materials\u003c/b\u003e and looking for novel and better bone-substitute materials is crucial in improving the clinical outcome. Experimental testing of various grafting materials requires establishing an appropriate biological model to conduct studies and evaluate their clinical effects on osteogenesis and healing. Animal models with simulated bone defects are considered appropriate as experimental models for testing clinical interventions.\u003csup\u003e(\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eNext to congenitally induced models, surgically created bone defects in animals also seem suitable for experimental studies regarding bone grafting material's histologic and biomechanical properties. Moreover, it is essential to allow proper timing for the healing of the defect and establish an alveolar cleft of appropriate width that mimics the human scenario of a skeletal defect extending to the nasal mucosa and the adjacent teeth, and is suitable for clinical testing.\u003csup\u003e(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe present study aims to assess bone healing histologically using an animal model developed to create bone defects that closely replicate clinical conditions for testing tissue-engineered bone substitute materials without compromising animal health.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003eForty-five\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erats, aged 8\u0026ndash;10 weeks, weighing approximately 250\u0026ndash;300 grams, were obtained from the animal house of Pharos University in Alexandria (PUA). The ethical approval was obtained from the Unit of Research Ethics Approval Committee (UREAC) of PUA (02202410273282), complying with the ARRIVE reporting guidelines. Rats were housed in standard experimental conditions and kept in well-ventilated plastic cages with wood shavings as bedding with a 12-hour light/dark cycle and ad libitum access to food and water for at least 7 days before the experiment. Animals are fasted for 6 hours before the surgery to reduce the risk of aspiration during anesthesia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThey were\u0026nbsp;allocated to\u0026nbsp;three equal\u0026nbsp;groups: Control (No-extraction) group:\u0026nbsp;Fifteen\u0026nbsp;rats\u0026nbsp;without\u0026nbsp;intervention. Fifteen rats underwent tooth extraction without a bone graft. Fifteen rats underwent tooth extraction followed by Xenograft bone graft placement.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eTime points for evaluation:\u0026nbsp;\u003c/strong\u003e2, 4, and 6 weeks post-extraction.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eEthical Considerations:\u0026nbsp;\u003c/strong\u003eAll procedures are carried out in\u0026nbsp;accordance with the ethical guidelines for animal research and approved by Pharos University (PUA 02202410273282).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e- Anesthetics and Analgesics: Ketamine (60 mg/kg) for general anesthesia. Xylazine (10 mg/kg) to enhance sedation by intraperitoneal injection for general anesthesia.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eBone Graft Materials:\u0026nbsp;\u003c/strong\u003eXenograft bone graft (OneGraft) (lab-made).\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eHistological Analysis\u003c/strong\u003e: 10% Formalin solution for tissue fixation. Decalcifying solution (e.g., EDTA-based solution) for bone decalcification. Hematoxylin and Eosin (H\u0026amp;E) staining kit for histological assessment of bone healing. Immunohistochemically (Masson\u0026apos;s trichrome stain) reagents for markers of osteogenesis (e.g., Osteocalcin, Runx2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSurgical Procedure:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eThe aseptic technique\u003c/strong\u003e is followed throughout the procedure.\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eTooth Extraction:\u003c/strong\u003e The tooth is extracted using forceps. Care is taken to avoid damage to the surrounding bone. The Extraction aimed to create a bone defect.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eBone Graft Placement:\u003c/strong\u003e After Extraction, the alveolar socket is debrided to remove any remaining soft tissue and bone debris. A bone graft is prepared and inserted into the socket to fill the defect using xenograft and finely minced before graft placement. The graft is compacted to ensure stable placement within the socket. The surgical site is irrigated with sterile saline to maintain cleanliness\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eClosure:\u003c/strong\u003e The mucosal incision is sutured using absorbable sutures (3-0 Vicryl) in a simple interrupted pattern\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003ePostoperative Care:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals are kept in a warm recovery area until they are fully awake from anesthesia. Postoperative analgesia is administered (Carprofen 5 mg/kg) for 3 days. An antibiotic (Enrofloxacin 10 mg/kg) was given for 5 days to prevent infection. Rats are monitored daily for signs of infection, pain, or complications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eII. \u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHistological procedure\u003c/strong\u003e\u003csup\u003e(28)\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eFixation:\u0026nbsp;\u003c/strong\u003eThe left half of the mandibles was fixed immediately in 10% neutral buffered formalin solution for 48 hours:\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003ePreparation of the 10% neutral buffered formalin solution:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e40% formalin (100 ml)\u003c/li\u003e\n \u003cli\u003eTap water (900ml)\u003c/li\u003e\n \u003cli\u003eSodium phosphate monobasic (4.0gm)\u003c/li\u003e\n \u003cli\u003eSodium phosphate dibasic anhydrous (6.5gm)\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e- \u003cstrong\u003eWashing:\u0026nbsp;\u003c/strong\u003eAfter fixation, the specimens were washed in running water to remove the formalin.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eDecalcification:\u0026nbsp;\u003c/strong\u003eThe specimens were decalcified in 8% trichloroacetic acid and were tested for complete decalcification by piercing the hard tissue with a needle far from the target socket. The tissue was ready for further treatment when the needle easily entered the bone. Then, the mandibles were washed in running water for at least 24 hours to remove all the acid.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eDehydration:\u0026nbsp;\u003c/strong\u003eThe specimens were gradually dehydrated by passing through increasing percentages of ethyl alcohol (40%, 60%, 80%, 95%, and absolute alcohol), remaining in each dish for several hours.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eClearance:\u0026nbsp;\u003c/strong\u003eThe specimen is passed from alcohol through two changes of xylene, a clearing agent that is miscible with both alcohol and paraffin.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eInfiltration:\u0026nbsp;\u003c/strong\u003eThe specimens were removed from the xylene to be infiltrated with paraffin. They were placed in a dish of melted embedding paraffin, and the dish was put into a constant-temperature oven regulated to about 60\u0026deg; C. The specimens were then left in the oven for 2 hours.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eEmbedding:\u0026nbsp;\u003c/strong\u003eWhen the specimen is completely infiltrated with paraffin, it is embedded in a small paper box filled with melted paraffin, and with warm forceps, the specimen is removed from the dish of melted paraffin and placed in the center of the box of paraffin in a direction to obtain a longitudinal bucco-lingual section.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eCutting:\u0026nbsp;\u003c/strong\u003eThe hardened paraffin block was mounted on a paraffin-coated wooden cube and then clamped on a precision rotary microtome, which was adjusted to cut serial sections of 5 \u0026mu;m in thickness.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eMounting:\u0026nbsp;\u003c/strong\u003eThe wax ribbons were mounted on the glass slide and then placed in a constant-temperature oven.\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eStaining:\u0026nbsp;\u003c/strong\u003eThe stains used in this study were H\u0026amp;E (Hematoxylin and Eosin) and trichrome stains.\u003c/p\u003e\n\u003cp\u003e\u0026middot; Haematoxylin and Eosin stain\u003csup\u003e(29)\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u0026middot; Erlish\u0026apos;s hematoxylin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStaining of the specimen\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026middot; Sections were deparaffinized in xylene and then transferred into descending grades of alcohol. (100%,90%,70%,50%)\u003c/p\u003e\n\u003cp\u003e\u0026middot; Sections were brought down to the water for 3 minutes.\u003c/p\u003e\n\u003cp\u003e\u0026middot; Then, sections were stained with hematoxylin for 5 minutes.\u003c/p\u003e\n\u003cp\u003e\u0026middot; After that, sections were washed in running tap water for 3 minutes.\u003c/p\u003e\n\u003cp\u003e\u0026middot; The excess stain was removed by decolorizing (differentiating) in 0.5-1% hydrochloric acid\u003c/p\u003e\n\u003cp\u003e\u0026middot; in 70% alcohol for a few seconds. Thus, the blue staining of the hematoxylin was changed to red by the action of the acid.\u003c/p\u003e\n\u003cp\u003e\u0026middot; The decolorization was stopped, and the blue color was regained by washing it in alkaline and running tap water for at least 5 minutes.\u003c/p\u003e\n\u003cp\u003e\u0026middot; The sections were stained in a 1% solution of Eosin for 1 minute and then washed in water.\u003c/p\u003e\n\u003cp\u003e\u0026middot; Dehydration in ascending grades of alcohol, clearing in xylene, and mounting with Canada Balsam were performed after staining and washing.\u003c/p\u003e\n\u003cp\u003eThen, the sections were ready for examination with the light microscope.\u003c/p\u003e\n\u003cp\u003eFollowing mandibular dissection, the left hemimandible of each specimen was processed for light microscopic (LM) analysis to assess histological alterations across experimental groups. Specimens were fixed in 10% neutral-buffered formalin and subsequently decalcified in 10% ethylenediaminetetraacetic acid (EDTA) solution (pH 7.0\u0026ndash;7.4) at room temperature, with frequent solution replacement to ensure effective decalcification. After thorough rinsing under running water, the tissues were processed routinely and embedded in paraffin blocks according to standard histological procedures⁽\u0026sup1;⁾. Serial sections of 5 \u0026mu;m thickness were obtained and stained with hematoxylin and eosin (H\u0026amp;E) and mason\u0026apos;s trichrome stains for general morphological assessment⁽\u003csup\u003e1\u003c/sup\u003e⁾. Histological examination was performed using a light microscope equipped with a digital imaging system (Leica ICC50 HD), and representative fields were photographed and appropriately labeled.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIII.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBlood sample examination\u0026nbsp;\u003c/strong\u003e\u003csup\u003e(3,28,30)\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were collected from the tail vein of the rats in glass tubes without anticoagulant and left at room temperature for 30 min for spontaneous clotting. Clotted blood was centrifuged at 2000 rpm for 5 min at 4\u0026deg;C, pipette off the top yellow serum layer without disturbing the white buffy layer. The serum was frozen at -80\u0026deg;C for analysis, and the serum was used for the determination of the following parameters: Some inflammatory markers and interleukins such as C-reactive protein (CRP), Procalcitonin, Tumor necrosis factor- \u0026alpha; (TNF-\u0026alpha;), interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-1\u0026beta; (IL-1\u0026beta;). Also, some Bone formation markers, such as Bone morphogenetic protein 1 (BMP1) and Osteocalcin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of inflammatory markers and interleukins in serum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCRP was measured using ELISA kits (Cat. No. CYT294) purchased from Millipore Laboratory Corporation (USA and Canada). Procalcitonin was measured using an ELISA kit (Cat. No. RK03873) purchased from Abclonal Technologies (Wuhan, China). TNF-\u0026alpha;, IL-6, IL-1\u0026beta;, and IL-10 were measured using ELISA kits (Cat. No. RK00029) for TNF-\u0026alpha;, (Cat. No. ER0042) for IL-6, (Cat. No. ER1094) for IL-1\u0026beta;, (Cat. No. ER0033) for IL-10, purchased from Finetest biotech Inc. (USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of some bone formation markers in serum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMP was measured using an ELISA kit (Cat. No. RE2765R) purchased from Reed Biotech Ltd (Wuhan, China). Osteocalcin was measured using an ELISA kit (Cat. No. RK09279) purchased from Abclonal technologies (Wuhan, China).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThis study included 45 rats and was divided equally into three groups: Control, Extraction without bone graft, and Extraction with bone graft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI.\u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHistological Results:\u0026nbsp;\u003c/strong\u003eFigure (1)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eHematoxylin and Eosin (H\u0026amp;E) Stainin\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eg\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eControl Group (No extraction):\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe alveolar crest was intact and positioned normally just below the cemento-enamel junction. The alveolar bone exhibited well-organized lamellar architecture from the crest to the apical region, including the interradicular areas adjacent to the periodontal ligament. Osteocytes were evenly distributed within the mature bone matrix, and no evidence of inflammation or tissue disruption was observed throughout the study period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction-Only Group\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 2 weeks, the extraction socket exhibited a loose connective tissue matrix with marked inflammatory infiltrate. Early granulation tissue was evident, and minimal new bone formation was observed at the periphery of the socket. By 4 weeks, a moderate degree of trabecular bone extended from the socket walls, while central fibrous connective tissue persisted. The inflammatory response had decreased compared with the earlier time point. At 6 weeks, bone trabeculae were more prominent, with areas of woven bone forming within the socket; however, healing remained incomplete, as residual fibrous tissue continued to occupy the central defect.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction + Bone Graft Group:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 2 weeks, graft particles were clearly identifiable and surrounded by early connective tissue infiltration. Mild inflammatory cell presence was noted, and early osteoid formation was observed adjacent to the graft surfaces. By 4 weeks, osteoblasts were lining the newly forming bone around the graft particles, and the amount of woven bone was greater than in the Extraction-only group. Inflammatory cells were markedly reduced, indicating progression of healing. At 6 weeks, the defect exhibited abundant newly formed bone with mature trabecular structures. The graft particles were partially resorbed and integrated with host bone. Bony trabeculae were dense, containing cellular, normally sized bone marrow spaces lined by endosteal cells with visible reversal lines. Osteocyte lacunae were of normal size and distribution, and the tissue architecture closely resembled native bone, indicating advanced bone healing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eMasson's Trichrome Staining\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Masson's Trichrome confirms (H\u0026amp;E) results and highlights improved collagen deposition and tissue organization in the grafted group. The maturation of bone matrix is more advanced at each interval compared to the extraction-only group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eControl Group:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStaining revealed dense collagen-rich connective tissue, and mature lamellar bone stained deep blue. The organization of collagen bundles was uniform and compact.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction-Only Group:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this group, collagen deposition progressed over time. At 2 weeks, predominantly red-stained areas indicated immature connective tissue and early granulation tissue, with sparse collagen. By 4 weeks, collagen content increased (light blue areas) and showed early organization around forming trabeculae. At 6 weeks, moderate blue staining suggested further collagen deposition, but the bone matrix remained immature compared with controls, with fibrous tissue still present within the socket.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction + Bone Graft Group:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe group showed accelerated collagen maturation. At 2 weeks, blue-stained collagen fibers surrounded graft particles, and the extracellular matrix showed early organization. By 4 weeks, staining intensified and was more widespread, indicating progressive collagen maturation, particularly at bone-graft interfaces. At 6 weeks, a well-organized, collagen-rich bone matrix was observed, with dense blue staining of newly formed trabeculae, complete resorption of graft remnants, evident reversal lines, and mature, parallel collagen fibers, indicating advanced remodeling and restoration of native bone architecture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eII.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHematological Results\u0026nbsp;\u003c/strong\u003e\u003cem\u003e(data are presented as mean ± SD)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eCRP (mg/L) Table (1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCRP levels differed significantly among the three groups at 4 and 6 weeks (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001). CRP decreased from 4 to 6 weeks in both Extraction-only (\u003cem\u003ep=.\u003c/em\u003e009) and Extraction + Bone Graft groups (\u003cem\u003ep=.\u003c/em\u003e024), with no change in the Control group (\u003cem\u003ep=.\u003c/em\u003e417). Pairwise comparisons showed CRP was lower in the grafted group than in the Extraction-only group (\u003cem\u003ep=.\u003c/em\u003e001), and lowest in the Control group (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001).\u003c/p\u003e\n\u003cp\u003e-\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cstrong\u003eBMP1 (pg/mL) Table (2)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMP-1 levels differed significantly among the three groups at 4 and 6 weeks (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001). Levels increased from 4 to 6 weeks in the Extraction-only group (\u003cem\u003ep=.\u003c/em\u003e037), while no significant changes were observed in the Control (\u003cem\u003ep=1.\u003c/em\u003e000) or Extraction + Bone Graft groups (\u003cem\u003ep=.\u003c/em\u003e471).\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eOsteocalcin (ng/mL) Table (3)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOsteocalcin levels differed significantly among the three groups at 4 and 6 weeks (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001). Levels increased from 4 to 6 weeks in the Extraction + Bone Graft group (\u003cem\u003ep=.\u003c/em\u003e040), while no significant changes were observed in the Control (\u003cem\u003ep=.\u003c/em\u003e800) or Extraction-only groups (\u003cem\u003ep=.\u003c/em\u003e106).\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eTNF-alpha (ng/mL) Table (4)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTNF-α levels differed significantly among the three groups at 4 and 6 weeks (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001). Levels decreased from 4 to 6 weeks in both the Extraction-only (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001) and Extraction + Bone Graft groups (\u003cem\u003ep=.\u003c/em\u003e001), while no significant change was observed in the Control group (\u003cem\u003ep=.\u003c/em\u003e715).\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eProcalcitonin (pg/mL)Table (5)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProcalcitonin levels differed significantly among the three groups at 4 and 6 weeks (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001). Levels decreased from 4 to 6 weeks in both the Extraction-only (\u003cem\u003ep=.\u003c/em\u003e017) and Extraction + Bone Graft groups (\u003cem\u003ep=.\u003c/em\u003e001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIL-6 (pg/mL)Table (6)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL-6 levels differed significantly among the three groups at 4 and 6 weeks (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001). Levels decreased from 4 to 6 weeks in both the Extraction-only and Extraction + Bone Graft groups (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001 for both), while no significant change was observed in the Control group (\u003cem\u003ep=.\u003c/em\u003e905).\u003c/p\u003e\n\u003cp\u003e- \u003cstrong\u003eIL-1B (pg/mL) Table (7)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL-1β levels differed significantly among the three groups at 4 and 6 weeks (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001). Levels decreased from 4 to 6 weeks in the Extraction-only group (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001), while no significant change was observed in the Control (\u003cem\u003ep=.\u003c/em\u003e197) or Extraction + Bone Graft groups (\u003cem\u003ep=.\u003c/em\u003e052).\u003c/p\u003e\n\u003cp\u003e-\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cstrong\u003eIL-10 (pg/mL)Table (8)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL-10 levels differed significantly among the three groups at 4 and 6 weeks (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001). Levels remained unchanged in the Control group (\u003cem\u003ep=.\u003c/em\u003e850), decreased in the Extraction-only group (\u003cem\u003ep=.\u003c/em\u003e001), and increased in the Extraction + Bone Graft group (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001) from 4 to 6 weeks.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study aimed to establish a reliable animal model simulating a clinical environment for evaluating bone regeneration following tooth extraction, and to assess the effect of bone grafting on the healing process through histological and histochemical analysis at various time intervals. Our findings demonstrate that bone grafting substantially accelerates and enhances alveolar bone regeneration compared to ungrafted defects.\u003c/p\u003e \u003cp\u003eHistological evaluation using H\u0026amp;E staining revealed that the extraction-only group exhibited a typical healing response characterized by initial granulation tissue formation, followed by progressive but incomplete bone regeneration over six weeks. This is in accordance with previous studies describing the natural sequence of socket healing, where inflammatory cell infiltration and fibrous tissue predominated in early stages, followed by delayed woven bone formation.\u003csup\u003e(31)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn contrast, defects treated with bone grafts showed significantly improved healing outcomes. As early as two weeks, the presence of osteoid formation and mild inflammatory response indicated early osteogenic activity around graft particles. By six weeks, grafted sockets demonstrated mature trabecular bone formation with clear evidence of graft integration and resorption. This suggests that the graft served as an osteoconductive scaffold, facilitating cellular migration and bone matrix deposition, consistent with the established biological role of bone substitutes in enhancing osteogenesis.\u003csup\u003e(3,32)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results were further substantiated by Masson\u0026rsquo;s Trichrome staining, which provided insight into the extracellular matrix and collagen maturation. Collagen deposition was sparse and disorganized in the early stages of healing in the extraction-only group. Although gradual organization was noted by six weeks, the extent of matrix maturation remained inferior to that observed in the grafted group. In grafted sockets, collagen fibers were more abundant and better organized throughout the healing period, with robust matrix formation clearly supporting advanced bone tissue development. The control group, as expected, showed normal histological architecture with mature lamellar bone and dense collagen matrix, providing a baseline for comparison. The striking differences in the healing trajectory between the grafted and non-grafted groups underscore the clinical importance of biomaterial application in enhancing socket healing, particularly in cases where natural healing is compromised or slow.\u003c/p\u003e \u003cp\u003eOur findings align with multiple studies reporting improved bone formation with various grafting materials, including xenografts and composite bioactive scaffolds.\u003csup\u003e(33\u0026ndash;35)\u003c/sup\u003e The accelerated healing observed in our grafted group may be attributed not only to the osteoconductive properties of the graft material but also to its potential to stabilize the clot and maintain space for tissue ingrowth.\u003c/p\u003e \u003cp\u003eTrauma from tooth extraction can trigger inflammation by activating immunocompetent cells such as macrophages and mast cells, which stimulate the production of pro-inflammatory cytokines, particularly TNF-α and IL-1.\u003csup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e)\u003c/sup\u003e Even after the extraction wound has healed, mechanical stimuli from mastication may reactivate these cytokines via neurogenic pathways, leading to continued alveolar bone resorption. \u003csup\u003e(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eProinflammatory cytokines such as IL-1, IL-2, IL-6, IL-8, and TNF-α are highly sensitive diagnostic markers since their levels can rise both systemically in the blood and locally in oral fluids. \u003csup\u003e(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e Acting as antigen-nonspecific defense factors, they play a central role in immune and inflammatory responses. In the context of dental implant surgery, an imbalance between pro- and anti-inflammatory cytokines may impair osseointegration and promote peri-implant tissue destruction. \u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/sup\u003e Moreover, uncontrolled cytokine alterations reflect heightened inflammatory and tissue-destructive activity in the oral cavity and maxillofacial region, as these mediators are released by lymphocytes, macrophages, serum transudate, and salivary gland secretions during inflammatory events or surgical interventions. \u003csup\u003e(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA thorough evaluation of inflammatory markers is crucial for planning dental implant and maxillofacial treatments. Markers such as IL-1, IL-2, IL-6, IL-8, TNF-α, and C-reactive protein are linked to systemic and oral disease pathogenesis and help predict treatment outcomes. Measuring these markers in blood or saliva is especially useful for patients with systemic conditions, as oral and systemic inflammation can mutually influence overall health.\u003c/p\u003e \u003cp\u003eThe present study revealed that all inflammatory markers (CRP, TNF-α, Procalcitonin, IL-6, and IL-1β) were elevated in both experimental groups compared with the control, consistent with the acute inflammatory response to surgical trauma and tissue injury. \u003csup\u003e(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e At four weeks, the extraction without bone graft group showed persistently higher levels of systemic inflammation compared to the extraction with bone graft group. By six weeks, both groups demonstrated a reduction, but inflammatory markers in the grafted group approached control values, whereas the ungrafted group remained elevated. These findings suggest that bone grafting not only preserves the alveolar ridge but also helps modulate the host inflammatory response, promoting a faster return to baseline systemic conditions.\u003c/p\u003e \u003cp\u003eThese findings suggest that bone grafting after tooth extraction not only preserves the alveolar bone but also modulates the post-extraction inflammatory response, promoting a faster return to baseline systemic inflammation. By stabilizing the extraction site and reducing tissue trauma, grafting may create a more controlled healing environment that limits the release of inflammatory mediators such as CRP, TNF-α, IL-6, IL-1β, and Procalcitonin. Consequently, patients receiving grafts tend to show a quicker reduction of systemic inflammation compared with those left ungrafted, whose inflammatory markers remain elevated for a longer period. CRP, IL-6, and TNF-α are well-established markers of systemic inflammation and have been widely studied in periodontal and peri-implant healing, while elevated Procalcitonin\u0026mdash;typically linked with systemic infection\u0026mdash;may also reflect the acute inflammatory burden of surgical trauma. \u003csup\u003e(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e The earlier reduction of these mediators in the grafted group highlights the potential systemic benefits of socket preservation, particularly in medically compromised patients where prolonged inflammation may impair healing or exacerbate comorbidities.\u003c/p\u003e \u003cp\u003eThese results suggest that bone grafting not only aids in ridge preservation but also stabilizes the wound environment, dampening prolonged cytokine release and facilitating faster normalization of systemic inflammation. Such modulation may be particularly beneficial in medically compromised patients, where persistent inflammation could impair healing or exacerbate comorbidities.\u003c/p\u003e \u003cp\u003eThe reduction in inflammatory markers observed in the bone-grafted group may be attributed to several mechanisms. Placement of a graft stabilizes the extraction socket, minimizes collapse of the alveolar walls, and reduces secondary trauma, thereby attenuating the release of pro-inflammatory cytokines. Avila-Ortiz et al. \u003csup\u003e(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e)\u003c/sup\u003e, in a systematic review and meta-analysis, confirmed that alveolar ridge preservation (ARP) through socket grafting effectively limits post-extraction bone loss in nonmolar sites, both horizontally and vertically. Furthermore, xenografts and demineralized freeze-dried bone matrices provide a scaffold for cell migration and angiogenesis, facilitating tissue repair with complete osseous integration. \u003csup\u003e(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)\u003c/sup\u003e This controlled environment may explain the quicker normalization of systemic inflammation in grafted sockets compared to prolonged cytokine release in ungrafted sites. Similarly, Canullo et al. (2021) \u003csup\u003e(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e)\u003c/sup\u003e reported that patients receiving bone grafts after extraction showed reduced inflammatory levels and fewer soft tissue healing complications, although the differences did not reach statistical significance. Together, these findings suggest that bone grafting not only preserves alveolar bone but also contributes to the modulation of the systemic inflammatory response.\u003c/p\u003e \u003cp\u003eInflammation is primarily driven by activated macrophages, which release pro-osteolytic mediators such as TNF-α, IL-1β, IL-6, and IL-8. These cytokines promote monocyte recruitment, inhibit osteoblastic differentiation, and induce osteoclastogenesis, making macrophage activation a central factor in peri-implant osteolysis.\u003csup\u003e(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u003c/sup\u003e The production of inflammatory cytokines by peripheral blood monocyte-derived macrophages (PBMMs) in response to biomaterials is therefore critical, as it significantly influences tissue integration and regeneration. Rani et al. (2024)\u003csup\u003e(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e)\u003c/sup\u003e investigated the immune response to demineralized and decellularized bovine bone substitutes and found that these materials did not trigger significant inflammatory responses in PBMMs, suggesting their potential to reduce inflammation during bone healing. In contrast, treatment with a demineralized bone (DMB) substitute resulted in markedly higher proinflammatory cytokine expression, with TNF-α increasing 3.4-fold (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and IL-1β mRNA rising 21.75-fold (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to controls and decellularized bone (DCC, 5.01-fold). Similarly, studies using murine air pouch models have demonstrated that different commercially available demineralized bone matrices (DBM) elicit variable inflammatory reactions, underscoring the importance of considering both the efficacy and immunological safety of graft processing methods. \u003csup\u003e(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMoreover, modifications in the physico-chemical properties of biomaterials can promote macrophage polarization favorable for tissue regeneration. \u003csup\u003e(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/sup\u003e For example, an in vitro pilot study by Panahipour et al. \u003csup\u003e(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/sup\u003e demonstrated that DBM reduced IL-1β and IL-6 expression in macrophage cells, suggesting that certain DBM preparations can modulate inflammatory responses. These conflicting results likely stem from differences in graft processing techniques, which alter the physico-chemical and topographical characteristics of the materials, as well as the potential presence of residual native cells capable of provoking inflammation.\u003c/p\u003e \u003cp\u003eInflammation triggered by tooth extraction is characterized by the release of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α, which impair osteoblastic differentiation, stimulate osteoclastogenesis, and contribute to alveolar bone resorption. Beyond localized bone changes, these cytokines may also influence systemic metabolism, contributing to hyperlipidemia and vascular dysfunction. Elevated levels of CRP and fibrinogen further amplify this systemic response, with CRP synthesized by the liver in reaction to inflammatory signals from oral infections such as periodontitis.\u003csup\u003e(31, 32)\u003c/sup\u003e Esteves-Lima et al.\u003csup\u003e(32)\u003c/sup\u003e reported that individuals with periodontitis exhibited significantly higher CRP levels, supporting the link between oral inflammation and systemic disease. Clinical data reinforce this connection: D\u0026rsquo;Aiuto et al. \u003csup\u003e(33)\u003c/sup\u003e observed a rise in CRP 24 hours after periodontal therapy or extractions, while Ide et al. \u003csup\u003e(34)\u003c/sup\u003e found rapid IL-6 and TNF-α elevations within two hours of subgingival scaling.\u003c/p\u003e \u003cp\u003eHowever, Bahrani-Mougeot et al. \u003csup\u003e(35)\u003c/sup\u003e found no clear association between inflammation severity and the 12 cytokines assessed, particularly IL-6 and TNF-α. Interestingly, individuals with healthier periodontal status tended to show higher circulating cytokine levels compared to those with poorer periodontal health, though the differences were not statistically significant. Consistent with these findings, our results also suggest that TNF-α shows little correlation with inflammatory status. Given that cytokines are often present at very low concentrations in plasma, the possibility of false-negative results must be considered.\u003c/p\u003e \u003cp\u003eUsing a xenograft for socket preservation helps maintain alveolar ridge volume, provides a scaffold for bone regeneration, and stabilizes healing by moderating inflammation. Ungrafted sockets, in contrast, show faster resorption, unpredictable bone healing, and potential complications for prosthetic rehabilitation. IL-1β and IL-6 amplify inflammation by promoting chemokine release, prostaglandin production, and connective tissue degradation. They recruit neutrophils, monocytes, and fibroblasts, supporting normal healing when regulated, but sustained elevations can delay wound repair. IL-6 in gingival tissues also aids healing by protecting open wounds from bacterial invasion. \u003csup\u003e(36)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eKarnes et al. \u003csup\u003e(37)\u003c/sup\u003e demonstrated that TNF-α enhances mesenchymal cell recruitment by upregulating intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), thereby improving intrinsic migration capacity. It also promotes the proliferation and differentiation of mesenchymal precursor cells into chondrogenic and osteogenic phenotypes, partly through the induction of early BMP-2 secretion in neighboring osteoblasts. This dual role\u0026mdash;supporting mesenchymal stem cell repopulation at physiological levels while driving osteoclastogenesis when elevated\u0026mdash;positions TNF-α as both a critical regulator of bone remodeling and a potential therapeutic target. However, Freitas et al. \u003csup\u003e(38)\u003c/sup\u003e reported that blocking TNF-α, while suppressing acute inflammation, impaired healing by delaying cell migration, reducing fibroblast activity and collagen maturation, and limiting angiogenesis. Similarly, Saim et al. \u003csup\u003e(39)\u003c/sup\u003e highlighted TNF-α\u0026rsquo;s role in post-odontectomy swelling through increased vascular permeability, with IL-1 and IL-6 further enhancing immune cell recruitment and fluid accumulation at the injury site.\u003c/p\u003e \u003cp\u003eWhen a socket is left ungrafted, extraction trauma leads to a pronounced and prolonged inflammatory response, reflected by elevated systemic and local markers such as CRP, TNF-α, Procalcitonin, IL-6, and IL-1β. These cytokines and proteins drive osteoclastic activity, suppress osteoblastic differentiation, and prolong tissue breakdown, resulting in accelerated bone resorption and delayed healing.\u003c/p\u003e \u003cp\u003eBone formation markers (BMPI, Osteocalcin, and IL-10) were initially lower in both experimental groups compared with the control. Over time, the extraction with bone graft group demonstrated a significant increase in all three markers, particularly at six weeks, whereas the extraction without bone graft group showed either minimal improvement (BMPI, Osteocalcin) or a decline (IL-10).\u003c/p\u003e \u003cp\u003eThese findings suggest that bone grafting positively influences the expression of bone formation markers. The significant increase in BMPI, Osteocalcin, and IL-10 in the grafted group indicates enhanced osteogenic activity and a more favorable anti-inflammatory environment, which supports faster and more effective bone regeneration. In contrast, the minimal improvement or decline observed in the non-grafted group highlights the limited regenerative potential of extraction sites without grafting, emphasizing the benefits of bone grafts in promoting both bone formation and immunomodulation during healing.\u003c/p\u003e \u003cp\u003eInflammation, on the other hand, can increase osteoclast numbers through the action of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β), as well as via the receptor activator of nuclear factor-κB ligand (RANKL) and its receptor RANK. \u003csup\u003e(40, 41)\u003c/sup\u003e The RANK\u0026ndash;RANKL system is a central regulator of bone remodeling, with TNF and the TNF receptor family further influencing cell proliferation and apoptosis. Elevated osteoclast activity driven by this pathway ultimately contributes to alveolar bone resorption.\u003csup\u003e(42)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eKresnoadi et al. \u003csup\u003e(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/sup\u003e reported that the DFDBBX\u0026thinsp;+\u0026thinsp;PEG group showed an increase in osteoblasts and a decrease in osteoclasts at 7 and 30 days compared with the PEG (control) group, indicating enhanced bone regeneration. A significant difference in RANKL expression (P\u0026thinsp;=\u0026thinsp;0.002\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was also observed between the groups, suggesting reduced inflammation due to socket preservation with DFDBBX. Additionally, osteocalcin levels in the DFDBBX-only group were higher than in the control at both time points, reflecting its osteogenic potential, as osteoblasts synthesize non-collagenous proteins such as osteocalcin. Consistently, Khan \u003csup\u003e(43)\u003c/sup\u003e reported that placing graft material into the extraction socket can promote osteoinduction and stimulate new bone formation.\u003c/p\u003e \u003cp\u003eMotamedian et al. \u003csup\u003e(44)\u003c/sup\u003e demonstrated that demineralized bovine bone matrix (DBBM) stimulates the expression of both early and late osteogenic genes in dental pulp stem cells (DPSCs) after one week in osteogenic medium and two weeks in standard medium, whereas Seebach et al.\u003csup\u003e(45)\u003c/sup\u003e reported no early osteogenic gene expression when mesenchymal stem cells were cocultured with DBBM for 10 days. In terms of bone formation, markers such as BMP1, osteocalcin, and IL-10 initially showed lower levels than the control, likely due to the immediate catabolic response following extraction. Over time, however, the grafted group exhibited significant increases in all three markers, particularly by week six. Osteocalcin, a non-collagenous protein secreted by osteoblasts during bone matrix formation, and BMP1, a critical regulator of extracellular matrix assembly, were markedly higher in grafted sockets, reflecting enhanced osteoblastic activity and accelerated bone remodeling. \u003csup\u003e(46)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eInterestingly, IL-10, an anti-inflammatory cytokine with immunomodulatory and pro-healing properties, increased significantly only in the grafted group, whereas the non-grafted group showed a decline. \u003csup\u003e(47)\u003c/sup\u003e This supports the notion that bone grafting fosters a balanced inflammatory response, promoting a faster transition from a pro-inflammatory to a regenerative phase. Short et al. \u003csup\u003e(48)\u003c/sup\u003e further highlighted IL-10\u0026rsquo;s role in regulating inflammation while preserving the integrity of the healing process. Socket preservation with a xenograft stabilizes the clot and protects the wound from excessive collapse, reducing the release of pro-inflammatory mediators and creating a more controlled, less destructive healing environment compared with ungrafted sockets.\u003c/p\u003e \u003cp\u003eSocket preservation with a xenograft moderates the post-extraction inflammatory response by stabilizing the clot and preventing excessive wound collapse, thereby reducing the release of pro-inflammatory mediators. Consequently, inflammatory markers tend to normalize faster in grafted sockets compared with ungrafted ones, creating a more controlled and less destructive healing environment.\u003c/p\u003e \u003cp\u003eSimultaneously, bone formation markers such as BMP1, osteocalcin, and IL-10 show a more favorable trend in grafted sites. Osteocalcin, secreted by active osteoblasts, reflects enhanced osteogenic activity, while BMP1 indicates early bone matrix synthesis. IL-10, an anti-inflammatory cytokine, supports tissue regeneration by downregulating excessive inflammation. In contrast, ungrafted sockets exhibit lower or declining levels of these markers, consistent with impaired bone regeneration.\u003c/p\u003e \u003cp\u003eOverall, xenograft use in socket preservation not only maintains alveolar ridge volume mechanically but also optimizes the biological environment by limiting inflammation and promoting bone formation and remodeling, highlighting its advantage over extraction without grafting.\u003c/p\u003e \u003cp\u003eSivolella et al. \u003csup\u003e(49)\u003c/sup\u003e found that xenografts promoted new bone formation and provided sufficient support for implant placement compared with extraction alone, with histologic outcomes indicating more favorable tissue repair and less connective tissue infiltration. Barbecket al.\u003csup\u003e(50)\u003c/sup\u003e demonstrated that the physical properties of bone substitute materials, including xenografts and synthetic grafts, influence immune cell responses, with macrophages and monocytes modulating the expression of inflammatory and regulatory cytokines such as IL-10, IL-6, IL-1β, IL-4, and TNF-α, ultimately affecting healing outcomes. Supporting this, Lyu et al.,\u003csup\u003e(51)\u003c/sup\u003e showed that IL-10 treatment drives macrophage polarization toward a reparative phenotype, decreasing inflammation and enhancing osteogenesis in mesenchymal stem cells, highlighting the critical role of IL-10 and regulatory pathways in bone regeneration.\u003c/p\u003e \u003cp\u003eTaken together, the findings indicate that Graft placement after tooth extraction preserves the alveolar ridge and modulates systemic inflammation, promoting faster bone formation and improved healing. This creates optimal conditions for future implants or prosthetics, especially in patients with systemic conditions. The accelerated healing likely reflects both the graft\u0026rsquo;s osteoconductive properties and its ability to stabilize the clot and maintain space for tissue ingrowth.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eXenograft socket preservation enhances bone regeneration and maintains alveolar ridge dimensions by promoting osteogenic activity and a balanced inflammatory response. Grafted sites showed organized bone and collagen formation with reduced systemic inflammation, whereas ungrafted sockets exhibited delayed healing and greater ridge resorption. These findings support the clinical potential of xenografts to improve post-extraction healing and optimize conditions for future restorative procedures.\u003c/p\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eRECOMMENDATIONS\u003c/h2\u003e \u003cp\u003eXenografts are recommended for socket preservation to enhance bone regeneration, maintain ridge dimensions, and modulate inflammation, especially in patients planning implants, with systemic risk factors, or in esthetically critical areas. Monitoring biomarkers such as CRP, TNF-α, IL-6, IL-1β, procalcitonin, BMP-1, osteocalcin, and IL-10 can guide individualized post-extraction care. Further clinical trials are needed to confirm these benefits and assess long-term implant and prosthetic outcomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eLIMITATIONS\u003c/h2\u003e \u003cp\u003eThis study has several limitations. The follow-up period of 2, 4, and 6 weeks provides only early insights into bone healing and may not reflect long-term maturation and stability. Evaluation was limited to histological analysis without assessing functional or mechanical properties of the regenerated bone. As an animal study, the findings may not fully translate to human clinical conditions. Only one type of graft material was tested, limiting comparison with other alternatives. Additionally, the sample size may affect the statistical power and generalizability of the results.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDFDBBX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDemineralized Freeze-Dried Bovine Bone Xenograft\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBone Morphogenetic Protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eC-reactive protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor Necrosis Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eWe declare and confirm that the work covered in this manuscript has been conducted only after receiving relevant institutional ethical approvals, PUA02202410273282, Unit of Research Ethics Approval Committee [UREAC], Pharos University in Alexandria. I declare that the submitted manuscript is original, has not been published before, and is not being considered for publication elsewhere. We understand that the Corresponding Author is the contact for the Editorial process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement:\u0026nbsp;\u003c/strong\u003eThe authors received no specific funding for this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e Aliaa A. Habib; surgical procedure, acquisition of data, analysis, and interpretation of data collected. Eman M. Salem; histological procedure and guarantor of the manuscript for final approval. Ghadir Elnawawy; hematological results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors acknowledge Prof. ElSayed Amr Basma for his effort in the statistical analysis of the work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVan der Weijden F, Dell'Acqua F, Slot DE. Alveolar bone dimensional changes of post-extraction sockets in humans: a systematic review. J Clin Periodontol. 2009;36(12):1048\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVittorini Orgeas G, Clementini M, De Risi V, de Sanctis M. Surgical techniques for alveolar socket preservation: a systematic review. Int J Oral Maxillofac Implants. 2013;28(4):1049\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllegrini S Jr., Koening B Jr., Allegrini MR, Yoshimoto M, Gedrange T, Fanghaenel J, et al. Alveolar ridge sockets preservation with bone grafting\u0026ndash;review. Ann Acad Med Stetin. 2008;54(1):70\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePagni G, Pellegrini G, Giannobile WV, Rasperini G. Postextraction alveolar ridge preservation: biological basis and treatments. Int J Dent. 2012;2012:151030.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim S, Kim S-G. Advancements in alveolar bone grafting and ridge preservation: a narrative review on materials, techniques, and clinical outcomes. Maxillofacial Plast Reconstr Surg. 2024;46(1):14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeo V, Bhongade M. Pathogenesis of periodontitis: role of cytokines in host response. Dent Today. 2010;29(9):60\u0026ndash;2. 4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawlor F, Camp R, Greaves M. Epidermal interleukin 1 alpha functional activity and interleukin 8 immunoreactivity are increased in patients with cutaneous T-cell lymphoma. J Invest Dermatol. 1992;99(4):514\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshimura A, Hara Y, Kaneko T, Kato I. Secretion of IL-1β, TNF‐α, IL‐8 and IL‐1ra by human polymorphonuclear leukocytes in response to lipopolysaccharides from periodontopathic bacteria. J Periodontal Res. 1997;32(3):279\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorv\u0026aacute;th A, Mardas N, Mezzomo LA, Needleman IG, Donos N. Alveolar ridge preservation. A systematic review. Clin Oral Investig. 2013;17(2):341\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBartee BK. Extraction site reconstruction for alveolar ridge preservation. Part 1: rationale and materials selection. J Oral Implantol. 2001;27(4):187\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIrinakis T. Rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. J Can Dent Assoc. 2006;72(10):917\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhojasteh A, Kheiri L, Motamedian SR, Nadjmi N. Regenerative medicine in the treatment of alveolar cleft defect: A systematic review of the literature. J Cranio-Maxillofacial Surg. 2015;43(8):1608\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaballero M, Morse JC, Halevi AE, Emodi O, Pharaon MR, Wood JS, et al. Juvenile swine surgical alveolar cleft model to test novel autologous stem cell therapies. Tissue Eng Part C: Methods. 2015;21(9):898\u0026ndash;908.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAd De R, Meijer G, Dormaar T, Janssen N, Van Der Bilt A, Slootweg P, et al. β-TCP versus autologous bone for repair of alveolar clefts in a goat model. Cleft Palate-Craniofacial J. 2011;48(6):654\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOryan A, Alidadi S, Moshiri A, Maffulli N. Bone regenerative medicine: classic options, novel strategies, and future directions. J Orthop Surg Res. 2014;9(1):18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCypher TJ, Grossman JP. Biological principles of bone graft healing. J foot ankle Surg. 1996;35(5):413\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoulet JA, Senunas LE, DeSilva GL, Greenfield MLV. Autogenous iliac crest bone graft: complications and functional assessment. Clin Orthop Relat Research\u0026reg;. 1997;339:76\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrunsvold MA, Mellonig JT. Bone grafts and periodontal regeneration. Periodontology 2000 1993;1(1):80\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNasr HF, Aichelmann-Reidy ME, Yukna RA. Bone and bone substitutes. Periodontol 2000. 1999;19:74\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllegrini S Jr, Koening B Jr, Allegrini M, Yoshimoto M, Gedrange T, Fanghaenel J, et al. editors. Alveolar ridge sockets preservation with bone grafting\u0026ndash;review. Annales Academiae Medicae Stetinensis; 2008.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKresnoadi U, Rahmania PN, Caesar HU, Djulaeha E, Agustono B, Ari MDA. The role of the combination of Moringa oleifera leaf extract and demineralized freeze-dried bovine bone xenograft (xenograft) as tooth extraction socket preservation materials on osteocalcin and transforming growth factor-beta 1 expressions in alveolar bone of Cavia cobaya. J Indian Prosthodont Soc. 2019;19(2):120\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen RE, Alsuwaiyan A, Wang B-Y. Xenografts and periodontal regeneration. J Orthod Endod. 2015;1(1):1\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePilanci O, Cinar C, Kuvat S, Altintas M, Guzel Z, Kilic A. Effects of hydroxyapatite on bone graft resorption in an experimental model of maxillary alveolar arch defects. Arch Clin Exp Surg (ACES). 2013;2:170\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaposo-Amaral CE, Kobayashi GS, Almeida AB, Bueno DF, Freitas FRS, Vulcano LC, et al. Alveolar osseous defect in rat for cell therapy: preliminary report. Acta Cirurgica Brasileira. 2010;25:313\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSawada Y, Hokugo A, Nishiura A, Hokugo R, Matsumoto N, Morita S et al. A trial of alveolar cleft bone regeneration by controlled release of bone morphogenetic protein: an experimental study in rabbits. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2009;108(6):812\u0026thinsp;\u0026ndash;\u0026thinsp;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDalia E-B, Smith SJ, Germane N, Sharawy M. New technique for creating permanent experimental alveolar clefts in a rabbit model. Cleft Palate-Craniofacial J. 1993;30(6):542\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl Deeb M, Horswell B, Waite DE. A primate model for producing experimental alveolar cleft defects. J Oral Maxillofac Surg. 1985;43(7):523\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSikorska E, Wołosz D, Kasarełło K, Koperski Ł, G\u0026oacute;rnicka B, Cudnoch-Jędrzejewska A. Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina. J Visualized Experiments (JoVE) 2024, (210):e66663.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSullivan-Brown J, Bisher ME, Burdine RD. Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin. Nat Protoc. 2011;6(1):46\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalem EM, Abdelfatah OM, Hanafy RA, El-Sharkawy RM, Elnawawy G, Alghonemy WYJBOH. Comparative study of pulpal response following direct pulp capping using synthesized fluorapatite and hydroxyapatite nanoparticles. 2025;25(1):17.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 8 are available in the Supplementary Files section.\u003c/p\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":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alveolar bone preservation, Xenograft, Osteogenesis, Tooth extraction, Inflammatory markers, Bone regeneration, Rat model","lastPublishedDoi":"10.21203/rs.3.rs-8941374/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8941374/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e \u003cp\u003eWhile tooth extraction is a routine dental procedure, it often leads to alveolar bone resorption, which changes the shape and size of the ridge. Over the past years, various bone graft materials have been investigated, with demineralized freeze-dried bovine bone xenograft (DFDBBX) being among the most widely used in dentistry. DFDBBX exhibits both osteoinductive and osteoconductive properties, thereby enhancing new bone formation.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eThis study aimed to evaluate bone healing and systemic biological responses following tooth extraction and socket preservation with a xenograft in an animal model simulating clinical conditions. Histological analysis assessed local bone regeneration, while hematological measurements of pro-inflammatory (CRP, TNF-α, IL-6, IL-1β), anti-inflammatory (IL-10), and bone metabolism markers (Osteocalcin, BMP-1) provided a comprehensive evaluation of local and systemic effects.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eForty-five white rats, animals were divided into three groups: Control (no extraction), Extraction without bone grafting, and Extraction with bone grafting. For histological analysis, tissues were fixed in 10% formalin, decalcified using an EDTA-based solution, and stained with H\u0026amp;E and Masson's trichrome to assess bone healing and osteogenic markers. Blood samples were collected and analyzed for inflammatory markers (CRP, Procalcitonin, TNF-α, IL-6, IL-1β, IL-10) and bone formation markers (BMP-1, Osteocalcin) using commercially available ELISA kits.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCRP, TNF-α, Procalcitonin, and IL-6 differed significantly among groups at 4 and 6 weeks (\u003cem\u003ep\u0026lt;.\u003c/em\u003e001) and decreased over time in both extraction groups. Histologically, the Extraction-only group showed granulation tissue and incomplete trabecular bone by 6 weeks, whereas the Extraction\u0026thinsp;+\u0026thinsp;Bone Graft group exhibited early osteoid at 2 weeks, osteoblast-lined new bone at 4 weeks, and mature trabecular bone with organized collagen and normal osteocytes by 6 weeks. Masson's Trichrome confirmed enhanced collagen deposition and matrix organization in the grafted group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eXenografts enhance post-extraction bone healing by promoting organized trabecular and collagen matrix formation while modulating systemic inflammation, supporting ridge preservation, and creating optimal conditions for future restorative procedures.\u003c/p\u003e","manuscriptTitle":"Evaluation of Xenograft-Enhanced Osteogenesis and Modulation of Systemic Inflammation in Post-Extraction Alveolar Bone Defects (An Experimental Rat Study)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-24 15:02:57","doi":"10.21203/rs.3.rs-8941374/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-02T00:17:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42443328189387432777499245769654068151","date":"2026-04-18T05:30:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T09:08:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-20T14:55:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-26T09:30:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-25T21:06:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2026-02-25T21:01:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cda32cc2-9611-4551-b69e-e52bae4eeab8","owner":[],"postedDate":"April 24th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-02T00:17:37+00:00","index":66,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T15:02:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-24 15:02:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8941374","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8941374","identity":"rs-8941374","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}