The preservation of the alveolus for jawbone health has been widely discussed in the scientific literature. In the majority of cases, when a tooth is extracted, the surrounding alveolar bone undergoes a series of resorptive processes1. Studies have shown that natural healing post-extraction is associated with a significant loss in vertical and horizontal bone23. Moreover, a variety of factors, including trauma and endodontic pathologies, contribute to the loss of alveolar bone and subsequently complicate restorative treatments such as dental implants and dentures4. Many studies have demonstrated the potential merits of socket preservation following tooth extraction. To date, various materials and techniques have been used to preserve bone after tooth extraction5. For instance, the use of dense hydroxyapatite (HA) and bovine bone mineral integrated in collagen matrix have been reported after tooth extraction67.

Extracted teeth can be used as grafting materials8, and dentin is of particular interest for this purpose due to its unique chemical composition9. Dentin is an acellular, avascular, collagen-rich tissue matrix, compared to bone that is a cellular tissue with vessels. However, dentin and bone have similar components; they both consist of 10% body fluid, 20% organic materials, and 70% minerals (mainly HA) and contain bone morphogenetic proteins (BMPs), insulin-like growth factor (IGF)-II, and transforming growth factor (TGF)-β. Cementum, a bone-like material surrounding tooth roots, also contains TGF-β, IGF-I, and type I and type III collagen1011121314. The osteoinductive potential of dentin was discovered in 196715. Since then, several lines of studies have shown that animal demineralized dentine matrix induces ectopic bone formation in subcutaneous and intramuscular pockets in rodents911. Regarding the biochemical properties of dentin, we sought to assess the regenerative properties of fresh dentin as a novel graft material for alveolar bone regeneration.

This study was conducted on four 2.5-year-old mongrel dogs (male; weight, 25 to 30 kg). These animals were housed in temperature-controlled rooms and lived under a standard 12-hour light/dark cycle. The protocol of this prospective, randomized controlled trial was approved by the ethics committee of the Dental Research Center at Shahid Beheshti University of Medical Sciences (Tehran, Iran). The animals were sedated with an intramuscular injection of 2% acepromazin (0.1 mg/kg; Ingelheim Vetmedica Inc., St. Joseph, MO, USA) and subsequently anesthetized with 10% ketamine hydrochloride (25 mg/kg; Parke-Davis, Morris Plains, NJ, USA) administered intravenously and maintained during anesthesia; 2% lidocaine with 1:80,000 epinephrine (Darouphakhsh, Tehran, Iran) was injected to control any hemorrhaging. The third mandibular premolars on both sides were carefully mobilized with a dental elevator and then removed using forceps without the elevation of a muco-periosteal flap or compromising the marginal gingiva. The sockets were checked after extraction and thoroughly debrided with a curette to remove the periodontal ligament. After irrigation with saline solution, the depth of each socket was measured with a probe. The pulp tissues of the roots were extirpated with a hand instrument, and the root surfaces were cleaned with sterile gauze and a soft periodontal curette. The crown portions of the extracted teeth were removed with cutters, and the root portions of the remaining teeth were collectively trimmed as closely as possible to 350 to 500 µm using a calibrated mesh filter. In one quadrant, dentin and cementum (DC) chips harvested from the extracted teeth were soaked in blood and packed into the fresh sockets (autograft) so that they were completely filled with graft material. In the other quadrant, no bone substitute was placed. The sockets were sutured (Seralene 4-0s; Serag-Wiessner, Naila, Germany), and the animals were observed once a day for any clinical abnormality. Antimicrobial prophylaxis (cephalosporin 15 mg/kg, twice a day) was continued for 48 hours after surgery. Postoperative pain control was achieved via carprofen (50 mg/os/day, Rimadyls; Pfizer Santé Animale, Orsay, France) for 13 days. The dogs were placed on a soft diet throughout the entire observation period. The sutures were removed under a brief period of general anesthesia two weeks after the surgery.

Experiments performed and conducted in accordance with Regional Committee of Ethic complied with the regulations of the European Convention on Vertebrate Animals protection (2005).

Histological examination of group DC at the end of the second week showed particles of unreacted ground dentin and some dilated vessels.(Fig. 2) On the control side, no specific reaction was noted. At the end of eight weeks in group DC, granular tissue with fibroblast cells and collagen fibers was the dominant histological feature, and sparse bone formation was noted in the vicinity of the particles.(Fig. 3) On the control side after eight weeks, the histological examination showed collagen fiber deposition, a few scattered osteoblasts, bone formation in a centripetal pattern, and venules packed with red blood cells.(Fig. 4, 5)

This animal study was conducted to assess the healing of tooth extraction sockets using fresh DC. The results showed that the application of DC induced no effects on the healing process before two weeks. However, after eight weeks, granular connective tissue was prominent in DC sockets. We used descriptive examination in conjunction with morphometric analysis to estimate the SA of newly formed bone. This method provides tangible and quantitative results from histological sections and therefore enables the precise comparison of data18. The results of morphometric analysis revealed that bone formation proceeded at a lower rate in sockets treated with fresh DC. The present results are likely the first report on the use of fresh DC in reconstructive surgery. The application of biomaterials such as dentin has been studied extensively in the field of reconstructive surgery. Our data suggest that the route of preparation is a crucial step in the osteoinductivity of biomaterials19. Studies have shown that demineralizing treatments of bone and dentin increase their osteoinductivity and decrease their antigenicity2021. Nampo et al.22 studied the effect of iliac bone and teeth on new bone formation in the jawbones of rats. The results of their study showed that both types of materials could be used as suitable grafting materials. Kim et al.21 reported that a mixture of heat-pulverized dentin and plaster repaired large jaw defects 57 months after surgery. They concluded that a dentin/plaster mixture was a useful biomaterial in the reconstructive surgery of jaw defects13. Register found that dentin allografts activated bone formation14, and Jeong et al.23 reported that autogenous teeth and bone could be used as good alternatives to autogenous bone. Collectively, the aforementioned studies have reported the potential of dentin as a grafting material. However, our results are not compatible with these studies. The discrepancies between our results and those of other studies may be explained by the wounds or defects, species, age, and method of material preparation. For instance, Nampo et al.22 used the extracted tooth with its pulp as a source of stem cells and neural crest cells. Devecioğlu et al.24 studied the effects of demineralized, freeze-dried DC on periodontal ligaments and fibroblasts. Their results implied that the applied materials had greater potential to form mineral-like nodules than did other applied materials. Gomes et al.25 studied the effects of demineralized dentin matrix on bone repair in diabetic rabbits and showed that dematerialized dentin was biocompatible in diabetic rabbits.

Given the results of these studies and our findings, fresh DC has little capability to induce significant bone formation in the alveolar socket. This result may be explained by the fact that such mineral acts as a barrier to chemotactic and morphogenic molecules of dentin, while acid initiates a demineralizing process that exposes osteoinductive molecules like BMPs. It seems that demineralization of biomaterials such as DC would be a key determinant factor in subsequent inductive/conductive potential of in vivo grafted materials192021. Studies have documented that demineralization of dentin exposes the sequestrated BMPs in the insoluble collagen of the dentin matrix. Decalcified dentin has revealed better bone induction activity due to the activation of BMPs, which bind to collagen matrices through the demineralization process171819202126. Recent data provide evidence that addition of BMP2 to demineralized dentin accelerates its osteoinductivity1626.

In conclusion, the strength of our pilot study is the evidence that fresh mineralized DC autografts have little to no effect on the induction of new bone in the alveolar socket milieu. We suggest using ultrastructural and molecular approaches in similar studies in order to determine the underlying mechanisms.