In the reconstruction of hard tissue defects, the most ideal bone graft material is the autogenous bone. The autogenous bone exhibits osteogenic, osteoinductive, and osteoconductive potentials simultaneously, while it causes no immune rejection and has the benefit of rapid healing. However, the downside is the limitation of collectible amount and the need for a donor site . To resolve the limitations of allogenic, heterologous, and alloplastic bones, there is an ongoing research and development on various methods, and furthermore, it is necessary to develop an ideal bone graft material like the autogenous bone [4,5,6].
Recently, a bone graft material based on extracted autogenous tooth has been developed and applied to clinical practice, as the material overcoming the demerits of allogenic, heterologous, and alloplastic bones. The autogenous tooth bone graft material (AutoBT) has been developed based on the close correspondence between dental components and bone components. The elaborate collagen structure of tooth provides an ideal environment for the differentiation and proliferation of cells essential in new bone formation, such as bone mesenchymal stem cells and osteoblasts. As an autogenous tissue, AutoBT ensures excellent biocompatibility without immune rejection, and together with the fact that there is no concern for disease infection or need for a donor site [2, 3], the emotional rejection patients used to display before allogenic or heterologous bone graft can be eliminated. AutoBT has also been reported to exhibit not only osteoconductive but also osteoinductive potential [9,10,11,12,13,14,15], and it can be produced in diverse sizes and forms to allow easy application.
The early research on the use of tooth as a graft material mainly focused on inorganic matter, especially on hydroxyapatite. The high-temperature treatment of tooth led to the collection of hydroxyapatite and creation of organic matter-removed particulate dentin that was reported to be suitable as a graft material for increasing the alveolar bone. The burnout of tooth removes the organic matter to leave the inorganic hydroxyapatite and β-TCP as the main components, and this is referred to as particulate dentin. The graft of particulate dentin mediates rapid resorption and substitution by a bone tissue at an early stage, followed by a pattern of reduced resorption. This is due to the composition of particulate dentin; the early rapid resorption is due to β-TCP and the later slow resorption pattern is mainly due to hydroxyapatite. Such burnout method resembles the method of processing heterologous bone, and a high-temperature treatment of tooth forms high crystalline carbonic apatite to cause low resorption in the body, which may have an adverse effect on bone formation [10, 11].
Hydroxyapatite is the main component of the inorganic matter that composes 95% of enamel and 70-75% of dentin, and it plays a crucial role in maintaining the volume of bone graft material and in osteoconduction. The organic matter in dentin is approximately 90% collagen, mostly type I collagen, which plays a key role in calcification. The rest of the organic matter comprises non-collagenous proteins, carbohydrate, lipid, citrate, and lactate . It is known that proteins have various bone growth factors including BMPs that play significant roles in the osteoinductive potential of AutoBT .
Recent studies have focused on the method of demineralization that removes the inorganic matter among the dental components of AutoBT while leaving the organic matter. Rather than the heat treatment method to produce particulate dentin, a reagent is used to demineralize the tooth to remove the inorganic matter, with the product being used in a form of powder or block. The reduced proportion of inorganic content in the graft material induces neonatal bone formation as well as the process of resorption and substitution of the grafted material by a bone tissue, which may improve the beneficial effect of the graft material on bone regeneration. However, the rapid resorption of demineralized graft material may lead to inadequate ability to maintain the space and a drawback of excessive resorption of the graft material .
This study thus set out to examine the osteogenic potential of AutoBT after the removal of organic matter in the dental tissue using sodium chlorate (NaOCl), ethyl alcohol, and chloroform, as well as that after the supplementary addition of type I collagen constituting over 90% of the organic matter of tooth. The in vitro cytological experiments showed that the affinity for cells was the highest in the control, followed by the OM-removed-collagen-treated group and the OM-removed group, while cell adhesion morphology also showed a decrease in adhesion in the above order. A similar result was obtained for the cell proliferation rate. This is a consequence of cell induction effect from the addition of type I collagen, and combining AutoBT with the addition of type I collagen was shown to have led to enhanced osteoinductive potential. The estimated degree of mineralization showed a contrasting result, where comparatively higher values were obtained for the OM-removed group and the OM-removed-collagen-treated group than the control group. This is determined to be due to the increased bone density from the removal of organic matter, which is likely to help AutoBT to enhance the osteoconductive potential and maintain the bone volume.
In addition, this study monitored the changes in rabbit calvarial tissue after having created four same size defects (8 mm) and applied AutoBT without OM and type I collagen. In the control, no treatment was given to the defect site, whereas in the experimental group A, an independent treatment of a graft using AutoBT without OM was given to the defect site, and in the experimental group B, a combined treatment of a graft using AutoBT without OM and a simultaneous addition of 100 mg/ml type I collagen was given to the defect site. At the end of weeks 1, 2, 4, and 6, the rabbits were sacrificed and the levels of bone formation were comparatively observed. Histopathologic and immunohistochemical analyses were also conducted.
The result of histopathologic analysis showed that, in the experimental group grafted with AutoBT without OM, the formation of connective tissue and blood vessel was observed around the graft material at an earlier period than the control, while neonatal bone formation was also detected at an early stage. In the experimental group grafted with AutoBT without OM and additionally administered with type I collagen, more intense vascularization was observed at an earlier period, compared to the other experimental group and the control, with neonatal bone formation detected at an earlier period. In week 4, a characteristic collagen expression in the compact connective tissue immersing the graft material began to be detected, and the expression intensified toward week 6. Also, in week 6, a high level of neonatal bone formation could be seen, compared to the other experimental group and the control, with a large number of osteoblasts observed in the area around the graft material. These findings indicated that, compared to the control, the experimental group grafted with AutoBT exhibits neonatal bone formation at an earlier period, and the additional collagen further promotes the formation. In week 6, the experimental group that received the treatment combining AutoBT and type I collagen led to the detection of a larger number of osteoblasts, which may implicate the possibility of further bone formation at a later stage. To conclude, the combined treatment of AutoBT without OM and type I collagen facilitates the neonatal bone formation while increasing the level of bone formation further, whereby the advantages of AutoBT as a graft material are maximized.
This study went on to conduct immunohistochemical analysis to monitor the expressed level of osteocalcin (OSC), a protein among BMPs, in the tissue of the control and each experimental group, in order to examine the effects of AutoBT without OM and type I collagen treatment on bone formation. OSC is a non-collagenous protein engaged in the accumulation of inorganic matter produced in bone or tooth . Previous studies have found OSC expression in osteoblasts of the periodontal ligament close to the alveolar bone upon tooth movement in rats, which was characterized by the expression of OSC in the bone formation area but a lack of OSC in the resorption area . In humans, OSC is found uniquely in osteoblasts and dentinoblasts, and considering this characteristic, OSC expression can serve as a marker for determining osteogenic potential in the expressed area [21,22,23]. In this study, OSC expression was not observed in the control, but the expression was detected in both experimental groups A and B in week 4. The expressed level was, however, far higher in the experimental group B with additional type I collagen treatment. This was indicative of the strong osteogenic potential by week 4 upon the combined treatment of AutoBT without OM and type I collagen, and the fall in OSC expression in week 6 showed that neonatal bone formation was accompanied with a gradual decrease in osteogenic potential.
The findings of this study suggested that AutoBT without OM played a sufficient role as a bone graft material in the spatial retention and osteoconduction, and that the osteogenic potential was enhanced by additional type I collagen treatment. Nevertheless, the difference in the ability as a bone graft material was not significant, which was presumed to be due to the low level of other bone morphogens such as BMPs. Thus, it is necessary that further studies focus on whether the supplementary addition of bone morphogens and AutoBT without OM could carry out the role of a mediator.
In this study, a graft material based on human autogenous tooth rather than rabbit tooth was used. For the animal experiments, human autogenous tooth bone graft material was used unlike the previous studies where animal tooth was used, because it was predicted that the graft of pre-treated human AutoBT without OM as in conventional cases of allogenic or heterologous bone graft, would not cause immune rejection in rabbit calvaria, in addition to the issue of nutrient supply due to extraction . Based on the findings of this study, AutoBT without OM and the addition of type I collagen is likely to play a sufficient role as a bone graft material in clinical practice. Nevertheless, studies should continue to focus on the immune rejection upon an allogenic graft of AutoBT without OM.